422

Brain Research, 166 (1979) 422-425 © Elsevier/North-HollandBiomedicalPress

Posterior pituitary adenylate cyclase: stimulation by dopamine and other agents

HO SAM AHN*, SUSAN C. FELDMAN** and MAYNARD H. MAKMAN Departments of Biochemistry and Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, 10461 (U.S.A.)

(Accepted December 28th, 1978)

The posterior lobe of the pituitary gland contains dopaminergic and noradrenergic terminalsS,6, t4 as well as moderate levels of dopamine (DA) and norepinephrine is. In addition, the presence of receptors for DA in sheep posterior pituitary was indicated by binding studies employing [3H]-dihydroergocriptineS although negligible specific binding of another ligand, [3H]spiroperidol, was reported in a study of calf posterior pituitaryL A number of studies have suggested that monoamines and their receptors may be involved in the control of secretion of neurohypophysial hormones such as antidiuretic hormones (ADH) and oxytocin. Thus, a variety of agents including norepinephrine, epinephrine, reserpine, morphine, lithium chloride and antipsychotic drugs (e.g., chlorpromazine, perphenazine) which exert direct or indirect effect on monoaminergic transmission4 influence ADH and oxytocin releasO1,12,15. Further, DA levels and release in the arcuate-median eminence DA cell group whose axons terminate in the posterior pituitary, are known to change following experimental manipulations (e.g., dehydration, suckling) affecting secretion of ADH and oxytocin~0,20. The levels of cyclic AMP in the posterior lobe are markedly elevated following acute treatment with reserpine, an amine depleting agent ~a, suggesting the possible presence of amine receptors linked to adenylate cyclase in the posterior pituitary. Also under conditions which increase ADH secretion in vivo, cyclic AMP levels in posterior pituitary are elevated 17. We report here the ability of DA, DA agonists and other agents to stimulate adenylate cyclase activity and a partial pharmacologic characterization of DA-stimulated adenylate cyclase in homogenates of rat posterior pituitary. Young adult male Sprague-Dawley rats weighing 200-300 g were sacrificed by decapitation and the posterior lobes of the pituitary glands rapidly dissected. The procedure used did not include separation of the intermediate lobe, although this tissue would represent only a minor component based on its relative tissue mass. The * Present address: Schering Corporation,Bloomfield,New Jersey, 07003, U.S.A. ** Present address: Department of Anatomy, Columbia University, College of Physicians and Surgeons, New York, N.Y. 10032, U.S.A.

423 posterior pituitary tissue was preincubated in physiological saline at 5 °C for 2-3 h before homogenization in 60 vol (wet wt/vol) of Tris.Maleate buffer (pH 7.4) containing 0.8 mM EGTA. For adenylate cyclase assay 1 20-40 #g homogenate protein per tube was incubated with the reaction mixture at 30 °C for 2.5 min. Each experiment involved at least triplicate adenylate cyclase incubations for every condition studied with single or replicate determinations of cyclic AMP. A paired Student's t-test was used to determine the significance of difference in mean values between basal and agent-stimulated activities. Studies were carried out to investigate the possible influence of catecholamines and other agents on adenylate cyclase activity of rat posterior pituitary. In preliminary studies we found negligible stimulation of enzyme activity by DA unless we first preincubated the posterior pituitary in physiological saline at 5 °C. Subsequent studies employed 2-3 h of preincubation of tissue before homogenization and adenylate cyclase assay. Also in these studies we found that cyclic AMP levels in rat posterior pituitary immediately after decapitation were approximately 100 pmol/mg protein, a value about 7 times that found by Guidotti et al. 13 after sacrifice by microwave irradiation. Furthermore, following decapitation there was in rat posterior pituitary a high basal adenylate cyclase activity which subsequently declined during the preincubation. The failure of the whole pituitary adenylate cyclase to respond to DA in an earlier study 19 may have been at least in part due to the initial activation of basal adenylate cyclase activity after sacrifice of the animal. It should also be noted that exposure of the posterior pituitary to cold in vitro has been shown to release vasopressin 9. Thus, it is also possible that more complex physiological changes during the preincubation used in the present study may have been responsible for revealing DA-stimulated adenylate cyclase activity. 200 -

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Fig. 1. Activation ofadenylate cyclase by dopamine and its agonists and effectsof blocking agents on the dopamine stimulated activity in rat posterior pituitary homogenates. Basal adenylate cyclase activity was 64 ± 4 pmol cyclic AMP formed/rag protein/2.5 rain (an average of 6 incubations). Unless otherwiseindicated stimulations werestatisticallysignificantat least atp < 0.05. NS, statistically not significantly different from the basal enzyme activity. The number of incubations is indicated in the columns. Abbreviations: DA, dopamine; PL, propranolol; PA, phentolamine; HAL, haloperidol; APO, apomorphine.

424 TABLE I Effect of catecholamines and Gpp ( NH)p on adenylate cyclase activity of fetal and adult rat posterior pituitaries

Each value represents the mean ± S.E.M. of 3 incubations. Per cent stimulation due to agent is in parenthesis. Stimulationsof adenylate cyclaseactivity by agents are statisticallysignificantat P < 0.05 ~0.0001. Agents added

pmol cyclic AMP formed]mg protein~2.5 min 21-day-oldfetal rats

None Dopamine, 100 #M Norepinephrine, 100/~M Isoproterenol, 100/~M Prostaglandin E1 Gpp(NH)p, 50/~M* or 100/~M** * 2 month old adults.

57 79 68 79

± ± ± ±

4 1 (+39) 1 (+19) 1 (+39)

246 ± 1 (+332)

2-month-oldrats

68:L3 184 ± 19 (+171) 222 ± 4 (+226) 313 ± 4 (+360) 301 ± 24 (+343) 265 -4- 7 (+290)

** 21 day old fetus.

Dopamine stimulated in a dose-dependent manner adenylate cyclase activity in homogenates of posterior pituitary of rat with maximal stimulation occurring at 50-100 #M DA (Fig. 1 and Table I). A significant stimulation (40 ~) was observed at 0.5 #M DA and half-maximal activation was produced by approximately 2.5 /~M DA. Enzyme activity in posterior pituitary was also significantly stimulated by the known DA receptor agonist, apomorphine (Fig. 1). The DA-stimulated activity was more effectively blocked by haloperidol, a relatively specific DA receptor blocking agent, than by phentolamine, an a-adrenergic receptor blocker or propranolol, a fladrenergic receptor antagonist (Fig. 1). These data suggest the presence of DA receptors coupled to adenylate cyclase in rat posterior pituitary. As shown in Table I, posterior pituitary adenylate cyclase was also stimulated by norepinephrine, isoproterenol, prostaglandin E1 and Gpp(NH)p, a GTP analogue. Unlike the anterior pituitary enzyme2,3 adenylate cyclase of 21 day fetal rat posterior pituitaries was significantly stimulated by DA, norepinephrine and isoproterenol. However, the response of adenylate cyclase to catecholamines but not to Gpp(NH)p was much lower in the fetal rat posterior pituitary than in that from 2 month old rats (Table I) indicating postnatal development of catecholamine receptoradenylate cyclase systems. These experiments show for the first time that DA and DA agonists stimulate adenylate cyclase in the posterior pituitary. The stimulatory effects of other catecholamines and of prostaglandin E1 indicate that other receptor systems not characterized pharmacologically as yet are also coupled to adenylate cyclase in this tissue. In this regard it may be noted that prostaglandin E1 when injected directly into posterior pituitary stimulates release of A D H 16. These studies together with previous findings provide a basis for further elucidation of the role of cyclic AMP in neuroendocrine control mechanisms in the posterior pituitary.

425 This w o r k was s u p p o r t e d by N I H N S 09649.

REFERENCES I Ahn, H. S. and Makman, M. H., Neurotransmitter-sensitive adenylate cyclase in the hypothalami of guinea-pig, rat and monkey, Brain Research, 138 (1977) 125-138. 2 Ahn, H. S., Feldman, S. C., Seeger, T., Bornstein, M. B. and Makman, M. H., Dopamine sensitive adenylate cyclase of anterior pituitary and cultured anterior pituitary explants, Fed. Proc., 37 (1978) 2772. 3 Ahn, H. S.. Gardner, E. and Makman, M. H., Anterior pituitary adenylate cyclase: stimulation by dopamine and other monoamines, Europ. J. Pharmacol., in press. 4 Barchas, J. D., Berger, P. A., Ciaranello, R. D. and Elliott, G. R., Psychopharmacology from Theory to Practice, Oxford University Press, New York, 1977. 5 Bjorklund, A. and Nobin, A., Fluorescence histochemical and microspectrofluorometric mapping of dopamine and noradrenaline cell groups in the rat diencephalon, Brain Research, 51 (1973) 193-205. 6 Bjorklund, A., Moore, R. Y., Nobin, A. and Stenevi, U., The organization of tubero-hypophysial and reticulo-infundibular catecholamine neuron systems in the rat brain, Brain Research, 51 (1973) 171-191. 7 Creese, I., Schneider, R. and Snyder, S. H., 3H-Spiroperidol labels dopamine receptors in pituitary and brain, Europ. J. PharmacoL, 46 (1977) 377-381. 8 Cronin, M. J., Roberts, J. M. and Weiner, R. I., Dopamine and dihydroergocryptine binding to the anterior pituitary and other brain areas of the rat and sheep, Endocrinology, 103 (1978) 302-309. 9 Douglas, W. W. and Ishida, A., The stimulant effect of cold on vasopressin release from the neurohypophyis in vitro. J. Physiol. (Lond.), 179 (1965) 185-191. 10 Fuxe, K., H6kfelt, T. and Nilsson, O., Factors involved in the control of the activity of the tuberoinfundibular dopamine neurons during pregnancy and lactation, Neuroendocrinology, 5 (1969) 257-270. 11 Ginsburg, M., Production, release, transportation and elimination of the neurohypophysial hormones. In B. Berde (Ed.), Handbook of Experimental Pharmacology, Vol. 23, Neurohypophysial Hormones and Similar Peptides, Springer-Verlag, New York, 1968, pp. 286-371. 12 Givant, Y. and Sulman, F. G., Endocrine effects psychotherapeutic drugs. In E. Usdin and I. S. Forrest (Eds.), Psychotherapeutic Drugs, Part 1 - - Principles, Marcel Dekker, New York, 1976, pp. 387-435. 13 Guidotti, A., Zivkovic, B. and Costa, E. Possible involvement of cyclic nucleotides in the stimulation of pituitary function elicited by reserpine. In N. Hatotani (Ed.), Psychoneuroendocrinology, Workshop Conf. Int. Soc. Psychonearoendocrinology, Karger, Basel, pp. 259-266. 14 Jacobowitz, D. M., Distribution of biogenic amines in the pituitary gland. In E. Zimmermann, W. H. Gispen, B. H. Marks and D. deWied (Eds.), Drug Effects on Neuroendocrine Regulation, Elsevier, New York, 1973, pp. 199-209. 15 Renaud, P., Neurophysiological organization of the endocrine hypothalamus. In R. J. Baldessarini and J. B. Martin (Eds.), The Hypothalamus, Raven Press, New York, 1978, pp. 269-301. 16 Ruoff, H. J., Gosbee, J. J. and Lederis, K., Substances affecting the release of neurohypophysial hormones. In K. Lederis and K. E. Cooper (Eds.), Recent Studies of Hypothalamic Function, Karger, Basel, 1974, pp. 67-79. 17 Ruoff, H. J., Mathison, R. and Lederis, K., Cyclic 3',5'-adenosine monophosphate in the hypothalamo-neurohypophysial system of normal, NaCl-treated and lactating rats, Neuroendocrinology, 22 (1976) 18-29. 18 Saavedra, J. M., Palkovits, M., Kizer, J. S., Brownstein, M. J. and Zivin, J. A., Distribution of biogenic amines and related enzymes in the rat pituitary, J. Neurochem., 25 (1975) 257-260. 19 Schmidt, M. J. and Hill, L. E., Effects of ergots on adenylate cyclase activity in the corpus striatum and pituitary, Life Sci., 20 (1977) 789-798. 20 Shoemaker, W. J. and Schlumpf, M. Diencephalic dopamine cell groups: correlations with endocrine status. In E. Costa and G. L. Gessa (Eds), Advanc. Biochem. Psyehopharmacol., Vol. 16, Raven Press, New York, 1977, pp. 359-367.

Posterior pituitary adenylate cyclase: stimulation by dopamine and other agents.

422 Brain Research, 166 (1979) 422-425 © Elsevier/North-HollandBiomedicalPress Posterior pituitary adenylate cyclase: stimulation by dopamine and ot...
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