Angiotensin II receptors in the gonads.

The presence of components of the renin-angiotensin system in ovaries and testes suggests that angiotensin II (AII) is involved in gonadal function, and thus we sought to characterize receptors for AII in rat and primate gonads. In the testes, autoradiographic studies showed receptors in the interstitium in all species. In rat interstitial cells fractionated by Percoll gradient, AII receptors coincided with hCG receptors indicating that AII receptors are located on the Leydig cells. In Leydig cells and membranes from rat and rhesus monkey prepuberal testes, AII receptors were specific for AII analogues and of high affinity (Kd=nM). During development, AII receptor content in rat testes decreases with age parallel to a fall in the ratio of interstitial to tubular tissue. In the ovary, the distribution of AII receptors was dependent on the stage of development, being high in the germinal epithelium and stromal tissue between five and 15 days, and becoming localized in secondary follicles in 20-and 40-day-old rats. No binding was found in primordial or primary follicles. In rhesus monkey ovary, AII receptors were higher in stromal tissue and lower in granulosa and luteal cells of the follicles. Characterization of the binding in rat and monkey ovarian membranes showed a single class of sites with a Kd in the nmol/L range and specificity similar to that of the adrenal glomerulosa and testicular AII receptors. Receptors for AII were also present in membrane fractions from PMSG/hCG primed rat ovaries. Infusion of AII (25 ng/min) or captopril (1.4 micrograms/min) during the PMSG/hCG induction period had no effect on ovarian weight or AII receptor concentration in the ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticotropin-releasing factor receptors and pituitary adrenal responses during immobilization stress.

The regulation of pituitary and brain CRF receptors and corticotroph responses during stress were studied in rats subjected to prolonged immobilization. Plasma ACTH levels showed the characteristic biphasic changes, with a rapid 23-fold increase in 15 min, followed by a decrease to about twice the basal levels after 6-h immobilization. In contrast, plasma corticosterone levels were markedly elevated throughout the duration of the stress. Pituitary CRF receptor content, measured by binding of [125I]Tyr-ovine CRF to pituitary membrane-rich fractions, was unchanged after 2.5 h, but was reduced by 28 +/- 2.7% (+/- SE) and 47.6 +/- 1.1% after 18 and 48 h of immobilization, respectively. These results were confirmed by autoradiography in slide-mounted frozen pituitary sections. In contrast, no changes in CRF receptor content were observed in brain areas, including olfactory bulb, frontoparietal cortex, hippocampus, amygdala, and lateral septum. A concomitant decrease in immunoreactive (ir) CRF content in the median eminence of rats immobilized for 48 h is consistent with the hypothesis that increased release of CRF into the portal circulation occurs during chronic stress. Despite pituitary CRF receptor loss and reduced in vitro responses to CRF, the increases in plasma ACTH and corticosterone in vivo after ether exposure or CRF injection were greater and more prolonged in rats immobilized for 48 h than in nonimmobilized controls. The decrease in pituitary CRF receptors was accompanied by decreased CRF-stimulated cAMP and ACTH release in cultured pituitary cells from 48-h restrained rats. However, concomitant incubation of cells with CRF and vasopressin restored cAMP and ACTH responses to control levels, suggesting that the simultaneous release of both regulators from the hypothalamus determines the plasma ACTH level. These findings indicate that the decrease in plasma ACTH during the adaptation phase to stress is accompanied by decreases in pituitary CRF receptors. However, the enhanced pituitary response to a superimposed stress or CRF injection implies that the decrease in plasma ACTH levels during prolonged stress may be due to adaptive changes at the central level. These findings emphasize the importance of the integrated actions of CRF and other regulators in the control of the pituitary adrenal-axis during stress.

Corticotropin-releasing factor (CRF) induces desensitization of the rat pituitary CRF receptor-adenylate cyclase complex.

The rise in circulating ACTH levels after adrenalectomy in the rat is associated with a decrease in CRF receptor-binding capacity in the anterior pituitary. To investigate the role of increased hypothalamic CRF release on pituitary CRF receptor regulation after withdrawal of glucocorticoid feedback by adrenalectomy, the effects of chronic CRF infusion and lesions in the medial basal hypothalamus were studied in the rat. Subcutaneous infusion of CRF at 10, 25, 50, and 100 ng/min for 48 h in intact rats caused dose-dependent increases in plasma ACTH levels from the control value of 32.1 +/- 4.3 to 58.0 +/- 4.9, 82.0 +/- 7.1, 135.5 +/- 11.6, and 149.2 +/- 13.2 pg/ml, respectively. In contrast, the pituitary CRF receptor concentration was reduced by 25.3 +/- 4.5%, 38.3 +/- 2.5%, 43.8 +/- 0.9%, and 45.8 +/- 2.0%, respectively. Intravenous infusion of increasing doses of CRF caused a similar increase in plasma ACTH levels, which became maximum at the lowest infusion dose (32.4 +/- 5.4, 138.5 +/- 12.3, 162.0 +/- 18.3, and 167 +/- 19.1 pg/ml for control and 10, 50, and 100 ng/min CRF, respectively). Pituitary CRF receptor concentration was again decreased after iv CRF infusion [by 42 +/- 6.2% with the lowest dose (10 ng/min)], with no further reduction after infusion of 50 and 100 ng/min (49.0 +/- 6.8% and 26.0 +/- 6.2%, respectively)]. The decrease in pituitary CRF receptors after CRF infusion was accompanied by a decrease in CRF-stimulated adenylate cyclase activity, with a 10- to 100-fold increase in the concentration of CRF required for threshold stimulation. In cultured pituitary cells prepared from animals infused with 50 ng/min CRF for 48 h, maximum CRF-stimulated ACTH release was reduced by 29 +/- 3.2% (P less than 0.01; n = 3), with no significant change in sensitivity to CRF (ED50, 0.6 +/- 0.5 and 1.0 +/- 0.5 nM CRF for control and CRF infusion, respectively). The role of endogenous CRF in adrenalectomy-induced pituitary CRF receptor down-regulation was also studied in rats with medial basal hypothalamic deafferentation. The marked loss of pituitary CRF receptors after adrenalectomy was completely prevented by such hypothalamic lesioning, indicating that receptor down-regulation was dependent on the release of CRF or/and other hypothalamic factors. The data demonstrate that while increased CRF levels result in down-regulation and desensitization of pituitary CRF receptors, the differences between adrenalectomy and CRF infusion indicate that additional regulatory factors are involved in the modulation of CRF receptor content and activity after adrenalectomy.

Phorbol 12-myristate 13-acetate and vasopressin potentiate the effect of corticotropin-releasing factor on cyclic AMP production in rat anterior pituitary cells. Mechanisms of action.

The potentiation of corticotropin-releasing factor (CRF)-stimulated cAMP production by vasopressin (VP) in the pituitary cell was investigated by studies on the interaction of CRF, VP, and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) on cAMP, adenylate cyclase and phosphodiesterase. Addition of VP or PMA (0.01-100 nM) alone did not alter cellular cAMP content, but markedly increased the effect of 10 nM CRF with ED50 of about 1 nM. Treatment of the cells with 200 ng/ml pertussis toxin for 4 h increased CRF-stimulated cAMP accumulation by 3.2-fold, an effect that was not additive to those of VP and PMA. Incubation of pituitary cells with 2 mM 1-methyl-3-isobutylxanthine increased CRF-stimulated cAMP accumulation and decreased the relative effect of VP and PMA, suggesting that the actions of VP and PMA are partially due to inhibition of phosphodiesterase. This was confirmed by the demonstration of a 30% inhibition of the low-affinity phosphodiesterase activity in cytosol and membranes prepared from cells preincubated with VP or PMA. In intact cells, following [3H]adenine prelabeling of endogenous ATP pools, measurement of adenylate cyclase in the presence of 1-methyl-3-isobutylxanthine showed no effect of VP and PMA alone, but did show a 2-fold potentiation of the effect of CRF. Measurement of adenylate cyclase in pituitary homogenates by conversion of [alpha-32P]ATP to [32P]cAMP showed a paradoxical GTP-dependent inhibition by VP of basal and CRF-stimulated adenylate cyclase activity, suggesting that the VP receptor is coupled to an inhibitory guanyl nucleotide-binding protein. Pertussis toxin pretreatment of the cells prevented the VP inhibition of adenylate cyclase activity observed in pituitary cell homogenates. These findings indicate that besides inhibition of phosphodiesterase, VP has a dual interaction with the pituitary adenylate cyclase system; a direct inhibitory effect, manifested only in broken cells, that is mediated by a receptor-coupled guanyl nucleotide-binding protein, and a physiologically predominant indirect stimulatory effect in the intact cell, mediated by protein kinase C phosphorylation of one of the components of the CRF-activated adenylate cyclase system.

Functional corticotropin releasing factor receptors in the primate peripheral sympathetic nervous system.

Corticotropin releasing factor (CRF) is a key hormone in the integrated response to stress, acting both as the major regulator of pituitary adrenocorticotropic hormone (ACTH) release and as a neuropeptide in the brain. The actions of CRF are mediated by specific plasma membrane receptors in the anterior pituitary gland and in discrete brain areas including the cerebral cortex and several regions related to the limbic system. In addition to the pituitary and central actions of CRF, systemic administration of the peptide in the rat, dog, monkey and man causes hypotension and tachycardia because of a decrease in peripheral vascular resistance. These observations, in conjunction with the finding of immunoreactive and bioactive CRF in peripheral tissues, suggest that the peptide is locally released in tissues to act as a neurotransmitter or paracrine hormone. As CRF is present in the adrenal medulla and the peptide is known to modulate the central activity of the autonomic nervous system, we investigated the possibility that CRF is involved in the regulation of the peripheral autonomic nervous system. Such an action of CRF is supported by our demonstration of specific CRF receptors in the monkey adrenal medulla and sympathetic ganglia. In the adrenal medulla, these receptors are coupled to adenylate cyclase and can stimulate the secretion of catecholamines and Met-enkephalin.

Effect of long-term feeding of soy-based diets on the pancreas of Cebus monkeys.

Feeding soy-based protein containing trypsin inhibitor causes pancreatic hypertrophy in the rat, and long-term feeding (up to 2 years) has revealed a high incidence of adenoma following hypertrophy. It was therefore of interest to determine whether the ingestion of soy-based protein has any adverse effects on the primate pancreas. A resource of 27 Cebus albifrons monkeys, previously used to evaluate the protein quality of several soy and milk proteins, has been maintained on semi-synthetic diets for 3 to 4 years; the protein sources for the diets were casein, lactalbumin, soy isolate and soy concentrate. In general the monkeys were in good physical health and their weights were appropriate for age and sex. Serum biochemical and hematological profiles were normal and there were no major differences between the groups. A pancreatic biopsy from both the head and tail region of the pancreas was taken from each monkey. Visual observation of the pancreas revealed no overt pathology; two independent histological examinations indicated no diet-related differences between groups, and biochemical analyses of trypsin, chymotrypsin, protein, DNA and RNA revealed no differences. It is concluded that feeding low level trypsin inhibitor-containing diets for up to 4 years caused no adverse effects in the pancreas of the Cebus nonhuman primate.

Receptor-mediated actions of corticotropin-releasing factor in pituitary gland and nervous system.

High-affinity corticotropin-releasing factor (CRF) receptors which mediate the actions of the hypothalamic peptide on adrenocorticotropic hormone (ACTH) release have been identified in the rat anterior pituitary gland. Occupancy of the pituitary receptor by CRF agonists stimulates ACTH release via activation of adenylate cyclase and cyclic adenosine monophosphate dependent protein kinase. In the regulation of ACTH secretion, the effects of CRF on the corticotroph are integrated with the stimulatory actions of cyclic adenosine monophosphate-independent stimuli such as angiotensin II, vasopressin and norepinephrine, and the inhibitory effects of glucocorticoids and somatostatin. In contrast to the major importance of the inhibitory effect of glucocorticoid feedback on ACTH secretion, somatostatin has relatively little effect on CRF-stimulated ACTH release in the normal rat corticotroph. Following adrenalectomy, the progressive elevation of plasma ACTH levels is accompanied by a concomitant decrease in pituitary CRF receptors. The postadrenalectomy loss of CRF receptors, which is prevented by dexamethasone treatment, is caused by a combination of occupancy and processing of the pituitary sites during increased secretion of the hypothalamic peptide. Recently, specific receptors for CRF have been localized in the rat and monkey brain and adrenal medulla, where they are also coupled to adenylate cyclase. Brain CRF receptors are most abundant in the cerebral and cerebellar cortices and in structures related to the limbic system and control of the autonomic nervous system. The actions of CRF on the central and peripheral nervous systems, as well as on the pituitary gland, emphasize the role of CRF as a key hormone in the integrated response to stress.

The effects of long-term soy protein and milk protein feeding on the pancreas of Cebus albifrons monkeys.

Twenty-seven 2- to 4-yr-old cebus monkeys (Cebus albifrons) were fed from infancy purified diets containing lactalbumin, soy isolate, casein or soy concentrate as the sole protein source. Hematologic and clinical chemistry values were similar for all groups. Head and tail portions of each pancreas were surgically removed for histopathologic evaluation and determination of protein, RNA and DNA content, and for trypsin and chymotrypsin activity. Hematoxylin and eosin-stained sections from 26 of 27 monkeys showed normal pancreatic tissue with occasional acinar vacuolation in all diet groups. The remaining animal, one of only two fed soy concentrate, had diffuse interstitial fibrosis of the pancreas associated with mild to moderate atrophy of acinar tissue. Biochemical analyses of the pancreatic biopsies indicated no group differences among animals fed lactalbumin, soy isolate or casein. One of two monkeys in the soy concentrate group showed decreased pancreatic protein, RNA and trypsin concentrations; this was probably due to the fibrosis in this animal. No evidence of pancreatic hypertrophy or hyperplasia, as measured by RNA/DNA and protein/DNA ratios, respectively, was seen in any diet group.

Regulation of corticotropin-releasing factor (CRF) receptors in the rat pituitary gland: effects of adrenalectomy on CRF receptors and corticotroph responses.

The stimulation of ACTH release from anterior pituitary cells by corticotropin-releasing factor (CRF) is mediated by specific, high affinity receptors with a Ka of 10(9) M-1 for ovine CRF. The relationship between ACTH secretion and CRF receptor activation was analyzed in normal and adrenalectomized rats by comparison of ACTH release with changes in CRF receptors and adenylate cyclase activity. The marked increase in plasma ACTH levels that occurred after adrenalectomy (from 71 to 478 pg/ml after 4 days) was accompanied by a progressive decrease in pituitary CRF receptor concentration [by 29 +/- 1%, 75 +/- 2%, 77 +/- 6%, and 80 +/- 4% (+/- SE) after 1, 2, 3, and 4 days, respectively]. Most of this decrease was due to receptor down-regulation rather than occupancy by endogenous CRF, since high dose infusions of CRF (300-500 ng/min) for 30 min before pituitary membrane preparation reduced CRF-binding sites by only 40%. The marked reduction in CRF receptors after adrenalectomy was accompanied by comparable decreases in maximal CRF-stimulated adenylate cyclase activity and sensitivity to CRF (ED50, 3.8 +/- 2.8 vs. 58 +/- 3.7 X 10-9 M CRF in control and 2-day-adrenalectomized rats, respectively). Fluoride-stimulated adenylate cyclase activity was unchanged at 24 h, but was decreased by 28 +/- 7% at later times. Such decreases in CRF receptors and adenylate cyclase activity in adrenalectomized rats were prevented by dexamethasone treatment. In cultured anterior pituitary cells from 4-day-adrenalectomized rats, CRF-stimulated cAMP production was decreased by 40%. However, in contrast to the decreases in CRF receptors and cAMP production, there was a 3-fold increase in CRF-stimulated ACTH release, with no change in sensitivity to CRF. The ability of corticotrophs to maintain increased ACTH release, in conjunction with reduced CRF receptors and CRF-stimulated adenylate cyclase, indicates that elevated ACTH secretion can be maintained by occupancy and activation of only a small number of CRF receptors. This finding also suggests that synergistic interactions between CRF and other regulators of ACTH release may contribute to the sustained increase in ACTH secretion that follows adrenalectomy.

Actions of growth hormone-releasing factor and somatostatin on adenylate cyclase and growth hormone release in rat anterior pituitary.

The interaction of growth hormone-releasing factor (GRF) and somatostatin (SRIF) on adenylate cyclase activity and growth hormone release was investigated in pituitary homogenates and 2-day cultured rat anterior pituitary cells. GRF stimulated growth hormone release by about 3-fold (ED50 1.6 X 10(-12) M) and caused a rapid 15-fold increase in cyclic AMP production (ED50 6.0 X 10(-12) M). The increase in cyclic AMP was due to direct stimulation of adenylate cyclase by GRF, which caused a 4-fold increase in the activity of the enzyme measured in anterior pituitary homogenates. GRF-induced cyclic AMP formation and GRF-stimulated adenylate cyclase activity were maximally inhibited to the extent of about 50% by 10(-8) M somatostatin. In contrast, GRF-stimulated growth hormone release was completely inhibited by somatostatin (ID50 3.2 X 10(-11) M), suggesting a second site of action of somatostatin. These studies demonstrate that GRF stimulates growth hormone release via activation of adenylate cyclase and a rise in intracellular cyclic AMP. In addition, these findings indicate that the inhibitory action of somatostatin on growth hormone release is exerted at two levels, one at the level of adenylate cyclase affecting the production of cyclic AMP, and the other beyond the formation of the nucleotide, at a site which modulates the release of growth hormone from the cell.

Mechanisms of action of corticotropin-releasing factor and other regulators of corticotropin release in rat pituitary cells.

The role of cyclic AMP in the stimulation of corticotropin (ACTH) release by corticotropin-releasing factor (CRF), angiotensin II (AII), vasopressin (VP), and norepinephrine (NE) was examined in cultured rat anterior pituitary cells. Synthetic CRF rapidly stimulated cyclic AMP production, from 4- to 6-fold in 3 min to a maximum of 10- to 15-fold at 30 min. Stimulation of ACTH release by increasing concentrations of CRF was accompanied by a parallel increase in cyclic AMP formation, with ED50 values of 0.5 and 1.3 nM CRF for ACTH and cyclic AMP, respectively. A good correlation between cyclic AMP formation and ACTH release was also found when pituitary cells were incubated with the synthetic CRF(15-41) fragment, which displayed full agonist activity on both cyclic AMP and ACTH release with about 0.1% of the potency of the intact peptide. In contrast, the CRF(21-41) and CRF(36-41) fragments were completely inactive. The other regulators were less effective stimuli of ACTH release and caused either no change in cyclic AMP (AII and VP) or a 50% decrease in cyclic AMP (NE). Addition of the phosphodiesterase inhibitor, methylisobutylxanthine, increased the sensitivity of the ACTH response to CRF but did not change the responses to AII, VP, and NE. In pituitary membranes, adenylate cyclase activity was stimulated by CRF in a dose-dependent manner with ED50 of 0.28 nM, indicating that the CRF-induced elevation of cyclic AMP production in intact pituitary cells is due to increased cyclic AMP biosynthesis. The intermediate role of cyclic AMP in the stimulation of ACTH release by CRF was further indicated by the dose-related increase in cyclic AMP-dependent protein kinase activity in pituitary cells stimulated by CRF with ED50 of 1.1 nM. These data demonstrate that the action of CRF on ACTH release is mediated by the adenylate cyclase-protein kinase pathway and that the sequence requirement for bioactivity includes the COOH-terminal 27 amino acid residues of the molecule. The other recognized regulators of ACTH release are less effective stimuli than CRF and do not exert their actions on the corticotroph through cyclic AMP-dependent mechanisms.

Somatostatin modulates effects of angiotensin II in adrenal glomerulosa zone.

The octapeptide angiotensin II is a major regulator of the adrenal glomerulosa zone, acting both as an acute stimulus of aldosterone secretion and as a trophic hormone which increases steroidogenic enzymes and angiotensin II receptors in glomerulosa cells. Angiotensin II also mediates the adrenal effects of altered sodium balance, and is essential for the aldosterone response to sodium restriction. However, the adrenal effects of angiotensin II are attenuated during sodium loading, suggesting that other local or humoral factors modulate its actions on adrenal glomerulosa function. Somatostatin, the somatotropin release inhibiting factor of the hypothalamus, has been shown to inhibit the secretion and action of several pituitary and non-pituitary hormones. Because somatostatin has been found in several non-neural tissues, and seems to act as a local regulator of endocrine function, we have now examined the possibility that it may also modulate the effects of angiotensin II in the adrenal glomerulosa cell. Our studies have shown that low concentrations of somatostatin specificity inhibit the production of angiotensin II-stimulated aldosterone, and that this action is mediated by specific, high-affinity receptors for somatostatin in the zona glomerulosa.

Lactogenic receptor regulation in hormone-stimulated steroidogenic cells.

Receptor sites for lactogenic hormones such as prolactin (PRL), human growth hormone (HGH), and placental lactogens, are widely distributed in mammalian tissues, including mammary glands, steroid-secreting cells of the adrenal, testis, and ovary, and target cells of steroid hormone action such as liver, prostrate, and kidney. Although the biological functions of lactogen binding sites remain uncertain, a relationship between prolactin and lipoprotein metabolism is implied by the occurrence of prolactin receptors in steroidogenic cells of the gonads and adrenal, and by the ability of prolactin to increase esterified cholesterol in the testis. Recently, loss of testicular prolactin receptors has been observed following elevation of circulating luteinising hormone (LH) concentrations by the gonadotropin releasing hormone (GnRH) and its agonist analogues. The hormone dependence of lactogen receptor sites in steroid-secreting cells was further analysed in rat testis, ovary, and adrenal glands after treatment with the respective trophic hormones, gonadotropin and ACTH. In each of these tissues, rapid and transient loss of lactogen receptors was observed after trophic hormone stimulation. These findings indicate that increased turnover of lactogen receptors is an important component of the target-cell response, and suggest that prolactin receptors might be involved in the transport of lipoprotein precursors for steroid biosynthesis.

Gonadotropin-releasing hormone analogue binds to luteal cells and inhibits progesterone production.

Although gonadotropin-releasing hormone (GnRH) is believed to mediate the hypothalamic control of pituitary gonadotropin secretion, continuous or repeated administration of GnRH or its agonist analogues has been shown to cause paradoxical antifertility effects in several species, including primates. GnRH-induced interruption of reproductive cycles and pregnancy is associated with diminished progesterone production, implying defective function of the corpus luteum. These luteolytic effects have been attributed to the well recognized desensitising actions of elevated luteinising hormone (LH) levels on ovarian LH receptors and steroidogenesis, subsequent to GnRH-induced gonadotropin release from the anterior pituitary. However, treatment with high doses of exogenous LH did not cause suppression of serum progesterone levels during early pregnancy in rats, whereas a highly active GnRH analogue was effective in this regard. These observations suggested that GnRH and its agonist analogues, given in high or sustained doses, can exert a direct action on the ovary that is independent of the pituitary. This hypothesis was further supported by the ability of GnRH and its agonists to inhibit human chorionic gonadotropin (HCG)-induced ovarian and uterine weight gain in hypophysectomised rats and to delay the onset of puberty in intact female rats. Also, GnRH and its agonist analogues have recently been shown to inhibit steroidogenesis induced by follicle-stimulating hormone (FSH) in cultured granulosa cells, confirming the direct action of such peptides on the ovarian follicle. The marked inhibitory effects of GnRH and its agonists on corpus luteum function suggest that these compounds could exert direct actions by binding to specific receptors on luteal cells. The present experiments, which examine the effects of GnRH agonists on luteal steroidogenesis, demonstrate the existence of such actions and their mediation by specific high-affinity receptor sites present in luteal cell membranes.