Direct stimulation of 132-adrenergic receptors in rat anterior pituitary ...

Proc. Natl. Acad. Sci. USA Vol. 80, pp. 6728-6731, November 1983

Neurobiology

Direct stimulation of 132-adrenergic receptors in rat anterior pituitary induces the release of adrenocorticotropin in vivo (isoproterenol/propranolol/glucocorticoids/stalk transection) EVA MEZEY*, TERRY D. REISINEt, MIKLOS PALKOVITS*, MICHAEL J. BROWNSTEIN*, ANDJULIUS AXELRODt *Laboratory of Cell Biology and tSection on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20205

Contributed by Julius Axelrod, August 12, 1983

ABSTRACT Previous work in our laboratory has shown that stimulation of g32-adrenergic receptors on mouse. anterior pituitary tumor cells causes the secretion of immunoreactive adrenocorticotropin (ACTH). The present study was designed to test the hypothesis that catecholamines can cause the release of ACTH in vivo by the direct stimulation of f32-adrenergic receptors in the rat anterior pituitary. Systemic administration of a 3-adrenergic receptor agonist (-)-isoproterenol resulted in an increase in plasma ACTH levels in intact animals and in rats with transected pituitary stalks. This effect could be blocked by the 8i-adrenergic receptor antagonist, propranolol, but not by the specific (31-adrenergic receptor antagonist, practolol. Salmefamol, a fi2-adrenergic receptor agonist also elevated plasma ACTH levels in stalk-sectioned animals. Dexamethasone, a glucocorticoid that inhibits the synthesis and release of ACTH from the anterior pituitary but not the intermediate lobe, prevented the elevation of ACTH secretion by (-)-isoproterenol in stalk-transected rats. These data indicate that 32-adrenergic receptors are present on anterior pituitary cells and suggest that catecholamines can directly stimulate ACTH secretion.

Adrenocorticotropic hormone (ACTH) can be released from the anterior pituitary by various stress stimuli. The release of ACTH is controlled by corticotropin-releasing factor(s) some of which originate from hypothalamic neurons. A major corticotropin-releasing factor has been isolated, and the -sequence has been determined by Vale et al. (1). Catecholamines also can induce the release of ACTH; however, it has not yet been established whether they act directly on the anterior pituitary or produce this effect indirectly by a central reflex mechanism (2). Peripheral administration of epinephrine increases plasma ACTH levels in rats (3-5). This increase is blocked by the 3-adrenergic receptor antagonist propranolol. Because (-)-isoproterenol, a f3-adrenergic agonist, only weakly effects ACTH secretion from primary cultures of the rat anterior pituitary, the direct a-adrenergic stimulation of the adenohypophysis to release ACTH has been discounted (6). Recent work in our laboratory has shown that (-)-isoproterenol is a potent stimulant of ACTH release from a homogenous population of corticotrophs (AtT-20/D1616) (7). It also was demonstrated that ACTH is released from these cells via a ,32-adrenergic receptor coupled to adenylate cyclase (7). These findings suggested that 3adrenergic agonists may directly regulate in vivo ACTH release from anterior pituitary cells. We now show that catecholamines can evoke the release of ACTH in rats in which all central control of the pituitary was severed by the pituitary stalk transection.

METHODS AND MATERIALS Animals. Male Sprague-Dawley rats (180-200 g) were used in all experiments. Food and water were provided ad lib. The rats were housed under diurnal lighting (lights on 6 a.m. to 6 p.m.) with 4-6 animals per cage. Surgery. Two kinds of surgical interventions were performed. In one group of animals, the pituitary stalk was transected by means of a parapharyngeal approach. In another group of rats the median eminence was destroyed by means of a dorsal approach, with a small Hala'sz knife (8) (1.5-mm radius) that was lowered into the brain in the midline until it touched the ventral surface of the cranial vault and then moved in the midsagittal line from the bregma level to the mamillary bodies while being rotated continuously. In this way the median eminence and arcuate nuclei were destroyed. The results of the surgical procedures were verified at the time that the animals were killed. Injection Protocol. The following drugs were dissolved in 0.2 ml of saline and administered intraperitoneally: (-)-isoproterenol, 25 ug; d,l-propranolol, 500 Ag; practolol, 500 pug; salmefamol, 25 Ag; dexamethasone, 400 pug. Isoproterenol, salmefamol, and saline (control) were injected 30 min before killing the rats. Propranolol and practolol were given 40 min before the isoproterenol injection. Dexamethasone was injected 16 hr prior to administration of the other drugs. Plasma Collection. The animals were decapitated 5 see after being removed from their cages. After decapitation, trunk blood was collected in ice-cold polystyrene tubes containing 100 A.l of 10% EDTA. The blood was centrifuged at 3,000 x g for 20 min, and the plasma was removed, centrifuged again, and stored at -250C. Determination of ACTH. ACTH was extracted from the plasma as described by van Dijk (9). Blood (1.5 ml) was extracted with Vycor glass powder (35 mg per tube) from which the ACTH was eluted with 50% acetone. The extracts were dried in a vacuum centrifuge and reconstituted in veronal buffer (10). ACTH was determined by radioimmunoassay as described (10). To determine the ACTH recovery, internal standards were added to plasma of hypophysectomized rats and extracted and assayed as described above. All values are corrected for recovery, which was about 50%. A sample of pooled plasma was run with each radioimmunoassay to determine the interassay variation (1518%). The immunoreactive ACTH detected by the radioimmunoassay will be referred to as ACTH in the text. RESULTS Stimulation of ACTH Release by (-)-Isoproterenol. To determine whether catecholamines can induce the release of ACTH from the pituitary directly, the pituitary was disconnected from

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviation: ACTH, adrenocorticotropin. 6728

Neurobiology: Mezey et al.

Proc. Natl. Acad. Sci. USA 80 (1983)

A

6729

* * *

I-

0 m

200

I

HQ

B

*r

0. 20 7 19 0

FIG. 1. Isoproterenol-stimulated ACTH release in intact and stalk transected animals. Intact (A) or stalk-transected (B) animals were injected intraperitoneally with 0.2 ml of saline (0) or (-)-isoproterenol (25 jtg) (03) 30 min before killing the rats. In another group of animals, propranolol (500 1g) was injected intraperitoneally 40 min prior to the (-)-isoproterenol injection. (ED) After decapitation, blood was collected and immunoreactive ACTH was measured. Values on the x axis represent the number of animals used in each treatment. The results are the means SEM plasma ACTH levels of each group. *, P < 0.05; **, P < 0.01 different from saline-injected controls using a Student t test. ±

the hypothalamus by stalk transection. In these animals, intraperitoneally administered (-)-isoproterenol increased plasma ACTH content, whereas saline injection itself did not affect plasma ACTH levels (Fig. 1). To exclude the possibility for a regeneration of the neurohumoral connections between the hypothalamus and the pituitary, the median eminence of rats was surgically destroyed. One day after this procedure, animals responded to (-)-isoproterenol with a 2-fold increase in ACTH release (saline injected, 99.1 11.6 pg/ml, n = 10; isoproterenol injected, 185.3 28.6 pg/ml, n = 9; P < 0.01). ±

±

Stimulation of fi6-Adrenergic Receptors Increases ACTH Release in Stalk-Transected Rats. From the experiments described above, it appeared that a f3-adrenergic receptor was involved in the stimulation of ACTH secretion from the pituitary. The class of the /adrenergic receptor was determined by the use of various types of /3adrenergic receptor agonists and antagonists. The (-)-isoproterenol-induced increase in plasma ACTH levels was blocked by propranolol (f3l- and f32-adrenergic antagonist) but not by practolol (a P/-adrenergic antagonist) (Fig. 2). Propranolol itself did not significantly affect ACTH secretion. The selective 82-adrenergic agonist, salmefamol, stimulated ACTH release to an even greater extent than that

6

6

FIG. 2. P2-Adrenergic receptor-mediated release of ACTH from pituitary stalk-transected animals. Lesioned animals were administered either saline (a), salmefamol (25 fig) (9), or (-)-isoproterenol (25 ,ug) ([1) as described in Fig. 1. In some rats, 40 min prior to the isoproterenol treatment, propranolol (500 pug) (011) or practolol (500 fig) (0) were injected. Immunoreactive ACTH was measured as described. Values on thex axis represent the number of animals used in each treatment. The results are the means ± SEM of plasma ACTH levels of each group. **, P < 0.01 different from saline; ***, P < 0.001 different from saline-injected controls.

observed for (-)-isoproterenol (Fig. 2). Selective Stimulation of ACTH Release from the Anterior Pituitary. The central corticotropin-releasing factors arising from the hypothalamus could release ACTH from both anterior and intermediate lobes of the pituitary. To distinguish whether the peripheral catecholamines release ACTH from the anterior lobe or the intermediate lobe or both, dexamethasone was used. Dexamethasone has been shown to block hormone release from the anterior but not the intermediate lobe (11). Pretreatment with dexamethasone prevented the rise in plasma ACTH induced by (-)-isoproterenol in stalk-transected animals (Fig. 3). Basal ACTH release was also reduced by this treatment. DISCUSSION Previous work has shown that catecholamines can elevate the levels of plasma ACTH (4, 12-14). The precise mechanism by which catecholamines can induce this effect has not been demonstrated. The experiments described in this report determined whether 83-adrenergic agonists could act directly on the anterior pituitary to affect ACTH secretion. To remove the influence of central factors on ACTH secretion, the pituitary stalk of rats were transected. Dye injection studies showed that no vascular connections from the brain to the pituitary existed after such lesions, and dopamine, which reaches the pituitary from neurons of the hypothalamus, was totally depleted in the pituitary after the median eminence lesions and after stalk transections (data not shown). The pituitary vasculature derived from the periphery (posterior hypophyseal artery) was intact, and no

6730

Proc. Natl. Acad. Sci. USA 80 (1983)

Neurobiology: Mezey et al.

To distinguish from which pituitary lobe the peripherally administered 83-adrenergic agonists stimulated ACTH secretion, dexamethasone, a glucocorticoid analogue that selectively prevents hormone release from the anterior but not from the in-

or

termediate lobe (20-22) where negligible amounts of glucocorticoid receptors are present (23), was utilized. Dexamethasone blocked the rise in plasma ACTH levels induced by (-)-isoproterenol, indicating a predominant involvement of the anterior lobe in the effect of circulating catecholamines on ACTH

200

secretion.