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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cultured rat granulosa cells have provided a useful model to examine the hormonal regulation of inhibin secretion. In the present study we have used the cloned rat inhibin alpha- and beta A-subunit cDNAs to characterize the influences of gonadotropins, growth factors, and GnRH on inhibin subunit mRNA levels in granulosa cells obtained from immature estrogen-treated rats. Cells were cultured in medium with or without added hormones. Total RNA from cultured cells was extracted and hybridized with 32P-labeled inhibin alpha- and beta A-subunit cRNA or beta-actin cDNA probes, and inhibin subunit mRNA levels were normalized with beta-actin mRNA levels. Treatment of granulosa cells with FSH increased inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Similarly, LH, but not PRL, increased alpha- and beta A-subunit mRNA levels in granulosa cells pretreated with FSH to induce functional LH and PRL receptors. The effects of FSH and LH on inhibin subunit mRNA levels were mimicked by forskolin, which increased alpha- and beta A-subunit transcripts in a dose- and time-dependent manner, suggesting involvement of the cAMP-dependent protein kinase-A pathway. Since several growth factors have been shown to influence inhibin secretion, their effects on inhibin subunit mRNA levels were also studied. Treatment of cells with transforming growth factor-beta 1 increased both basal and FSH-stimulated inhibin alpha- and beta A-subunit mRNA content, whereas insulin-like growth factor-I had no significant effect. In contrast, both epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) markedly suppressed both basal and FSH-stimulated inhibin subunit transcript levels. The inhibitory effects of EGF and basic FGF were dose dependent and persisted from 12-72 h of incubation. The regulatory peptide GnRH, which decreases inhibin secretion, was also found to suppress FSH-stimulated inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Furthermore, the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting the involvement of specific GnRH-binding sites in GnRH action. These studies indicate that, except for insulin-like growth factor-I, the effects of gonadotropins, growth factors (EGF, basic FGF, and transforming growth factor-beta 1), and GnRH on inhibin secretion are related to their regulation of inhibin alpha- and beta A-subunit mRNA levels.
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PMID:Regulation of inhibin subunit messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells. 211 34

This report presents findings pertaining to the role of protein kinase-Cs in the release of PRL and liberation of arachidonate from PRL-secreting cells. In our experiments, protein kinase-C activators increased PRL release and arachidonate liberation from anterior pituitary cells and from the PRL-secreting cell line MMQ. In cells depleted of pituitary protein kinase-Cs by chronic exposure to protein kinase-C activators, such as phorbol dibutyrate or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, TRH, angiotensin-II, and neurotensin each increased PRL release and [3H]arachidonate liberation in a normal manner. In addition, the PRL-releasing activities of protein kinase-C activators and those of TRH appeared to be synergistic, an unexpected effect if these substances were functioning through the same intracellular pathways. It, therefore, appears that phorbol diester-sensitive protein kinase-Cs may not be involved in the increased secretion of PRL or liberation of arachidonate that is caused by TRH, angiotensin-II, or neurotensin.
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PMID:Evidence that phorbol diester-sensitive protein kinase-C(s) may not be directly involved in secretagogue-stimulated prolactin release and arachidonate liberation. 250 62

The biochemical mechanisms underlying the direct stimulatory action of dopamine (DA) withdrawal on PRL release and on the potentiation of TRH stimulation are not known. These actions can be mimicked by pretreatment of lactotrophs with the protein kinase-C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate. Previous studies have shown that administration of TRH or withdrawal of DA stimulates polyphosphoinositide breakdown, although to different degrees. We have tested whether the acute withdrawal of DA activates PKC and determined if the prior removal of DA modifies the activation of PKC by TRH. Primary cultures of dispersed anterior pituitaries from estradiol-treated rats consisting of approximately 80% lactotrophs were maintained overnight in 500 nM DA. Activation of PKC was assayed immunochemically as translocation of PKC to a membrane fraction and by in situ phosphorylation of an acid-soluble heat-stable 80K substrate. Acute withdrawal of DA induced translocation of immunoreactive PKC to the membrane fraction (25-250%) and enhanced phosphorylation (40-100%) of an 80K protein. These effects were detected within 5-15 sec of DA withdrawal and were prolonged (10-30 min). TRH induced a rapid and transient activation of both parameters. The duration and magnitude of the action of TRH were increased by prior removal of DA. These results are consistent with a role for PKC activation in transduction of the stimulation of PRL release by the withdrawal of DA. The longer lasting activation of PKC may explain at least in part the potentiation of the PRL-releasing action of TRH by the withdrawal of DA.
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PMID:Dopamine withdrawal and addition of thyrotropin-releasing hormone stimulate membrane translocation of protein kinase-C and phosphorylation of an endogenous 80K substrate in enriched lactotrophs. 250 64

To determine the rat PRL (rPRL) promoter sequences that mediate pituitary-specific and cAMP-induced gene expression in vivo, various lengths of the rPRL promoter were ligated to the luciferase reporter gene and introduced into pituitary and non-pituitary cell lines. A 30-fold increase in rPRL promoter activity was observed in GH4 rat pituitary tumor cells compared to nonpituitary Rat2 fibroblast and HeLa cervical carcinoma cells. About 45% of this cell-specific promoter activity was competed by a plasmid containing the -67 to -45 rPRL promoter region, which is the most proximal binding site for a lactotroph-specific factor. Compared to a -425 rPRL construct, transfection with rPRL 5'-end points of -212, -178, and -127 contained 23%, 45%, and 1%, respectively, of luciferase activity. Forskolin stimulation resulted in a 10-fold induction of all the rPRL promoter fragments tested. Of note, a -127 deletion which was devoid of any basal promoter activity was also induced 10-fold by forskolin. The forskolin effect was abolished when GH4 rat pituitary cells were cotransfected with a plasmid encoding a protein kinase A inhibitor, indicating protein kinase A is involved in the activation mechanism. These data document that both positive and negative effectors influence basal rPRL promoter activity. Furthermore, the minimum sequences required for pituitary-specific rPRL promoter activity are altered by intracellular cAMP levels. Taken together, the data indicate that hormone-activated and cell-specific factors may interact to establish a particular setpoint for rPRL gene expression.
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PMID:Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3',5'-cyclic adenosine monophosphate-regulated gene expression in vivo. 254 56

The PRL gene is expressed at a high basal level in rat pituitary tumor GH3 cells, and this basal level enhancement of PRL gene expression is maintained through a Ca2+-calmodulin-dependent mechanism. We have now examined whether the enzyme, DNA topoisomerase II, which has been shown to be phosphorylated by a Ca2+-calmodulin-dependent protein kinase, plays a role in the Ca2+-calmodulin-dependent basal level enhancement of PRL gene expression. The topoisomerase II inhibitor, novobiocin, at concentrations in the range of 35-140 microM, effectively blocked the ability of Ca2+ to increase PRL mRNA levels. Examination of the effects of novobiocin on the levels of protein synthesis, glucose-regulated protein (GRP) 78 mRNA, histone 3 mRNA, and 18S ribosomal RNA indicated that the drug selectivity inhibited PRL gene expression. Two other topoisomerase II inhibitors, m-AMSA and VM26, also diminished the Ca2+-induced levels of PRL mRNA at concentrations (100-400 nM) that did not lower total mRNA levels. We then examined whether topoisomerase II interacted nonrandomly with DNA from the 5' transcribed and 5'-flanking region of the rat PRL gene by in vitro mapping of topoisomerase II DNA cleavage sites. In initial assays with a 10.5 kilobase (kb) PRL genomic DNA fragment containing 3.5 kb of 5'-transcribed DNA and 7 kb of 5'-flanking DNA, we detected 4 major cleavage sites in the following regions: site 1, +1500 to +1600; site 2, +1 to -100; site 3, -1200 to -1300; and site 4, -2900 to -3000.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for a role of topoisomerase II in the Ca2+-dependent basal level expression of the rat prolactin gene. 284 May 67

Although dopamine inhibits PRL release from the normal anterior pituitary lactotroph, a conclusive demonstration of the mechanisms involved in this response has been impeded by the presence of other cell types in the anterior pituitary. To circumvent this problem, we have isolated a clonal cell line, designated MMQ, from the 7315a rat pituitary tumor. The MMQ cell is an exemplary model for our use because it only secretes PRL. Our studies show that dopamine inhibits secretagogue-induced PRL release from these cells. In addition, dopamine decreases the intracellular cAMP concentration in MMQ cells that have been exposed to forskolin, cholera toxin, or vasoactive intestinal polypeptide, each a stimulator of cAMP generation. This inhibition is, in turn, reversed by the dopamine antagonist haloperidol and by pertussis toxin, an inactivator of the GTP-binding coupling protein. Dopamine also decreases the uptake and fractional efflux of 45Ca2+ by MMQ cells that have been exposed to the calcium channel activator maitotoxin. It seems, therefore, that dopamine decreases PRL release from MMQ cells at least in part by decreasing intracellular cAMP levels and calcium uptake. In additional experiments, we have found that MMQ cells are responsive to somatostatin, estrogen, progesterone, and acetylcholine, but not to TRH, angiotensin II, neurotensin, or bombesin. Furthermore, these cells possess a functional protein kinase-C system, as evidenced by the increase in PRL release and decrease in stimulated intracellular cAMP levels that occur in response to treatment with phorbol diesters. We suggest that the MMQ cell line will prove a useful model system for study of the biochemical effects of dopamine and other factors that modify PRL release.
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PMID:Characterization of the MMQ cell, a prolactin-secreting clonal cell line that is responsive to dopamine. 284 8

The purpose of this study was to characterize the action of phorbol 12-myristate 13-acetate (PMA), a tumor-promoting agent, on rat anterior pituitary gland, focusing the attention on prolactin secretion. PMA elicited a significant increase in prolactin secretion without affecting phosphatidylinositol turnover, considered as an early post-receptor event controlling PRL secretion. However incubation of anterior pituitary glands with PMA caused a loss of protein kinase-C activity in cytoplasm concomitant with an increased enzyme activity in the membrane. The action of PMA on prolactin secretion seems to be mainly dependent from the redistribution and activation of protein kinase-C. In fact, the phorbol ester did not affect pituitary cAMP and cGMP metabolism either in basal conditions or after theophylline.
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PMID:Phorbol 12-myristate 13-acetate induces prolactin secretion from rat anterior pituitary gland by the activation of protein kinase-C. 309 41

In the present study, we investigated the ability of phorbol esters to potentiate Ca2+-dependent depolarization-induced release of tritium-labeled dopamine ([3H]DA) from median eminence and striatal synaptosomes. Phorbol esters potentiated [3H]DA release in a concentration-dependent manner in both kinds of dopaminergic nerve terminals and with a potency series similar to that reported for stimulation of protein kinase-C (PKC) activity in other cell systems. Evoked [3H]DA release was increased by 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) M) after 1, 3, 5, and 10 sec of depolarization. The effect of TPA was suppressed by sphingosine, a PKC inhibitor. TPA enhanced [3H]DA release evoked by high K+, veratridine or the Ca2+ ionophore A23187. Phorbol ester potentiation was found to be depolarization dependent, as it was present from 30-75 mM, but not at 5-20 mM external K+. Potentiation was seen at all external Ca2+ concentrations studied between 0.01-3 mM. However, in the absence of external free Ca2+ (i.e. with 0.1 mM EGTA), the phorbol effect was not present. These data indicate that an increase in intrasynaptosomal Ca2+ concentration is necessary for the enhancement of [3H]DA release by phorbol esters to occur. The combination of TPA and the Ca2+ ionophore A23187 does not show the marked synergism observed in some other systems, that is maximal release was not reinstated. This suggests that in dopaminergic nerve terminals, activation of PKC has a modulatory, rather than a mediating, effect on release. Recently, we have shown that hyperprolactinemia stimulated [3H]DA release from median eminence synaptosomes by an external Ca2+-independent mechanism which might involve the PKC pathway. However, in the present work we found that the TPA and PRL effects on evoked [3H]DA release were additive, suggesting that two independent mechanisms are involved. A marked difference in the sensitivity of median eminence and striatal synaptosomes to calcium ionophore was discovered. The concentration of A23187 required to support significant [3H]DA release from median eminence synaptosomes was 3-fold greater than that in striatal synaptosomes. This suggests that some difference in calcium homeostatic processes exists, such as a higher resting striatal Ca2+ concentration, in these two kinds of dopaminergic nerve terminals. These data support the hypothesis that PKC activation potentiates the intrasynaptosomal stimulus-secretion coupling mechanism(s) and that nigrostriatal and tuberoinfundibular dopaminergic nerve terminals are affected by phorbol esters in a similar manner.
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PMID:Phorbol esters potentiate rapid dopamine release from median eminence and striatal synaptosomes. 313 Nov 21

TRH, epidermal growth factor (EGF), and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulate PRL synthesis by GH4C1 rat pituitary cells. Recent evidence suggests that TPA activates directly phospholipid- and calcium-dependent protein kinase C in other cell types and that TRH might act analogously by altering phospholipid metabolism in GH4C1 cells. To examine the pathways by which these three agents stimulate PRL synthesis, we determined their calcium dependencies as well as their combined effects on PRL production. By equilibration of GH4C1 cells in a protein-free medium for 24 h, the free cytosolic calcium concentration ([Ca2+]i) was found to increase (from 90 to 360 nM) when the extracellular calcium concentration ([Ca2+]e) was varied from 15 to 800 microM. Basal PRL production increased in parallel (from 1 to 4 micrograms/ml X 24 h). TPA-stimulated PRL production was highly calcium dependent and required 180 nM [Ca2+]i for maximal enhancement. TRH-stimulated PRL production was constant between 10 and 660 microM [Ca2+]e, whereas EGF stimulated PRL production to a similar extent as TRH at 10 microM [Ca2+]e, but continued to enhance production with increasing [Ca2+]e. TRH elevated [Ca2+]i acutely, and at [Ca2+]e greater than 100 microM caused both a burst and a plateau phase in elevated [Ca2+]i. At lower [Ca2+]e, at which TRH still caused a maximal stimulation of PRL production, only the burst phase of [Ca2+]i occurred. When cultures were treated with any combination of maximally effective concentrations of TPA, TRH, or EGF, PRL production was increased by additive increments. The additive actions of TPA and TRH could not be explained by a calcium-promoted increase in TPA-stimulated PRL production. We conclude that TPA stimulates PRL production by a highly calcium-dependent pathway and that TRH and EGF stimulate PRL production by a different pathway(s) requiring lower [Ca2+]i.
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PMID:Thyrotropin-releasing hormone and epidermal growth factor stimulate prolactin synthesis by a pathway(s) that differs from that used by phorbol esters: dissociation of actions by calcium dependency and additivity. 393 Feb 23

A protein kinase activity fraction was defined in cytosols and membranes of mammary tissue isolated from rats during pregnancy lactation, and weaning. By partial purification on DEAE-cellulose columns, it was shown that this protein kinase activity is cAMP independent and that its preferential substrate is casein and not histone. This protein kinase activity is inhibited by the bioflavonoid quercetin at doses that do not inhibit cAMP-dependent protein kinase activity. The enzyme requires Mg2+ and is inactive in the presence of 10 mM Ca+2; these properties distinguish this activity from casein kinase activity found in the Golgi fraction and involved in milk protein processing. By following the physiological cycle of mammary gland development during pregnancy, lactation, and weaning, we found a close correlation between proliferation, expressed as the DNA content per gland, and quercetin-inhibited cytosolic protein kinase activity. Moreover, changes in this phosphorylating activity preceded the glandular growth changes. There was a less significant correlation between the growth process and protein kinase activity in the membrane fraction. The cytosolic cAMP-dependent protein kinase activity showed (only partial) correlation with growth only during pregnancy. Cytosolic progesterone receptor levels in mammary tissue were used as an estrogenic marker. Tissue growth correlated with progesterone receptor levels during pregnancy, where estrogens are the predominant hormones affecting tissue proliferation. However, no such correlation was found during lactation and weaning, when PRL is the major hormone affecting mammary gland growth. These results suggest that quercetin-inhibitable protein kinase activity is not merely another estrogenic marker, but represents more general regulatory activity which might be connected to growth processes of breast tissue.
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PMID:Protein kinase activity in the rat mammary gland during pregnancy, lactation, and weaning: a correlation with growth but not with progesterone receptor levels. 609 41


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