Changes in responsiveness of the hypothalamic-pituitary-adrenocortical axis to 2-deoxy-D-glucose in developing rats.

During the first 2-3 weeks of postnatal life, the hypothalamic-pituitary-adrenocortical axis in rats exists in a relatively dormant state, termed the stress-hyporesponsive period. The development of the hypothalamic-pituitary-adrenal axis in young rats was examined by testing the ability of the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG) to stimulate CRF in vitro and ACTH in vivo. Intraperitoneal injection of 2-DG into rats 11-12 days of age or into adult rats resulted in significant hyperglycemia by 60 min that was greater in magnitude in the adults. This response was accompanied by a significant increase in plasma ACTH to levels more than 500% of the noninjected or saline-injected control values in adults. A much smaller (approximately 200%) but still significant ACTH response was observed 60 min after 2-DG injection in the neonates. The drug had no effect on the ACTH response to exogenous CRF in the neonates. The pattern of corticosterone secretion paralleled that of ACTH, with a very moderate rise (from less than 1 to 2 micrograms/dl) seen in the neonate. To test the hypothesis that CRF was driving the ACTH response to glucoprivation induced by 2-DG in the neonate and to determine the ontogeny of hypothalamic responsiveness to this stressor, complete hypothalami were existed from rats 10-35 days of age and incubated in a defined buffer containing 5.5 mM glucose with or without 22 mM 2-DG. There was no effect of the analog on CRF secretion until day 35, at which time the magnitude of the response resembled that previously reported to occur in adult tissue. To determine if the failure to observe a CRF response was due to heightened sensitivity to the negative feedback effects of glucocorticoids, 8- to 10-day-old pups were adrenalectomized and returned to their mothers for 3 days, at which time the hypothalami were removed and tested for CRF secretion. No difference was observed between basal CRF secretory rates in the control or adrenalectomized groups, and there was still no significant response to 2-DG. Moreover, adrenalectomy did not potentiate the ACTH response to injection of 2-DG in vivo. The results suggest that during neonatal life in the rat, the hypothalamic glucostat/CRF cell mechanism is incapable of promoting a normal secretory response to glucoprivation. This deficit is probably not related to the increased sensitivity to negative feedback that has been proposed to account in part for the attenuated ACTH responses to stress in the neonatal animal.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of the pituitary-adrenocortical axis in day-old rats by insulin-induced hypoglycemia.

Most newborn mammals, including humans, are susceptible to neonatal hypoglycemia shortly after birth, especially if fasted. Glycogenolysis provides short term benefit, but gluconeogenesis is required for glucose homeostasis at this stage of development. A functional hypothalamic-pituitary-adrenocortical (HPA) axis is essential for glucose homeostasis in adults, but little is known of the importance of this endocrine axis in this regard during the neonatal period. The neonatal rat is an excellent model for study of both glucose metabolism and the HPA, and shares many similarities with other mammals. For the first 2 weeks of postnatal life, the rat HPA appears to be relatively unresponsive to stress, however, which would seem maladaptive during the critical first 24 h. The present study examined the ability of day-old rats to respond to hypoglycemia with activation of the HPA system. Each of three doses of insulin resulted in hypoglycemia (glucose, less than 40 mg/dl), with the greatest decrease in glucose 120 min after an insulin dose of 2.5 U/kg BW. At this dose, plasma corticosterone was increased to levels comparable to those observed in adults. The steroid response was associated with a small but significant increase in ACTH, and both endocrine responses were delayed compared to those in adults. There was no difference in adrenal response time to ACTH in incubations of adrenal cells isolated from day-old or adult rats, however. The stimulatory effect of insulin was progressively diminished in older animals (2-6 days postnatal). Insulin had no direct effect on adrenal steroidogenesis either alone or in combination with ACTH when tested on dispersed cells in vitro. We conclude that 1) the adrenal gland is very sensitive to the stress of hypoglycemia for 1 day after birth, but rapidly declines in activity over the first 6 days of life; 2) the response time of the HPA axis to hypoglycemia stress is delayed in day-old rats, and this delay occurs above the level of the adrenal; and 3) there may be factors in addition to ACTH that promote steroidogenesis on the first postnatal day, or there may be bioactive or immunoreactive differences in the ACTH molecule during this time.

The interaction of estradiol and daylength in modifying serum prolactin secretion in female hamsters.

Changes in serum levels of PRL have been implicated in the development of seasonal reproductive quiescence in male hamsters, yet the role of serum PRL in seasonal anestrus in the female has not been examined. The present studies were designed to examine the secretory patterns of serum PRL and the impact of estradiol on PRL secretion in female hamsters exposed to stimulatory and nonstimulatory photoperiods. In the first study, anestrous short day [6 h of light, 18 h of darkness (LD 6:18)] hamsters or metestrous cycling long day (LD 16:8) hamsters were decapitated at one of six timepoints over the 24-day. PRL was never detectable in the serum of anestrous females in short days; in cycling metestrous females, PRL was elevated during the light phase of the 24-h day. In a second experiment, LD 16:8 females were ovariectomized and implanted with 10-mm Silastic capsules that were either empty or filled with estradiol benzoate. Animals were then maintained in long days or transferred to short days and decapitated 4 weeks later. Estradiol dramatically enhanced serum PRL in both long and short day ovariectomized females, but levels were lower in short day females. Ovariectomy without estradiol treatment led to low levels of serum PRL, but levels were consistently lower in short day females than in long day females. The results suggest that low levels of serum PRL in the short day anestrous hamster result both from a loss of estradiol facilitation and from extraovarian effects of the short day photoperiod and may play a critical role in the loss of estrous cyclicity. (Endocrinology 108: 371, 1981)

Calmodulin-binding proteins in plasma membranes from adrenocortical cells.

Highly purified plasma membranes from Y-1 mouse adrenal tumor cells and those from bovine fasciculata cells were shown by [125I]iodocalmodulin overlay to contain five calmodulin-binding proteins of 240,000, 150,000, 66,000, 60,000, and 51,000 mol wt (Mr). Three of these proteins were also detected by affinity chromatography on calmodulin-Sepharose. Calmodulin binding was inhibited by competition with unlabeled calmodulin and by an inhibitor of calmodulin (trifluoperazine). Binding to each of the proteins was Ca2+ dependent. The relative proportion of binding to each of the five proteins was very different for Y-1 and bovine membranes. In Y-1 membranes as much as 50% of total binding was to the 51,000 Mr protein, whereas in bovine membranes more than 50% of binding occurred with the 150,000 Mr protein. Three of the five proteins were tentatively identified as follows: the 240,000 Mr protein is alpha-spectrin, the 60,000 Mr protein is the A subunit of the Ca2+/calmodulin-dependent protein phosphatase called calcineurin and the 51,000 Mr protein is the major subunit of a Ca2+/calmodulin-dependent protein kinase. The kinase was shown to act on specific substrates. It is concluded that calmodulin, by binding to the kinase and phosphatase, is capable of influencing the degree of phosphorylation of specific substrates in the plasma membranes of adrenal cells, and by binding to alpha-spectrin it may influence the cytoskeletons of these cells. These effects of calmodulin are likely to be important in the regulation of steroid synthesis in the adrenal cortex.

The effects of corticotropin-releasing factor on adrenocorticotropin secretion from perifused pituitaries in vitro: rapid inhibition by glucocorticoids.

The effect of synthetic corticotropin-releasing factor (CRF) and glucocorticoids on ACTH secretion from rat anterior pituitaries was examined in vitro. Pituitaries were cut into 12 fragments and perifused at 37 C with a modified Krebs buffer. CRF produced a rapid (1-min), Ca2+-dependent rise in ACTH secretion, with a half-maximally effective concentration of about 1 nM. ACTH secretion declined after 90 min of continuous perifusion with CRF. In vivo, glucocorticoids can inhibit stress-induced ACTH secretion with a latency of a few seconds. In our experiments, coperifusion with 10(-7) M cortisol, corticosterone, or dexamethasone, but not cortisone, inhibited the effect of CRF by nearly 50%. The latency of the inhibition was between 10 and 20 min, and the inhibition persisted for as long as the steroid was present. Basal secretion was unaffected by steroid treatment for 30 min. These results indicate that rapid inhibition of stimulated ACTH secretion can occur at the level of the pituitary in vitro, but that the feedback differs from that observed in vivo in terms of the longer latency and the persistence of the response.

Phosphorylation of three proteins in the plasma membrane of Y-1 adrenal cells by a membrane-bound adenosine 3',5'-monophosphate-dependent protein kinase.

Highly purified plasma membranes from Y-1 adrenal tumor cells were incubated with [gamma-32P]ATP with and without cAMP to determine whether endogenous protein substrates are phosphorylated by a cAMP-dependent protein kinase. Three membrane proteins (mol wt 270,000, 35,000, and 17,000) were phosphorylated without cAMP and, to a greater extent, with the nucleotide (0.05-20 microM). Phosphorylation was rapid (less than 60 sec), specific for cAMP, and occurred exclusively at serine residues. Two of the three proteins (35,000 and 17,000) were phosphorylated in whole cells under the influence of cAMP when the cells were incubated with [32P]orthophosphate. The cAMP-dependent protein kinase of these plasma membranes was not extracted by Triton X-100 (0.5% wt/vol) nor by KCl (0.4 M) but was almost completely extracted by the two agents together. By means of photoactivation of 8-azido-[32P]cAMP, it was found that both regulatory subunits RI and RII are present in the membranes. To the extent that the second messenger acts only by way of protein kinase enzymes, these changes in the three proteins are likely to be important in the responses of the plasma membranes of adrenal cells to ACTH.

Free fatty acids activate the hypothalamic-pituitary-adrenocortical axis in rats.

Intravenous administration of Intralipid 10% increases blood levels of essential free fatty acids. In rats and man, this is associated with an inhibition of GH secretion from the anterior pituitary. Because GH is lipolytic, the inhibition of its secretion may represent a negative feedback action of the fats on pituitary sensitivity to GH-releasing hormone. Since corticosterone, the final secretory product of the rat hypothalamic-pituitary-adrenocortical (HPA) axis, is also lipolytic, we tested the hypothesis that FFA would inhibit the HPA axis. Rats were cannulated via the jugular vein and infused with different doses of heparin-Intralipid 10% or heparin-saline; sequential blood samples were obtained and analyzed for ACTH, corticosterone, FFA, and glucose. Intralipid at 2.85 ml/kg increased plasma FFA to over 3 meq/liter by 15 min, with a return to baseline by 60-90 min. There was no effect of the infusion on plasma osmolarity or pH. At 60 min, plasma ACTH levels were significantly elevated to over 1500 pg/ml in Intralipid-infused rats, but were unchanged in saline controls. This dose of Intralipid increased corticosterone levels by nearly 20-fold at 120 min. At 180 min, corticosterone levels were still significantly greater than those in saline controls. Lower doses of Intralipid also significantly elevated both FFA and corticosterone levels, but by 180 min, levels of both were similar to those in controls. The effects of Intralipid on corticosterone secretion could not be attributed to the presence of glycerol in the suspension, since glycerol infusions had no significant effect on steroid levels compared to those in saline controls. In dexamethasone-pretreated rats, there was no significant rise in plasma corticosterone after either of two Intralipid doses, suggesting that the action of Intralipid was at a site within the HPA axis above the adrenal gland. This finding also suggested that the high steroid levels after Intralipid treatment were not due to interference with the corticosterone RIA. This was verified by the finding that there was no increase in plasma immunoreactive corticosterone after Intralipid infusion into adrenalectomized rats. Intralipid also caused an increase in plasma glucose levels that was first significant at 60 min and declined to baseline by 180 min, possibly reflecting increased autonomic activity or peripheral insensitivity to insulin. The results suggest that high circulating FFA levels activate, rather than inhibit, the HPA axis in rats. Since stress activates glucocorticoid production and increases FFA levels due to lipolysis, it is possible that FFA and the HPA axis constitute a previously unrecognized positive feedback loop.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of polymyxin B on steroidogenesis from adrenocortical cells.

The action of the cyclic peptide polymyxin B (a well known inhibitor of protein kinase C) on adrenal steroid synthesis was examined with Y-1 adrenal tumor cells. Polymyxin B produces a biphasic effect on the stimulation of steroid synthesis by 2 nM ACTH in these cells, with inhibition at low concentrations (less than 10 microM) and a return to control levels at high concentrations (greater than 100 microM). Polymyxin B does not inhibit the stimulatory effect of Bu2cAMP on steroidogenesis. Inhibition of the steroidogenic response to ACTH by a fixed concentration (20 microM) of polymyxin B is overcome by high concentrations of ACTH. Polymyxin B causes a concentration-dependent stimulation of steroid synthesis by Y-1 cells and, over the same concentration range, increases the production of cAMP by these cells. Polymyxin B also partially inhibits the increased production of the cyclic nucleotide produced by ACTH. In addition, polymyxin B inhibits binding of [125I](Phe2,Nle4)ACTH-(1-38) to Y-1 cells. Polymyxin B, like ACTH, promotes rounding of Y-1 cells and partially inhibits rounding produced by ACTH. These effects of polymyxin B are specific to the extent that polymyxins E1 and E2 do not exert similar effects. The actions of polymyxin B are not confined to transformed cells, since responses similar to those seen with Y-1 cells were also observed with cultured rat fasciculata cells. On the other hand, the effect of polymyxin B is specific for adrenal cells, since the cyclic peptide does not influence the steroidogenic response of rat Leydig cells to LH. It is concluded that polymyxin B is a partial agonist of ACTH which is likely to prove useful in studying the molecular basis of the interaction between ACTH and its adrenal receptor.

The role of calmodulin in the responses to adrenocorticotropin of plasma membranes from adrenal cells.

Inhibitors of calmodulin [trifluoperazine, chlorpromazine, pimozide, and calmidazolium N-(6-aminohexyl)5-chloro-1-napthalenesulphonamide (W7)] and calmodulin antibodies were used to investigate the role of calmodulin in the response of Y-1 mouse adrenal cells to ACTH, with particular reference to events in the plasma membrane. In whole cells it was found that two responses (production of steroids and cAMP) to two stimulating agents (ACTH and forskolin) were inhibited by trifluoperazine at concentrations consistent with those involved in binding of the inhibitor to pure calmodulin (10-25 microM). The steroidogenic responses were also inhibited by the three other inhibitors of calmodulin (chlorpromazine, calmidazolium, and W-7). Trifluoperazine and pimozide (1-500 microM) did not inhibit binding of an [125I]ACTH analog to highly purified plasma membranes of Y-1 cells or to the cells themselves. With Y-1 plasma membranes it was found that trifluoperazine, pimozide, W-7, and calmodulin antibodies inhibited the increase in adenylate cyclase activity in response to ACTH, but not the cyclase responses to cholera toxin or forskolin. Moreover, the effect of cholera toxin on the ADP-ribosylation of specific membrane substrates was independent of the presence or absence of endogenous and/or exogenous Ca2+/calmodulin. The response of adenylate cyclase to ACTH was also decreased in plasma membranes from which calmodulin was removed by washing, and exogenous calmodulin partly reversed this decrease. Anti-calmodulin immunoglobulin inhibited the stimulation of adenylate cyclase produced in plasma membrane by ACTH, but was without effect on the responses to cholera toxin and forskolin. Exogenous calmodulin partly reversed the inhibition of stimulation by ACTH of adenylate cyclase produced by the antibody. It is concluded that calmodulin influences the events taking place in the plasma membrane in response to ACTH, after the binding of the hormone to its receptor and before the action of the G protein (Gs). That is, calmodulin is involved in coupling the occupied receptor to Gs. The effects of inhibitors of calmodulin in whole cells must involve some additional effect(s) requiring the intact cell.

The influence of plasma membrane cholesterol on the response of adrenal cells to adrenocorticotropin.

The concentration of cholesterol in plasma membranes of Y-1 cells was altered between values of the molar ratio of cholesterol to phospholipids (C/P) of 0.6:1.6 to study the influence of plasma membrane cholesterol on the response to ACTH. Increasing concentrations of membrane cholesterol (C/P) were associated with an increase in the number of ACTH receptors and, hence, production of cAMP and steroids without affecting the nature of binding (Kd) of ACTH to its receptor. Binding studies revealed spare ACTH receptors at all concentrations of membrane cholesterol. However, production of cAMP and steroids by Y-1 cells were tightly coupled. Studies with cholera toxin showed that extreme changes in C/P were without influence on the activity of Ns or that of the cyclase itself. It was also shown that production of steroids in the absence of ACTH and that in response to cholera toxin involve some effect of membrane cholesterol not dependent on cAMP.

Secretion of corticotropin-releasing factor from cultured rat hypothalamic cells: effects of catecholamines.

An understanding of the regulation of CRF secretion in rats is currently incomplete, in part due to the lack of sensitive in vitro models available for studying this neuropeptide. In particular, the effects of catecholamines on CRF secretion, and the receptor subtypes mediating these actions have long been the subject of much debate. A cultured cell model has been adapted for studying secretory responses of hypothalamic cells of 1-week-old rats. Between 7-16 days in monolayer culture the cells secreted detectable levels of immunoreactive CRF, and this release was paralleled by the appearance of punctate bead-like regions of immunoreactivity along fine cellular processes. CRF secretion was increased up to 4-fold by norepinephrine (EC50, approximately 0.5 microM). The increase in CRF secretion produced by norepinephrine was blocked by the beta-receptor antagonist propranolol, but not by the alpha-antagonist prazosin. Moreover, the beta-receptor agonist isoproterenol significantly elevated CRF secretion, whereas the alpha-agonist phenylephrine was without effect, except at high concentrations. Addition of phenylephrine, however, potentiated the effect of isoproterenol, but this response was still significantly less than that produced by norepinephrine. Forskolin (EC50, approximately 0.7 microM) and the active phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (EC50, approximately 40 nM) also increased CRF secretion by 3- to 4-fold. Inactive phorbol derivatives had no effect on CRF release from these cultures. The results indicate that cultured neonatal rat hypothalamic cells are a powerful model for the study of CRF release in vitro, and that norepinephrine acts directly at the isolated cell level to stimulate secretion of this peptide, primarily by activating beta-adrenoceptors. The results also suggest that at least two functional second messenger systems (adenylate cyclase and protein kinase-C) are involved in CRF secretion and are already functional in the neonatal hypothalamus.

Developmental expression of the peripheral-type benzodiazepine receptor and the advent of steroidogenesis in rat adrenal glands.

Although the precise mechanism whereby cholesterol is transported across the outer mitochondrial membrane is uncertain, a multimeric receptor complex termed the peripheral-type benzodiazepine receptor (PBR) appears essential for this process. We therefore predicted that adrenal cells at different developmental stages would express PBR coincidentally with the advent of steroidogenesis. Adrenals of neonatal rats demonstrate greatly reduced sensitivity to ACTH that gradually increases after the first 2 weeks of life. Thus, neonates have lower circulating corticosterone levels following exposure to stress. We examined mitochondrial PBR ligand binding activity, immunoreactive (ir) PBR content, and adrenal sensitivity to ACTH in vivo and in vitro. Ontogeny of both mitochondrial PBR ligand binding capacity and irPBR directly paralleled that of ACTH-inducible steroidogenesis in isolated rat adrenal cells and in rats injected with ACTH. In addition, neonatal PBR had approximately 2-fold higher affinity for PK11195, a synthetic ligand that binds with high affinity to PBR. No correlation was observed during neonatal life between ir-steroidogenic acute regulatory (StAR) protein content and steroidogenesis. These results are consistent with the hypothesis that PBR is an absolute prerequisite for adrenocortical steroidogenesis, and suggest that the stress hyporesponsive period of neonatal rats may result from decreased PBR expression. In addition, the higher affinity of neonatal PBR and the relatively high basal expression of StAR protein in neonatal adrenals may partly explain the high constitutive steroidogenesis characteristic of neonatal rat adrenal cells.

Normal suppression of the reproductive axis following stress in corticotropin-releasing hormone-deficient mice.

The hypothalamic neuropeptide CRH has been postulated to inhibit LH secretion by a central action within the brain. To characterize the physiological significance of CRH in stressor-induced inhibition of LH secretion, CRH-deficient and wild-type mice were subjected to restraint or food withdrawal, and plasma LH levels were determined. The proestrus LH surge of female mice was equally suppressed by restraint in both genotypes, and central administration of a CRH antagonist did not alleviate this suppression in either genotype. Male mice of both genotypes also demonstrated suppression of both LH and testosterone secretion following restraint. Furthermore, food withdrawal caused similar suppression of LH secretion in both female and male mice regardless of CRH status. These data demonstrate that CRH is not necessary to inhibit LH secretion following either restraint or food withdrawal and that other molecules are able to suppress LH secretion during the response to stress in the context of CRH deficiency.

Reversible changes in adrenocorticotropin (ACTH)-induced adrenocortical steroidogenesis and expression of the peripheral-type benzodiazepine receptor during the ACTH-insensitive period in young rats.

We previously demonstrated decreased adrenal content of a mitochondrial cholesterol transporter [peripheral-type benzodiazepine receptor (PBR)] during the first postnatal week in rats, when ACTH-inducible steroidogenesis is low. Here we report that the expression of PBR protein and mRNA increases throughout the neonatal/juvenile period in rats in parallel with ACTH-inducible steroidogenesis in vitro. We also previously reported that chronic stimulation of rat pups with daily ACTH injections augmented the steroidogenic response of the developing adrenal cortex. We therefore tested the hypotheses that the change in phenotype induced by ACTH was permanent, and that the effects of ACTH were mediated by increased PBR expression. Pups were injected with ACTH or saline from postnatal d (pd) 2-8 or 2-14. Another group of pups received ACTH from pd 2-7, followed by saline from pd 8-14. On the final day, all pups were challenged with a test injection of ACTH or saline. Exposure to ACTH, but not saline, for 1 wk significantly increased adrenal mass, the corticosterone response to ACTH, and the expression of PBR protein and mRNA. Continued ACTH treatment for a second week maintained adrenal mass, steroidogenesis, and PBR mRNA expression. When ACTH was withdrawn after 1 wk and replaced with daily saline injections, however, adrenal mass, ACTH-inducible steroidogenesis, and PBR expression returned to levels comparable to those in age-matched saline controls (i.e. animals that had not received ACTH injections during the first 2 wk). Thus, although ACTH was capable of inducing increased steroidogenic capacity of the juvenile rat adrenal, its effects were only manifest when pups were exposed regularly to high plasma ACTH levels. ACTH, therefore, has significant, but reversible, effects on the development of adrenocortical function, possibly mediated in part by increased expression of PBR.

Developmental expression and regulation of adrenocortical cytochrome P4501B1 in the rat.

A 57-kDa protein whose expression in rat adrenocortical microsomes is increased after weaning has been identified as cytochrome P4501B1 (CYP1B1). Levels of CYP1B1 protein were moderately expressed in late gestation fetuses and on postnatal day 1 (pdl), but were nearly undetectable on pd6 and pd1O. CYP1B1 expression initially increased in the late preweaning period (pd17-19) and again immediately postweaning (pd21-24). The temporal coincidence of CYP1B1 expression and weaning was not due to transition from suckling to solid food, as neonates that were prematurely weaned showed no increase in adrenal CYP1B1 compared with normally weaned littermates. The pattern of CYP1B1 expression paralleled changes in microsomal metabolism of 7,12-dimethylbenz[a]anthracene (DMBA), a marker of CYP1B1 activity. Twice daily injections of ACTH to rat pups (pd3-10) failed to significantly increase the expression of CYP1B1 in pd 10 adrenals, although the injections weakly stimulated steroidogenesis. Adrenocortical cells from pd17 neonates and adult cells, when cultured for 3 days, responded similarly to ACTH induction, although neonates showed more than 4-fold less basal activity. It is concluded that rat adrenal CYP1B1 may be developmentally suppressed, and its expression is independent of diet or the presence of a dam. This suppression is retained in cell culture, but is not due to deficient ACTH signaling. These results may explain the reported resistance of neonatal rat adrenals to the toxic effects of polycyclic aromatic hydrocarbons, which are metabolized by CYP1B1 into mutagenic by-products.

Impaired basal and restraint-induced epinephrine secretion in corticotropin-releasing hormone-deficient mice.

CRH is thought to play a role in responses of the adrenocortical and adrenomedullary systems during stress. To investigate the role of CRH in stress-induced secretions of corticosterone and epinephrine, we subjected wild-type (WT) and CRH-deficient (knockout, KO) mice to restraint, and analyzed plasma corticosterone, plasma catecholamines, and adrenal phenylethanolamine N-methyltransferase (PNMT) gene expression and activity before and during 3 h of restraint. Plasma corticosterone increased over 40-fold in WT mice, but minimally in CRH KO mice. Adrenal corticosterone content tended to increase in CRH KO mice, although to levels 5-fold lower than that in WT mice. CRH KO mice had significantly lower plasma epinephrine and higher norepinephrine than WT mice at baseline, and delayed epinephrine secretion during restraint. Adrenal PNMT messenger RNA content in CRH KO mice tended to be lower than that in WT mice, though the degree of induction was similar in both genotypes. PNMT enzyme activity was significantly lower in CRH KO mice. Pharmacological adrenalectomy abolished restraint-induced corticosterone secretion and PNMT gene expression in WT mice, consistent with an absolute requirement of glucocorticoids for PNMT gene expression. We conclude that glucocorticoid insufficiency in CRH KO mice leads to decreased basal and restraint-induced plasma epinephrine and adrenal PNMT gene expression and enzyme activity.

Leptin receptor expression increases in placenta, but not hypothalamus, during gestation in Mus musculus and Myotis lucifugus.

In addition to effects on appetite and metabolism, the hormone leptin is required for reproduction in mammals. Maternal plasma leptin is increased above non-pregnant levels in all mammals thus far examined, including humans. The increase in plasma leptin appears to result in part from upregulation of adipose leptin secretion (e.g., in mice), or from production and secretion of leptin from the placenta (e.g., in humans and some bats). The placenta may also modulate maternal leptin levels via production of a plasma leptin-binding protein (mice, humans). Thus, the placenta plays a coordinating role in regulation of maternal leptin during pregnancy. In this study, the hypothesis that the placenta is also a target organ for leptin in diverse taxa was tested by examining the expression of leptin receptors (Ob-R) in placentae from species of distantly related mammalian taxa, Mus musculus (the laboratory mouse) and Myotis lucifugus (the little brown myotis, also called the little brown bat). A partial sequence of M. lucifugus Ob-R cDNA was first obtained and found to share approximately 78-88% homology at the nucleotide level with known mammalian Ob-R cDNAs. Using probes and primers designed from this sequence, receptor expression was detected in numerous tissues of M. lucifugus, including placenta, which expressed two major receptor isoforms as judged by molecular size. In both species, Ob-R mRNA expression in placenta significantly increased from early to late gestation. Expression of Ob-R mRNA was not affected by cAMP treatment in vitro. The increase in Ob-R mRNA expression in placenta was specific, since Ob-R mRNA expression did not change during gestation in either species in hypothalamus, the major site of the central actions of leptin. Thus, Ob-R is expressed in placenta throughout gestation in mice and bats, and its expression increases over the course of gestation, which raises the possibility that leptin may exert temporally distinct effects on placental growth or function throughout gestation. Because similar placenta-specific changes in leptin receptor expression occurred in species from distantly related mammalian taxa which collectively comprise approximately 70% of all known mammalian species, it is possible that placental actions of leptin are conserved across mammals, even in those species (such as the Swiss-Webster strain of mouse) in which the placenta does not itself produce leptin.

Steroidogenesis in isolated adrenocortical cells during development in rats.

After postnatal day 1 (d1), the hypothalamo-pituitary-adrenal axis of neonatal rats becomes less responsive to certain stimuli for up to 2 weeks. The present study was designed to quantify the development of adrenocortical cell responsiveness to its normal secretagogue, adrenocorticotropic hormone (ACTH), and to better localize intracellular sites of adrenal cell hyporesponsivity. Maximum steroidogenic responses of collagenase-dispersed adrenocortical cells (using two isolation methods) to ACTH varied significantly in the order adult > d1 > d10. The response pattern to dibutyryl cAMP ((Bu)2cAMP) was identical to that observed for ACTH (adult > d1 > d10), suggesting that neonatal adrenal responsiveness is limited by a site distal to cAMP formation. Sensitivity (EC50) of adult cells to ACTH was approximately 3-fold greater than in neonatal cells, but there was no age-dependent shift in sensitivity to (Bu)2cAMP. 20 alpha-Hydroxycholesterol (20 alpha-OHCHOL), a membrane permeable analog of cholesterol, also failed to normalize the d10 adrenal response to ACTH. This result indicates that one site of refractoriness is apparently distal to cholesterol transport, and strongly suggests possible differential cytochrome P450 enzyme expression or activity in neonatal rat adrenal cells. Finally, although stimulated secretion was lower in neonatal cells, basal corticosterone secretion was significantly greater in neonatal adrenals, suggesting that constitutive activity of neonatal adrenal cells is high compared to that of adult cells.

Protein kinase C in adrenal cells: possible role in regulation of steroid synthesis.

Y-1 adrenal tumor cells and rat fasciculata cells were shown to possess an enzyme with the properties of protein kinase C. Activity was stimulated by Ca2+ and phospholipid (specifically phosphatidylserine). Enzyme activity was stimulated by addition of phorbol ester to a cell homogenate (ED50 10 nM) and inhibited by trifluoperazine (ID50 10 microM). ACTH and cyclic AMP added to Y-1 cells increased the activity of protein kinase C. Dose-response curves with ACTH showed that the hormone was effective in stimulating protein kinase C at lower concentrations than those required to increase steroid synthesis. When phorbol ester was added to Y-1 cells, total kinase C activity was diminished. Neither phorbol ester nor ACTH causes redistribution of protein kinase C between membranes and cytosol. Phorbol ester also stimulates steroid production by Y-1 cells. Protein kinase C phosphorylates 5 proteins in Y-1 cells (67, 61, 32, 16 and less than 14.4 kDa). Puromycin and cycloheximide increase the activity of protein kinase C in adrenal cells. It is concluded that protein kinase C may play an ancillary role in regulation of adrenal steroid synthesis but does not mediate the classical steroidogenic response that results from activation of adenylate cyclase by ACTH.

Ontogeny of immunoreactive and bioactive microsomal steroidogenic enzymes during adrenocortical development in rats.

The functional development of the neonatal rat adrenal cortex is characterized by a triphasic response to adrenocorticotropic hormone (ACTH), with a nadir in responsiveness around neonatal day 10 (d10). In this study, the hypothesis was tested that hyporesponsiveness to ACTH partly results from deficiencies in steroidogenic enzyme content. Immunoreactive (ir) levels of mitochondrial cytochrome P450 enzymes (side chain cleavage (P450scc) and 11 beta-hydroxylase (P450c11)) did not change during neonatal development. Immunoreactive levels of microsomal 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD), however, were significantly and comparably lower in both day 1 (d1) and d10 neonates compared to adult rats. Activity of 3 beta-HSD did not parallel changes in ir 3 beta-HSD content. Enzyme activity was low on d1 (approximately 39% of adult activity), but by d10 was statistically equivalent to that of microsomes from adult adrenal glands. Immunoreactive levels of microsomal cytochrome P450 21 alpha-hydroxylase (P450c21) were significantly lower in d1 glands than in adult glands (by approximately 50%), but by d10 were statistically indistinguishable from adults. On the other hand, P450c21 activity was equivalent on d1 and d10 and both were significantly lower compared to adults (approximately 62% of adult activity). ACTH injections from d3-d10 facilitated the adrenocortical steroidogenic response to ACTH on d10. This treatment increased levels of ir 3 beta-HSD, but not ir P450c21. The results suggest that rat adrenocortical 3 beta-HSD and P450c21 are developmentally and differentially regulated, and that ir levels of the proteins are not correlated with enzyme activity during the neonatal period. One possible explanation for these observations is that multiple isoforms of the two enzymes, with different antigenic and enzymatic properties, may be expressed during development at different times. In addition, the combined decreased activities of these two enzymes can almost entirely account for the decreased steroidogenic output of rat adrenocortical cells on d1, but not during the later neonatal period.