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Biomedical subjects

W W Vale

Publications and source records attributed to W W Vale.

At least 199 records · Page 11Linked to original sources
PubMed1983

Solubilization of the gonadotropin-releasing hormone receptor from bovine pituitary plasma membranes.

The plasma membrane receptor for GnRH in bovine anterior pituitaries has been solubilized with the cholic acid derivative detergent 3([3-cholamidopropyl)dimethylammonio]propane sulfonate. The soluble receptor in the supernatant from a 100,000 x g x 90 min centrifugation displays high affinity, saturability and specific binding to the agonist, [DA1a6,N alpha MeLeu7, Pro9-NEt]-GnRH. The dissociation constant, KD, for the soluble receptor is 0.56 +/- 0.07 nM (mean +/- SEM) and the number of sites, Ro, is 85 +/- 17 fmols/mg protein. For the membrane-bound receptor the KD is 0.50 +/- 0.04 nM and Ro is 300 +/- 18 fmols/mg protein. The relative potencies of GnRH and the potent antagonist, [Ac-delta 3Pro1,pFDPhe2, DTrp3,6]-GnRH are similar for both the soluble and membrane-bound receptor.

Animals↗
PubMed1983

Phorbol diesters stimulate somatostatin secretion from cultured brain cells.

Phorbol diester tumor promoters are potent cocarcinogens which also possess activity in a variety of biological assays. We have examined the effect of phorbol diesters on secretion of somatostatin-like immunoactivity (SRIF-LI) by dispersed cells of fetal rat brain maintained in long term culture. Phorbol-12-myristate-13-acetate (PMA) and phorbol 12, 13-dibutyrate stimulate SRIF-LI secretion in a dose-dependent fashion. 4-O-Methyl-PMA is approximately 100 times less potent than phorbol 12, 13-dibutyrate. 4-beta-Phorbol was inactive. Treatment with a nonphorbol irritant, teleocidin, also was associated with significantly augmented release of SRIF-LI. Significant stimulation is seen within 7.5 min of treatment and response is linear over 1 h. Administration of phorbol diesters in low calcium buffer (0.1 mM) with or without cobalt or pretreatment with verapamil are associated with significantly diminished secretion. Substitution for sodium ion by choline or pretreatment with tetrodotoxin (10(-7) M) also inhibits response to PMA. gamma-Aminobutyric acid (50 microM) or the gamma-aminobutyric acid agonist muscimol (5 microM) decrease response to PMA as does sodium pentobarbital (IC50 approximately 30 microM). Phenobarbital is less potent as an inhibitor; significant suppression is not seen until approximately 300 microM. The data are consistent with an action for phorbol diesters mediated at least in part by voltage dependent sodium channels and calcium influx into excitable cells. Inhibition by hyperpolarizing agents is compatible with this mechanism. Phorbol diesters may thus mimic endogenous modulator substances active at the nerve cell membrane.

Alkaloids↗
PubMed1983

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.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Stimulation of adenosine 3',5'-monophosphate production by growth hormone-releasing factor and its inhibition by somatostatin in anterior pituitary cells in vitro.

The stimulation of GH secretion from the anterior pituitary by synthetic GRF (hpGRF) is associated with a rapid increase in cAMP production. Within 5 min of the addition of 1 nM hpGRF to cultured rat anterior pituitary cells, intracellular cAMP levels are elevated 6-fold, with a maximal response being observed at 30 min. cAMP accumulation in the extracellular medium is also enhanced by this peptide. Comparison of the two cellular responses (GH secretion and cAMP formation) at various concentrations of hpGRF indicates that 10 times more hpGRF is required to obtain half-maximal stimulation of cAMP production than for GH secretion. Somatostatin totally blocks hpGRF-stimulated GH release, but only partially attenuates cAMP production in the presence or absence of a phosphodiesterase inhibitor. Verapamil also inhibits GH release in response to hpGRF, but, unlike somatostatin, this effect is not associated with an attenuation of cAMP production. In fact, intracellular cAMP levels are slightly augmented in the presence of verapamil, indicating that Ca2+ is required for hormone release but not for the activation of adenylate cyclase. Consistent with this is the observation that the release of GH due to 8-bromo-cAMP is also blocked by verapamil. A requirement for Ca2+ is further indicated by the inhibitory effects of CoCl2 and CdCl2 on both basal and hpGRF-stimulated GH release. These results suggest that cAMP may play a role as an intracellular mediator of GRF action in somatotrophs and that Ca2+ is required for the release process. Somatostatin may exert its inhibitory effects on GH secretion either by interfering with cAMP production or by an action on the secretory process subsequent to cAMP production.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Steroid secretion in polycystic ovarian disease after ovarian suppression by a long-acting gonadotropin-releasing hormone agonist.

The principal glandular source of increased serum androgens in polycystic ovarian disease (PCO) is controversial), since complete separation of ovarian from adrenal function has not been achieved. The purpose of this study was to determine whether a long-acting GnRH agonist could be used to selectively inhibit ovarian steroid secretion in PCO and ovulatory women. Each of five typical PCO patients and six ovulatory subjects on day 2 of their menstrual cycles received D-Trp6-Pro9-NEt-LHRH (GnRH-a; 100 micrograms) for 28 consecutive days. Their results were compared to basal serum hormone values in eight oophorectomized women. In response to GnRH-a, PCO and normal subjects exhibited sharp and sustained rises of LH and gradual decreases in FSH. These levels were clearly less than basal levels seen in oophorectomized women. Episodic LH release was significantly attenuated in both groups at the end of GnRH-a treatment. After the administration of agonist, serum estradiol (E2), estrone (E1), androstenedione (A), and testosterone (T) were suppressed to castrate levels in both groups. The decrements of E2 and E1 in PCO were gradual and continuous compared to initial dramatic rises, which reached peaks at 14 days, and subsequent abrupt falls in the ovulatory controls. Serum A and T declined steadily in both groups. Basal serum dehydroepiandrosterone and dehydroepiandrosterone sulfate, but not cortisol, levels were elevated in PCO subjects. The 24-h secretion patterns and responses to ACTH of these hormones in PCO and ovulatory subjects were unaltered by GnRH-a administration. These data demonstrate that 1) in PCO subjects, GnRH-a induced complete suppression of ovarian steroid secretion, as circulating levels at the end of treatment were comparable to those seen in our oophorectomy subjects; 2) elevated A and T levels in PCO patients were derived primarily from the ovary; and 3) adrenal steroid secretion was unaltered by GnRH-a in both PCO and normal women.

Adrenal Cortex Hormones↗
PubMed1981

Short-term treatment of idiopathic precocious puberty with a long-acting analogue of luteinizing hormone-releasing hormone. A preliminary report.

The uncoupling of pituitary stimulation and response observed in adults during administration of the luteinizing hormone-releasing hormone analogue, D-Trp6-Pro9-NEt-LHRH (LHRHa) suggested that this drug might be useful in treating precocious puberty. We treated five girls with idiopathic precocious puberty (ages two to eight) for eight weeks with daily subcutaneous injections of LHRHa. The patients had Tanner II to IV pubertal development, advanced bone age, an estrogen effect on vaginal smear, measurable basal gonadotropin levels with pulsed nocturnal secretion, and a pubertal gonadotropin response to LHRH. Irregular vaginal bleeding was present in three patients. LHRHa significantly decreased basal (P less than 0.025) and LHRH-stimulated (P less than 0.01) gonadotropin levels as well as serum estradiol (P less than 0.05). The vaginal maturation-index score, which reflects the estrogen effect, fell by 25 per cent. Eight weeks after stopping treatment, all hormonal values and the vaginal maturation index had returned to pretreatment levels. These favorable short-term results will need further study before the benefits and risks of chronic treatment with LHRHa can be adequately assessed.

Child↗
PubMed1981

Desensitization to gonadotropin-releasing hormone observed in superfused pituitary cells on Cytodex beads.

Chronic exposure to gonadotropin-releasing hormone (GnRH) in vivo eventually results in a failure to maintain maximal gonadotropin secretion, suggesting a loss of pituitary responsiveness. To examine changes in pituitary sensitivity in vitro, we have developed a superfusion technique in which dissociated rat anterior pituitary cells are attached to Cytodex beads. In this system, continuous administration of 30 nM GnRH caused an initial 20-fold increase in LH and FSH secretion rates. However, hormone secretion declined rapidly, reaching basal levels within 12 h. Both the calcium ionophore A23187 and the cocarcinogen phorbol myristate acetate were effective in releasing additional LH and FSH from cells made refractory to 30 nM GnRH, whereas higher doses of GnRH and 8-bromo-cAMP were minimally active. A rapid loss of response was also seen with a superactive GnRH agonist but was not observed with a GnRH antagonist. Desensitization to prolonged superfusion with phorbol myristate acetate was also observed, although these cells remained sensitive to GnRH. In contrast to the development of tolerance produced by continuous exposure to GnRH, repeated delivery of GnRH in a pulsatile fashion maintained pituitary responsiveness for more than 24 h. A priming effect was observed for both LH and FSH. Such results demonstrate that continuous, but not intermittent, exposure to GnRH is capable of producing desensitization in superfused anterior pituitary cells.

Animals↗
PubMed1980

Superfusion of rat anterior pituitary cells attached to Cytodex beads: validation of a technique.

A superfusion method consisting of fully recovered, dissociated pituitary cells adhering to Cytodex beads has proved useful in monitoring the dynamics of hormone secretion over time. Male rat anterior pituitaries were dissociated with collagenase and Viokase, then cultured in the presence of Cytodex beads for 3-5 days, during which time the cells attached firmly to the surface of the beads. The bead-attached cells were stable and could be transferred to any vessel without the need for centrifugation or further trypsinization. For this application, the bead-attached cells were packed in a column and superfused with a low bicarbonate buffer requiring no CO2 gassing. Viability was more than 95% after 48 h in the column. The cells responded in a normal physiological manner to hypothalamic releasing and inhibitory peptides. The ED50 was 0.3 nM for somatostatin and 1.2 nM for gonadotropin-releasing hormone. A postinhibitory rebound of GH secretion was observed after the discontinuation of large doses of somatostatin. LH secretion reached maximal levels within 6 min after 10 nM gonadotropin-releasing hormone, but started declining after 2 h of continuous stimulation and dropped close to baseline within 18 h. GH release was significantly increased by prostaglandin E2, 3-isobutyl-1-methylxanthine, and 8-bromo-cAMP. LH secretion increased 5-fold in response to 1 mM 8-bromo-cAMP, but showed little increase during prostaglandin E2 or 3-isobutyl-1-methylxanthine stimulation. The cocarcinogen phorbol myristate acetate (12-O-tetradecanoyl-phorbol-13-acetate) induced secretion of all pituitary hormones and continued to do so for hours after a short pulse. The superfusion system is simple to operate and has proven effective in studying transient phenomena, desensitization, and short term kinetics of secretagogues.

Animals↗
PubMed1980

Radioligand assay for gonadotropin-releasing hormone: relative potencies of agonists and antagonists.

A radioligand assay employing tritiated gonadotropin-releasing hormone, [3H-Pro9]GnRH or [3H-pGlu1]GnRH is used to investigate the binding of GnRH, its agonists, and its antagonists to male rat anterior pituitary homogenates. The tritiated GnRH purified by high pressure liquid chromatography and stored in 10 mM HOAc is stable for binding for at least 14 weeks. It is found that there is at least one high affinity site with an observed Kd of congruent to 2 nM and another low affinity site whose Kd is congruent to 1 microM. Only approximately 25% of the total specific binding is to the low affinity site. At room temperature, the binding is reduced to 50% of that at 0 C, and at 37 C, there is no measurable binding. Bacitracin has no effect on the binding at any temperature. Maximum binding occurs between pH 7.5--8.5. Quantitative relative binding potencies of several agonists and antagonists are given. These potencies closely parallel their biological potencies, but all antagonists have higher absolute binding affinities when compared to their potencies to inhibit GnRH-mediated LH secretion in vitro.

Binding, Competitive↗