INFLUENCE OF RESERPINE ON THE PITUITARY CONTENT OF MELANOCYTE-STIMULATING HORMONE AND ON HYPOTHALAMIC FACTORS WHICH AFFECT ITS RELEASE

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1 INFLUENCE OF RESERPINE ON THE PITUITARY CONTENT OF MELANOCYTE-STIMULATING HORMONE AND ON HYPOTHALAMIC FACTORS WHICH AFFECT ITS RELEASE MARIA E. TOMATIS and S. TALEISNIK Instituto de Investigación Médica Mercedes y Martín Ferreyra* Casilla de Correo 389, Córdoba, Argentina (Received 18 March 1968) SUMMARY The melanocyte-stimulating hormone (MSH) content of whole toad pituitary glands decreased upon treatment with reserpine. With daily injections the values remained low for 1 week but regained normal levels after 2 weeks in spite of an increased secretion of MSH as indicated by darkening of the skin. In rats a drop in pituitary MSH content also occurred after reserpine injection but normal values were found after 7 and 14 days of treatment. MSH-releasing factor found in stalk-median eminence tissue of normal male rats was not present in the reserpine-injected animals, but after 7 days of treatment an increase in MSH-release-inhibiting factor (MSH-R-IF) was demonstrated. MSH-R-IF was also found to have increased in female castrated rats after 2 days of treatment with reserpine. It is concluded that reserpine permits the secretion of pituitary MSH by blocking the release of MSH-R-IF, which accumulates in the hypothalamic neurones. INTRODUCTION It is well established that in amphibians the central nervous system exerts an inhibitory control on the release of melanocyte-stimulating hormone (MSH). Inter ruption of the connexions between the hypothalamus and the pituitary by trans plantation of the gland (Etkin, 1941, 1943), section of the pituitary stalk (Etkin, 1962) or lesion of the hypothalamus (Stoppani, 1941; Voitkevich, 1962; Kastin & Ross, 1965) results in a darkening of the skin because of the increased release of MSH. The concept that this inhibitory control is exerted through a neurosecretory sub stance has received new support by the demonstration in toad hypothalamic extracts of an agent which inhibits the release of MSH (Taleisnik & Tomatis, 1967 a). Reserpine has been shown to be a valuable tool in studying the hypothalamohypophysial relationship of several pituitary hormones. This drug exerts a depressing * Supported by Grant III from the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina.

2 action on the hypothalamus and has been shown to cause darkening of the skin in frogs and toads (Khazan & Sulman, 1961; Enemar & Falck, 1965; Iturriza, 1966). In the present paper the effect of reserpine on the MSH content of the pituitary and and on the activity of the hypothalamic neurosecretory principles which control the release of MSH in rats and toads has been studied. MATERIALS AND METHODS Male albino rats ( g.) and toads (Bufo arenarum Hensel) ( g.) were used. The animals were injected subcutaneously with reserpine at a dose level of 0-2 mg./kg. body weight/day unless otherwise stated. The injections were repeated every 24 hr. from 1 up to 14 days and the animals were killed 24 hr. after the last injection, except for the groups of rats killed at 6 hr. The rats were killed with ether, which does not affect the pituitary content of MSH, and the toads by decapitation after the spinal cord had been destroyed with a stylet. The hypophyses were im mediately removed, weighed, suspended in an appropriate volume of distilled water and kept frozen at 20 until MSH was assayed. Each group consisted of a pool of two pituitaries of rats or of three toad pituitaries. MSH activity in the pooled pituitaries was measured by an in-vitro assay method (Taleisnik & Orias, 1965). The activity of MSH in each group was compared with that of a control group (two pituitaries from normal animals killed at the same time as the experimental groups were pooled). Control and experimental pituitaries were assayed for MSH at two dose levels and the activity of MSH was expressed as % of the control groups per mg. gland in the case of rats and per gland in the case of toads. Potency and confidence limits were calculated according to accepted statistical methods (Bliss, 1952). The results of duplicate assays were combined by the procedure of Sheps & Moore (1960). Crude acidic extracts were prepared with 0-1 N-HC1 from stalk-median eminence (SME) or posterior lobe tissue of rats killed with ether as previously described (Taleisnik & Orias, 1965). The tissue from two to four animals was pooled for each extract. The extracts were injected intravenously into male rats weighing g., under ether anaesthesia, and the animals killed 20 min. later. Each extract was in jected into two animals, whose hypophyses were then pooled, suspended in a measured volume of distilled water and kept frozen until MSH activity was assayed. The de crease in MSH activity in the pituitary induced by the injection of the extracts was taken as an index of MSH-releasing factor (MSH-RF) activity. MSH-release-inhibiting factor was measured by the capacity of extracts to block the depletion of pituitary MSH induced by SME extracts from normal male rats. The controls of the different groups were pools of two pituitaries from animals injected with a volume of solvent equal to that of the hypothalamic extracts. RESULTS Effect of reserpine on pituitary MSH concentration The results of assays of pituitary MSH activity from rats or toads injected with reserpine are summarized in Fig. 1 and are expressed as a percentage of the level in control non-injected animals, taken as 100%.

3 In both species reserpine induced a drop in pituitary MSH. In the rat, 6 hr. after the injection of reserpine, a small but significant decrease in pituitary MSH con centration was observed. The values were still lower at 24 hr. and reached the lowest level on the 2nd day. There was a small recovery after 4 days of daily reserpine injec tion and the values returned to normal in the animals injected for 1 or 2 weeks. In toads there was also a drop in the pituitary MSH content of up to 50 % of the normal values. This low level was still evident in the animals injected for 7 days but Fig. 1. Pituitary melanocyte-stimulating hormone (MSH) activity of rats or toads injected daily with 0-2 mg./kg. reserpine. Each point is the MSH potency of two pooled rat pituitaries (O-O) or three toad pituitaries ( - ). The values are expressed as % of MSH potency of pituitaries from control animals per mg. gland of rats and per gland of toads. The horizontal line is the weighted mean of each group and the vertical line indicates the 95 % limits. a normal hormone content was found in toads treated for 14 days. All injected toads showed intense darkening of the skin. No statistically significant difference was found between the weights of the pituitary glands of the control and the reserpine-injected animals. Effect of reserpine on MSH-BF content in rat stalk-median eminence or posterior lobe extracts The results obtained with rat SME extracts from normal or reserpine-injected animals are shown in Table 1. As shown repeatedly extracts of SME from normal rats induced a significant depletion in pituitary MSH concentration. In contrast, SME extracts from rats injected with 0-2 mg./kg. reserpine daily for 7 days did not modify the pituitary MSH content. The duplicate assays of three extracts from three different groups of animals gave reproducible values. The mean pituitary MSH potency of the

4 _ three assays was ± 8-4 (s.e.) % while that of the controls was %. The difference between both groups was highly significant (P < 0-01). The MSH-RF content of extracts of posterior lobes taken from reserpine-injected animals was also studied. Table 1 shows that extracts of the neural lobes of control animals induced a drop in pituitary MSH in each of three assays. On the other hand, extracts prepared from animals injected with reserpine had an activity similar to that found in the normal animals on two out of three occasions. Each extract from the reserpine-treated animals was injected into two groups of rats and gave reproducible values. Table 1. Effect of reserpine (0-2 mg./kg. for 7 days) on MSH-releasing activity of stalk-median eminence or posterior lobe extracts MSH activity in pituitary (% of controls)*,-a-.-, Intact rats Reserpine-injected rats Expt,-*-«no. Mean of group Individual assays P Stalk-median eminence extracts ( )t 95-7 ( ) { ** ^ttjlltl} < ' ( ) 116-7( ) {"JJ JJÍÍÍm} NS ( ) 88-5 ( ) j ^-5 ( ) < 0.Q1 Over-all mean 62-9 ± _ + S.E. Posterior lobe extracts < ( ) 57-5 ( ) / 49'7 ( ) vrg ' \ 60-2 ( / ( ) ' 102-8( ) V ' f100'0 ( ) < ( / ( ) 48-4 ( ) Í 45' v ' (25,4~57-9l Ng \ 49-8 ( / Over-all mean 58-9± S.E. NS * Each animal was injected with an amount of extract containing an equivalent of one SME or one posterior lobe. f Estimated potency (95% confidence limits). NS = not significant. Effect of reserpine on MSH-R-IF activity in stalk-median eminence extracts Since the SME extracts of reserpine-injected animals did not depress the pituitary MSH content of the recipient animals as occurred with the extracts from control animals, the possibility was studied that the SME extract from reserpinized animals contained an MSH-R-IF. Consequently the ability of such extracts to inhibit the drop of pituitary MSH induced by SME extracts from normal male rats was determined. Table 2 shows that when SME extracts of reserpine-injected animals were added to SME extracts from normal rats the decrease in pituitary MSH was blocked. While the difference between the single extract and the effect seen after simultaneous treat ment with an extract of animals injected with 0-2 mg./kg. reserpine for 7 days was

5 highly significant (P < 0-01), the blocking effect of extracts from animals injected for 2 days did not attain significance (P > 0-05). The MSH-R-IF activity in the SME of rats injected with 0-2 mg./kg. reserpine Table 2. Content of MSH-release-inhibiting factor in extracts of stalk-median eminence (SME) of rats injected with reserpine Treatment SME extract of normal rats (A) A plus extract of one SME of rats injected with reserpine (1 mg./kg. for 2 days) Expt no. SME extract of normal rats (A) A plus extract of one SME of rats injected with reserpine (0-2 mg./kg. for 7 days) * Referred to that per mg. pituitary t Compared with rats injected MSH activity in pituitary (%)* 95% limits / of controls. with SME extract of normal rats. Mean MSH activity 95% limits Pi NS < 0-01 NS = not significant. A. Male -Reserpine, 2 days Reserpine, 7 days B. Spayed female o-control - Reserpine, 2 days.--"i Antilog. M = 1-48 P = N.S. n = 12 I Antilog. M = 309 P<001 n = L. 0-6 Log dose of SME Fig. 2. In A the melanocyte-stimulating hormone-release-inhibiting factor (MSH-R-IF) potency of stalk-median eminence (SME) extracts from male rats treated for 2 and 7 days with reserpine is compared. In B the MSH-R-IF potency of an extract of the SME from spayed females is compared with that of spayed females injected with reserpine. Each point is the mean melanocyte index of an aliquot from two pooled pituitaries from rats injected with a dose of SME extract of normal males to which two different doses of the SME from groups A and B were added. The vertical lines are the standard errors of the mean, n is the number of pieces of toad skin used for each point.

6 daily for 2 and 7 days was compared in one experiment. Extracts from treated rats were added at two dose levels (equivalent to 0-2 and 0-6 SME) to an extract of SME from normal animals and the combined extracts injected into pairs of recipient animals. The pooled pituitaries of each group were assayed for MSH activity at one dose level and the mean melanocyte index was determined in 12 pieces of skin at each point. The mean response is shown in Fig. 2. The activity in the SME of animals injected for 7 days was 148 % ofthat of animals injected for 2 days but the difference was not significant. Since detectable levels of MSH-R-IF are present in SME extracts from ovariectomized female rats, it was interesting to know whether these levels could be increased by reserpine. Therefore the activity of extracts of animals injected with 0-2 mg./kg. reserpine for 2 days was compared with that of non-treated animals. Figure 2 shows that in one experiment, in which equivalents of 0-2 and 0-8 of SME were added to a dose of SME from normal males, 3-09 times more inhibitory activity was found in the reserpine-injected group. This increase in activity was statistically significant (P < 0-01). DISCUSSION Reserpine has been shown to cause darkening of the skin when injected into frogs or toads, a result confirmed in the present study. Since this effect of reserpine is lacking in hypophysectomized animals (Iturriza, 1966) it is assumed that reserpine promotes MSH release. This increased release is accompanied by a decrease in the pituitary content of the hormone, as the results presented indicate. The fact that MSH in the pituitary also decreased in rats after the injection of reserpine leads to the assumption that a similar mechanism for the release of MSH may be operative in mammals. No direct evidence is as yet available to account for the fact that the pituitary MSH content returns to normal despite the sustained administration of reserpine. Since an intense darkening of the skin was evident in toads after 14 days' treatment with reserpine and the pituitary MSH content was normal, there was presumably an increase in MSH synthesis to compensate for the increased release. In rats a drop gland MSH in content on the first day after disconnexion of the pituitary gland from the central nervous system by lesions in the median eminence was followed 14 days later by almost normal values (Taleisnik, de Olmos, Orias & Tomatis, 1967). This suggests that the MSH content on the hypophysis is to some degree regulated autonomously. Since reserpine appears to depress the hypothalamus (Gaunt, Chart & Renzi, 1963), the increased release of MSH after reserpine administration implies that the secre tion of MSH is held in check by the central nervous system. This effect of reserpine is similar to that observed after disconnexion of the pituitary by section of the stalk, transplantation of the gland or lesions of the hypothalamus. Tranquillizing drugs also cause the release of MSH, as shown by skin darkening in frogs (Scott, 1959; Scott & Nading, 1961) and by the decrease in the MSH content of the rat pituitary (Kastin Schally, & 1966). An inhibitory factor for the release of MSH has been demonstrated in hypothalamic extracts of toads (Taleisnik & Tomatis, 1967 a) and rats (Taleisnik & Tomatis, 19676)

7 and may be the agent that physiologically restrains the release of MSH. The experi ments reported in the present work show that MSH-R-IF activity not detectable in normal male SME was present after reserpine administration. This change in activity could be assigned to an increase in the amount of this principle, a fact which is supported by the unequivocal increase in ovariectomized rats after reserpine injection. It can be postulated that under physiological conditions a certain amount of IF is tonically released, thereby inhibiting MSH from the pituitary. Reserpine permits MSH secretion by blocking the release of the inhibitory agent which accumu lates in the hypothalamic neurones. Since reserpine affects the content of norepinephrine and 5-hydroxytryptamine in nerve endings (Shore, 1962), it might be assumed that monoamines mediate the nervous influence which maintains a tonic secretion of MSH-R-IF under normal conditions. It should be noted that the hypothalamus also chronically inhibits the release of prolactin. The injection of reserpine induces pseudopregnancy in rats (Barraclough & Sawyer, 1959) and a lactogenic effect has been reported in rabbits (Meites, 1957) and rats (Benson, 1958). Furthermore, after reserpine treatment a drop in pituitary prolactin content in rats (Moon & Turner, 1959) and in ovariectomized, oestrogenprimed rabbits (Kanematsu, Hilliard & Sawyer, 1963) has been described. These facts indicate that a similar mechanism may be involved in the control of secretion of MSH and prolactin. However Ratner, Talwalker & Meites (1965) found that reserpine depleted the rat hypothalamus of PIF when administered in vivo, whereas the results of the present study show that, by contrast, reserpine injection caused an increase of MSH-R-IF content in the hypothalamus by blocking its release. This work was supported by Grant NB from the U.S. Public Health Service. REFERENCES Barraclough, C. A. & Sawyer, C. H. (1959). Induction of pseudopregnancy in the rat by reserpine and chlorpromazine. Endocrinology 65, Benson, G. K. (1958). Effect of reserpine on mammary gland involution, and on other organs in the rat. Proc. Soc. exp. Biol. Med. 99, Bliss, C. I. (1952). The statistics of biossay. New York: Academic Press. Enemar, A. & Falck, B. (1965). On the presence of adrenergic nerves in the pars intermedia of the frog, Rana temporaria. Gen. comp. Endocr. 5, Etkin, W. (1941). On the control of growth and activity of the pars intermedia of the pituitary by the hypothalamus in the tadpole. J. exp. Zool. 86, Etkin, W. (1943). The developmental control of pars intermedia by brain. J. exp. Zool. 92, Etkin, W. (1962). Hypothalamic inhibition of pars intermedia activity in the frog. Gen. comp. Endocr. Suppl. 1, Gaunt, R., Chart, J. J. & Renzi, A. A. (1963). Interactions of drugs with endocrines. A. Rev. Pharmac. 3, Iturriza, F. C. (1966). Monoamines and control of the pars intermedia of the toad pituitary. Gen. comp. Endocr. 6, Kanematsu, S., Hilliard, J. & Sawyer, C. H. (1963). Effect of reserpine on pituitary prolactin content and its hypothalamic site of action in the rabbit. Ada endocr., Copenh. 44, Kastin, A. J. & Ross, G. T. (1965). Melanocyte-stimulating hormone activity in pituitaries of frogs with hypothalamic lesions. Endocrinology 77, Kastin, A. J. & Schally, A. V. (1966). MSH activity in pituitary glands of rats treated with tranquilizing drugs. Endocrinology 79, Khazan, N. & Sulman, F. G. (1961). Melanophore-dispersing activity of reserpine in Rana frogs. Proc. Soc. exp. Biol. Med. 107, Meites, J. (1957). Induction of lactation in rabbits with reserpine. Proc. Soc. exp. Biol. Med. 96,

8 Moon, R. C. & Turner, C. W. (1959). Effect of reserpine on oxytocin and lactogen discharge in lactating rats. Proc. Soc. exp. Biol. Med. 101, Ratner, A., Talwalker, P. K. & Meites, J. (1965). Effect of reserpine on prolactin-inhibiting activity of rat hypothalamus. Endocrinology 77, Scott, G. T. (1959). Melanophore dispersing action of ataraxic drugs. Biol. Bull. mar. biol. Lab., Woods Hole 117, 400. Scott, G. T. & Nading, L. K. (1961). Relative effectiveness of phenothiazine tranquilizing drugs causing release of MSH. Proc. Soc. exp. Biol. Med. 106, Sheps, M. C. & Moore, E. A. (1960). Methods for combining the results of two biological assays. J. Pharmac exp. Ther. 128, Shore, P. A. (1962). Release of serotonin and catecholamines by drugs. Pharmac. Rev. 14, Stoppani, A. O. M. (1941). Estudios fisiológicos y farmacológicos sobre los melanóforos de los batracios. Buenos Aires: El Ateneo. Taleisnik, S., de Olmos, J., Orias, R. & Tomatis, M. E. (1967). Effect of hypothalamic lesions on pituitary melanocyte-stimulating hormone. J. Endocr. 39, Taleisnik, S. & Orias, R. (1965). A melanocyte-stimulating hormone-releasing factor in hypothalamic extracts. Am. J. Physiol. 208, Taleisnik, S. & Tomatis, M. E. (1967a). Antagonistic effect on melanocyte-stimulating hormone release of two neural tissue extracts. Am. J. Physiol. 212, Taleisnik, S. & Tomatis, M. E. (19676). Melanocyte-stimulating hormone-releasing and inhibiting factors in two hypothalamic extracts. Endocrinology 81, Voitkevich, A. A. (1962). Neurosecretory control of the amphibian metamorphosis. Gen. comp. Endocr. Suppl. 1,