Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Addition of [D-Trp6]gonadotropin-releasing hormone (GnRHa) to alpha T3-1 cells induced a very rapid response upon gonadotropin alpha-subunit mRNA which was detected after 30-60 min and was abolished by pretreatment with actinomycin D. A similar response was obtained with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), or the Ca2+ ionophore, ionomycin. GnRHa (10 nM) also stimulated a secondary rise in alpha-subunit mRNA levels between 12 and 24 h of incubation. No additivity was obtained (at 60 min) upon the combined addition of GnRHa and PMA, GnRHa and ionomycin, or PMA and ionomycin. The effect of GnRHa upon alpha-subunit mRNA was blocked by the PKC inhibitors staurosporine or GF 109203X. Down-regulation of endogenous PKC activity resulted in inhibition of the stimulatory effect of gonadotropin-releasing hormone (GnRH), PMA and ionomycin. Removal of extra-cellular Ca2+ abolished the effect of GnRHa and PMA upon alpha-subunit mRNA levels. Interestingly PMA and ionomycin had no effect on alpha-subunit mRNA levels at 24 h of incubation; however, the combined addition of the drugs mimicked the late phase of GnRHa (10 nM) action. The data provide evidence that PKC and Ca2+ are involved in mediating the early and the late responses of GnRHa upon alpha-subunit mRNA elevation and that differential cross-talk exists between the messengers.
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PMID:Mechanism of action of gonadotropin-releasing hormone upon gonadotropin alpha-subunit mRNA levels in the alpha T3-1 cell line: role of Ca2+ and protein kinase C. 754 47

The role of persistent protein phosphorylation upon gonadotropin releasing hormone (GnRH) stimulated luteinizing hormone (LH) release was investigated by the use of the selective inhibitors of protein phosphatase type 1 (PP1) and 2A (PP2A), okadaic acid (OA) and calyculin A. Pre-incubation of cultured rat pituitary cells with OA (24 h) or calyculin A (30 min) resulted in inhibition of GnRH-stimulated LH release with significant inhibition being detected at 10 nM and 30 nM for OA and calyculin A, respectively. The inactive OA analog norokadone and the protein tyrosine phosphatase inhibitor vanadyl hydroperoxide had no significant effect on GnRH-induced LH release. The stimulatory effects of the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA, 50 ng/ml) or the Ca2+ ionophore, ionomycin (1 micron), upon LH release were also abolished by pretreatment with OA (10-20 nM) or calyculin A (30 nM). Stimulation of LH release by high K+ (28 mM) or residual LH release stimulated by GnRH in Ca(2+)-free medium were also blocked by OA. These observations indicate that protein dephosphorylation is involved positively in GnRH-stimulated LH release. The site of action of the protein phosphatases PP1 and PP2A is most likely downstream to Ca2+ elevation and PKC activation by GnRH.
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PMID:Involvement of protein phosphatases in gonadotropin releasing hormone regulated gonadotropin secretion. 764 55

We recently demonstrated that immortalized GT1-7 neurons co-express luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor and gonadotropin releasing hormone (GnRH) genes. Treatment of GT1-7 neurons with LH/hCG resulted in a transcriptional inhibition of GnRH gene. In the present study, we investigated the signaling and transacting factors involved in the action of hCG. Eight-bromo-cyclic AMP can mimic the down-regulating action of hCG on GnRH mRNA levels. H-89, a protein kinase (PK) A inhibitor, but not bisindolylmaleimide, a PKC inhibitor, blocked the down- regulating actions of hCG as well as of 8-bromocyclic AMP. Treatment with the PKA inhibitor alone modestly decreased GnRH mRNA levels suggesting that PKA signaling also controls the basal expression of the GnRH gene. The direct measurement of PK activities revealed that hCG treatment of GT1-7 neurons increased the PKA but not the PKC activity. New protein synthesis is required for the down-regulating action of hCG on GnRH mRNA levels. Since some of the new proteins could be nuclear transcription or transacting factors, we investigated the effects of hCG on cyclic AMP response element binding protein (CREB), c-Fos and c-Jun protein levels. Treatment of GT1-7 neurons with hCG resulted in an increase of 43 kDa phosphorylated CREB, 50 kDa c-Fos and 40 kDa c-Jun proteins compared to the corresponding controls. The kinetics of increases were different and in all cases the increases of the proteins preceded the decrease of GnRH mRNA levels. In summary, PKA signaling and transacting factors such as CREB, Fos and Jun are probably involved in transcriptional inhibition of GnRH gene by hCG in GT1-7 neurons.
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PMID:Signaling and transacting factors in the transcriptional inhibition of gonadotropin releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons. 766 77

Transient transfection studies using gonadotrope-derived, alpha T3-1 cells were used to determine the DNA sequences of the mouse glycoprotein hormone alpha-subunit gene that mediate the transcriptional response to gonadotropin releasing hormone (GnRH). The roles of phorbol esters and cyclic AMP in mediating the GnRH response were also investigated. The initial studies demonstrated that a construct containing approximately 500 base pairs of alpha-subunit flanking sequence was sufficient to mediate responses to a GnRH agonist (GnRHa), phorbol myristate acetate (PMA) and a cAMP analog. Responses to combinations of cAMP and GnRHa or cAMP and PMA were approximately additive, whereas the response to the combination of GnRHa and PMA was similar to that seen with either of the agents alone. Cotransfection studies with an expression vector for the heat-stable inhibitor of the cAMP-dependent protein kinase demonstrated that GnRHa and PMA responses are not dependent on the cAMP-dependent kinase. Deletion analysis indicated that sequences between -507 and -205 were involved in mediating responses to GnRHa and PMA. To determine if this region alone could support responses to these agents, the -507 to -205 region was linked to a minimal promoter and tested in transient transfections. The results demonstrated that this region supports responses to GnRHa, PMA, and cAMP. Clustered point mutations of this region were used to further characterize sequences involved in the GnRH response. Mutations in two regions, one at positions -406 to -399 and one at positions -337 to -330, resulted in decreased responses to GnRH and PMA. There is no obvious sequence similarity between the two regions that are required for the GnRH response. An enhancer test demonstrated that multimers of the -416 to -385 region were able to function as a GnRH-responsive element when linked to a minimal promoter, although a single copy of this region was not sufficient to permit a response to GnRH. In contrast, multimers of the -344 to -300 region did not permit a response to GnRH, but enhanced basal transcription. These findings are consistent with the identification of a two-component GnRH response unit, which probably involves the functional cooperation of two different transcription factors. The observation that GnRH responsiveness appears to co-localize with PMA responsiveness suggests that GnRH effects on the alpha-subunit transcription are likely mediated by the protein kinase C pathway.
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PMID:Two different DNA elements mediate gonadotropin releasing hormone effects on expression of the glycoprotein hormone alpha-subunit gene. 768 35

The action mechanism of gonadotropin-releasing hormone (GnRH) on the cytosolic free calcium concentration ([Ca2+]i) and high-threshold voltage-dependent Ca2+ channel activity was studied in human nonsecreting (NS) pituitary adenoma cells. [Ca2+]i was monitored in individual cells by dual emission microspectrofluorimetry using indo 1 as intracellular fluorescent Ca2+ probe. The whole-cell recording patch-clamp technique was used to study Ca2+ channels. A short application of GnRH (1 to 100 nM) induced an increase in [Ca2+]i due to Ca2+ entry through plasma membrane voltage-sensitive L-type Ca2+ channels. Protein kinase C (PKC) depletion induced by a pretreatment with 1 microM PMA for 24 h abolished spontaneous Ca2+ transients and the action of GnRH on [Ca2+]i and Ca2+ channels. Phloretin (250 microM) and staurosporine (20 nM), two protein kinase C inhibitors, inhibited Ca2+ channel activity, thereby suppressing the effect of GnRH. On the other hand, activation of PKC by a short application of phorbol myristate acetate (10 nM) stimulated Ca2+ influx through Ca2+ channels. These findings demonstrate that, in human NS adenoma cells, GnRH (1 to 100 nM) induces an increase in [Ca2+]i, principally due to Ca2+ entry through L-type voltage-activated Ca2+ channels. PKC regulates this mechanism as well as basal ion channel activity, thus exerting both positive and negative control of [Ca2+]i in stimulated and unstimulated NS adenoma cells.
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PMID:Gonadotropin-releasing hormone induced Ca2+ influx in nonsecreting pituitary adenoma cells: role of voltage-dependent Ca2+ channels and protein kinase C. 770 45

GT1-7 cells, a clonal line derived from specific tumours of gonadotropin-releasing hormone-secreting neurons from mouse hypothalamus, were used as a model system to investigate the cellular mechanisms underlying the histamine H1 receptor-mediated desensitisation. GT1-7 cells contain H1 receptors, acute stimulation of which leads to the desensitisation of histamine-mediated calcium mobilisation and is manifest as a concurrent reduction in both the magnitude of the calcium transient and of the sustained phase. Acute pretreatment of the cells with the phorbol ester, phorbol 12-myristate 13-acetate, can also ablate the histamine-stimulated calcium mobilisation. In addition, acute H1-receptor stimulation and acute phorbol ester treatment result in the attenuation of histamine-mediated inositol phosphate production. Receptor desensitisation resulting from acute stimulation with histamine is not affected by inhibiting protein kinase C (PKC) activity with Ro 31-7549 or staurosporine. In contrast, the desensitisation of H1-receptor responses induced by direct activation of protein kinase C is preventable by PKC inhibitors. Thus, these results imply that a PKC-dependent mechanism and PKC-independent mechanism are involved in the H1-receptor desensitisation cascade in GT1-7 cells and do not support the involvement of PKC in the receptor-mediated desensitisation of H1 receptor-stimulated calcium and inositol phosphate responses.
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PMID:Receptor-mediated desensitisation of histamine H1 receptor-stimulated inositol phosphate production and calcium mobilisation in GT1-7 neuronal cells is independent of protein kinase C. 779 Aug 57

One important role of the junctional communication in the ovarian follicle is to mediate transmission of cAMP, the regulatory signal that maintains the oocyte in meiotic arrest. Luteinizing hormone (LH) interrupts cell-to-cell communication within the ovarian follicle, leading to a decrease in intraoocyte concentrations of cAMP followed by resumption of meiosis. Our experiments were directed at exploration of mechanisms involved in the LH-induced communication breakdown in the preovulatory ovarian follicle. Immunofluorescence and Western blot analysis, using highly specific antibodies, showed that connexin-43 (Cx43), the ovarian gap junction protein, is present in the cytoplasmic membranes of the follicular cells in multiple phosphorylated forms. The relative amounts of the different forms of Cx43 vary in response to LH: short time exposure (10 min) stimulated phosphorylation of Cx43 followed by immediate dephosphorylation, while longer incubations (8 and 24 h) with this hormone resulted in elimination of the protein. Forskolin mimicked the LH-induced phosphorylation/dephosphorylation, as well as the decrease of Cx43 protein level. A gonadotropin-releasing hormone analog (GnRHa) also induced an immediate phosphorylation/dephosphorylation of Cx43 and a later reduction of the amount of Cx43. The direct PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced phosphorylation of Cx43 that was completely blocked by the protein kinase C inhibitor, staurosporine. This kinase inhibitor partially interfered with LH, but not forskolin-induced phosphorylation of Cx43. Analysis of the effect of LH on Cx43 gene expression revealed a significant decrease (45%) in Cx43 mRNA level at 24 h of incubation. A drop of Cx43 mRNA was also induced by GnRHa. Our results suggest that the LH-induced gating mechanism of the gap junctions in rat ovarian follicles is comprised of two steps: the immediate response is represented by a change in the phosphorylation state of the Cx43 protein, and the later response is manifested by a reduction of Cx43 protein level, due to attenuation of its gene expression. Phosphorylation of Cx43 may occur through PKA-, as well as PKC-dependent pathways.
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PMID:Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. 798 67

The gonadotroph-derived alpha T3-1 cell line was used to investigate the effect of gonadotropin-releasing hormone (GnRH) upon conventional protein kinase C sub-types (cPKCs) gene expression. Addition of the stable analog [D-Trp6]GnRH (GnRH-A, 0.1 nM) resulted in a rapid increase (30 min) of the steady state levels of PKC beta, but not PKC alpha, mRNA levels, while PKC gamma is not expressed in the cells. The rapid stimulatory effect of GnRH-A was blocked by pretreatment with actinomycin D or with the GnRH antagonist (D-pGlu1, pC1Phe2,D-Trp3,6)GnRH and was not mimicked by thyrotropin-releasing hormone. Addition of the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted also in a rapid (30 min) and selective increase in PKC beta, but not PKC alpha, mRNA levels. In contrast, the calcium ionophore, ionomycin, increased rapidly (30 min) both PKC alpha and PKC beta mRNA levels, and its stimulatory effect on PKC beta was not additive with that of TPA. The rapid stimulatory effect of GnRH-A was blocked by the PKC inhibitor bisindolylmaleimide (GF 109203X) or by down-regulation of endogenous PKC. Similarly, the rapid effect of GnRH-A was abolished by the intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) or by removal of extracellular Ca2+. Stimulation of PKC beta mRNA levels by ionomycin was only reduced by GF 109203X and was not affected by down-regulation of PKC. In contrast the effect of TPA on PKC beta mRNA levels was reduced by BAPTA and abolished by removal of Ca2+. We conclude that Ca2+ and PKC act sequentially during GnRH-A-induced PKC beta gene expression and that PKC beta gene expression induced by GnRH-A is autoregulated by PKC.
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PMID:Activation of protein kinase C beta gene expression by gonadotropin-releasing hormone in alpha T3-1 cell line. Role of Ca2+ and autoregulation by protein kinase C. 798 40

The effects of two cryptic peptides from pro-TRH: Ps4 (160-169) and Ps5 (178-199) were investigated on basal and secretagogue (GRH and TRH)-induced releases of GH from perifused fragments of rat adenohypophysis. Validation of the perifusion system was done by measuring: (1) the dose-dependent effect of GRH and TRH on GH release; and (2) the stimulation of that release by forskolin (to mimic the adenylate cyclase pathway) or by phorbol ester (to mimic the protein kinase C pathway). We show that: (1) Ps4 and Ps5 (1 microM) do not modify basal GH release; (2) Ps4 (1 microM) changes neither GRH (10 nM)- nor TRH (100 nM)-induced release of GH; (3) Ps5 (100 nM and 1 microM) significantly decreases the release of GH induced by equimolar concentrations of TRH but not that induced by GRH.
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PMID:A cryptic peptide of TRH prohormone inhibits TRH-induced GH release. 799 14

Desensitization of gonadotropin release by the pituitary gland in response to gonadotropin-releasing hormone (GnRH) agonists has clinical applications in the treatment of gonadal-hormone-dependent disorders. We therefore investigated possible desensitization of inositol phosphate (IP) responses of GNRH receptors. No short-term homologous desensitization of the IP response to GnRH was observed in either alpha T3 gonadotrope cells line or GH3 cells transfected with GnRH receptor cDNA. The absence of homologous desensitization is unusual among G-protein-coupled receptors, and may be due to the absence of a C-terminal cytoplasmic tail, a unique feature of the GnRH receptor. Several potential protein kinase C phosphorylation sites which might mediate heterologous desensitization are present on the GnRH receptor. In both alpha T3 cells and GnRH-receptor-transfected Cos-1 cells, activation of protein kinase C by pretreatment with phorbol ester caused a 35-53% decrease in the IP response to GnRH. However, phorbol ester also inhibited guanosine 5'-[gamma-thio]triphosphate-stimulated IP production in permeabilized Cos-1 cells, suggesting that this inhibition is mediated at a post-receptor site.
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PMID:Absence of rapid desensitization of the mouse gonadotropin-releasing hormone receptor. 800 31


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