Transmembrane signals mediating adrenocorticotropin release from mouse anterior pituitary cells

The effect of arginine vasopressin (AVP) and corticotropin releasing factor (CRF) an adrenocorticotropin (ACTH) secretion, phosphatidylinositol breakdown and cAMP accumulation was examined in primary cultures of mouse anterior pituitary cells. AVP and CRF added alone stimulated ACTH secretion in a dose-dependent manner. At 10(-8) M concentration of peptide, AVP and CRF stimulated ACTH secretion 2.8- and 4.6-fold, respectively. AVP and CRF added in combination at equal doses gave an additive effect. CRF enhanced cAMP accumulation, but AVP had no effect on basal or CRF-induced cAMP accumulation. Both forskolin (10(-5) M) and 8-bromo-cAMP (10(-3) M) increased ACTH secretion in these cells by 2.8- and 1.7-fold, respectively. AVP induced the breakdown of phosphoinositides, and CRF alone, or in combination with AVP did not modify this effect. Phorbol 12-myristate 13-acetate (10(-7) M), dioctanoylglycerol (10(-4) M) and phospholipase C (100 mU/ml) also stimulated ACTH secretion in these cells by 4.2-, 2.4-, and 3.7-fold, respectively. Depletion of intracellular and extracellular Ca2+ decreased ACTH secretion, but had no significant effect on CRF-induced cAMP accumulation. However, AVP-induced phosphoinositide breakdown was dependent on extracellular Ca2+. These results indicate that CRF stimulates ACTH secretion via the cAMP-dependent pathway and AVP via the phosphoinositide breakdown-phospholipase C pathway. In the presence of AVP and CRF, both pathways appear to operate independently to produce an additive effect on ACTH secretion.     

Glucocorticoids inhibit corticotropin-releasing factor-induced production of adenosine 3',5'-monophosphate in cultured anterior pituitary cells

The effect of corticotropin-releasing factor (CRF) on ACTH secretion and cAMP production by cultured anterior pituitary cells was investigated. Intracellular cAMP production was stimulated over 13-fold within 10 min in response to 10 nM CRF, reaching a plateau between 20 and 30 min. This was followed by a gradual decline in cellular cAMP, which was associated with its release into the medium. The release of both cAMP and ACTH followed similar time courses and remained linear over a 4-h incubation. CRF caused a dose-dependent increase in both cAMP production and ACTH release over a similar concentration range during short (30-min) incubations when cellular cAMP was maximal. During longer (4-h) incubations, however, significantly higher concentrations of CRF were required to stimulate cAMP production than those needed to stimulate ACTH release. Pretreatment of these cells with 20 nM dexamethasone for 18 h prevented CRF-stimulated ACTH secretion and significantly attenuated cAMP production at all time points during the 4 h. Concentrations of dexamethasone required to prevent CRF-stimulated ACTH release and cAMP production as well as 8-bromo-cAMP-stimulated ACTH release were similar. Equivalent results were obtained with corticosterone. These results suggest that changes in intracellular cAMP levels may, at least in part, mediate the action of CRF on ACTH secretion in the anterior pituitary gland and that one mechanism by which glucocorticoids exert their inhibitory action on corticotrophs may involve an effect on cAMP production.     

Angiotensin II potentiates corticotropin-releasing activity of CRF41 in rat anterior pituitary cells: mechanism of action

In this work the ability of angiotensin II (AII) to potentiate the corticotropin-releasing activity of ovine CRF41 (CRF) and the intracellular mechanism responsible for this effect are described. In perfused rat anterior pituitary cells, AII (10(-8)M) was found to potentiate the corticotropin-releasing activity of CRF producing a parallel shift of the dose-response curve. Similar results for ACTH release were observed in monolayer cell cultures. In this system, the concentration of cAMP was measured and was shown to be increased in the presence of CRF with a maximal value (2.5-fold greater than control) after 5-10 min incubation. On the other hand, AII at a dose inducing ACTH release (10(-7)M), had no effect on basal cAMP concentration, but when given simultaneously with CRF, potentiated the CRF-induced cAMP production (1.9-fold greater than CRF value). These results indicate that AII potentiates the corticotropin-releasing activity of CRF and that this effect is preceded by a similar increase in the CRF-induced cAMP production.     

Posttranslational modulation of glucocorticoid feedback inhibition at the pituitary level

Diagnostic tests of hypothalamic-pituitary-adrenocortical function in psychiatric illness largely report the interaction of hypothalamic secretagogues with glucocorticoids at the pituitary level. This study investigated whether the efficiency of glucocorticoid inhibition is subject to modulation by intracellular processes that enhance cAMP accumulation and/or facilitate membrane depolarization. The secretion of ACTH induced by corticotropin-releasing factor (CRF; 0.1 nM) in primary cultures of rat anterior pituitary cells was markedly inhibited upon a 2-h exposure to 100 nM corticosterone. Arginine vasopressin (2 nM) enhanced the cAMP as well as the ACTH responses to CRF and reduced the efficiency of glucocorticoid inhibition of ACTH release. The action of arginine vasopressin was mimicked by rolipram, an inhibitor of cyclic nucleotide phosphodiesterase type 4. Application of the broad specificity K(+) channel blockers clofilium and astemizole produced minor or no significant enhancement of CRF-induced ACTH release, respectively, but opposed the inhibitory effect of corticosterone. Specific blockers of HERG, KCNQ, and Isk channels had no effect on ACTH release under any condition examined. In summary, these data reveal multiple sites of posttranslational modulation of adrenal corticosteroid action at the level of the pituitary gland, which appear important for the outcome of diagnostic tests of hypothalamic-pituitary- adrenocortical function.     

Pituitary corticotrope tumor (AtT20) cells as a model system for the study of early inhibition by glucocorticoids.

The utility of the established ACTH secreting mouse pituitary tumor cell line AtT20 for investigating early glucocorticoid inhibition was examined. Three different strains of the cell line D1, D16v, and D16:16, respectively, were analyzed. In initial studies CRF and phorbol esters were used as secretagogues to examine the properties of hormone secretion. In a perifusion system (cells in suspension) D1 cells failed to respond to the secretagogues, whereas both D16v and D16:16 cells were responsive. However, hormone release declined upon repeated exposure to secretagogue in both D16v and D16:16 cells and similar data were obtained when cells adhering to cover slips were perifused. In static incubation D16:16 cells gave more consistent results especially with respect to inhibition by glucocorticoids and were used in all subsequent studies. Synthetic glucocorticoids acting through the type II receptor inhibited CRF-induced ACTH release within 45 min; at 120 min, stimulated release was strongly (80-90%) suppressed. In contrast, no consistent inhibition by corticosterone could be found. In the presence of glycyrrhetinic acid, an inhibitor of 11 beta-hydroxysteroid dehydrogenase, a high concentration of corticosterone (10 microM) did produce a slight inhibition of ACTH release. Dexamethasone also inhibited ACTH release induced by the calcium channel activator compound (+)202-791. The accumulation of cAMP in response to CRF was not altered by dexamethasone. The inhibitory effect of synthetic glucocorticoids on ACTH release was prevented by blockers of messenger RNA (actinomycin D, dichlorobenzimidazole ribofuranoside) or protein (puromycin) biosynthesis, indicating the induction of new proteins. Immunoblotting for lipocortin I (annexin I) and chromogranin A revealed no induction by dexamethasone of any of these proteins in D16:16 cells. Messenger RNA encoding lipocortin I was not detectable and was not induced by treatment with dexamethasone in D16:16 cells. These data show that the AtT20 D16:16 strain is a useful model for early glucocorticoid action, which is mediated by type II receptors and involves the induction of new protein(s). Notably, induction of lipocortin I messenger RNA or protein could not be detected at a time when the inhibitory effect of glucocorticoids on stimulated hormone secretion was maximal.     

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

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

Growth hormone-releasing factor desensitization in rat anterior pituitary cells in vitro

Preincubation of rat pituitary cells in primary culture with rat GH-releasing factor (rGRF) resulted in substantial desensitization to subsequent GRF stimulation. rGRF-directed GH release and intracellular cAMP accumulation decreased in the desensitized cells. Whereas prior treatment of rat pituitary cells caused partial depletion of intracellular GH levels, diminished cellular reserves could not entirely account for the decreased GH release. Cells that had been preexposed to 10 nM rGRF for 4 h demonstrated at 30-50% depletion of intracellular GH; subsequent stimulation of those cells with 10 nM rGRF elicited GH release which was only 5% of that seen in cells that were not desensitized [control, 112 +/- 3.2 ng/well (+/- SEM); GRF-stimulated, 435 +/- 32 ng/well; GRF-pretreated, control, 63 +/- 3 ng/well, GRF-pretreated, GRF-stimulated, 73 +/- 3.4 ng/well]. Despite the marked depletion of cellular GH stores and the greatly diminished rGRF-stimulated GH release in cells that had been preexposed to rGRF, both forskolin and (Bu)2cAMP were able to induce a 2-fold stimulation of GH release. Incubation of the rGRF-pretreated cells with fresh medium which lacked rGRF resulted in gradual recovery of the ability of rGRF to stimulate GH release without complete reconstitution of the intracellular GH stores. These results indicate that exposure of rat pituitary cells to rGRF results in 1) partial depletion of intracellular GH stores; 2) a diminished ability of a subsequent rGRF challenge to elicit GH secretion and intracellular cAMP accumulation, and 3) a sustained ability of forskolin and (Bu)2cAMP to stimulate GH release, indicating that rGRF desensitization occurs in vitro.     

Corticotropin-releasing factor stimulates phospholipid methylation and corticotropin secretion in mouse pituitary tumor cells.

The 41-residue synthetic ovine corticotropin-releasing factor (CRF; corticoliberin) has been shown to stimulate release of corticotropin (adrenocorticotropic hormone; ACTH) and beta-endorphin from AtT-20/D16-16 mouse pituitary tumor cells. Phospholipid methylation of phosphatidylethanolamine to phosphatidylcholine with S-adenosylmethionine as methyl donor has been suggested as a possible membrane transduction mechanism for some receptor-induced events. CRF increased phospholipid methylation in pituitary tumor cells at concentrations that also stimulated immunoreactive ACTH secretion, and both processes increased linearly and in parallel with time. The methionine sulfoxide derivative of CRF was less potent than CRF was in stimulating both phospholipid methylation and hormone secretion, and the COOH-terminal free acid analogue of CRF had no effect on either process. CRF-induced increases in phospholipid methylation and ACTH secretion were reduced when cells were treated with the phospholipid methyltransferase inhibitors 3-deazaadenosine and L-homocysteine thiolactone. These CRF-stimulated effects were also blocked by the glucocorticoid dexamethasone. It is suggested that phospholipid methylation may be a CRF receptor-mediated event associated with ACTH release in pituitary tumor cells.     

Glucocorticoid enhancement of adrenocorticotropin-induced 3',5'-cyclic adenosine monophosphate production by cultured ovine adrenocortical cells

The present study examines the effect of chronic treatment of glucocorticoids on ACTH1-24- or forskolin-induced cAMP output of cultured sheep adrenocortical cells. Cells cultured for 2 days in the presence of 1 microM dexamethasone released more cAMP in response to ACTH1-24 than did untreated cells, both in the absence and presence of 0.5 mM 1-methyl-3-isobutylxanthine. Such an enhancing effect required greater than or equal to 21 h of treatment and was both concentration-dependent and steroid specific. The ED50 of dexamethasone was about 10 nM, while that of cortisol and corticosterone was about 1 microM; testosterone at concentrations less than or equal to 10(-5) M had no enhancing effect. Glucocorticoids enhanced the cAMP response to ACTH1-24 without altering its ED50. Treatment of cultures with aminoglutethimide or the antiglucocorticoid RU 38486 for 48 h resulted in a dose-dependent decrease in ACTH1-24-induced cAMP output. Moreover, RU 38486 antagonized the enhancing effect of dexamethasone. Glucocorticoids did not increase the cAMP response to forskolin. These results suggest that chronic exposure to glucocorticoids is necessary for the full expression of the cAMP response to ACTH1-24 of adrenocortical cells from adult sheep.     

Calcium-dependent control of corticotropin release in rat anterior pituitary cell cultures

The role of calcium in the stimulation of ACTH secretion by CRF and other regulators was studied in rat anterior pituitary cells. Incubation of cultured pituitary cells in normal calcium with CRF, vasopressin, angiotensin II, or norepinephrine increased the rate of ACTH release for up to 45 min and then became constant for up to 3 h. In the absence of extracellular calcium, the initial rate of stimulated secretion was unaffected, but after 45 min the secretion rate decreased by 40% for CRF and to a greater extent for the other stimuli. Addition of calcium after 90 min in calcium-free medium restored the CRF-stimulated ACTH release rate to the control value. The absence of extracellular calcium had no effect on CRF-stimulated cAMP accumulation, but intracellular calcium depletion by preincubation of the cells with EGTA completely inhibited CRF-stimulated cAMP production and ACTH release. The voltage-dependent calcium channel antagonist nitrendipine and the calcium channel agonist BK 8644 had little effect on the CRF-stimulated ACTH release rate, while they, respectively, inhibited and enhanced the stimulation by vasopressin and high potassium. In calcium-depleted cells incubated with the calcium ionophore A23187, CRF stimulation of cAMP production and ACTH release were dependent upon extracellular calcium concentrations from 0.1-100 microM. These findings have defined two phases in the stimulation of ACTH release by CRF and cAMP-independent stimuli in cultured pituitary cells: an early phase with a rapid increase in the ACTH release rate which is independent of extracellular calcium, and a late phase of constant secretion rate, with partial extracellular calcium dependence for the stimulation by CRF and complete calcium dependence for the stimulation by non-cAMP-mediated stimuli.     

Effects of adrenalectomy and dexamethasone administration on the level of prepro-corticotropin-releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin/beta-lipotropin precursor mRNA in the pituitary in rats

RNA blot hybridization analysis with cloned rat CRF precursor (prepro-CRF) cDNA as a probe showed that prepro-CRF mRNA existed in rat hypothalamic and extrahypothalamic brain tissue, whereas it was undetectable in the pituitary and adrenal. To study the effect of glucocorticoid on the level of prepro-CRF mRNA in the hypothalmus and that of ACTH/beta-lipotropin (beta LPH) precursor mRNA in the pituitary, effects of adrenalectomy and dexamethasone administration were studied in rats. Adrenalectomy markedly raised mRNA coding for ACTH/beta LPH precursor in the anterior pituitary, but not in the neurointermediate pituitary lobe. Hypothalamic pre-pro-CRF mRNA increased only to 152% of the control value, 7 days after adrenalectomy. The administration of dexamethasone (200 micrograms/day for 7 days) started immediately after adrenalectomy lowered the ACTH/beta LPH precursor mRNA level in the anterior pituitary to 19% of the intact control value, whereas the level of prepro-CRF mRNA in the hypothalamus decreased only to 102%. These results suggest that glucocorticoids exert their feedback effect at the level of gene expression on both hypothalamic CRF neurons and pituitary corticotropes. Although the possibility that CRF neurons insensitive to glucocorticoid in the hypothalamus might blunt the change in the prepro-CRF mRNA could not be ruled out, it is also possible that the effect of glucocorticoids on the pituitary is dominant.     

Characteristics of the ACTH response to repeated pulses of corticotrophin-releasing factor and arginine vasopressin in vitro

A multi-column perifusion system was used to investigate the dynamics of the dose-response relationships of ACTH release by ovine pituitary cells when stimulated by both corticotrophin-releasing hormone (CRF) and arginine vasopressin (AVP) given alone and in combination. A dose-response relationship was obtained when 10-min pulses were given at 60-min intervals over the range of 0.002-2000 nmol CRF/1 and 1-2000 nmol AVP/1, with a minimum effective concentration of 0.02 nmol CRF/1 or 1 nmol AVP/1. When AVP was given together with CRF, the expected potentiation of the ACTH response occurred when compared with the summed response of these secretagogues given separately. At the higher concentrations of CRF and AVP used, the ACTH responses to repeated pulses decreased with time during the experiment. The rate of this loss of responsiveness was significantly correlated to the size of the response to the first pulse (for CRF: r = 0.89, P less than 0.01; for AVP: r = 0.95, P less than 0.01), being greatest when the response was potentiated by adding the secretagogues together (for CRF plus AVP: r = 0.95, P less than 0.01). Reduced availability of receptors or changes in intracellular transduction processes may contribute to this desensitization. Reduced levels of secretable ACTH do not appear to be implicated because desensitization to pulses of one secretagogue did not cause equivalent desensitization to the other. In addition, cells stimulated continuously with submaximal levels of either secretagogue showed desensitization while more ACTH was still available for release to higher levels of stimulant.(ABSTRACT TRUNCATED AT 250 WORDS)     

The hormonal actions of corticotropin-releasing factor in sheep: effect of intravenous and intracerebroventricular injection

The 41-residue ovine corticotropin releasing factor (CRF) was administered iv and intracerebroventricularly (icv) to merino sheep. A significant rise in plasma ACTH, beta-lipotropin (beta LPH) and cortisol was demonstrated after the administration of 200 micrograms, iv. A highly significant correlation between the increments in plasma ACTH and beta LPH was observed. The plasma ACTH rise was evident within 5 min and was abolished by the prior administration of 0.4-4.0 mg dexamethasone. No significant rise in plasma GH, LH, PRL, insulin, glucagon, pancreatic polypeptide, met-enkephalin, angiotensin II, aldosterone, or vasopressin could be demonstrated. Although smaller doses of CRF (50 ng to 5 micrograms) were effective when given icv, the ACTH response was more delayed. It is concluded that CRF stimulates a rapid increase in the secretion of ACTH and beta LPH in sheep. Suppression of this response by dexamethasone indicates that glucocorticoids are capable of acting on the pituitary to inhibit the ACTH response to CRF. The delayed response when CRF is given icv may be due to diffusion. The action of CRF appears to be relatively specific, in that the plasma concentrations of the other pancreatic, pituitary, and adrenal hormones measured were not affected.     

Effects of cyproheptadine, reserpine, and synthetic corticotropin-releasing factor on pituitary glands from patients with Cushing's disease

Direct effects of cyproheptadine, reserpine, synthetic ovine corticotropin-releasing factor (CRF), dexamethasone, and lysine-8-vasopressin (LVP) on the secretion of immunoreactive ACTH and beta-endorphin from the adenoma and the nonadenomatous tissue of patients with Cushing's disease were examined using a superfusion system. Cyproheptadine and reserpine (10(-9)-10(-7) M of each) suppressed immunoreactive ACTH and beta-endorphin secretion from both tissues. CRF (10(10)-10(7) M) stimulated the secretion of both peptides from the nonadenomatous tissue, but only a high dose of CRF could stimulate the secretion of these peptides from some adenomas. Such CRF-induced secretion was partially suppressed by dexamethasone. LVP (10(-9)-10(-7) M) stimulated peptide secretion from both types of tissue. These results suggest direct inhibitory effects of cyproheptadine and reserpine on the secretion of these peptides from the pituitary of patients with Cushing's disease, a different stimulatory mechanism of LVP from that of CRF in these tissues, and low sensitivity of the adenoma to CRF.     

Rapid glucocorticoid inhibition of vasoactive intestinal peptide-induced cyclic AMP accumulation and prolactin release in rat pituitary cells in culture.

Vasoactive intestinal peptide (VIP) stimulates both adenosine 3',5'-cyclic monophosphate (cAMP) accumulation and prolactin release in normal rat pituitary cells in culture. cAMP accumulation is significant (P less than 0.01) at VIP concentrations as low as 1 nM and reaches a maximum with 0.1 microM. Addition of dexamethasone as early as 15 min before VIP inhibits VIP stimulation of both cAMP production and PRL secretion. The rapid inhibition is dose-dependent: it appears at doses as low as 0.01 pM and is complete at 1 pM dexamethasone. Increasing concentrations of dexamethasone induce a noncompetitive type of inhibition, as shown by the decrease in Vmax with no change in the apparent Km for VIP. Cycloheximide (1 mM) counteracts the inhibitory effect of dexamethasone on VIP-induced cAMP production, which suggests the involvement of a rapid protein synthesis mechanism. Ru-26988, a specific glucocorticoid devoid of any mineralocorticoid activity and which does not bind to intracellular transcortin-like component, also produces an inhibition of VIP-induced cAMP accumulation. Corticosterone also inhibits VIP-induced cAMP production but at concentrations higher than those of dexamethasone. In contrast, aldosterone, progesterone, estradiol, and testosterone have no effect. These results demonstrate that, in normal rat pituitary cells in culture, glucocorticoids at physiological concentrations rapidly inhibit the cAMP production and prolactin release induced by VIP by acting through specific glucocorticoid receptors.     

G protein-coupled receptor kinase 2 involvement in desensitization of corticotropin-releasing factor (CRF) receptor type 1 by CRF in murine corticotrophs

Hypothalamic CRF stimulates synthesis and secretion of ACTH via CRF receptor type 1 (CRFR1) in the anterior pituitary gland. After agonist-activated stimulation of receptor signaling, CRFR1 is down-regulated and desensitized. Generally, it is thought that G protein-coupled receptors may be desensitized by G protein-coupled receptor kinases (GRKs). However, the role of GRKs in corticotropic cells has not been determined. In this study we focused on involvement of GRKs in desensitization of CRFR1 by CRF in corticotropic cells. We found that GRK2 (but not GRK3) mRNA and protein were expressed in rat anterior pituitary cells and AtT-20 cells (a line of mouse corticotroph tumor cells). To determine the role of GRK2 in CRF-induced desensitization of CRFR1 in mouse corticotrophs, AtT-20 cells were transfected with a dominant-negative mutant GRK2 construct. CRF desensitized the cAMP-dependent response by CRFR1. Desensitization of CRFR1 by CRF was significantly less in AtT-20 cells transfected with the dominant-negative mutant GRK2 construct compared with desensitization in control (an empty vector-transfected) AtT-20 cells. Furthermore, pretreatment with a protein kinase A inhibitor also partially blocked desensitization of CRFR1 by CRF. These results suggest that GRK2 is involved in CRF-induced desensitization of CRFR1 in AtT-20 cells, and the protein kinase A pathway may also have an important role in desensitization of CRFR1 by CRF seen in corticotropic cells.     

Glucocorticoid modulation of beta-adrenergic receptors of cultured rat arterial smooth muscle cells

Since both glucocorticoids and catecholamines are involved in the regulation of normal blood pressure, we investigated the modulation of beta-adrenergic receptors of cultured rat arterial smooth muscle cells by glucocorticoids. The synthetic glucocorticoids dexamethasone and RU 28362, at 10(-8) M concentration, increased maximum beta-adrenergic binding but had no effect on the dissociation constant (Kd). Each steroid caused an increase in maximum [3H]dihydroalprenolol binding over the concentration range of 10(-8) to 10(-6) M, but not at 10(-9) M. The glucocorticoid effect on beta-adrenergic receptors of arterial smooth muscle cells required a minimum of 20 hours of incubation in the presence of the steroid and was significantly inhibited by cycloheximide (10 micrograms/ml), indicating that the glucocorticoid effect required protein synthesis. The effect of dexamethasone on [3H]dihydroalprenolol binding was significantly inhibited by the glucocorticoid antagonist RU 38486. Basal and agonist-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels in arterial smooth muscle cells, before and after glucocorticoid treatment, were measured as an indicator of the physiological significance of the observed glucocorticoid-induced increase in beta-adrenergic receptor binding. While causing no change in the basal cAMP level, treatment of arterial smooth muscle cells with 10(-6) M dexamethasone for 24 hours increased the 10(-6) M isoproterenol-stimulated cAMP levels.     

Defective glucocorticoid regulation of proopiomelanocortin gene expression and peptide secretion in a small cell lung cancer cell line

A human small cell lung cancer cell line (COR L103) that actively expresses the proopiomelanocortin (POMC) gene has been used as a model of extrapituitary ACTH-secreting tumors to investigate the phenomenon of resistence of ACTH production to glucocorticoids. After both short term (24 h) and long term (10 days) exposure to hydrocortisone at concentrations of 500 and 1000 nM, the accumulation of intracellular POMC mRNA, ACTH, and ACTH precursor peptides in the culture medium was not suppressed. These finding contrast with those in the pituitary corticotroph cell line AtT20, in which POMC mRNA, ACTH, and ACTH precursors were suppressed under the same conditions. Two other genes that are regulated by glucocorticoids in other cell types, the tyrosine amino transferase gene and the glucocorticoid receptor gene, were expressed in COR L103 cells. However, neither gene appeared to be regulated by hydrocortisone in this small cell lung cancer cell line. Further studies demonstrated that glucocorticoid receptor binding could be detected in the nucleus and cytoplasm, with a Kd of 5 X 10(-9) M. It is concluded that nonsuppression of POMC by glucocorticoids is probably part of a more global defect of glucocorticoid signaling in these cells, but that this defect lies distal to steroid binding in the nucleus.     

Rapid as well as delayed inhibitory effects of glucocorticoid hormones on pituitary adrenocorticotropic hormone release are mediated by type II glucocorticoid receptors and require newly synthesized messenger ribonucleic acid as well as protein

Glucocorticoid hormones suppress the release of ACTH by the anterior pituitary gland: rapid feedback inhibits hormone secretion within 30 min of steroid application, delayed feedback is most effective at 1-2 h, and slow feedback becomes manifest in several hours. The aim of the present study was to determine the type of glucocorticoid receptor that mediates the rapid and delayed feedback actions of glucocorticoids and whether genomic activation occurs during the rapid and delayed time domains. Rat anterior pituitary cell columns were perfused with Dulbecco's minimum essential medium, 41-residue CRF (10(-9) M) was used as the secretagogue, which stimulated ACTH secretion to a peak of about 8- to 10-fold of basal release. The amount of ACTH released upon repeated 5 or 10 min stimulation with CRF was constant. Treatment with 10(-7) M corticosterone for 20 min immediately before and for 10 min during stimulation with CRF reduced ACTH release by about 50% (rapid feedback), while at 1 h and 2 h after the initial exposure to corticosterone the secretory response was 33% and 15% of control, respectively. The effect of corticosterone was prevented by the type II glucocorticoid/progesterone antagonist RU 38486 (10(-6) M). The selective type II receptor agonist RU 28362 (10(-7) M) was even more potent than corticosterone in inhibiting ACTH release; the time course of action was similar. When actinomycin D (10(-4) M) was applied in conjunction with RU28362 or corticosterone, no inhibitory effects appeared up to 2 h after the exposure to steroid. Puromycin (10(-4) M), given during and for 1 h after the administration of the steroid prevented the rapid as well as the delayed (1 h) inhibitory action of RU28362. When puromycin was removed from the system, a 75% inhibition of stimulated ACTH release developed at 2 h after the application of the steroid, indicating that translatable messenger RNA (mRNA) was still present in the cells. Cycloheximide (10(-4) M) was only partially effective at inhibiting rapid or delayed feedback, and increasing its concentration impaired the ACTH response to CRF-41. In summary, at the pituitary level the rapid as well as the delayed feedback inhibition of ACTH secretion by adrenal corticoids is exerted via type II glucocorticoid receptors. Furthermore, both rapid and delayed feedback require the synthesis of new mRNA and protein.     

A superfusion system technique for the study of the sites of action of glucocorticoids in the rat hypothalamus-pituitary-adrenal system in vitro. II. Hypothalamus-pituitary cell-adrenal cell superfusion.

Basal and stimulated CRF release by hypothalamic blocks was studied by coupling the effluent of superfused hypothalamus tissue to a joint pituitary cell-adrenal cell superfusion system and measuring corticosterone production. Log dose-response curves of the adrenal cells for ACTH and of the pituitary cell-adrenal cell system for CRF were linear over the ranges used. Ca++-independent basal CRF release by the hypothalamus could be blocked in vitro by 0.2 mug/ml dexamethasone in the medium, or in vivo by treating the hypothalamus donor rats with corticosterone, 1 mg/rat ip 30 min before decapitation. These treatments did not impair CRF release caused by Veratridine (5 x 10(-6)M or by electrical stimulation. Adrenalectomy increased only basal but not stimulated CRF release. These results indicate that glucocorticoids have a hypothalamic site of action.