Central neural mechanisms mediating excitation of sympathetic neurons by hypoxia

Progress in Neurobiology Vol. 44, pp. 197 to 219, 1994

Copyright :~ 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0301-0082/94/$26.00

Pergamon

CENTRAL N E U R A L MECHANISMS M E D I A T I N G EXCITATION OF SYMPATHETIC N E U R O N S BY HYPOXIA M I A O - K U N S U N a n d D O N A L D J. R E I S Department of Neurology and Neuroscience, Cornell University Medical College, 411 East 69th Street, New York, N Y 10021, U.S.A.

CONTENTS I. Introduction 2. Sympathetic responses to stimulation of arterial chemoreceptors 2.1. Arterial chemoreceptors 2.2. Transmitters and chemoafferents 2.3. Central pathways 2.3.1. Afferent pathways 2.3.2. Central integration: RVL-spinal vasomotor neurons 3. RVL-spinal vasomotor neurons as central oxygen detectors 3.1. RVL-spinal vasomotor neurons 3.1.1. Hypoxic excitation of RVL-spinal vasomotor neurons in vivo after peripheral chemodenervation 3.1.2. Effect of cyanide on RVL-spinal vasomotor neurons in vivo 3.1.3. Membrane responses of the medullary pacemaker neurons to hypoxia and cyanide in vitro 3.2. Other medullary neurons 3.3. Neurons in the spinal cord 4. Chemotransductive mechanism 4.1. Metabolic hypothesis 4.2. Intracellular Ca"+ mobilization 4.3. O_,-sensitivechannels 4.3.1. K + channels 4.3.2. Ca2+ channels 4.4. NADPH oxidase 5. Sympathetic excitation elicited by cerebral ischemia 5.1. Role of RVL-spinal vasomotor neurons 5.2. Signals triggering the sympathetic CIR 5.2.1. Hypoxia 5.2.2. Acidosis and hypercapnia 5.2.3. Neurotransmitters 5.2.4. Energy failure 5.2.5. Calcium and enzyme activation 5.2.6. Other factors 6. Conclusions Acknowledgements References

1. INTRODUCTION A major function of the autonomic nervous system is to initiate and sustain reflex responses of the circulation so as to preserve tissue in the face of reductions in oxygen, either as an environmental threat or, more commonly, in response to reduced tissue perfusion initiated by hemorrhage or ischemia. Such responses consist of relatively stereotyped patterns of activation of the sympathetic nervous system, leading to marked and somewhat differentiated sympathetically mediated patterns of vasoconstriction in muscular, cutaneous, renal and mesenteric vascular beds, vasodilation in the cardiac and cerebral circulations, and release of adrenomedullary catecholamines (Adachi et al., 1976; Alexander, 1945; De Geest et al., 1965; Downing et al., 1963; Fletcher et al., 1992;

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Fukuda et al., 1989; Guyton, 1948; Hachinski and Norris, 1985; Heistad and Abboud, 1988; Hoka et al., 1989; Raichle, 1983). Under many conditions, when ventilation is reduced (e.g. the diving response or response to cerebral ischemia) or controlled (artificial ventilation) the response is accompanied by a marked vagal bradycardia. The pattern of response has been viewed as representing an optimal physiological adjustment to oxygen deprivation, serving to preserve blood flow (and hence oxygen) to heart and brain at the expense of tissues capable of sustaining ischemia for more prolonged periods such as muscle and viscera. It is recognized that neurons in the medulla oblongata play a pivotal role in initiating and sustaining the physiological adjustments of the

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circulation to oxygen lack. While it has been known for many years that the medulla was essential in integrating the reflex adjustments to stimulation of arterial chemoreceptors, the principal oxygen sensors in the periphery, more recent evidence has indicated that medullary neurons mediating vasomotor components of arterial chemo- and other reflexes may also be directly and selectively stimulated by hypoxia, either by hypoxemia or the reduced local perfusion associated with local ischemia (Dampney and Moon, 1980; Dampney et al., 1979; Guyenet and Brown, 1986; Kumada et al., 1979; Rohlicek and Polosa, 1983b; Sun and Reis, 1991; Sun et al., 1991, 1992). The observations suggest that central neurons may themselves be oxygen sensors, serving to detect changes in cerebral perfusion and/or blood gas tensions and initiating, in addition to appropriate respiratory reflexes (Lee and Millhorn, 1975; Lipscomb and Boyarsky, 1972; Mitchell et al., 1963; Morrill et al., 1975; Neubauer et al., 1990; Schlaefke, 1981), marked excitation of the sympathetic nervous system. An understanding of the neural mechanism involved in hypoxic sympathoexcitation thus seems a necessary step in interpretation of the respiratory and cardiovascular responses to hypoxic responses associated with such diverse medical conditions as hemorrhagic shock, brain-stem compression and/or ischemia, sleep apnea, and chronic altitude sickness. In this paper we critically review the current state of knowledge of the sensors, pathways and stimuli which activate sympathetic nervous system in response to hypoxia, concentrating on recent physiological evidence of the important role played by reticulospinal vasomotor neurons of the rostral ventrolateral reticular nucleus (RVL) in peripheral and central oxygen chemoreception. Specifically, we shall discuss arterial chemoreflexes, sympathoexcitatory responses initiated from stimulation of the medullary chemosensots, and how that knowledge can help to understand mechanisms underlying the powerful excitation of the sympathetic nervous system elicited by brain-stem ischemia (the cerebral ischemic reflex).

2. SYMPATHETIC RESPONSES TO STIMULATION OF ARTERIAL CHEMORECEPTORS 2.1. Arterial Chemoreceptors It is now widely accepted that the principal chemoreceptors in the periphery initiating reflex respiratory and circulatory responses (Marshall, 1987; Verna et al., 1975) to hypoxia are the catecholaminecontaining type I glomus cells (Biscoe and Duchen, 1990a), localized in the carotid and sometimes aortic bodies, and probably a number of mini-glomera along the course of the common carotid artery which lie in close apposition to the carotid sinus nerve afferents (Biseoe, 1971; Howe and Neil, 1972; Matsuura, 1973). The type I glomus cells in the carotid body detect changes in arterial blood gas tensions, particularly of oxygen (Calvelo et aL, 1970; Heistad and Abboud, 1988),although pH and carbon dioxide may modulate their response. Stimulation by hypoxia initiates

cellular events which result in release of dopamine, which in turn excites afferent fibers of the carotid sinus nerve. In addition, the chemoreceptors can be stimulated by NaCN, K + and carbonyl cyanide-ptrifluomethoxyphenyl hydrazone (FCCP) and a range of other drugs (Eyzaguirre and Fidone, 1980). Chemoreceptors other than those associated with carotid and aortic bodies have also been described in other peripheral beds. Chemoreceptors have been detected within the heart, many in close association with the coronary circulation, which have ill-defined receptive properties, although many appear to subserve a chemosensitive function (Sinclair, 1987). In the abdominal vagus of the rat, chemoreceptor-like activity has been reported to be induced by a few minutes of extreme hypoxic hypoxia (Howe e t a [ . , 1981). However, no evidence has been provided whether the evoked activity would lead to changes in respiration and cardiovascular function. There are also receptors with chemosensitive function in the arteries supplying to the kidneys, and perhaps other organs, that may contribute to cardiovascular control (Anderson et al., 1980; Sinclair, 1987). Within skeletal muscle, receptors with afferent fibers in the group III and IV categories sense the metabolic state during exercise (Kniffke et al., 1981) to initiate appropriate reflex cardiovascular and respiratory adjustments (Wall et al., 1991). However, these receptors may not be involved in the acute responses to hypoxia, at least in rats, since a bilateral denervation of the carotid sinus nerves eliminates hypoxic increases in phrenic nerve activity (Fig. 1). In rats, the aortic depressor nerves do not have functional chemoafferents (Sapru and Krieger, 1977), while chemoreceptors innervated by vagal and glossopharyngeal afferents do not respond to hypoxia until several days after carotid chemodenervation (Sinclair, 1987). 2.2. Transmitters and Chemoafferents Arterial chemoreceptors of the carotid and aortic bodies (Lahiri et al., 1980) are innervated by the carotid sinus branch of the IXth and aortic depressor branch of the Xth cranial nerves, respectively. At normal arterial blood P, O2 (100mmHg), P~CO2 (40 mmHg) and pH (7.4), the chemoafferents fire at low frequency (<2impulses/see/fiber) and their discharges are relatively independent of systemic blood pressure within a certain range (Johnson et al., 1968), probably due to autoregulation of local blood flow. The discharge rate increases when arterial blood PO2 falls below 60 mmHg, which is roughly the threshold at which reflex-induced increases in respiration are observed, and/or when PCO2 or [H +] increases. It is generally accepted that of several putative neurotransmitters, chemoreceptor cells release dopamine in proportion to the intensity of hypoxic stimulation, and that the release response is paralleled by the electrical activity in the carotid sinus nerve (Fidone et al., 1982; Gonzalez and Fidone, 1977; Rigual et al., 1986, 1991; Donnelly, 1993) and may represent a final output of sensory transduction initiated by chemosensors in the type I chemoreceptor cells. While it is not clear whether membrane conductance changes are essential to the hypoxic chemotransduc-

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50 20s Fig. 1. Effect of bilateral carotid chemodenervation on arterial chemoreception as monitored on hypoxic responses of the phrenic nerves in the anesthetized and ventilated rat. In the intact rat, hypoxia produced a rapid increase in activity of the phrenic nerves and arterial pressure (left panel). Bilateral carotid chemodenervation eliminated the hypoxic increase in activity of the phrenic nerves. The hypoxic response of the phrenic nerves was a small decrease in the resting activity (right panel), indicating the effectiveness of the denervation and hypoxic depression of the central respiratory center under the experimental conditions. After the denervation, hypoxia induced a decrease-increase response pattern of arterial pressure. The effects were examined in the same rat. PND: phrenic nerve discharges. tion of the glomus cells in the carotid body (Biscoe and Duchen, 1989, 1990a, 1990b; Duchen et al., 1988; also see below), there is evidence that about 30% of these cells are electrotonically coupled (Monti-Boch et al., 1993). The meaning of the coupling in terms of chemoreception is not clear though the coupling-uncoupling process may be related to their function as secreting cells. While afferent impulses generated at the chemoreceptors are transmitted via myelinated (A-) and unmyelinated (C-) fibers, C-fibers dominate with respect to numbers and reflexogenic actions. For example, the carotid sinus nerve of rats contains an average of 625 axons, of which 86.3% are unmyelinated (McDonald, 1983a,b). In cats, a total of 2000--4000 axons are estimated in the sinus nerve, of which 67.8% are unmyelinated (Eyzaguirre and Uchizono, 1961). Among the unmyelinated axons, 17% of them are estimated to be chemosensitive, 29% barosensitive and the rest are sympathetic and other types of axons. Among the myelinated axons, about two-thirds are chemosensitive, whereas the remainder are barosensitive. The aortic nerve of rats and rabbits does not contain a significant amount of functionally active chemoreceptor fibers (Numao et al., 1983, 1985; Sapru and Krieger, 1977). Studies with retrograde labeling of the carotid sinus nerve with fluoro-gold (Ichikawa et al., 1993) in rats reveal that the majority (94.5%) of carotid sinus nerves arise from neurons of the petrosal ganglion with the remainder localized to jugular (5.2%) or nodose ganglia (0.3%). The neurotransmitter released by chemoreceptor afferents in the brain-stem, however, is not certain. In the rat petrosal ganglion (Ichikawa et al., 1993), neurons giving rise to the carotid sinus nerve are

immunoreactive for galanin (43 %), calcitonin gene-related peptide (25%), substance P (16.7) and vasoactive intestinal polypeptide (VIP) ( < 1%). The presence of tyrosine hydroxylase (30%, Katz and Black, 1986; 4%, Ichikawa et al., 1993) suggests that some neurons may synthesize catecholamines. Whether any of these agents participate in chemoreceptor initiated reflexes is not known although evidence suggests that substance P may be involved (McQueen, 1980; Monti-Boch and Eyzaguirre, 1985; Prabhakar et al., 1984; Srinivasan et al., 1991). There is also functional evidence that L-glutamate may be the key neurotransmitter released by peripheral chemoreceptor stimulation (Hoop et al., 1990; Housley and Sinclair, 1988; Vardhan et al., 1993; Mifflin, 1993): local application of excitatory amino acid receptor antagonists in the nucleus tractus solitarii (NTS), where the arterial chemoafferents terminate, abolishes the chemoreflex (Vardhan et al., 1993; Zhang and Mifflin, 1993), though it remains to be determined whether the essential neurotransmitter is released from interneurons or primary afferent fibers.

2.3. Central Pathways 2.3.1. Afferent Pathways Afferent fibers of the carotid chemoreceptors terminate primarily within the NTS. While the chemoreceptor afferents travel in close approximation to the arterial baroreceptors in the sinus nerve they appear to terminate in distinct zones within the NTS. The chemoreceptor afferents are concentrated within the commissural and medial subnuclei of the caudal NTS (Claps and Torrealba, 1988; Donoghue et al., 1982, 1984; Finley and Katz, 1992;

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Kalia and Sullivan, 1982), while the baroreceptor afferents terminate more rostrally. Although there is a topographic segregation within the NTS of the chemo- and baroreceptor inputs, the possibility for an integrative interaction between the two major reflexes at the level of the NTS is still possible (Mifflin, 1993). That the region of the NTS where the chemoreceptor afferents terminate may mediate the arterial chemoreceptor reflex is supported by observations of Vardhan et al. (1993) who found that microinjections of muscimol or antagonists of excitatory amino acid receptors into commissural nucleus of the NTS blocked carotid chemoreceptor responses, and by Zhang and Mifflin (1993), who found that the pressor responses to intra-carotid injections of CO2-saturated solution or electrical stimulation of the carotid sinus nerve in rats were eliminated by microinjections of kynurenate into, mostly, the ipsilateral commissural NTS. Electrophysiological studies agree, by and large, with neuroanatomical studies, although the exact group(s) of neurons mediating the sympathoexcitatory response of chemoreflexes has not been defined. The difficulty involved is partly due to the fact that, like the baroreceptor inputs (Sun and Reis, 1993a; Sun and Spyer, 1991a), the arterial chemoreceptors innervate neurons that are not directly involved in the cardiorespiratory regulation. In anesthetized cats, about 1/4 of the NTS neurons that respond to electrical stimulation of the carotid sinus nerves with an excitatory post-synaptic potential (Mifflin, 1992) receive an excitatory input from the carotid sinus baroreceptors. These neurons are therefore not involved in the regulation of sympathetic nerve discharge or respiration since effects of activation of the baroreceptors and the chemoreceptors on sympathetic nerve discharge and respiration are opposite (Kubin et al., 1985; Millhorn et al., 1982; Trzebski et al., 1975). The carotid sinus nerve also projects to brain-stem areas other than the NTS, including the area postrema, the dorsal motor nucleus of the vagus, the nucleus of ambiguus, the reticular formation just ventral to the solitary complex, the external cuneate nucleus (Claps and Torrealba, 1988; Crill and Reis, 1968; Donoghue et al., 1984; Finley and Katz, 1992; Kalia and Sullivan, 1982; M iura and Reis, 1969). The contribution of these pathways to the chemoreflex sympathoexcitation is not known. 2.3.2. Central Integration: R V L - S p i n a l Vasomotor Neurons

Sympathoexcitation elicited from stimulation of the arterial chemoreceptors is mediated by excitation of RVL-spinal vasomotor (sympathoexcitatory or cardiovascular) neurons (Koshiya et al., 1993; Sun and Spyer, 1991a; Sun and Reis, 1993a). These neurons are spontaneously active (Brown and Guyenet, 1984, 1985; Morrison et al., 1988; Sun, 1992; Sun and Guyenet, 1985, 1987; Sun and Spyer, 1991b,c). Their discharges are pulse-modulated (Fig. 2C and D) and very sensitive to arterial baroreflex-mediated inhibition (Fig. "2A). These

neurons project to the spinal cord, demonstrated by antidromic collision tests (Fig. 2B) and innervate only autonomic centers of the spinal cord and monosynaptically the sympathetic preganglionic neurons in the spinal cord (Ross et al,, 1984a,b; Milner et al., 1987). They are contained in a restricted zone of the RVL containing a subpopulation of neurons expressing phenylethanolamine N-methyltransferase (PNMT) (Milner et al., 1987; Morrison et al., 1988; Sun et al., 1988a,b,c) and overlying the chemosensitive zones of the ventral medullary surface (Benarroch et al., 1986). The RVL spinal vasomotor neurons are believed to represent the so-called 'tonic vasomotor center' of the medulla oblongata. When excited, they activate the sympathetic preganglionic and postganglionic neurons in the spinal cord while their inhibition results in a reduction of sympathetic nerve activity and a corresponding fall of arterial pressure (Willette et aL, t984). That the RVL-spinal vasomotor neurons mediate the sympathoexcitation from chemoreceptor stimulation is supported by several lines of evidence. First, this sympatho-excitatory region of the RVL corresponds with specific chemoresponsive areas of the ventral surface of the medulla oblongata variously designated the intermediate chemosensitive area (IA), the glycine-sensitive area or the rostral ventrolateral pressor area (Feldberg and Guertzenstein, 1972, 1976; Guertzenstein and Silver, 1974). Cooling (Millhorn et al., 1982; Prabhakar et al., 1986), electrolytic lesions of (Ciretto and Calaresu, 1977), or glycine applied to, this region (Marshall, 1986) attenuate the cardio-respiratory responses to carotid sinus nerve stimulation or systemic hypoxia/ ischemia. Second, neurons in the RVL are excited by electrical stimulation of the carotid sinus nerve (Caverson et al., 1984; McAllen, 1992; Terui et al., 1986). Third, the RVL-spinal vasomotor neurons are rapidly excited by intra-carotid injection of H2PO4 (Sun and Spyer, 1991a) or systemic hypoxia (Koshiya et al., 1993; Sun and Reis, 1992a, 1993a, 1994a) in anesthetized rats (Fig. 3A). Indeed, substitution of 100% N2 for 100% O2 for 20 sec which reduces the PaO2 to 27 mmHg on average (Sun and Reis, 1992b) activates RVL-spinal vasomotor (Fig. 3A) and sympathetic nerves in parallel (Fig. 4A), preceding elevations in arterial pressure by about 2 sec (Sun and Reis, 1993a, t994a). The latencies for and short duration of the responses rule out the possibility that vasoactive molecules such as vasopressin, angiotensin, and endothelin are released by the brief period of hypoxia in contributing to the elevations of arterial pressure. These same RVL vasomotor neurons are inhibited (Fig. 2; Sun and Guyenet, 1985, 1986) by stimulation of the arterial baroreceptors with an identical sensitivity to that of the sympathetic nerves, satisfying the requirement that the neuronal response characteristics to different biological stimuli are therefore suitable for encoding the autonomic information (Sun and Reis, 1993a, 1994a; Sun and Spyer, 1991a). The chemo-sympathetic reflexes involve a rapid activation of the RVL-spinal vasomotor neurons (Sun and Reis, 1992a, 1993a, 1994a; Sun and Spyer,

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Fig. 2. Characteristics of RVL-spinal vasomotor neurons. A: Effect of change in arterial pressure on the neuronal discharge rate. Neuronal activity is displayed in the form of integrated rate histogram. Arterial pressure was elevated via descending aortic constriction (started at the first arrow and released when the neuron became silent) and reduced by i.v. injection of l0 #g sodium nitroprusside (at second arrow). B: Spinal cord projection of the neuron. The antidromic spikes (arrow, top trace) evoked by the spinal cord stimulation (asterisks) failed to occur at the recording site (bottom trace) when the stimulation was applied within a critical period after spontaneously occurring spikes. C: Pulse-synchronous discharge of a RVL-spinal vasomotor neuron. The arterial pressure trace (middle trace) and ECG signal (bottom) represent a single sweep, while the trace of the neuronal discharge represents twelve consecutive sweeps, all triggered on ECG signals. D: ECG-triggered time histograms of the neuronal activity (300 sweeps, 3 msec/bin). The top and middle traces represent averaged arterial pressure and ECG signals, respectively (50 sweeps each). (After J. Physiol., Lond. 436, 685-700, 1991.)

1991a). The transmitter involved is probably L-glutamate since bilateral microinjections ofkynurenate into the RVL blocked the chemo-sympathetic reflexes but not the baro-sympathetic reflexes (Koshiya et aL, 1993; Sun and Reis, 1994e). It is not known whether the NTS neurons mediating the sympathetic chemoreflex directly project to the RVL, making monosynaptic connections with the reticulospinal vasomotor neurons. Although, the sympathoexcitatory responses to acute hypoxia appear mediated entirely by the RVL vasomotor neurons, whether the neural circuit mediating the chemoreflex sympathoexcitatory responses is confined to the brain-stem has not been entirely established, since activation of the carotid chemoreceptors also excites vasopressin-secreting neurons in the hypothalamus (Harris, 1979), possibly relayed from the caudal ventrolateral medulla (CVL) (Jamieson and Harris, 1989; Li et al., 1992) and evokes the visceral component of the defence reaction (Marshall, 1986). Release of vasopressor substances, such as vasopressin, angiotensin, and endothelin, may be involved if hypoxia lasts. It is well established that reflex regulation of activities of the reticulospinal vasomotor neurons of the RVL and the sympathetic nerves are often relayed in neurons located in the CVL. These include the

baroreflex (Agarwal et al., 1990; Biscoe et al., 1970; Blessing, 1989; Jeske et al., 1993; Masuda et al., 1991) and somato-sympathetic inhibitory reflex (Masuda et al., 1992). So far no evidence has been presented which indicates an involvement of CVL neurons in sympathetic chemoreflexes. It has been reported that in anesthetized rabbits (Gieroba et al., 1992), excitation of the arterial chemoreceptors by hypoxia alters discharge rate of many CVL neurons which are barosensitive. These neurons are all antidromically stimulated from the RVL so that their axons must either terminate or pass through the RVL. However, from all the examples shown, it appears that the changes in activity of these neurons in response to hypoxia becomes apparent after the evoked changes in arterial pressure. The neuronal response during hypoxia is thus more likely a secondary event elicited by the changes in arterial pressure rather than a primary chemoreceptor-mediated event. Different brain-stem pathways mediating the baroreflex and the chemoreflex have recently been suggested (Koshiya et al., 1993) since microinjection of kynurenate, a wide spectrum antagonist of excitatory amino acid receptors (Perkins and Stone, 1985), or muscimol, a GABAA sub-receptor agonist, into the CVL is found to block the baroreflex but not the chemoreflex in rats.

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Fig. 3. Effect of bilateral sino-aortic denervation on hypoxic excitation of RVL-spinal vasomotor neurons, Hypoxia (N2, 20 sec) excited the neuron both before (A) and after (B) bilateral sino-aortic denervation. These were tested on the same neuron. The aortic nerves were also denervated to eliminate possible contribution of a change in the arterial baroreflex to the responses. The effectivenessof the baro-denervation is indicated by a total loss oftbe neuronal sensitivityto changes in arterial pressure. The hypoxicincreasesin the neuronal activity and arterial pressure could not result from activation of the arterial chemoreceptors and a blockade of the arterial baroreflexes. Arrows indicate the starting of aortic constriction. Nitroprusside: 10/~gsodium nitroprusside, intravenously administered.

3. RVL-SPINAL VASOMOTOR NEURONS AS CENTRAL OXYGEN DETECTORS It has long been recognized that following denervation of the arterial chemoreceptors, hypoxia can still excite the sympathetic preganglionic neurons, often resulting m vasoconstriction and elevation of arterial pressure and release catecholamines (Cantu et al., 1966; Heistad and Abboud, 1988). The observation implies that hypoxia can stimulate neurons within the central nervous system which are related to sympathetic outflow. However, the observations pose a number of questions: is the response to hypoxia the result of stimulation of 'receptors' entirely contained within the central nervous system or to receptors lying, like the arterial chemoreceptors, outside of the neuraxis? Are the elements which sense hypoxia neurons and, if so, where are they located? Is the signal for sympathetic activation hypoxia itself or some associated metabolic consequence of oxygen depletion? Is hypoxia 'detected' or does it reflect a non-specific response of neurons to depletion of energy supplies? What is the pathway by which hypoxic excitation of 'detectors'

projects to excite the sympathetic nervous system? What are the cellular events which transduce hypoxia into neuronal activity? A distinction should also be made at this juncture between neuronal networks which signal hypoXia and those which respond to hypoxia as a pathological event. In the former, the signal (hypoxia) is triggered by cellular mechanisms specialized to detect reduction in PO2 at levels which do not deplete energy reserves (e.g. ATP), do not damage the sensing cell and which will usually occur within a few seconds. These are physiological responses. The latter will be those changes which can be attributed to energy toss (e.g. pump failure, non-specific inhibition of oxidases, etc.) attributable to loss of energy stores. These will take many seconds or minutes and can be considered as pathological responses. Until recently, there has been little information available to answer many of these questions. While hypoxia has been demonstrated to modify the activity of neurons in disparate parts of the central nervous system (Haddad and Jiang, 1993; Krnjevi~ and Leblond, 1989), the stimulus has usually been

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so Fig. 4. Effect of bilateral sino-aortic denervation on hypoxic excitation of the sympathetic nerves. Hypoxia (N2, 20 sec) excited the sympathetic nerves both before (A) and after (B) bilateral sino-aortic denervation. These were tested in the same rat. The aortic nerves were also denervated to eliminate possible contribution of a change in the arterial baroreflex to the responses. The effectivenessof the baro-denervation is indicated by a total loss of the nerve activity to changes in arterial pressure. The hypoxic increase in the sympathetic nerves and arterial pressure could not result from activation of the arterial chemoreceptors and a blockade of the arterial baroreceptors. Arrows indicate the starting of aortic constriction. Nitroprusside: i 0 #g sodium nitroprusside, intravenously administered. sufficiently prolonged so as to be pathological However, recently we have obtained evidence that the RVL-spinal vasomotor neurons may in fact themselves be oxygen sensors and are capable of driving sympathetic activity in response to hypoxia in animals deprived of arterial chemoreceptors. These studies will be summarized. 3.1. RVL-Spinal Vasomotor Neurons

3.1.1. Hypoxic Excitation of RVL-Spinal Vasomotor Neurons In Vivo After Peripheral Chemodenervation In our studies in rats with intact chemoreceptors, we observed that hypoxia, elicited by substitution of 100% N2 for 02 for 20sec, which lowered PaO2 to approximately 27 mmHg, increased activities of the RVL-spinal vasomotor neurons and sympathetic nerves, elevated arterial pressure, and profoundly excited phrenic nerve activity, the usual integrated response (Figs 1, 3, 4; also see Sun and Reis, 1994a, c).

The evoked increases in activities of the RVL-spinal vasomotor neurons and sympathetic nerves and arterial pressure are associated with marked oscillations (Figs 3, 4), probably due to the imbalance between chemoreflex-mediated excitation and baroreflex-mediated inhibition into the same system (Sun and Reis, 1994a). Following selective denervation of the arterial chemoreceptors [produced in rats by selective denervation of carotid sinus nerves, leaving the arterial baroreceptors in the aortic depressor nerve intact (Sapru and Krieger, 1977)], a comparable period of hypoxia resulted in a somewhat different response: phrenic nerve activity is inhibited (Fig. 1), while sympathetic nerve activity gradually increases (Sun and Reis, 1992b, 1994a,b). Interestingly, after the chemodenervation, despite elevation of sympathetic nerve activity, arterial pressure first falls, undergoes a secondary rise associated with a burst of sympathetic nerve activity and then, following a tertiary drop which recovers during reoxygenation. The dis-

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sociation between the elevation of sympathetic nerve activity and the decrease of arterial pressure, as noted by others (Bower, 1977; Fukuda et al., 1989; Hoka et al., 1989; Rohlicek and Polosa, 1983a), results from a marked vasodilation consequent to release from blood vessels of nitric oxide (Sun and Reis, 1992b). The fall of arterial pressure is not, as a result of baroreceptor unloading and/or production of cerebral ischemia, the stimulus for sympathetic excitation since lowering arterial pressure to a comparable extent by intravenous administration of nitroprusside does not stimulate the big changes in sympathetic nerve activity and arterial pressure and, alternatively, sympathetic activation persists when the fall of arterial pressure is blocked by aortic constriction (Sun and Reis, 1994a). Also the same response pattern was observed in rats after bilateral sino-aortic denervation (Fig. 4). There are several reasons for believing that the excitation of the sympathetic nerves by hypoxia in chemodenervated rats results from activation of the same reticulospinal sympathoexcitatory vasomotor neurons in the RVL, which mediate sympathoexcitatory responses to stimulation of the arterial chemoreceptors (Sun and Reis, 1992a, 1994a; Sun and Spyer, 1991a). First, in chemo-denervated (Sun and Reis, 1993a, 1994a,b) or sino-aortic denervated rats, hypoxia excites the RVL-spinal vasomotor neurons (Fig. 3) in advance of the secondary rise of arterial pressure. Second, the responses of RVL-spinal vasomotor neurons to hypoxia do not appear to result from secondary pathological consequences of 02 deprivation since the responses are reversible and reproducible over many trials. Third, within the RVL, the excitatory response to hypoxia is restricted to reticulospinal vasomotor neurons: adjacent neurons, including barosensitive neurons which do not project to the spinal cord (Sun and Reis, 1993a), respiratory neurons of the ventral respiratory group, and local nonrespiratory non-cardiovascular neurons are either inhibited or unaffected (Sun and Reis, 1993a, 1994a). Interestingly, while vagal motoneurons are not excited during the 20 sec of hypoxia, a marked bradycardia appears when the hypoxic episode is prolonged (thereby further reducing the PaO2). This presumably reflects excitation of cardiovagal motoneurons whose sensitivity for excitation by hypoxia is less than that of the RVL-spinal vasomotor neurons. The response of the RVL-spinal vasomotor neurons to systemic hypoxia appears to be chemically selective. While systemic hypoxia will excite the RVL vasomotor neurons in chemodenervated rats, hypercapnia and the associated acidosis produced by 40sec of ventilation with 5% CO2 does not modify the discharge rate of these nor of sympathetic neurons. Arterial pressure and heart rate are not altered by the CO2 inhalation even though the stimulus excites, as expected, respiratory neurons. Excitation of the RVL-spinal vasomotor neurons in chemodenervated rats in vivo does not appear to result from enhanced liberation of excitatory amino acids or reduction in GABAergic neurotransmission, stimuli which will both increase their discharge (Sun and Guyenet, 1985, 1986), nor can it be

attributed to elevations of K t, release of adenosine, and/or formation of nitric oxide in the RVL by the hypoxic stimulus (Sun and Reis, 1993c). Thus while hypoxia, when prolonged, releases L-glutamate and/or aspartate (Benveniste et al., 1984: Clark and Rothman, 1987; Drejer et al., 1985), by processes which are partly Ca2+-dependent and independent (Ikeda et al., 1989), hypoxic excitation of RVL-spinal vasomotor neurons is not attenuated by kynurenate (a wide spectrum antagonist of excitatory amino acid receptors (Collingridge and Lester, 1989; Perkins and Stone, 1985)), applied directly by iontophoresis or intracisternally in doses which eliminate excitatory responses to L-glutamate (Sun et al., 1992) or N-methyl-o-aspartate in chemo-denervated rats (Sun and Reis, 1993c). In addition, while hypoxia depresses GABAergic transmission, consequent to a decline in GABA release (Krnjevi6 et al., 1991), desensitization of GABA receptors by intracellular Ca -,+ (Akaike et aL, 1988; Dubinsky and Rothman, 1991; Duchen et al., 1990; Inoue et al., 1986; Leblond and Krnjevi6, 1989; Stelzer et al., 1988), and/or desensitization of GABAA receptors by depletion of ATP (since these receptors require phosphorylation to maintain functional integrity (Stelzer et al., 1988)), diminution of GABAergic transmission does not appear to occur during the brief stimulation period since the inhibition of the RVL-spinal vasomotor neurons by baroreceptor stimulation, a response dependent upon local release of G A B A (Sun and Guyenet, 1985, i987), is preserved (Sun et al., 1992). Preservation of the baroreceptor reflex during hypoxic excitation also indicates that during hypoxia, the RVL-spinal vasomotor neurons are still capable of responding to synaptic inputs, presumably a necessary feature of a system designed to respond to systemic hypoxia. It is also unlikely that excitation of the RVL-spinal vasomotor neurons can be attributed to accumulation of [K]o. As first demonstrated by Morris (1974) and subsequently confirmed by others (Hansen, 1985), even a few seconds of anoxia can elevate brain [K]o. However, increases in [K]o, though very consistent, within the first few minutes typically do not exceed 1-2 mM in the hippocampal slices (Hansen et al., 1982; Morris et al., 1991; Sick et al., 1987). Such small increases in [K]o would have a minimal direct depolarizing effect at most. Moreover, the fact that only vasomotor neurons are excited by hypoxia while the discharge of many respiratory neurons are depressed and other RVL neurons are not affected is inconsistent with the mechanism. While hypoxia/ischemia will also release adenosine (Hagberg et al., 1987; Merrill et al., 1986; Phillis et al., 1987; Simpson and Phillis, 1991; Winn et al., 1981 ) and generate nitric oxide by activation of nitric oxide synthase (Bredt et al., 1990; Garthwaite et al., 1989; Moncada et al., 1992), which conceivably participate in ischemic-hypoxic responses of neurons (Garthwaite et al., 1988; Moncada et al., 1992; Snyder and Bredt, 1991), such mediators do not appear to mediate the excitation of RVL-spinal vasomotor neurons to hypoxia. Perfusion of adenosine deaminase at a concentration at which it eliminates neuronal response to adenosine does not alter the membrane response of the medullary neurons to cyanide (Sun and Reis,

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Excitation of Sympathetic Neurons by Hypoxia 1994d). Moreover intracisternal administration of NG-nitro-L-arginine, a potent and specific inhibitor of nitric oxide synthase (Ishii et al., 1990; Moore et al., 1990; Snyder and Bredt, 1991) does not attenuate the hypoxic sympathoexcitatory responses (Sun and Reis, 1993c). Finally, superfusion of the ventral medulla has been proposed as a mechanism by which respiratory drive is inhibited in the hypoxic brain (Lee and Millhorn, 1975). The hypothesis is based on the supposition that increased local blood flow will wash out tissue CO2 and H ÷ concentrations when metabolism is not increased in parallel and consequently lead to ventilatory depression (Lee and Millhorn, 1975; Morrill et al., 1975; Neubauer et al., 1985). However, such a mechanism can not explain the hypoxic increase in sympathetic nerve activity, since responses of the reticulospinal vasomotor neurons and the sympathetic nerves to hypoxia are excitatory and occur prior to hemodynamic changes (Sun and Reis, 1993a, 1994a). 3.1.2. Effect o f Cyanide on R VL-Spinal Vasomotor Neurons In Vivo That the RVL-spinal vasomotor neurons may themselves be central oxygen sensors and thereby initiate the hypoxic sympathoexcitatory response is supported by observations (Sun et al., 1991, 1992; Sun and Reis, 1991, 1992a) that RVL-spinal vasomotor neurons are selectively excited by local microiontophoresis of cyanide in vivo (Fig. 5). Cyanide is a powerful stimulant of the arterial chemoreceptors (Choi, 1988; Eyzaguirre and Koyano, 1965) and has been used as a model for producing chemical hypoxia (Di Rodriguez and Bazan, 1983; Goldberg et al., 1987). The possibility that cyanide might excite sympa-

thoexcitatory neurons of the RVL was first observed in a study in which microinjection of cyanide (300 pmol/site, pH 7.3 in 50 nl) into the cardiovascular portion of the RVL, the region containing reticulospinal vasomotor neurons in rats, with the arterial chemoreceptors intact, elicited a dose-dependent and site specific elevation of arterial pressure and depression of phrenic nerve discharge (Sun et al., 1992; Sun and Reis, 1994a). In contrast, lactic acid (3 nmol/50 nl/site, pH 3.3) while transiently increasing phrenic nerve activity was without effect (Sun and Reis, 1994a). To define the cellular responses more precisely, cyanide was iontophoresed onto RVL-spinal vasomotor neurons (Sun et al., 1992; Sun and Reis, 1994a). Cyanide elicited a dose-dependent and reproducible increase in the discharge rate of RVL-spinal vasomotor neurons. The excitatory responses, like those elicited by hypoxia (Sun and Reis, 1994a), were neuronally selective since the discharges of respiratory neurons of adjacent ventral respiratory groups and non-cardiovascular non-respiratory neurons in the region were either inhibited or not affected (Sun et al., 1992) by cyanide. The excitation by cyanide is not the result of blockade of inputs to these neurons from arterial baroreflex mechanisms since the increase in neuronal activity was significantly larger than that observed during baroreceptor unloading by i.v. injection of nitroprusside to lower arterial pressure, and the baroreflex-mediated inhibition of the neurons remains intact during the cyanide-induced excitation (Fig. 5). The rapidity and reversibility of the response with preservation of neuronal excitability to L-glutamate, inhibition by GABA or baroreceptor stimulation, indicate that the responses to cyanide cannot be attributed to depletion of ATP stores, a general failure of the cellular energy supplies or irreversible neuronal

NaCN lOOnA

20s

50 Nitroprusside Fig. 5. Effect of microiontophoretically applied cyanide on activity and baroreflex-mediated inhibition of RVL-spinal vasomotor neurons. Note that microiontophoresis of cyanide rapidly excited the RVL-spinal vasomotor neuron but did not attenuate baroreflex-mcdiated inhibition of the neuron. The hypoxic increase in discharge rate of the neuron is much bigger than that evoked by baroreceptor-unloading elicited by i.v. injection of sodium nitroprusside to lower arterial pressure. Arrows indicate the starting of the aortic constriction. (After Am. J. Physiol. 262, RI82-RI89, 1992.) JPN

44/2~

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damage. More likely, the response and sensitivity of these neurons to cyanide reflects a more selective mechanism. The rapid response of RVL-spinal vasomotor neurons to iontophoresis of cyanide in vivo appears to depend upon Ca -'+, probably Ca 2+ influx since it was blocked by co-iontophoresis of Co 2+ (Sun et al., 1992). While an influx of Ca -,+ could result, as mentioned above, from a cyanide-initiated release of L-glutamate, activation of N-methyl-D-aspartate receptors (Eyzaguirre and Koyano, 1965; Rothman, 1984) and consequently-enhanced Ca -,+ influx (Goldberg et al., 1987; MacDermott et al., 1986; Rothman, 1984; Zorumski and Thio, 1992), coapplication of kynurenate did not attenuate the response to cyanide yet abolished the neuronal response to iontophoreticallyapplied L-glutamate (Sun et al., 1992). The excitatory responses of RVL-spinal vasomotor neurons to cyanide contrasts with their relative insensitivity to extracellular H + (Sun and Reis, 1993b), HCO3-, or lactate (Sun and Reis, 1994a), though extracellular H + ions have modulatory effects on responses of the neurons to G A B A and cyanide (Sun and Reis, 1993b). This is consistent with the relative insensitivity of RVL-spinal vasomotor neurons, sympathetic nerves, and arterial pressure of rats to brief periods of hypercapnia (Guyenet and Brown, 1986; Hoka et al., 1989; Sun and Reis, 1994a). The neuronal responses to cyanide were transient and reproducible over many trials, indicating that cyanide did not 'poison' the cells. The observations were of interest for several reasons. First, they demonstrated that cyanide replicated the integrated cardio-respiratory pattern of hypoxia generated in

chemodenervated rats by hypoxia. Thus chemodenervation could not explain the pattern of response to systemic hypoxia described above. Second, they indicated that either the RVL-spinal vasomotor neurons themselves or neural structures immediately adjacent to these neurons were detecting cyanide, and by extension, hypoxia, and hence the 'sensor', is not at a site remote from the RVL-spinal vasomotor neurons. Third, they suggest that cyanide and hypoxia act upon cardiovascular and respiratory neurons in the RVL by comparable intracellular processes (Gonzalez et al., 1992).

3.1.3. Membrane Responses o f The Medullary Pacemaker Neurons to Hypoxia and Cyanide In Vitro

In an in vitro preparation, RVL vasomotor neurons are excited by hypoxia and cyanide. Such stimuli induce a rapid inward current associated, as in the glomus cells (Hayashida and Eyzaguirre, 1979), with increases in noise indicating channel activity (Sun and Reis, 1992a, 1994d). No excitatory post-synaptic potentials of the medullary neurons can be detected. The underlying mechanism of excitation is a rapid membrane depolarization, associated with significant increases in membrane conductance (Fig. 6) caused by an inward Ca -~÷ membrane current (Sun and Reis, 1993c, 1994b,d). This response is not blocked by a high concentration of tetrodotoxin (TTX), thereby indicating that it is not due to synaptic release of neurotransmitters. In addition, the membrane response to cyanide is not

300pM NaCN

-0.1 nA -0.4

-$$ mV

-!!0

40s Fig. 6. Effect of cyanide on membrane potential and resistance of the medullary pacemaker neuron in vitro. In the presence of 10/z~ tetrodotoxin, which blocked spontaneously occurring and evoked action potentials, cyanide evoked a reversible membrane depolarization of the neuron. The evoked membrane depolarization was associated with an increase in the membraneConductance since during the response the same intensity of current pulses ( - 0.3 hA, 2 ~ m s e c ) elicited much smaller changes in the membrane potential, as examined when the membrane potentials were restored to the pre-cyanide level by further injectinghyporpotarizing current to ~tvoid influence from voltage-dependent changes in the membrane conductance(After J. Physiol., Lond. 476, 101-! 16, 1994,)

Excitation of Sympathetic Neurons by Hypoxia

/

2 mM Co 2÷

gM CN 75%N2 300 laM CN

I~ 'd 2 min

- 70 mV

Fig. 7. Effect of hypoxia and cyanide on the membrane current of the medullary pacemaker neuron in vitro. The responses were observed when the membrane was voltage-clamped at -70 mV and after tetrodotoxin (10/aM) treatment. Hypoxia and cyanide evoked the same pattern of inward currents, whose magnitudes depended on the cyanide concentrations applied. The hypoxic inward currents were abolished by extracellular Co2+. (After NeuroscL Lett. 157, 219-222, 1993.) attenuated by extracellular replacement of Na ÷ ions (Sun and Reis, 1994d). The in vitro observations are consistent with those obtained in vivo, namely that excitation of RVL-spinal vasomotor or sympathetic neurons induced by hypoxia or cyanide are blocked by extracellular Co 2÷ (Fig. 7; Sun and Reis, 1992a, 1993c, 1994d), a nonselective Ca 2+ channel blocker. Similarly, the hypoxic membrane responses of these neurons were not changed by the application of an effective dose of kynurenate and 2-amino-5-phosphovaleric acid to block the excitatory amino acid receptors (Sun and Reis, 1994d). This indicates that the neuronal responses could not result from synaptic release or non-synaptic leakage of excitatory amino acids from the cytosol of damaged cells, consistent with the in vivo observation (Sun et aL, 1992). Consistent with the in vivo results (Sun and Reis, 1993c), the membrane response of the medullary pacemaker neurons is not attenuated by 30 min perfusion of NG-nitro-L-arginine, a potent and specific inhibitor of nitric oxide synthase (Sun and Reis, 1994d), indicating that the formation of nitric oxide is not involved in the neuronal response to acute hypoxia. Similarly, extracellular application of adenosine deaminase has no effect on the neuronal response to cyanide while abolishing the effect of adenosine, indicating that release of adenosine (Simpson and Phillis, 1991; Winn et al., 1981) is not involved in the hypoxic excitation of the medullary neurons (Sun and Reis, 1994d). The evidence, taken together, indicates that the RVL-spinal vasomotor neurons are 02 sensors. Like the peripheral chemoreceptors, neurons in the RVL are often in very close apposition to blood vessels and in fact may be penetrated by capillaries (Milner et al., 1987). Since the RVL-spinal vasomotor neurons are in the central place in the neural circuit involved in regulation of activity of the sympathetic nerves, and changes in activity of these neurons are now well known to have significant effects on activity of the sympathetic nerves and arterial pressure, it is not entirely unexpected that these neurons themselves are actually PO2 chemo-sensitive and provide a direct link

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between brain tissue oxygenation and blood/O2 supply, though the relative contribution of reflexive and central components to the integrated autonomic responses and the threshold difference of the various oxygen sensors to hypoxia remains to be determined. 3.2. Other Medullary Neurons

Activity of other medullary neurons is known to be affected by hypoxia. These include the respiratory neurons (the dorsal and ventral groups) and vagal motoneurons. Systemic hypoxia depolarizes and hyperpolarizes neurons in the dorsal motor nucleus of the vagus (Donneily et al., 1992; Dean et al., 1991) and increases cardiac vagal efferent activity (Potter and McCloskey, 1986). Hypoglossal neurons (Haddad and Donnelly, 1990; Jiang and Haddad, 1991) and neurons in the nucleus of solitary tract (Dean et al., 1991) and in the K611iker-Fuse nucleus (Haddad and Jiang, 1993) are also reported to be depolarized by hypoxia. However, these neurons are unlikely involved in the rapid excitatory responses of the RVL-spinal vasomotor and sympathetic neurons, since responses of the medullary vasomotor neurons to hypoxia remain intact when synaptic inputs are largely attenuated (Sun and Reis, 1993c) or blocked (Sun and Reis, 1994d). In addition, these medullary neurons are not as sensitive as the RVL-spinal vasomotor neurons to hypoxia and it usually takes a few minutes to develop the responses (Jiang et al., 1991; Haddad and Jiang, 1993). Therefore, the responses of these neurons to hypoxia are more likely nonspecific. Energy failure may play an important role in the responses of these neurons since addition of ATP in the recording pipette reduces in a major way the hypoxia-induced changes in membrane currents studied with patch electrodes (Haddad and Jiang, 1991, 1993). Cowan and Martin (1992) reported that in slices, a few minutes of anoxia produced membrane depolarization or hyperpolarization of the rat dorsal vagal motoneurons, though most of these neurons may not innervate cardiac muscle. Interestingly, responses of the medullary pacemaker neurons to hypoxia are different from other neurons in the central nervous system, i.e. neurons with obviously different physiological functions are actually affected differently by hypoxia. Most central neurons respond to hypoxia with decreases in activity and membrane hyperpolarization after a relatively longer delay (Krnjevi6 and Leblond, 1989). RVL neurons with respiratory discharge frequencies (the ventral respiratory group) are silenced by hypoxia in peripherally chemodenervated rats and iontophoresis of cyanide, while RVL-spinal vasomotor neurons are rapidly excited. It is not clear, however, whether hypoxic inhibition of the respiratory neurons results from a specific oxygen-sensing mechanism expressed in these neurons or the response represents a general response to hypoxia in vertebrate brains, i.e. reducing energy expenditure early in hypoxia by reducing neuronal activity. The specificity of the neuronal responses to hypoxia is restricted to neurons with very similar discharge pattern. For example, RVL neurons with similar sensitivity to baroreflexes and pulsemodulation activity but without spinal projections are not excited by hypoxia in peripherally chemodener-

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vated animals (Sun and Reis, 1993a), suggesting that chemotransductive mechanisms are specifically expressed in the RVL-spinal vasomotor neurons.

activation of oxygen-sensitive channels; (d) sensing by NADPH oxidase. 4.1. Metabolic Hypothesis

3.3. Neurons in the Spinal Cord

The spinal cord may represent an additional hypoxic neurogenic regulator of vascular tone, as first suggested by Alexander (1945). In the acute spinal cat, a few minutes of severe hypoxia increase activity of the cervical sympathetic nerves (Rohlicek and Polosa, 1981, 1983a). The hypoxic response may result from direct effects of oxygen lack on the sympathetic preganglionic neurons in the spinal cord. However, due to low sensitivity and the small magnitude of the response to a relatively long period of hypoxia, the sympathetic nerves may not contribute a large part of the hypoxic response in central nervous system-intact animals. This is supported by the observations that microinjections of muscimol into the RVL or intrathecal administration of kynurenate eliminate the increases in arterial pressure and sympathetic nerve activity evoked by the short period of hypoxia (Sun and Reis, 1994c). It has been reported that after section of the cervical spinal cord, systemic hypercapnia or superfusion of the first thoracic spinal segment with acidic solutions can induce an increase in both arterial pressure and the activity of the sympathetic preganglionic neurons in rats (Lioy and Trzebski, 1984) and cats (Accili et al., 1988; Johnson et al., 1968; Szulczyk and Trzebski, 1976; Zhang et al., 1982). However, unlike the responses of the RVL-spinal vasomotor neurons to hypoxia, COs-mediated responses of the sympathetic preganglionic neurons were observed 3-4 hr after the transection (Rohlicek and Polosa, 1986), and were not present immediately after the spinal section (Lioy et al., 1978). However, others reported no change in postganglionic nerve activity in response to hypercapnia in spinal dogs (Alexander, 1945) and cats (Meckler and Weaver, 1985).

The metabolic hypothesis proposes that the respiratory chain of mitochondria is the principal sensor of hypoxia, triggering the PO_,-dependent transmitter release from glomus cells and by extension altering membrane channel conductances in the central neurons. Hypoxia decreases oxidative phosphorylation (Mulligan et al., 1981) and/or ATP content (Anichkow and Belinki, 1963; Joels and Neil, 1963), which in turn increases the accumulation of free Ca 2÷ ions in the cytosol and thereby increases release of transmitters from glomus cells or neuronal discharge. In support are observations that inhibition of oxidative phosphorylation with oligomycin completely blocks carotid chemoreceptor response to 02 (Shirahata et al., 1987) and that PO2 chemoreception in the carotid body is not possible without a functioning respiratory chain (Mulligan et al., 1981). In rat CA1 hippocampal neurons, hypoxia evokes a K+-conductance-mediated hyperpolarization, which can be prevented by including ATP (2 mM) in the solution filling the whole-cell recording electrodes (Zhang and Krnjevi6, 1993). The metabolic hypothesis, however, can not explain the rapidity of the responses since ATP content of the carotid bodies changes rather little after a brief period of hypoxia (Acker and Starlinger, 1984; Verna et al., 1990) or exposure to cyanide (Obeso et al., 1989), and responses of the cells to cyanide occur regardless of the presence of intracellular ATP (Biscoe and Duchen, 1989; Peers and O'Donnell, 1990). As for the RVL-spinal vasomotor neurons, iontophoretic application of cyanide or systemic hypoxia evokes an excitatory response of the neurons within seconds (Sun et aL, 1992), a response too rapidly produced to be explained by the metabolic mechanism (see below). 4.2. IntraceHular Ca 2÷ Mobilization

4. CHEMOTRANSDUCTIVE MECHANISM The prototypical oxygen sensor in vertebrates is beleived to be the type I glomus cells of the carotid body. In response to hypoxia or to cyanide, these specialized cells release transmitter to trigger a powerful and graded discharge of the afferent nerves which innervate the organs thereby eliciting the integrated autonomic responses. The fact that RVL-spinal vasomotor neurons are also excited by comparable periods of hypoxia raises the question whether the cellular mechanisms which signal hypoxia in the type I glomus cells of the carotid body and RVL-spinai vasomotor neurons are comparable despite the fact that the glomus cells require afferent fibers of another cell to initiate responses while RVL-spinal vasomotor neurons are both sensor and effector. Before considering mechanisms by which hypoxia stimulates RVL-spinal vasomotor neurons, it is useful to review the principal hypotheses which have been proposed to explain signalling in type I glomus cells. Four have been proposed: (a) the metabolic hypothesis; (b) the Ca 2+ mobilization hypothesis; (c)

The Ca 2+ mobilization hypothesis proposes that mitochondria sense hypoxia and in response mobilize and increase intracellular Ca 2÷. It is the elevated Ca `-+ rather than reduced ATP that is critical since the elevations of intracellular Ca -'+ occur before intracellular ATP concentrations are reduced. The hypothesis is entirely based on studies on the type I cells of the carotid body in response to cyanide, an inhibitor of electron transfer (Eyzaguirre and Koyano, 1965), which elicits a dose-dependent augmentation in chemoreceptor afferent discharge (Biscoe et al., 1970). Studies in the glomus cells dissociated from adult rabbits have demonstrated that cyanide produces a paradoxical hyperpolarization (Biscoe and Duchen, 1989; Duchen et aL, 1988), resulting from an increased K ÷ conductance and an increase of intracellular Ca 2+ (Biscoe and Duchen, 1989, 1990a; Duchen et al., 1988; Heschler et al., 1989; Lopez-Barneo et aL, 1988). Based on these observations, Biscoe and Duchen (1989, 1990a) proposed that the rapid intracellular Ca -'+ mobilization from mitochondria, which they view as the O., sensors, is the

Excitation of Sympathetic Neurons by Hypoxia underlying mechanism of chemoreception. Since cyanide and hypoxia attenuate C a 2+ currents but enhance a voltage-dependent outward K + current (Biscoe and Duchen, 1989, 1990a), Biscoe and Duchen further proposed that electrophysiological response of the carotid cells to cyanide and hypoxia is not central to the transduction process, rather it is the hypoxic release of mitochondrial Ca 2+, which results in the rapid release of dopamine from the type I carotid cells to stimulate chemoreceptor afferents. However, others have disputed this hypothesis, arguing that in healthy resting cells (Carafoli, 1987; McCormack et al., 1990), the mitochondria may not have levels of Ca 2+ high enough to produce significant rises in [Ca2+]i when released. Contrary results have also been obtained in studies of the type I glomus cells in adult rats, which have shown that hypoxia decreases intracellular calcium concentration (Donnelly and Kholwadwala, 1992) and that hypoxia or cyanide produce membrane depolarization due to an inhibition of membrane K ÷ conductance (Delpiano et al., 1988; Lopez-Barneo et al., 1988; Ganfornina and Lopez-Barneo, 1992a,b; Gonzalez et al., 1992; Peers and O'Donnell, 1990). In anesthetized rats in vivo, we demonstrated that the rapid excitatory responses of the RVL-spinal vasomotor neurons to iontophoresis of cyanide depend on extracellular Ca 2÷, since the responses are attenuated by iontophoretically applied Co 2+, a Ca 2+ channel blocker (Sun et al., 1992). In these hypoxia-sensitive neurons, the membrane electrophysiological response to hypoxia, membrane depolarization due to increased Ca 2÷ influxes, is central to the neuronal response, suggesting that extracellular Ca 2+ is crucial.

4.3. O2-sensitive Channels Membrane channels or channel complexes may directly sense changes in the 02 tension. Changes in the channel conductance then lead to alterations in the neuronal activity or transmitter release. Two types of channels, potassium and calcium, have been proposed to act as O2-sensitive sensors. 4.3.1. K + Channels There are contrary reports as to whether the voltage-dependent outward K + currents are enhanced by hypoxia and cyanide (Biscoe and Duchen, 1989, 1990a) or suppressed by hypoxia (Delpiano et al., 1988; Lopez-Barneo et al., 1988) and cyanide (Gonzalez et al., 1992; Peers and O'Donnell, 1990). Ganfornina and Lopez-Barneo (1992a,b) reported that a type of K ÷ channel, which is steeply voltage dependent and not affected by [Ca2+]i, may be the O2-sensitive channels. The reversible inhibition of the K + channel activity by low PO 2 does not desensitize and is not related to the presence of F - , ATP, and GTP-y-S at the internal face of the membrane, suggesting that the O2-K + channel interaction occurs either directly or through an 02 sensor intrinsic to the plasma membrane, closely associated with the channel molecule. A working hypothesis has been proposed (Gonzalez

209

et al., 1992), based on the O~.-K + channel interaction,

that low PO2, by reducing the opening probability of O2-sensitive K + channels, produces the initial depolarization required to activate the voltagedependent Ca 2+ channels. Simultaneous activation of Na + channels provides a fast recruitment of Ca 2+ channels, potentiating the entry of Ca 2+ and the release of neurotransmitters, while Ca 2+dependent K + channels contribute to cell repolarization. It is not clear what is the cause for the opposite observations: activation or inhibition of the voltagedependent K ÷ channel activity by hypoxia in the same type of cells. 4.3.2. Ca 2+ Channels On the other hand, hypoxia can produce an increase in cytosolic Ca 2+ concentrations by activating a number of mechanisms including voltage-dependent Ca 2÷ channels (membrane depolarization) and ligand-mediated changes in Ca 2+ concentrations (Meyer, 1989). In the type I carotid chemoreceptors, early studies showed that the extracellular calcium activity decreased under hypoxia (Acker, 1980) and that hypoxia induced calcium influx (Pietruschka, 1985). However, a majority of the recent studies did not find a hypoxia-induced increase in calcium inward currents in the type I cells of the carotid bodies, though this was challenged by Buckler and VaughanJones (1994), who reported that hypoxia induced calcium influx in cultured rat carotid body type I cells. In the reticulospinal vasomotor neurons, our data suggest that a large part of the neuronal response to cyanide originates from Ca 2+ influx (Sun and Reis, 1992a, 1994d; Sun et al., 1992), blocked by extracellular application of Ca 2+ channel blockers. The mechanism might therefore differ from that of the arterial chemoreceptors, in which it has been reported that cyanide reduces the amplitude of the Ca 2÷ current (Biscoe and Duchen, 1989), hypoxia does not affect Na ÷ o r C a 2+ currents (Heschler et al., 1989; Lopez-Lopez et al., 1989), and the cellular response to cyanide is not affected by extraceilular application of Ca 2+ channel blockers (Biscoe and Duchen, 1990b). However, it remains to be determined whether the Ca -*+ channel or channel complex of the RVL-spinal vasomotor neurons is O2-sensitive or if the response involves a second messenger(s). 4.4. NADPH Oxidase It has also been proposed (Acker, 1989; Acker et al., 1989; Cross et al., 1990) that an 02 sensor of chemoreceptor cells is a heine-linked NADPH oxidase. According to this proposal, a reduction in hydrogen peroxide produced by the oxidase during low /'02 stimulation changes the glutathione redox state, which in turn alters the configuration of membrane proteins and hence the conductivity in ionic channels. In the medullary pacemaker neurons, our preliminary results suggest that the oxidase may not be involved in the hypoxic excitation, since application of diphenylene iodonium (10 gM, 15-20 min), the specific inhibitor of the NADPH oxidase, has no effect on the

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responses of the neurons to cyanide (unpublished observation).

5. SYMPATHETIC EXCITATION ELICITED BY CEREBRAL ISCHEMIA The cerebral ischemic response (CIR) consists of stereotyped autonomic response, made up of a rapid and potent increase in sympathetic nerve activity, peripheral vascular constriction, increased arterial pressure, bradycardia and apnea elicited by rendering the brainstem ischemic (Dampney et al., 1979; Dampney and Moon, 1980; Guyenet and Brown, 1986; Guyton, 1948; Haselton et al., 1985; Kumada et al., 1979). Experimentally, this is usually accomplished by occluding a patent carotid artery in animals in which the other carotid and both vertebral arteries are occluded. The CIR therefore consists of a pattern of reaction similar to that elicited in vertebrates by submersion (the diving reflex; Irving et al., 1942; McCulloch and West, 1992), by brain-stem distortion (the Cushing reflex; Doba and Reis, 1972) and by hypoxia, when elicited in the absence of arterial chemoreceptors. The initiators of the CIR that act as a last line of defense in protecting the brain from terminal metabolic damage are believed to be located in the RVL.

5.1. Role of RVL-spinal Vasomotor Neurons The CIR results from stimulation of receptors within the parenchyma of the medulla oblongata (Dampney et al., 1979; Kumada et al., 1979) and depends upon activity of neurons within the medulla for its expression (Cross et al., 1990; Dampney and Moon, 1980; Guyenet and Brown, 1986; Haselton et al., 1985; Prabhakar et al., 1986; Rohlicek and Polosa, 1983b). In support is evidence that: (a) chemical stimulation of the RVL with excitatory amino acids simulates the CIR by increasing arterial pressure and inhibiting phrenic nerve discharge (Baradziej and Trzebski, 1989; Lawing et al., 1987); (b) lesions of the RVL abolish the CIR (Dampney and Moon, 1980); and (c) cerebral ischemia excites RVL-spinal vasomotor neurons in advance of the rise of arterial pressure (Guyenet and Brown, 1986). The fact that cerebral ischemia may reduce neuronal activity in other brain regions (Guyenet and Brown, 1986; Hansen, 1981, 1985) suggests that excitation of the RVL-spinal vasomotor neurons is not a non-selective excitatory event and raises the possibility that they may sense and possibly initiate the event.

5.2. Signals Triggering the Sympathetic CIR Cerebral ischemia can, particularly when sustained, result in a variety of metabolic events including local hypoxia, hypercapnia, acidosis, accumulation of such cellular products as K ÷, H +, lactic acid, adenosine, ATP, products of arachidonic acid metabolism, and a variety of neurotransmitters notably L-glutamate (Bazan et al., 1986; Hagberg et al., 1985; Hansen, 1981, 1985; Harris and Symon, 1984; Kraig et al., 1985; Nicholls, 1989; Siesj6, 1978; Silver and Erecinska,

1990; Walter et al., 1988). Given the panoply of cellular signals, the question is posed: What is the cellular signal which triggers the response? One of the hallmarks of the sympathetic CIR is its brief latency. In rats, the increase in arterial pressure can begin within or even less than 2 sec following arterial occlusion (Guyenet and Brown, 1986) and coincident with the first reduction in medullary blood flow measured by laser-doppler flowmetry (Reis and Maiese, unpublished). The very brief latency tbr the sympathetic CIR would suggest that the response is a consequence of a 'sensor' detecting an immediate signal elicited by a brief reduction in brainstem blood flow, and not to the biochemical consequences of depriving brain of its blood flow (Siesj6, 1978). Hypoxia seems to be the, at least the major, signal triggering the sympathetic CIR. 5.2.1. Hypoxia Hypoxia is one of the most potent stimuli for activation of the sympathetic nervous system and may be the early signal of ischemia which triggers the sympathetic CIR, since the autonomic responses elicited by brainstem hypoxia are comparable to the pattern of the CIR, a rapid activation of the sympathetic nervous system, inhibition of the respiratory center, and bradycardia (H0ka et al., 1989; Neubauer et aL, 1990; Sun and Reis, 1993a,c, 1994a; Takeuchi et al., 1992; Washicko et al., 1990). Direct activation of the RVL-spinal vasomotor neurons by hypoxia during ischemia may, therefore, represent one of the major actions of acute ischemia in evoking the rapid sympathoexcitatory response. This is supported by the observations (Sun and Reis, 1993b, 1994a,d) that other possible metabolic triggers in ischemia, including H ~, CO_,, lactate, adenosine, are not potent stimuli for activation of the RVL-spinal vasomotor neurons and the sympathetic nerves (see below), while changes in extracellular K + concentrations, though rapid, are too small to have a significant effect on the neuronal activity and on the sympathetic nervous system, as discussed above. 5.2.2. Acidosis and Hypereapnia One of the earliest events associated with ischemia in the cerebral cortex is the accumulation of H + (Hansen, 1981; Harris and Symon, 1984; Siesj6, 1984; Siesj6 et al., 1990), caused by ischemic accumulation of bicarbonate (Arita et al., 1989; Ljunggren et aL, 1974a) and lactic acid (Katsura et al., 1991; Ljunggren et al., 1974b; Paschen et al., 1987; Siesj6 et a l , 1990; Smith et al., 1986). The pH shifts during ischemia can be large enough to influence channel and enzyme functions (Chesler and Kaila, 1992; HiIlered et aL, 1984), especially when the ischemia lasts. A variety of ion channels can be influenced by modest shiftsin [H +] and might thereby modulate neuronal excitability (Gruol et al., 1980; Konnerth et al., 1988; Krishtat and Pidoplichko, 1980; Moody, 1984; Sun and Reis, 1993b; Tang et al., 1990; Traynelis and Cull-Candy, 1990). Low pH can cause denaturation of protein molecules in cell membrane and cytosol, e.g.

Excitation of Sympathetic Neurons by Hypoxia membrane channels and metabolic enzymes (Chesler, 1991). Intracellular acidosis may cause depolarization in mammalian neurons and glial cells (Lehmenkohler et al., 1989; Dean et al., 1989) or produce an increase in cytosolic free Ca 2+ concentration (Bums et al., 1991). It has been proposed that accumulation of H + ions is the stimulus for central chemosensors located on the ventral surface in an area corresponding to the RVL (Fukuda, 1983; Fukuda et al., 1978; Loeschcke, 1982). Administration of hypercapnic gas mixtures to anesthetized dogs is found (Downing and Siegel, 1963) to increase the discharge rate of the inferior cardiac nerve even after sino-aortic denervation, and perfusing the cephalic circulation with hypercapnic and/or hypoxic blood results in increases in systemic arterial pressure, heart rate, total peripheral resistance and myocardial contractility (De Geest et al., 1965; Downing et al., 1963; Hainsworth et al., 1984; Hanna e t al., 1979; Kao et al., 1962). While the application of solutions of varying pH to the ventral medullary surface has modified sympathetic activity (Lioy et al., 1981; Millhorn and Eldridge, 1986; Szulczyk and Trzebski, 1976), others have found it to have no effect (Cragg et al., 1977). Generally speaking, the arterial pressure changes to acidosis and/or hypercapnia tend to be small in magnitude and inconstant. In our study, we found (Sun and Reis, 1993b, 1994a) that hypercapnia and extracellular application of H ÷ did not have profound effects on arterial pressure, sympathetic nerve activity, and activity of the RVL-spinal vasomotor neurons in anesthetized and artificially respired rats. Local iontophoretically applied H ÷, however, modifies responses of the RVL-spinal vasomotor neurons to iontophoretically applied GABA and baroreflex inhibition of the neurons (Sun and Reis, 1993b), indicating that the effect of acidosis is more of modulating the neuronal responses to other transmitters. Therefore, the evidence suggests that acidosis and hypercapnia may not be the major signals evoking the sympathetic CIR. However, extracellular application of H ÷, acidosis, can significantly modify sympathoexcitatory responses of hypoxic chemoreception. 5.2.3. Neurotransmitters It has been firmly established that L-glutamate-mediated excitotoxic mechanisms play a pivotal role in at least some forms of hypoxic/ischemic neuronal injury (Meldrum, 1989; Rothman and Olney, 1986, 1987), which are directly relevant to the human setting of brain injury following cardiac arrest (Petito et al., 1987), probably to focal ischemic stroke. L-Glutamate accumulates to high levels because ischemic depolarization of neurons causes a release of L-glutamate into the extracellular space (Benveniste et al., 1984) and re-uptake systems are blocked by the ischemic energy failure. It has been suggested that excitatory amino acids are toxic because they open postsynaptic calcium channels and allow entry of extracellular calcium into the cells and the calcium ions induce lipolytic and proteolytic reactions in the vulnerable neurons (Choi, 1988; McDonald and Johnston, 1990;

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Rothman and Olney, 1986, 1987). This is supported by the evidence that antagonists of specific receptors for L-glutamate diminish the degree of cellular damage caused by ischemia (Ozyurt et al., 1988; Simon et al., 1984), especially antagonists of N-methyl-o-aspartate (NMDA) receptors (Gill et al., 1987), though others have reported otherwise (Shearman, 1989). It is, therefore, interesting to find out whether the early ischemic excitation of the RVL-spinal vasomotor neurons (Guyenet and Brown, 1986) could result from a rapid release of excitatory amino acids, which then excite the neurons. Our investigation of the hypoxic response of the RVL-spinal vasomotor neurons indicates that a rapid release of excitatory amino acids could not mediate the neuronal response to brainstem hypoxia. First, application of kynurenate at a dose at which it blocks responses of the neurons to microiontophoretically applied L-glutamate does not affect the hypoxic sympathoexcitation in vivo and kynurenate does not attenuate the membrane responses of the neurons to cyanide in vitro (Sun and Reis, 1993c). This is also consistent with the observation that the membrane response of the neurons is not eliminated by TTX (Sun and Reis, 1994d), which should block synaptic inputs into the neurons and any effect depending on activation of synaptic inputs.

5.2.4. Energy Failure It is well known that the primary effect induced by ischemia is inhibition of mitochondrial function and inhibition of energy production, causing rapid depletion of ATP (Hansen, 1985). In the carotid chemoreceptors, it has been proposed that energy failure may be the underlying mechanism initiating the hypoxic chemo-responses (see above). However, the responses of the reticulospinal vasomotor neurons (Guyenet and Brown, 1986), the sympathetic nerves and of arterial pressure appear too quickly to have resulted from energy failure. It is well known that brain neurons contain potential sources of energy-rich phosphate groups which allow a normal rate of ATP regeneration for almost 1 min, but the RVL-spinal vasomotor neurons react to interruption of the blood oxygen supply and cyanide within seconds (Guyenet and Brown, 1986; Sun et al., 1992; Sun and Reis, 1994a), and such rapid responses would be inconsisent with the suggestion that a general energy failure of the neurons may be the underlying mechanism responsible for the rapid excitation of these neurons in response to ischemia. This view is supported by the observation that a brief period of hypoxia in fact causes little change in the ATP levels in rabbit carotid body (Verna et al., 1990), and a reduction of no more than 10-20% in guinea pig hippocampus (Lipton and Whittingham, 1982). 5.2.5. Calcium and Enzyme Activation Calcium ions function widely as cellular messengers and intracellular regulators (Carafoli, 1987). Marked increases in intraceUular cytoplasmic calcium ion concentrations occur in the course of ischemia and

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may be crucial in irreversible cell injury (Choi, 1988; Hass, 1981; Raichle, 1983; Siesj6, 1981). By lowering ATP supply, ischemia can be expected to slow down processes that normally maintain a very low cytosolic free Ca 2+, but this may not be relevant to the early response of the RVL-spinal vasomotor neurons to ischemia. Calcium channel blockers have been evaluated in a variety of experimental models ofischemia with only mixed success in averting the consequences of brain ischemia (Ginsberg et al., 1980; Kobayashi et al., 1988; Mohamed et al., 1985). It is unresolved whether intracellular release of calcium may be involved in the ischemic excitation of the RVL-spinal vasomotor neurons. However, in an in vivo study, cyanide-induced rapid increases in activity of the RVL-spinal vasomotor neurons were abolished by extracellular Co > (Sun et al., 1992), a Ca 2÷ channel blocker. In an in vitro study, the medullary pacemaker neurons were excited by hypoxia and cyanide by activating membrane Ca 2+ channels (Sun and Reis, 1993c, 1994d). These results strongly suggest that a calcium mechanism may be involved in the rapid sympathetic CIR. The deleterious sequelae of ischemia-induced increases in intracellular cytosolic calcium are thought to be mediated via activation of phospholipases (phospholipases A~, A2, and C), the breakdown of membrane phospholipids, and the accumulation of free fatty acids, lyso-phospholipids, and diacylglycerols (Bazan, 1970, 1976; Edgar et al., 1982; Rordorf et al., 1991). Phospholipases A t and A 2 liberate the fatty acids at the first and second carbons of the glycerol moiety. Phospholipase C hydrolyzes the phosphatidyl base group from phospholipids, liberating diacylglycerol. It has been suggested (Bazan, 1970, 1976) that the activation of a phospholipase A2 is involved during ischemic insult. It is becoming increasingly evident (Glaser et al., 1993; Hsueh and Needleman, 1979) that mammalian tissues contain two groups of calcium-dependent phospholipase A2. The highly specific phospholipase A2 is hormone or agonist sensitive (such as to bradykinin), involves a receptor-mediated mechanism, and selectively liberates arachidonic acid from membrane phospholipids (Aveldafio and Bazan, 1975; Di Rodriguez and Bazan, 1983). This enzyme may be involved in ischemic injury at a early period, and response produced by such a mechanism is reversible. The other phospholipase A2 non-specifically responds to noxious stimuli (ischemia and trauma) and releases all fatty acids from the C-2 carbon of phospholipids. Under ischemic insult the latter type of phospholipase A2 is probably activated (Hsueh and Needleman, 1979), and damage caused by the stimulation of this enzyme is irreversible. It is of great interest to examine whether activation of phospholipases and/or the formation of as well as metabolites of free fatty acids and diacylglycerols may represent ischemic signals initiating excitatory responses of the RVL-spinal vasomotor neurons and the sympathetic nerves. Arachidonic acid and diacylglycerols are activators of protein kinase C and activation of this enzyme results in facilitation of calcium influx as reported in other neurons (DeRiemer et al., 1985; McPhail et al., 1984).

5.2.6. O t h e r Factors Interestingly, increases in extracellular K + occur during the earliest phases (first 10 sec) of an ischemic episode (Hansen, 1981, 1985). However, the induced changes in extracellular K ÷ concentrations are generally too small to produce a significant neuronal response (see above). Adenosine is another metabolic indicator of ischemia-hypoxia and metabolic rate. However, its role as a neuronal excitant, or a trigger, in the sympathetic CIR is not clear. In explaining how regional blood flow within muscle tissue can be regulated according to metabolic demand, several investigators proposed the 'adenosine hypothesis' (Berne, 1980; Dobson et al., 1971; Olsson and Bunger, 1987; Spaks and Bardenheuer, 1986). This hypothesis suggests that tissues consuming oxygen can 'signal' smooth muscle in blood vessels to relax, by an action of adenosine released from the muscle. It has been shown that the extracellular adenosine concentration increases dramatically during increased nerve activity, hypoxia and ischemia (Hagberg et al., 1987; Phillis et aL, 1987; Simpson and Phillis, 1991; Zetterstr6m et al., 1982). Adenosine, which can cross cell membranes, is generated in increasing amounts as a shift in the distribution of adenosine-containing chemicals occurs (ATP --, ADP ---, AMP ~ adenosine) when ATP regeneration is prevented by inadequate oxygen supply. This hypothesis is supported by evidence from several laboratories that adenosine antagonists and adenosine deaminase treatment can reduce coronary metabolic vasodilation (Headrick and Willis, 1990; Merrill et al., 1986; Sato et al., 1985), but not by others (Klabunde et al., 1988). However, adenosine is generally considered an inhibitory neuromodulator (Biaggioni, 1992; Prince and Stevens, 1992). It produces hyperpolarization of neurons, decreases nerve firing, inhibits the release of a variety of neurotransmitters and voltage-activated Ca 2÷ channels (Dolphin et al., 1986; MacDonald et aL, 1986), and has central depressor actions. Recent study indicates that the actions of adenosine are mediated by distinct receptors (Biaggioni, 1992): A~ receptors are coupled to G~ proteins and their activation results in inhibition of adenylate cyclase and opening of potassium channels; A2 receptors are coupled to Gs proteins and their activation results in stimulation of adenylate cyclase. An A2 receptor-mediated central vasodepressor action of adenosine has been reported (Barraco et al., 1991; Tseng et aL, 1988). Furthermore, adenosine has excitatory effects on the arterial chemoreceptors (Biaggioni, 1992). Intra-carotid injections of adenosine in cats (McQueen and Ribeiro, 1986) and rats (Monteiro and Ribeiro, 1987) increase carotid sinus nerve activity. This effect is independent of the vascular effects of adenosine and is also observed in the isolated superfused cat carotid (Runold et aL, 1990a) and aortic (Runold et al., 1990b) bodies. A recent study by Mogul et al. (1992) has revealed that adenosine selectively inhibits and excites different subtypes of Ca 2+ channels via activating different subceptors of adenosine. Canine coronary vasoditation induced by systemic hypoxia was reported (Merrill et al., 1986) to be attenuated by adenosine deaminase. However, our investigation indicates that

Excitation of Sympathetic Neurons by Hypoxia adenosine is unlikely to be the signal mediating the ischemic activation of the RVL-spinal vasomotor neurons since (i) adenosine does not mimic the effect of hypoxia and cyanide on the medullary pacemaker neurons (Sun and Reis, 1994d), and (ii) the membrane response of the medullary neurons to cyanide is insensitive to perfusion of adenosine deaminase at a concentration at which adenosine deaminase abolishes the membrane response induced by adenosine (Sun and Reis, 1994d).

6. CONCLUSIONS RVL-spinal vasomotor neurons play a critical and integrative role in initiating and probably sustaining the cardiovascular adjustments to hypoxia and also to local ischemia. They are critical in the reflex responses initiated by stimulation of the peripheral chemoreceptors but themselves may also be biological sensors of oxygen. Together, these chemosensitive mechanisms provide a link between sympathetic nerve activity and oxygenation of arterial blood and act to maintain arterial PO2, PCO2, and pH within physiological limits. A model that is consistent with most of the data available today is presented in Fig. 8. Hypoxia rapidly excites the arterial chemoreceptors, the peripheral O2 RVL Reticulospinal

/.

NTS neurons

[

VaSOmOtor neurons I

~+

/

/

/

Sensory neurons in petrosal ganglion, etc. / /

/ ~ +

IML

/ / / chemoreceptors

213

sensors, triggering the early hypoxic sympathoexcitatory responses in vertebrates. Activation ofthe arterial chemoreceptors induces a rapid release of transmitters, probably catecholamines which, in turn, excite the afferent terminals of the sensory neurons with cell bodies located in the petrosal or nodose ganglia. The signal is transferred to neurons in the NTS, which then project to the RVL-spinal vasomotor neurons, monosynaptically or multisynaptically via unidentified relays. In addition, hypoxia can directly excite the RVL-spinal vasomotor neurons. Changes of activity of the RVL-spinal vasomotor neurons elicit dramatic responses of the sympathetic nerves and vascular beds, through their neural connections with the sympathetic preganglionic neurons in the spinal cord. The pathway illustrated in Fig. 8, however, does not describe the possible existence of central PO2 sensors involved in regulation of respiration. It is not clear whether the respiratory and sympathetic nervous systems share the same sensors and whether the two system interact in their chemoreceptive responses. However, there are obvious differences between the two systems. The central sympathetic PO2 chemoreceptors are mainly located in the RVL since restricted inhibition of the RVL eliminates the centrally-initiated sympathetic responses to hypoxia while the ventilatory chemoreceptors, according to Coates et al. (1993) are widespread in the brainstem. Longer period ( < 25 sec) of hypoxia also stimulate the vagal center, eliciting severe bradycardia. It is not known whether separate sensors are involved in initiating hypoxic responses of these centers. The oxygen chemotransductive mechanism of the RVL-spinal vasomotor neurons appears to involve O2-sensitive calcium influx and may be different from that of the arterial chemoreceptors, in which intracellular calcium mobilization or a rapid inactivation of the O2-sensitive K ÷ channels may be the underlying mechanism. The oxygen chemoreceptive responses of the RVL-spinal vasomotor neurons may also underlie the mechanism involved in initiation of the sympathetic component of the CIR. Acknowledgements--This work was supported by National

preganglionic neuronsI

2. i Sympatheticpostganglionic neurons

I

I Fig. 8. Schematic diagram showing proposed pathways providing the basis for oxygen chemoreceptive sympathoexcitation. An effective stimulus (a fall in PO2) stimulates the arterial chemoreceptors. Activation of the receptors induces sympathoexeitatory responses via the afferent neurons ---,the NTS neurons -- the reticulospinal vasomotor neurons ---,the sympathetic preganglionic neurons ---, the sympathetic postganglionic neurons. Hypoxia also directly excites the reticulospinal vasomotor neurons in the rostral ventrolateral medulla, resulting in central sympathoexcitatory response. IML; intermediolateral cell column: NTS; nucleus solitary tract: RVL; rostral ventrolateral medulla.

Heart, Lung and Blood Institute Grant HL-18974.

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