The sensory-efferent function of capsaicin-sensitive sensory neurons

Gen. Pharmac. Vol. 19, No. I, pp. 1-43, 1988 Printed in Great Britain. All fights reserved

0306-3623/88 $3.00+0.00 Copyright © 1988 PergamonJournals Ltd

REVIEW THE SENSORY-EFFERENT FUNCTION OF CAPSAICIN-SENSITIVE SENSORY N E U R O N S CARLO ALBERTO MAGGI and ALBERTOMELI Pharmacology Department, A. Menarini Pharmaceuticals, Via Sette Santi 3, Florence 50131, Italy (Received 2 March 1987)

Abstraet--Capsaicin-sensitive sensory neurons convey to the central nervous system signals (chemical and physical) arising from viscera and the skin which activate a variety of visceromotor and neuroendocrine reflexes integrated at various levels (intramurally in peripheral organs, at level of prevertebral ganglia, spinal and supraspinal level). Much evidenceis now available that peripheral terminals of certain sensory neurons, widely distributed in skin and viscera have the ability to release, upon adequate stimulation, their transmitter content. In addition to the well-known "axon reflex" arrangement, the capsaicin-sensitive sensory neurons have the ability to release the stored transmitter also from the same terminal which is excited by the environmental stimulus. The efferent function of these sensory neurons is realized through the direct and indirect (i.e. mediated by activation of other cells) effects of released mediators. The action of released transmitters on postjunctional elements covers a wide range of effects which may have a physiological or pathological relevance. Development of drugs capable of controlling the sensory-efferent functions of the capsaicin-sensitivesensory neurons represent a new and very promising area of research for pharmacological treatment of various human diseases. l. INTRODUCTION

believe that it may selectively affect a certain population of sensory neurons have been extensively debated and reviewed (Virus and Gebhart, 1979; Nagy, 1982; Fitzgerald, 1983; Szolcsfinyi, 1982, 1983, 1984a, b; Russell and Burchiel, 1984). A recent review (Buck and Burks, 1986) presents a detailed analysis of the extensive literature which appeared on this topic between 1978 and 1983. To quote those Authors: "There can be little doubt that capsaicin is remarkably specific for primary afferent neurons. This holds true for the initial direct excitatory effect of the compound as well as for the subsequent desensitization and biochemical changes" (Buck and Burks, 1986). The capsaicin-sensitive neuron (Fig. 1) represents a peculiar type of sensory cell able to release the stored transmitters both peripherally and in the central nervous system: at these levels the released transmitters determine the "sensoryefferent" functions (Fig. 1). The discovery that: (a) a distinct population of small, dark, type "B" sensory neurons undergoes a lifelong degeneration and death following the systemic administration of large doses of s.c. capsaicin to newborn rats (Jancs6 et al., 1977, 1981, 1985a; Jancs6 and Kiraly, 1980) and (b) the content of substance P-like immunoreactivity (SP-LI) in sensory areas of the central nervous system (CNS) is depleted following capsaicin desensitization (Jesseil et al., 1978, 1979; Gamse et al., 1980, 1981, 1982a, b) led to the appealing hypothesis that capsaicin may be a specific neurotoxin for SP-neurons. Enthusiasm about capsaicin usefulness as a tool in neuroscience research was dampened by the observation that: (a) SP-LI containing neurons of non-sensory origin are unaffected by capsaicin (see for instance Holzer et al., 1980); (b) in addition to SP, various other neuropeptides may be stored in capsaicin-sensitive sensory

In recent years interest for the dual "sensoryefferent" functions mediated by the capsaicinsensitive sensory neurons (Jancs6 et al., 1967, 1968; Jancsr, 1968; Szolcsfinyi, 1982, 1983, 1984a, b; Maggi and Meli, 1986a) has accumulated, adding new dimensions to the potential pathophysiological role of these mechanisms at both cutaneous and visceral level (Fig. 1) and, also with regard to their ability to play a role on neuroendocrine functions and exert a trophic effect on various tissues. The aim of this article is to review the evidence suggesting that the pharmacological control, modulation or mimicry of the "sensory-efferent" function of capsaicin-sensitive sensory neurons offers a range of pathophysiological targets for development of new drugs to be used for control of pain, inflammation (at both cutaneous and visceral level), bronchial asthma, gastroduodenal ulcers and, more generally speaking, for the control of certain disturbances of visceral motility at respiratory, genitourinary, gastrointestinal and vascular level. In this review, particular emphasis will be given to the role of capsaicin-sensitive nerves in regulating visceral and autonomic function. The aspects related to the role of capsaicin-sensitive nerves in the control of pain and thermoregulation. have been extensively reviewed previously (see Section 3 for references). After acceptance of this paper, a number of contributions appeared in the literature which are relevant for many of the topics discussed here. A selected list of references is presented in the Note added in proof. 2. CAPSAICIN:A TOOL FOR EXPLORING

THE FUNCTIONOF SENSORY NEURONS The pharmacology of capsaicin and reasons to G.P. 19/I--A

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SENSORY NEURON Fig. I. Schematic drawing illustrating the evolution of knowledge about the sensory-efferent functions mediated by the capsaicin-sensitivesensory neurons. In the upper part of the scheme is depicted a classical pseudounipolar sensory neuron which in addition to conveying sensory impulses to the central nervous system, may also release the transmitter in the periphery thorugh an axon reflex arrangement. In this case the release of transmitter is thought to occur at a distinct terminal, specialized for secretion of transmitter and distinct from that which is activated by the environment. The studies of N. Jancs6 et al., (see Szolcs~nyi, 1984a) demonstrated that certain effect which occur following activation of the capsaicinsensitive nerve terminals are produced by secretion of transmitter from the same terminal which is activated by the environmental stimulus. This new discovery has added a new dimension to the question of the sensory-efferent function of sensory neurons, which forms the object of this article. neurons (see for instance Jancs6 et al., 1981, and Section 7 of this review) and (c) various studies failed to demonstrate a correlation between changes in SP levels and time course of functional impairment following systematic capsaicin desensitization (Gamse et al., 1980; Lembeck and Donnerer, 1981; Miller et al., 1982a; Buck et al., 1982; Bittner and Lahann, 1984). These observations led to skepticism about the selectivity of action of capsaicin and, consequently, its usefulness as a tool for exploring sensory neuron function. A further element to be considered is the fact that, in certain instances, the capsaicin-pretreated animals exhibit a variation in tissue levels of certain transmitters which are not thought to have a sensory function. Examples for this are: increased levels of monoamines in rat carotid body (McQueen and Mir, 1984) and thoracic spinal cord (Virus et al., 1983) and depletion of fl endorphin in the hypothalamus (Panerai et al., 1983). The significance of these latter biochemical changes is not immediate: an impressive body of evidence indicates that, in different organs, from various species, capsaicin desensitization spares various types of innervation of non-sensory origin (see Szolcs~nyi and Barth6, 1978; Cervero and McRitchie, 1982; Szolcs/tnyi, 1984a; Martling et al., 1984; Santicioli et aL, 1986; Maggi et aL, 1984a, 1985a, 1986a-d, 1987a, b), supporting the idea that this type of treatment selectively affects only a certain class of sensory nerves. Assuming that the action of capsaicin is really selective for certain sensory fibers, changes in tissue levels of transmitters of non-sensory origin may represent

an epiphenomenon reflecting adaptive changes to deafferentation (see also Buck and Burks, 1986). Likewise, when used in test systems for which a separate assessment of sensory and efferent branch of reflex visceromotor (Cervero and McRitchie, 1982; Maggi et al., 1984a, 1986d) or neuroendocrine responses (Stoppini et al., 1984) was possible, the effects of systemic capsaicin desensitization were confined to a very specific impairment of certain sensory components of the integrated response, suggesting capsaicin as an extremely selective and useful tool in neuroscience research. The "specific" action of capsaicin on sensory nerves is characterized by a well defined pattern of events which on a temporal scale (see Szoics~inyi, 1985) involve first an excitation of the sensory terminal and consequent activation of both sensory and efferent functions. Thereafter, just following the end of the excitatory phase produced by a maximally effective dose of capsaicin, the sensory terminal becomes inexcitable to both natural and artificial (ineluding capsaicin itself) stimuli (see Fig. 2). At this stage, the sensory terminals still contain large amounts of sensory transmitters but they cannot longer transmit sensory information nor produce a local release of the transmitter (see Maggi et al., 1987d). At a later stage, neuropeptide depletion occurs in both peripheral and central terminals of these sensory neurons: this effect may involve multiple mechanisms including a blockade of axonal flow of transmitters from the neuronal body to the terminals (Jancs6 et al., 1980; Gamse et al., 1982c) and also, possibly, blockade of the transport of trophic

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Fig. 2. Typical tracings showing the visceromotor response produced by antidromic activation (field stimulation, F.S.) of the capsaicin-sensitive nerves of the rat isolated ureter (Maggi et al., 1986c, 1987a). A series of rhythmic contraction was induced by addition of kassinin (0.2/~M). Field stimulation (10 Hz, 1.0 msec, 60 V for 5 sec) induced a transient inhibition of motility. Capsaicin (3 #M) has a much more marked inhibitory effect. Note that after acute exposure to capsaicin F.S. did not more inhibit ureteral motility while isoprenaline was effective. Blockade of the action of F.S. could be observed within min from capsaicin administration supporting the idea (Szolcs~.nyi,1985) that the sensory terminals are functionally impaired by capsaicin just after the end of the stimulatory phase.

factors from innervated tissues to the neuronal body (Miller et al., 1982b; Johnson and Yip, 1985; Taylor et aL, 1985). In adult rats a fourth phase occurs after which is characterized by a gradual (and possibly incomplete) recovery of function and parallels the recovery in tissue levels of sensory neuropeptides (Maggi et al., 1987d). On the other hand, following systemic capsaicin desensitization of newborn rats, there is an irreversible cell destruction of about 50% of cells in sensory ganglia (Jancs6 et al., 1977, 1985a). The exact mechanism through which sensory cells undergo degeneration and/or death following exposure to large doses of capsaicin is unknown. In rats desensitized to capsaicin as adults, signs of degeneration were observed at mitochondrial level (Joo et al., 1969; Chiba et al., 1986). Administration of capsaicin to newborn animals produced a marked accumulation of Ca 2+ in damaged ganglion cells, particularly at mitochondriai level (Jancs6 et al., 1984). Accumulation of Ca 2÷ into sensory cells may be consequent to the prolonged depolarization induced by the drug (Williams and Zieglgansberger, 1980): in sensory cells, the occurrence of degenerative and neurotoxic changes (Schanne et al., 1979; Jancs6 et al., 1984) may be facilitated by the peculiar incapacity of these elements (as compared to other neurons) to buffer an elevated concentration of intracellular Ca 2+ determined by repetitive activation (Jia and Nelson, 1986). Capsaicin is certainly not a "pure" tool: nonspecific effects on smooth muscle contractility can be observed in isolated organs excised from capsaicinpretreated animals (Duckies, 1986; Maggi et al., 1987b). An important feature of the non-specific effects of capsaicin seems to be their lack of susceptibility to desensitization. An example of this behaviour is shown in Fig. 3: in the isolated rat proximal urethra, capsaicin produces, in low concentrations, both excitatory and inhibitory effects on motility, ascribable to activation of sensory nerves

(see Section 4.3). These effects were absent in preparations excised from capsaicin-pretreated animals and also following extrinsic denervation (pelvic ganglionectomy) (Maggi et al., 1986d and unpublished data). However, in high concentrations (30-100/~M) capsaicin produces a second type of inhibitory effect on urethral motility: in Fig. 3 is shown the inhibition toward KCl-induced contraction of the urethra. This action was unaffected by systemic capsaicin desensitization or extrinsic denervation (removal of pelvic ganglia) and represents, most likely, a direct effect of capsaicin on contractility. Other non-specific effects of capsaicin on visceral motility were observed in intact animals: in rats, i.v. capsaicin produces a complex series of cardiovascular effects including a transient pressor phase, still present in capsaicinpretreated rats, which was attributed to a direct vasoconstrictor action on vascular smooth muscle (Donnerer and Lembeck, 1982). We have proposed that desensitization of the acute responses to capsaicin is an useful functional marker to distinguish specific effects consequent to activation of sensory nerves from non-specific effects of this substance (Maggi et al., 1987b). However, the molecular basis of the eapsaicin desensitization phenomenon are still unknown, reflecting our lack of knowledge on the mechanisms of action of capsaicin at cell membrane level (see below). Therefore, caution is needed before drawing firm conclusions in this field: much work on various types of viscera is needed the test the hypothesis that desensitization is a specific marker of the action of this substance on sensory nerves, although a large mass of experimental data supports this assumption. At this stage, the study of the effects of capsaicin following extrinsic chronic denervation of the tissue (Szolcsfinyi, 1984a; Maggi et al., 1986b, d, e; Santicioli et al., 1986) seems the most suitable criterion for establishing the role of specific (on sensory nerves) and non-specific components in the acute viscerometer response to this substance.

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Fig. 3. Non-specific inhibitory action of capsaicin on KCl-induced tonic contraction of the rat isolated proximal urethra (see Maggi et al., 1987a for details of method). In these conditions a high concentration (I00/IM) of capsaicin induced an almost complete relaxation of the preparation. This effect which could not be ascribed to the vehicle (ethanol) was reproducible when a second administration of capsaicin was made 1 hr later. The lack of desensitization (cf. Maggi et al., 1987b) indicates that, in this case, the effect of capsaicin does not involve sensory nerves but is most likely ascribable to a direct action on muscle cells. In the same preparation, specific (exhibiting desensitization) visceromotor responses to capsaicin, (ascribable to neuropeptide release from sensory nerves) can be observed in # M concentrations (cf. Maggi et al., 1987a and Fig. 8 of this review).

Nowadays the major obstacle which, in our opinion, prevents the definitive acceptance of capsaicin as a tool to be confidently used for exploring sensory function is the fact that capsaicin-sensitive sensory neurons, as defined by anatomical, neurophysiological, biochemical and functional studies (see Nagy, 1982; Szolcsfinyi, 1982, 1984 and Buck and Burks, 1986 for reviews) cannot be unequivocally identified by any other characteristic than the fact that they are "capsaicin-sensitive". This type of circular argument, reflecting the lack of a specific marker to distinguish a priori the capsaicin-sensitive from the capsaicinresistant sensory structures, can be circumvented only by functional means, i.e. by demonstrating, in any given system, as to whether or not the action of this substance is really selective in affecting sensory function(s). The lack of knowledge about the potential existence of a capsaicin "receptor", hypothesized by Szolcsfinyi (1982, 1984) on the basis of structureactivity relationship of various capsaicin congeners is, accordingly, the most important missing point in capsaicin story. Definition of the molecular target of capsaicin action on sensory nerves is, by consequence one of the most appealing goals for future research in this field. Little more is known about the action of capsaicin at post-receptorial level. Available evidence indicates that this substance may affect K + conductance (Dubois 1982; Heyman and Rang, 1985; Erdelyi and Such, 1984): this action may be relevant for the direct excitatory (depolarizing) action of capsaicin on sensory nerves. Desensitization of the acute excitatory response on K ÷ conductance following repeated ap-

plication of capsaicin was verified on rat dorsal root ganglion (d.r.g.) cells (Heyman and Rang, 1985; see also Williams and Zieglgansberger, 1982). 3. THE SENSORY FUNCTIONAND ITS ROLE IN THE REGULATIONOF VISCERALMOTILITY The "sensory-efferent" function of the capsaicinsensitive neuron is, from a theoretical point of view, a single event activated by the environmental stimulus. The natural stimulus depolarizes the sensory nerve ending, produces secretion of transmitters in the periphery and, by generating a propagated action potential determines the release of transmitters at the various terminals of the neurons both peripherally and in the CNS: it is not known, at this stage, whether a graded receptor potential below threshold for inducing an action potential is p e r se sufficient to induce a certain secretion of transmitter from the peripheral terminal of the sensory neuron (see Section 4.1). To date, the only way to label some effects as "sensory" or "efferent" would be that transmitter release occurs at different sites (terminals) in the central nervous system (CNS) and prevertebral ganglia ("sensory" functions) or in the periphery ("efferent" functions) but there are few unequivocal reasons to do so: in fact, peptides released in the periphery may activate directly or indirectly other sensory fibers, thus leading to a further increase of the afferent discharge (see Maggi and Meli, 1986a and Maggi et al., 1984a, 1985b-d, 1986e, f, 1987c; Prabhakar et aL, 1984; see also Section 10.2). On the other hand afferent impulses influence the peripheral functions not only through the activation of organ-

Capsaicin-sensitive neurons specific reflexes (see Table 1), but also determine the release of hormones (Section 3.5) which have profound and widespread effects on peripheral functions. Therefore, any division a priori of the "sensory" from "efferent" components of the function exerted by the capsaicin-sensitive neurons is somehow arbitrary: however, we will discuss separately these two aspects tbr the sake of convenience. In the "sensory" section we will review those aspects which, in a classical sense, deal with activation of reflexes organized at CNS and prevertebral ganglia level. In the "efferent" section we will review those effects which involve a peripheral action of sensory transmitters which do not need participation of the CNS (see Section 11). The study of the action of capsaicin-desensitization on pain perception and thermoregulation (see Nagy, 1982; Fitzgerald, 1983; Szolcs~nyi, 1982 and Hori, 1986 for reviews) forms the core of the earlier literature of the pharmacology of this substance (see also Buck and Burks, 1986). In particular, studies on the antinociceptive effect of systemic capsaicin desensitization were a major source of enthusiasm and disappointment about the use of this drugs as a tool for physiopharmacological research. Conflicting results about intensity, duration and even occurrence of an antinociceptive effect following capsaicin desensitization in various species and animals of various age, in response to chemical, thermal or mechanical noxious stimuli, have contributed to delay: (a) the acceptance of capsaicin as a tool to explore sensory function and (b) the extension of studies on capsaicin action on sensory function in other systems. Recent investigations by Szolcs~nyi (1985) have provided an important clarifying contribution to these topics: when administered to adult rats the antinociceptive effect observed following systemic capsaicin desensitization is highly dependent upon type and intensity of nociceptive stimulus and also, with different modalities in relation to nociceptive stimuli, upon time elapsed from capsaicin administration. Systemic capsaicin desensitization (400 mg/kg s.c.) completely abolished xylene-induced nociception for at least 1 week and recovery of chemosensitivity was incomplete up to 25 days. Threshold for noxious heat "was shifted higher by 3-4°C, but no complete thermal analgesia was observed . . . . The recovery was slow but faster than in the case of chemonociception..."(Szolcsfinyi, 1985). Finally, an increased threshold for mechanonociception could be observed at 1-2 days after systemic capsaicin desensitization, but recovery was complete within 1 week. With the exception of chemosensitivity, data of Szolcsfinyi clearly show that if a suprathreshold thermal or mechanical stimulus is used, no antinociceptive effect could be observed even if the test was done 1 day after capsaicin desensitization (see also Abbott et al., 1984). These observations demonstrate that, among the various modalities of pain induction, chemonociception is the one which most critically depends upon anatomo-functional integrity of this type of sensory fibers. From our point of view, one of the most interesting findings in these data (Szolcsfinyi, 1985) is the unequivocal demonstration that for certain types of stimuli (mechanical and thermal in

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this case) the capsaicin-sensitive sensory fibers may operate as the low-threshold component of a twopart sensory system in which, at high stimulus intensities, function is expleted through mechanisms which, in adult rats, are capsaicin-resistant. It seems noteworthy that a similar arrangement was found by us with regard to the effects of systemic capsaicin desensitization in adult rats on threshold for reflex micturition (see Maggi et al., 1984a, 1986g; Maggi and Meli, 1986a, b). In fact the effect of capsaicin-desensitization on bladder function (increased bladder capacity) may be appreciated only when threshold for reflex micturition is determined at a low, physiological-like filling rate. If the bladder is filled at a high, non-physiological rate, no difference in volume threshold was observed between capsaicindesensitized and control animals (Maggi et al., 1985b 1986g). These findings indicate that, in adult rats, at least two distinct sensory systems transmit volume information from the urinary bladder to CNS (see Maggi and Meli, 1986a, b see Section 7. l). Therefore, at both cutaneous and visceral level, those fibers which are capsaicin-sensitive in adult rats are activated at a "low" threshold stimulus intensity. This statement does not contradict the notion that, at cutaneous level, the capsaicin-sensitive polymodal nociceptors behave as high-threshold mechanoreceptors which are not activated by a light, innocuous touch (Szoics~nyi, 1977, 1984a). On the other hand, available evidence indicates that at both visceral and somatic level, the capsaicin-sensitive mechanoreceptors operate in such a way that their functional impairment following systemic capsaicin desensitization shifts the threshold for eliciting reflex responses (pain and micturition, respectively) to higher-than-normal values. As will be further discussed in Section 7.1, a part of those capsaicinsensitive fibers which are capsaicin-resistant in adult rats are capsaicin-sensitive in newborn animals. Thus, a comparison of the effects of capsaicin desensitization in newborn vs adult rats provides, when using.methods which quantitate threshold for reflex responses, a method of exploring the function of different subpopulations of sensory neurons (see also Jancs6 et al., 1985a; Maggi et al., 1987d). At present, we have no information as to whether or not this arrangement is a general feature of the sensory functions mediated by this class of sensory neurons. In the next sections we will attempt to summarize current knowledge on the sensory function of the capsaicin-sensitive sensory neurons at visceral level. Two main criteria should be satisfied for the functional demonstration that capsaicinsensitive fibres are involved in a certain reflex response, that is: (a) in subthreshold conditions for eliciting the reflex response, the acute application of low concentrations of capsaicin at the sites where the sensory fibers which initiate the natural response are located, should activate the reflex (Maggi et al., 1984a, 1985a, 1986d) (b) in animals desensitized to capsaicin, threshold for eliciting the reflex using a natural stimulus should be elevated as compared to vehicle-treated animals and (c) appropriate testing of the neural pathways subserving the efferent arm of the reflex response should produce normal responses in capsaicin-pretreated animals.

3.1 Capsaicin-sensitive and respiratory system

nerves in the cardiovascular

A number of studies clearly indicate the existence of a population of capsaicin-sensitive sensory fibers, of vagai or sympathetic origin, widely distributed in the cardiovascular and respiratory systems, which can activate a variety of reflex responses (see for instance, Jancs6 and Such, 1983; Clozel et al., 1985; Ordway and Pitetti, 1986). This topic has been the object of several excellent review articles (Coleridge and Coleridge, 1980, 1981, 1984) and will not be further discussed here. We want only to outline that the major part of studies in cats and dogs explored only the acute stimulatory action of capsaicin on cardiorespiratory reflexes. Jancs6 et al. (1985b) reported that administration of a large dose of capsaicin to newborn dogs produces a selective degeneration of small-to-medium sized primary sensory neurons located in cranial and spinal sensory ganglia. In addition, the distribution pattern of degenerating argyrophylia within the spinal cord and medulla closely resembles that obtained in the rat. Various studies aimed to exploring the potential significance of capsaicin-sensitive afferents in initiating cardiovascular and respiratory reflexes in rats or guinea pigs. Even in these species the acute administration of capsaicin, either centrally or peripherally, activates a number of reflex responses at cardiovascular and respiratory level (Makara et al., 1967a; Hausler and Osterwalder, 1980; Donnerer and Lembeck, 1982, 1983; Gamse et al., 1986b). Anatomical and biochemical evidence indicates that neuropeptide-containing nerve fibers are widely distributed throughout the cardiovascular and respiratory system of various species and that these neuropeptides are, at least in part, stored in capsaicin-sensitive nerves (Papka et al., 1981; Holzer et al., 1982; Furness et al., 1982a, b; Urban and Papka, 1985; Lundberg et al., 1983a, 1984a; Terenghi et al., 1983; Dalsgaard and Lundberg, 1984). In addition, capsaicin-sensitive, neuropeptides containing nerve fibers are present in certain areas of the CNS, Such as the nucleus Tractus Solitarius (nTS): functional evidence suggests that substance P may be one of the transmitters released from primary afferent fibers of the baroreceptor reflex arch in the nTS and application of capsaicin at this level activates the reflex response (Hausler and Osterwalder, 1980; Lorez et al., 1983; Helke et al., 1980). In spite of the large number of studies in this field, it is somewhat disappointing that functional evidence about the potential role of capsaicin-sensitive sensory nerves in the regulation of cardiovascular and respiratory homeostasis is still limited. Lower resting values of blood pressure were reported in capsaicin-desensitized animals anaesthetized with barbiturates (Donnerer and Lembeck, 1982; Lembeck and Donnerer, 1983) and in conscious, freely moving rats (Virus et al., 1981) but these findings were not confirmed by other authors (Furness et al., 1982b; Bennett and Gardiner, 1985a, b). Lorez et al. (1983) found similar blood pressure but lower resting heart rate values in capsaicin-pretreated as compared to control rats. Another area in which conflicting reports have been presented is that relative

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to baro- and chemoreceptor reflexes. Two groups failed to detect any change in baroreceptor reflexes in rats or guinea-pigs desensitized to capsaicin (Furness et al., 1982; Lorez et al., 1983). On the other hand, Bond et aL (1982) reported an impairment in various chemo- and baroreceptor reflexes in rats desensitized to capsaicin as newborns. Both pressor and respiratory response to bilateral carotid occlusion was dampened by systemic capsaicin desensitization. Also the increase in respiratory minute volume induced by i.v. sodium cyanide or hypoxia was significantly lowered by capsaicin pretreatment (Bond et al., 1982). Some alterations in resting respiratory parameters have been described in rats desensitized to capsaicin as newborns (Bond et al., 1982; Towle et al., 1985; Hedner, 1985). Some discrepancies were observed with regard to type of parameter affected, which were likely to be influenced by methodological differences, including the type of anaesthetic used (Hedner, 1985). However, both groups (Bond et al., 1982; Towle et al., 1985) reported that basal respiratory function was depressed by neonatal capsaicin desensitization. Loss of afferents of vagal origin may be involved, since any difference between control and capsaicin pretreated animals disappeared following bilateral cervical vagotomy (Towle et al., 1985). Since centrally-administered SP stimulates respiratory activity (Hedner et al., 1982) these findings may indicate that capsaicin-sensitive afferents convey to CNS an input which stimulates respiratory function and this effect may be mediated by release of substance P and related sensory neuropeptides at CNS sites which regulate breathing. 3.2 Capsaicin-sensitive nerves in the gastrointestinal system and genitourinary tract

Anatomical and biochemical evidence indicates that capsaicin-sensitive nerve fibers are widely distributed through the genitourinary tract of various species (Holzer et aL, 1982; Traurig et aL, 1984a, b; Sharkey et al., 1983; Gibbins et al., 1985; Su et al., 1986). Likewise, capsaicin-sensitive fibers are present in the gastrointestinal tract (Matthews and Cuello, 1984; Sharkey et al., 1984; Rodrigo et al., 1985; Sternini and Brecha, 1985) but in this ease the major part of tissue levels in sensory neuropeptides (such as SP) remains following systemic capsaicin desensitization, because of the presence of populations of intrinsic neurons (Holzer et aL, 1980; see Barth6 and Holzer, 1985 for a review). The importance of sensory stimuli, particularly of chemical origin, arising from the gastrointestinal tract for the initiation of reflexes organized at intraand extramural level was reviewed recently (Ewart, 1985; Mei, 1985; see also Melone, 1986). In the gastrointestinal tract and various other abdominal organs (gallbladder, pancreas, liver) of dogs and eats, the topical application or close intraarterial injection of capsaicin activates reflex cardiovascular responses (Ashton et al., 1982; Longhurst et aL, 1980, 1984; Ordway et aL, 1983; Ordway and Longhurst, 1983). The natural stimuli for these afferent fibers and, consequently, pathophysiological relevance of these mechanisms is largely unknown. On the other hand, experiments in rats suggested that the capsaicin-sensitive sensory fibers are involved

in the initiation of various reflex responses arising from the gastrointestinal and genitourinary tract: examples supporting this statement are listed in Table 1. At the gastrointestinal level, Holzer (1986) found that neither gastric emptying nor gastrointestinal transit or defecation in relation to food intake were significantly affected in adult rats systemically pretreated with capsaicin, 1 week before the experiment. Essentially similar findings, with regard to gastric emptying, were obtained in our laboratory, using rats desensitized to capsaicin as newborns (Evangelista and Santicioli, unpublished data). Likewise, intestinal peristalsis, as studied in isolated segments from the guinea-pig small intestine is not altered in capsaicintreated animals (Donnerer et al., 1984). More recently, Holzer et al. (1986) reported that the inhibition of intestinal motility induced by irritation of peritoneal chemonociceptive fibers (cf. Cervero and McRitchie, 1982) as well as postoperative ileus were prevented in capsaicin-pretreated rats. Holzer et al. (1986) concluded that postoperative ileus or that following chemical peritoneal irritation is, at least in part, due to activation of a reflex whose afferent limb involves the capsaicin-sensitive nerves while the efferent limb involves postganglionic sympathetic neurons. The fact that capsaicin-sensitive afferents may activate a sympathetic reflex inhibiting intestinal motility is also supported by the observation that in anaesthetized guinea-pigs (Maggi et al., 1987b) i.v. capsaicin produced a transient relaxation of the distal colon which was abolished by tetrodotoxin, hexamethonium or guanethidine as well as by systemic capsaicin desensitization. Since the response was observed also following topical application of capsaicin on the colon we hypothesized that the response may be physiologically activated by intramural sensory nerves which initiate this colonic inhibitory sympathetic reflex (Table 1). Previous studies showed that mechanoreceptors in the guinea-pig distal colon, activated by distension, provide synaptic input to sympathetic neurons in the inferior mesenteric ganglion (i.m.g.) which in turn inhibit motility by re= leasing catecholamines at colonic level (Crawcroft et al., 1971; Szurszewki and Weems, 1976; Weems and Szurszewski, 1977, 1978; Kreulen et al., 1983; King and Szurszewski, 1984). In addition, capsaicin produces a depolarization of postganglionic neurons in the i.m.g, mediated by release of SP by collaterals of the capsaicin-sensitive afferents (Tsunoo et al., 1982; Matthews and CueUo, 1982, 1984; Gamse et al., 1981). Recent findings indicate also that the synaptic input to i.m.g, activated by colonic distension was abolished in capsaicin pretreated guinea-pigs (Kreulen and Peters, 1986). 3.3 Capsaicin-sensitive nerves in the urinary bladder

The reader is referred to other review articles for detailed listing of evidence supporting the idea that capsaicin-sensitive nerves regulate threshold for initiation of reflex micturition, and more generally speaking, of reflexes regulating vesicourethral motility (Maggi and Meli, 1986a, b). A schematic representation of the organization of pathways which regulate bladder voiding is presented in Fig. 4. Available evidence indicates that the capsaicin-sensitive

8

CARLO ALBERTO MAGG1 a n d ALBERTO M E L I CONSCIOUS MICTURITION THALAMUS.CORTEX AND OTHER SUPRAPONTINE CENTERS

PONTINE MICTURITION CENTER /

REFLEX

/ /

MICTURITION

I I /

I

[

I DORSAL ROOT GANGLION

- -- -- -t>

SACRAL PARASYMPATHETIC --I> NUCLEUS

I DETRUSORMusCLE

ACTIVATION OF BLADDER

/

MECHANORECEPTORS

~

I DISTENSION

Fig. 4. Schematic drawing which illustrates the major neural pathways subserving "conscious" and "reflex" micturition in rats (see Maggi and Meli, 1987a, b for a detailed discussion of this topic). Available evidence indicates that capsaicin-sensitive nerves are essential for initiating "reflex" micturition at both spinal and supraspinal level, while a capsaicin-resistant sensory input may play a role in "conscious" micturition.

nerves are involved in regulating reflex micturition at both spinal and supraspinal level while conscious bladder voiding may be initiated also through a capsaicin-resistant mechanism (Maggi and Meli, 1986a, b; Conte et al., unpublished data). In rats, two distinct types of vesico-vesical reflexes can be elicited in response to bladder filling, which are organized at spinal and supraspinal level (Maggi et al., 1986h, i, 1987d). Both of them can be initiated by capsaicinsensitive afferents (bladder mechanoreceptors) and systemic capsaicin desensitization produces an increase in threshold for evoking the reflex response to the natural stimulus (Maggi et al., 1986d, g, 1987c; Maggi and Meli, 1986a, b). Likewise, in acute spinal rats, topical application of capsaicin on the outer surface of the urinary bladder activates a transient pressor and tachycardic response (Maggi et al., 1987d). Interestingly, the content of substance P-like immunoreactivity (SP-LI) of the rat urinary bladder is almost depleted by systemic capsaicin desensitization, both in newborn and adult animals (Holzer et al., 1982; Maggi et al., 1987d, e). Extrinsic bladder denervation induced an almost complete depletion in bladder SP-LI content, indicating that the peptide is entirely stored in neural structures (Maggi et al., 1987d, e). Recent findings indicate that tile sensory function of the capsaicin-sensitive fibers is directly proportional to SP-LI in the bladder: the higher the SP-LI, the lower the volume threshold for eliciting

reflex bladder contractions (Maggi et al., 1987e). A similar correlation was found also for the "efferent" function of the capsaicin-sensitive fibers (tetrodotoxin-resistant capsaicin-induced contraction). This is, to our knowledge, the first example for which a positive correlation was demonstrated, quantitatively, between sensory function ofcapsaicinsensitive nerves in individual normal animals and tissue levels of sensory neuropeptides. At somatic level, changes in tissue levels of sensory neuropeptides were observed in parallel to induction of a pathological-like condition. Lembeck et al. (1981) reported in rats with adjuvant-induced polyarthritis, an increase in SP-LI, in neural tissue subserving the inflamed area (see also Lembeck and Gamse, 1982 and Colpaert et al., 1983). This latter, was interpreted as due to adaptive changes in the synthesis of substance P induced by the chronic noxious event (Lembeck and Gamse, 1982). Likewise, Levine et al. (1984) reported that, in rats, those joints (ankles) having the greatest susceptibility to develop experimental arthritis were those containing more SP-LI, having the lower nociceptive threshold and the greater projection to the dorsal spinal cord. The close correlation between SP-LI in the bladder and the ability of sensory nerves to perceive the amount of fluid present into the viscus strongly suggests a functional significance (Maggi et al., 1987d, e). We do not know, at this time, whether the animals with the greatest SP-LI bladder content have a greater number of

Capsaicin-sensitive neurons SP-LI fibers or a greater peptide content per nerve fiber. Irrespective of this, these findings demonstrate that measurement of tissue levels in sensory neuropeptides is a valid biochemical marker of function, providing that measurements are done, as in the case of rat bladder, in a tissue where the peptide content is entirely ascribable to the presence of sensory nerves (Maggi et al., 1987d, e; see also Senapati et al., 1986). The connection between bladder SP-LI content and volume threshold for reflex micturition which was observed in normal animals was disrupted following systemic administration of a large dose of s.c. capsaicin to adult rats. In fact, 1 hr later, when the bladder content of SP-LI was still unaffected by capsaicin, both sensory and efferent functions mediated by these sensory neurons were blocked (Maggi et al., 1987d). This was interpreted as a consequence of the capsaicin-induced blockade of the sensory nerve terminals (cf Section 2) which prevents generation of the sensory impulse and secretion of transmitter (both centrally and peripherally) even at a stage when peptide content was unaffected (Maggi et al. 1987d). 3.4. Capsaicin-sensitive nerves in the renal pelvis and ureters Recordati et al. (1978, 1980, 1982) described, in

rats, the presence of two populations of renal chemoreceptors: R1 elements, which are not spontaneously active and are activated by renal ischemia and R2 elements which have a resting discharge, inversely proportional to diuresis, and are stimulated by renal ischemia and, also by backflow of urine. This latter excitation was determined by changes "in chemical environment of the renal interstitium, as modified by ions crossing the pelvic epithelium, by leakage of ions out of ischemic cells..." (Recordati et al., 1980). Subsequent investigations (Recordati et al., 1982) showed that these chemoreceptors can activate rentrenal sympathetic reflexes which may be integrated at both spinal and supraspinal level. In a separate series of experiments, Szolcsfinyi (1984a) showed that R2 chemoreceptors are capsaicin-sensitive and can be activated by bradykinin or 5-HT. By contrast, R1 receptors were not excited by capsaicin and could be readily activated by renal ischemia, in capsaicin-pretreated animals. More recently we presented evidence for the presence of a capsaicin-sensitive innervation in the rat ureter whose stimulation produces a powerful inhibitory effect on ureteral motility (Maggi et al., 1986c, e; 1987a, f). Interestingly, an increased tendency toward spontaneous motility was observed in ureters excised from capsaicin-pretreated animals, suggesting that these preparations were, at least, more excitable than controls. From the anatomical point of view, degenerating axons were observed in the ureters from adult rats systemically pretreated with capsaicin (Hoyes and Barber, 1981; Chung et al., 1986). Taken together, these findings indicate that the capsaicin-sensitive innervation of the rat renal pelvis and ureters subserves a dual sensory-efferent function. The sensory function, which may involve pain perception during ureteral obstruction, can also activate sympathetic reflexes directed toward the kidney

(Recordati et al., 1982). The functional significance of these mechanisms awaits further investigation: R2 chemoreceptors behave, functionally, as osmoreceptors and are sensitive to intrarenal extracellular dehydration e.g. they monitor the ionic concentration and composition of the renal interstitium (Recordati et al., 1982). SP-containing nerves were observed in the wall of the ureter, renal pelvis, around blood vessels and close to proximal and distal tubules in the rat kidney (Ferguson and Bell, 1985). Efferent sympathetic activity to the kidney influences Na ÷ and H20 reabsorption and also renin secretion (Zanchetti and Stella, 1975; Colindres et al., 1980; Di Bona and Rios, 1980). Thus, capsaicin-sensitive R2 renal chemoreceptors (Recordati et al., 1980, 1982; Szolcs/myi, 1984) may have a profound influence on body fluid composition and blood pressure regulation. A dysfunction of these mechanisms may be important for the development of hypertension. Renal denervation prevents or delays various types of experimental hypertension in rats (Liard, 1977; Katholi et al., 1980; Winternitz et al., 1980). 3.5 Neuroendocrine reflexes and capsaicin-sensitive primary afferents

In recent years important advancements in research about sensory functions activated by capsaicin-sensitive neurons has been done with regard to their role in activating certain neuroendocrine reflexes such as: secretion of luteotropic hormone (Traurig et al., 1984a, b); secretion of both ACTH and adrenal catecholamines in response to stressful situations (Makara et al., 1967b; Khalil et al., 1984, 1986; Lembeck and Amann, 1986); secretion of B endorphin from the pituitary (Mueller, 1981; Koenig et al., 1986) secretion of arginin-vasopressin in response to acute hypotension (Bennett and Gardiner, 1984, 1985a, b) or chemical stimuli in the portal vein (Stoppini et aL, 1984). We want to draw the attention of the reader to these findings because some neuroendocrine reflexes may be initiated by capsaicinsensitive fibers located at visceral level (see Stoppini et al., 1984). Capsaicin-sensitive fibers have now been described to be present in a variety of organs and tissues, such as blood vessels or the urinary tract (see Sections 3.1 and 3.2) and, through their contact with body fluids and perceive their chemical composition and also signals representing physiological or pathological-like events. Initiation of neuroendocrine reflexes may thus be a general function of these sensory systems having the scope of activating feedback loops significant for the maintenance of chemical homeostasis of the body (see Section 6). The peculiar chemosensitivity of capsaicin-sensitive fibers makes them most likely candidates for monitoring the "internal" chemical environment. 3.6 Sensory nerves and visceral pain: a role f o r capsaicin-sensitive fibers

An interesting field in which relatively few studies have been thus far performed is that relative to the role of capsaicin-sensitive fibers in the genesis of visceral pain. The hypothesis has been considered, for instance, that cardiac afferents which mediate the pain of myocardial ischemia are of peptidergic nature (Furness et al., 1982b). The peculiar chemosensitivity

10

CARLO ALBERTO MAGGI and ALBERTO MEL1

of capsaicin-sensitive nerves suggests that changes in tissue levels of certain chemicals resulting as byproducts of cellular damage (lowering of pH, increase of extracellular K + ) could activate these sensory nerves. The branching of certain sensory nerves in the periphery may further provide an anatomical basis to the phenomenon of referred pain (Langford and Coggeshall, 1981; Pierau et al., 1984a, b; Alles and Dom, 1985; Coggeshall, 1986). Anyway, the study of the potential role of capsaicin-sensitive nerves in the genesis of visceral pain has been, until now, rather neglected (see Lembeck and Skofitsch, 1982 and Cervero and McRitchie, 1982). An important step for advancement in this field would be represented by development of adequate animal models of visceral pain in which threshold could be accurately measured. Certain sensory neurons may innervate both somatic and visceral structures (see also Section 4.2): at which extent pathological or pathological-like events on one side of these multipolar sensory elements (see Section 11) may influence the sensoryefferent functions at other nerve terminals, is a matter open to investigation which may lead to important advances in the pathophysiology of human disease having both somatic and visceral component. 4. THE EFFERENT FUNCTION OF CAPSAICIN-SENSITIVE SENSORY NEURONS

As efferent functions of sensory neurons we may consider those actions which are produced by transmitters released from peripheral terminals of sensory neurons (Fig. 1) as defined by Szolcs~nyi (1984a; cf. Section 11). The concept that sensory neurons may release the stored transmitters from peripheral terminals through an axon reflex arrangement is a well established physiological principle (Bayliss, 1901; Langley, 1923; Lewis and Marvin, 1927). According to the classical view, the mediator of antidromic vasodilatation is released from a nerve ending which is specialized in releasing the transmitter, being distinct from the sensory receptors which, on the other hand, are specialized in initiating afferent impulses (see Szolcsfinyi, 1984a). Recent findings, summarized in Section 4.1, indicate that, in addition to the axon reflex mechanism, the capsaiein-sensitive fibers may release transmitters also from the same terminal activated by the environmental stimulus. 4.1 The sensory receptor potential-coupled "efferent" response In 1968 Nicolas Jancs6 et al. presented the first

evidence indicating that certain sensory neurons, identified by their responsiveness and/or sensitivity to capsaicin (and other chemical irritants) may release the stored transmitter(s) in the periphery also from the same sensory terminal which is activated by environmental stimuli. This concept has received strong experimental support in recent years (Szolcsfinyi, 1983, 1984a; Maggi et al., 1984a, 1986c, 1987a; Maggi and Meli, 1986a). The reader is referred to the paper of Szolcs/myi (1984a) for a detailed analysis of data supporting the view that chemical stimulation in the generative region of the sensory terminal may release the stored

neuropeptide(s) through a tetrodotoxin-resistant depolarization. The key observation leading to this proposal was that certain biological responses produced by: (a) local application of capsaicin (or other chemical irritants) in vivo or (b) visceromotor responses to capsaicin in isolated organs are neurogenic in origin (abolished by tissue denervation) while being resistant to agents which, like local anaesthetics or tetrodotoxin block the fast Na ÷ channel and, consequently, axonal conduction. Accordingly, a tetrodotoxin-resistant release of sensory neuropeptides was induced by capsaicin in various systems (see for instance Gamse et al., 1979 and Saria et al., 1983a). On the other hand, transmitter secretion produced by ortho- or antidromic activation of sensory nerves are abolished by local anaesthetics or tetrodotoxin, even following infiltration of the terminal portion of the nerve (Szolcsfinyi, 1984a, b). In these sensory neurons transmitter secretion was prevented by tetrodotoxin or local anesthetics providing that it was consequent to transmission of the impulse from one terminal to another: for instance, distension of the colon produces a tetrodotoxin-sensitive depolarization of sympathetic neurons in guinea-pig inferior mesenteric ganglion which is prevented by systemic capsaicin desensitization (Kreulen and Peters, 1986): this response seems ascribable to release of sensory neuropeptides such as SP (Gamse et al., 1981) from collaterals of the sensory neurons which terminate in the ganglion (Matthews and Cuello, 1982, 1984). This means that activation of a peripheral terminal (in the colonic wall) produces a propagated action potential (tetrodotoxin-sensitive) which transmits the information at another terminal (in the ganglion) (c.f. Section 11). At the level of the terminal, the ionic basis of depolarization-coupled transmitter secretion are tetrodotoxin-resistant: in fact capsaicin produces a tetrodotoxin-resistant depolarization of sympathetic neurons in the guinea-pig inferior mesenteric ganglion (Dun and Kiraly, 1983). The hypothesis that the sensory receptor itself is the main site of action of capsaicin has received further experimental support by our recent observations on the rat isolated ureter (Maggi et al., 1986c, 1987a). In this preparation either field stimulation (FS) or capsaicin produce a transient inhibition of rhythmic contractions activated by exogenous transmitters. The inhibitory response to FS was abolished by tetrodotoxin while the effect of capsaicin was almost completely tetrodotoxin-resistant. However the response to both FS and capsaicin was prevented by (a) systemic capsaicin desensitizations, as well as, (b) acute exposure to capsaicin in vitro indicating that the response to FS involves antidromic activation of capsaicin-sensitive sensory terminals (Maggi et al., 1986c, 1987a). The response to FS or capsaicin was also abolished by tissue denervation achieved by cold storage (at 4°C). Blockade of the response to FS was observed within min from capsaicin administration, indicating that the capsaicin-induced blockade of the sensory terminal (Fig. 2) occurs a short time and possibly just after the termination of the excitatory effect, as proposed by Szolcs~inyi (1985). Antidromic activation of capsaicin-sensitive nerves by nerve stimulation was reported to induce efferent

Capsaicin-sensitive neurons responses (motor and inflammatory) in a number of

studies at cutaneous, tracheobronchial, intestinal, urinary and cardiac level (Jancs6 et al., 1967, 1968; Szolcsfinyi and Barth6 1982; Barth6 and Szolcs~myi, 1980; Saria et al., 1983a; Lundberg et al., 1984b; Saito et al., 1986) and was abolished by capsaicin desensitization. To quote Davis (1961) "The concept of the neur o n . . , recognizes the dendrite as a functional entity quite different from the axon. The dendrite is chemically excitable and responds with graded postsynaptic potentials and n o t . . , with all-or-none nerve impulses". Accordingly, environmental stimuli induce a receptor action on sensory cells which is analogous to chemical excitability of the dendrites, e.g. a receptor process which includes the generation of an electric potential, is interposed between the environmental stimulus and the initiation of a nerve impulse (Davis, 1961). The receptor potential is a local event (non-propagated) and for this reason, resistant to local anaesthetics or tetrodotoxin. Receptor potentials should summate (no refractory period) if one or more of them occur in close succession, thus leading to initiation of the sensory impulse. Electrophysiological experiments indicate that the stimulusinduced depolarization of the sensory receptor is unaffected by local anaesthetics, while being Ca 2÷ dependent (Lowenstein et al., 1963; Eyzaguirre et al., 1970; Eyzaguirre and Nishi, 1974; Eyzaguirre, 1981; Hayashida et al., 1980). According to these observations, the capsaicin-induced depolarization of the rat sciatic nerve (an action which exhibited marked desensitization) was found to be tetrodotoxinresistant (Hayes et al., 1984). It should be noted that capsaicin "receptors" should be found also at axonal level in sensory nerves: in fact, topical application of capsaicin on nerve trunks produces both functional and biochemical changes indicating a blockade of impulse transmission and impairment of axonal flow of neuropeptides (Jancs6 et al., 1980; Gamse et al., 1982c). This latter produces a time dependent decrease in tissue levels of sensory neuropeptides which parallels the time-dependent decrease of the efferent functions mediated by these sensory nerves (Gamse et al., 1982c). Thus, following systemic capsaicin administration, the biochemical and functional changes are most likely consequent to an action at two sites, the nerve terminal and the axon. Anyway, blockade of the sensory terminal by capsaicin-desensitization should be per se sufficient to induce a functional impairment of both sensory and efferent functions (cf. Maggi et al., 1987d). Therefore, orthodromic activation of the sensory receptor produces: (a) Na÷-dependent action potential which transmits the information to the central nervous system and prevertebral ganglia and, by invading collaterals of the same sensory neuron, produces antidromic release of transmitters in the periphery (axon reflex) and (b) through a Na+-independent mechanism, secretion of stored transmitters from the depolarized sensory terminal (local "efferent" function) (Fig. 1). In principle, the capsaicin-sensitive sensory neurons seem able to exert an efferent function every time the receptor is stimulated (Jancs6, 1968) through a depolarization-

11

coupled secretion of transmitter. For the sake of convenience the tetrodotoxin-resistant component of "efferent" responses will be thereafter referred to as the sensory receptor potential ( S R P )-coupled efferent response. The anatomical and structural basis of this response are largely unexplored. In this respect an important observation from Yokokawa et al. (1985) indicates the presence of synaptic vesicle~ in nerve terminals containing the SP-LI in the rat urinary bladder. Since the SP-LI of the rat bladder is almost depleted by systemic capsaicin pretreatment in either newborn (Hoher et al., 1982) or adult animals (Maggi et al., 1987d, e) we may assume that the findings from Yokokawa et al. (1985) indicate the presence of synaptic vesicles in peripheral terminals of capsaicin-sensitive sensory neurons. This latter would provide a structural and morphological basis for the proposed SRP-coupled response due to tetrodotoxin-resistant release of substance P in the rat bladder (Maggi et al., 1984a, 1985b; see also Gulbenkian et al., 1986). It is not known whether a depolarization of the sensory receptor which per se is insufficient to activate an action potential (initiation of the sensory impulse) may produce a graded secretion of transmitter(s) in the periphery through the SRP-coupled mechanism. 4.2 The relative contribution o f axon reflex and S R P coupled response to the "efferent" actions o f the capsaicin-sensitive nerves The relative contribution of the axon reflex arrangement and of the SRP-coupled mechanism to the "efferent" responses mediated by the capsaicinsensitive fibers may differ in various tissues. At cutaneous levels the flare component of the inflammatory response to capsaicin or other irritants is prevented by local anaesthetics (Jancs6 et al., 1968; Foreman and Jordan, 1984; Szolcs~inyi, 1984a, b): this observation supports the idea that axon reflexes play a major role in determining neurogenic inflammation at cutaneous level. This may not be the case for certain viscera: for instance, in the rat urinary bladder, the capsaicininduced plasma extravasation is almost unaffected by topical application of tetrodotoxin (Maggi et al., 1987a). There is some evidence that the effector element of the axon reflex at cutaneous level involves the release of mediators from mast cells. SP is a potent activator of mast cell degranulation in both rat and human skin (Fewtrell et al., 1982; Piotrowski and Foreman, 1985). SP, released antidromically from sensory nerves through an axon reflex arrangement was proposed as a main mediator of antidromic vasodilatation and neurogenic inflammation (Lembeck and Hoher 1979). Recent anatomical evidence indicates a close spatial relationship between nerve fibers containing SP-LI and mast cells (Skofitsch et al., 1985), supporting the idea of a functional unit which may induce mast cell degranulation following stimulation of sensory nerves, thus leading to vasodilatation and plasma extravasation. In various viscera, the motor response to capsaicin are totally resistant to tetrodotoxin or local anaesthetics (Szolcs~inyi, 1983; Maggi et al., 1984a, 1985b, 1986a, b,c, 1987a; Santicioli et al., 1986),

CARLO ALBERTO MAGGI and ALBERTO MELI

12

be due to transmitter release from sensory nerves. The main criterion for inclusion in this list is, for the reasons given in Section 2, the susceptibility of these effects to capsaicin desensitization. In a number of cases, the neurogenic nature of these responses has been demonstrated by denervation experiments. A variety of visceromotor responses to capsaicin in isolated organs (guinea-pig ileum, colon and trachea, rat urinary bladder and iris sphincter muscle) (see Table 2 for References) may be ascribed to release of SP from these sensory nerves. In all these tissues, exogenous SP produced a response which closely mimicked that of capsaicin. In a number of cases, evidence for an involvement of endogenous SP was presented either by using desensitization of SP receptors or SP antagonists (see for instance Chahl, 1982; Barth6 et al., 1982; Bjorkroth, 1983; Maggi et al., 1985b). However, certain visceromotor effects of capsaicin (chrono- and inotropic effect in atrial preparations, inhibition of spontaneous motility of rat uterus (Lundberg et al., 1984c; Zernig et al., 1984) could not be readily explained through an action of endogenous SP, mainly because this peptide (and other tachykinins) were ineffective or produced an effect opposite to that of capsaicin. It should be noted that the effect of capsaicin in the heart and uterus exhibited desensitization. However, at that time, the possible involvement of an unspecific action of capsaicin could not be ruled out (Zernig et al., 1984; Lundberg et al., 1984c). Since 1983, other sensory neuropeptides were discovered and many of them are reportedly present in capsaicin-sensitive nerves: first, tachykinin family ex-

suggesting that release of transmitters occurred mainly or solely through SRP-coupled mechanisms. On the other hand, in certain organs (guinea-pig ileum, rat duodenum) a component of the motor response to capsaicin is sensitive to tetrodotoxin. In these systems, sensory neuropeptides, released through the SRP-coupled response, activate intramural efferent neurons (cholinergic in the guinea-pig ileum, nonadrenergic noncholinergic (NANC) in the rat duodenum, Fig. 5). In vivo experiments in cats led to the hypothesis that mucosal nociceptive stimuli can activate an axon reflex arrangement confined to splanchnic afferents which, via SP release leads to regional gastric contractions (Delbro et al., 1984)i This mechanism is also activated by bradykinin a known activator of C fibers (Delbro et al., 1986). Whether these intramural neuronal circuitries (guinea-pig ileum, rat duodenum, cat stomach) represent the "visceral" counterpart of t h e axon reflex arrangement described at cutaneous level, remains to be established. At both levels, an intrinsic effector cell (mast cell in the skin, cholinergic or N A N C neuron in the intestine) contributes to the final response (inflammation or change in motility) following stimulation by a sensory neuropeptide. A similar mechanism may account also for the capsaicin-induced N A N C neurogenic inhibition of motility of the guinea-pig isolated distal colon (Maggi et al., 1987b). 4.3 Visceromotor responses to capsaicin in isolated organs

In Table 2 we have listed a number of preparations in which the visceromotor response to capsaicin may

GUINEA PIG ILEUM PRIMARY AFFERENT FIBER C>

+

INTRAMURAL CNOLINERGIC NEURON

SP

CAPSAICIN

l p+ I

~

"~" ACh

MUSCLE CELL

I

RAT DUODENUM PRIMARY AFFERENT FIBER I

.~

~

INTRAMURAL NANC NEURON

~> CGRP?

! CAPSAICIN

~;i I

.UBC,E

ATP? D CELL

I

Fig. 5. Schematic drawing illustrating the proposed intramural neuronal circuitry which would be responsible for the capsaicin-induced contraction of the isolated guinea-pig ileum (upper panel) and relaxation of the isolated rat duodenum (lower panel). In both cases capsaicin releases neuropcptides from sensory fibers of extrinsic origin and the neuropeptides produce a visceromotor response partly through a direct action on muscle cells and partly through activation of an intramural "effector" neuron

(cholinergic in the guinea-pig ileum, non-adrenergic non-cholinergic in the rat duodenum). Substance P and CGRP have been proposed as the major determinants of the response in the two preparations (from Chahl, 1982; Barth6 et al., 1982; Maggi et al., 1986a, b).

Capsaicin-sensitive neurons

Preparation Gastrointestinal tract Oesophagus Duodenum Ileum Ileum Colon Colon Rectum Genitourinary tract Vas deferens Ureter Ureter Urinary bladder Urinary bladder Urethra Urethra Uterus Tracbeobronchial tree Main bronchi Segmental bronchi Blood vessels Carotid artery Thoracic aorta Others Iris sphincter Iris sphincter NT = Not Tested.

13

Table 2. Visceromotor responses to capsaicin in isolated organs Susceptibility to Effect of capsaicin Capsaicin Species desensitization on motility TTX Denervation

Reference

Opossum Rat Guinea-pig

Contraction Inhibition Contraction

Yes Yes Yes

NT Partial Partial

NT Yes Yes

Rabbit Guinea-pig Guinea-pig Rat

Contraction Contraction Inhibition Inhibition

Yes Yes Yes Yes

Partial NT Partial NT

NT NT NT NT

Robothamet al. (1985) Maggi et al. (1986b) Szolcsfinyi and Barth6 (1978) Barth6 and Szolcs/myi(1979) Barth6 and Szolcs~nyi(1980) Szolcsanyi and Barth6 (1979) Maggi et al. (1987b) Maggi, unpublished data

Rat Rat Guinea-pig Rat Guinea-pig Rat Rat Rat

Inhibition Inhibition Contraction Contraction Contraction Contraction Inhibition Inhibition

Yes Yes Yes Yes Yes Yes Yes Yes

No No NT No NT NT NT NT

Yes Yes NT Yes NT Yes Yes NT

Maggi et al. (1987f) Maggi et aL (1986c; 1987a) Hua and Lundberg, (1986) Santicioli et al. (1986) Lundberget al. (1983b) Maggi el al. (1987a) Maggi et aL unpublished data Zernig et al. 0982)

Guinea-pig Human

Contraction Contraction

Yes Yes

No NT

Yes NT

Szolcs/myi 0983) Lundberget al. (1983b)

Guinea-pig Guinea-pig

Inhibition Inhibition

Yes Yes

NT NT

NT NT

Duckies (1986) Duckies (1986)

Rabbit Rat

Contraction Contraction

Yes Yes

NT NT

NT NT

Bjorkrotn(1983) Banno et al. (1985)

panded with the discovery of the new kassinin-like mammalian tachykinins (neurokinins), termed neurokinin A and neurokinin B (see Maggio, 1985 for a detailed account of their discovery) and more recently with neuropeptide K and a novel eledoisin-like peptide (Theodorsson-Norheim et al., 1984, 1985; Tatemoto et al., 1985; H u a et al., 1985, 1986a). In 1983, also the calcitonin gene-related peptide ( C G R P ) (see G o o d m a n and Iversen, 1986 for a review) was discovered and this peptide seems very important for the responses determined by capsaicin. In fact, various novel tachykinins and also C G R P are co-stored with SP in capsaicin-sensitive sensory nerves both in the central and peripheral nervous system (Hua et al., 1985, 1986a; Lee et al., 1985; Dalsgaard et al., 1985; Gibbins et al., 1985; Y o k o k a w a et al., 1986; Gulbenkian et al., 1986; Su et a k , 1986). Further, recent evidence indicates that multiple neuropeptides are co-released by capsaicin both centrally and peripherally (Saria et al., 1986; Hua et al., 1986a). In this new scenario, the problem relative to the nature of certain "strange" (e.g. not ascribable to an action of endogenous SP) visceromotor responses to capsaicin was re-evaluated. Lundberg et al. (1985a, b) showed that in guinea-pig atrium exogenous C G R P has very potent stimulatory effect on chrono- and inotropism and the action of capsaicin is prevented by C G R P desensitization: this supports the idea that the response to capsaicin is mainly determined by release of e n d o g e n o u s C G R P from sensory nerves (Franco-Cereceda and Lundberg, 1985; Lundberg et al., 1985a, b). A similar conclusion was reached by Saito et al. (1986) who also demonstrated that the

capsaicin-sensitive nerves in guinea-pig atrium could also be activated antidromically by field stimulation and proposed a role of these sensory nerves to the genesis of vagal tachycardia. Further, in various organs, (rat duodenum, ureter and vas deferens) we observed an inhibitory effect of capsaicin on spontaneous or stimulated motility: these effects were prevented by capsaicin desensitization or tissue denervation (ganglionectomy or cold storage) indicating the specific involvement of these sensory nerves (Maggi et al., 1986a-e, 1987a, f). Again, in these tissues, SP and other tachykinins have an effect which is o p p o s i t e to that of capsaicin while exogenous C G R P closely mimicks the effect of capsaicin. In the duodenum (Fig. 6) and ureter, desensitization to exogenous C G R P prevents the effects o f capsaicin suggesting the involvement of e n d o g e n o u s C G R P in capsaicin's action (Maggi et al., 1986b; Santicioli and Maggi, unpublished data). Similar results were obtained by H u a et al. in the guinea-pig ureter in vivo (Hua, 1986; H u a et al., 1986b; Hua and Lundberg, 1986) with the exception that at high doses of i.v. capsaicin, the action turned from inhibitory to excitatory, presumably due to the prevailing release of tachykinin-like material. Therefore we have now a second group of preparations (rat duodenum, vas deferens and ureter, guineapig atrium and ureter) in which certain responses to capsaicin are mainly ascribable to the effect of C G R P rather than to tachykinins. Also the capsaicininduced vasodilation of certain blood vessels (Duckles, 1986) may fall in this category (see Edvinsson et al., 1985). To further complicate the picture we have now

14

CARLO ALBERTO MAGGI and ALBERTO MELI i,

1 rnin

,t

lg

t OMPP

t CORP

t CIDRP

t CGRP

t CAPSAICIN

t N A

Fig. 6. Typical tracing illustrating the effect of CGRP desensitization (0.1/~M, three consecutive challenges at 2 min intervals) on capsaicin-induced NANC relaxation of the rat isolated duodenum. In control preparations capsaicin induced a transient relaxation which amounted to 60-80% of the response produced by DMPP (0.1mM) or noradrenalinee (1/~M). CGRP desensitization does not modify amplitude of DMPP or noradrenaline induced relaxations (data are from Maggi et al., 1986b). examples of tissues in which capsaicin can produce, at the same time, opposite effects on motility. Two examples of this are the guinea-pig distal colon and ileum: in the isolated colon, capsaicin produced first an atropine-sensitive contraction followed by an atropine- and guanethidine-resistant but tetrodotoxin-sensitive inhibition of motility (Maggi et al., 1987b). Likewise, in the guinea-pig ileum, the well-known capsaicin-induced contraction, mediated by SP (Chahl, 1982; Barth6 et al., 1982) is followed by a transient inhibition of amplitidue of twitches (see Fig. 7): as could be noted exogenous CGRP produces a transient contraction of the ileum (cf. Tippins et al., 1984) but also an inhibition of twitches while SP did not affect amplitude of twitches (Fig. 7). Another example of a tissue in which capsaicin produced both excitatory and inhibitory effects on motility is the rat isolated proximal urethra (Fig. 8). At this level capsaicin (1 #M) produced a small and transient contraction in about 50% of cases. This effect was closely mimicked by low concentration of exogenous tachykinins (Maggi et al., 1987a). However, if the preparation was concomitantly field stimulated, an inhibitory effect on amplitude of the nerve mediated responses was observed in all preparations tested (see Fig. 8). Both effects of capsaicin were absent in urethral rings from capsaicin-pretreated or ganglionectomized animals. In some cases such as the example shown in upper panel of Fig. 8, the excitatory and inhibitory effect on urethral motility occurred concomitantly. Exogenous CGRP inhibits the nerve-mediated contraction of the rat proximal urethra and also slightly inhibits the motor effect of exogenous neurokinins. Taken as a whole the visceromotor response to capsaicin in various tissues seems to involve various neuropeptides and is greatly influenced by a variety of factors including the presence or not of a spontaneous activity. The important points which warrant specificity of action of capsaicin are: (a) desensitization following administration of a large dose of capsaicin to the intact animal or following exposure to a high concentration of capsaicin in vitro and (b) blockade of the response by extrinsic denervation of the tissue. In the guinea-pig colon and rat proximal

urethra capsaicin in high concentrations (ECs0> 10/~M) produced also an unspecific inhibitory effect on motility and this action was still observed in preparations excised from desensitized or ganglionectomized animals (cf. Maggi et al., 1978b). 4.4 Neurogenic inflammation and other types o f efferent actions o f capsaicin-sensitive nerves The efferent function of capsaicin-sensitive sensory neurons determines not only a variation in motility but also an inflammatory response which can be quantitated as plasma extravasation e.g. increased vascular permeability following injection of a dye which, like Evans blue, does not normally escape from the blood stream. As mentioned above, neurogenic inflammation involves, at cutaneous level, the axon reflex arrangement (e.g. the response is reduced by tetrodotoxin or local anesthetics) as well as degranulation of mast cells. However, neither the axon reflex arrangement nor secretion of autacoids from mast cells are a pre-requisite for the neurogenic inflammation phenomenon. For instance, in the rat urinary bladder, topical capsaicin induced a marked plasma extravasation which was unaffected by topical tetrodotoxon or antihistaminics but was abolished by bladder denervation or systemic capsaicin desensitization (Maggi et al., 1987a). Therefore, the relative contribution of the axon reflex arrangement, mast cell degranulation and SRP-coupled response to neurogenic inflammation may exhibit noticeable species-, system- and organ-related differences. Either systemic or topical administration of capsaicin produced plasma extravasation both in the skin (see Szolcsfinyi, 1984b) and viscera. An interesting feature of this response is its marked regional distribution (Saria et al., 1983, 1984; Lundberg et al., 1984). Generally speaking, the intensity of the reaction seems greater in those organs or part of an organ which might more easily come into contact with the external environment. In female rats, the inflammatory response to capsaicin was much greater in the vagina than in the uterus (Saria et al., 1983b, 1984). In the urinary bladder, the reaction was much greater in the bladder neck and proximal urethra than

Capsaicin-sensitive neurons 5

15

rnin

1.r t

CAPSAICIN I

t

SUBSTANCE 10 nM

pM

t

P

CGRP 10 nM

Fig. 7. Typical tracings illustrating the effect of capsaicin, substance P or calcitonin gene-related peptide on the motor activity of the longitudinal muscle from guinea pig isolated ileum. The preparations were field stimulated (0.1 Hz, 0.3 msec, 60V). Capsaicin produced a transient contraction followed by a sustained depression of twitches. At the concentrations tested substance P induced a contraction and barely affected twitches. CGRP produced a small contraction and induced a prolonged depression of twitches. These findings suggest that in this organ, the visceromotor response to capsaicin could be best explained by the action of SP and CGRP than by the action of SP alone.

5 rnin

t

CAPSAICIN 1 ~JM

t

CAPSAICIN 1 ~JM

t

CAPSAICIN I pM

t

CAPSAICIN 1 ~JM

Fig. 8. Specific (exhibiting desensitization) visceromotor responses to capsaicin in the rat isolated proximal urethra. In both instances a second administration of capsaicin (1/zM) 1 hr later did not affect motility. The rhythmic contractions observed before and during capsaicin administration were determined by field stimulation (10 Hz, 60 V, 0.5 msec for 3 sec). See the text for details.

16

CARLO ALBERTO MAGG1 and ALBERTO MEL!

in the bladder dome (Maggi et al., 1986e, 1987a). In the intestinal tract, an intense reaction was observed in the oesophagus and anal mucosa but not in the small intestine or colon (Saria et al., 1983b, 1984). A notable exception seem to be represented by the rat proximal duodenum where a small but clear blueing was observed following capsaicin administration (Maggi et al., 1987g). In various organs from guinea-pigs intensity of the capsaicin-induced plasma extravasation was proportional to tissue levels of SP-LI (Lundberg et al., 1984b). Likewise, in the rat isolated urinary bladder, the intensity of the capsaicin-induced tetrodotoxinresistant contraction is directly proportional to tissue levels of SP-LI (Maggi et al., 1987e). A particular form of efferent function mediated by the capsaicin-sensitive nerves was described in the rabbit maxillary sinus in vivo (Lundberg 1986; Lindberg and Mercke, 1986a, b). In this system, i.v. capsaicin or SP as well as antidromic stimulation of the maxillary nerves or exposure to cigarette smoke accelerated mucociliary activity. The response to capsaicin was inhibited by atropine, hexamethonium or a SP antagonist: likewise, the response to cigarette smoke was abolished by these drugs as well as by previous capsaicin desensitization. The Authors proposed that, at this level, the capsaicin-sensitive afferents participate to a secretory reflex mediated by cholinergic effector neurons: this reflex may have a protective significance by regulating mucociliary activity in response to irritants. The real significance of the "efferent" component of responses mediated by the capsaicin-sensitive sensory neurons needs to be established: certain responses such as the bronchoconstriction to irritants may have pathophysiological significance (Lundberg and Saria, 1982, 1983; Lundberg et al., 1983b, c, 1984b). In other tissues, such as the rat proximal duodenum, capsaicin activates an efferent response (NANC relaxation of the longitudinal muscle) which closely mimicks the response of the tissue to a natural stimulus (distension of the lumen leading to an increase of intraluminal pressure) (Holman and Hughes, 1965). This raises the possibility that, at this level, capsaicin-sensitive nerves participate to certain motility patterns which also occur physiologically. And yet, in the urinary bladder, the neuropeptide released from the sensory nerves following chemical but possibly also physical (distension) stimulation affect muscle cells and nerves (both sensory and efferent) in such a way to produce a further recruitment of afferent units, activate micturition and potentiate bladder voiding (Maggi and Meli, 1986a, b). Other types of "efferent" actions produced by release of sensory neuropeptides in the periphery will be briefly discussed in Section 5. The question relative to physiological and pathophysiological significance of the "efferent" component of function of sensory nerves will be further discussed in Sections 5, 6, 8 and 9 of this review. 5. THE "TROPHIC" ACTION OF CAPSAICIN-SENSITIVE SENSORY NEURONS

The term "trophic" action may have, when referred to endogenous molecules, various meanings

(see Varon and Bunge, 1978; Burnstock, 1983; McArdle, 1983). Among the "trophic'" effects mediated by the capsaicin-sensitive sensory neurons we may identify those actions of sensory transmitters which, apart from inducing an immediate response in target structures (contraction or relaxation of muscle cells, changes in blood flow and vascular permeability, degranulation of mast cells etc.) participate, on a long-term basis, to development and maintenance of the anatomofunctional integrity of tissues and are involved in their ability to react and repair in response to environmental factors. Quite obviously, this area is of extreme interest, not only for basic science but also for its practical implications. If a tonic, "trophic" action of sensory neuropeptides exists in the nervous tissue, viscera and skin, what could be the impact of drugs which block or antagonize these actions on normal function? A response to this question seems relevant for a rational strategy of drug development in this area. 5.1 Capsaicin-sensitive mechanism in the skin and eye

It is well-known that a lesion of sensory nerves may produce distrophic and/or atrophic changes in the skin. Accordingly, it is a common experience of anyone working with rats desensitized to capsaicin as newborns that these animals show signs of cutaneous lesions and/or dystrophia. Gamse et al. (1982b) reported that affected rats exhibit " . . . w o u n d s , particularly around their nostrils and behind the ears" and that this effect was never observed in Sprague-Dawley rats but the nature of these lesions and cause of differences was not determined. Such an effect does not seem to be present in rats desensitized to capsaicin as adults (Santicioli and Maggi, unpublished observations). This difference could be related to the fact that, in rats, multiple populations of capsaicin-sensitive nerves exist among which certain are resistant to capsaicin desensitization in the adult age (see Section 7.1). Likewise, corneal lesions with opacities have been observed in mice and rats (Wistar strain) desensitized to capsaicin as newborns: these were attributed to a neurotoxic effect on sensory fibers of the trigeminal nerve (Shimizu et al., 1984). Further studies showed that, in the cornea of capsaicin-pretreated animals, the proliferating basal layer of epithelial cells and the normal geometry of cell renewal were severely impaired (Fujita et al., 1984). On the other hand, in guinea-pigs high doses of capsaicin produced skin lesions (scabs with hair loss in the wound, limited to the neck and facial area) and corneal opacities also when the animals were treated as adults (Buck et al., 1983). Thus adult guinea-pigs seem to be more dependent than adult rats upon integrity of capsaicin-sensitive afferents for trophism at skin level (see Section 7.1). The nature of these lesions of the skin and eye is unknown: one possibility, which would need experimental proof, is that capsaicin-sensitive nerves exert a "tonic" trophic action on skin and eye (Szolcsfinyi, 1984a; Hanley, 1985). This may depend upon the ability of released neuropeptides to increase local blood flow (see Brain et al., 1986) and capillary permeability, facilitating and modulating the tissue reaction to injury. In addition, experimental evidence

Capsaicin-sensitive neurons indicates that sensory neuropeptides such as SP or NKA may activate a variety of cellular functions (phagocitosis, chemotaxis, mitosis etc.) (Bar-Shavit et al., 1980; Payan et al., 1983a, b; Payan, 1985; Nilsson et al., 1985; Hanley, 1985; Roch-Arveillier et al., 1986) which are important in determining tissue reaction and repair to injury. As support to this theory, an increased number of SP fibers was demonstrated in the guinea-pig skin (after an initial decrease) during burn wound healing (Kishimoto, 1984). Interestingly, recent findings indicated a depletion of sensory neuropeptides (SP, CGRP, somatostatin) in rat skin during wound healing but the origin of these changes (increased release, decreased synthesis etc.) has not been established (Senapati et al., 1986). Helme and McKernan (1985) reported a decrease in neurogenic flare reaction to topical capsaicin in aged human skin and proposed that a loss in SP afferent nerve fibers may play a role in changes in structure and function of the skin observed in the elderly. It should be noted that application of capsaicin or antidromic stimulation of sensory nerves produces neurogenic inflammation at both cutaneous and ocular level (Jancs6 et al., 1967, 1968; Lembeck and Holzer, 1979) and also a release of SP-LI in response to injury (Camras and Bito, 1980; Jonsson et al., 1986; Helme et aL, 1986 a, b). To date, the mechanisms responsible for the genesis of cutaneous and ocular lesions in animals desensitized to capsaicin have not been systematically investigated. The "trophic" action of sensory transmitters on the peripheral tissue represents a particular form of "efferent" function. This system could operate tonically at a "low" level in such a way that "normal" sensory stimuli produce a continuous outflow of sensory transmitters whose action maintains integrity of the tissue. When the stimuli are particularly intense the reaction, activated at a higher degree, takes the form of "neurogenic inflammation". 5.2 Trophic action on the gastrointestinal mucosa

Immunohistochemical evidence indicates that a variety of neuropeptides is present in the nerve fibers in the stomach wall and some of these are contained in capsaicin-sensitive structures (Schultzberg et al., 1980; Ekblad et al., 1985; Sharkey et al., 1984; Clague et aL, 1985; Sternini and Brecha, 1985). The functional role of this peptidergic innervation is largely unexplored. Many data point to the fact that neuropeptides play an important role in regulating gastric acid secretion (g.a.s.) at CNS level (Morley et al., 1982): in recent years evidence has accumulated indicating that various neuropeptides may modulate g.a.s, by acting also at peripheral level (see Konturek and Kitler, 1986 for a review). A "tonic" trophic action of released neuropeptides may play a role in preventing ulceration at the level of the gastrointestinal mucosa. Formation of gastrointestinal ulcers may be considered as the adverse outcome of a dynamic state in which "normal" trophism of the mucosa is maintained by a balance between aggressive and protective factors. Accordingly, removal of some "gastrointestinal defence mechanism" by systemic capsaicin desensitization G.P. 19/I--B

17

may lead to an increased susceptibility to the ulcerogenie action of aggressive factors. In 1981 Szolcsfinyi and Barth6 reported that in rats desensitized to capsaicin as adults there is an increased susceptibility to formation of gastric ulcers induced by pylorus ligation or acid distension. Subsequent studies extended and confirmed this observation (Szolcsfinyi and Mozsik, 1984; Holzer and Sametz 1986a, b; Evangelista et al., 1986) and showed an aggravation of gastric ulcers induced by a variety of ulcerogenic agents in rats desensitized to capsaicin as newborns or as adults. In addition, a capsaicinsensitive defence mechanism affords antiulcer protection also at duodenal and small intestinal level (Evangelista et al., 1987; Maggi et al., 1987g). Szolcsfinyi and Barth6 (1981) reported that a single oral administration of a low dose of capsaicin to rats has a protective effect toward development of gastric ulcers. This finding was interpreted as an indication that capsaicin stimulates sensory nerves to release their mediator content which induce local vasomotor changes responsible for reduction in ulcers formarion. The neurogenic origin of the protective effect induced by a low-dose of capsaicin was not demonstrated in these experiments: clarification of this point seems crucial to rule out the possibility that unspecific actions of capsaicin, not necessarily related to its action on sensory nerves may be involved. An acute antiulcer effect of oral capsaicin has been reported recently by Holzer and Sametz (1986b) in the ethanol-induced gastric ulcer model. In this study, acute denervation of the stomach did not modify the acute antiulcer effect of a low-dose of capsaicin. However, in other systems such as the rat duodenum or bladder, at 48-72 hr time lag is necessary, following extrinsic denervarion, to observe the disappearance of acute visceromotor response to capsaicin (this time lag is presumably required to allow complete degeneration of the intramural sensory nerves) (Maggi et al., 1986b, d; Santicioli et al., 1986). Chronic denervation of the rat stomach, (bilateral subdiaphragmatic truncal vagotomy) induced some atropic changes of the gastric mucosa (Hakanson et al., 1984). Whether or not the loss of atrophic action of sensory neuropeptides released from capsaicinsensitive nerves was involved in this latter phenomenon remains to be established. Anyway, the acute protective effect of oral capsaicin toward ethanolinduced gastric ulcers was not observed in rat pretreated with capsaicin as newborns (Holzer and Sametz, 1986): this observation strongly supports the hypothesis of an involvement of a specific action of this substance on sensory nerves (see Section 2). It is interesting to note that systemic capsaicin desensitization facilitates the formation of experimental ulcers whose pathogenesis is either dependent or independent upon g.a.s. This observation is important when considering the possible mechanisms through which capsaicin-sensitive nerves exert their effect on gastrointestinal mucosa (see below). Resting g.a.s, seems unaffected by systemic capsaicin desensitization (Szolcsfinyi and Barth6, 1981). Likewise, the ability of rat gastric tissue to produce PGE2 and PGI2 is not impaired by systemic capsaicin desensitization (Hoizer and Sametz, 1986). Various m e c h a n i s m s seem to play a role in the

CARLO ALBERTOMAGGIand ALBERTOMEL!

18

genesis of the gastric defence mechanisms mediated by the capsaicin-sensitive fibers. Szoles~nyi and Barth6 (1981) proposed that release of neuropeptides from sensory fibers facilitates the removal of ulcerogenie stimuli and prevents their adverse action on the mucosa by increasing mucosal blood flow. Induction of some plasma extravasation may also have a buffering effect in the interstitial spaces of the mucosa (Szolcsfinyi and Barth6, 1981). Acute administration of capsaicin may increase mesenteric blood flow in the dog small intestine (Rozsa et al., 1984, 1985a) ascribable to release of various neuropeptides, including SP and vasoactive-intestinal polypeptide (VIP) (Rosza et al., 1985b). Likewise, endoluminai perfusion with SP produced a selective mucosal/ submucosal hyperaemia in the cat jejunum, ascribable to a direct effect on vascular smooth muscle (Gronstad et al., 1986). The amount of SP-LI released in the gut lumen (cat jejunum) under basal conditions was enhanced following vagai nerve stimulation (Gronstad et al., 1985, 1986). Although the putative sensory origin of these vagal nerve fibers has not been assessed, it is possible that, in response to physiologically-relevant events e.g. a meal, SP and/or other neuropeptides might be released from sensory nerves in the gastrointestinal wall and safeguard mucosal integrity by enhancing blood flow particularly to the mucosal/submucosal layers. Loss of this trophic influence may render the animals more susceptible to aggressive environmental stimuli, In this scenario, the adequate stimulus activating the

CENTRAL NERVOUS SYSTEM

~')

defensive response may be represented by factors present in digesta among which certain nutrients could be considered as putative candidates (see Section 6 and Mei, 1985). At duodenal level, lowering of pH is an effective stimulus to activate capsaicinsensitive fibers in such a way to inhibit gastric motility (entero-gastric reflex) (Cervero and McRitchie, 1982). In capsaicin-desensitized rats there is an aggravation of duodenal ulcers induced by chemicals (cysteamine, dulcerozine) which act by increasing g.a.s, and motility (Maggi et al., 1987g). In addition, i.v. capsaiein produces a small but evident plasma extravasation in the rat proximal duodenum, just at the same site where cysteamine or dulcerozine induce ulceration (Maggi et al., 1987g). In Fig. 9 are schematically shown the sensory-efferent functions mediated by the capsaicin-sensitive nerves in the rat duodenum/small intestine. Some of these functions may be involved in the capsaicin-sensitive "duodenal defence mechanism". In addition to vascular changes, other mechanisms may be involved in activating the capsaicin-sensitive gastrointestinal defence mechanism. Capsaicinsensitive sensory fibers may activate a sympathetic reflex organized at prevertebral ganglia level, which could inhibit ulcers formation (Holzer and Sametz, 1986a). A third possibility stems from the observation that many neuropeptides have a potent inhibitory effect on g.a.s. (Konturek and Kitar, 1986). We have recently shown that very low doses of peripherally administered CGRP have a marked pro-

ENTERO- GASTRIC INHIBITORY REFLEX

pain ? activation of cardiovascular ref lexes

INTESTINO - INTESTINAL INHIBITORY REFLEX s*

d

PREVERTEBRAL ] GANGLIA

~ Vasoactive peptides

Z

PLASMA EXTRAVASATION

CAPSAICIN - SENSITIVE SENSORY FIBER

L

distension nutrients?

CGRP ~ NEURONAL BODY IN DORSAL ROOT GANGLIA

"

INTRAMURAL I ATP? L NANC NEURON J

_

i MUSCLE CELLS lI

Fig. 9. Schematic drawing illustrating the various types of effect concerned with the sensory-efferent function of the capsaicin-sensitive fibers of the rat duodenum-small intestine. The reaction involves: (a) activation of intramurally organized motility pattern (NANC relaxation of the longitudinal muscle) (Maggi et al., 1986a, b); (b) increase in vascular permeability and induction of plasma protein extravasation (Maggi et al., 1987gi); (c) activation of the enterogastric inhibitory reflex (Cervero and McRitchie, 1982); (d) activation of intestino-intestinal inhibitory reflexes determining ileus paraliticus (Holzer et al., 1986); (e) transmission of pain and activation of cardiovascular reflexes in the CNS (Lembeck and Skofitsch, 1982; Lembeck and Donnerer 1983). Various of these mechanisms may be involved in the "duodenal defence mechanism" (Maggi et al., 1987gi) which affords antiulcer protection in the rat proximal duodenum.

Capsaicin-sensitive neurons tective effect toward gastric and duodenal ulcers produced through an hypersecretion of acid (Maggi et aL, 1987h). Nerve fibers containing CGRP-LI are present in the rat stomach which disappear following systemic capsaicin desensitization (Sternini and Brecha, 1985). Therefore, we hypothesized that endogenous CGRP, if released by ulcerogenic stimuli from sensory fibers in the gastric wall may exert an antiulcer effect by reducing g.a.s. (Maggi et al., 1987h). A fourth mechanism may involve the participation of adrenals (Evangelista et al., 1986a): in fact, aggravation of indomethacin-induced gastric ulcers produced by systemic capsaicin desensitization in adult rats was prevented by adrenalectomy. This finding implies that, in normal rats, the capsaicin-sensitive nerve may antagonize ulcers formation also through a mechanism involving hormone release from the adrenal medulla. Taken together these findings indicate that the trophic action of capsaicin-sensitive nerves on the gastrointestinal mucosa may involve a complex series of mechanisms whose relative contribution may vary from one model of experimental ulcers to another. All the evidence discussed thus far suggests a protective role of capsaicin-sensitive mechanisms toward ulcers formation. However, some findings indicate that the picture may be more complex: Alfoldi et al. (1986) reported that in rats desensitized to capsaicin as adults (total dose 300 mg/kg in divided doses during 5 days), g.a.s, response to histamine was markedly reduced while the response to pentagastrin (25-250#g/kg) or carbachol was unaffected. More recently, Dugani and Glavin (1986) reported a decreased g.a.s, response to pentagastrin (6#g/kg) in adult rats receiving 65 mg/kg of s.c. capsaicin, 6 days before. In both studies, determination of g.a.s, was made in conscious rats with an indwelling gastric cannula. The discrepancy about the effect of pentagastrin is most likely ascribable to some methodological factors, but the important point is that both studies report a reduced secretory response to certain stimulants in capsaicin-desensitized animals: this suggest that under certain circumstances, these sensory nerves may be involved in a mechanism which is potentially aggressive for the integrity of the gastric mucosa. The physiological relevance of these findings is, at present, unclear: in principle, not all the actions mediated by capsaicin-sensitive sensory nerves at this level are directed toward production of an antiulcer effect. The altered susceptibility to experimentally-induced ulcers in laboratory animals must thus be interpreted as the net effect resulting from removal or functional impairment of several intra- and extramural mechanisms, among which some action could be per se pro-ulcerative. 5.3 Trophic action o f capsaicin -sensitive sensory nerves on neuromuscular structures

"Conventional" transmitters (acetylcholine, monoamines, GABA etc.) induce rapidly ensuing and short-lasting changes in membrane excitability thought to be relevant for fast communication between cells (neuron-to-neuron or neuron-to-effector). On the other hand, neuropeptides can induce changes in neuronal excitability (at both pre- or postsynaptic level) which are less intense but of longer duration

19

than those produced by "conventional" transmitters (see Iversen 1984). This led to the speculation that certain neuropeptides have a "trophic" action on neuronal excitability by inducing long-term changes e.g. a "tuning" effect on neural excitability, modulating the responsiveness of target cells to the action of other, fast-signalling transmitters (see Iversen, 1984). A further point is that, in certain tissues, neuropeptides are present in nerves from which could be released during synaptic events: in spite of this, electrophysioiogical recordings failed to detect any potential change ascribable to the effect of peptides. Again, a "trophic" effect of neuropeptides was postulated (see for instance Bowers et al., 1986): atrophic action of neuropeptides on neural structures may be demonstrated in tissue culture studies (Brenneman et al., 1985). In the neural tissue the trophic action of sensory neuropeptides may be involved "in the growth and development of nerves.., and nerve interconnections" (Morley, 1986). As an extension of the preceding concept, may be that "trophic" actions mediated by the capsaicinsensitive primary afferent fibers influence postnatal development and maturation of neuromuscular structures (Maggi et al., 1984b, 1986e). At present, the concept that sensory fibers exert a trophic action on neuromuscular structures is a very speculative one which needs much experimental work. However, available evidence indicates that neuropeptides may modulate the expression of certain receptors on the muscle membrane. For instance, CGRP may be a motoneurone-derived trophic factor that increases the synthesis of acetylcholine receptors at vertebrate neuromuscular junction (New and Mudge, 1986; Fontaine et al., 1986). Further, SP stimulates proliferation of embryonic rat aortic smooth muscle cells, an effect observed even at nM concentrations (Payan, 1985). Similar findings have been obtained with SP and neurokinin A (NKA) on cultures of smooth muscle cells from the aortic media of adult rats (Nilsson et al., 1985). Whether CGRP, SP and other neuropeptides, released in very small amounts from sensory nerves could produce similar effects on other systems, remains to be established. 6. T H E ADEQUATE STIMULI FOR ACTIVATING T H E SENSORY-EFFERENT FUNCTION OF CAPSAICIN-SENSITIVE SENSORY NEURONS

According to classical physiological principles, sensory fibers should be classified on the basis of the adequate natural stimuli for their activation. At cutaneous levels, capsaicin possesses a very selective action on polymodal nociceptors (Szoics~inyi 1977, 1984a). These fibers can be activated by a variety of chemical and physical stimuli, including mechanical irritation and thermal injury (Saria et al., 1984; Helme et al., 1986b). Certainly, capsaicin cannot be regarded as an adequate stimulus for classification of sensory afferents: according to Szolcs~myi (1985) "capsaicin is by no means a sensory C fiber neurotoxin in general". Therefore, there would be no need to rename a well defined class of afferents such as cutaneous polymodal nociceptors as capsaicin-sensitive (Szolcshnyi,

20

CARLO ALBERTO MAGGI and ALBERTO MELI

1984a). However, at visceral level, other sensory modalities, not necessarily involving pain perception, operate physiologically to stimulate the capsaicinsensitive fibers (see Sections 3.2 and 3.3). For this reason, the use of the term capsaicin-sensitive sensory fibers, as defined by Szolcsfinyi and Barth6 (1978) is still justified, at this stage of knowledge, for a collective labeling of these sensory afferents. A better knowledge on the natural stimuli which activate these sensory neurons seem mandatory for evaluation of their relevance to pathophysiology of human diseases and, consequently, of the possible significance and use of drugs which modulate these functions. Accordingly, the definition of adequate natural stimuli capable of activating capsaicin sensory fibers in various organs and systems is a major trend of research in this area (Maggi et al., 1987f). At both somatic and visceral level capsaicin-sensitive elements behave as polymodal receptors, e.g. various types of environmental stimuli are able to activate these elements but very little is known about the intimate mechanisms through which physical stimuli such as distension are able to excite the sensory terminal. Physiological studies led to two interpretations about the type of natural stimuli capable of activating visceral sensory fibers. Coleridge, Coleridge et al. (1981, 1984) studied extensively the vagal and sympathetic C afferent fibers in the heart lung and vessels and concluded that chemical signals are the major if not the sole natural stimulus for these fibers. On the other hand Paintal (1969, 1986) showed that physical events such as increase in interstitial volume following increase in pulmonary capillary pressure can activate C fibers. Paintal postulated that C fibers (termed J receptors) were surrounded by collagen fibers which, in the presence o f an increased interstitial fluid (lung edema), would swell and distort afferent nerve endings. This hypothesis is supported by recent observations (Roberts et al., 1986) indicating that dog pulmonary C fibers are reversibly activated by lung edema. A list of environmental stimuli capable of activating the capsaicin-sensitive sensory nerves is given in Table 3. In certain viscera both chemical or physical stimuli may activate responses of physiological significance, i.e. lowering of pH in the duodenum activates the enterogastric reflex (Cervero and McRitchie, 1982), fluid distension activates micturition reflex in the urinary bladder (Maggi et al.,

1984a, 1986d) or intestino-intestinal inhibitory reflexes regulating motility (Kreulen and Peters, 1986; Maggi et al., 1987b; Holzer et al., 1986). Likewise, in other viscera, both chemical or physical stimuli can activate pathological-or pathological-like responses mediated by capsaicin-sensitive fibers, i.e. mechanical or thermal injury induces plasma extravasation in the skin and airways (Helme et al., 1986a, b; Lundberg and Saria, 1983), and lung edema activates, through distension of the interstitium, vagal afferent C fibers (Paintal, 1969, 1986; Roberts et al., 1986). Likewise, various chemical irritants stimulate plasma extravasation in the airway and urinary tract, and also induce bronchoconstriction (Lundberg and Saria, 1983) and cystitis with detrusor hyperreflexia (Abelli, Giuliani et al., unpublished data). Therefore, also in the case of the capsaicin-sensitive fibers, the relative role played by chemical and physical stimuli in activating the sensory impulse (see Paintal 1986; Roberts et al., 1986) cannot be easily distinguished. It should be noted, that, in principle, the problem is non-dichotomizing in nature. In fact, physical factors, such as distortion of the cell membrane are able to activate biochemical events leading to production of bioactive substances, such as prostanoids (Piper and Vane, 1971; Gryglewski and Vane, 1972). Likewise Roberts et al. (1986) noted that "although... the mechanical consequences of vascular engorgement and extravascular water accumulation were largely responsible for C-fiber stimulation" during experimentally-induced lung edema, the possibility cannot be excluded that "chemicals released in edematous lung may have played a part". For instance, prostacyclin is released during pulmonary edema (Grondelle et al., 1984) and this autacoid stimulates both pulmonary and bronchial C fibers (Roberts et al., 1985). In the isolated rabbit iris sphincter muscle, indomethacin reduced the SP-mediated component of contraction produced by antidromic activation of capsaicin-sensitive nerves which had no effect on responses to exogenous SP (Ueda et al., 1985). This and other observations suggested that in resting conditions prostanoids produced in the tissue modulate the excitability of these sensory nerves. In addition, certain effects of exogenous prostanoids such as induction of plasma extravasation in rat skin (Arvier et al., 1977) and activation of reflex micturition in rats (Maggi et al., unpublished data) are almost abolished following systemic capsaicin desensi-

Table 3. Stimuli of physiological or pathophysiological relevance having the ability to activate the sensory-efferent function of the capsaicin-sensitive sensory neurons at vesceral level Adequate stimuli Preparation

Physical

Gastrointestinal tract Lower urinary tract

Distension Distension

Tracheobronchial tree

Mechanical irritation Pulmonary edema Distension?

Cardiovascular system

Chemical pH, CCK-8, Nutrients? Constitutents of urine Bradykinin, 5-HT, irritants Cigarette smoke Nicotine Anoxia? Bradykinin

References I, 2, 3 4, 5, 6 7, 8, 9 10, I1, 12

(I) Lembeek and Skofltsch (1982); (2) Cervero and McRitchie (1982); (3) McLean (1985); (4) Maggi et al. (1984) (1986d) and unpublished data; (5) Szolcs~nyi (1984); (6) Recordati et al. (1980), (1982); (7) Lundberg and Saria (1983); (8) Lundberg et al. (1983), (1984); (9) Roberts et al. (1986); (10) Bond et al. (1982); (11) Lindefors et al. (1986); (12) Stoppini et al. (1984).

Capsaicin-sensitive neurons tization. Similarly, also certain responses to bradykinin or histamine are prevented following systemic capsaicin desensitization (see Table 8). Therefore, the possibility should be verified that certain physical stimuli (distension, heat etc.) stimulate the capsaicin-sensitive fibers indirectly, by generating bioactive substances (prostanoids, bradykinin etc.) produced at demand in target tissues. For the capsaicin-sensitive fibers the problem is intimately linked to the existence of a capsaicin "receptor" as proposed by Szolcs~inyi (1982, 1984a). If this receptor exists, what is its physiological ligand? Are physical stimuli capable of generating a substance(s) physiologically deputed to activate the "capsaicin" receptor? 6. I The peculiar chemosensitivity o f capsaicin -sensitive nerves: functional implications in relation to external and internal environment

As discussed in Section 5 the pathological-like reaction (neurogenic inflammation, plasma extravasation etc.) produced by activation of capsaicinsensitive nerves in the skin and mucosa could be viewed as an intense response of a physiologically relevant defense reaction, that is, a t r o p h i c mechanism which, in normal conditions operates at a much lower level. This concept may be tenable when looking at capsaicin-sensitive fibers in the skin and certain hollow viscera (stomach, tracheobronchial tree, urinary bladder and urethra) which come easily into contact with chemicals from the external environment. Much different seems the problem when looking at sensory fibers in the cardiovascular system or, anyway, to sensory fibers which are into contact with body fluids (kidney, renal pelvis etc.). Anoxia or hypoxia seems to be an adequate stimulus to activate certain reflex responses mediated by the capsaicinsensitive sensory fibers (Bond et al., 1982) or to induce release of sensory neuropeptides (Lindefors et al., 1986). Further, renal ischemia and backflow of urine can activate the capsaicin-sensitive fibers in the rat kidney and renal pelvis which regulate diuresis, renin secretion and blood pressure (Recordati et al., 1980, 1982; Szolcs~inyi, 1984a). Thus, a main function of this second type of capsaicin-sensitive fibers (those monitoring the internal environment) could be that of preserving chemical homeostasis in body fluids. 7. HETEROGENICITYOF CAPSAICIN-SENSITIVE SENSORY NEURONS There is evidence that somatic and visceral sensory neurons can be activated by a variety of stimuli, but the anatomofunctional determinants of this heterogenicity are largely unexplored. Evidence is accumulating that certain subpopulations of sensory cells may be distinguished from each other on the basis of anatomical, neurophysiological and biochemical criteria (Dodd et al., 1983, 1984; Rambourg et al., 1983; Sharkey et al., 1983; Tuchscherer and Seybold, 1985; Kai-Kai et al., 1986; Matsuyama et aL, 1986; Rose et al., 1986). Electrophysiological experiments indicate that action potentials (APs) recorded from sensory neurons

21

in dorsal root ganglia (d.r.g.) have various shapes~ presumably reflecting the specific activation of certain ionic channels (Yoshida et al., 1978; Harper and Lawson, 1985a, b; Matsuda et al., 1976, 1978; see also Higashi, 1986). It was suggested that spike shape, recorded in neuronal somata, correlates with the type of receptor innervated by peripheral fibers (Belmonte and Gallego, 1983; Strauss and Duda, 1982). Recently, Rose et al. (1986) showed that, when recording APs from somata of cat L7-S1 d.r.g, there was a characteristic spike duration for each receptor type (Golgi tendon organ, D-hair etc.) and also that somata whose axons supply low threshold cutaneous receptors are characterized by spikes of shorter duration than those innervating high-threshold cutaneous receptors. Anatomically, 6 different types of sensory neurons have been detected in d.r.g. (Rambourg et al., 1983). Among these, the capsaicin-sensitive elements belong to the small dark, type B sensory neurons but no anatomical or electrophysiological marker has been identified thus far to label a priori a given sensory neuron as being a capsaicin-sensitive one (see Szolcsfinyi, 1984a). The presence of multiple neuropeptides in these elements and the observation that certain populations of sensory neurons contain a given neuropeptide raises the possibility that expression of a given type of immunoreactivity in the cytoplasm may be a marker to distinguish between functionally distinct populations of sensory neurons. Studies in cats failed to observe a correlation between the type of sensory modality capable of activating a sensory neuron and presence of a particular type of peptide-like immunoreactivity in the cytoplasm (Leah et al., 1985). On the other hand, Kuraishi et al., (1985a) reported that application of a specific type of sensory stimulus to the skin elicited a stimulus-specific peptide release in dorsal horns of rabbit spinal cord: SP-LI and somatostatin-LI (SSTLI) were specifically released following application of noxious mechanical and thermal stimuli, respectively. Wiesenfeld-Hallin (1986) reported that in spinal rats intratechal SP increased excitability of the cord to both mechanical and thermal stimuli while the effect of SST was restricted to thermal stimuli. Thus, the common point of these two studies (Kuraishi et al., 1985a; Wiesenfeid-Hallin, 1986) was that, in two different species, SST seems specifically associated with nociceptive thermal stimuli. 7.1 Evidence for the existence o f multiple populations o f sensory neurons innervating the rat urinary bladder

In rats, capsaicin treatment at birth or in the adult age provides a mean to distinguish between two specific subpopulations of sensory nerves (Jancs6 et al., 1977, 1980, 1985a; Jancs6 and Kiralyi, 1980; see also Maggi et al., 1987d). It seems conceivable that these two populations of sensory neurons may have different biochemical markers, anatomical characteristics and may also play different role on a functional ground. On the basis of anatomical, biochemical and functional studies, (Holzer et al., 1982; Sharkey et al., 1983; S u e t al., 1986; Yokokawa et al., 1986; Maggi et al., 1987d, e; Santicioli et al., 1985, 1987) we proposed that three distinct sets of sensory cells

22

CARLO ALBERTO MAGGI and ALBERTO MELI Table 4. Subpopulations of sensory cells innervating the rat urinary bladder

Population I Population 2 Population 3

% of sensory cells ~-20 -~30 ~ 50

Sensitivityof capsaicin In all ages of life Only in newborn animals NO

innervate the rat urinary bladder (Maggi et al., 1987d). These three populations, hereafter referred to as P1, P2 and P3 cells (see Table 4) can be distinguished on the basis of their sensitivity to capsaicin, neuropeptide content and function. From the functional point of view, the crucial observation is that in rats desensitized to capsaicin as adults the maximal impairment in reflex micturition can be obtained with s.c. capsaicin 50 mg/kg, 4 days before. In these animals there is an increase in micturition threshold but reflex micturition occurs otherwise normally (Fig. 10). This dose of capsaicin produces a complete depletion of SP-LI bladder content in adult rats (Maggi et al., 1987d, e) and is also maximally effective in inducing neuronal degeneration in sensory cells of d.r.g. (Jancs6 et al., 1985a). A larger dose of s.c. capsaicin did not produce a greater functional impairment of reflex micturition than the lower one (Maggi et al., 1986g) nor increased the number of affected cells in d.r.g. (Jancs6 et al., 1985a). SP-LI + "bladder" sensory cells (e.g. those d.r.g, elements labelled following injection into the bladder wall of a retrogradely transported tracer) represent about 20% of total population in d.r.g. L6-SI (Sharkey et al., 1983; Yokokawa et al., 1986)

l

CONTROL

Transmitter content

SP, CGRP CGRP? ?

Functional role Reflex micturition, low threshold Reflex micturition, high threshold Conscious micturition?

that is about the same % of sensory cells which in adult rats: (a) show signs of degeneration following s.c. capsaicin (Jancs6 et al., 1985a) and (b) are depolarized following exposure to capsaicin in vitro (Heyman and Rang, 1985). On the other hand, in rats desensitized to capsaicin as newborns, reflex micturition is either abolished or impaired more severely than in rats desensitized as adults (Santicioli et al., 1985; Maggi et al., 1987c; Fig. 10). In these animals, loss of "bladder" sensory cells in L6-SI approaches 50% of total elements (Sharkey et al., 1983). This allows to identify two sets of capsaicin-sensitive sensory cells innervating the urinary bladder that is: (a) P1 cells (about 20% of the total) which contain SP-LI, are sensitive to capsaicin in both newborns and adult rats; (b) P2 cells (about 30% of the total), are sensitive to capsaicin only in newborn rats. P2 cells do not contain SP-LI since bladder content of this neuropeptide is depleted by capsaicin desensitization of adult animals (Maggi et al., 1987d, e). P1 cells contain not only SP-LI but also CGRP-LI as shown by Yokokawa et al. (1986). P2 cells may contain CGRP-LI: in fact, some CGRPLI + "bladder" sensory cells in d.r.g. L6-S1 were S P - L I - (Yokokawa et al., 1986) and CGRP-LI

M

M

+

i°l i°l

CAPSAICIN

AS

ADULT

M

/ .

__~..~ . . . . . .

...a

.

~ ~

Jr'-*-: ~" '--'-"~..-4~-

0 ~-

CAPSAICIN

AS

NEWBORN

OVERFLOW

INCONTINENCE

0 J-

START OF SALINE

INFUSION

0.046 ml/min

Fig. 10. Typical tracings illustrating the bladder response (micturition, M) to transvesical saline filling in a control rat (upper panel) or in animals desensitized to capsaicin (50 mg/kg s.c.) as adult (4 days before) or as newborn (2nd day of life). In the first case capsaicin desensitization increased bladder capacity while in the second ease reflex micturition was abolished and overflow incontinence ensued at a certain degree of bladder filling.

Capsaicin-sensitive neurons content of the rat bladder was depleted in rats desensitized to capsaicin as newborns (Su et al., 1986). Therefore, the C G R P - L I + b u t S P - L I elements may form a distinct population (P2) of sensory cells innervating the rat bladder. That CGRP-LI + but SP-LI - sensory cells form a distinct subpopulation of sensory elements was also proposed by Matsuyama et al. (1986) on the basis of observations in the rat cerebral arteries. However, in this system, the CGRP-LI +elements were capsaicinresistant also in newborn rats. More important, no information is available until now to allow a differentiation of these elements on a functional ground. PI and P2 cells activate reflex micturition but in the "conscious" state, micturition was largely preserved following desensitization to capsaicin (see HolzerPetsche and Lembeck, 1984; Conte and Giuliani, unpublished data). P3 ceils are, by exclusion, totally capsaicin-resistant, do not contain SP-LI or CGRPLI and may subserve "conscious" micturition (Maggi and Meli, 1986a, b; Maggi et al., 1987d). The proposed scheme is to our knowledge, the first example of an integration of anatomical, biochemical and functional data with regard to heterogenicity of sensory cells subserving a particular function. Our interpretation is based on the assumption that in this system: (a) all the deficits observed in capsaicintreated animals are selectively ascribable to a blockade of certain sensory neurons (see Maggi and Meli, 1986a, b; Maggi et al., 1987d) and (b) P2 cells are capsaicin-resistant in adult animals. Assumption (b) is supported by anatomical and functional data indicating that in adult rats, maximal effects on neuronal degeneration and bladder voiding were obtained with a dose of 50 mg/kg s.c. (Jancs6 et al., 1985; Maggi et aL, 1986g). Interestingly, systemic capsaicin desensitization in adult guinea-pigs produces a marked impairment in reflex micturition which seems more severe than that determined in adult rats: again, these changes were paralleled by an almost complete (> 90%) depletion of bladder SP-LI (Maggi, Geppetti et al., unpublished data). Likewise, guinea-pigs desensitized to capsaicin as adults exhibit a marked insensitivity to thermal nociceptive stimulation (Buck et al., 1981a) while this was not observed in rats desensitized as adults (Hayes and Tyers, 1980; Gamse, 1982) possibly because in these animals determination of nociceptive threshold is important to observe an effect of capsaicin (Szolcsfinyi, 1985). At both visceral (reflex micturition) and somatic (thermal nociception) level, adult guinea-pigs seem much more sensitive to the sensory neuron-blocking action of capsaicin than adult rats. According to the schematization proposed in Table 4, this may be interpreted as indication that PI cells are much more important for guinea-pigs than rats. As a matter of fact, in guinea-pigs almost all ( > 9 0 % ) sensory neurons containing CGRP-LI also contain SP-LI (Gibbins et al., 1985). According to our scheme, this would mean that P2 cells (SP-LI--, CGRP-LI + , capsaicin-sensitive only in newborn animals) are not present in this species or represent a very minor population of sensory elements. Interestingly, SP-LI content in some viscera (including the ureter and the urinary bladder) was much greater in

23

guinea-pigs than rats (Bucsics et al., 1983; see also Buck et al., 1981; Wharton et aL, 1986). Likewise, we may anticipate that, in certain viscera, the CGRPLI/SP-LI ratio should be much greater than unity in rats and in any case, much greater than the corresponding value in guinea-pigs. Accordingly, the CGRP-LI/SP-LI ratio is 0.78 in the guinea-pig ureter (calculated from data of Hua, 1986) and 15-17 in the rat ureter (Santicioli et al., 1986). In the superior mesenteric artery, the CGRP-LI/SP-LI ratio was 6.7 and 1.9 for rats and guinea-pigs, respectively (calculated from Wharton et al., 1986). Further studies are needed to test the validity of the P1-P2-P3 cells hypothesis and further, the possible application of this scheme to other systems. Data in the literature indicate that in different d.r.g, the percent of capsaicin-sensitive cells (see Arvidsson and Ygge, 1986) as well as the percent of elements containing SP-LI and other neuropeptides (Tuchscherer and Seybold, 1985; Dockray and Sharkey, 1986) may vary within rather large limits. In rats, a separate population of d.r.g, elements (about 10% of all neuronal cell bodies) contains SST-LI and these elements are distinct from those containing SP-LI (Hokfelt et al., 1976). These neurons are sensitive to neonatal capsaicin desensitization but do not innervate the rat urinary bladder (Sharkey et al., 1983). Whether capsaicin-sensitive SST-LI neurons form a distinct subset of elements possibly involved in thermonociception (Kuraishi et al., 1985a; WiesenfeldHallin et al., 1986) remains to be established.

8. FUNCTIONAL SIGNIFICANCE OF NEURoPEPTIDE COEXISTENCE IN CAPSAICIN-SENSITIVE SENSORY NEURONS

Once the hypothesis that capsaicin is a selective "substance P neurotoxin" is abandoned, the picture is now clear with regard to the fact that many neuropeptides (see Jancs6 et al., 1981 and Table 5) and possibly also non-peptide transmitters (Nagy et al., 1984) are present in capsaicin-sensitive sensory neurons, both in the central and peripheral nervous system. This marked neurochemical heterogenicity is certainly due, at least in part, to the fact that the entities we call "capsaicin-sensitive sensory neurons" are an heterogeneous populations of cells (see the preceding section). To date, at least 12 different types of peptide-like immunoreactivities were reported to be present in capsaicin-sensitive nerves (Table 5). In several instances, evidence came from immunohistochemical studies showing the presence of a particular type of immunoreactivity, stained by "specific" antisera which disappears following systemic capsaicin desensitization. Recent observations call for caution in this field since certain antisera may have little or no cross-reactivity with family-related peptides but have important cross-reactivity with other, familyunrelated peptides. Examples of this type were described for CCK-CGRP (Ju et aL, 1986) and CRF-SP (Berkenbosch et aL, 1986; Table 5). Both biochemical (Hua, 1986; Hua et aL, 1986a; Saria et al., 1984b, 1986) and anatomical obser-

24

CARLO ALBERTO MAGGi a n d ALBERTO MELI Table 5. Neuropeptides described to be present in capsaicin-sensitive sensory neurons Neuropeptide

Reference

Substance P Neurokinin A Neuropeptide K Eledoisin-like peptide Somatostatin Vasoactive intestinal polypeptide Choleeystokinin-oetapeptide* Calcitonin gene-related peptide Galanin Cor tieotropin-releasing factort Arginin vasopressin Bombesin-like peptides

Jessell et al. (1978) Hua et al. (1985), (1986a) Hua et al. (1985), (1986a) Hua et al. (1985), (1986a) Nagy et aL (1981); Gamse et al. (1982b) Jancs6 et aL (1981) Jancs6 et al. (1981) Gibbins et aL (1985); Lundberg et al. (1985) Skofitsch and Jacobowitz (1985) Skofitsch et al. (1984) Kai-Kai et aL (1986) Decker et al. (1985)

*Recent observations (Ju et al., 1986) indicate that CCK-LI in primary sensory neurons may represent CGRP or a similar peptide. tRecent observations (Berkenbosch et al., 1986) indicate that presence of CRF-LI in primary sensory neurons may be due to cross-reactivity of some anti-CRF antisera with SP.

vations (Yokokawa et al., 1985, 1986; Gulbenkian et al., 1986) indicate that multiple neuropeptides can be simultaneously released from capsaicin-sensitive nerves both in the central and peripheral nervous system. The functional significance of the presence of multiple transmitters in the same sensory neuron is very attractive and promising topic: available evidence indicates that the system seems constructed in such a way to cope with a variety of functional requirements, having different significance when moving from one system to another. As shown in Table 6 at least four types of interactions have been described thus far. For the sake of convenience we will limit our analysis to the interactions between SP and CGRP. However, the picture is certainly much more complex than depicted in Table 6. For instance, both neurokinin A, neuropeptide K and a not yet identified eledoisin-like peptide are present in SP-LI + nerves from which can be released by capsaicin (Hua et al., 1985, 1986a; Tatemoto et al., 1985; Theodorsson-Norheim et al., 1984a; Hua, 1986; Saria et al., 1986). The question relative to the functional significance of neuropeptide co-existence in sensory nerves has several implications. Firstly, not all the peptides in capsaicin-sensitive nerves are necessarily co-released (see Saria et al., 1986 and Maggi et al., 1987f for a discussion). Secondly, an adequate tool should be

developed to study concomitantly the biological response produced by sensory neuropeptides and the relative amounts released. Capsaicin is certainly a good tool but as already mentioned in Section 7, capsaicin-sensitive neurons are probably heterogeneous and caution is necessary when trying to assess the functional significance of the capaicininduced responses. Thirdly, the possibility exists that, at different degrees of stimulus intensity, certain neuropeptides are preferentially released from sensory nerves (see Hua and Lundberg, 1986). In spite of this and other limitations discussed in extenso elsewhere (Maggi et al., 19870, the four types of interactions described in Table 6 cover almost entirely the spectrum of possible interactions which could, theoretically, occur between two of these substances at postjunctional level. In the first condition only one transmitter is effective on a given function: for SP and CGRP both combinations have been described (Table 6). In the second condition, one transmitter (SP) is effective in stimulating its own receptor while the other (CGRP) does not produce per se any effect, but potentiates markedly the effect of SP (Table 6). The reverse (SP is ineffective but potentiates CGRP) has not been reported. The potentiating effect by CGRP of the SP-induced responses may involve a interference with metabolic breakdown of the latter (Le Greves et al., 1985).

Table 6. Functional interactions between substance P and calcitonin gene-related peptide (CGRP) (A) One peptide has very poor activity, or is ineffective SP + CGRP - rat urinary bladder SP - CGRP + guinea-pig atrium (B) Potentiation --plasma extravasation in rat or rabbit skin - - r a t biting and scratching syndrome --salivary secretion in rats (C) Physiological antagonism - - r a t duodenum - - r a t vas deferens - - r a t or guinea-pig ureter --human uterus (D) Co-operation ---cat cerebral blood vessels --gunea-pig ileum?

References Maggi el aL unpublished

Franco-Cereceda and Lundberg (1985) Gamse and Saria (1985) Brain and Williams (1985) Wiesenfeld-Hallin et aL (1985) Ekman et al. (1986) Maggi et al. (1986b) (1987f) Maggi et al. (1987f) Maggi et al. (1987), (1987f); Hua 0986) Samuelson et al. (1985) Edvinsson et al. (1985)

Capsaicin-sensitive neurons In the third type of interaction, both neuropeptides produce an effect, but this is of opposite sign on the same function (Table 6). Assuming a concomitant release of the peptides from the same sensory terminal in response to natural stimuli, we may have an example of a true physiological antagonism. To date, in all the viscera in which this interaction was described, CGRP had a very potent inhibitory effect on visceral motility which may overcome the weak excitatory effect of SP. The low potency of SP in inducing a response in these tissues (see for instance Fig. 11) is largely accounted by the presence, on target structures of an SP-E type of tachykinin receptor (as described by Lee et al., 1982). Whether the repetitive occurrence of this arrangement in various organs from three different species was casual or reflects a more general type of "solution" which copes with particular functional requirements of certain organs remains to be established. It should be noted that in all these tissues N K A (and other kassinin-like peptides) are much more potent than SP in producing an excitatory effect on motility. In these tissues the visceromotor response to capsaicin was of an inhibitory type suggesting that the action of CGRP prevailed on that of tachykinins co-released from sensory nerves. The fourth possible type of interaction is cooperation of peptides in determining the same final response. For instance both SP and CGRP produce a contraction of the guinea-pig ileum (Fig. 7) and also a relaxation of vascular smooth muscle (Table 6). This latter point seems of extreme interest: Regoli et al. demonstrated that in the same vessel, different types of tachykinin receptors mediate opposite effects on vascular tone (D'Orleans-Juste et al., 1986). The vasodilation was induced following activation of an NK-P receptor type present on endothelial cells which in turn produced a potent vasodilating substance. However, a second receptor of the N K - A type (Regoli et al., 1985, 1986, 1987), present on muscle cells, mediates a contraction of endothelium-deprived preparations (D'Orleans-Juste et al., 1985). RAT

~

DUODENUM

loo

CGRP

CONCENTRATION

25

Capsaicin-sensitive nerves in blood vessels contain both SP and N K A (which are the most potent natural tachykinins at the NK-P and N K - A receptors, respectively). However, also CGRP-LI is present in sensory nerve fibers innervating blood vessels (Uddman et al., 1985; Saito and Goto, 1986; McCullouch et al., 1986; Jansen et al., 1986) and this substance exerts a potent and prolonged vasodilatatory action in vivo (Fisher et al., 1983; Brain et al., 1985). In rat aorta, the GRP-induced vasodilation was endothelium-dependent (Brain et al., 1985) but in other systems this effect is ascribable to a direct action on vascular smooth muscle (Edvinsson et al., 1985, 1986; McCulloch et al., 1986) presumably through accumulation of cAMP (Edvinsson et al., 1985). Thus, at vascular smooth muscle level, CGRP may co-operate with SP in producing vasodilatation while both peptides may act as physiological antagonists of N K A which per se may induce vasoconstriction. Few data are available on the effect of capsaicin on isolated vascular smooth muscle (Toda, 1972; Duckies, 1986). Duckies (1986) observed that capsaicin induces an endotheliumindependent vasodilatation and this effect was not observed in vessels from desensitized animals. In the same tissue, SP induced an endothelium-dependent vasodilatation. Therefore, sensory neuropeptides may interact in a variety of manner. Available data indicate that postjunctional sensitivity is a major determinant of responses observed (Maggi et al., 19870 but much work is needed to establish the possible pathophysiological implications of neuropeptide coexistence in sensory nerves. Most examples listed in Table 6 deal with interaction between these neuropeptides in determining visceromotor responses. However, as outlined in Section 5, accumulating evidence indicates that neuropeptides can determine important effects on certain cell populations (leucocytes, fibroblasts etc.) and interaction between sensory neuropeptides at this level is unknown. This consideration is particularly important when analyzing the condition described in point A (Table 6), that is, one peptide has a very poor action or is ineffective, in such a way that the action of the other peptide is prevailing. It is clear that, in any given tissue, one sensory neuropeptide may have important actions on a particular function while being ineffective on other tissue components. This implies that, in the same tissue, one or more of the conditions listed in Table 6 may occur with regard to different functions. For instance, CGRP has little or no activity on contractility of the rat bladder muscle (Maggi et aL, unpublished) but may contribute with SP to induce vascular changes and maintain tissue trophism. 9. PATHOPHYSIOLOGICAL ROLE OF CAPSAICIN-SENSITIVE SENSORY NEURONS IN HUMAN DISEASES

(riM)

Fig. 11. Concentration-response curves for the contractile response to Kassinin or substance P and the relaxant response to CGRP in the longitudinal muscle of the rat isolated duodenum. Each point is mean + SE of at least 5 experiments.

The basic science advancements reported in previous chapters provide the ground for speculating that sensory neurons may play a pathophysiological role in various systems and possibly in various types of human disease (see Table 7). At present there is

26

CARLO ALBERTO MAGGI and ALBERTO MELI Table 7. Human diseases in which the capsaicin-sensitive neurons have been shown or proposed to play a pathogenic role Cluster headache, Migraine Asthma, Bronchial hyper-reactivity Rheumatoid arthritis, Rheumatic inflammatory disease Psoriasis Skin Allergic reactions Cold urticaria Thermal injury of the skin Detrusor instability, Detrusor hyper-reflexia

little direct evidence that the capsaicin-sensitive sensory neurons are involved in the pathogenesis of a particular human disease (see T6th-Kasa et al., 1983; Lundblad et aL, 1985) but circumstantial evidence indicates that this may be the case in various instances. More basic clinical work is needed to define at which extent drugs expected to antagonize, interfere or mimick with functions exerted by this class of sensory neurons may have a therapeutic application in certain human diseases. Experimental evidence indicates that great caution is needed when extrapolating from one species to another with regard to the functional significance of the capsaicin-sensitive nerves in a given system. To date, a comparison between animal data and clinical observations allows to hypothesize the potential involvement of capsaicin-sensitive nerves in human diseases in three areas which will be discussed below. 9. I Capsaicin-sensitive nerves and skin disease

The human skin contains capsaicin-sensitive structures. Topical application of capsaicin on human skin produces flare (Jancs6 et al., 1968; Helme and McKernan 1985; Carpenter and Lynn, 1981; Wallengren and Moiler, 1986) but this effect is absent on denervated skin (Jancs6 et al., 1968). Following topical capsaicin desensitization, the human skin no longer (for various d~ys) develop a flare reaction around a small injury'(Carpenter and Lynn, 1981). The same procedure prevents also the flare response to histamine and various neuropeptides (Bernstein et al., 1981; Foreman et al., 1983; Anand et al., 1983). More recently, Helme and McKernan (1985) have shown that in humans the capsaicin-induced flare exhibits a proximal-to-distal gradient and also that intensity of reaction declines with age. Due to easy procedure, there is a relatively abundant information about the effect of topical capsaicin desensitization on spontaneous manifestations of skin disease in humans. Jancs6 et al. (1983) reported that axon reflex vasodilatation in response to topical application of mustard oil (which stimulates capsaicin-sensitive fibers) was severely impaired by herpes zoster. An interesting finding of that study was an observation made in patients suffering from postherpetic neuralgia. The burning sensation produced by topical mustard oil was much more intense (up to painful sensation) in the affected skin region while only a moderate burning sensation was perceived in the

Moskowitz et al. (1979) Hardebo (1984) Sicuteri et al. (1985) Lundberg and Saria (1983) Barnes (1986) Levine et aL (1984), (1985h) Devillier et al. (1986) Hanley (1985) Farber et aL (1986) Lundblad et al. (1985) T6th-Kasa et al. (1983) Saria (1984) Maggi and Meli (1986), (1987)

contralateral unaffected skin area. Capsaicinsensitive neurons may play some pathogenic role in certain pain syndromes such as cluster headache (see Table 7 for References). Thus, the interesting hypothesis emerges that an irritative condition of capsaicinsensitive neurons may be involved in the genesis of certain forms of postherpetic neuralgia. T6th-Kasa et al. (1983) showed that in patients with acquired heat or cold urticaria, topical capsaicin prevented the appearance of flare reaction in response to heat or cold stimulation, suggesting a possible pathogenetic link between capsaicin-sensitive nerves and spontaneous manifestations of the disease. Likewise, Lundblad et al. (1985) reported that in humans, capsaicin-pretreatment abolished the flare component and itching sensation of the cutaneous allergic reaction while the wheal was unaffected. The ability of SP to stimulate mitosis led Hanley (1985) to speculate that an "overreactivity of these sensory nerves could have a pathogenic role in psoriasis". The hypothesis was elaborated more in e x tenso by Farber et al. (1986) (see also Section 5.1 of this review). Recently, T6th-Kasa et al. (1986) reported that topical application of capsaicin on the human skin produces a reversible marked reduction of histamineinduced itching suggesting that these sensory fibers may be responsible for spontaneous itching and pruritus associated with skin diseases. 9.2 Capsaicin-sensitive nerves and rheumatic disease

Various animal studies implicate the capsaicinsensitive nerves in joint inflammation (Lembeck et al., 1981; Colpaert et al., 1983; Hara et aL, 1984; Gamillscheg et al., 1984; Levine et al., 1984, 1985a, b; Ferrell and Russell, 1985). SP and possibly, related sensory peptides, may be involved in the pathogenesis of both central (pain) and peripheral (inflammation) components of rheumatic disease (Iversen, 1985; Levine et al., 1984). Recently, Levine et al. (1985a, b) reported that in rats, capsaicin-sensitive afferents determine, via connections across the spinal cord a reflex form of neurogenic inflammation e.g., hyperalgesia and swelling were produced contralateral to the injured paw through an entirely neurogenic pathway. This mechanism did not show any sign of tachyphylaxis at the uninjured paw. Thus repeated injury on one site w a s able to induce a sustained inflammation at a remote site through a mechansim involving capsaicin-

Capsaicin-sensitive neurons sensitive afferents. This type of response may allow the seeding of other cellular and humorai components of acute and chronic inflammation at widespread and possibly somatotopically organized sites in the body (Levine et al., 1985a, b). To date, only one study in humans supports the idea that neuropeptides may have a pathogenic role in rheumatic disease. Devillier et al. (1986) reported that in patients with rheumatic inflammatory disease (including rheumatoid arthritis) or osteoarthritis, the inflammatory synovial fluid contains more tachykinin-LI than non-inflammatory one. 9.3 Asthma and bronchial hyperreactivity

An impressive body of experimental data suggest that neuropeptide-containing sensory nerves may have a pathogenic role in asthma and bronchial hyperreactivity through the involvement of both sensory (cough, reflex bronchoconstriction, activation of various reflexes at cardiovascular and respiratory level) and efferent (bronchoconstriction from locally released sensory neuropeptides, neurogenic inflammation, modulation of the immune response etc.) function of these sensory nerves (Lundberg and Saria, 1983; Widdicombe, 1986; Payan and Goetzl, 1986; Joos et al., 1986a; Persson, 1986; Barnes, 1986). Capsaicin inhalation in man produces coughing which is abolished by application of local anesthetics to the larynx (Collier and Fuller, 1984). Further, paroxysmal coughing can be induced, in volunteers, by i.v. capsaicin. This response is blocked by local anaesthetic aerosol (Winning et al., 1986). These experiments established the presence, in human airways of capsaicin-sensitive nerves capable of reacting to irritants. Further, exposure of isolated human bronchi to capsaicin produced a contractile effect exhibiting desensitization (Lundberg et al., 1983, Table 2). Recently Joos et al. (1986) reported that inhalation of SP or NKA had no bronchoconstrictor effect in normal, non-smoking humans but produced a prompt and prolonged bronchoconstriction in asthmatics (Joos et al., 1986b). These findings suggest the existence of a disease-related hyperreactivity to neurokinins in human airways which supports the idea of pathogenic role of sensory nerves in asthma and bronchial hyperreactivity (see Barnes, 1986). 10. T H E PHARMACOLOGY OF CAPSAICIN-SENSITIVE SENSORY NEURONS

The pharmacology of sensory neurons has been reviewed previously (Douglas and Ritchie, 1962; Paintal, 1964; Ginzel, 1974; Higashi, 1986). Certain special characteristics of "afferent" vs "efferent" pharmacology were analyzed by Ginzel (1974): particularly worth of mention are, in our opinion, the special relevance which in "afferent" pharmacology play factors such as type and depth of anesthesia, the occurrence of dramatic species-related differences and, finally the fact that application of very low doses of stimulating agents to restricted "target" areas may have profound and widespread effects in various organs and systems. In addition, a special mention is deserved to the fact that "afferent" pharmacology is almost directly linked to physiology of the system

27

under study. This raises the question of the physiological (and pathophysiologicai) significance of the responses under study (Ginzel, 1974). Recent experiences with capsaicin, summarized in Section 3 indicates that in many systems these drug-activated responses are physiologically relevant as indicated by changes in threshold for activating reflex responses in capsaicin-pretreated animals. Accordingly, pharmacological studies using capsaicin as a tool or a probe to activate certain sensory neurons stand at the borderline between physiology and pharmacology. Paintal (1964), in reviewing the pharmacology of vertebrate mechanoreceptors proposed that the net effect of drugs on sensory nerves may be used for classificative purposes and proposed the following 5 cathegories for drug action on sensory nerves: (1) stimulation; (2) sensitization; (3) stimulation and sensitization; (4) stimulation and desensitization; (5) depression. In this review we propose a classification of drugs modulating capsaicin-sensitive sensory neurons based on a functional criterion, e.g. considering the final effect exerted by the drug on: (a) excitability and, presumably, release of transmitters from central and peripheral terminals (see Sections 10.1 and 10.2) and (b) receptors for sensory transmitters at postjunctional level (see Sections 10.3 and 10.4). An incomplete list of drugs and transmitters acting on or through capsaicin-sensitive sensory is shown in Table 8. The main purpose of this Table is to draw attention to the fact that various substances and, even more important, drugs used in human therapy, may produce some of their effects by modifying the excitability of sensory nerves. An example illustrating this point is that relative to the presence of both GABA A and GABA B receptors on central terminals of the capsaicin-sensitive primary afferents at spinal cord level (Singer and Placheta, 1980; Price et al., 1984). It seems conceivable that, at this site, GABA modulates excitability of these neurons and consequently transmitter release. Accordingly, we may hypothesize that some of the clinically relevant effects of drugs which potentiate or mimick GABAergic transmission, such as benzodiazepines or baclofen, may involve an action on excitability of capsaicin-sensitive sensory neurons. According to a functional criterion we want to distinguish three types of drugs affecting the function of capsaicin-sensitive sensory neuron, e.g.: (a) drugs which block the function; (b) drug (or transmitters) which modulate the function, either in an inhibitory or excitatory fashion; (c) drugs (or transmitters) which mimick the function and (d) drugs which prevent the action of released transmitters. As could be noted, in category (b) and (c) we will include not only drugs, but also transmitters and/or other endogenous modulators influencing excitability of sensory neurons. In this respect, our classification embraces not only the pharmacological (in a classical sense), but also the physiological chemical modulation of the function. 10.1 Drugs which block the capsaicin-sensitive neurons

At present the drugs known to produce this effect e.g. permanent or transitory functional blockade of the capsaicin-sensitive sensory neurons are capsaicin

28

CARLO ALBERTO MAGGI and ALBERTO MELI Table 8. Drugs or transmitters which may affect the excitability of capsaicin-sensitive primary afferent nerves

(A) Substances which increase excitability or stimulate the capsaicin-sensitive sensory nerves Prostaglandins Arvier et al. (1977); Ueda et al. (1985) Bradykinin Jancs6 et al. (1980); Baccaglini and Hogan (1983) Histamine Jancs6 et al. (1980); Toth-Kasa et aL (1986) 5-HT (SHT3 receptors) Jancs6 et al. (1980); Richardson and Engel (1986) Leukotriens? Stewart et al. (1984) Noradrenaline, adrenaline (in the periphery) Devor (1983); Blumberg and Janig (1984); Levine et al. (1986) Acetylcholine (nicotinic receptors) Juan et aL (1980); Juan et aL (1982) Neurokinins? Maggi et al. (1986e), (1987c); Lew and Longhurst (1986) BAY K 8644? Perney et al. (1986) McLean (1985); Ritter et al. (1986) Cholecystokinin octapeptide Pentagastrin? Dugani and Glavin (1986) (B) Substances which decrease excitability of capsaicin-sensitive sensory nerves Sodium cromoglycate? Dixon et al. (1980) Somatostatin Brodin et al. (1981); Gazelius et al. (1981) Morphine, other opioids Bentley et al. (1981); Barth6 and Szolcsanyi (1981); Lembeck et al. (1982) Dihydropyridines? Perney et al. (1986) Corticotropin releasing factor (CRF) Kiang and Wei (1985); Wei et al. (1986) Theophylline? Manzini et al. in press Monoamines (noradrenaline, dopamine) in the CNS Kuraishi et aL (1985b)

itself and its congeners or analogs which are able to induce desensitization and consequently functional impairment of both sensory and efferent functions (Szoicsfinyli, 1982). To date, it is unclear if the acute stimulatory effects of capsaicin responsible for its pain-inducing properties is necessarily coupled to its sensory neuron blocking properties. If this were not .~he case then development of a capsaicinoid devoid of pungent properties may offer new therapeutic possibilities in various types of human disease, including pain, inflammation asthma and others. Recently Levine et al. (1986a) reported that prolonged treatment with gold salts administered at doses which effectively inhibited adjuvant induced arthritis in rats, produced a neurotoxic effect on peripheral nerves involved in neurogenic inflammation (both sensory and sympathetic) and proposed that a similar mechanism determines the therapeutic effect of gold salts in humans. 10.2 Drugs or transmitters which modulate the sensory-efferent function of capsaicin-sensitive neurons

These substances may be subdivided into two categories e.g. those which act by exciting and those which act by inhibiting the function of the sensory neurons (Table 8). Much information can be found in previous reviews (Douglas and Ritchie, 1962; Paintal, 1964; Ginzel, 1975) about substances (phenyldiguanide, veratrum alkaloids etc.) or irritants which stimulate sensory nerves. In most instances, no information was given as to whether or not these substances acted on capsaicin-sensitive elements (but see Skofitsch et al., 1983). Among transmitters which may activate the capsaicin-sensitive nerves we want to outline three examples: CCK-8, noradrenaline and neurokinins. Peripherally administered CCK-8 causes suppression of food intake in many mammals, inlcuding humans. In rats this effect occurs as a part of a behavioural sequence which is similar to that induced by prior ingestion of food (reduced locomotor and exploratory activity, sleep) (Autin et al., 1973). The effects of CCK-8 were prevented by sub-

diaphragmatic vagotomy, suggesting that a specific group of vagal afferents was involved (Smith et al., 1981; Crawley et al., 1981). Recent findings indicate that the effect of CCK-8 on food intake as well as the subsequent "satiety" syndrome are prevented by capsaicin desensitization (MacLean, 1985; Ritter et al., 1986; Ritter and Ladenheim, 1986). Whether food-induced gastric distension releases endogenous CCK-8 which in turn activates the satiety behaviour by stimulating (either directly or indirectly) the capsaicin-sensitive vagal afferents is an interesting possibility. The important point of these findings is, in our opinion, that peripheral activation of this class of afferents by an endogenous substance is capable of inducing a very complex behavioral sequence. In view of the peculiar chemosensitivity of these fibers it is tempting to speculate that other examples of this type occur physiologically e.g. neurohormones and transmitters released at demand in the periphery could influence even the most integrated (behavioural) responses in the CNS, through the mediation of sensory fibers. For instance, selective destruction of capsaicin-sensitive afferents in the rat area postrema and nTS produces an overconsumption of palatable foods suggesting an involvement of these afferents in the control of preferred food intake (South and Ritter, 1983). The concept that endogenous substances or drugs which do not cross the blood brain barrier may induce complex behavioural responses by influencing CNS activity through an activation of peripheral sensory fibers represents a new type of approach for developing drugs capable of influencing certain CNS disorders. Noradrenaline (and possibly other monoamines) exert an antinociceptive effect by inhibiting the release of sensory transmitters from primary afferent fibers in the CNS (Yaksh et al., 1981; Kuraishi et al., 1985b; Sugimoto et al., 1986). However, there is evidence that the opposite occurs at peripheral level e.g. sympathetic influences enhance the excitability of the capsaicin-sensitive fibers: this may explain the pathogenic influence at peripheral level of sympathetic nerves in certain pain syndromes such as causalgia (Devor, 1983; Biumberg and Janig, 1984).

Capsaicin-sensitive neurons Levine et al. described recently (1986b) that guanethidine-pretreatment significantly attenuated the "reflex" neurogenic inflammation. The positive interaction between sympathetic and capsaicinsensitive nerves is of further interest when considering that both types of nerves complete, during development, for limited amounts of nerve growth factor (NGF) which limits the relative density of these types of nerves (Nielsch and Keen, 1986). The mechanisms underlying the facilitatory effect of sympathetic nerves on excitability of capsaicin-sensitive neurons at peripheral level have not yet been elucidated: earlier studies suggest the involvement of ct-adrenoceptors, putatively located on sensory nerves (Devor, 1983; Blumberg and Janig, 1984). More recently Levine et al. (1986b) reported that the noradrenaline-induced hyperalgesia may involve the synthesis of prostanoids. Sympathetic postganglionic innervation is important for reflex regulation of visceral motility at both gastrointestinal (Furness and Costa, 1974; Holzer et al., 1986; Maggi et al., 1987b) and urinary bladder level (De Groat and Theobald, 1976; Maggi et al., 1985e). To date, there is no information about the interesting possibility that sympathetic nerves interact with the capsaicin-sensitive nerves at visceral level. Various neurokinins are present in the peripheral terminals of capsaicin-sensitive nerves and consequently may be released in the periphery in response to natural stimuli (see Sections 4.1 and 4.3). Among the possible targets of sensory neuropeptides in peripheral tissues there are the sensory nerves themselves. Sensory neuropeptides could activate or depress excitability of the sensory nerves indirectly, by inducing a contraction of muscle cells or the release of other transmitters/autacoids such as histamine release from mast cells. In addition, recent findings indicate the possibility that at least in certain systems, neurokinins, including substance P may activate directly certain sensory nerves (Maggi et al., 1985c, d, 1986e, f, 1987c; Lew and Longhurst, 1986). In the rat urinary bladder, indirect evidence from functional experiments indicates that certain effects of exogenous neurokinins could be explained by admitting the presence of a NK-B type of receptor on sensory nerves (Maggi et al., 1987c; 1987i). Whether or not a distinct type of neurokinin receptor exists on sensory nerves in the urinary bladder, the ability of exogenous neurokinins to activate the micturition reflex was preserved in rats desensitized to capsaicin as adults but was lost in rats desensitized as newborns (Maggi et aL, 1986e, 1987c). According to the scheme proposed in Table 4, this would mean that the ability of neurokinins to stimulate reflex micturition depends upon integrity of P2 sensory cells, which are sensitive to the action of capsaicin only at birth. Interestingly, the SP-LI content of the rat bladder seems restricted to P1 cells (Maggi et al., 1986d, e) e.g. the low-threshold element deputed to perceive intravascular volume for activation of reflex micturition. Accordingly, neurokinins released at intramural level from terminals of P1 cells may recruit other P1 and P2 afferents indirectly (e.g. by inducing small myogenic contractions) and possibly also sensitize directly P2 afferents thus reinforcing the stimulus

29

to void. Also other transmitters and autacoids may produce their effects by activating sensory cells which are sensitive to capsaicin in newborn but not adult rats: for instance, Jancs6 et aL (1980) reported that plasma extravasation induced by histamine, serotonin or bradykinin was prevented in rats desensitized to capsaicin as newborns but not as adults. Availability of potent and selective NK-B receptor antagonists is required to establish: (a) whether NKB receptors mediate these effects of neurokinins on sensory nerves and (b) the possible physiological significance of this mechanism in initiating reflex micturition. 10.3 Drugs or transmitters which mimic the sensoryefferent function o f capsaicin -sensitive neurons As a matter of fact, transmitters belonging to this category are the various neuropeptides whose presence has been reported in the capsaicin-sensitive sensory neuron and fibers both in central and peripheral nervous system (Table 5). Obviously, the presence of a given type of immunoreactive material in a neural structure does not warrant that: (a) the substance(s) detected plays a role as neurotransmitter and (b) the immunoreactive material has the same chemical composition of that which had been used to raise the antiserum (cross-reactivity) (cf. Ju et al., 1986; Berkenbosch et al., 1986). To date, release of substance P, neurokinin A, calcitonin gene-related peptide and somatostatin and an eledoisin-related peptide was shown in response to capsaicin (Hua et aL, 1986a; Saria et al., 1986). The putative transmitter role of the other neuropeptides listed in Table 5 remains to be established. For instance VIP-like immunoreactivity has been described in the capsaicinsensitive structures in the CNS (Jancs6 et aL, 1981) and certain functional responses to capsaicin may be blocked by a VIP-antiserum (Rosza et al., 1985b) but no release of VIP has been reported from central terminals of capsaicin-sensitive nerves (Yaksh et al., 1982). 10.4 Drugs which prevent the action o f transmitters released from capsaicin-sensitive neurons In this category we may include those drugs or substances which, by acting at postjunctional level, prevent the action of transmitters released from central or peripheral terminals of these sensory neurons. Under a certain point of view, CGRP may fall in this category, since in certain systems it acts in such a way to prevent the actions of endogenous neurokinins, putatively co-released by capsaicin or other stimuli (physiological antagonism, see Section 8). In this category we can certainly include those substances which prevent the actions of sensory neuropeptides by interacting with their specific receptor sites (sensory neuropeptide antagonists). To date, a great number of antagonists for substance P have been synthesized and characterized (see Regoli et al., 1984 for a review). The major drawbacks of these drugs are as follows: (1) some of these antagonists possess, at certain concentrations and in certain systems, unspecific effects unrelated to their SP antagonistic properties (Post et al., 1984; Lembeck et al., 1986); (2) the affinity of these drugs for the SP

30

CARLO ALBERTO MACrGI and ALBERTO MELI

receptors is still much lower than that of the natural agonists (Regoli, 1985); (3) some of the antagonists are also antagonists of bombesin (see Regoli, 1985); (4) generally speaking, the antagonists described thus far are mainly active at the NK-P receptor: although some antagonist exhibited a little preferential activity at the N K - A or NK-B site, much work remains to be done in this field (Regoli, 1985; Regoli et al., 1985). In spite of these drawbacks, this generation of SP antagonists has been used in a number of instances to support a neurotransmitter role of endogenous substance P released from sensory nerves (see, among the others, Stoppini et al., 1983; Prabhakar et al., 1984; Maggi et al., 1985).

I1. CONCLUDING REMARKS: THE EMERGING PICTURE OF SENSORY-EFFERENT FUNCTIONS OF THE CAPSAICIN-SENSITIVE SENSORY NEURONS

The overall picture of the capsaicin-sensitive sensory neurons indicates that these elements play a crucial role in the connections between central nervous system and the periphery. Impulses of various origin arising in the periphery are transferred through the various branches of the neuron at various sites, both peripherally and centrally. When the propagated wave of depolarization invades a given terminal, the transmitter is secreted and produces its action on target structures. Again, it becomes obvious that any distinction between "sensory" and "efferent" functions is merely a consequence of our tendency to separate, for classificative purposes, phenomena which occur almost simultaneously at the various branches of the same sensory neuron. According to classical anatomo-functional principles, sensory cells in d.r.g, are pseudounipolar e.g. have a stem process which divides in two branches (bipolar sensory neuron) and sends one axon in the periphery where it receives the sensory input and the other one in the CNS where it transmits the information to secondorder sensory neurons. Although some arborizations of both central and peripheral process are usually considered, classical neuroanatomy describes the central process of primary cells as a single channel throughout the length of the dorsal root and the peripheral process as a single channel throughout much of the length of the peripheral nerve (Coggeshall, 1986). This picture has undergone major changes: sensory axons may have arborizations or collaterals in prevertebral ganglia which give synaptic input for modulation of excitability and/or activation of reflexes organized outside of CNS (see Gamse et al., 1981; Tsunoo et al., 1982; Bulygin, 1983). In the past few years evidence has been presented suggesting that axons of sensory neurons may have an extensive branching in the periphery (Pierau et al., 1984; Taylor and Pierau, 1982; Taylor el al., 1984; Mizutani et al., 1983; Pierau and Taylor, 1985; Alles and Dora, 1985; see also Coggeshall, 1986). The incidence of somatosensory fibers which dichotomize into different nerves exceeds the small number of cases in which action potentials are propagated from one nerve to another (Devor et al., 1984; Pierau et al., 1985). Whether this type of anatomical arrangement may account for phenomena such as the axon reflex or referred pain

(see Sinclair, 1948 an also Strassman et al., 1986) remains to be established. In addition, branching of sensory axons may occur at end-organ level: here, one sensory terminal could terminate in close contact with an effector cell (mast cell in the skin, cholinergic or N A N C neurons in the intestine) in such a way that activation of sensory receptors produces, a certain response through a propagated action potential in the same organ or tissue where the impulse was generated (axon reflex arrangement). As mentioned in Section 3, 4.3 and 10.2 there are reasons which make difficult an unequivocal separation of the "sensory" and "efferent" component of function mediated by capsaicin-sensitive sensory nerves. At this stage of knowledge, we may propose that as "sensory" functions should be considered those which involve a transmission of information from the sensory receptor to another terminal of the sensory neuron, located either centrally or peripherally, where released transmitters acts on a receptive element (second-order sensory neuron in the CNS, postganglionic neurons in prevertebral ganglia, mast cells in the skin, intramural effector neurons in the intestine) which in turn produces a certain biological effect by releasing its own transmitter(s). The common point which links together all these "sensory" functions is that transmission of the stimulus occurs from one terminal to another: this process requires a propagated action potential (Na + dependent, sensitive to tetrodotoxin and local anesthetics). On the other hand, the "efferent" functions would be those exerted by the transmitters released from the same terminal which is activated by the environment. Accordingly, the efferent function does not require a propagation of the information but is an entirely local phenomenon (receptor potential, Na + independent, resistant to tetrodotoxin and local anesthetics). As compared to previous interpretations of the literature (see Szoicsfinyi, 1983, 1984; Maggi et al., 1986b) this schematization considers those manifestations ascribable to an axon reflex arrangement as a "sensory" function of these neurons even if they occur independently from the CNS. It is now clear that the capsaicin-sensitive neurons must be considered as multipolar elements capable of releasing the stored transmitters at multiple sites. Indeed capsaicin can induce a tetrodotoxin-resistant release of neuropeptides from the various branches of these multipolar sensory neurons e.g. in the CNS (Gamse et al., 1979) prevertebral ganglia (see for instance Dun and Kiraly, 1983) and peripheral terminals (Saria et al., 1983a). Thus, the ionic basis for transmitter secretion may be similar at all terminals of the branches of these sensory elements. This further justifies the distinction between "sensory" and "efferent" functions of these elements, as discussed above. In principle, all terminals of these neurons located either centrally or peripherally rely upon a similar (tetrodotoxin-resistant, Ca 2+ dependent) ionic mechanism for transmitter secretion and, putatively for production of a sensory receptor depolarization which initiates a propagated action potential. Transmission of the information from one terminal to another is the marker of the "sensory" function which occurs through a mechanism having

Capsaicin-sensitive neurons an ionic basis different from the "efferent" function. This latter occurs, in principle, every time that the sensory receptor is stimulated. The relative contribution of reflex responses integrated at prevertebral ganglia level or in the CNS (both spinal and supraspinal sites) to the overall response produced by these nerves may have a great variability in terms of organ-, system- and speciesspecificity. As depicted in Fig. 12 a given environmental stimulus may produce transmitters release from at least 4 different terminals in the same neuron, thus leading to a wide range of short- and long-term biological effects. Neuropeptides, synthesized in the neuronal body are transported to both central and peripheral endings of this sensory neuron where they are stored in synaptic vesicles, ready to be released. A wide range of receptors for autacoids and transmitters is expressed on the membrane of these sensory neurons. Activity (membrane potential, excitability, amount of transmitter released) of sensory nerve terminals may be modulated in a very complex fashion, both centrally and peripherally. In addition, peripheral terminals of these sensory neurons possess the adequate characteristics (receptors ?) to be excited by chemical and physical environmental stimuli. Among these, chemical stimuli are markedly heterogeneous and, possibly, organ-specific. However, the picture may be even more complicated. The central terminals of the capsaicin-sensitive sensory neurons are provided with receptors regulating excitability of these sensory nerves. This raises a question which could add a new dimension to the whole problem: could chemical signals arising at CNS level or at terminals where the "sensory" function is produced (in the sense described above) be transferred to the periphery and there induce a secretion of transmitter ? Or in other words, is the capsaicin-sensitive neuron functionally bidirectional? Available data do not allow to obtain an unequivocal response to/this question. Impulses originating in the central terminals of afferent systems have been described in a few vertebrate sensory systems. These include the trigeminal system (Baker and Llinas, 1971) the vestibular system

31

(S. Highstein, quoted by Slesinger and Bell, 1985) and the somatosensory system. At this level the response has been described as the "dorsal root reflex" (Toennies, 1938) in which an efferent volley is elicited in somatosensory nerves by electrical stimulation of the same or a different somatosensory nerve. Some evidence indicates that this type of response can be elicited also by natural stimuli (Decima, 1969; Miyamoto, 1976). Accordingly, efferent impulses can be elicited in primary sensory afferents by low-level "physiological" stimuli applied centrally (Slesinger and Bell, 1985). Conduction to the periphery of impulses arising in the CNS may also be involved in the phenomenon of "reflex neurogenic inflammation" described by Levine et al. (1985a). The possibility that capsaicin-sensitive neurons may activate responses at peripheral level following stimulation of central terminals is, theoretically a simple extension of the notion that the "efferent" function of these sensory nerves can be activated by antidromic nerve stimulation. Accordingly, the real question is: do centrally-originating stimuli have sufficient intensity to overcome the stimuli generated in the periphery and induce secretion of transmitter at this level? It seems conceivable to assume that, in normal conditions, the number of sensory impulses arising in the peripheral receptive fields of the neuron may be more numerous than those which could originate centrally. Collision of impulses generated in different terminals of the neurons would be a mechanism capable of suppressing almost all the sensory input which could be generated in the "central" sensory terminals. However central activation of sensory terminals by local chemical events and consequent transmitter secretion in the CNS through the tetrodotoxin-resistant mechanism may be an important pathogenic mechanism for certain pain syndromes characterized by functional deafferentation and denervation supersensitivity (see Sicuteri, 1986). Jancs6 (1981) and Gamse et al. (1984) reported that intracisternally administered capsaicin produces, in rats, effects which may be explained by an activation of central terminals of afferent fibers and consequent release of SP and/or other peptides from both

CENTRAL TERMINAL {~) I

COLLATERAL IN PREVERTEBRAL GANGLIA (~)

NEURONAL BODY IN DORSAL ROOT GANGLIA THE

CAPSAICIN

-

PERIPHERAL

'b -91--.-- STIMULUS (~)

SENSITIVE NEURON

Fig. 12. Schematic drawing illustrating the four sites at which the multipolar capsaicin-sensitive sensory neuron may release its transmitter content. See the text for details.

CARLO ALBERTO MAGGI and ALBERTO MELI

32

central and peripheral endings of the sensory neuron. The peripheral responses were defined as "chcmicaUy-evoked dorsal root vasodilatation" (Gamse et al., 1984). However, recent investigation in guinea-pigs (Gamsc et al., 1986a, b) emphasizes the concept that certain peripheral effects of centrally administered capsaicin may be due to its systemic absorption and direct action on peripheral sensory terminals. Therefore, at this stage, no conclusion can be drawn on this topic: the possibility that certain peripheral effectsmediated by capsaicin-sensitivcsensory neurons are produced through an antidromic activation of their central terminals is an attracting hypothesis which deserves further investigation. Acknowledgements--We acknowledge the collaboration of Drs S. Evangelista, S. Giuliani and P. Santicioli (Menarini Firenze) L. Abelli and B. Conte (Menarini Sud, Pomezia Rome) for performing many of the experiments described in this paper. We wish to thank Drs P. Geppetti (Department of Internal Medicine, University of Florence) and S. Manzini (Department of Pharmacology, Malesci Pharmaceuticals) for reading the manuscript and pertinent criticism. A special thanks is directed to Dr Paolo Santicioli for helpful advice and suggestions and Dr P. Rovero for careful reading of the proofs. This work was in part supported by IMI, Rome (Progetto di Ricerca: Farmaci per il trattamento a lungo termine della incontinenza urinaria: VES, grant No. 46287).

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133, 129-136, 1987.

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