Somatostatin receptor subtypes 2 and 5 inhibit corticotropin-releasing hormone-stimulated adrenocorticotropin secretion from AtT-20 cells

Somatostatin (SRIH) regulates pituitary adrenocorticotropin (ACTH) secretion by interacting with a family of homologous G protein-coupled membrane receptors. The SRIH receptor subtypes (sst(1)-sst(5)) that control ACTH release remain unknown. Using novel, subtype-selective SRIH analogs, we have identified the SRIH receptor subtypes involved in regulating ACTH release from AtT-20 cells, a model for cell line pituitary corticotropes. Radioligand-binding studies with (125)I-SRIH-14 and (125)I-SRIH-28 showed that SRIH-14 and SRIH-28 recognized specific, high-affinity and saturable membrane-binding sites. Nonpeptidyl agonists with selectivity for the sst(2) (L-779,976; compound 2) or sst(1)/sst(5)) (L-817,818; compound 5) receptor subtypes potently displaced (125)I-SRIH-28 from AtT-20 cell membranes, while agonists selective for the sst(1) (L-779,591; compound 1), sst(3) (L-796,778; compound 3) or sst(4) (L-803,087; compound 4) subtypes were inactive. Tyr(11)-SRIH-14, compound 2 (sst(2)) or compound 5 (sst(5)) inhibited forskolin and corticotropin-releasing hormone (CRH)-induced increases in intracellular cAMP. Furthermore, the sst(2) and sst(5) agonists potently inhibited CRH-induced ACTH release from AtT-20 cells. These results provide the first evidence that sst(2) and sst(5) receptor subtypes, but not sst(1), sst(3) or sst(4), inhibit cAMP accumulation and regulate ACTH secretion in the AtT-20 cell model of the rodent corticotrope.     

Free fatty acids inhibit adrenocorticotropin and cortisol secretion stimulated by physical exercise in normal men

BACKGROUND: The basal circulating levels of ACTH and cortisol, but not the ACTH/cortisol response to hCRH, are significantly reduced by free fatty acid (FFA) infusion. OBJECTIVE: To verify whether FFA infusion modifies the ACTH/cortisol response to physical exercise, a well-known activator of the HPA axis at suprapituitary level. DESIGN: Exercise tests on a bicycle ergometer during infusion of a lipid-heparin emulsion (LHE) (experimental test) or normal saline (NaCl 0.9%) (control test). SETTING: Department of Cardiology at the University-Hospital. SUBJECTS: Seven healthy male subjects aged 25-33 years. INTERVENTIONS: On two mornings, at weekly intervals, LHE or saline were infused for 60 min; infusion started 10 min before exercise test on a bicycle ergometer, which lasted about 15 min. MAIN OUTCOME MEASURES: Circulating ACTH/cortisol levels and physiological variables during physical exercise. RESULTS: FFA levels (0.4 +/- 0.1 mEq/l) remained constant during control test, whereas they progressively rose (peak at 60 min, 2.7 +/- 1.0 mEq/l) during LHE infusion. Neither basal nor exercise-induced changes in physiological variables were modified by LHE infusion. Both ACTH and cortisol increased during exercise, with peak levels at 20 min and 30 min (control test: 103% and 42%, P < 0.001; experimental test: 28.5% and 18.6%, P < 0.05 higher than baseline, respectively). Both ACTH and cortisol responses were significantly lower in the experimental than in the control test (at 20 min P < 0.002 and at 30 min P < 0.05 for ACTH; at 20 min P < 0.05 and at 30 min, 40 min and 50 min P < 0.001 for cortisol). CONCLUSIONS: These data represent the first demonstration of an inhibitory action of increased circulating FFA levels on the HPA axis under stimulatory conditions (i.e. physical exercise, a challenge acting at suprapituitary level). In contrast, previous studies did not show FFA effects on the CRH-induced ACTH/cortisol response. Therefore, our data suggest negative effects of FFAs on the HPA axis at hypothalamic or higher centres in the central nervous system.     

Cytokines act within the brain to inhibit luteinizing hormone secretion and ovulation in the rat

Alterations of immune activity are often accompanied by reproductive disorders. Because interleukins mediate the host's response to immune activation, we first examined the effect of the central injection of several lymphokines on LH secretion by gonadectomized rats. We then studied the ability of the most potent lymphokine in this system, interleukin-1 beta (Il-1 beta), to interfere with the proestrous LH surge and ovulation in the intact female rat as well as the dependence of this effect on the activation of opiate receptors. Finally, we investigated the possibility that increased brain levels of Ils, as induced by the central administration of a bacterial endotoxin, might also alter the normal ovulatory process. After intracerebroventricular (icv) injection, Il-1 beta, Il-6, and tumor necrosis factor all lowered plasma LH levels in castrated rats. On a molar basis, Il-1 beta was the most potent inhibitor of LH secretion. In gonadectomized animals, 2.5 and 10 ng Il-1 beta administered icv significantly (P less than or equal to 0.01) decreased plasma LH levels for 6 h, while the effect of 40 ng lasted up to 12 h. By contrast, FSH secretion was only measurably altered by 40 ng Il-1 beta 8 and 12 h posttreatment. In intact cycling female rats, icv injection of 40 ng Il-1 beta or 400 ng Il-1 alpha at 0830 h on the morning of proestrus significantly (P less than 0.01 and P less than 0.05, respectively) interfered with the proestrous surge of LH. In contrast, the peripheral administration of either lymphokine was without measurable effect. Centrally injected Il-1 beta (40 ng at 0830 h on proestrus) also blocked ovulation in most animals, while a dose of 25 ng injected iv was ineffective. Finally, we observed that the icv injection of endotoxin (a lipopolysaccharide), also interfered with ovulation. The possible involvement of opiate-dependent pathways in mediating the inhibitory action of Il-1 beta on reproductive processes was tested by implanting naloxone pellets 16-24 h before lymphokine treatment. The pellets, which released 200, 400, or 800 micrograms naloxone/h (corresponding to approximately 0.6, 1.32, or 2.64 mg/kg BW.h), had no effect on ovulation by themselves. When given before icv Il-1 beta, all naloxone regimens countered the effect of the cytokine, with the 800-micrograms/h dose restoring ovulation in eight of nine rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hypophysiotropic regulation of adrenocorticotropin secretion in response to insulin-induced hypoglycemia

The hypophysiotropic coding of ACTH secretion resulting from insulin-induced hypoglycemia was investigated in urethane-anesthetized fasted rats. The participation of corticotropin-releasing factor (CRF), arginine vasopressin (AVP), and catecholamines in the ACTH response was first investigated by systemic administration of CRF antiserum, an AVP pressor antagonist, or a ganglionic blocking agent. These treatments were without effect on the hypoglycemic response, which was characterized by a 67% fall in systemic glucose levels within 30 min of insulin administration. ACTH secretion in response to insulin-induced hypoglycemia was differentially affected by these pharmacological treatments. Administration of antiserum to CRF abolished the ACTH response, whereas ganglionic blockade was without significant effect. However, administration of a vasopressinergic pressor antagonist significantly attenuated ACTH secretion after insulin treatment. These observations suggested the participation of both CRF and AVP in mediation of the ACTH secretory response to hypoglycemia. Infusion of glucose to counter the hypoglycemia action of insulin injection prevented the ACTH secretory response. Measurement of immunoreactive (ir) CRF, irAVP, and ir-oxytocin in sequential collections of hypophysial portal plasma revealed a significant elevation of irAVP concentration without concomitant elevation of irCRF or ir-oxytocin levels. We propose that CRF functions in a permissive role, maintaining a relatively constant portal concentration and thereby allowing expression of the weaker ACTH-releasing activity of AVP and other secretagogues. Thus, AVP, not CRF, appears to represent the dynamic mediator of ACTH secretion accompanying insulin-induced hypoglycemia. These observations provide additional support for the hypothesis of multifactor stimulus-specific hypophysiotropic coding of ACTH secretion.     

Inhibition of brain prostaglandin endoperoxide synthase-2 prevents the preparturient increase in fetal adrenocorticotropin secretion in the sheep fetus

Maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. Recently, we proposed that prostaglandin generation within the fetal central nervous system is critical for the modulation of hypotension-induced fetal ACTH secretion. The present study was designed to test the hypothesis that the preparturient increase in fetal ACTH secretion is dependent upon fetal central nervous system prostaglandin synthesis mediated by the activity of prostaglandin endoperoxide synthase (PGHS)-2 (cyclooxygenase-2) in the fetal brain. We performed two studies in chronically catheterized fetal sheep. In the first study, we infused nimesulide or vehicle intracerebroventricularly (i.c.v) into singleton fetal sheep and collected blood samples until spontaneous parturition. Nimesulide significantly delayed parturition, and inhibited fetal ACTH and proopiomelanocortin secretion but did not prevent the preparturient increase in fetal plasma cortisol concentration. In the second study, we used twin fetuses. One fetus received intracerebroventricular nimesulide and the other intracerebroventricular vehicle. Nimesulide reduced brain tissue concentrations of prostaglandin estradiol, while not affecting plasma prostaglandin E(2) concentrations, demonstrating an action restricted to the fetal brain. Nimesulide reduced PGHS-2 mRNA and increased PGHS-2 protein, while not altering PGHS-1 mRNA or protein in most brain regions, suggesting an effect of the inhibitor on PGHS-2 turnover and relative specificity for PGHS-2 in vivo. We conclude that the preparturient increase in fetal ACTH and proopiomelanocortin is dependent upon the activity of PGHS-2 in the fetal brain. However, we also conclude that the timing of parturition is not solely dependent upon ACTH in this species.     

Physiological concentrations of atrial natriuretic factors with intact N-terminal sequences inhibit corticotropin-releasing factor-stimulated adrenocorticotropin secretion from cultured anterior pituitary cells

The effectiveness of atrial natriuretic factors (ANFs) as inhibitors of CRF-stimulated ACTH secretion was examined in cultured rat anterior pituitary cells. alpha ANF-(1-28) significantly inhibited ACTH release stimulated by 1-5 nM CRF. At the most effective concentration of 100 pM, alpha ANF-(1-28) inhibited ACTH release by 40.1% (P less than 0.001). This effect was manifested after 3 h, but not after 0.5 or 1 h, of incubation, suggesting that ACTH synthesis may have been reduced. Conversely, alpha ANF-(5-28), at concentrations of 10 pM to 10 nM, had no effect on ACTH secretion after 0.5, 1, or 3 h. alpha ANF-(1-11) weakly inhibited ACTH secretion at concentrations of 100 and 1000 pM. Again, these effects were observed after 3 h, but not after 0.5 or 1 h, of incubation. These results suggest that ANF-induced inhibition of CRF-stimulated ACTH release requires 1) an intact N-terminal sequence of the ANF peptide, 2) low concentrations, and 3) more than 1 h of incubation. The results explain previous negative evidence and suggest that alpha ANF-(1-28) may be a physiological inhibitor of ACTH secretion.     

Studies on the site of action of vasopressin in inducing adrenocorticotropin secretion.

Hypophysectomized rats bearing three transplanted pituitaries under the kidney capsule responded to synthetic lysine vasopressin or pitressin with a significant elevation of plasma corticosterone, whereas hypophysectomized rats with no grafts did not. This response was completely abolished by pretreatment of animals with dexamethasone but was unaltered by central hypothalamic destruction. Corticotropin-releasing factor content of the hypothalamic median eminence, hypophyseal stal-, or pars nervosa of the posterior pituitary of intact rats was unchanged 5 or 10 min after ip injection of vasopressin compared to the basal level. We conclude that vasopressin and dexamethasone act directly on the adenohypophysis in vivo to exert their stimulatory or inhibitory effect on ACTH secretion.     

Role of central epinephrine on the regulation of corticotropin-releasing factor and adrenocorticotropin secretion

Epinephrine (EPI) has been described to stimulate the hypothalamic-pituitary-adrenal axis. However, whether central EPI neuronal systems play a major physiological role in the regulation of ACTH secretion and whether that role is primarily stimulatory or inhibitory in nature is still controversial. The present study addressed these questions using different inhibitors of phenylethanolamine-N-methyltransferase (PNMT), which were either active peripherally or were both peripherally and centrally active. Male rats received either vehicle or a PNMT inhibitor at various times before further experimental procedures. A large decrease in hypothalamic EPI levels was observed in rats given central PNMT inhibitors, whereas these treatments did not affect hypothalamic norepinephrine (NE) levels. Plasma EPI, but not NE, was decreased to similar levels after treatment with peripheral or central PNMT inhibitors. Basal plasma ACTH decreased slightly during the 12 h after central PNMT inhibition. Central, but not peripheral, inhibition of PNMT significantly decreased the plasma ACTH response to ether vapor stress at 5 and 15 min. This effect was seen 3 or 12 h after PNMT inhibition. The suppression of the stress response was not due to a change in responsiveness of the pituitary to CRF. The hypothalamic content of CRF was significantly decreased 9 and 12 h after inhibition of central PNMT. Blockade of the stress response actually preceded the changes in CRF levels. The content of arginine vasopressin, another potent ACTH secretagogue, was not affected 3, 6, 9, or 12 h after that treatment. The effect on CRF was not observed in rats treated with the peripheral PNMT inhibitor, nor was it caused by manipulation and stress of the animals 12 h before death. The dat demonstrate that central inhibition of PNMT, which produces a selective decrease in hypothalamic EPI levels, blunts the response of plasma ACTH to ether vapor stress, and at later times also causes a selective decrease in CRF content. Furthermore, the altered ACTH response to ether stress is not due to a change in responsiveness of the pituitary to CRF or to an alteration in arginine vasopressin levels. Thus, an endogenous EPI neuronal system appears to stimulate CRF neurons responsible for the increase in ACTH after ether vapor stress.     

Role of the second-messenger cyclic-adenosine 5'-diphosphate-ribose on adrenocorticotropin secretion from pituitary cells

We examined the role of the second-messenger cyclic-ADP-ribose (cADPR) on the regulation of ACTH secretion using AtT20 corticotroph tumor cell line. We found that the cADPR antagonist, 8-Br-cADPR, substantially diminished the secretion of ACTH induced by CRH and potassium in these cells, whereas xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, had no effect. In addition, the cADPR agonist, 3-deaza-cADPR, augmented ACTH secretion. The presence of the components of the cADPR system, namely ryanodine receptor, CD38, and cADPR itself, was determined in AtT20 cells. Furthermore, we observed that antagonists of the ryanodine channel and cADPR system can decrease the potassium-induced Ca2+ transients in these cells. These results suggest that cADPR is a second messenger in pituitary cells and regulates ACTH secretion by a mechanism dependent on activation of the ryanodine channel by extracellular Ca2+.     

Corticosterone can act at the posterior paraventricular thalamus to inhibit hypothalamic-pituitary-adrenal activity in animals that habituate to repeated stress

Glucocorticoids released by stress bind to glucocorticoid (GR) and/or mineralocorticoid receptors (MR) to exert negative feedback of subsequent hypothalamic-pituitary-adrenal (HPA) responses to stress. Feedback inhibition is implicated in habituation of HPA activity to repeated exposure to the same (homotypic) stressor. We hypothesized that the posterior paraventricular thalamus (pPVTh) is a site where corticosterone acts to exert negative feedback during repeated stress and that is important for habituation. As previously reported, the pPVTh inhibits HPA responses to homotypic and heterotypic stressors in repeatedly, but not acutely, stressed rats. We conducted a series of experiments involving intra-pPVTh administration of MR and/or GR agonists or antagonists during different time frames over 8 d of restraint. MR exist in the pPVTh, as do GR as shown by our immunocytochemical results. Acute intra-pPVTh injection of MR and/or GR antagonist before the eighth restraint did not alter expression of habituation. Because habituation may develop before d 8, we manipulated GR and MR in the pPVTh throughout 8 d of stress using intra-pPVTh corticosterone implants, which enhanced habituation on d 8 without affecting acute stress responses. Conversely, daily intra-pPVTh injections of GR and MR antagonists on d 1-7 of restraint prevented habituation on d 8. These data suggest that corticosterone released during repeated stress can act at GR and MR in the pPVTh to inhibit HPA responses to homotypic stress. We also found that some GR-containing cells in the pPVTh project to the medial prefrontal cortex and basolateral amygdala, suggesting that pPVTh-induced inhibition of HPA activity is potentially mediated by its projections to these select limbic structures.     

Influence of adrenocorticotropin and adrenalectomy on gonadotropin secretion in immature rats

We examined the effects and mechanisms of action of ACTH and ACTH fragments on gonadotropin secretion in immature rats. ACTH administered by daily injection or continuous infusion (osmotic minipumps) attenuated the postcastration rise in serum LH. Pituitary LH concentration was either unchanged or increased in ACTH-treated rats and pituitary sensitivity to gonadotropin-releasing hormone (GnRH) was reduced by ACTH treatment. A fragment of ACTH (ACTH 4-10), which is less steroidogenic, did not alter levels of serum LH, and ACTH did not reduce LH secretion in adrenalectomized castrates. Serum and pituitary concentrations of prolactin were normal in ACTH-treated animals. These studies demonstrate that the suppression of gonadotropin secretion by ACTH is mediated by the adrenal gland. This mechanism causes a decreased pituitary sensitivity to GnRH, but LH synthesis does not appear to be affected. Prolactin does not play a role in this mechanism.     

Mutually independent effects of adrenocorticotropin on luteinizing hormone and testosterone secretion

In this study, we examined the effect of ACTH on the sensitivity of the testes to gonadotropin and determined the role of the testosterone (T) negative feedback system in mediating the inhibitory effect of ACTH on LH secretion in adult male rats. ACTH infusion for 3 days reduced basal levels of serum T and the T response to GnRH, but did not alter basal levels of serum LH (immunoreactive) or the LH response to GnRH. These effects required the presence of the adrenal glands. Infusion of corticosterone (B) at a dose that increased serum B concentrations 9-fold had an effect similar to that of ACTH on basal serum T levels and the serum T response to GnRH. Basal levels of serum LH and the serum LH response to GnRH were not affected by B administration. These data suggest that ACTH administration reduces the sensitivity of the testes to LH, resulting in a lower basal level of T and a reduced T response to GnRH. This effect was independent of basal serum LH levels or the LH response to GnRH. It appears that B mediates the effect of ACTH on testicular sensitivity to gonadotropin. In another experiment, ACTH administration for 4 days did not alter serum LH values, but reduced serum T levels in sham-castrated male rats. In contrast, ACTH treatment blunted the increase in serum LH after castration by day 2 of treatment, despite the absence of detectable levels of serum T within 6 h after castration. These data suggest that T is not essential for the inhibitory effect of ACTH on LH secretion to occur. They do not support the hypothesis that ACTH enhances the sensitivity of the hypothalamus and/or pituitary to the negative feedback effects of T.     

Starvation enhances the ability of insulin to inhibit its own secretion

To examine whether decreased insulin secretion during starvation is related to a change in the ability of insulin to inhibit its own secretion, plasma C-peptide was measured after plasma insulin levels were acutely raised by intravenous (IV) insulin infusion in a dose of 40 and 80 mU/M2/min in obese subjects before and after a 72 hour fast. Plasma glucose concentration was maintained +/- 4% of basal levels by a variable glucose infusion. During the 80 mU infusion, at plasma insulin levels of 200 microU/mL, plasma C-peptide fell by 0.17 pmol/mL in the fed state. In the fasted state, despite basal levels that were 36% lower, C-peptide decreased by 0.21 pmol/mL. Highly significant increases in percent suppression after fasting were noted during both 40 mU and 80 mU studies. The plasma C-peptide response was related to the insulin infusion dose in both the fed and fasted state. In contrast, alpha cell suppression by insulin, as determined by plasma glucagon levels, was not altered by fasting. It is concluded that enhanced inhibitory influences of insulin on the beta cell during starvation may be a physiologically important mechanism for diminished insulin secretion during the transition from the fed to the fasting state.     

Adrenocortical secretion of dehydroepiandrosterone in healthy women: highly variable response to adrenocorticotropin

Excess adrenal androgen (AA) levels are observed in 25--50% of women with the polycystic ovary syndrome (PCOS), and AA excess in PCOS may represent selection bias. Thus, it is possible that AA secretion among the general population is highly variable, and that those women who are predisposed to secreting greater amounts of AA have a greater probability of having PCOS. We now hypothesize that the levels of AAs are highly variable among normal nonhyperandrogenic women, and that this heterogeneity is the result of a variable response of AAs to ACTH stimulation. To test this hypothesis we prospectively studied the response of dehydroepiandrosterone (DHA) and cortisol (F) to a 60-min acute stimulation with ACTH-(1--24) in 56 healthy eumenorrheic nonhirsute healthy women with a mean age of 28.9 yr (range, 20--37 yr.) and a mean body mass index (BMI) of 29.2 kg/m(2) (18.2--46.2 kg/m(2)). Baseline samples and poststimulation samples were assayed for DHA and F. The basal and ACTH-stimulated levels of DHA, but not those of F, were negatively correlated with age, although neither the basal nor ACTH-stimulated responses of DHA and F varied with BMI. After controlling for age, the basal F level was negatively correlated to its net increment (i.e. Delta F; r = -0.54; P < 0.001), whereas there was no significant relationship between basal DHA and Delta DHA. We also compared the intersubject variability (coefficient of variation) for basal and stimulated levels of DHA and F. For basal (DHA(0)), 60 min (DHA(60)), and net increment in (Delta DHA) DHA levels, the coefficients of variation were 67.9%, 61.4%, and 76.0%, respectively; for F(0), F(60), and Delta F, they were 40.4%, 16.9%, and 31.3%, respectively. The variance in Delta DHA was significantly higher, and the variance in F(60) was significantly lower than that in all other variables; DHA(0), DHA(60), F(0), and Delta F had similar variances. In conclusion, in our population of healthy reproductive-aged women we observed that both basal and ACTH-stimulated levels of DHA after ACTH-(1--24) stimulation had significantly greater intersubject variance (approximately 60--70%) compared with the basal and poststimulation levels of F (approximately 15--40%). These data support the hypothesis that among normal women, AA (i.e. DHA) levels are highly variable compared to those of F. In addition, the intersubject variability in DHA levels is at least in part due to a variable response of AAs to ACTH stimulation. Whether the AA excess frequently observed in PCOS is due to the greater risk of those women with higher AA levels, basally and after ACTH stimulation, remains to be confirmed.     

Increased cortisol secretion after small hemorrhage is not attributable to changes in adrenocorticotropin

Adrenal secretory rates of cortisol and corticosterone and arterial concentrations of ACTH and cortisol were measured in conscious trained dogs subjected to 10 ml/(kg . 3 min) or 20 ml/(kg . 3 min) hemorrhage. All four variables increased substantially after 20 ml/(kg . 3 min) hemorrhage. Secretion rates of cortisol and corticosterone increased significantly after 10 ml/(kg . 3 min) hemorrhage, without a change in ACTH. The responses of ACTH and the secretion rates of cortisol to 10 ml/(kg . 3 min) hemorrhage and iv infusion of ACTH were compared. Infusions of ACTH required to match the secretory response of cortisol after 10 ml/(kg . 3 min) hemorrhage resulted in concentrations of ACTH significantly higher than those observed after 10 ml/(kg . 3 min) hemorrhage. These results suggest that 10 ml/(kg . 3 min) hemorrhage induces an acute increase in adrenocortical sensitivity to ACTH.