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K J Catt

Publications and source records attributed to K J Catt.

At least 271 records · Page 15Linked to original sources
PubMed1984

Arachidonic acid metabolism in gonadotroph-enriched pituitary cells.

Control of pituitary hormone secretion by hypothalamic-releasing peptides appears to involve unidentified products of the cyclooxygenase and lipoxygenase pathways, as well as the adenylate cyclase system. To identify the patterns of arachidonic acid metabolism in specific pituitary cell types, the labeled products formed from [14C]-arachidonic acid were analyzed in rat pituitary cells separated by centrifugal elutriation into fractions enriched in gonadotrophs, somatotrophs and lactotrophs. Gonadotroph-enriched cell fractions metabolized arachidonic acid to 11-, 12- and 15-HETE, HHT, PGD2, PGE2 and TXB2. The products were characterized by high performance liquid and thin-layer chromatography, together with gas chromatographic-mass spectrometric identification of 12- and 15-HETE. In cells preincubated with indomethacin, the formation of 11-HETE, HHT, PGD2, PGE2 and TXB2 was markedly reduced. In gonadotroph-enriched cell fractions, the production of cyclooxygenase metabolites was 3 to 4 times greater than that of lipoxygenase products. The somatotroph- and lactotroph-enriched cell fractions produced only very small amounts of oxygenated arachidonic acid metabolites under the conditions studied, but all cell fractions incorporated [14C]-arachidonate into mono-, di- and tri-glycerides, as well as into phospholipids. These results demonstrate the differential capacities of the individual pituitary cell populations for metabolizing arachidonic acid, and emphasize the relative prominence of the oxidation pathways for arachidonate metabolism in the gonadotroph-enriched cell fraction of the rat pituitary gland.

Animals↗
PubMed1984

GnRH agonist-induced inhibitory and stimulatory effects during ovarian follicular maturation.

The in vivo regulation of ovarian gonadotropin and prolactin receptors and adenylate cyclase activity by FSH, and the potent GnRH agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa), was studied in immature hypophysectomized diethylstilbestrol-implanted rats. During FSH treatment over a 48 h period, FSH receptors increased 2-fold with the maximum response during the first 12 h, whereas LH and prolactin receptors increased by 10-fold and 6-fold with the maximum response from 12 to 48 h. Administration of GnRHa at any time during the 48 h period of FSH treatment inhibited the subsequent development of gonadotropin and PRL receptors. In contrast, administration of a single dose of 10 micrograms GnRHa after 48 h of FSH treatment stimulated follicular luteinization and caused increases in basal adenylate cyclase activity, ovarian weight and PRL receptor content, and concomitant decreases in gonadotropin receptors and adenylate cyclase responses. In the immature follicles of animals not primed with FSH, GnRHa caused progressive inhibition of FSH-sensitive adenylate cyclase activity, with a decrease in FSH receptors, but increased both basal and GMP-P(NH)P-stimulated adenylate cyclase activities. These results demonstrate that GnRHa causes marked inhibition of gonadotropin receptor expression in the basal and FSH-stimulated ovary. This decrease in gonadotropin receptors is an important component of the mechanism by which GnRH agonists inhibit ovarian gonadotropin-sensitive adenylate cyclase activity. In addition, these peptides exert stimulatory effects upon ovarian weight and basal adenylate cyclase activity, and cause an increase in PRL receptors and luteinization of mature ovarian follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1984

Autoradiographic localization of angiotensin II receptors in rat brain.

The 125I-labeled agonist analog [1-sarcosine]-angiotensin II ( [Sar1]AII) bound with high specificity and affinity (Ka = 2 X 10(9) M-1) to a single class of receptor sites in rat brain. This ligand was used to analyze the distribution of AII receptors in rat brain by in vitro autoradiography followed by computerized densitometry and color coding. A very high density of AII receptors was found in the subfornical organ, paraventricular and periventricular nuclei of the hypothalamus, nucleus of the tractus solitarius, and area postrema. A high concentration of receptors was found in the suprachiasmatic nucleus of the hypothalamus, lateral olfactory tracts, nuclei of the accessory and lateral olfactory tracts, triangular septal nucleus, subthalamic nucleus, locus coeruleus, and inferior olivary nuclei. Moderate receptor concentrations were found in the organum vasculosum of the lamina terminalis, median preoptic nucleus, medial habenular nucleus, lateral septum, ventroposterior thalamic nucleus, median eminence, medial geniculate nucleus, superior colliculus, subiculum, pre- and parasubiculum, and spinal trigeminal tract. Low concentrations of sites were seen in caudate-putamen, nucleus accumbens, amygdala, and gray matter of the spinal cord. These studies have demonstrated that AII receptors are distributed in a highly characteristic anatomical pattern in the brain. The high concentrations of AII receptors at numerous physiologically relevant sites are consistent with the emerging evidence for multiple roles of AII as a neuropeptide in the central nervous system.

Angiotensin II↗
PubMed1984

Inhibitory actions of adenosine on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells.

To determine the effects of adenosine on follicle-stimulating hormone (FSH)-induced differentiation, granulosa cells isolated from the ovaries of diethylstilbestrol-treated immature rats were cultured with increasing concentrations of the nucleoside and modulators of adenosine action. Although adenosine had no effect on basal granulosa cell function during 48 h of culture, concentrations of the nucleoside from 10 microM to 1 mM progressively inhibited FSH-induced responses, including progesterone production and expression of FSH and luteinizing hormone (LH) receptors. Adenosine had biphasic effects on FSH-stimulated cAMP accumulation, causing inhibition of cAMP production at 10 to 100 microM and stimulation at higher concentrations. The enhancement of cAMP production by 1 mM adenosine occurred during the first 24 h of culture, while both 100 microM and 1 mM adenosine reduced FSH-stimulated cAMP production from 24 to 48 h. The inhibitory effects of adenosine were prevented by adenosine deaminase and dipyridamole, an inhibitor of adenosine transport, and were antagonized by 1-methyl-3-isobutylxanthine. The inhibition of cAMP and progesterone production by adenosine was partially overcome when cells were washed and reincubated with forskolin, but not with FSH. Adenine, guanosine, and inosine at concentrations of 100 microM did not modify FSH-induced cAMP formation or LH receptor induction. These results indicate that adenosine exerts predominantly inhibitory actions on hormone-induced granulosa cell differentiation, as manifested by prominent reductions in steroidogenesis and gonadotropin receptor expression.

1-Methyl-3-isobutylxanthine↗
PubMed1984

The role of angiotensin II receptors in vascular regulation.

Plasma-membrane receptors for angiotensin II (AII) have been identified in many AII-responsive tissues involved in the control of blood pressure via direct or indirect actions on vascular contractility. The specific, high-affinity receptors for AII in adrenal zona glomerulosa, vascular smooth muscle, kidney, brain, and anterior pituitary gland exhibit generally similar binding properties. However, the AII receptors in adrenal zona glomerulosa and vascular smooth muscle undergo reciprocal regulatory changes during alterations in sodium intake. These appear to be mediated by changes in circulating AII and are accompanied by parallel changes in sensitivity to AII. The AII receptors in the anterior pituitary gland are located in lactotrophs and corticotrophs and mediate the stimulatory actions of AII upon prolactin and ACTH secretion, acting in conjunction with other hypothalamic regulators. The anterior pituitary receptors are not affected by changes in sodium balance or AII infusion, in contrast to adrenal and vascular AII receptors, but exhibit similar ligand-binding properties to the sites present in other tissues. In the brain, AII receptors are present in several discrete regions and are particularly concentrated in the circumventricular organs. During dehydration, AII receptors are increased in the subfornical organ, but show no significant changes in the other circumventricular organs. The increase in subfornical-organ receptors is analogous to the up-regulation of AII sites in the adrenal cortex during sodium deficiency and may have a potentiating action upon the dipsogenic role of AII during dehydration. Mapping of AII receptors of the brain by topical autoradiography has revealed a highly characteristic pattern of distribution in brain regions concerned with drinking, adrenergic control, blood-pressure regulation, and hypothalamic control of pituitary-hormone secretion. In the rat kidney, AII receptors have been localized in the cortex and medulla by topical autoradiography with 125I-[Sar1]AII. The renal cortical receptors appear to be localized in glomeruli, whereas the AII receptors in the renal medulla are distributed diffusely in medullary tissue and also as localized radiating stripes which correspond to the vasa rectae bundles. The location of the renal receptors for AII in cortical and medullary sites emphasizes the multiplicity of actions of the octapeptide upon the individual compartments of the kidney.

Adrenal Glands↗
PubMed1984

Dopaminergic modulation of aldosterone secretion in the rat.

The dopamine antagonist metoclopramide (MCP) has been shown to acutely stimulate aldosterone secretion in vivo. To determine whether a dopaminergic mechanism is involved in the regulation of aldosterone secretion, we examined the effect of minipump infusion of MCP (iv) and/or angiotensin II (AII;sc) upon plasma aldosterone, adrenal capsular AII receptors, and 18-hydroxylase activity in rats maintained on high sodium intake. During normal sodium intake, plasma aldosterone was elevated from 8.3 +/- 1.3 to 35.4 +/- 3.2 ng/dl after 2-day infusion of a nonnatriuretic dose of AII (25 ng/min) and to 15.0 +/- 1.8 ng/dl after the infusion of 1.2 micrograms/min MCP. AII receptors were unchanged by MCP infusion, and rose from 1014 +/- 98 to 1638 +/- 98 fmol/mg after AII infusion. During high sodium intake, the infusion of either AII or MCP alone produced no change in plasma aldosterone or AII receptors. However, after simultaneous infusion of AII and MCP, plasma aldosterone rose from 4.5 +/- 1.2 to 32.5 +/- 2.7 ng/dl, AII receptors increased from 969 +/- 35 to 1607 +/- 280 fmol/mg, and 18-hydroxylase activity, measured as the conversion of corticosterone to aldosterone by isolated mitochondria, rose from 29.5 +/- 1.67 to 40.6 +/- 2.9 pmol/mg . min. These adrenal responses induced by the combined treatment with AII and MCP were similar to the effects of AII infusion during normal sodium intake, indicating that MCP exerts a permissive action upon the trophic effects of AII on the adrenal cell during high sodium intake. These actions of MCP were completely abolished by the simultaneous infusion of dopamine (2 micrograms/min), suggesting that the effects of MCP on adrenal function are due to its dopaminergic antagonist properties. In collagenase-dispersed adrenal glomerulosa cells, only supraphysiological concentrations of dopamine in the incubation medium (10-100 microns) inhibited basal, AII-stimulated, and ACTH-stimulated aldosterone production, and these inhibitory effects were not reversed by high concentrations of MCP. Also, MCP itself inhibited both basal and stimulated aldosterone production. These results suggest that the stimulatory actions of MCP in vivo are exerted through liberation of other local regulators, rather than directly upon the adrenal glomerulosa cell. These findings have defined a mechanism by which the primary regulatory action of AII upon aldosterone secretion can be modulated during high sodium intake by dopaminergic inhibition of adrenal glomerulosa function.

Adrenal Glands↗
PubMed1984

Ontogeny of gonadotropin receptors in the fetal and neonatal rat testis.

To evaluate the extent to which LH receptors may participate in the regulation of testosterone production by the fetal and neonatal rat testis, the gonadal content of LH receptors was determined and correlated with the gonadal content of testosterone and other steroids at daily intervals throughout the period of sexual differentiation and early neonatal life. Also, to determine if FSH has a potential role during fetal life, FSH receptors were measured from 14.5 until 21.5 days of gestation, just before birth. LH receptors were first detected in the fetal rat testis at 15.5 days of gestation, at a concentration of 0.072 +/- 0.016 fmol/gonad. This is the time at which previous studies have shown a biological response to LH stimulation with increases in cAMP and testosterone production. LH receptor content remained constant from 15.5-17.5 days of gestation and then increased sharply at 18.5 days of gestation to 0.706 +/- 0.046 fmol/gonad, coincident with a surge in the testosterone content of the fetal testis. LH receptor content continued to rise until birth, reaching a maximum level of 2.00 +/- 0.24 fmol/gonad at 21.5 days of gestation, and did not significantly differ from this value through 5 days after birth, whereas intratesticular testosterone content decreased after birth. FSH receptors could be measured from 17.5 days of gestation, but remained very low through 19.5 days of fetal life. At 20.5 days, FSH receptor levels began to rise sharply and continued to increase through 21.5 days of gestation, just before birth. These data show that LH receptors are measurable at the time when the fetal testis becomes responsive to LH stimulation. It is possible that the increase in LH receptors may act as a mechanism for amplification of the stimulatory effect of low circulating LH levels on the fetal testis being concomitant with the intratesticular testosterone increase at the same stage. The decrease in testosterone production after birth cannot be attributed to a decrease in LH receptors in the neonatal gonad. Since 5 alpha-reduced androgens increase after birth, other possibilities, such as alternative steroid pathways, are a more likely explanation for the decrease in testosterone content. The presence of FSH receptors in the fetal testis imply that this hormone may have a function in the developing gonad, particularly in the last 2 days of fetal life.

Aging↗
PubMed1984

Induction of granulosa cell differentiation by forskolin: stimulation of adenosine 3',5'-monophosphate production, progesterone synthesis, and luteinizing hormone receptor expression.

The effects of forskolin on the acquisition of differentiated functions in cultured ovarian granulosa cells were compared with the actions of FSH and prostaglandin E2 (PGE2). In 48-h granulosa cell cultures from immature diethylstilbestrol-treated rats, 100 microM forskolin caused a 45-fold increase in cAMP accumulation and stimulated progesterone production from undetectable levels (less than 0.2 ng/ml) to 80 ng/ml. The forskolin-induced increase in cAMP was similar to the maximum response to FSH, and progesterone production was about 50% of that elicited by FSH. PGE2 also enhanced cAMP and progesterone production in a concentration-dependent manner, with a maximum 8-fold increase in cAMP accumulation and an increase in progesterone to 5.6 ng/ml when the PGE2 concentration was 10 micrograms/ml. The time course of forskolin-stimulated cAMP production was notable for its rapid rise to the maximum level during the first 24 h of culture, followed by a plateau for up to 72 h. This contrasted with FSH-stimulated cAMP production, which increased progressively for up to 72 h when measured at 24-h intervals. LH receptor levels were low in untreated cells and after exposure to the various stimuli for 24 h, but increased 9- to 11-fold after culture with FSH or forskolin for 48-72 h. PGE2-induced LH receptor formation was about 20% of that seen after FSH stimulation. Forskolin enhanced cAMP and progesterone production in response to FSH and choleragen, but impaired the effects of these ligands on LH receptor formation. Exposure of the cultured cells to a potent GnRH agonist inhibited forskolin-induced progesterone and LH receptor synthesis, but did not influence forskolin-stimulated cAMP production. These results demonstrate the ability of forskolin to serve as a nonhormonal stimulator of granulosa cell differentiation and indicate the importance of cAMP in this process, as well as the ability of GnRH agonists to exert inhibitory effects on post-cAMP steps in cellular maturation.

Animals↗
PubMed1984

Gonadotropin-releasing hormone agonist analog-induced steroidogenic lesion in the neonatal rat testis: evidence for direct gonadal action.

In this study we sought to determine whether a GnRH agonist analog (GnRH-A) could influence steroidogenesis by a direct effect on the neonatal rat testis. Five-day-old male rats were given a single sc injection of hCG (600 IU/kg BW), GnRH-A [D-Ser(tBu)6]des-Gly10-GnRH N-ethylamide (4 micrograms/kg BW), or their combination. Testicular testosterone (T) was increased (3-fold) only 6 h post-GnRH-A treatment, whereas after hCG administration testicular T remained elevated (3 to 6-fold) for 48 h. Testicular progesterone (P) increased by 40% 6-72 h after hCG treatment, but was not raised after GnRH-A injection. In vitro T production by testes from control and GnRH-A-treated (injection 24 h earlier) animals was stimulated 5 to 8-fold by hCG (7.9 nM) or 8-bromo-cAMP (8-Br-cAMP; 1 mM). hCG and 8-Br-cAMP did not further stimulate T production from testes of animals treated with hCG in vivo 24 h earlier. While hCG and 8-Br-cAMP had only a small stimulatory effect (1.5 to 2-fold) on in vitro P production by testes from control or hCG-treated animals, their stimulation of P production from testes of GnRH-A-treated animals was dramatic (20 to 30-fold). In vitro P production from testes of animals receiving combined treatment with hCG and GnRH-A in vivo reached a high hCG-stimulated rate similar to that found after GnRH-A treatment alone; the unstimulated values were also considerably elevated (5-fold) compared to those of untreated animals. The ability of GnRH-A treatment to stimulate testicular P production in the presence of a high concentration of hCG strongly suggests a direct gonadal action of the peptide. The possibility of such action was corroborated by the finding of abundant GnRH receptors in the neonatal testis. These results indicate that the steroidogenic lesion seen in adult rat testis after gonadotropic stimulation (blockade of C21 steroid side-chain cleavage with compensatory accumulation of P) can be reproduced in neonatal rat testes by a direct action of GnRH-A, but not by hCG.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Estrogens enhance the adenosine 3',5'-monophosphate-mediated induction of follicle-stimulating hormone and luteinizing hormone receptors in rat granulosa cells.

The effects of estrogens on cAMP-induced FSH and LH receptor expression were studied in granulosa cells isolated from immature diethylstilbestrol-implanted rats. Although estradiol alone had negligible effects on granulosa cell maturation, estradiol concentrations from 10(-11)-10(-8) M progressively enhanced cAMP production and gonadotropin receptor formation in choleragen-stimulated cells. During 48 h of culture, estradiol augmented cAMP levels by 2-fold, LH receptors by 4- to 6-fold, and FSH receptors by 20-40%. Estradiol also enhanced the extent of LH and FSH receptor formation by other cAMP-inducing ligands, including FSH, prostaglandin E2, and forskolin. The stimulatory action of 8-bromo-cAMP on gonadotropin receptors was also increased by estradiol, indicating that part of the estrogenic effect was exerted on cAMP-activated processes. Scatchard analyses indicated that estradiol increased the number of choleragen-induced FSH receptors from 2,600 to 3,200/cell and of LH receptors from 13,000 to 86,000/cell with no changes in receptor binding affinity. Choleragen-stimulated cAMP accumulation was enhanced by estradiol during the later stages of culture (after 30 h), while increased LH receptors were detected by 30 h and FSH receptors by 43 h. The stimulatory effects of estradiol were not due to increased cellular proliferation and were also exerted by other estrogens, including estrone and diethylstilbestrol. Androgens, including testosterone and androstenedione, also amplified choleragen action. This effect was largely through conversion to estrogens, since dihydrotestosterone, a nonaromatizable androgen, did not markedly enhance LH receptor formation by choleragen. In contrast, progestins and pregnenelone had no facilitative effect on choleragen-induced responses. Although cortisol and dexamethasone increased choleragen-induced cAMP accumulation, only cortisol elevated LH receptors, and dexamethasone inhibited FSH receptor formation. These results demonstrate that estrogens enhance both ligand-induced cAMP production and cAMP-activated responses during granulosa cell differentiation. In particular, estrogens exert a major effect on the levels of gonadotropin receptors expressed in response to FSH and other cAMP-inducing ligands.

Androgens↗
PubMed1984

Dependence of cyclic nucleotide production and luteinizing hormone receptor formation upon ribonucleic acid synthesis in cultured rat granulosa cells.

The role of newly synthesized RNA in the differentiation of granulosa cells isolated from diethylstilbestrol-treated immature rats was studied during culture with actinomycin D. Choleragen-induced LH receptor formation and cGMP production at 48 h were completely inhibited by actinomycin D (greater than or equal to 100 ng/ml) added as late as 20 h after the initiation of culture and were partially reduced by addition of the antibiotic from 30-48 h. In contrast, addition of actinomycin D to freshly prepared cells enhanced choleragen-stimulated cAMP accumulation during the 48-h culture period. This effect was caused by both elevation of adenylate cyclase activity at 3 and 6 h of culture and inhibition of choleragen-induced phosphodiesterase activity during culture. The increase in cAMP production by actinomycin D was confined to the first few hours of culture, since the antibiotic did not enhance cAMP levels when added after 3 h and significantly reduced cAMP accumulation when added from 20-48 h of culture. Actinomycin D inhibited choleragen-stimulated incorporation of [3H]uridine into RNA of freshly prepared cells by 65% and reduced both RNA synthesis and incorporation of [3H]leucine into protein at 20 and 48 h of culture by approximately 90%. In untreated cells, RNA and protein synthesis and phosphodiesterase activity increased to a larger extent from 20-48 h than after choleragen treatment, but did not lead to elevated cAMP levels or LH receptors. These results suggest that the cAMP-induced syntheses of RNA and protein that are specific for increases in cGMP production and LH receptor formation occur predominantly during the second day of granulosa cell culture. In contrast, cAMP production can be markedly altered by changes in RNA and protein syntheses during the first hours of culture.

Adenosine Triphosphate↗
PubMed1984

Inhibitory actions of calmodulin antagonists on steroidogenesis in zona glomerulosa cells.

The stimulation of aldosterone (Aldo) production in the adrenal glomerulosa cell by angiotensin II (AII) and ACTH is highly dependent on calcium. To determine the role of calmodulin (CM) as a regulator of intracellular calcium during hormonal activation of steroidogenesis, the actions of a series of CM inhibitors on the stimulation of Aldo biosynthesis by AII, the calcium ionophore A23187, ACTH, and cAMP were examined in isolated adrenal glomerulosa cells. The CM antagonists, pimozide, W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and W5 [N-(6-aminohexyl)-1-napthalenesulfonamide] caused dose-dependent inhibition of hormone-stimulated Aldo production, in proportion to their reported binding affinities for CM. The maximum inhibitory concentration (5 microM) of the most potent CM antagonist, pimozide, had no effect on the conversion of progesterone to Aldo. However, submaximal inhibitory concentrations of W7 and W5 also reduced conversion of progesterone to Aldo, indicating that their inhibitory action is not only due to their CM antagonist properties. The relative sensitivities of the stimuli of Aldo release to inhibition by all three CM inhibitors were A23187 = AII greater than ACTH greater than 8-bromo-cAMP (8-Br-cAMP). Aldo responses to ACTH were more sensitive to inhibition by pimozide than those to 8-Br-cAMP, suggesting that ACTH-induced cAMP formation is also dependent on CM. Pimozide (5 microM) completely inhibited the Aldo responses to AII and A23187, whereas the Aldo responses to ACTH and 8-Br-cAMP were only partially inhibited. At submaximal inhibitory concentrations, pimozide caused a decrease in the maximum response to AII without changing the concentration of the peptide required for half-maximum stimulation of Aldo production. The inhibition of Aldo production by pimozide was accompanied by parallel inhibition of pregnenolone accumulation measured in the presence of cyanoketone. No effect of pimozide (5 microM) was observed on the binding of 125I-AII to adrenal glomerulosa cells, placing the CM-dependent site between the receptor and the side-chain cleavage enzyme. These findings indicate that CM-mediated regulation is essential for the steroidogenic response to calcium ionophore and AII, but is only partially required for the responses to ACTH and 8-Br-cAMP.

Adrenal Cortex↗
PubMed1984

Biphasic modulation of pituitary sensitivity to GnRH by oestrogens: the effects of A- and D-ring substitution on LH release in cultured pituitary cells.

The sensitizing effect of oestrogens on GnRH-stimulated LH release was evaluated in pituitary cells from adult female rats, cultured for 2 days in the presence of 10(-13) to 10(-6) M concentrations of oestradiol and selected A- and D-ring modified oestrogens. With almost all steroids tested, bell-shaped dose-response curves with comparable LH-maxima but different ED max values were obtained for the LH response to a submaximal GnRH stimulus (5 X 10(-10) M). Maximal LH response to 5 X 10(-10) M GnRH were found at the following oestrogen concentrations: oestradiol and 4-hydroxyoestradiol = 10(-11) M; 2-methyloestradiol = 10(-9) M; 2-hydroxyoestradiol = 10(-8) M; with 4-methyloestradiol no significant maximum was observed. When cells were pretreated with 10(-13), 10(-11) and 10(-9) M oestradiol, or 4-hydroxyoestradiol, or 2-hydroxyoestradiol, and exposed to increasing concentrations of GnRH (10(-11) to 10(-7) M), an almost 10-fold decrease in the ED50 for GnRH was observed after pretreatment with 10(-11) M oestradiol and 4-hydroxyoestradiol. With 2-hydroxyoestradiol at this concentration, the sensitizing effect was much less pronounced. Increasing the steroid concentration to 10(-9) M slightly decreased the effect of oestradiol and 4-hydroxyoestradiol, whereas it increased the effect of 2-hydroxyoestradiol. Thus, at the target cell 4-hydroxyoestradiol has the same potency as oestradiol, while 2-hydroxyoestradiol is significantly less active. The sensitizing effect of oestradiol is only slightly decreased by the presence of a methyl group in position 2, but is markedly reduced by a methyl group in position 4. Our results also demonstrate the high sensitivity of the pituitary to oestrogen-induced enhancement of GnRH-stimulated gonadotrophin release, as well as the decrease of the positive effect at high oestrogen concentrations. The bell-shaped dose-response curves for oestrogen action should be taken into account when evaluating the effects of oestrogens and their derivatives upon gonadotrophin release from the pituitary gland.

Animals↗
PubMed1983

Hormonal regulation of a plasma membrane phosphodiesterase in differentiating granulosa cells. Reciprocal actions of follicle-stimulating hormone and a gonadotropin-releasing hormone agonist on cAMP degradation.

The activity of a plasma membrane cAMP-phosphodiesterase in cultured ovarian granulosa cells was regulated by follicle-stimulating hormone (FSH) and the gonadotropin-releasing hormone (GnRH) agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa). Degradation of cAMP was similar in cultures treated with FSH alone or FSH plus GnRHa when the labeled cyclic nucleotide was added from 24 to 42 h of culture. However, at 48 h and subsequent times of incubation, cAMP phosphodiesterase activity was significantly higher in cells incubated with FSH plus GnRHa. Phosphodiesterase activity was progressively increased by GnRHa concentrations between 10(-13) and 10(-10) M, and was maximally stimulated by 10(-9) M GnRHa. In comparison with control cells, FSH lowered the Vmax of cAMP catabolism by the high (1 microM cAMP substrate) and the low (50 microM) affinity phosphodiesterase, while GnRHa raised enzyme activity toward control levels. These actions of FSH and GnRHa were specific for a plasma membrane phosphodiesterase that was accessible to extracellular cAMP, since extracellular substrate was hydrolyzed, no intracellular uptake of [3H]cAMP was observed, and only a small fraction (10%) of cAMP was catabolized in the incubation medium in the absence of cells. Further, the actions of FSH and GnRHa on the membrane enzyme were the opposite of those observed when total phosphodiesterase activity was measured in cellular sonicates. Hormonal changes in phosphodiesterase activity were not due to leakage of the enzyme from damaged cells since a constant percentage of cAMP hydrolysis in the medium was observed during culture. Analysis of cAMP catabolites in granulosa cells indicated that the phosphodiesterase reaction product, 5'-AMP, was rapidly converted to adenosine by a plasma membrane 5'-nucleotidase, independent of the cellular hormonal status. These results indicate that the opposing actions of FSH and GnRHa upon granulosa cell differentiation include modulation of cAMP degradation at the plasma membrane level.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Agonist-induced regulation of pituitary receptors for gonadotropin-releasing hormone. Dissociation of receptor recruitment from hormone release in cultured gonadotrophs.

The regulation of receptors for gonadotropin-releasing hormone (GnRH) by the homologous decapeptide ligand was analyzed in cultured rat anterior pituitary cells. Assay of GnRH receptors in both intact and disrupted cells showed that GnRH binding to gonadotrophs was rapidly followed by dose-dependent loss of sites that was maximal within 1 h. This early loss of GnRH receptors was not dependent on protein synthesis, and was attributable to ligand-induced processing of the peptide binding sites. No loss of GnRH sites was observed after receptor occupancy by a GnRH antagonist, or after target cell activation by exposure to a depolarizing concentration of KCl to stimulate luteinizing hormone release. After their initial down-regulation, GnRH receptors returned to normal and subsequently increased in concentration after 6 h of incubation. The delayed phase of receptor up-regulation was prevented by treatment with cycloheximide or actinomycin D and was calcium-dependent, being induced by 50 mM KCl and by low concentrations of the calcium ionophore, A23187. Conversely, calcium antagonists such as verapamil and MgCl2 impaired the agonist-induced increase of GnRH receptor sites. These findings have demonstrated that pituitary GnRH receptors undergo two distinct phases of regulation after interaction with the homologous ligand. The initial phase of agonist-dependent receptor loss is followed by a postsecretory phase of receptor recruitment that is dependent on protein synthesis. The expression of GnRH receptors can be completely dissociated from gonadotropin secretion, indicating that fusion of luteinizing hormone secretory granules with the plasma membrane is not a major pathway for transport of GnRH receptors to the cell surface in cultured gonadotrophs. Such changes in cell surface GnRH receptors during activation by the peptide agonist are relevant to the alterations in gonadotroph sensitivity that occur in vivo during physiological regulation of the pituitary gland by GnRH.

Animals↗
PubMed1983

Mechanisms of action of corticotropin-releasing factor and other regulators of corticotropin release in rat pituitary cells.

The role of cyclic AMP in the stimulation of corticotropin (ACTH) release by corticotropin-releasing factor (CRF), angiotensin II (AII), vasopressin (VP), and norepinephrine (NE) was examined in cultured rat anterior pituitary cells. Synthetic CRF rapidly stimulated cyclic AMP production, from 4- to 6-fold in 3 min to a maximum of 10- to 15-fold at 30 min. Stimulation of ACTH release by increasing concentrations of CRF was accompanied by a parallel increase in cyclic AMP formation, with ED50 values of 0.5 and 1.3 nM CRF for ACTH and cyclic AMP, respectively. A good correlation between cyclic AMP formation and ACTH release was also found when pituitary cells were incubated with the synthetic CRF(15-41) fragment, which displayed full agonist activity on both cyclic AMP and ACTH release with about 0.1% of the potency of the intact peptide. In contrast, the CRF(21-41) and CRF(36-41) fragments were completely inactive. The other regulators were less effective stimuli of ACTH release and caused either no change in cyclic AMP (AII and VP) or a 50% decrease in cyclic AMP (NE). Addition of the phosphodiesterase inhibitor, methylisobutylxanthine, increased the sensitivity of the ACTH response to CRF but did not change the responses to AII, VP, and NE. In pituitary membranes, adenylate cyclase activity was stimulated by CRF in a dose-dependent manner with ED50 of 0.28 nM, indicating that the CRF-induced elevation of cyclic AMP production in intact pituitary cells is due to increased cyclic AMP biosynthesis. The intermediate role of cyclic AMP in the stimulation of ACTH release by CRF was further indicated by the dose-related increase in cyclic AMP-dependent protein kinase activity in pituitary cells stimulated by CRF with ED50 of 1.1 nM. These data demonstrate that the action of CRF on ACTH release is mediated by the adenylate cyclase-protein kinase pathway and that the sequence requirement for bioactivity includes the COOH-terminal 27 amino acid residues of the molecule. The other recognized regulators of ACTH release are less effective stimuli than CRF and do not exert their actions on the corticotroph through cyclic AMP-dependent mechanisms.

Adrenocorticotropic Hormone↗