Role of cyclic AMP in corticotropin releasing factor mediated ACTH release

To elucidate the role of cAMP in the secretion of ACTH, the effect of (1) three phosphodiesterase inhibitors, (2) forskolin, and (3) 8Bromo-cAMP, on CRF mediated ACTH release was studied in rat pituitary cell culture. The action of glucocorticoids on CRF induced cAMP accumulation and ACTH release was investigated. Isobutyl-methylxanthine (IBMX), caffeine, and forskolin augmented the release of ACTH induced from CRF 1.0 nM by 17%, 39%, and 20%, respectively. Also IBMX and caffeine potentiated CRF 10 nM stimulated ACTH release by 32% and 20%. Doses of forskolin and 8Bromo-cAMP, which alone stimulate large amounts of ACTH release, did not increase the amount of ACTH released from CRF 100 nM stimulated cells. Cortisol (500 nM) and corticosterone (500 nM) inhibited CRF induced intracellular cAMP by 39% and 26% while inhibiting pituitary ACTH release by 40% and 52%. In conclusion, cAMP plays an important role in the mechanism of ACTH secretion and it appears the final intracellular mechanism of CRF stimulated ACTH is via cAMP. Also, glucocorticoids exert their inhibitory influence prior to cAMP generation.     

Human phosphoserine 31 corticotropin1-39. Isolation and characterization

Two distinct forms of corticotropin1-39 (ACTH) were isolated and purified from an extract of three adult human pituitaries by reversed-phase chromatographic techniques. Structural studies indicated that the more polar form of ACTH was phosphorylated at serine residue 31. Approximately 30% of the ACTH was found in the phosphorylated form. A similar proportion of phosphorylated ACTH was observed in extracts of three pituitaries from human fetuses of 15, 17, and 20 weeks gestation. Phosphorylated and nonphosphorylated human ACTH1-39 were found to be steroidogenically equipotent using both an isolated rat adrenal cell bioassay and a cultured human fetal adrenal cell bioassay.     

Ultrastructural and biochemical evidence for a steroid-containing secretory organelle in the perfused cat adrenal gland

A correlative study of the ultrastructural and biochemical effects of ACTH on fasciculata cells was carried out on the isolated cat adrenal gland perfused in situ with Locke's solution. The outstanding morphologic feature of cortical cells exposed to microunit ACTH concentrations for 40 min was the abundance of electron-dense granules (0.2-0.4 mum). These organelles were observed in small groups in close proximity to the Golgi region and to the cell membrane. Morphometric and biochemical analysis of control and ACTH-treated glands demonstrated that ACTH stimulation was associated with a fourfold increase in the number of these granules and a comparable increase in the corticosteroid content of the gland. By contrast, ACTH failed to augment cortical lysosomal enzyme activity. These findings, which link steroid release to the appearance of intracellular granules, extend further the parallels between the mechanism of release of newly synthesized steroid and the release of preformed hormones stored in secretory organelles. These results also lend support to the concept that a process related to exocytosis may be the underlying mechanism for extruding steroid from the cortical cell.     

Production of antiserum to CRF associated with adrenal atrophy in a rabbit

Corticotropin releasing factor (CRF) was recently isolated from ovine hypothalami by its ability to stimulate adrenocorticotropin (ACTH) and β-endorphin release from dispersed rat pituitary cells. Intramuscular injection of synthetic ovine CRF conugated to bovine thyroglobulin with 1-ethyl-3(3-dimethylaminopropyl) carbodiimide and emulsified with Freund's complete adjuvant into a random bred New Zealand white rabbit resulted in antiserum production to CRF associated with adrenal atrophy. A decrease in the level of plasma coticosteroids was associated with an increase in mean total binding of ¹²⁵I-N-Tyr-CRF. Upon sacrifice, a decrease in pituitary content of ACTH and a decrease in adrenal weight and content of corticosteroids was observed in the rabbit producing antiserum to CRF. Adrenal atrophy was histologically verified with an observed decrease in the adrenal cortical zone not reflected in the zona glomerulosa. Individual cells were relatively larger either with more abundant pale cytoplasm or with distinctly vacuolated cytoplasm. The results presented here are consistent with a physiologically necessary role for this CRF or peptides with similar structures in the hypothalamic-pituitary-adrenal axis.     

Evidence for 6-keto-PGF1 alpha in adrenal cortex of the rat and effects of 6-keto-PGF1 alpha and PGI2 on adrenal cAMP levels and steroidogenesis.

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF1 alpha, the hydrolysis metabolite of PGI2, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF1 alpha was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF2 alpha or PGE2. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI2 metabolite. Studies in vitro, using isolated cortical cells exposed to 6-keto-PGF1 alpha (10(-6)-10(-4)M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI2 (10(-6)-10(-4)M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering cortico-sterone production, ACTH (5-200 microU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI2 produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused in situ with PGI2 showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF1 alpha is present in the rat adrenal cortex, its precursor, PGI2, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

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

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

Toluene inhalation-induced adrenocortical hypertrophy and endocrinological changes in rat

Rats were exposed to toluene (1,500 ppm for 4 hr per day) for 7 days. The body weight of the rats was significantly lower and the weight of the adrenal gland was significantly higher in the toluene inhalation group compared to the controls. Microscopically, there was no obvious change in the medulla, but hypertrophy of the cortex was observed in the toluene inhalation group. And, the size of adrenocortical cells in treated-rats was also significantly enlarged than the control. Immunohistochemical staining did not show a clear difference in localization of aldosterone-positive cells between the control and inhalation groups. Expansion of the corticosterone-positive area consistent with the cortical hypertrophy was recognized in the inhalation group. Enhancement of 72 kD-heat-shock protein (HSP70)-expression in the toluene inhalation group was not observed. Neither stress nor damage to cortical cells due directly to toluene exposure was observed in the cortex. Also, there was no obvious difference in the anti-proliferating cell nucleus antigen (PCNA)-immunostaining between control and inhalation groups. Thus, it is suspected that cortical hypertrophy was the result of cell enlargement due to the stimulation of the cortical cells. Corticotropin-releasing factor (CRF) immunoreactivity in the paraventricular nucleus (PVN) was increased in the inhalation group. Concentration of plasma ACTH was elevated significantly by toluene exposure. The amounts of mRNA of adrenocortical steroid metabolism gene, cytochrome side-chain cleavage (P450scc), was also increased by toluene inhalation. Toluene exposure might induce adrenocortical hypertrophy via the hypothalamus-pituitary-adrenal gland (HPA) axis.     

Characterization of the genomic corticotropin-releasing factor (CRF) gene from Xenopus laevis: two members of the CRF family exist in amphibians.

In mammals, the release of pituitary ACTH is stimulated by CRF. Two related peptides exist in nonmammalian vertebrates, sauvagine from frog skin and urotensin-I from the urophysis of teleost fish. Their related structures (approximately 50%) and capacity to stimulate the release of ACTH from mammalian and fish pituitaries has led to the proposal that sauvagine and urotensin-I are homologs of mammalian CRF. However, sauvagine does not appear to stimulate ACTH release in amphibians, although mammalian CRF (ovine) induces a potent response from amphibian pituitaries. This could indicate that the main function of sauvagine does not involve ACTH regulation and suggests that an additional CRF-like peptide exists in Amphibia. We report here the isolation of two highly homologous CRF-like genes from the frog, Xenopus laevis. Analysis of the expression pattern of these CRF-like genes revealed mRNA in splenic tissue and in the preoptic nucleus and paraventricular organ of the brain. The amino acid sequence of the mature peptide regions (1-41) of both X. laevis genes is strikingly conserved, sharing more than 93% homology with mammalian CRFs, yet only 50% homology with sauvagine. In view of the fact that these new amphibian CRF-like genes share far greater homology with mammalian CRF than that exhibited by sauvagine, we propose that the new Xenopus CRF-like genes are the amphibian counterparts to mammalian CRF. Thus, two members of the CRF family have now been identified in the Amphibia, namely CRF and sauvagine.     

The stimulation by ACTH of 17 alpha-hydroxylation in cultures or rabbit adrenal cells

Adrenal cells from control rabbits (control-cells) and from rabbits that had been injected twice daily for 3 days prior to sacrifice with 25 IU ACTH (ACTH-cells) were cultured both in the absence and presence of 100 mIU ACTH. Culture durations varied from 24 to 120 h in 24 h increments. The culture medium was changed daily and fresh ACTH added to appropriate vessels. At the time of the final media change 0.1 muCi [4-14C]pregnenolone was added. Twenty-four hours later the cultures were terminated and the products formed from the pregnenolone were isolated, quantified and identified by solvent extraction, chromatography and crystallization to constant specific activity. After 72 h ACTH-cells cultured in the presence of ACTH converted 18.5% of the pregnenolone substrate to 17-hydroxycorticosteroids (cortisol plus 11-deoxycortisol) while ACTH-cells cultured in the absence of ACTH converted a maximum of 1.6%. A similar but smaller difference, 10.9 vs 2.1%, was recorded with control cells cultured in the presence and absence of ACTH. Corticosterone production from [4-14C]pregnenolone in the 72-h cultures was increased to a lesser degree by ACTH exposure. In ACTH-cells the conversion climbed from 5.9 to 10.5% and from 9.6 to 12.0% in control cells. Microscopic examination of parallel cultures showed no significant differences in cell density between cells cultured in the presence or absence of ACTH. 17 alpha-Hydroxylase activity arising from the in vivo stimulation did not survive cell division in culture, but required the continual presence of ACTH. In conclusion, the data show that ACTH is capable of stimulating 17-hydroxycorticoid formation in rabbit adrenal cell cultures.     

Regulation of low density lipoprotein receptors by adrenocorticotropin in the adrenal gland of mice and rats in vivo

Membranes prepared from the adrenal gland of mice and rats possess high affinity binding sites that recognize 125I-labeled human low density lipoprotein (LDL). These binding sites resemble the functional LDL receptors that mediate the uptake of LDL by cultured mouse and bovine adrenal cells. The number of LDL binding sites per mg of membrane protein increased 2- to 5-fold over 24 h when mice or rats were treated with adrenocorticotropin (ACTH). In rats, this increase was accompanied by a similar ACTH-induced increase in the adrenal uptake of intravenously administered 125I-LDL, suggesting that the LDL binding sites mediate the uptake of LDL by the adrenal in the intact animal. The number of LDL binding sites on adrenal membranes rose by 5-fold when animals were rendered lipoprotein-deficient, either by treatment of mice with 4-aminopyrazolopyrimidine or by treatment of rats with 17 alpha-ethinyl estradiol. This increase was prevented when endogenous ACTH secretion was blocked by administration of dexamethasone, suggesting that ACTH was required. The current experiments suggest that LDL receptors provide one source of cholesterol for the mouse and rat adrenal in vivo and that the number of LDL receptors of this organ is regulated by ACTH.     

Microscopic study of cell death in the adrenal glands of mouse and chick embryos

Dying cells of both chromaffin and cortical cell types were found scattered throughout the adrenal gland of 14-18 day mouse embryos and 17-19 day chick embryos. The ultrastructural appearance of these dying cells was unlike that of cells undergoing apoptosis and there was no evidence of macrophages or other phagocytes removing these cells from the adrenal. Possible morphogenetic functions of cell death in the developing adrenal are discussed.     

Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells

The roles of human low density lipoprotein (LDL)- cholesterol and high density lipoprotein (HDL)- cholesterol on adrenal steroidogenesis were investigated using cultured human adult and fetal adrenocortical cells and the findings were then compared to those obtained with bovine adrenocortical cells. The secretion of cortisol in both human and bovine adrenocortical cells was dose-dependently increased by the administration of LDL- or HDL-cholesterol in the presence of adrenocorticotropin (ACTH). LDL-cholesterol was utilized to a greater extent than HDL-cholesterol in both human and bovine adrenal steroidogenesis in the presence of ACTH. Exogenous lipoprotein-derived cholesterol was less utilized in human adrenal steroidogenesis than in bovine adrenal steroidogenesis, compared to the endogenous cholesterol. An increase in the secretion of cortisol and dehydroepi androsterone sulfate (DHEA-S) continued for the 5-day culture period, in the presence of lipoprotein cholesterol and ACTH in both human adult and fetal adrenocortical cells. The secretion of aldosterone increased on the first day of the culture period, then gradually decreased for the 5-day culture period in human adult adrenocortical cells, but not in human fetal adrenocortical cells in the presence of lipoprotein cholesterol and ACTH. These findings demonstrate that exogenous cholesterol utilized in the biosynthesis of steroids is mainly from LDL-cholesterol in both human adult and fetal adrenals and bovine adrenal and the proportion of cholesterol synthesized de novo is significantly larger in the human adult adrenal than in the bovine adrenal.     

Differentiation of embryonic mouse GH cells and ACTH cells in vitro

The ability of cells that produce growth hormone (GH) and cells that produce adrenocorticotropic hormone (ACTH) to differentiate in various culture media was analyzed by means of ultrastructural immunocytochemistry on 13-day embryonic mouse pituitaries that were maintained in organ culture for 3-11 days. At the time of culture, relatively undifferentiated nongranulated or poorly granulated cells that were unreactive with anti-growth-hormone serum (anti-GH) and anti-adrenocorticotropic-hormone serum (anti-ACTH) were present in the pituitary. After 10-11 days in culture, immunoreactive GH cells were obtained only in media that were supplemented with cortisol, whereas ACTH cells were obtained in all media tested, including Medium 199 alone. In cortisol-supplemented media, the GH cells showed ultrastructural features typical of those that occur in vivo, and anti-GH immunoreactivity was obtained after as little as 3 days in culture, i.e., at a stage comparable to that which occurs in vivo. The results indicate that mouse GH cells are capable of differentiating in Medium 199 supplemented only with cortisol, without the addition of fetal calf serum or insulin; cortisol therefore appears to be an essential component of the embryonic milieu for the production of GH-secretory granules.     

Effects of ACTH and estradiol on steroid production by cultured adrenal cells from an anencephalic fetus and from normal adults

Dispersed adrenal cells from a 16 1/2 week anencephalic fetus, 7 fetuses with intact pituitaries and 3 adult subjects undergoing renal transplants were maintained in tissue culture and the steroidogenic responses to ACTH (0-10(3) pg/ml), with or without added estradiol (0-10(4) ng/ml) were evaluated. In the anencephalic preparation the response to ACTH was delayed, but by the fifth day production of cortisol, dehydroepiandrosterone (DHA) and DHA-sulfate was similar to that in the other cultured fetal adrenal cells. The addition of estradiol caused dose-related inhibition of cortisol production and concomitant increase in DHA and DHA-sulfate production. The adult adrenal cells in the presence of ACTH showed a much higher cortisol/DHA secretion ratio, but the addition of estradiol markedly reduced this ratio as in fetal cells. The data support the suggestion that the major factors which interact to impose the characteristic fetal pattern of adrenal steroidogenesis are ACTH and the synergistic effects of placental and intra-adrenal steroids (such as estradiol) which act to inhibit 3 beta-hydroxysteroid dehydrogenase activity.     

Regulation of adrenal scavenger receptor-BI expression by ACTH and cellular cholesterol pools.

Scavenger receptor BI (SR-BI) mediates selective uptake of high density lipoprotein (HDL) cholesteryl ester in the liver and adrenal gland. Adrenal SR-BI is increased both in adrenocorticotropic hormone (ACTH)-treated mice and also in apolipoprotein A-I knock-out (apoA-I0) mice which have depleted adrenal cholesterol stores. The goal of the present study was to determine whether adrenal cholesterol stores and ACTH have independent effects on SR-BI expression in adrenal gland. Adrenal SR-BI levels were 5-fold higher in apoA-I0 than wild-type mice when killed under low stress condition, and plasma ACTH levels were similar in both strains. After male apoA-I0 or wild-type mice were treated with dexamethasone to suppress ACTH release, adrenal SR-BI protein levels were decreased in both groups but remained 13-fold higher in apoA-I0 than in wild-type mice. By contrast, uncontrolled stress or supplemental ACTH treatment increased SR-BI levels but narrowed the difference in SR-BI expression between apoA-I0 and wild-type. Cholesterol depletion by beta-cyclodextrin in cultured Y1-BS1 adrenal cells also led to a rapid 2- to 3-fold increase in SR-BI mRNA and protein levels, in association with a significant depletion of cellular free cholesterol.These results indicate that depletion of adrenal cholesterol stores can act independently from ACTH to increase SR-BI expression, but in vivo this effect is diminished under high ACTH conditions. Both stimuli may increase selective uptake via increased SR-BI as a means of replenishing cholesterol stores for steroid hormone synthesis.     

From integrative signalling to metabolic disorders

The adrenal cortex undergoes constant dynamic structural changes, a key element in ensuring integrative functionality of the gland. Studies have shown that the cellular environment can modulate cell functions such as proliferation and steroid secretion. For example, 3-day treatment with angiotensin II promotes protein synthesis with a concomitant decrease in proliferation of glomerulosa cells, when cultured on fibronectin, but not on collagen IV or laminin. These effects involve close interaction between cytoskeleton-associated proteins and activation of p42/p44mapk and p38 MAPK pathways. On the other hand, adrenocorticotropin hormone (ACTH), which is clearly the most potent stimulus of fasciculata cells, induces specific modulation of targeted proteins, when cells are cultured on collagen IV, but not on fibronectin or laminin. In particular, ACTH treatment leads to increased expression of Seladin-1 and induces the relocalization of Seladin-1 from the cytoplasm to the nucleus, both in vivo and in culture conditions, in adult rats and in human fetal adrenal glands. As a whole, these results indicate that Seladin-1, together with collagen IV, is able to modulate ACTH responsiveness. Hence, Seladin-1 may participate in the regulation of steroidogenesis when localized in the cytoplasm, while conversely protecting cells against oxidative stress generated by intense ACTH stimulation when massively localized in the nucleus.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE ACTH-INDUCED ULTRASTRUCTURAL TRANSFORMATION OF MITOCHONDRIA OF CORTICAL CELLS OF RAT ADRENALS IN TISSUE CULTURE

The ultrastructure of the mitochondria of the cultured cortical cells of rat adrenals was studied. In vivo it was found that the zona fasciculata mitochondria have vesicular internal structure. 600-A vesicles appear free in the matrix or as protrusions of the inner mitochondrial membrane. In tissue cultures of the fetal and newborn rat adrenal cortex it was seen that ACTH induces transformation of the tubulo-vesicular internal structure of the mitochondria to 600-A vesicles. Actinomycin D and puromycin inhibited this transformation if they were added with ACTH. When added alone, these inhibitors of protein synthesis induced no change in the ultrastructure of the mitochondria in cultured cortical cells of rat adrenals.     

Thymosin fraction 5 (TF5) stimulates secretion of adrenocorticotropic hormone (ACTH) from cultured rat pituitaries

In vivo administration of a partially purified thymic hormone-containing extract of the thymus gland, TF5, causes an increase in serum glucocorticoids. The lack of a direct effect of TF5 on adrenal corticosterone secretion suggests that it is mediated at the level of the pituitary. Cultured rat pituitary monolayers were used to determine if the effect is mediated by stimulation of ACTH secretion from the pituitary. Two lots of TF5, BPP100 and C114080-01, caused a dose dependent secretion of ACTH from cultured pituitary monolayers. There was a synergistic effect when the cells were treated with both TF5 and corticotropin-releasing factor (CRF). Immunoneutralization studies were done in which the cells were treated with TF5 or CRF and an antibody to CRF. The antibody completely blocked CRF induced ACTH release, but had no effect on TF5 stimulated ACTH release, suggesting that the activity is not due to a CRF-like peptide in TF5. A number of peptides isolated from TF5, and certain other peptides produced by the immune system were evaluated for their ability to stimulate ACTH secretion. These included thymosin (TSN) alpha 1, alpha 11, and beta 4, prothymosin alpha (PT alpha, thymopoeitin 5 (TP5), factuer thymique serique (FTS), interferon alpha (INF alpha), INF gamma, interleukin 1 (IL-1), and interleukin 2 (IL-2). None of these factors had any effect on pituitary ACTH secretion. These results demonstrate that some peptide component of TF5 causes an increase in serum corticosteroids by stimulating pituitary ACTH release.     

Regulation of the hypothalamic-pituitary-adrenal axis in birth

In sheep an increase in fetal pituitary-adrenal function, reflected in rising concentrations of plasma ACTH and cortisol, is important in relation to fetal organ maturation and the onset of parturition. This review presents evidence that implicates the hypothalamic-pituitary-adrenal axis in the control of parturition and describes recent experiments that explore in detail the maturation of the fetal hypothalamus and pituitary in relation to fetal adrenal function. Recent improvements for the measurement of ACTH in unextracted plasma and the ability to maintain vascular catheters in chronically catheterized fetal sheep have enabled subtle changes in fetal ACTH concentrations to be detected. As a result of these advances it has now been established that the terminal rise in cortisol, which is responsible for the onset of parturition in sheep, is preceded by an increase in fetal plasma ACTH concentrations. This has led to the hypothesis that birth results from the sequential development of the fetal hypothalamic-pituitary-adrenal axis with the signal originating from the fetal brain. This increase in trophic drive to the fetal adrenal may result from changes in the responsiveness of the fetal pituitary gland to factors that stimulate the release of ACTH. Corticotropin releasing factor (CRF) and arginine vasopressin are two such factors that stimulate the secretion of ACTH and cortisol secretion in the chronically catheterized fetal sheep. The response to these factors increases with gestational age and is sensitive to glucocorticoid feedback. Furthermore, repeated administration of CRF to immature fetal sheep results in pituitary and adrenal activation and in some cases may lead to premature parturition.(ABSTRACT TRUNCATED AT 250 WORDS)     

The biosynthesis and secretion of adrenocorticotropin by the ovine anterior pituitary is predominantly regulated by arginine vasopressin (AVP). Evidence that protein kinase C mediates the action of AVP

This study was undertaken to define the roles of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of adrenocorticotropin (ACTH) release and biosynthesis in cultured ovine anterior pituitary cells and to define the intracellular mechanisms responsible for their action. At 4 h, CRF and AVP increased both ACTH release and total ACTH content, with AVP clearly the more potent agonist (maximal ACTH release: AVP, 22.8-fold; CRF, 7.6-fold; maximal increment in total ACTH content: AVP, 1.9-fold; CRF, 1.1-fold; EC50 for ACTH release: AVP, 2.3 +/- 0.5 nM; CRF, 9.2 +/- 5.0 nM). The increase in total ACTH content was interpreted to reflect an augmentation of ACTH biosynthesis since it was abolished by 10 microM cycloheximide. Exposure of the anterior pituitary cells to increasing concentrations of forskolin or 8-bromo-cAMP elicited increases in ACTH release and total ACTH content that were similar to those caused by CRF. A 30-min incubation with phorbol 12-myristate 13-acetate (PMA) caused a dose-related translocation of protein kinase C from the cytosol to the cell membrane; after 4 h, the increases in ACTH release and total ACTH content in response to increasing concentrations of PMA were similar to those caused by AVP. Chronic (24 h) exposure to 150 nM PMA caused an almost total depletion of both cytosolic and membrane-bound protein kinase C activities. When protein kinase C-depleted cells were subsequently exposed to AVP, the increases in ACTH release and total ACTH content were markedly attenuated, but the responses to CRF were preserved. Finally, the combination of CRF and AVP, CRF and PMA, or AVP and 8-bromo-cAMP increased ACTH release and total ACTH content in a synergistic manner. We conclude that: 1) in ovine anterior pituitary cells, AVP is the predominant regulator of ACTH secretion and biosynthesis; 2) the action of AVP is predominantly mediated by activation of protein kinase C, whereas the action of CRF is likely to be mediated by activation of the cAMP-dependent protein kinase (protein kinase A); and 3) the ability of CRF and AVP to increase total ACTH content and secretion in a synergistic manner provides a demonstration in normal pituitary cells that protein kinases C and A may interact in a unidirectional manner to regulate ACTH biosynthesis in addition to ACTH release. This interaction may take place within, or between, individual corticotropes.