Parathyroid hormone and adenosine-3',5'-monophosphate acutely increase phospholipids of the phosphatidate-polyphosphoinositide pathway in rabbit kidney cortex tubules in vitro by a cycloheximide-sensitive process

Parathyroid hormone (PTH) rapidly increased to concentrations of phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide during incubations of rabbit kidney cortical tubules in vitro. These effects were preceded by increases in cAMP, which also induced virtually identical increases in these phospholipids. Pretreating the tubules with cycloheximide inhibited these phospholipid effects of PTH and cAMP. These findings are similar to those reported for ACTH and cAMP in the adrenal cortex. Hormones that utilize cAMP as their "second messenger" may influence membrane structure and function via stimulation of the phosphatidate-polyphosphoinositide pathway.     

A23187 inhibits adrenal protein synthesis and the effects of adrenocorticotropin (ACTH) on steroidogenesis and phospholipid metabolism in rat adrenal cells in vitro: further evidence implicating phospholipids in the steroidogenic action of ACTH

We examined the effects of ACTH and the Ca++ ionophore, A23187, on steroidogenesis and phospholipid metabolism during incubation of dispersed rat adrenal cells. Increasing doses of ACTH elicited nearly parallel increases in corticosterone production and adrenal inositide (mono- and di-) concentrations. As reported previously by other investigators in Y1 cells, A23187 inhibited ACTH- and cAMP-stimulated, but not basal or pregnenolone-stimulated, corticosterone production. A23187 also inhibited ACTH-induced increases in phosphatidic acid, phosphatidylinositol, and diphosphoinositide, and this was attended by inhibition of [3H]leucine incorporation into protein. These findings support our previous contentions that: 1) a labile protein is required for ACTH-induced increases in adrenal phospholipids in the phosphatidate-polyphosphoinositide-polyglycerophospholipid pathway; and 2) these phospholipids are involved in the steroidogenic action of ACTH.     

Adrenocorticotropin and adenosine 3',5'-monophosphate stimulate de novo synthesis of adrenal phosphatidic acid by a cycloheximide-sensitive, CA++-dependent mechanism

We tested further our postulate that enhanced de novo synthesis of phosphatidic acid is responsible for ACTH- and cAMP-induced increases in adrenal phospholipids in the phosphatidate polyphosphoinositide pathway. During incubation of adrenal sections or cells in vitro, ACTH and cAMP increased the concentrations of and incorporation of [3H]glycerol and [14C]palmitate into phosphatidylcholine and phosphatidylethanolamine, two major phospholipids which are derived from phosphatidic acid, but are extrinsic to the inositide pathway. Thus, it is unlikely that ACTH and cAMP increase inositide phospholipids at the expense of other phospholipids. Similar to previously reported effects on phosphatidic acid and inositide phospholipids, cycloheximide blocked the effects of ACTH and cAMP on phosphatidylcholine and phosphatidylethanolamine. In addition, Ca++ was required for these effects, as well as for cAMP-induced increases in phosphatidic acid, inositide phospholipids, and steroidogenesis. Our findings strongly suggest that ACTH, via cAMP, stimulates de novo phosphatidate synthesis by a cycloheximide-sensitive, Ca++-dependent process, and this stimulation causes a rapid generalized increase in adrenal phospholipids. Moreover, the increased incorporation of labeled glycerol and palmitate into phospholipids suggests that ACTH and cAMP may stimulate the glycerol-3'-PO4 acyltransferase reaction. This stimulatory effect may play a central role in the steroidogenic and trophic actions of ACTH and cAMP.     

Studies on the dynamics of adrenocortical responses to ACTH in the rat

The transient dynamics of plasma ACTH, adrenal cyclic AMP, adrenal corticosterone and plasma corticosterone were evaluated in male Sprague-Dawley rats, whose endogenous release of ACTH had been blocked by dexamethasone: (1) 40 min after single injections of ACTH ranging from 2 to 300 ng ACTH/100 g B.W., i.V.; (2) at time intervals after single injections of 9, 37 and 300 ng ACTH/100 g B.W.; (3) during and after prolonged infusion of 4 ng ACTH/min/100 g B.W. Plasma corticosterone concentration was still at a nearly maximal level 40 min after the injection of ACTH at a dose level for which the adrenal cyclic AMP content had fallen back to a value that was scarcely above the control one; a narrow window, defined by a 2-fold increase in the dose of ACTH, represents the transition between a minimal and a maximal adrenal cyclic AMP content. The adrenal cyclic AMP transient response after injection of graded doses of ACTH increased rapidly to a peak whose amplitude was dose-dependent; the duration of the cyclic nucleotide response, however, appeared to be independent of the ACTH dose level. The adrenal corticosterone content rose rapidly, and the eventual fall was delayed by increasing doses of ACTH. The time course of the early plasma corticosterone concentrations exhibited a similar rate of increase after any dose of ACTH; in any case, a steady state whose duration was dose-dependent was eventually reached and the ensuing fall therefrom occurred at a time when the adrenal cyclic AMP had fallen to very low levels. The adrenal cyclic AMP content showed an overshoot at a time when ACTH and corticosterone had reached a constant steady state, during a prolonged infusion of ACTH; adrenal cyclic AMP stabilized during the later phase of the infusion. After removal of the infusion, plasma ACTH levels fell relatively slowly as compared with adrenal cyclic AMP, whereas corticosterone remained at a maximal level for at least 120 min.Our results, derived from experiments in vivo, support the recent proposal by Bristow et al. (1980), derived from studies in vitro, that ACTH can act via either of two types of receptor: binding to one receptor elicits steroidogenesis via cyclic AMP production whereas binding to the other receptor elicits steroidogenesis through some other mechanism.     

Rapid glucose-dependent increases in phosphatidic acid and phosphoinositides in rat pancreatic islets

Glucose effects on islet phospholipids were examined during direct incubation or after 3 days of 32P prelabeling in primary culture. In both cases, glucose increased the 32P content of phosphatidic acid (PA), phosphatidylinositol (PI), and polyphosphoinositides (PPI). Glucose-induced increases in PA, PI, and PPI in the culture-prelabeling experiments were evident within 1 min, dose related, and reflective of increases in phospholipid mass, which was confirmed in direct incubations by measurement of PI phosphorus. Thus, in addition to increasing PI-PPI hydrolysis, glucose increases de novo phospholipid synthesis in pancreatic islets. The latter may result from enhanced glycolysis and substrate availability for PA-PI-PPI synthesis, since glyceraldehyde and pyruvic acid also increased PI levels. Our findings raise the possibility that increases in PA, PI, and PPI synthesis could serve as a mechanism to enhance the generation of intracellular mediators, which are purported to regulate insulin secretion.     

Rapid effects of angiotensin-II on polyphosphoinositide metabolism in the rat adrenal glomerulosa

Angiotensin-II (A-II) provoked a rapid decrease in 32p in triphosphoinositide (TPI) in 32p-prelabeled rat adrenal glomerulosa cells. This effect (presumably reflecting TPI hydrolysis) of A-II was nearly maximal at 5 sec of incubation and appeared to precede increases in labeling of phosphatidic acid and phosphatidylinositol. Other aldosterone-stimulating agents (ACTH, K+ and serotonin) did not provoke this effect. Since this effect appeared to be independent of Ca++, it is possible that TPI hydrolysis may be important for Ca++ mobilization during A-II action in glomerulosa tissue.     

Gonadotropin-releasing hormone (GnRH) rapidly stimulates the formation of inositol phosphates and diacyglycerol in rat granulosa cells: further evidence for the involvement of Ca2+ and protein kinase C in the action of GnRH

GnRH provokes a phospholipid response in rat granulosa cells that has been characterized by increased incorporation of radioactive precursors into phosphatidic acid and phosphatidylinositol, and by depletion of 32P-prelabeled polyphosphoinositides. In this report, rat granulosa cells from mature Graafian follicles were incubated with GnRH under various conditions to follow the hydrolysis of phosphoinositides and the generation of the metabolic byproducts of phospholipase C action. Granulosa cells were prelabeled for 3 h with myo[2-3H]inositol. GnRH provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol monophosphates, inositol bisphosphates, and inositol trisphosphates (IP3). Time-course studies revealed that IP3 was formed more rapidly than inositol bisphosphate and inositol monophosphate after GnRH treatment. The response to GnRH was concentration dependent (maximal at 10 ng/ml) and was prevented by a specific GnRH antagonist. Lithium chloride (1-10 mM) greatly enhanced the GnRH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. No changes were observed in the levels of free [3H] inositol and [3H]phosphatidylinositol in GnRH-treated cells. However, treatment with both lithium and GnRH for 30 min significantly reduced the levels of free [3H]inositol and [3H] phosphatidylinositol. In the presence of lithium, the rate of hormone-stimulated inositol phosphate formation was not altered by 30 min of prior treatment with GnRH, indicating that phospholipase C activity is not readily desensitized. GnRH also increased the formation of diacylglycerol (DAG), another product of phospholipase C action. In cells prelabeled with [3H] arachidonic acid, GnRH significantly increased levels of DAG in incubations lasting 2-5 min. Concomitant increases in [3H] phosphatidic acid were also observed in GnRH-treated cells. In conjunction with these studies, intracellular free Ca2+ levels were measured by Quin 2 fluorescence. GnRH and its agonistic analog rapidly increased (5 sec) cytosolic free Ca2+ levels (approximately double). The results demonstrate that an early event in the action of GnRH is the hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism. The products resulting from this action of GnRH, i.e. IP3 and DAG, may serve as intracellular mediators for the mobilization of intracellular calcium, or the activation of protein kinase C and arachidonic acid release.     

Polyphosphoinositides:stimulator of mitochondrial cholesterol side chain cleavage and possible identification as an adrenocorticotropin-induced, cycloheximide-sensitive, cytosolic, steroidogenic factor

ACTH treatment in vivo enhances the activity of adrenal cytosol for supporting pregnenolone synthesis in adrenal mitochondria. Upon fractionation of adrenal cytosol by gel filtration through Sephadex G200, pregnenolone synthesis-stimulating activity is eluted just after 3H2O(Kd = 1.1--2.1). This late eluting steroidogenic activity is enhanced by ACTH treatment and diminished by cycloheximide treatment, which also inhibits adrenal protein synthesis and ACTH-induced steroidogenesis. Chromatography on Sephadex G-10 yields even further separation of steroidogenic factor(s) from inactive substances; it appears that the active factor(s) adsorbs to Sephadex and that such adsorption is enhanced by a greater degree of cross-linking in the gel. The phospholipids, cardiolipin, diphosphoinositide, and triphosphoinositide, are recovered partly in adrenal cytosol and, by virtue of their polyphosphorylated head group, specifically stimulate mitochondrial pregnenolone synthesis. It seems likely that the ACTH effect on cytosolic steroidogenic activity is due to changes in polyphosphoinositides, since these phospholipids are specifically stimulated by ACTH, cycloheximide blocks the ACTH-induced increase in polyphosphoinositides, and these phospholipids comigrate with steroidogenic activity during gel filtration of adrenal cytosol. These findings along with other findings from our laboratory suggest that polyphosphoinositides may function as a cycloheximide-sensitive mediator in the steroidogenic action of ACTH.     

Effects of nutrient secretagogues upon phospholipid metabolism in rat pancreatic islets

Rat pancreatic islets labelled with [32P]Pi were used to investigate the effects of nutrient secretagogues upon phospholipid metabolism. Stimulatory concentrations of glucose, 4-methyl-2-oxopentanoate, 3-phenylpyruvate and 2-endo-aminonorbornane-2-carboxylic acid caused a marked and specific stimulation of labelling with [32P]Pi of phosphatidylinositol, phosphatidic acid and the polyphosphoinositides. The effects of glucose were concentration-related and were inhibited by mannoheptulose and menadione. Enhanced phospholipid labelling persisted in the absence of added calcium ions but was abolished by excess EDTA. The time-course of labelling of these phospholipids in response to glucose contrasted with that previously observed using carbamylcholine in that the accumulation of radioactivity in phosphatidic acid and in phosphatidylinositol occurred in parallel. We conclude that glucose, in common with other nutrient secretagogues or those which promote the metabolism of endogenous nutrients, causes a marked stimulation of turnover in the phosphatidylinositol cycle. This effect requires the integrity of nutrient catabolism and is probably not dependent upon the stimulation of Ca2+ uptake from the extracellular medium.     

Effects of alpha-melanocyte-stimulating hormone on the cyclic AMP and phospholipid metabolism of rat adrenocortical cells

Results on the effects of peptides on the phospholipid metabolism and steroid and cyclic AMP (cAMP) outputs of rat adrenal capsular cells (96% zona glomerulosa, 4% zona fasciculata) were obtained in a series of three batch experiments. Their significance was examined by analysis of variance. Incorporation of [32P] into phosphatidylcholine, phosphatidic acid and phosphatidylinositol was measured. Production of [3H]inositol-1 monophosphate, inositol-1,4 bisphosphate and inositol-1,4,5 tris-phosphate was estimated after prelabelling with [3H]inositol followed by 1 min incubation with a steroidogenic stimulus. Angiotensin II (0.25 nmol/l to 0.25 mumol/l) highly significantly (P less than 0.01) stimulated aldosterone and corticosterone outputs, [32P] incorporation into phosphatidic acid and phosphatidylinositol (but not into phosphatidylcholine) and the production of the three [3H]inositol phosphates. Aldosterone and corticosterone outputs were stimulated by alpha-MSH (above 0.1 nmol/l). However, incorporation of [32P] was not significantly increased until 10 mumol alpha-MSH/l but, unlike with angiotensin II, incorporation into phosphatidylcholine was also then stimulated. Also, the production of the inositol phosphates was not increased significantly (P greater than 0.05) by any dose of alpha-MSH (10 nmol/l, 1 mumol/l and 0.1 mmol/l) used. Therefore, it can be concluded that alpha-MSH does not stimulate phospholipase C in rat zona glomerulosa cells. In further experiments, it was also found that there were significant increases in cAMP as well as in steroid outputs above 1 nmol alpha MSH/l (highly significant above 10 nmol alpha-MSH/l). There were plateaux of the outputs of both steroids and cAMP from 0.1 to 1 mumol alpha-MSH/l. However, there were further increases in steroid and cAMP outputs of the capsular cells at higher doses. Concomitant results on the stimulation of corticosterone output by zona fasciculata-reticularis cells indicate that this additional increase was mostly due to the stimulation of the contaminating zona fasciculata cells. It was also confirmed that alpha-MSH preferentially stimulates steroidogenesis by the zona glomerulosa. However, under our conditions, alpha-MSH highly significantly increased the output of cAMP by both zona fasciculata and glomerulosa cells.     

Prostaglandin F2 alpha stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis and mobilizes intracellular Ca2+ in bovine luteal cells.

The present studies were conducted to determine whether prostaglandin F2 alpha (PGF2 alpha) stimulates the production of "second messengers" derived from inositol phospholipid hydrolysis and increases intracellular free Ca2+ ([Ca2+]i) in isolated bovine luteal cells. PGF2 alpha provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol mono-, bis-, and trisphosphates (InsP, InsP2, and InsP3, respectively). InsP3 was formed more rapidly than InsP2 or InsP after PGF2 alpha treatment. In addition, PGF2 alpha increased inositol phospholipid turnover, as evidenced by increased 32PO4 incorporation into phosphatidic acid and phosphatidylinositol. LiCl (1-20 mM) enhanced inositol phosphate accumulation in response to PGF2 alpha. Maximal increases in InsP3 occurred at 1 microM PGF2 alpha, with half-maximal stimulation occurring at 36 nM. The acute effects of PGF2 alpha on InsP3 levels were independent of reductions in extracellular calcium. Prostaglandins E1 and E2 also stimulated increases in inositol phosphate levels, albeit to a lesser extent. PGF2 alpha also induced rapid and concentration-dependent increases in [Ca2+]i as measured by quin-2 fluorescence. The PGF2 alpha-induced increases in [Ca2+]i were maximal within 30 sec (approximately 2- to 3-fold), and [Ca2+]i remained elevated for 8-10 min. The PGF2 alpha-induced increases in [Ca2+]i were also independent of extracellular calcium. These findings demonstrate that the action of PGF2 alpha is coupled to the phospholipase C-InsP3 and diacylglycerol second messenger system in the corpus luteum.     

Phospholipids stimulate phosphorylation of vinculin by the tyrosine-specific protein kinase of Rous sarcoma virus.

The phosphorylation of vinculin by a highly purified tyrosine-specific protein kinase was enhanced more than 10-fold by anionic phospholipids: the phosphorylation of casein, actin, and alpha-actinin was inhibited. The effect of phospholipid was dependent on the divalent cation used. Stimulation was observed by phosphatidylinositol or phosphatidylglycerol in the presence of either 0.5 mM Mn2+ of 5 mM Mg2+; with either phospholipid, more enzyme activity was observed with Mn2+. Maximal stimulation by phosphatidylinositol was observed at about 400 micrograms/ml. In contrast, marked stimulation by phosphatidylserine was observed only with Mn2+ and marked stimulation by phosphatidic acid was observed only with Mg2+. These results raise the possibility that phospholipids modulate vinculin phosphorylation in Rous sarcoma virus-transformed cells.     

A 3',5'-cyclic adenosine monophosphate-dependent pathway is responsible for a rapid increase in c-fos messenger ribonucleic acid by adrenocorticotropin.

ACTH rapidly and transiently increases c-fos mRNA in the rat adrenals in vivo. The present investigation was undertaken in order to determine what kind(s) of second messenger systems is involved in this increase. Rat adrenal cells were grown in monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. After 2 days of culture, cells were treated with ACTH and various agents alone or in combination. The amount of c-fos mRNA was determined by dot blot hybridization and corticosterone levels in the media were measured by RIA. ACTH (300 pg/ml) increased c-fos mRNA transiently with a peak level after 60 min. A similar increase was observed when (Bu)2cAMP (1 mM) was substituted for ACTH. Pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), a selective inhibitor of cAMP-dependent protein kinase, suppressed both basal and ACTH-increased c-fos mRNA. H-89 also suppressed corticosterone production. On the other hand, neither 12-O-tetradecanoyl-phorbol-13-acetate (100 ng/ml) nor elevated potassium ion (50 mM) affected the amount of c-fos mRNA and corticosterone production. Furthermore, pretreatment with cycloheximide (5 micrograms/ml) increased both basal and ACTH-increased c-fos mRNA. These results indicate that ACTH increases c-fos mRNA by phosphorylation of preexisting trans-acting factor(s) via cAMP-dependent protein kinase in common with steroidogenesis.     

Steroidogenic pathways and trophic response to adrenocorticotrophin of cultured adrenocortical cells in different states of differentiation.

Adrenocortical cells obtained from adult rats were propagated in monolayer culture. Depending on culture conditions, they grew either as lipid-containing epithelial-like cells with a high level of steroid production, or as fibroblast-like cells with a low level of steroid production. The major fluorogenic steroid secreted by both morphologic forms of adrenal cortical cell was corticosterone as determined by chromatography and acid fluorometry. Basal fluorogenic steroid production per 10(6) cells over 24 h was: epithelial-like cells, 5.0-mug; fibroblast-like cells, 0-014 mug. Stimulation with ACTH for 5 days increased fluorogenic steroid production and induced morphologic changes in both adrenal cell forms. ACTH stimulation of fluorogenic steroid production by both cell forms reached a maximum after 3 days, then dropped to a refractory state after 5 days. With maximal ACTH stimulation, production increased 25-fold in fibroblast-like cells and five-fold in epithelial-like cells. The latter rate of corticosterone production is similar, per cell, to ACTH-stimulated adrenal glands in vivo. Progressive morphologic changes were observed with ACTH stimulation: epithelial-like cells retracted from the substratum and lost lipid inclusions; fibroblast-like cells became more epithelial-like. Both adrenal cell types formed intermediates from [4-(14)C] pregnenolone including pregn-5-ene-3 phi, 20 alpha-diol and 20 alpha-hydroxy-pregn-4-en-3-one. Control cultures of muscle fascia fibroblasts did not produce corticosterone or intermediates from [4-(14)C[ pregnenolone and did not respond to ACTH functionally or morphologically.     

Splanchnic nerve stimulation modulates steroid secretion in hypophysectomized dogs

To test whether or not splanchnic neural input to the adrenal gland affects secretion of steroids from the adrenal cortex, the thoracic splanchnic nerve was electrically stimulated in pentobarbital-anesthetized dogs after hypophysectomy and replacement with physiological concentrations of ACTH. An adrenal vein cannula was placed to permit measurement of cortisol, corticosterone, 11-deoxycortisol, epinephrine and norepinephrine secretion rates and adrenal blood flow. Plasma ACTH was measured and the presentation rate of ACTH was calculated as the product of plasma ACTH concentration and adrenal plasma flow. Dogs were infused initially with ACTH for 60 min at 2 ng/min followed by infusion for 60 min at 10 ng/min. Within each infusion period, the distal end of the nerve was stimulated (20 V; 0.5-ms pulse duration) at 4 and at 20 Hz for 10 min each. Nerve stimulation resulted in a frequency-dependent increase in mean arterial pressure, in epinephrine and norepinephrine secretion and in adrenal blood flow. Arterial ACTH remained constant during nerve stimulation; however, increased adrenal blood flow resulted in increased presentation rate of ACTH to the adrenal. Cortisol secretion increased in response to nerve stimulation at 4 and 20 Hz during infusion of 2 and 10 ng/min ACTH and occurred prior to changes in presentation rate of ACTH. Corticosterone secretion also increased after stimulation at both frequencies, but the response was observed only during infusion of 10 ng/min ACTH. In contrast, 11-deoxycortisol decreased after nerve stimulation at 4 Hz but showed no response after stimulation at 20 Hz during infusion of 2 and 10 ng/min ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of diazepam binding inhibitor in rat adrenal gland by adrenocorticotropin.

Diazepam binding inhibitor (DBI) is a 9-kDa polypeptide that was initially isolated from rat brain and subsequently found to be present in several peripheral tissues. DBI is particularly abundant in steroidogenic tissues, such as the adrenal glands and testes, which also contain a high concentration of peripheral/mitochondrial benzodiazepine receptors (MBRs). Because occupancy of adrenal MBRs with DBI results in increased steroidogenesis, we have investigated the relation between ACTH, DBI, and the MBR in the rat adrenal glands. Evidence presented here indicates that both the amount of DBI and its rate of synthesis in the adrenal cortex are under the control of ACTH. Seven and 9 days after hypophysectomy, the amount of DBI-like immunoreactivity (DBI-LI) in rat adrenal glands decreased dramatically from approximately 80 to 15 ng/mg tissue. The administration of single dose of ACTH (ACTH residues 1-39; 200 mU/kg, iv) or repeated doses of ACTH-R (ACTH in saline containing 16% gelatin; 15 U/kg, sc, twice daily) reduced the decrease in adrenal DBI-LI caused by hypophysectomy. In hypophysectomized rats (7 days after hypophysectomy) the increases in both adrenal DBI-LI and plasma corticosterone induced by ACTH 1 h after a single injection (200 mU/kg, iv) were inhibited by injection of cycloheximide (40 mg/kg, ip) 10 min after ACTH. However, cycloheximide at this dose had no effect on the ACTH-induced increase in adrenal cAMP concentration or the number of affinity of MBRs for 4'-[3H]chlorodiazepam.     

Stimulation of adrenocorticotropin secretion by insulin-induced hypoglycemia in the developing rat involves arginine vasopressin but not corticotropin-releasing factor.

In the neonatal rat, the response of the hypothalamo-pituitary-adrenal axis to stressful stimuli is markedly decreased during the first 2 weeks of life. This peculiar period was named "stress hyporesponsive period." In this report, we studied the effect of insulin-induced hypoglycemia, known as a strong stimulator of the corticotroph function in the adult rat. Rats (8- or 20-day-old) were injected ip with 3 IU/kg synthetic insulin and were killed at various times. In 20-day-old rats, hypoglycemia induced a rapid drop in blood glucose concentrations accompanied by a stimulation of ACTH and corticosterone secretion which reached maximal values within 30 min. On the opposite, in 8-day-old rats, despite a rapid decrease in blood glucose levels, insulin injection induced a gradual rise of plasma ACTH and corticosterone concentrations which peaked at 90 min. This delayed response of the hypothalamo-pituitary-adrenal axis to hypoglycemia in the youngest rats does not seem to be due to a difference of sensitivity to insulin-induced hypoglycemia since injection of increasing doses of insulin (0.3, 0.75, or 3 IU/kg body wt) induced a dose-related decrease of blood glucose concentrations and a rise in plasma ACTH and corticosterone levels, comparable in the two age group studied. Basal or hypoglycemia-stimulated absolute corticosterone values were much lower in 8-day-old rats than in 20-day-old animals, suggesting an immaturity of the adrenal glands in the youngest animals. Daily ACTH injection, starting 3 days before the experiment, had a trophic effect on the adrenal glands leading to a more important increase of corticosterone levels after hypoglycemia in 8-day-old rats. Our results confirm that there is an immaturity of the adrenal glands in young rats, probably due to the low plasma ACTH levels during the neonatal period. To determine the respective role of the two major hypothalamic ACTH secretagogues, we studied the effect of passive immunization against CRF or arginine vasopressin (AVP) on plasma ACTH response after hypoglycemia. Passive immunization against AVP decreased significantly hypoglycemia-stimulated ACTH secretion in both 8- and 20-day-old rats, while no change of plasma ACTH response to insulin injection was observed after passive immunization against CRF. This results suggest that CRF does not seem to be involved in the regulation of ACTH secretion after hypoglycemia in the young rat while AVP seems to be the main hypothalamic stimulatory factor for anterior pituitary corticotrophs response to hypoglycemia during the postnatal period.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhancement of corticosterone release by repeated injections of ACTH in the dexamethasone pre-treated rat

To examine the possibility that pulsatile secretion of adrenocorticotrophic hormone (ACTH) enhances the responsiveness of the adrenal, the blood corticosterone response to repeated injections of ACTH was determined in the dexamethasone-nembutal pre-treated rat. Treatment with dexamethasone (100 micrograms/100 g body weight) at 13.00 h for 2 days decreased corticosterone levels and completely abolished these daily variations in both the blood and adrenal. Under these conditions, four or five successive iv injections of 0.2 or 2 mIU ACTH were given at 1/2 or 1 h intervals. Blood samples were taken immediately before and at frequent intervals after injection. A significant increase of blood corticosterone levels was observed 10 min after the second injection of 0.2 mIU ACTH. Further increases in blood corticosterone levels were observed after the subsequent injections. In addition, repeated injections of 2 mIU ACTH augmented the responsiveness of the adrenal to ACTH. The second or the third injections of 2 mIU ACTH produced a greater increase in adrenal corticosterone content than did the first ACTH injection. These results suggest that when ACTH acts on the adrenal gland in a pulsatile fashion, the steroidogenic response of the adrenal to ACTH increases markedly.     

The rapid release of corticosterone from the adrenal induced by ACTH is mediated by nitric oxide acting by prostaglandin E2

The adrenal cortex is a major stress organ in mammals that reacts rapidly to a multitude of external and internal stressors. Adrenocorticotropin (ACTH) is the main stimulator of the adrenal cortex, activating corticosteroid synthesis and secretion. We evaluated the mechanism of action of ACTH on adrenals of male rats, preserving the architecture of the gland in vitro. We demonstrated that both sodium nitroprusside (NP), a nitric oxide (NO) donor, and ACTH stimulate corticosterone release. NO mediated the acute response to ACTH because Nomega-nitro-l-arginine methyl ester, a NO synthase inhibitor, and hemoglobin, a NO scavenger, blocked the stimulation of corticosterone release induced by ACTH. NP stimulated prostaglandin E release, which in turn stimulated corticosterone release from the adrenal. Additionally, indomethacin, which inhibits cyclooxygenase, and thereby, prostaglandin release, prevented corticosterone release from the adrenal induced by both NP and ACTH, demonstrating that prostaglandins mediate acute corticosterone release. Corticosterone content in adrenals after incubation with ACTH or NP was lower than in control glands, indicating that any de novo synthesis of corticosterone during this period was not sufficient to keep up with the release of the stored hormone. The release induced by ACTH or NP depleted the corticosterone content in the adrenal by approximately 40% compared with the content of glands incubated in buffer. The mechanism of rapid release is as follows: NO produced by NO synthase activation by ACTH activates cyclooxygenase, which generates PGE(2), which in turn releases corticosterone stored in microvesicles and other organelles.