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

The acute and the long-term (24 h) effects of protein kinase C activators, phorbol 12 myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol, and the calcium ionophore A23187 on cultured pig Leydig cell functions were investigated. None of these drugs modified basal cAMP production, but they induced a small (3-4-fold) increase in testosterone secretion. The stimulatory effects of human choriogonadotropin (hCG; 1 nM) on both cAMP and testosterone productions were inhibited by short-term incubation with these drugs. In addition, they suppressed the stimulation of testosterone output by forskolin and 8-bromo-adenosine 3',5'-monophosphate, whereas the forskolin-dependent cAMP production was unaffected. The inhibitory effects of PMA on hCG stimulation of both cAMP and testosterone were due mainly to a decrease of the Vmax without modification of the ED50. Moreover, PMA did not modify the binding of 125I-hCG. Pretreatment of Leydig cells with the three drugs for 24 h induced more pronounced modifications, such as a reduction in the number of hCG binding sites and a decreased responsiveness to hCG and forskolin, the testosterone production being drastically reduced. The effects of PMA were dose- and time-dependent; however, the concentration of PMA required to induce half-maximal effects on hCG receptors (10 nM) was about one order of magnitude higher than those required to reduce cAMP and testosterone productions. Further, the inhibitory effects on cAMP and testosterone secretions appeared within the first 3 h, whereas the hCG receptor number remained constant for at least 8 h. It appears therefore, that the main alteration responsible for the steroidogenic refractoriness of PMA-treated Leydig cells is located beyond cAMP formation. Moreover, since conversion of exogenous pregnenolone to testosterone by control and PMA-treated cells was similar, the alteration was probably located before pregnenolone formation. Kinetic studies with 125I-hCG showed that the rate of internalization of the hormone-receptor complexes was similar in control cells and in PMA-treated cells, suggesting that the decline in receptor number observed in the latter group after an 8-h delay is not due to an increased rate of internalization nor to sequestration of the internalized receptors inside the cells. Since cycloheximide blocked the effects of PMA on hCG down-regulation, it is likely that the phorbol esters and 1-oleoyl-2-acetyl-sn-glycerol induce the synthesis of some proteins which blocked the recycling of internalized receptors. A similar hypothesis has been put forward recently to explain the hCG-induced down regulation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Stimulatory and inhibitory effects of protein kinase C activation and calcium ionophore on cultured pig Leydig cells. 302 94

To explore the mechanism of gonadotropin-releasing hormone (GnRH) action on Leydig cell steroidogenesis the effects of a GnRH analog (GnRHa) were compared to those of 12-O-tetradecanoylphorbol 13-acetate (TPA). Both compounds acutely stimulated androgen production 2-4 fold with EC50's of 9 nM (TPA) and 0.2 nM (GnRHa). The effects of TPA and GnRHa were not additive and neither compound acutely altered the luteinizing hormone (LH) concentration-response relationship. After 24 h of exposure to TPA or GnRHa the ability of LH to stimulate androgen production was impaired. The parallel effects of TPA and GnRHa on Leydig cell steroidogenesis suggest that they are acting via similar mechanisms; presumably the activation protein kinase C.
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PMID:Stimulation and inhibition of Leydig cell steroidogenesis by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate: similarity to the effects of gonadotropin-releasing hormone. 389 59

It has been demonstrated that erythropoietin (EPO) influences rat and human Leydig cell steroidogenesis, stimulating testosterone production through a direct and specific receptor-mediated mechanism. The aim of this study was to investigate the mechanism by which recombinant human erythropoietin (rHuEPO) exerts its stimulatory effect on rat Leydig cells. Recombinant human EPO did not induce, at any dose tested (10(-10) to 10(-13) mol/l), an increase in either cAMP or cGMP, suggesting that in Leydig cells the effect of rHuEPO does not involve the adenylate or guanylate-cyclase systems. The role of transmembrane calcium flux in rHuEPO-stimulated steroidogenesis was studied by evaluating the effect of calcium channel blocker, verapamil, and by the 45Ca2+ uptake method. Verapamil did not influence rHuEPO-induced testosterone secretion and rHuEPO did not modify calcium recycling, indicating that calcium transmembrane flux is not involved in the rHuEPO effect. The protein kinase C inhibitor staurosporine (10, 30, 100 and 300 nmol/l) inhibited rHuEPO-stimulated testicular steroidogenesis in a dose-dependent manner. This indirect evidence suggests that the stimulatory effect of rHuEPO on rat Leydig cells may involve protein kinase C activation.
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PMID:Erythropoietin and testicular steroidogenesis: the role of second messengers. 785 3

In this study the localization and regulation of steady-state follistatin messenger ribonucleic acid (mRNA) levels in testicular cell cultures were examined with a solution-hybridization assay using a specific 32P-labelled cytosolic RNA antisense probe for follistatin and a 35S-labelled cytosolic RNA antisense probe for cyclophilin as internal standard. Testes from immature rats were dispersed with collagenase and fractionated in Sertoli and Leydig cell-enriched cultures. Follistatin mRNA was mainly localized to the Sertoli cell-enriched fraction and the expression of follistatin mRNA could be stimulated in vitro with fetal calf serum, epidermal growth factor or phorbol-12-myristate-13-acetate (an activator of protein kinase C), whereas follicle-stimulating hormone and forskolin (an activator of protein kinase A) had no effect. Neither prostaglandin E2, the synthetic glucocorticoid RU 28362 or all-trans-retinoic acid, which all regulate follistatin mRNA levels in non-testicular cell types, nor extracellular adenosine triphosphate (a purinergic receptor agonist) or testosterone had any obvious influence on follistatin mRNA levels in Sertoli cell-enriched cultures. From this study it is concluded that Sertoli cells are likely to be the source of follistatin expression in the rat testis, that follistatin mRNA levels in Sertoli cell-enriched cultures are subjected to regulation by epidermal growth factor and the protein kinase C-dependent pathway but are not regulated by extracellular adenosine triphosphate, follicle-stimulating hormone, all-trans-retinoic acid, prostaglandin E2, forskolin, testosterone or the glucocorticoid RU 28362 and that the regulation of follistatin mRNA is sex- and tissue-specific.
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PMID:Expression of follistatin messenger ribonucleic acid in Sertoli cell-enriched cultures: regulation by epidermal growth factor and protein kinase C-dependent pathway but not by follicle-stimulating hormone and protein kinase A-dependent pathway. 810 86

Corticotropin-releasing factor (CRF), the key neuropeptide in the stress cascade, has major inhibitory actions on testicular function in addition to its known antireproductive effects at the central level (inhibition of sexual behavior and LH secretion). CRF is secreted by the Leydig cells of the testis and acts through high-affinity receptors at the Leydig cell membrane as a potent negative regulator of LH action, inhibiting gonadotropin-induced cAMP generation and androgen production. CRF is also a primary stimulus of beta-endorphin secretion by the Leydig cells, which in turn exerts paracrine inhibition of FSH action in the tubular compartment of the testis through high-affinity receptors in the Sertoli cells. CRF action in the Leydig cells involves a pertussis toxin-insensitive guanyl nucleotide regulatory unit. In contrast to CRF receptors in the brain, pituitary, and other peripheral tissues, those in the Leydig cell are not coupled to Gs. The inhibitory action of CRF in the Leydig cell is exerted through protein kinase C, at the level of the catalytic subunit of adenylate cyclase. The secretion of CRF by the Leydig cell is stimulated by LH, acting via release of serotonin (5HT) and autocrine activation of 5HT2 receptors. Serotonin acts on 5HT2 receptors in the Leydig cell to stimulate CRF secretion via a pertussis toxin insensitive G-protein and presumably through activation of phosphoinositide hydrolysis. The diversity of the biochemical responses to CRF and 5HT2 receptor activation (i.e., inhibition of adenylate cyclase at the cytoplasmic aspect of the cell membrane vs. stimulation of CRF release from secretion granules) may reflect the stimulation of different protein kinase C isoenzymes. The LH-->5HT-->CRF inhibitory loop serves to continuously buffer the stimulation of androgen production by gonadotropin. 5HT, the immediate stimulus of testicular CRF secretion, is released during stress and is locally increased in the testis in pathological conditions associated with impaired testicular function (i.e., orchitis, varicocele). Also, propranolol, the beta-adrenergic antagonist frequently used in the control of blood pressure in patients with hypertension and often associated with impotence, acts via a serotonergic mechanism to stimulate CRF secretion and causes marked inhibition of LH-induced cAMP production and steroidogenesis in cultured Leydig cells. These basic studies of 5HT and CRF are relevant to the pathogenesis of testicular dysfunction and for the development of antagonist therapies to block CRF production and its local antireproductive effects.
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PMID:Corticotropin-releasing factor: an antireproductive hormone of the testis. 838 38

We have investigated the role of protein kinase C (PK-C) in luteinizing hormone-releasing hormone (LHRH)-induced testosterone secretion from purified rat Leydig cells (70-80-day old Sprague-Dawley rats) by pretreating the cells in vitro with 200 mM phorbol 12,13-dibutyrate (PDBu) (a known procedure to down-modulate this enzyme in most cell types) and 1 muM [D-Ala6,Des-Gly10]-LHRH ethylamide, an LHRH agonist (LHRH-A). Following pretreatment we measured PK-C activity and secretion of testosterone in response to subsequent challenges with the PK-C activator PDBu (20-2000 nM) and with LHRH (0.001-1.0 muM) and the Ca(2+)-mobilizing secretagogue A23187 (0.1-100 microM) in the same cell preparation. PDBu and LHRH-A pretreatments caused a reduction in testosterone secretion in response to subsequent exposure to PDBu or LHRH. Both pretreatments decreased PK-C activity in crude and purified extracts of the same cells. The magnitude of reduction of the secretory response was greater than that of enzyme activity for both PDBu and LHRH-A pretreatment (68.9% reduction of testosterone secretion vs 54.7% reduction of PK-C activity in PDBu-pretreated cells and 78.6% reduction of testosterone production vs 36.6% reduction of PK-C activity in LHRH-A-pretreated cells). The effect of phorbol ester pretreatment on PDBu- or LHRH-stimulated testosterone secretion and PK-C activity was specific (no measurable effect with 4 alpha-PDBu, an inactive phorbol ester). While PDBu and LHRH-A pretreatment reduced Leydig cell responsiveness to PDBu or LHRH, the secretion of testosterone in response to the Ca2+ -mobilizing secretagogue A23187 was similar in PDBu- and LHRH-A-pretreated and in control (non-pretreated) cells. We conclude that down-modulation of protein kinase C by prolonged exposure of Leydig cells to phorbol esters or LHRH-A results in decreased PK-C activity and testosterone secretion. These results provide the first evidence that pretreatment with LHRH-A, which does not enter the cell, can affect the steroidogenesis and PK-C activity responses to PDBu (the intracellular ligand of PK-C).
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PMID:Pretreatment with phorbol ester and an LHRH agonist reduces testosterone production and protein kinase C activity in rat Leydig cells challenged with PDBu and LHRH. 919 61

Previous reports have demonstrated that corticotropin-releasing hormone (CRH) treatment of primary cultures of mouse Leydig cells and MA-10 mouse Leydig tumor cells results in a dose-dependent stimulation of steroidogenesis, probably by acting through the cAMP/protein kinase A second messenger pathway. Based on this observation, the mechanism of CRH-stimulated steroidogenesis is now further investigated and compared to trophic hormone stimulation. Both cell types were treated with human chorionic gonadotropin (hCG) or CRH in the absence and presence of the following agents: the translation inhibitor cycloheximide, the transcription inhibitor actinomycin D, the protonophore carbonyl cyanide m-chlorophenylhydrozone (mCCCP), which disrupts the mitochondrial electrochemical gradient or the phorbol ester, phorbol-12-myristate 13-acetate (PMA), a stimulator of protein kinase C. Cortico-releasing hormone-stimulated steroidogenesis was completely blocked by cycloheximide in both cell types, indicating that CRH-stimulated steroidogenesis in mouse Leydig cells requires ongoing protein synthesis. Actinomycin D had profound inhibitory effects on CRH-stimulated steroidogenesis in MA-10 cells, and this inhibition was greater than that seen in mouse primary Leydig cells. mCCCP severely inhibited CRH-stimulated steroid production in both cell types, indicating that an electrochemical gradient across the inner mitochondrial membrane is required for CRH-stimulated steroidogenesis. In addition, PMA inhibited hCG- and CRH-stimulated steroidogenesis in MA-10 cells and CRH-stimulated steroidogenesis in primary Leydig cells, suggesting that activation of the protein kinase C pathway can influence protein kinase A stimulated steroidogenesis. Results of these studies suggest that the mouse Leydig cell steroidogenic response to CRH shares many similarities to that of the LH response.
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PMID:The cellular mechanisms of corticotropin-releasing hormone (CRH)-stimulated steroidogenesis in mouse Leydig cells are similar to those for LH. 934 51

The immunostimulant tumor necrosis factor-alpha (TNF alpha), produced by monocytes/macrophages in response to inflammatory disorders, regulates gene expression in part through induction of mitogen-activated protein kinases (MAPKs), including the stress-activated protein kinase (SAPK) (c-Jun N-terminal kinase [JNK]) and the extracellular signal-regulated kinases (ERKs). In testicular Leydig cells, the induction of steroidogenesis by cAMP is inhibited by TNF alpha. To examine the potential mechanisms governing the mutual inhibition between cAMP and TNF alpha in Leydig cells, the intracellular signaling pathways that contribute to AP-1-dependent gene expression were examined in the mouse MA-10 Leydig cell line. TNF alpha induced SAPK activity sixfold at 15 min, and the PKC inhibitor calphostin C reduced the induction of SAPK by 30%. cAMP induced SAPK activity twofold but reduced TNF alpha-induced SAPK activity. ERK activity was inhibited by both cAMP and TNFa. TNFa increased c-Jun protein, but only weakly induced FOS proteins (c-Fos, FosB, Fra-1, and Fra-2) whereas cAMP increased the abundance of several FOS proteins (c-Fos, FosB, Fra-1, and Fra-2), with little effect on c-Jun levels. AP-1 binding activity, assessed using electrophoretic mobility shift assays, was increased twofold by TNF alpha and fivefold by cAMP. Cyclic AMP alone induced AP-1-responsive reporter (p3TPLUX) activity threefold after 2 h with peak effect of 4-fold at 4 hr. AP-1 reporter was not induced by TNF alpha alone but in the presence of cAMP, TNF alpha induced AP-1 reporter activity 12-fold. In conclusion, TNF alpha and cAMP induce distinct components that separately contribute to the modulation of AP-1 activity in MA-10 cells.
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PMID:The effect of tumor necrosis factor-alpha and cAMP on induction of AP-1 activity in MA-10 tumor Leydig cells. 936 89

Long-term (78 weeks) administration of lacidipine, a dihydropyridine calcium antagonist, increased the incidence of Leydig cell tumors (LCTs) in Sprague-Dawley rats. Lacidipine also increased and decreased the plasma luteinizing hormone (LH) and testosterone levels, respectively. Leydig cells from lacidipine-treated rats showed increases in luteinizing hormone-releasing hormone (LHRH) receptor expression, protein kinase C (PKC) activity, expression of proto-oncogenes, and 5-bromodeoxyridine uptake; whereas their calcium level, LH receptors, and testosterone content decreased. These data suggest that LHRH receptors play an important role in the development of rat LCTs induced by lacidipine, which activates a cascade of cell cycle-regulatory genes via PKC. When isolated Leydig cells were cultured with lacidipine or nicardipine, these changes in rat Leydig cells were not demonstrable in mice and monkeys, species having many fewer testicular LHRH receptors than rats. Thus, lacidipine may pharmacologically induce LCTs in rats but not in mice, with the difference depending on the presence or absence of testicular LHRH receptors. The induction of LCTs by lacidipine in rats is unlikely to occur in humans, since their Leydig cells lack LHRH receptors.
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PMID:Induction of Leydig cell tumors by lacidipine via up-regulation of the LHRH receptor on Leydig cells in rats. 951 20

The mechanism of arginine vasopressin (AVP) action in Leydig cells was investigated, and compared to the effects of phorbol-13-myristate-12-acetate (PMA) and interleukin-1 beta (IL-1 beta). Previous reports suggested that AVP inhibits Leydig cell testosterone production at the level of 17 alpha-hydroxylase/C17-lyase activity. The present study confirms and extends these observations, and contrasts the effects of AVP to IL-1. In all experiments, macrophage-depleted Leydig cells were isolated from mice and maintained in primary culture for 5 d prior to initiation of treatments. Leydig cells were treated with 8-Br-cAMP plus increasing concentrations of AVP or IL-1 beta. AVP caused a significant and dose-dependent inhibition of cAMP-stimulated testosterone production and P450c17 mRNA expression. IL-1 beta completely inhibited cAMP-stimulated testosterone production and P450c17 mRNA expression. PMA is a known activator of protein kinase C (PKC) and has been reported to inhibit Leydig cell steroidogenesis. Leydig cells express type V1 vasopressin receptors, which are coupled to PKC activation. The mechanism of IL-1 action in Leydig cells is not understood, but activation of the PKC pathway has been suggested for IL-1 action in other systems. Therefore, the effects of PMA on cAMP-stimulated steroidogenesis were compared to AVP and IL-1. Similar to the effects of AVP, PMA inhibited cAMP-stimulated testosterone production and P450c17 mRNA expression. To assess the possible involvement of PKC in AVP and IL-1 action in Leydig cells, the PKC inhibitor Calphostin C was tested. cAMP-stimulated testosterone production and P450c17 mRNA expression were significantly inhibited by 10 nM AVP (p < 0.05), and this inhibition was reversed by treatment with Calphostin C. Analogous experiments were performed to assess the role of PKC in IL-1 action. In contrast to the results for AVP, Calphostin C did not reverse the inhibitory effects of IL-1 on cAMP-stimulated P450c17 mRNA expression. To assess further PKC activation, myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation was analyzed. Only AVP and PMA, but not IL-1 beta, caused an increase in MARCKS phosphorylation. These results confirm that AVP and PMA activate PKC and indicate that IL-1 likely does not activate PKC in Leydig cells. The implications of AVP-mediated inhibition of steroidogenesis and potential role of MARCKS phosphorylation are discussed.
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PMID:Arginine vasopressin inhibition of cytochrome P450c17 and testosterone production in mouse Leydig cells. 966 41


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