Inhibition of corticotropin release in vitro by dexamethasone, aldosterone and spironolactone

Dexamethasone, aldosterone and spironolactone inhibited the release of immunoreactive corticotropin (ACTH) from primary culture of the rat anterior pituitary cells. The steroids inhibited only the ACTH release stimulated by Pitressin and not the basal ACTH release by non-stimulated cells. On a molar basis, aldosterone appears to be the most efficient inhibitor of ACTH release while the effect of spironolactone is similar to the effect of dexamethasone. Simultaneous incubation with aldosterone and spironolactone inhibited the ACTH release to the same extent as spironolactone alone. This indicates that aldosterone's effect on ACTH release is also inhibited by spironolactone at the pituitary level.     

Effect of ACTH and prolactin on dehydroepiandrosterone, its sulfate ester and cortisol production by normal and tumorous human adrenocortical cells

The effect of ACTH and prolactin on the synthesis of dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) was studied in cell suspensions of "normal" and tumorous (adenoma) human adrenal cortex. A stimulation of DHEA and no response of DHEAS production by ACTH in "normal" adrenocortical cell suspension was observed. However ACTH stimulated both DHEA and DHEAS synthesis in tumorous adrenocortical cells. Prolactin did not influence either the basal or the ACTH stimulated DHEA and DHEAS production of adrenocortical cells irrespective of their origin. Our results are compatible with the concept that the biosynthesis of DHEA is under ACTH control, while other factor(s) regulate(s) the sulfate pathway of DHEA secretion under normal conditions. In tumorous adrenocortical cells DHEA may be regulated--at least partly--by ACTH. Prolactin seems to have no direct effect on DHEA and DHEAS synthesis. It is postulated that the relationship between serum prolactin and DHEAS (or DHEA) levels observed by several authors might be an extraadrenal effect of prolactin on adrenal androgens.     

ACTH stimulates the release of alkaline phosphatase through Gi-mediated activation of a phospholipase C and the release of inositolphosphoglycan

We have previously reported that ACTH activates a phospholipase C that hydrolyzes glycosylphosphatidylinositol (GPI), which would release inositolphosphoglycan (IPG) to the extracellular medium, and that an IPG purified from Trypanosoma cruzi is able to inhibit ACTH-mediated steroid production in adrenocortical cells. In the present paper, it was found that anti-inositolphosphoglycan antibodies (anti-CRD) increased ACTH-mediated corticosterone production, which indicates that an endogenous IPG is a physiological inhibitor of ACTH response. On the other hand, we investigated the release to the extracellular medium of the GPI-anchored enzyme, alkaline phosphatase, by ACTH. We found that: (a) the released enzyme appeared in the aqueous phase after Triton X-114 partitioning, consistent with loss of the GPI, (b) the phospholipase C inhibitor, U73122, impaired the release of the enzyme by the hormone and (c) two inhibitors of IPG uptake, inositol 2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium. These results suggest that ACTH releases alkaline phosphatase by activation of a phospholipase C. Dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) was able to increase the release of alkaline phosphatase from adrenocortical cells and this effect was inhibited by U73122, suggesting that cAMP is involved in the activation of phospholipase C. In addition, it was found that a pertussis-toxin sensitive G-protein is required for ACTH- and db-cAMP-mediated release of alkaline phosphatase and that incorporation of anti-Gi antibodies in adrenocortical cells inhibited the release of alkaline phosphatase by ACTH. Our results suggest that ACTH increases the release of alkaline phosphatase by activation of a phospholipase C through cAMP and Gi which would contribute to produce IPG It was also found that the two inhibitors of IPG uptake, inositol-2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium of ACTH-treated cells more than in control cells, indicating that ACTH also stimulates the uptake of IPG These data support a role of GPI and the involvement of Gi in ACTH action.     

Enhanced 17 alpha-hydroxylation of pregnenolone and increased androgen production by rabbit adrenocortical cells stimulated chronically with corticotropin

The postulated chronic stimulatory effect of corticotropin (ACTH) on pregnenolone production and on 17 alpha-hydroxylase activity was evaluated on adrenocortical cells obtained from control and chronically ACTH-treated rabbits. The cells were incubated with various concentrations of ACTH added alone or together with trilostane, so as to inhibit further conversion of pregnenolone and dehydroepiandrosterone. The maximal steroidogenic effect of ACTH (determined in the absence of trilostane) was increased 2-fold in adrenocortical cells from ACTH-treated animals; furthermore, cortisol production was increased whereas that of corticosterone decreased. While the generation of pregnenolone was of comparable magnitude for cells from both experimental groups, chronic in vivo treatment with ACTH was followed by a 40-fold enhancement in 17-hydroxypregnenolone production. Concomitantly, maximal DHEA production documented in the presence of ACTH and trilostane was enhanced more than 200-fold, from 0.45 +/- 0.20 pmol in control rabbits to 147 +/- 67 pmol in cells from ACTH-treated animals. The corresponding values of DHEA-sulphate production were 0.86 +/- 0.12 and 432 +/- 334 pmol, respectively. Thus, a prolonged stimulatory effect of ACTH on rabbit adrenocortical cells consists in an enhancement of the capacity to generate pregnenolone, and to convert this compound into 17-hydroxylated steroids.     

Involvement of GABAB receptors in the regulation of the hypothalamo-pituitary-adrenocortical (HPA) axis in rats

Previous experiments have shown that the GABA_B receptor agonist -baclofen given subcutaneously to male rats significantly enhanced plasma concentrations of adrenocorticotropic hormone (ACTH) and the adrenocortical hormones corticosterone and aldosterone. The goal of the present study was to investigate whether the stimulatory effects on adrenocortical steroids elicited by -baclofen in vivo could be reversed by the selective GABA_B antagonist CGP 35 348. One hour before subcutaneous administration of 3 mg/kg -baclofen, a dose of 600 mg/kg CGP 35 348 or saline was administered intraperitoneally. The stimulatory effect of -baclofen on ACTH, corticosterone and aldosterone was significantly reduced by 60% after pretreatment with CGP 35 348. The GABA_B antagonist CGP 35 348 by itself had no effect on ACTH or the adrenocortical hormones. These results indicate that GABA_B receptors are involved in the -baclofen-induced activation of the HPA axis in rats. In vitro, however, neither -baclofen nor CGP 35 348 had any effects on corticosterone and aldosterone release from perifused adrenal cells. These results suggest that the participation of GABA_B receptors in the activation of the HPA axis induced by -baclofen in vivo does not occur at the level of the adrenal gland, and therefore must occur at the level of the pituitary or the brain.     

Glucocorticoid control of steroidogenesis in isolated rat adrenocortical cells

The role of end-product glucocorticoids in the regulation of corticosteroidogenesis in isolated adrenocortical cells was investigated. Trypsin-isolated cells from male rat adrenal glands were incubated with or without corticotropin (ACTH) and with or without corticosterone. Endogenous corticosterone production was determined by radioimmunoassay at the end of incubation. Cessation of ACTH-induced corticosterone production was apparent after 2-4 h of incubation. The suppression occurred later with lower cell concentrations. Corticosterone production was partially restored after washing the suppressed cells. Supernatant fluid from suppressed cell suspensions also suppressed steroidogenesis of a fresh population of cells. However, the suppressing property of the supernatant fluid was abolished after the removal of corticosterone by charcoal-dextran treatment, suggesting that corticosterone or other steroids caused the suppression. Exogenous corticosterone induced suppression over a wide range of ACTH concentrations, but did not change the half-maximal steroidogenic concentration of ACTH, indicating that the suppression does not change the sensitivity of the cells to ACTH. Suppression occurred within 30-60 min after corticosterone had been added to the incubation medium either at the start of incubation or while steroidogenesis was in progress. Suppression varied directly with the concentration of exogenous corticosterone. These data indicate that glucocorticoids can directly and acutely suppress corticosteroidogenesis and thus control adrenocortical function in concert with other regulators such as ACTH and Ca2+.     

Interactions between CRF, epinephrine, vasopressin and glucocorticoids in the control of ACTH secretion

The secretion of ACTH by corticotrophs in the anterior lobe of the rat pituitary gland is under the stimulatory influence of at least three receptors, namely that for peptidic CRF (corticotropin-releasing factor), vasopressin and alpha 1-adrenergic agents. CRF is a potent stimulator of cyclic AMP accumulation as well as adenylate cyclase activity in the rat adenohypophysis, thus suggesting an important role of cyclic AMP as mediator of CRF action on ACTH secretion. Vasopressin causes a 2-fold increase of the stimulatory effect of CRF on ACTH release in rat anterior pituitary cells in culture. The potentiating effects of vasopressin on CRF-induced ACTH release are accompanied by parallel changes of intracellular cyclic AMP levels. Vasopressin, while having no effect on basal cyclic AMP levels, causes a 2-fold increase in CRF-induced cyclic AMP accumulation without affecting the ED50 value of CRF action. ACTH secretion is also stimulated by a typical alpha 1-adrenergic receptor. Epinephrine causes a marked stimulation of ACTH release which is additive to that of CRF. Epinephrine, in analogy with vasopressin, although having no effect alone on basal cyclic AMP levels, causes a marked potentiation of CRF-induced cyclic AMP accumulation. Glucocorticoids cause a near-complete inhibition of epinephrine-induced ACTH secretion within 4 h with the following order of ED50 values: triamcinolone acetonide (0.2 nM) greater than dexamethasone (1.0 nM) much greater than cortisol (11 nM) greater than corticosterone (22 nM). Similar effects are observed for CRF- and vasopressin-induced ACTH release. Although the activity of the pituitary-adrenocortical axis in the rat is highly dependent upon sex steroids, 17 beta-estradiol, 5 alpha-dihydrotestosterone and the pure progestin R5020 have no detectable effect on basal or epinephrine-induced ACTH release, thus illustrating the high degree of specificity of glucocorticoids in their feedback control of ACTH secretion. Moreover, glucocorticoids have no effect on CRF-induced cyclic AMP accumulation, thus indicating that their inhibitory effect is exerted at a step following cyclic AMP accumulation.     

Steroid control of steroidogenesis in isolated adrenocortical cells: molecular and species specificity

The molecular and species specificity of glucocorticoid suppression of corticosteroidogenesis was investigated in isolated adrenocortical cells. Trypsin-isolated cells from male rat, domestic fowl and bovine adrenal glands were incubated with or without steroidogenic agents and with or without steroids. Glucocorticoids were measured by radioimmunoassay or fluorometric assay after 1-2 h incubation. Glucocorticoids suppressed ACTH-induced steroidogenesis of isolated rat cells with the following relative potencies: corticosterone greater than cortisol = cortisone greater than dexamethasone. The mineralocorticoid, aldosterone did not affect steroidogenesis. Suppression by glucocorticoids was acute (within 1-2 h), and varied directly with the glucocorticoid concentration. Testosterone also suppressed ACTH-induced steroidogenesis. Glucocorticoid-type steroids have equivalent suppressive potencies, thus suggesting that these steroids may induce suppression at least partly by a common mechanism. Although corticosterone caused the greatest suppression, testosterone was more potent. The steroid specificity of suppression of cyclic AMP (cAMP)-induced and ACTH-induced steroidogenesis were similar, suggesting that suppression is not solely the result of interference with ACTH receptor function or the induction of adenylate cyclase activity. Exogenous glucocorticoids also suppressed ACTH-induced steroidogenesis of cells isolated from domestic fowl and beef adrenal glands, thus suggesting that this observed suppression may be a general mechanism of adrenocortical cell autoregulation.     

Suppression of ACTH-induced steroidogenesis by supernatants from LPS-treated peritoneal exudate macrophages

The results from a number of clinical and experimental studies have suggested that during endotoxemia, suppression of adrenocortical steroidogenesis may occur. We have examined the possibility that macrophages are the source of a factor that suppresses adrenocortical steroidogenesis. Resident and peptone-elicited peritoneal exudate macrophages (PEM) from C3HeB/FeJ mice were incubated for 4 hr at 37 degrees C in the presence or absence of T cell hybridoma-derived lymphokine (LK) that contained high concentrations of MAF activity (assessed by induction of nonspecific tumoricidal activity in PEM). The LK was removed by rinsing, and fresh medium was added, followed by Salmonella minnesota R595 LPS (final concentration 10 micrograms/ml). After 18 hr at 37 degrees C the PEM supernatants and control medium from flasks without cells were harvested and stored at -20 degrees C. Explanted rabbit adrenocortical cells in 96-well plates were exposed to 30 microliters of PEM supernatant or control medium and ACTH (10 or 100 mU/ml) in a final volume of 120 microliters for 3 consecutive days. The adrenocortical cell supernatants were harvested each day, followed by replenishment of medium, PEM supernatant, and ACTH. Fluorogenic steroid production in wells that received control medium or supernatants from PEM not treated with LPS was normal (0.22 microgram +/- 0.010 (SD) per 5 X 10(4) cells). However, as much as 75 to 95% suppression of steroidogenesis was observed in wells that received supernatants from PEM treated with LK and LPS, compared to 40% suppression in wells that received supernatant from PEM treated with LPS alone. Continued exposure (over 3 days) of adrenocortical cells to supernatants from LPS-treated PEM resulted in progressively decreasing response to ACTH. Comparable suppressive activity was observed in supernatants from LPS-treated bone marrow-derived macrophages. In further experiments, suppression was observed in wells that were pretreated (22 hr) with the appropriate PEM supernatant, and evidence was obtained that the suppressive activity was not due to carry-over LPS. Finally, results from control experiments demonstrated that suppressive PEM supernatants neither inactivate ACTH nor interfere with the assay of fluorogenic steroids. Thus, these results suggest that during endotoxemia, products from LPS-stimulated macrophages may suppress adrenocortical function.     

Intranasal administration of ACTH(1-24) stimulates catecholamine secretion.

Previously, we reported that intranasal (IN) ACTH(1-24) administration stimulates adrenocortical steroid secretion in normal subjects. To determine the efficiency of transmucosal absorption of ACTH into the adrenal medulla, we measured serum cortisol, aldosterone, epinephrine, norepinephrine and dopamine levels after IN vs. intravenous (IV) administration of 250 microg ACTH(1-24) in 7 healthy adult men (mean age 21.7 +/- 1.2 yr; range, 21 - 24 yr). Blood was collected at 0, 30, 60 and 120 min after administration of ACTH(1-24), and the levels of adrenocortical steroids and catecholamines were measured by specific RIA and HPLC methods, respectively. There were no side effects associated with IN or IV ACTH administration. Consistent with the previous study, serum cortisol and aldosterone increased after IN administration of ACTH(1-24), peaking 30 min after administration. Sixty minutes after IN and IV administration of ACTH, epinephrine levels increased by 41.9 +/- 13.1 % and 63.3 +/- 11.8 %, respectively, and remained elevated throughout the sampling period. Thirty minutes after IN or IV administration of ACTH(1-24), plasma norepinephrine levels increased by 55.9 +/- 13.4 % and 73.7 +/- 15.0 %, respectively, peaking 30 min after ACTH(1-24) administration, and decreasing to basal levels within 60 min. Plasma dopamine levels did not change after IN administration of ACTH(1-24). Adrenocortical steroid and catecholamine levels did not increase after IN administration of saline. These results demonstrate that IN administration of ACTH(1-24) not only stimulates adrenocortical steroids, but also epinephrine and norepinephrine.     

Involvement of dopaminergic receptors in action of corticotropin releasing factor (CRF) under in vitro conditions.

ACTH release under the effect of median eminence extract (ME) was studied in both incubation and superfusion experiments. ACTH content of the incubation medium was measured by radioimmunoassay or by the corticosterone production of trypsinisolated adrenocortical cells. Dopamine at low concentration led to a slight increase of basal ACTH secretion, while at higher concentration failed to influence ACTH release. The dopamine agonist CB-154 produced a significant rise of ACTH secretion and augmented the ME extract-induced increase of pituitary ACTH release. Chlorpromazine and haloperidol suppressed basal ACTH secretion and inhibited the ME extract-induced release. The simultaneous administration of CB-154 and haloperidol into the incubation medium prevented the haloperidol-induced inhibition of ACTH release. The observations indicate that dopaminergic receptors play a role in the activation of CRF-induced ACTH secretion under in vitro experimental conditions.     

Polyhormonal regulation of avian and mammalian corticosteroidogenesis in vitro

1. The combined actions of ACTH, corticosterone and prolactin (PRL) in the acute regulation of corticosteroidogenesis were investigated using isolated adrenocortical cells from intact and hypophysectomized (hypox) rats (Rattus norvegicus) and from intact male domestic fowl (Gallus gallus domesticus). 2. Exogenous corticosterone suppressed to about 50% ACTH-induced corticosterone production of cells from either species. This suppression, in part, was due to corticosterone degradation. 3. oPRL, in the presence or absence of ACTH, raised corticosterone production of hypox rat cells, but not intact rat and domestic fowl cells. 4. In addition, oPRL counteracted the corticosterone-induced suppression of net ACTH-stimulated corticosterone production of hypox rat and intact domestic fowl cells, but not intact rat cells. 5. The potency of oPRL with domestic fowl cells was 4 times that with hypox rat cells. 6. Furthermore, in domestic fowl cells, the effect of oPRL was Ca2+-dependent.     

Biphasic effect of ACTH on growth of rat adrenocortical cells in primary culture

Summary The proliferation rate of differentiating fetal rat adrenocortical cells was studied in primary culture. In this system, stimulation with ACTH induces differentiation of zona glomerulosa-like cortical cells into zona fasciculata-like cells. Incorporation of bromodeoxyuridine (BrdU) was studied immunocytochemically by use of anti-BrdU antibody, and the proliferation rate was counted from the monolayer colonies of adrenocortical cells. After 21 days of cultivation in the absence of ACTH, the proliferation rate of zona glomerulosa-like cells was 10%. The rate slowly declined to 1% at the age of 100 days during continuous cultivation in the absence of ACTH. Stimulation with ACTH induced a strong inhibition in the proliferation rate (down to 2% during the first 24 h). Treatment with ACTH during the following 48 h led to an extremely intense proliferation of adrenocortical cells at a proliferation rate of 25%. Continuous treatment with ACTH up to 100 days led to a persistent growth of adrenocortical cells, and a proliferation rate over 2-fold higher than in control cells cultivated in the absence of ACTH. Thus, ACTH is the principal growth-promoting factor also in vitro, as has been found in in vivo studies. This growth effect is mediated by a biphasic course; at the beginning of differentiation the effect is inhibitory and is followed by a persistent stimulation of the growth of adrenocortical cells.     

ACTH and growth hormone relationship in fetal rat adrenal steroidogenesis in vitro.

The effects of growth hormone and ACTH, alone or in combination, on fetal rat adrenal steroidogenesis in vitro were examined on the last day of intrauterine development. ACTH increased, while growth hormone did not affect fetal adrenal weight. ACTH increased fetal rat adrenal steroidogenesis, hydroxylation of 4-14C-progesterone to corticosterone, 18-hydroxy-11-deoxycorticosterone, 11-hydroxycorticosterone and aldosterone. Growth hormone alone had no effect on fetal adrenal steroidogenesis. ACTH and growth hormone administered together increased the conversion of progesterone to the above mentioned steroids to a greater extent than ACTH alone. The results indicate that growth hormone may participate in the fetal rat adrenal steroidogenesis potentiating the effects of fetal pituitary ACTH.     

Comparison of the structural features of corticotropin required for stimulation of steroidogenesis and cAMP production in rat and rabbit adrenocortical cells

The steroidogenic activities of ACTH, alpha-MSH, beta-MSH as well as analogs of the hormones have been compared in rat and rabbit adrenocortical cells. ACTH is equally active in both species and the melanotropins have very low steroidogenic potency in either species. The steroidogenic potencies of the peptide analogs are strikingly similar in the two species, suggesting that the structural requirements for eliciting steroidogenesis are the same in rat and rabbit adrenocortical cells. The analog NPS-ACTH has low, comparable steroidogenic activity in both species. NPS-ACTH is a potent antagonist of ACTH-induced cAMP production in rat adrenocortical cells but acts as a weak partial agonist in rabbit adrenocortical cells. These results suggest that steroidogenesis may be mediated by receptors different from those involved in the cAMP response observed at supraphysiological concentrations of ACTH.     

Endogenous cholesterol synthesis is sufficient for ACTH-induced differentiation of rat adrenocortical cells in primary culture

Summary To define the role of endogenously synthesized cholesterol in the differentiation of adrenocortical cells in primary culture, fetal rat adrenal cells were cultured in the presence of exogenous cholesterol (serum-supplemented medium) or in the absence of it (serum-free medium or lipoprotein-free medium). Ultrastructurally the cells had features of glomerulosa cells: mitochondria were oval or rod shaped with lamellar inner membranes. The amount of smooth endoplasmic reticulum was small, and lipid droplets were few. When the cells were cultured in serum-free medium some intracytoplasmic vacuoles were seen. The undifferentiated zona glomerulosa-like cells secreted low amounts of corticosterone and 18-OH-deoxycorticosterone (18-OH-DOC) in all three media (serum-supplemented medium, serum-free medium, and lipoprotein-free medium). Stimulation of the adrenocortical cells with ACTH induced the ultrastructural features of differentiated zona fasciculata-like cells. Mitochondrial inner membranes were well developed in lipoprotein-free medium, but not in serum-free medium. The amount of intracellular lipids was increased in both media devoid of cholesterol. In the ACTH stimulated cultures the presence of exogenous cholesterol resulted in increased secretions of corticosterone and 18-OH-DOC. In the absence of an exogenous source of cholesterol, the amounts of steroids secreted were only half of that secreted in the presence of serum-supplemented medium. Endogenously synthesized cholesterol is sufficient for the morphologic differentiation of fetal rat adrenocortical cells under ACTH stimulation. However, without exogenously provided cholesterol, the steroid production accounts only for half of the maximal output achieved using serum-supplemented medium. This work was supported by Finnish Culture Foundation.     

Angiotensin II increases ACTH release in the absence of endogenous arginine-vasopressin

Recent studies from our laboratory indicate a primary central site of action of Angiotensin II (AII) to release ACTH. The present studies were designed to test whether AII is able to release ACTH in vivo in a similar fashion in intact, cannulated, freely moving Long-Evans (LE) or in vasopressin (AVP)-deficient, Brattleboro (DI) female rats. The in vivo response to AII was compared with that elicited by synthetic CRF. AII injected i.v. (0.4 or 2 micrograms/100 g BW) induced a significant, dose-related increase in plasma ACTH values 5 and 15 min after injection, in both LE and DI rats. CRF given to LE and DI rats at 0.4 micrograms/100 g BW elicited a larger increase in ACTH plasma values than a similar dose of AII, 5 or 15 min after the injection. Moreover, ACTH levels after CRF in DI rats were significantly greater than those obtained in LE controls. In vitro studies using dispersed anterior pituitary cells indicate that the response of cells from either LE or DI rats to AII or AVP (both at 10(-9) and 10(-8)M) was similar. Cells from DI donors were hyperresponsive to CRF (2 X 10(-11) and 10(-10)M) in terms of ACTH release when compared with the response of cells from LE rats. The present results suggest that the presence of AVP is not essential to mediate the central response to AII and that AII may act centrally to stimulate CRF release from the hypothalamus in vivo, which would then enhance ACTH output. The results in the DI rat indicate that the increased response to CRF may be an important compensatory mechanism involved in the regulation of adrenocortical function in the DI rat.     

Induction of lactogenic receptors. II. Studies on the liver of hypophysectomized male rats and on rats bearing a growth hormone secreting tumor.

The role of growth hormone (GH), as well as prolactin and ACTH, in the induction of the PRL receptor was investigated both in hypophysectomized male rat livers and in the livers of male rats bearing a GH secreting tumor. After 7 days of s.c. injections, specific binding (% SB) of PRL in controls and rats treated with oPRL, hGH, ACTH, hCG, estradiol (E2), or testosterone (T) was approximately 1%. Treatment with oPRL plus ACTH increased SB to 4%; adding E2 to this combination produced a further increase to 8%, whereas the addition of T decreased hepatic binding to 1%. Combination of hGH with ACTH was most effective, giving a SB of 33%, which is similar to that observed in the liver of rats bearing a GH secreting tumor (36%). These studies suggest that GH acts synergistically with PRL and/or ACTH to increase lactogenic binding sites in the male rat liver and that sex steroids have a modulating effect on this action.     

Morphine stimulates prolactin release in normal but not in castrated male rats

Morphine (200 micrograms/rat) was injected intraventricularly (i.v.t.) into normal and into long-term castrated (4 weeks) adult male rats. Animals were killed 10, 20, 40 and 60 min after treatment. In normal animals, the treatment with morphine resulted in a significant increase of serum prolactin concentrations at all time intervals considered. However, the i.v.t. injection of 200 micrograms morphine/rat into castrated rats did not exert any significant effect on prolactin release at any time interval considered. When morphine (200 micrograms/rat) was administered i.v.t. together with the specific opioid receptor blocker naloxone (7.5 or 15 micrograms/rat) the stimulatory effect of morphine on prolactin release was diminished at 10 min, and totally blocked at 20 min. Naloxone given alone did not influence serum prolactin concentrations. The results suggest that the presence of endogenous androgens is essential to permit the stimulatory effect of morphine on prolactin release.