Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Stimulation of rat granulosa cell aromatase activity by FSH has recently been used as a sensitive biological end point to develop an in vitro FSH bioassay. The present report provides a detailed validation and application of this assay. In the presence of androstenedione and diethylstilbestrol, FSH stimulated estrogen production in a dose-dependent manner. Although addition of high doses of a phosphodiesterase inhibitor [1-methyl-3-isobutyl xanthine (MIX)] decreased maximal estrogen production, treatment with 0.125 mM MIX increased the sensitivity of granulosa cells to FSH, presumably by minimizing endogenous cAMP breakdown. Addition of insulin and human CG (hCG) further synergistically enhanced granulosa cell sensitivity to FSH. Although inclusion of gonadotropin-free serum obtained from hypophysectomized male rats decreased the assay sensitivity, pretreatment of serum with polyethylene glycol [(PEG) 10-14%] resulted in a dose-dependent decrease in the serum-interfering effect. Studies using exogenous [125I]iodo-rat FSH or RIA measurement indicated recovery of 94-98% FSH after pretreatment of serum with 12% PEG. In the presence of the PEG-pretreated gonadotropin-free serum (4%), ovine, rat, and human FSH preparations induced parallel dose-response curves for estrogen production with minimal detectable doses of 0.12 ng, 0.12 ng, and 0.12 mIU/culture, respectively. In contrast, treatment with GH, PRL, TSH, and ACTH did not affect estrogen production. The apparent stimulatory effect of high doses (greater than 60 ng/culture) of LH and hCG could be attributed to FSH contamination or intrinsic FSH activity in these preparations. Changes in serum bioactive FSH levels were studied in adult male rats after GnRH administration. GnRH (5 micrograms/rat) treatment significantly elevated FSH levels within 30 min after injection. Maximal increases (approximately 2.8-fold) in serum bioactive FSH were observed between 60-120 min. At 8 h after treatment, FSH levels decreased to control levels. Comparison between granulosa cell aromatase bioassay and RIA results indicated no apparent changes in the bio- to immuno- ratio of FSH after GnRH treatment. In conclusion, extreme sensitivity of the bioassay allows the measurement of circulating levels of bioactive FSH. Since rat granulosa cells respond to FSH preparations from different species, the in vitro assay should also provide valuable information on FSH levels in many animal species including those lacking a specific RIA. Measurement of serum levels of bioactive FSH should provide insight regarding the role of FSH in various physiological and pathological conditions.
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PMID:Granulosa cell aromatase bioassay for follicle-stimulating hormone: validation and application of the method. 242

In an attempt to delineate the mechanism(s) of PRL secretion from human lactotrophs, the effects of dopamine and somatostatin on PRL release from adenomatous and nonadenomatous human pituitary cells in culture was studied. High K+ and the divalent cation ionophore A23187 both elevated PRL secretion, which was blocked by dopamine and somatostatin. When the cells were incubated in low calcium medium, PRL secretion was significantly inhibited. The addition of dopamine or somatostatin to low calcium medium further decreased PRL release. The stimulatory action of ionophore A23187 on PRL release was found even in the absence of extracellular calcium. Theophylline and isobutylmethylxanthine, when added to the incubation medium, increased PRL secretion, and dopamine as well as somatostatin again inhibited PRL release induced by phosphodiesterase inhibitors. No qualitative difference in these PRL responses was found in adenomatous and nonadenomatous human lactotrophs. In prolactinoma cells obtained from three different patients, cAMP generation was correlated with hormone release. Exposure of the cells to dopamine or somatostatin resulted in a parallel decrease in intracellular cAMP content and PRL secretion. The inhibitory effect of dopamine on PRL secretion and cAMP accumulation was blocked by coincubation of the cells with haloperidol. These results suggest that an increase in cytosol calcium caused by either mobilization from intracellular calcium pools or influx from the extracellular compartment and intracellular cAMP accumulation may be involved in the mechanism of PRL secretion from human lactotrophs, and dopamine and somatostatin may influence these two messengers to suppress PRL secretion.
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PMID:Mechanism of the inhibitory action of dopamine and somatostatin on prolactin secretion from human lactotrophs in culture. 257 87

In GH4C1 cells, the calmodulin antagonist trifluoperazine (TFP) showed a dose-dependent, biphasic effect on the basal release of PRL. An inhibition of PRL release was observed with 15-50 mumol/l TFP, whereas a concentration of 100 mumol/l and above had a stimulatory effect. The increase in basal hormone release evoked by TRH (1 mumol/l) and high extracellular concentration of K+ (50 mmol/l) was eliminated by 30 mumol/l TFP. The stimulatory effect of 100 mumol/l TFP on basal hormone release was not affected by addition of TRH (1 mumol/l) or K+ (50 mmol/l). The Ca2+ antagonists Co2+ (5 mmol/l) and verapamil (100 mumol/l), and the Ca2+ chelator EgTA (4 mmol/l) abolished the stimulatory effect of TRH (1 mumol/l) and of K+ (50 mmol/l) on PRL release, whereas only Co2+ inhibited the stimulation caused by 100 mumol/l TFP. TFP (75 mumol/l) caused a transient increase in the concentration of cellular cAMP. Incubation of intact GH4C1 cells with TFP (75 mumol/l), had an inhibitory effect on both the low and the high affinity form of cAMP phosphodiesterase. Basal as well as TRH-stimulated adenyl cyclase activity were inhibited by TFP, and this effect was counteracted by addition of calmodulin.
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PMID:Effects of trifluoperazine on prolactin release and cyclic AMP formation and degradation in GH4C1 pituitary cells. 282 17

The hormonal regulation of inhibin production by cultured granulosa cells from immature hypophysectomized, estrogen-treated rats was examined using a specific RIA which detects the N-terminal portion of the inhibin alpha-chain. The RIA measured bioactive inhibin of Mr about 32,000 in granulosa cell conditioned media fractionated by fast protein liquid chromatography. In the presence of 10(-7) M androstenedione, FSH stimulated inhibin production in a dose-dependent manner during a 2-day culture. Inclusion of a phosphodiesterase inhibitor decreased the EC50 for FSH from 2.6 to 0.8 ng/ml (n = 3). The stimulatory effect of FSH could be mimicked with forskolin (an adenyl cyclase activator) and with a cAMP analog, (Bu)2cAMP, consistent with FSH action mediated through a cAMP dependent pathway. Intracellular levels of inhibin were unmeasureable, suggesting that inhibin is not stored to any great extent by the granulosa cells. This finding was consistent with in vivo studies which showed that whereas FSH treatment for 2 days doubled serum inhibin levels when compared with basal levels, there was no increase in the concentration of extractable inhibin in ovarian tissue. Granulosa cells which had been exposed to 20 ng/ml FSH for 2 days to induce LH receptors produced inhibin in response to both LH and human CG during the subsequent 2-day culture, with the levels of inhibin equalling the amount inducible by FSH. In contrast, neither PRL nor terbutaline, a beta 2-adrenergic agonist, had any effect on inhibin production even though receptors for these hormones are also induced by FSH. GnRH was found to inhibit the FSH-stimulated production of inhibin (IC50, 10(-7) M), consistent with previous observations that GnRH can act at the ovarian level to inhibit granulosa cell differentiation. This inhibition by GnRH could be reversed by inclusion of a specific GnRH antagonist. On the other hand, another regulatory peptide, vasoactive intestinal peptide, slightly stimulated inhibin production. The effect of several growth factors was also tested. Insulin-like growth factor I raised not only FSH-stimulated inhibin levels, but basal levels as well. Insulin was also effective, but only at 100-fold higher concentration. Epidermal growth factor inhibited FSH-stimulated inhibin production (IC50 = 0.1 ng/ml), whereas fibroblast growth factor had no effect. Thus, granulosa cell inhibin secretion is regulated by FSH and LH but not by PRL, presumably via a cAMP-mediated pathway.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Hormonal regulation of granulosa cell inhibin biosynthesis. 302 19

Enzymatically dispersed testis cells derived from 7-day-old male rats maintained their gonadotropin-stimulated testosterone production for 18 days in culture. Treatment with hCG or LH stimulated androgen production in a dose-dependent manner, with ED50 values of 0.030 +/- 0.007 and 1.0 +/- 0.4 ng/ml for hCG and LH, respectively. Concomitant treatment with a phosphodiesterase inhibitor further enhanced LH action. In contrast, treatment with FSH, GH, or PRL was without effect. Treatment with forskolin, cholera toxin, or 8-bromo-cAMP induced dose-dependent increases in testosterone biosynthesis; this was accompanied by stimulation of 3 beta-hydroxysteroid dehydrogenase activity after treatment with hCG, forskolin, or 8-bromo-cAMP. RIA measurement of different androgens in HPLC-fractionated medium revealed that the main androgen secreted by the neonatal testis cells was testosterone, with lower production of 5 alpha-androstane-3 alpha,17 beta-diol and negligible 5 alpha-dihydrotestosterone, androstenedione, and androsterone. Treatment with epidermal growth factor, GnRH, and arginine vasopressin (AVP) decreased hCG-induced testosterone biosynthesis. Since the inhibitory actions of GnRH and AVP were blocked by concomitant addition of specific hormone antagonists, their inhibitory actions were probably mediated by specific testis receptors. In contrast, treatment with several potent synthetic steroid hormone analogs [diethylstilbestrol (an estrogen), dexamethasone (a glucocorticoid), R5020 (a progestin; 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), R1881 (an androgen; 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one), or cyproterone acetate (an antiandrogen; 17 alpha-acetyloxy-6-chloro-1,2-dihydro-(1 beta,2 beta)3'-H-cyclopropa-(1,2) pregna-1,4,6-trien-3,20-dione)] did not affect testosterone biosynthesis in hCG-treated cells. These results demonstrate that testosterone production by neonatal testis cells is maintained by gonadotropins during prolonged culture; the ability of cAMP-generating drugs and a cAMP analog to mimic gonadotropin actions on testosterone biosynthesis and 3 beta-hydroxysteroid dehydrogenase activity suggests a mediatory role of cAMP in gonadotropin action; and AVP, epidermal growth factor, and GnRH, through their putative testis receptors, directly inhibit gonadotropin-stimulated testosterone synthesis, while various steroids (androgens, estrogens, progestins, and glucocorticoids) do not affect Leydig cell function in the neonatal testis. The present culture system offers a unique model for elucidating the hormonal control of Leydig cell androgen biosynthesis during neonatal development.
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PMID:Hormonal regulation of androgen biosynthesis by primary cultures of testis cells from neonatal rats. 388 12

Both somatostatin (SRIF) and urotensin II, a dodecapeptide from the teleost caudal neurosecretory system, inhibit PRL release from the organ-cultured rostral pars distalis of the tilapia, Sarotherodon mossambicus, in a dose-related manner. The inhibitory action of SRIF on PRL release was completely prevented by the presence of the calcium ionophore A23187. PRL release was also blocked when Ca++ was excluded from the incubation medium, even in the presence of the ionophore. Both dibutyryl cAMP (dbcAMP) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, alone or in combination, stimulated PRL release during incubation in high osmotic pressure medium. The effect of dbcAMP appeared to be dose related. Together, dbcAMP and 3-isobutyl-1-methylxanthine were also effective in preventing the inhibition of PRL release by SRIF. These results are consistent with the notion that Ca++, and possibly cAMP, may be important mediators of PRL secretion, and it is likely that SRIF may inhibit PRL release by blocking a Ca++- or cAMP-mediated mechanism.
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PMID:Effects of somatostatin and urotensin II on tilapia pituitary prolactin release and interactions between somatostatin, osmotic pressure Ca++, and adenosine 3',5'-monophosphate in prolactin release in vitro. 617 10

The intermediary role of cAMP in the mechanism of action of FSH was reinvestigated in vitro using forskolin, a highly specific adenylate cyclase probe. Granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were cultured for 3 days in the absence or presence of forskolin. Treatment with increasing concentrations (10(-7)-10(-4) M) of forskolin led to dose-dependent increments in the accumulation of extracellular cAMP, with an apparent median effective dose of 1.6 +/- 0.5 X 10(-5) M. Concomitant blockade of cAMP phosphodiesterase activity further enhanced the forskolin effect. Treatment with forskolin also brought about dose- and time-dependent increments in progesterone and estrogen accumulation. Granulosa cells not pretreated with forskolin displayed negligible LH/hCG binding and remained unresponsive to luteotropic (LH/hCG), beta 2-adrenergic (terbutaline), or lactogenic (PRL) stimulation. In contrast, forskolin (10(-5) M)-pretreated granulosa cells displayed significant increases over controls in LH/hCG binding (46-fold) as well as in progesterone accumulation stimulated by hCG (3.3-fold), terbutaline (1.9-fold), and PRL (1.8-fold). Furthermore, concomitant treatment with a functionally inert low dose (10(-7) M) of forskolin, substantially potentiated the FSH-stimulated accumulation of extracellular cAMP, progesterone, and estrogen as well as the FSH-mediated increase in LH/hCG binding. Taken together, our findings indicate that forskolin, like FSH, is capable of inducing the differentiation of cultured rat granulosa cells by itself, and that a functionally inert low dose of forskolin can potentiate FSH hormonal action. Inasmuch as forskolin-simulated and forskolin-potentiated hormonal action are acceptable as novel criteria of cAMP dependence, our findings provide new evidence in support of the notion that cAMP may be an intracellular second messenger of FSH.
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PMID:Forskolin-induced differentiation of cultured rat granulosa cells: new evidence for an intermediary role of adenosine 3',5'-monophosphate in the mechanism of action of follicle-stimulating hormone. 632 47

TRH was found to rapidly influence 32PO4 incorporation into phospholipids of PRL-secreting GH pituitary cells. Analogs of TRH were found to exert similar effects, with potencies related to receptor-binding affinity. Additional PRL-releasing agents were also tested. Bombesin exerted a similar effect, whereas vasoactive intestinal polypeptide, 8-bromo cAMP, phosphodiesterase inhibitors, 50 mM K+, and scorpion venom toxin had no influence. Cationophore A23187 stimulated phospholipid labeling in a manner distinguishable from that of TRH. Chromatographic analysis showed the action of TRH to be restricted to the labeling of phosphatidylinositol and phosphatidic acid. Kinetic studies indicated a rapid influence of TRH on phosphatidylinositol breakdown, with subsequent accelerated 32PO4 incorporation into phosphatidylinositol and phosphatidic acid. These studied identified a rapid, receptor-mediated, cAMP-independent action of TRH on phospholipid metabolism. Similar effects of other hormones are believed to be involved in promoting cellular Ca2+ translocation. The rapid onset of the response reported here suggests that this event may play a role in mediating the PRL-releasing effects of TRH and bombesin in GH cells.
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PMID:Thyrotropin-releasing hormone (TRH) selectively and rapidly stimulates phosphatidylinositol turnover in GH pituitary cells: a possible second step of TRH action. 680 Jul 71

Studies were carried out to determine the possible roles of the polyamines, cyclic nucleotides, icosanoid products, and protein kinase C in the prolactin regulation of amino acid transport in cultured mammary gland explants derived from 12-14 day pregnant mice. Elevated cyclic AMP concentrations impaired the PRL stimulation of AIB transport. DBcAMP as well as the phosphodiesterase inhibitors theophylline and methyl isobutylxanthine, when added to the cultures, attenuated or abolished the PRL responses. 8-Bromo cyclic GMP elicited a modest stimulation of AIB transport. Ongoing polyamine synthesis appears to be necessary for PRL to effect a stimulation of AIB transport since methylglyoxal bis(guanyl hydrazone), an inhibitor of S-adenosyl methionine decarboxylase, abolishes the PRL response; specificity of this effect was established by its reversal with the addition of spermidine to the culture medium. Ongoing icosanoid product synthesis also appears to be required for the PRL stimulation of AIB transport since indomethacin abolishes the PRL response. Finally, the inhibition of the PRL response by the protein kinase C inhibitor H-7 suggests that the activation of kinase C activity may also be involved in the PRL stimulation of AIB transport.
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PMID:Studies on the mechanism by which prolactin stimulates alpha-aminoisobutyric acid uptake into cultured mouse mammary tissues. 769 65

The neuroendocrine system plays a key role in the regulation of the secretion of the thymic peptide, thymulin, but it remains to be determined whether thymulin exerts reciprocal regulatory actions on the functional activity of the hypothalamo-pituitary axis. In the present study, we have used a well established in vitro preparation to examine the influence of thymulin on cyclic nucleotide formation and hormone secretion by the rat anterior pituitary gland. Thymulin-Zn2+ (0.5-50 pM) stimulated the release of immunoreactive corticotrophin (ir-ACTH), producing effects which were maximal at 10 pM (p < 0.01). At the two highest concentrations tested (10 and 50 pM), it also produced small but significant increases in immunoreactive luteinising hormone (ir-LH) release (p < 0.05), but the secretion of immunoreactive growth hormone (ir-GH) was unaffected by the peptide (p > 0.05) while that of immunoreactive prolactin (ir-PRL) was reduced (p < 0.01). The ACTH responses to thymulin were accompanied by increased cyclic nucleotide formation. Thus, thymulin (0.5-50 pM) raised the cyclic 3',5'-adenosine monophosphate (cyclic AMP) content of the pituitary tissue (p < 0.01). At high concentrations (10-50 pM), it also increased cyclic 3',5'-guanosine monophosphate (cyclic GMP; p < 0.01) accumulation, although lower concentrations of the peptide were ineffective in this regard. The increases in ir-ACTH release provoked by thymulin-Zn2+ (0.5-5.0 pM) were potentiated markedly by rolipram (1 microM; p < 0.01), a selective inhibitor of the cyclic-AMP-specific phosphodiesterase enzyme. By contrast, zaprinast (10 microM), a selective inhibitor of cyclic-GMP-specific phosphodiesterase, attenuated the corticotrophic responses to higher concentrations of the peptide (10 and 50 pM; p < 0.05). Neither rolipram (1 microM) nor zaprinast (10 microM) influenced the release of ir-LH, ir-PRL or ir-GH in the presence or absence of thymulin-Zn2+ (0.5-50 pM; p > 0.05). The results suggest that thymulin modulates the secretion of ACTH and possibly LH by the anterior pituitary gland and that its actions are associated with increased cyclic nucleotide formation; in addition, it appears to exert an inhibitory influence on ir-PRL release.
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PMID:Thymulin stimulates corticotrophin release and cyclic nucleotide formation in the rat anterior pituitary gland. 948 96


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