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Biomedical subjects

G Aguilera

Publications and source records attributed to G Aguilera.

At least 109 records · Page 6Linked to original sources
PubMed1989

Angiotensin II receptors in the gonads.

The presence of components of the renin-angiotensin system in ovaries and testes suggests that angiotensin II (AII) is involved in gonadal function, and thus we sought to characterize receptors for AII in rat and primate gonads. In the testes, autoradiographic studies showed receptors in the interstitium in all species. In rat interstitial cells fractionated by Percoll gradient, AII receptors coincided with hCG receptors indicating that AII receptors are located on the Leydig cells. In Leydig cells and membranes from rat and rhesus monkey prepuberal testes, AII receptors were specific for AII analogues and of high affinity (Kd=nM). During development, AII receptor content in rat testes decreases with age parallel to a fall in the ratio of interstitial to tubular tissue. In the ovary, the distribution of AII receptors was dependent on the stage of development, being high in the germinal epithelium and stromal tissue between five and 15 days, and becoming localized in secondary follicles in 20-and 40-day-old rats. No binding was found in primordial or primary follicles. In rhesus monkey ovary, AII receptors were higher in stromal tissue and lower in granulosa and luteal cells of the follicles. Characterization of the binding in rat and monkey ovarian membranes showed a single class of sites with a Kd in the nmol/L range and specificity similar to that of the adrenal glomerulosa and testicular AII receptors. Receptors for AII were also present in membrane fractions from PMSG/hCG primed rat ovaries. Infusion of AII (25 ng/min) or captopril (1.4 micrograms/min) during the PMSG/hCG induction period had no effect on ovarian weight or AII receptor concentration in the ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1989

Distribution of angiotensin II receptors and renin in the mouse fetus.

To investigate the ontogeny of the renin-angiotensin system we studied the characteristics and location of angiotensin II (AII) receptors in mouse fetuses and examined sites of renin mRNA expression by in situ hybridization and Northern blot analysis. Autoradiographic analysis of the binding of 125I-[Sar1,Ala8]AII to slide-mounted frozen sections of 17-day-old DBA/2N mice revealed abundant AII receptors widely distributed throughout the body. High receptor density was found in primitive mesenchymal tissue under the epidermis and surrounding muscle and cartilage, in skeletal and smooth muscle, and in all layers of the adrenal cortex. Lower receptor density was seen in the kidney, liver, and lungs. The autoradiographic staining was abolished by incubation of the sections with excess unlabeled AII. Scatchard analysis of the binding of 125I-[Sar1,Ala8,]AII to membrane-rich fractions of eviscerated fetuses showed a single type of high affinity receptors with a Kd of 2.9 x 10(-9) M and a receptor concentration of 3300 fmol/mg protein. Localization of renin mRNA was analyzed by in situ hybridization using an antisense 35S-labeled riboprobe transcribed from a mouse renin2 cDNA clone. Hybridization to fetal tissue sections showed high intensity staining in the kidney and adrenal cortex. Northern blot analysis confirmed the high expression of renin mRNA in the fetal kidney. The presence of an active renin-angiotensin system in the fetus was confirmed by the demonstration of renin-like activity and bioactive AII in fetal extracts. The widespread distribution of AII receptors in the fetus, compared to the discrete localization to specialized tissues in the adult, may indicate a unique role for the peptide during development.

Angiotensin II↗
PubMed1989

Protein kinase C mediates the effect of vasopressin in pituitary corticotrophs.

The role of protein kinase C (PKC) on vasopressin (VP) action was investigated by inhibition of endogenous PKC using prolonged incubation of the cells with phorbol ester, and by direct measurement of PKC activity in pituitary cells. Preincubation of the cells for 6 h with 100 nM TPA at 37 C resulted in a 90% decrease in total PKC activity. In the PKC-depleted cells, cAMP responses to stimulation with 100 nM CRF for 30 min were normal, but the potentiating effects of VP and PMA on CRF-stimulated cAMP production were abolished. The stimulation of ACTH secretion by VP and PMA alone was also abolished in PKC- depleted cells. PKC activity in cytosolic and detergent-solubilized membrane fractions from enriched pituitary corticotrophs obtained by centrifugal elutriation, was directly measured by enzymatic assays and by immunoblotting techniques. Basal PKC activity was higher in the cytosol than in the membranes (8.43 +/- 0.47 and 1.93 +/- 0.11 pmol 32P incorporated/10 min, respectively). After incubation of the cells with VP for 15 min or [3H] phorbol-12-myristate-13-acetate (PMA) for 30 min, PKC activity in cytosol was decreased by 40% and 89%, respectively, while the activity in the membrane was increased by 138% and 405%, respectively. Such VP- and PMA-induced translocation of PKC was also observed when the enzyme content in the cytosol and the membranes was measured by immunoblotting using a specific anti-PKC antibody and [125I]protein A. Autoradiographic analysis of immunoblots revealed an 80 kilodalton band characteristic of PKC, with OD higher in the cytosolic than in the membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Influence of the thymus on steroidogenesis by rat ovarian cells in vitro.

Thymic hormones and factors have been shown to modulate the function of other endocrine glands including the gonads. Absence of the thymus during development results in ovarian dysgenesis characterized by a decrease in the number of follicles and corpora lutea, bringing about severe changes in reproductive function. To examine whether thymic secretions might affect ovarian activity, whole dispersed ovarian cells obtained from immature rats pretreated with pregnant mare serum gonadotrophin were exposed to a thymus fraction of approximately 28 kDa and also to the media from incubated thymuses (TIM) and the conditioned media from cultured thymic reticuloepithelial cells (TCM). The thymic fraction caused a dose-dependent decrease in human chorionic gonadotrophin (hCG)-stimulated production of progesterone, oestradiol and testosterone, but had no effect on their synthesis in the absence of hCG. Similarly, hCG-induced production of these steroids was decreased by TIM and TCM. Progesterone secretion was the most markedly affected. These results suggest: (1) that the thymus contains a factor with a molecular weight of approximately 28 kDa which interacts with hCG in ovarian cells, (2) that the thymus can release active substances which modify steroid secretion by the ovary in vitro and (3) that the reticuloepithelial cells of the thymus are involved in the secretion of factors which modulate the stimulation by hCG of steroidogenesis in ovarian cells.

Animals↗
PubMed1988

Control of glomerulosa cell function by angiotensin II: transduction by G-proteins and inositol polyphosphates.

1. The receptor-activated mechanisms that mediate the steroidogenic actions of angiotensin II (AII) have been characterized in rat and bovine adrenal glomerulosa cells. In rat adrenal cells, the AII receptor is coupled to a guanine nucleotide inhibitory protein which reduces adenylate cyclase activity and cyclic AMP production. However, receptor-mediated stimulation of aldosterone production by AII is exerted through a separate pertussis-insensitive nucleotide regulatory protein that subserves coupling of activated receptors to phospholipase C. 2. In AII-stimulated glomerulosa cells, hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase C yields diacylglycerol and inositol 1,4,5-trisphosphate (Ins-P3), which act as second messengers by activating calcium-calmodulin and calcium-phospholipid dependent protein kinase pathways. Ins-1,4,5-P3 is a potent stimulus of intracellular calcium mobilization, and is promptly inactivated by two major routes of metabolism. Direct degradation of Ins-1,4,5-P3 by a 5-phosphatase gives inositol 1,4-bisphosphate which in turn is metabolized to inositol-4-monophosphate. The latter product can be derived only from higher inositol phosphates, and thus serves as a specific marker of polyphosphoinositide breakdown in agonist-stimulated cells. In contrast, inositol-1-phosphate is largely derived from phosphatidylinositol hydrolysis, which is not increased during the initial phase of AII action. 3. Ins-1,4,5-P3 formed in AII-stimulated glomerulosa cells is also phosphorylated by a calcium-calmodulin dependent 3-kinase to form inositol 1,3,4,5-tetrakisphosphate (Ins-P4), which is rapidly dephosphorylated to the biologically inactive Ins-1,4,5-P3 isomer, Ins-1,3,4-trisphosphate. The latter metabolite, like Ins-1,4,5-P3, is both degraded to lower phosphates (Ins-3,4,P2 and Ins-1,3-P2) and phosphorylated to form a new tetrakisphosphate isomer (Ins-1,3,4,6-P4). Ins-1,4,5-P3 formed during AII action is bound with high affinity to specific intracellular receptors through which InsP3 causes calcium mobilization during the initiation of cellular responses to AII and other calcium-dependent ligands.

Angiotensin II↗
PubMed1988

Corticotropin-releasing factor (CRF) induces desensitization of the rat pituitary CRF receptor-adenylate cyclase complex.

The rise in circulating ACTH levels after adrenalectomy in the rat is associated with a decrease in CRF receptor-binding capacity in the anterior pituitary. To investigate the role of increased hypothalamic CRF release on pituitary CRF receptor regulation after withdrawal of glucocorticoid feedback by adrenalectomy, the effects of chronic CRF infusion and lesions in the medial basal hypothalamus were studied in the rat. Subcutaneous infusion of CRF at 10, 25, 50, and 100 ng/min for 48 h in intact rats caused dose-dependent increases in plasma ACTH levels from the control value of 32.1 +/- 4.3 to 58.0 +/- 4.9, 82.0 +/- 7.1, 135.5 +/- 11.6, and 149.2 +/- 13.2 pg/ml, respectively. In contrast, the pituitary CRF receptor concentration was reduced by 25.3 +/- 4.5%, 38.3 +/- 2.5%, 43.8 +/- 0.9%, and 45.8 +/- 2.0%, respectively. Intravenous infusion of increasing doses of CRF caused a similar increase in plasma ACTH levels, which became maximum at the lowest infusion dose (32.4 +/- 5.4, 138.5 +/- 12.3, 162.0 +/- 18.3, and 167 +/- 19.1 pg/ml for control and 10, 50, and 100 ng/min CRF, respectively). Pituitary CRF receptor concentration was again decreased after iv CRF infusion [by 42 +/- 6.2% with the lowest dose (10 ng/min)], with no further reduction after infusion of 50 and 100 ng/min (49.0 +/- 6.8% and 26.0 +/- 6.2%, respectively)]. The decrease in pituitary CRF receptors after CRF infusion was accompanied by a decrease in CRF-stimulated adenylate cyclase activity, with a 10- to 100-fold increase in the concentration of CRF required for threshold stimulation. In cultured pituitary cells prepared from animals infused with 50 ng/min CRF for 48 h, maximum CRF-stimulated ACTH release was reduced by 29 +/- 3.2% (P less than 0.01; n = 3), with no significant change in sensitivity to CRF (ED50, 0.6 +/- 0.5 and 1.0 +/- 0.5 nM CRF for control and CRF infusion, respectively). The role of endogenous CRF in adrenalectomy-induced pituitary CRF receptor down-regulation was also studied in rats with medial basal hypothalamic deafferentation. The marked loss of pituitary CRF receptors after adrenalectomy was completely prevented by such hypothalamic lesioning, indicating that receptor down-regulation was dependent on the release of CRF or/and other hypothalamic factors. The data demonstrate that while increased CRF levels result in down-regulation and desensitization of pituitary CRF receptors, the differences between adrenalectomy and CRF infusion indicate that additional regulatory factors are involved in the modulation of CRF receptor content and activity after adrenalectomy.

Adenylyl Cyclases↗
PubMed1988

Angiotensin II receptors in testes.

Receptors for angiotensin II (AII) were identified and characterized in testes of rats and several primate species. Autoradiographic analysis of the binding of 125I-labeled [Sar1,Ile8]AII to rat, rhesus monkey, cebus monkey, and human testicular slide-mounted frozen sections indicated specific binding to Leydig cells in the interstitium. In rat collagenase-dispersed interstitial cells fractionated by Percoll gradient, AII receptor content was parallel to that of hCG receptors, confirming that the AII receptors are in the Leydig cells. In rat dispersed Leydig cells, binding was specific for AII and its analogs and of high affinity (Kd, 4.8 nM), with a receptor concentration of 15 fmol/10(6) cells. Studies of AII receptors in rat testes during development reveals the presence of high receptor density in newborn rats which decreases toward the adult age (4934 +/- 309, 1460 +/- 228, 772 +/- 169, and 82 +/- 12 fmol/mg protein at 5, 15, 20, and 30 days of age, respectively) with no change in affinity. At all ages receptors were located in the interstitium, and the decrease in binding was parallel to the decrease in the interstitial to tubular ratio observed with age. AII receptor properties in membrane-rich fractions from prepuberal testes were similar in the rat and rhesus monkey. Binding was time and temperature dependent, reaching a plateau at 60 min at 37 C, and was increased by divalent cations, EGTA, and dithiothreitol up to 0.5 mM. In membranes from prepuberal monkey testes, AII receptors were specific for AII analogs and of high affinity (Kd, 4.2 nM) with a receptor concentration of 7599 +/- 1342 fmol/mg protein. The presence of AII receptors in Leydig cells in rat and primate testes in conjunction with reports of the presence of other components of the renin-angiotensin system in the testes suggests that the peptide has a physiological role in testicular function.

Aging↗
PubMed1988

Corticotropin-releasing factor receptors and pituitary adrenal responses during immobilization stress.

The regulation of pituitary and brain CRF receptors and corticotroph responses during stress were studied in rats subjected to prolonged immobilization. Plasma ACTH levels showed the characteristic biphasic changes, with a rapid 23-fold increase in 15 min, followed by a decrease to about twice the basal levels after 6-h immobilization. In contrast, plasma corticosterone levels were markedly elevated throughout the duration of the stress. Pituitary CRF receptor content, measured by binding of [125I]Tyr-ovine CRF to pituitary membrane-rich fractions, was unchanged after 2.5 h, but was reduced by 28 +/- 2.7% (+/- SE) and 47.6 +/- 1.1% after 18 and 48 h of immobilization, respectively. These results were confirmed by autoradiography in slide-mounted frozen pituitary sections. In contrast, no changes in CRF receptor content were observed in brain areas, including olfactory bulb, frontoparietal cortex, hippocampus, amygdala, and lateral septum. A concomitant decrease in immunoreactive (ir) CRF content in the median eminence of rats immobilized for 48 h is consistent with the hypothesis that increased release of CRF into the portal circulation occurs during chronic stress. Despite pituitary CRF receptor loss and reduced in vitro responses to CRF, the increases in plasma ACTH and corticosterone in vivo after ether exposure or CRF injection were greater and more prolonged in rats immobilized for 48 h than in nonimmobilized controls. The decrease in pituitary CRF receptors was accompanied by decreased CRF-stimulated cAMP and ACTH release in cultured pituitary cells from 48-h restrained rats. However, concomitant incubation of cells with CRF and vasopressin restored cAMP and ACTH responses to control levels, suggesting that the simultaneous release of both regulators from the hypothalamus determines the plasma ACTH level. These findings indicate that the decrease in plasma ACTH during the adaptation phase to stress is accompanied by decreases in pituitary CRF receptors. However, the enhanced pituitary response to a superimposed stress or CRF injection implies that the decrease in plasma ACTH levels during prolonged stress may be due to adaptive changes at the central level. These findings emphasize the importance of the integrated actions of CRF and other regulators in the control of the pituitary adrenal-axis during stress.

Adrenal Glands↗
PubMed1987

Phorbol 12-myristate 13-acetate and vasopressin potentiate the effect of corticotropin-releasing factor on cyclic AMP production in rat anterior pituitary cells. Mechanisms of action.

The potentiation of corticotropin-releasing factor (CRF)-stimulated cAMP production by vasopressin (VP) in the pituitary cell was investigated by studies on the interaction of CRF, VP, and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) on cAMP, adenylate cyclase and phosphodiesterase. Addition of VP or PMA (0.01-100 nM) alone did not alter cellular cAMP content, but markedly increased the effect of 10 nM CRF with ED50 of about 1 nM. Treatment of the cells with 200 ng/ml pertussis toxin for 4 h increased CRF-stimulated cAMP accumulation by 3.2-fold, an effect that was not additive to those of VP and PMA. Incubation of pituitary cells with 2 mM 1-methyl-3-isobutylxanthine increased CRF-stimulated cAMP accumulation and decreased the relative effect of VP and PMA, suggesting that the actions of VP and PMA are partially due to inhibition of phosphodiesterase. This was confirmed by the demonstration of a 30% inhibition of the low-affinity phosphodiesterase activity in cytosol and membranes prepared from cells preincubated with VP or PMA. In intact cells, following [3H]adenine prelabeling of endogenous ATP pools, measurement of adenylate cyclase in the presence of 1-methyl-3-isobutylxanthine showed no effect of VP and PMA alone, but did show a 2-fold potentiation of the effect of CRF. Measurement of adenylate cyclase in pituitary homogenates by conversion of [alpha-32P]ATP to [32P]cAMP showed a paradoxical GTP-dependent inhibition by VP of basal and CRF-stimulated adenylate cyclase activity, suggesting that the VP receptor is coupled to an inhibitory guanyl nucleotide-binding protein. Pertussis toxin pretreatment of the cells prevented the VP inhibition of adenylate cyclase activity observed in pituitary cell homogenates. These findings indicate that besides inhibition of phosphodiesterase, VP has a dual interaction with the pituitary adenylate cyclase system; a direct inhibitory effect, manifested only in broken cells, that is mediated by a receptor-coupled guanyl nucleotide-binding protein, and a physiologically predominant indirect stimulatory effect in the intact cell, mediated by protein kinase C phosphorylation of one of the components of the CRF-activated adenylate cyclase system.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Angiotensin II receptors and mechanisms of action in adrenal glomerulosa cells.

The plasma-membrane receptors, coupling mechanisms, and effector enzymes that mediate target-cell activation by angiotensin II (AII) have been characterized in rat and bovine adrenal glomerulosa cells. The AII holoreceptor is a glycoprotein of Mr approximately 125,000 under non-denaturing conditions. Photoaffinity labeling of AII receptors with azido-AII derivatives has shown size heterogeneity among the AII binding sites between species and target tissues, with Mr values of 55,000 to 79,000. Such variations in molecular size probably reflect differences in carbohydrate content of the individual receptor sites. The adrenal AII receptor, like that in other tissues, is coupled to the inhibitory guanine nucleotide inhibitory protein (Ni). However, studies with pertussis toxin have shown that stimulation of aldosterone production by AII is not mediated by Ni but by a pertussis-insensitive nucleotide regulatory protein of unidentified nature. Although Ni is not involved in the stimulatory action of AII on steroidogenesis, it does mediate the inhibitory effects of high concentrations of AII upon aldosterone production. The actions of AII on adrenal cortical function are thus regulated by at least two guanine nucleotide regulatory proteins that are selectively activated by increasing AII concentrations. The principal effector enzyme in AII action is phospholipase C, which is rapidly stimulated in rat and bovine glomerulosa after AII receptor activation. AII-induced breakdown of phosphatidylinositol bisphosphate (PIP2) and phosphatidylinositol phosphate (PIP) leads to formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). These are metabolized predominantly to inositol-4-monophosphate, which serves as a marker of polyphosphoinositide breakdown, whereas inositol-1-phosphate is largely derived from phosphatidylinositol hydrolysis. The AII-stimulated glomerulosa cell also produces inositol 1,3,4-trisphosphate, a biologically inactive IP3 isomer formed from Ins-1,4,5-trisphosphate via inositol tetrakisphosphate (IP4) during ligand activation in several calcium-dependent target cells. The Ins-1,4,5-P3 formed during AII action binds with high affinity to specific intracellular receptors that have been characterized in the bovine adrenal gland and other AII target tissues, and may represent the sites through which IP3 causes calcium mobilization during the initiation of cellular responses.

Adrenal Glands↗
PubMed1987

Synthetic atrial natriuretic factors (ANFs) stimulate guanine 3',5'-monophosphate production but not hormone release in rat pituitary cells: peptide contamination with a gonadotropin-releasing hormone agonist explains luteinizing hormone-releasing activity of certain ANFs.

The effects of atrial natriuretic factors (ANFs) on anterior pituitary hormone secretion and cyclic nucleotide production were investigated in cultured rat pituitary cells. ANF had no effect on ACTH, GH, PRL, and TSH release or on cAMP production either on basal hormone levels or during stimulation of their secretion by the appropriate releasing factor. However, ANF markedly stimulated cGMP production in both mixed anterior pituitary cells and enriched anterior pituitary cell populations fractionated by centrifugal elutriation. Unexpectedly, certain ANF preparations, Bachem rat ANF-(5-28) and rat ANF-(5-25), markedly stimulated LH release from cultured anterior pituitary cells and gonadotroph-enriched elutriated pituitary cells. The same ANFs also displaced [125I-D-Lys6]GnRH ethylamide from binding to anterior pituitary membranes with potencies similar to their LH-releasing activities. Immunoprecipitation of ANF with a specific antiserum abolished the effect of ANF on cGMP production, but did not change the effect of ANF on LH release. In conclusion, ANF did not affect anterior pituitary hormone secretion or cAMP production, but stimulated cGMP formation. The effect of certain ANF preparations on LH release appears to be attributable to peptide contamination with a potent GnRH agonist.

Adrenal Glands↗
PubMed1987

Differential effects of atrial natriuretic factor on angiotensin II- and adrenocorticotropin-stimulated aldosterone secretion.

The possible role of atrial natriuretic factor (ANF) in the regulation of adrenal sensitivity to angiotensin II (AII) was investigated in vivo and in vitro by analyzing the characteristics of the inhibitory effect of ANF on aldosterone production stimulated by AII and other stimuli. In isolated adrenal glomerulosa cells, ANF caused a dose-dependent inhibition of basal and stimulated aldosterone production by submaximal concentrations of ACTH, AII, and potassium, with an ED50 of about 1 nM for ANF and complete inhibition with 10 nM ANF. ANF increased the ED50 for ACTH from 14.6 +/- 3.2 to 376 +/- 104 pM with no significant decrease in the maximum aldosterone response. In contrast, ANF inhibited the aldosterone responses to all doses of AII, decreasing maximal aldosterone production by 75%, with a small increase in the ED50 for AII. In conscious rats, ANF infusion (100 ng/min) markedly decreased the plasma aldosterone response to AII infusion (5-10 ng/min). With higher AII doses (50 and 100 ng/min), which increased plasma corticosterone (and presumably ACTH secretion), the inhibitory effect of ANF was less marked. When the rise in ACTH secretion was prevented by dexamethasone treatment, ANF decreased the aldosterone response to 100 ng/min AII by 85%. Similarly, ANF had a minor although significant inhibitory effect on the primary ACTH-mediated increases in plasma aldosterone after stress by immobilization for 15 min. The data demonstrate a prominent inhibitory effect of ANF on AII-stimulated aldosterone production in vivo and in vitro. Since plasma ANF levels are increased during atrial distension, these observations support a regulatory role of ANF in the control of the adrenal sensitivity to AII during alterations of extracellular volume.

Adrenal Glands↗
PubMed1987

Differential regulation of brain and pituitary corticotropin-releasing factor receptors by corticosterone.

The regulatory actions of CRF during the neuroendocrine response to stress are mediated by specific receptors within the nervous system and the anterior pituitary gland. Glucocorticoids exert negative feedback inhibition on ACTH secretion by interacting at the pituitary corticotrophs and the central nervous system. To determine whether glucocorticoids influence ACTH secretion by regulating the concentration of CRF receptor sites, binding of [125I]Tyr-oCRF to pituitary and brain membrane-rich particles was studied after glucocorticoid treatment. Corticosterone administration (0.5-150 mg/day) for 1-4 days in adult male rats caused a dose-dependent decrease in the number of CRF receptors in the anterior pituitary in parallel with the reduction in ACTH secretion. In the brain, binding studies in membrane-rich fractions or by autoradiography in slide-mounted frozen sections revealed no changes in CRF receptors in the cortex, hippocampus, amygdala, septal area, and olfactory bulb, although circulating corticosterone levels were higher than during stress. The selective down-regulation of anterior pituitary CRF receptors after corticosterone administration, without alterations in brain CRF receptors, is similar to the change in CRF receptors previously reported after adrenalectomy and indicates that receptor regulatory mechanisms in secretory cells differ from those in neural tissue. Furthermore, the decrease in pituitary CRF receptors after physiological increases in circulating glucocorticoids may contribute to the inhibitory effects of adrenal steroids on ACTH secretion.

Adrenocorticotropic Hormone↗
PubMed1987

Control of aldosterone production by angiotensin II is mediated by two guanine nucleotide regulatory proteins.

The involvement of guanine nucleotide regulatory proteins in the steroidogenic response of the adrenal glomerulosa to angiotensin II (AII) was investigated by analyzing the effects of Bordetella pertussis toxin (PT) on several aspects of AII action. These included receptor binding, stimulation of aldosterone production and GTPase activity, inhibition of cAMP production, and attenuation of the aldosterone response at high angiotensin concentrations. Pretreatment of glomerulosa cells with PT abolished the inhibitory effects of both AII and somatostatin (SRIF) on ACTH-stimulated cAMP production. Under the same incubation conditions, the stimulation of aldosterone secretion by submaximal and maximal steroidogenic concentrations of AII was completely unaffected by the toxin. However, the attenuation of steroid responses seen with supramaximal concentrations of AII was abolished. In addition, the ability of SRIF to inhibit AII-stimulated steroid production was markedly reduced by PT treatment. The binding of [125I]AII to high affinity sites in intact cells and particulate fractions, and modulation of the binding by guanine nucleotides, were unaffected by toxin pretreatment, even under conditions where a 40-41K protein was completely ADP ribosylated. In contrast, the toxin substantially diminished the binding of [125I]Tyr0-SRIF to SRIF receptors in glomerulosa cells (by 50% after 5 h and by 90% after 20 h). These results indicate that Ni or a similar protein probably mediates the inhibition of cAMP formation by AII and the attenuation of the steroid response by high concentrations of AII as well as the inhibitory actions of SRIF in the adrenal glomerulosa cell. Furthermore, the lack of effect of PT on AII binding and stimulation of GTPase activity suggests the existence of an additional pertussis-insensitive guanine nucleotide-regulatory protein that is activated by lower concentrations of AII and mediates the stimulation of aldosterone production.

Adenosine Diphosphate Ribose↗
PubMed1987

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.

Adrenocorticotropic Hormone↗