The effects of reserpine on hypothalamo-pituitary adrenocortical function

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THE E F F E C T S O F RESERPINE ON HYPOTHALAMO-PITUITARY ADRENOCORTICAL FUNCTION SANDRA V. VELLUCCI Department of Pharmacology. The School of Pharmacy, University of London, 29/39 Brunswick Square, London WCIN lAX. U.K. (Received 6 February

The adrenal cortex secretes a group of steroid hormones, the glucocorticoids, among which are corticosterone and cortisol. The synthesis and secretion of corticosterone from the adrenal cortex is under the control of a polypeptide hormone from the anterior lobe of the pituitary gland. This hormone is known as the adrenocorticotrophic hormone (ACTH) or corticotrophin, The release of ACTH is in turn intluenced by a neurohumoral peptide from the hypothalamus, known as the corticotrophin releasing factor (CRF). CRF travels from the hypothalamus to the anterior lobe of the pituitary gland, via a series of portal blood vessels (the hypothalamo-hypophysial portal vessels), which effectively link the hypothalamus to the anterior lobe of the pituitary gland. Thus. the entire system forms a functionally linked unit known as the hypothalamo-pituitary adrenocortical (HPA) axis (see Fig. 1). Many substances are capable of blocking the release of corticotrophin in response to specific stress fui stimuli: for example, promethazine and glucose can inhibit the release of ACTH which occurs in response to histamine and insulin, respectively. Some general anaesthetics (e.g. pentobarbitone) are also capable of inhibiting pituitary corticotrophic function in response to stress. However, there are only very few substances which can be classed as true neuroendocrine-blocking drugs, i.e. drugs that inhibit the release of ACTH in response to a variety of stressful stimuli. Major candidates which have been proposed for this role are the naturally-occurring and synthetic glucocorticoids, morphine, chlorpromazine and reserpine. The effects of reserpine on HPA function in the rat are the subject of this review. Reserpine is the main pharmacologically active alkaloid extracted from the root of R a u w o l f i a Serpentina (a climbing shrub found mainly in India). The alkaloid was first isolated by Miiller et al. (1952). Its chemical structure was established in 1954 (Schlittler et al., 1954) and the compound was synthesized in 1956 (Woodward et al., 1956). When administered in suitable doses to man and animals the drug causes sedation and exerts an antihypertensive action (Plummer et al., 1954). Reserpine has therefore been used in the treatment of certain psychiatric disorders and hypertension. In addition, because reserpine causes

depletion of cerebral 5-hydroxytryptamine (5-HT) (Paasonen & Vogt, 1956: Pletscher et al., 1956; Brodie & Shore, 1957) and catecholamines (Holzbauer & Vogt. 1956; Brodie et al., 1957; Carlsson et al.. 1957) and is also capable of inhibiting certain hypothalamic functions and of influencing the release of ~ hormones from the anterior lobe of the pituitary ! gland, the drug has been widely employed as a research tool in the study of the various neuroendocrine mechanisms and pathways which are involved in the complex process of regulating HPA activity. Although the effect of reserpine on HPA function has been extensively studied, the action of this alkaloid on the secretion ofcorticotrophin is still not fully understood. Some reports indicate that the drug reduces the activity of the HPA system, probably by depressing the hypothalamic regions which control the output of ACTH (Mason & Brady, 1956; Wells et al., 1956; Mahfouz & Ezz, 1958), whereas others suggest that it increases the functional activity of the system (Gaunt et al., 1954; Egdahl et al., 1956; Harwood & Mason, 1957; Saffran & Vogt, 1960; Montanari & Stoekham, 1962). The aim of this article is to review the relevant literature and to make some attempt to explain some of the discrepancies which exist. The earliest investigations of the effect of reserpine on adrenocortical function were carried out by Gaunt et al. (1954) in order to determine whether the drug affected endocrine function through its inhibitory action on the hypothalamus. However, after a fairly extensive study, these investigators found no conclusive evidence that reserpine exerts an inhibitory action, indeed the observations which they put forward were all consistent with the idea that the drug stimulates the adrenal cortex. Similar effects were reported by Egdahl et al. (1956). These authors administered reserpine to dogs, collected the adrenal venous blood at different time-intervals thereafter, and then determined its 17ohydroxycorticosteroid concentration. After administration of the drug they found a marked increase in adrenal corticosteroid secretion, with the highest values occurring between 0.5-3.0 hr after the injection. The maximum corticoid values were found to be of a similar magnitude to those obtained after the intravenous injection of large 275

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of a single dose of the drug. Howe',or. the failure of these authors to obtain adrenal ascorbic acid depletion may partly be explained on the basis that the dose of drug used was extremely small (8.0 /lg/kg) and that the time-interval between drug administration and removal of the adrenal glands for a.~orbic acid determination was comparatively short (30 min). Many investigators have attempted to stud) the mechanism(s)" whereby reserpine exerts its effect on HPA function. The long duration of action was thought not to be due directl) to the presence of the drug itself as it was believed that the drug did not accumulate in the bod)(Brodic et al.. 1961). l-lowevcr. more recently the presence of trace a m o u n t s of[H3] reserpine has been demonstrated in the brains of experimental animals, for up to 5 days after drug administration (Shcppard c t a l . . 1958; Maggiolo & Hale',', 1964; Manara et al., 1972: StitzcL 19771. During this time a relatively small fraction of the total reserpine administered becomes irreversibly associated with the monoaminergic granular membranes resulting in a persistent inhibition of E

Fig. 1. The hypothalamo-pituitary-adrenocortical (HPAi axis.

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doses of ACTH, though comparatively much delayed. With the use of only indirect indices of HPA function the possibility that the drug exerts a direct stimulant action on the adrenal cortex cannot easily be excluded. However, there is no evidence of adrenocortical overactivity in hypophysectomized rats that have been treated with reserpine (Wells et al., 1956; Montanari & Stockham, 1962). Results obtained by using direct estimates of plasma ACTH for the assessment of pituitary adrenocorticotrophic activity, in addition to the indirect indices which have been almost exclusively employed in the past, also indicated that reserpine is capable of powerfully stimulating the secretion of ACTH when administered acutely (Hodges & Vellucci, 1975). Thus, a single intraperitoneal injection of the drug (dose 2.5 mg/kg) was found to induce prolonged hypersecretion of ACTH which persisted for at least 24 hr (Fig. 2). A persistent and prolonged hypersecretion of ACTH after a single injection of the drug has been observed by other investigators (Wells et al., 1956; Harwood & Mason, 1957; Kitay et al., 1959; Saffran & Vogt, 1960; Maickel et al., 1961 ; Eechaute et al., 1962; Montanari & Stockham, 1962; Bhattacharya & Marks, 1969; Scapagnini et al., 1976), although reports of the exact time for which hypersecretion of the hormone lasts tend to vary. However, these differences may easily be accounted for on the basis of the use of different doses of the drug, different routes of administration and the use of different, indirect, indices of pituitary adrenocorticotrophic function. What is important is that all investigators except one group (Mahfouz & Ezz, 1958) have demonstrated that a single injection of the drug is capable of inducing a prolonged hypersecretion of ACTH. Using adrenal ascorbic acid measurements, Mahfouz & Ezz (1958) found no evidence of increased HPA activity 30 rain after an intramuscular injection

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Fig. 3a. Plasma ACTH concentrations in rats subjected to cold stress after adaptation to reserpine (2.5 mg/kg). Rats were killed immediately after cold exposure. Solid columns-untreated controls: dotted columns, seven daily injections of vehicle: open columns, seven daily injections of reserpine (2.5 mg/kg); C--before stress: T--5 min at 4°C. Each column represents the mean (+ S.EM.) of at least 6 determinations. From Hodges & Vellucci, 1975.

the Mg 2 " - A T P dependent monoamine uptake mechanism (Dahlstr6m et al., 1965). This inhibition of monoaminergic neurone function continues until sufficient quantities of newly-synthesized granules reach the nerve terminals to replace those whose function has been permanently impaired by reserpine. Thus, reserpine causes a marked decrease in the concentrations of 5-HT and catecholamines (noradrenaline and dopamine) in the CNS (Shore et al., 1955; Holzbauer & Vogt, 1956; Brodie et al., 1957). 5-HT and noradrenaline are putative neurotransmitter substances in the CNS pathways

that are involved in the regulation of corticotrophin release (Naumenko, 1968; Scapagnini et al., 1970; Ganong, 1972; Scapagnini et al., 1972; Telegdy & Vermes, 1973; Vernikos-Danelfis et al., 1973; Jones, et al., 1976a). Thus, it is possible that altered brain monoamine metabolism is closely associated with the observed effects of reserpine on the secretion of ACTH. Brodie et al. (1961) tested this hypothesis by studying the effects of a number of different Rauwolfia alkaloids on HPA function. Only those compounds which caused sedation and depleted brain monoamines were found to stimulate HPA activity. Of par-

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Fig. 3b. Plasma ACTH and corticosterone concentrations in rats subjected to cold stress after adaptation to reserpine (2.5 mg/kg). Rats were killed immediately after cold exposure. Solid columns-untreated controls; dotted columns--seven daily injections of vehicle only; open columns--seven daily injections of reserpine (2.5 mg/kg); C--before stress; T--I hr at 4°C. Each column represents the mean (+ S.E.M.) of at least 6 determinations.

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ticular interest was the finding that the pharmacologically inactive stereo-isomcr of reserpine (isorcserpine) did not cause prolonged hypersecretion of ACTH. These investigators also found that low doses of reserpine which failed to elicit sedation or to lower brain amine levels by 500'oalso failed to produce an increase in HPA activity. Westermann et al. (1962) subsequently showed that the release of ACTH produced by the drug was related to the blockade of monoamine storage and that the maximum release of the hormone occurred after a dose of reserpine that completely blocked monoamine storage. Although it is certain that the central actions of reserpine are associated with monoamine depletion it is not clear whether these actions are associated with the depletion of noradrenaline or of 5-HT, or both. Some investigators (Karki & Paasonen. 1959) believe that the central actions of the drug are mediatcd by noradrenaline whereas others (Garattini & Valzelli. 1958: Brodie et al., 1960, Sulser & Brodie, 1960) have put forward strong evidence which indicates that the central actions of the drug are associated with changes in cerebral 5-HT. Indeed, the bulk of available evidence strongly suggests that the central actions of reserpine are mediated by 5-HT. certainly as far as the sedative effects of this drug are concerned (Garattini & Valzelli, 1958; Brodie et al., 1960; Sulser & Brodie. 1960). The problem of whether the reserpine-induced hypersecretion of ACTH is related to changes in brain noradrenaline or 5-HT has also been extensively studied. Use has been made of the amino acid, :t-methyl-metatyrosine (~-MMT). This depletes noradrenaline by inhibiting tyrosine hydroxylase (Hess et al.. 1961 ; Specfor et al., 1965). If rats pretreated with zt-MMT were given reserpine, the same changes in adrenal a~orbic acid and plasma corticosterone concentrations occurred as in animals which had not been pretreated with the amino acid. Costa et al. [1962) found that the central actions of reserpinc, hypersecretion of ACTH and the blockade of 5-HT storage were all related and not dependent on brain noradrenaline concentrations. This suggested that the action of reserpine on the anterior pituitary gland was due to a sustained action on neural pathways in the brain in which 5-HT acted as the transmitter (i.e. tryptaminergic pathways). A more direct contirmation of the fact that the central actions of reserpine are related directly to changes in cerebral 5-HT could be obtained by the use of a compound which selectively depletes 5-HT without affecting catecholamine concentrations. p-Chlorophenyl-alanine (pCPA) was attributed with these properties (Koe & Weissman, 1966) and was thought to selectively deplete 5-HT, by inhibiting its biosynthesis. Sincc the original report of a marked, long-lasting depletion of cerebral 5-HT the drug has been widely used in the study of tryptamincrgic mechanisms. However, more recent cvidcncc has indicated that pCPA appears not to exert a selective effect on 5-HT. After the administration of pCPA a simultaneous decline in the concentrations of brain 5-HT and noradrenaline has been observed (McGeer et al., 1968; Welch & Welch, 1968) and it has been shown that the synthesis of noradrenaline and dopamine is also reduced (Tagliamontc et al., 1973). Despite this, most of the evidence which was available at that time tended to indicate that the hypersecretion of ACTH

induced by reserpine is associated with a sustained action on tryptaminergic pathways, and indeed, the assumption may be supported by the fact that 5-HT is thought to be an excitatory transmitter involved in regulating the release of ACTH (Burden et al.. 1974; Jones et al.. 1976b). in practice the problem of whether or not the hypersecretion of ACTH induced by reserpine can be ascribed to one particular monoamine i's far more complicated. This is partly because both noradrenaline and 5-HT have been shown to play a role in the central mechanisms which regulate the release of ACTH. and also partly because the precise role of each monoamine in the regulation of ACTH release is still the subject of dispute. [In general noradrenaline is believed to be an inhibitory transmitter in the release of ACTH (Ganong. 1972; Scapagnini et al.. 1972). whereas 5-HT is believed to be excitatory (Burden et al.. 1974: Jones et al.. 1976b)]. The discharge of ACTH is controlled by both inhibitory and stimulatory pathways which converge on the corticotrophin releasing factor (CRF)producing neurones in the hypothalamus. The hyper,secretion of ACTH may result from activation of stimulatory pathways or depression of inhibitory pathways. It is more probable that it is the precise ratio of these transmitter substances and their relative rates of turnover, rather than the absolute amount of each that is pre~nt, which is the important factor in determining whcther or not hypersecretion of ACTH occurs after reserpine treatment (McKinney et al., 1971). Unfortunately few, if any, detailed studies involving the correlation of central noradrenaline and 5-HT turnover with CRF and ACTH synthesis and release have been carried out in reserpine treated animals. The interpretation of the effects of reserpine on HPA function is further complicated by the fact that as well as depleting 5-HT and catecholamines, the drug has also been shown to exert effects on other putative neurotransmitter substances. For example, re~rpine has been shown to cause a marked increase in the content of acetylcholine in the hypothalamus (Malhotra & Pundlik, 1959). Acetylcholine is believed to stimulate the release of ACTH (Bradbury et al.. 1974). The drug has also been shown to deplete 7-aminobutyric acid (GABA) in this region (Balzer et al., 1961). This transmitter is believed to inhibit ACTH release (Makara & Stark, 1974). The possibility that the hypersecretion of ACTH induced by acute treatment with reserpine is due to a direct stimulant effect of the drug on the pituitary gland, rather than to its amine-depleting properties is unlikely. Martel et al. (1962), found that pretreatment of rats with a monoamine oxidase inhibitor, not only prevented monoamine depletion but also blocked the sedative effects and ACTH hypersecretion which are normally evident after reserpine administration, thus indicating that the effect of the drug on the pituitary adrenocortical system is an integral part of its pharmacological effect on the CNS. With the use of indirect indices of HPA function it has been shown that the marked pituitary adrenocorticotrophic effect produced by a single injection of the drug is reduced, and ultimately disappears, if the injections are repeated at daily intervals (Wells et al., 1956; Khazan et al., 1961; Scapagnini et at., 1976). Thus, some form of "adaptation" to the drug

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oL Fig. 4. Adrenal ascorbic acid and plasma corlicosterone concentrations in rats adapted to cold and killed I hr after an intrapcritoneal injection of reserpine (2.5 mg/kg) or vehicle, administered immediately after the [inal exposure to cold. Solid columns--untreated controls: dotted columns--cold exposure. 1 hr at 4 (" daily for 9 days; open columns cold exposure. I hr at 4 C daily for 9 days + injection of vehicle immediately after the final cold-exposure: hatched columns---cold exposure, I hr at 4 C dailx for 9 days + injection of rcscrpine (2,5 mg:kg) immediately after the final cold exposure. Each column represents the mean ( _+ S.E.M.) of at least 6 determinations. occurs. The results of Hodges & Vcllucci (1975) and Vellucci (1975) in which direct estimates of circulating ACTH were made, confirmed and extended the results of investigators who used only indirect indices of H P A activity. Furthermore, Hodges & Vellucci (1975) also confirmed the findings of Wells et al. (1956) and Kitay et al. (1959) who used adrenal ascorbic acid depletion and Maickel et al. (1961) who used plasma corticosterone concentrations as the indices of HPA activity, that the stress-induced release of corticotrophin may be inhibited after adaptation to the; drug has occurred. Thus, although exposure to cold stress (4:C) causes hypersecretion of ACTH. the response no longer occurs in rats that have been adapted to reserpine (Fig. 3a and 3b). In contrast in cold-adapted rats a subsequent injection of either reserpine or the vehicle elicits profound activation of the pituitary-adrenocortical system (Vellucci, 1975) (Fig. 4). Some investigators (Eechaute et al., 1962; Montanari & Stockham, 1962) failed to demonstrate the fact that pretreatment with reserpine can ultimately inhibit the normal HPA response to stress. However, this was probably due to their lack of recognition of the need for their experimental animals to become adapted to the effects of reserpine-treatment before the application of stressful stimuli. Thus, some of the divergent reports in the literature may be explained on this basis. O f great importance also is the finding that there is a lack of agreement between some of the data obtained using changes in adrenal ascorbic acid concentrations as the index of A C T H release and data obtained using changes in plasma corticosterone or plasma ACTH concentrations (Hodges & Vellucei, 1975). A dissociation between these indices has been reported previously (Slusher, 1958: Eskin & Mikhailova, 1968) and some of the discrepancies in the literature may possibly be due to the use of different

indices for the assessment of HPA activity. A decrease in the concentration of adrenal ascorbic acid is a reliable index of H P A function only for a single increase in A C T H release which occurs when the gland is "at rest" and contains a normal (high) concentration of ascorbic acid. U n d e r conditions in which there is prolonged release of ACTH, the index becomes unreliable (Vogt, 1965). The use of changes in the concentrations of plasma corticosterone as a measure of /~ 5

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pituitary-adrenocorticotrophic function also has cer- well established (Stark et al., 1963: Jones & Stocktain limitations (Stockham, 1964). Thus, a short stress ham. 1966; Stark et aL. 1968: Hodges & Vcllucci. such as a one minute exposure to ether vapour can 1975). Possible explanations for thc absence of raise the corticosterone concentration in the plasma adrenocortical hyperfunction during and immediately almost to the level seen after the administration of after repeated exposure to stress include the followa supramaximal dose of ACTH. so that a superiming: posed second stress produces no, or only a very small, (1) Prolonged exposure to stress is no longer increment in the plasma concentration of this steroid. accompanied by hypersecrction of A('TH. Therefore, a rise in the concentration of plasma cor(2) Prolonged exposure to stress is accompanied by ticostcrone is a reliable index of the amount of ACTH prolonged hypersecretion of ACTH. but the adrenal released, only if the initial concentration corresponds gland fails to respond with a corresponding increase to a low "'resting" level. The obvious limitations of in corticosterone secretion. the use of indirect indices for the assessment of pitui13) The rate of breakdown of corticostcroids b'. the tary-adrenocorticotrophic activity can be overcome body may be increased. by the use of direct estimates of ACTH, obtained, Direct estimates of circulating A C T H in rats for example, by the use of a bioassay (Chayen et al., adapted to reserpine (Hodges & Vellucci, 1975: Vel1972: Alaghband-Zadch et al.. 1974). Thus, the ability lucci. 1975) rule out the possibility that the inhibition of reserpine to block the release of AC'TH induced of stress-induced ACTH release is duc solely to a liiilby cold stress has been confirmed (Hodges & Vellucci, ure of the adrenal glands to respond to the hormone. 1975; Vellucci. 1975). In addition, Maickel et al. (1961) have reported that, The manner in which a substance may modif? the in rats given repeated injections of reserpine, thc rerelease of corticotrophin from the pituitary gland is sponse of the adrenals to exogenous ACTH remains complicated b~ the "non-specific'" effects evoked by normal. Furthermore. Stark et al. (1963) have shown the administration of the substance under investiga- that prolonged exposure to A C T H considerably intion. Pituitary corticotrophin release is increased by creases adrenal responsiveness both in r i r o and m handling of the animals and by intrapcritoneal injec- vitro. This being so, a reduced ability of the adrenal tions of saline (Barrett & Stockham, 1963). This. cortex to produce glucocorticoids in response to therefore, presents some problems when the effects ACTH cannot be invoked to explain the absencc of of injected substances on HPA function are being adrenal hyperfunction after repeated exposure to assessed. One of the most dilticult and persistent rescrpine.,stress. The third explanation is also untenproblems is to ensure that all animals are in a nonable, as an increase in adrenal corticosteroid output stressed condition before the beginning of an experi- does not cause a rise in the rate of breakdown of ment. Simple procedures such as handling or the subthese steroids [Kraicer & Logothetopoulos. 1963: jection to a strange environment, or to unaccustomed Stark et al., 1965). Thus. it appears that lailure to noise may all result in activation of the HPA system observe an increase in HPA activity after prolonged (Barrett & Stockham. 1963: Ader & Friedman. 1968). exposure to a given stress or after repeated injections It is not always possible to house the animals in a of reserpine is due to the fact that hypersecrction of completely noise-free room. However, if all the aniA C T H no longer occurs. mals, including the controls are subiectcd to the same Although it is evident that reserpine can. under cerenvironment for some days prior to their use in an tain circumstances, inhibit the normal pituitaryexperiment they will be affected in the same way. It adrenocorticotrophic response to an additional stressis. however, necessary to reduce to a minimum the ful stimulus, the exact mechanism whereby this occurs effects of any "'non-specific" stresses occurring during is still uncertain and has been the subject of dispute. the experimental procedures. The most effective way The absence of normal pituitary adrenocorticotrophic of reducing the "non-specific'" effects of injections is activation may be due to the fact that the drug has to handle the animals frequently for some days prior in some way altered or delayed the time-course o1 to the commencement of treatment and to use a carethe stress response. This idea was prompted by obserful injection technique (Rerup, 1961; Hodges & vations put forward by Brodish (1964, 1969) who Mitchley, 1970), rather than by using complicated showed that, although rats bearing hypothalamic "'training" procedures involving courses of either lesions did not release A C T H soon after stress, they saline injections or needle insertions, as some other did show a delayed hypersecretion of ACTH, which workers have suggested (Barrett & Stockham, 1965; elevated the concentration of plasma corticosterone Gosbee & Kraicer, 1969). Of grcat importance also, to levels which were comparable to those observed when the manner in which injcctcd substances may in intact animals subjected to the same stress. Thus, modify the release of corticotrophin from the pitui- in view of the fact that the drug is known to exert tary gland is being studied, is the use of proper con- effects on the hypothalamus, studies were made of trols, particularly as it has been shown that the reser- the time-course of the pituitary-adrenocorticotrophic pine vehicle (e.g. 1';,, glacial acetic acid in deionized response after " ' a d a p t a t i o n " to reserpine (Vellucci, water, adjusted to pH 4 with 4 M N a O H ) is capable 1975: Hodges & Vellucci, 1975). It was shown that of producing important changes in HPA function even up to 40 min after the linal injection of the alkawhich differ quantitatively from those produced by loid there was no evidence of increased HPA activity the injection of ~ l i n e or deionized water alone (Fig. 5). (Hodges & Vellucci, 1974: Vellucci, 1975). Another possible explanation for the lack of a stress The fact that repeated exposure to a given stressful response "after " ' a d a p t a t i o n " to reserpine was put forstimulus can decrea~ the pituitary-adrenocorticotroward by Kitay et al. (1959) who demonstrated that phic response to a subsequent stressful stimulus is a single injection of reserpine caused hypcrsecretion

Reserpine on hypothalamo-pituitary adrenocortical function of ACTH with concomitant depletion of the pituitary stores of the hormone. Repeated injections of the drug produced a sustained hypersecretion of ACTH associated with a decrease in the pituitary ACTH stores ranging from 36 to 73~ of the normal amount. As a result of this observation Kitay et al. (1959) suggested that the rapid release of ACTH in response to an additional stressful stimulus (e.g. ether) will be diminished or absent, depending upon the degree of pituitary ACTH depletion existing prior to stress. They did not believe that the drug exerted its inhibitory effect at the hypothalamic level. Westermann et al. (1961) carried out a similar investigation and came to the same conclusion. In addition, Saffran & Vogt (1960) demonstrated that a single intraperitoneal injection of the drug in the rat produced a fall in the pituitary ACTH content to 309~o of the normal amount. At the time, the explanation put forward by Kitay et al. (19591 seemed adequate to account for the observation that reserpine initially stimulated the release of ACTH and then appeared to inhibit it. However, more recent data (Hodges & Vellucci, 1975; Vellucci, 1975) indicated that the inhibition of ACTH release in reserpine-adapt.ed rats following cold stress occurs at a time when the pituitary stores of the hormone are not significantly different from those of the corresponding vehicle-treated controls and that a single intraperitoneal injection of reserpine does not cause severe depletion of pituitary ACTH stores. Several other objections can also be raised against the plausible hypothesis put forward by Kitay et al. (1959). Firstly the amount of ACTH remaining in the pituitary gland after chronic reserpine-treatment is far in excess of that required to produce the type of pituitary-adrenocorticotrophic activation that is normally observed after acute stress. Van Peenen & Way (19571 showed that 12-15 hr after a single large dose of reserpine, at a time when considerable pituitary ACTH depletion must have undoubtedly occurred, the ACTH response to the stress of histamine or aspirin injections appeared to be unimpaired, although the response to adrenaline and morphine was considerably reduced. It is also difficult to explain the reason for the adrenal enlargement reported to occur after continued reserpine administration (reviewed by Munson, 1963) if the stress-induced secretion of ACTH is seriously impaired by reduction of the pituitary stores of the hormone. Certainly a cause-andeffect relationship between pituitary ACTH depletion and a failure to respond to stress has not been established and there are numerous reports which indicate that a decrease in the pituitary ACTH content cannot be directly correlated with a failure to respond to an additional stressful stimulus. As early as 1949, Sayers & Cheng (1949) demonstrated that a stressful stimulus, such as adrenalectomy, can reduce the ACTH content of the rat pituitary gland by 80% without there being any evidence of a refractoriness of ACTH release in response to subsequent stress. Buckingham (1974) found that the adrenocorticotrophic overactivity observed in adrenalectomized rats occurs at a time when the pituitary A C T H content is normal, thus suggesting that the absolute amount of ACTH in the gland is not necessarily a reflection of its capacity to release ACTH. This confirmed the earlier findings of Hodges & Jones (1964) who showed that

281

stress-induced adrenocorticotrophic overactivity developed within 8 hr of adrenalectomy, at a time when the pituitary stores of the hormone were falling rapidly. Results obtained by Vernikos-Danellis (1963) also indicated that the absolute amount of ACTH stored in the pituitary gland does not play a significant role in the ability of the gland to respond to stress. Indeed, this author found that the release of ACTH in response to stress was accompanied by a significant and very rapid increase in pituitary ACTH stores, irrespective of whether they were low (as seen 4 hr after sham adrenalectomy) or high (as seen 30 days after adrenalectomyl. It therefore seems that the stress-induced release of ACTH is not dependent on the size of the pre-existing stores of the hormone in the pituitary, but on the ability of the gland to synthesize ACTH (Marks & Vernikos-Danellis, 1963), unlike the basal or resting level of the hormone which does appear to be dependent on the pituitary ACTH content. The basal ACTH output does not, however, appear to be affected by treatment with reserpine (Hodges & Vellucci, 1975; Vellucci, 1975). The inability of animals "adpated" to reserpine to release ACTH in response to an additional stressful stimulus cannot, therefore, be ascribed to lack of pituitary stores of the hormone, but rather to inhibition of rapid synthesis of the hormone, probably due to a decrease in the output of corticotrophin releasing factor (CRF) from the hypothalamus. There is no evidence in the literature to support the idea that reserpine exerts its effect by directly inhibiting the synthesis of ACTH. It is important to consider the possibility that the observed suppression of ACTH release may be due to a "feedback" effect exerted by the high concentrations of corticosterone previously evoked by the drug (Giuliani et al., 1966). Large doses of exogenous corticosteroids are known to inhibit the stressinduced release of ACTH and do so more effectively 18-24 hr after administration, than in the first few hours (Barrett, 1961). Other investigators (Hodges & Jones, 1963; Smelik, 1963a and b) have shown that if a stressful stimulus is applied at a time when the plasma concentration of an exogenously-administered steroid is high there will be no inhibition of stressinduced ACTH secretion. However, if the stressful stimulus is applied when the corticosteroid level is rapidly falling or has already fallen, then inhibition will occur (Stark & Fachet, 1963). Although it has been demonstrated that the inhibition of stressinduced ACTH secretion by naturally-occurring or synthetic steroids lasts for many hours after the blocking agent has disappeared from the circulation, this explanation seems unlikely as it has been shown by direct estimates of plasma ACI'H that inhibition of the stress response occurred at a time' when the plasma corticosterone concentration was normal and had been so for more than 24 hr (Hodges & Vellucci, 1975). It is considered unlikely that any feedback effect would persist for this length of time; however, definite proof that a feedback effect cannot be invoked in order to explain the observed results can only be obtained by carrying out further experiments in which plasma ACTH levels are measured in adrenalectomized, reserpine-treated animals. Taking into account all the available evidence, the

2S2

SANI)RA V. VI LI.U('CI

indication is that the inhibitory effects of reserpine 19671. Hence the increased tyrosine hydroxylase acon stress-induced HPA activation arc exerted at a tivity in the brain stem which was noted by Scapaglevel in the CNS higher than the pituitary gland. It nini et al. (1976) could be responsible for the restois well-known that the drug depletes brain' noradrenaration of tonic noradrenergic inhibition of line and 5-HT, particularly in thc hypothalamus. C R F - A C T H secretion at the hypothalamic level, These monoamincs are putativc neurotransmittcrs in- where most of the corresponding noradrenergic nerve volved in rcgulating the s',nthesis and release of CRF. endings arc located (Anden et al.. 19661. Thus, ScaConscqucntl,, the drug may exert a neural inhibitory pagnini et al. (19761 considered that the disappearance effect on C R F and ultimatcl', A C T H release b 3 alter- of adrenocortical activation following long-term ing the availability of these monoamines in the brain. treatment with reserpine is due to the stimulated forBhattacharya & Marks (19691 have shown that reser- mation of a small functional pool of noradrenaline pine is capable of greatly reducing the C R F activity which is then availablc for the inhibition of of thc h,,pothalamus. The effect may bc mediated C R F - A C T H secretion. In order to define more preeither by an alteration in neurotransmitter function cisely the effects of reserpine on HPA activity these or by a direct action on the CRI- storage mechanism, studies need to be extended further to include direct particularly as it is known that the drug is capable estimates of HPA activity (e.g. estimates of plasma of blocking the intraneuronal storage of monoamines. and pituitary ACTH concentrations and of hypothalaHowever. the first possibility seems far more likely, mic C R F activity) coupled with the investigation of the action of reserpine is relatively slow to develop central monoamine metabolism and synthetic enzyme and the pattern of neurotransmittcr depiction induced activity, during and after adaptation to reserpine and by the drug is parallclcd closcly by the time-course following the application of stressful stimuli to reserof the decrease in C R F activity. This idea has been pine-adapted animals. Thus. despitc numerous studies challcngcd by Smclik 119671 who found that hypothathe exact mechanism whereby reserpine exerts its lamic implants of reserpine did not block the response effects on HPA activity has not yet been fully elucito various stressful stimuli. However, the evidence for dated and clearly there is still scope for further investhe amine-depicting action of the drug put forward tigation. by Smelik (19671 was ba,~d solcl,, on fluorescence histochemical methods and it is unlikeb that the quantiREFERENCES tative significance of this method is adequate to define Ao~-:r R. & Fm~t)MAN S. B. (19681 Plasma corticosterone precisely the degree of monoamine depletion caused response to environmental stimulation: effects of by the drug. The most consistent iheory to emerge duration of stimulation and the 24 hr adrenocortical appears to be that reserpine exerts its effect on HPA rhythm. Neuroendocrinoloqy. 3. 378-386. activity b~ affecting monoamines which act as putaALAGHBAND'-ZADEHJ., DALY J. R.. BITENSKYL. & CHAYEN tive neurotransmitters in the regulation of the synJ. (1974)The cytochemical section assay for corticotrothesis and release of CRF. This action may take place phin. Clin. endocr. 3. 319 327. at the hypothalamus or at higher sites in the ( ' N S AyJoE:-: N. E.. DAm.SrROM A.. Ft:xt- K.. OL~Y L. & U~(Lengv~iri & Halzisz, 19721 which communicate neurGERSrEI)I U. (19661 Ascending noradrenaline neurons from the pans and the medulla oblongata. Experientia, ally with the CRF-containing neurones of the hypoth22, 44-45. alamic median eminence. Another possible explanation for the fact that reser- BALZER H. HOLTZ P. & PALM D. (19611 Reserpine and 7-aminobutyric acid content of brain. Experientia, 17. pine initially stimulates HPA activity and then in38-40. hibits it has been put forward by Scapagnini et al. BARRETTA. U. (19611 The effect of cortisone and hydro(1976). These authors obtained evidence that the cortisone on the plasma levels of corticotrophin in the stimulation of the HPA axis after reserpine treatment. rat, after an acute stress. J. Pharm. Pharmac. 13, 20-25. as evidenced by changes in the levels of plasma cor- BARRETT A. M. & STOCKHAMM. A. (1963) The effect of ticosterone, may be due to the removal of a central housing conditions and simple experimental procedures noradrenergic tonus in the hypothalamus that toniupon the corticosterone level in the plasma of rats. J. cally inhibits C R F - A C T H secretion. Prolonged treatendocr. 26, 97-105. ment with reserpine has been shown to cause a pro- BARRETT A. M. & SIOCKH^M M. A. (1965) The response of the pituitary-adrenal system to a stressful stimulus: gressive increase in central tyrosine hydroxylasc acthe effect of conditioning and pentobarbitone treatment. tivity (Segal et al.. 1971)especially in the brain stem J. endocr. 33. 145 152. (Scapagnini et al., 19761. This increased tyrosine hyBHAI'rACItARVAA. N. & MARKS B. H. (1969) Reserpinedroxylase activity is thought to be due to the synand chlorpromazine-induced changes in hypothalamothesis of new enzyme (Mueller et al.. 1969; Segal et hypophyseal-adrenal system in rats in the presence and al., 1971) and could perhaps be responsible for de absence of hypothermia. J. Pharmac. exp. Ther. 165, noco formation of a small functional pool of norad108 116. BRADBURYM. W. B., BURDt!~ J.. HILLHOUS[E. W. & JONI!S renaline which is directly involved in the mediation M. T. (19741 Stimulation electrically and by acetylchoof neuronal activity (Glowinski, 19701. This small line of the rat hypothalamus in vitro. J. Physiol. 239. functional pool of noradrenaline could restore the 269 -283. tonic noradrenergic inhibitory effect on C R F release which was lost after the initial massive reserpine- BRODIL B. B. & SHORI: P. A. 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