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)

Using the fluorescent probe, BCECF, the changes in intracellular pH (pHi) in rat peritoneal mast cells were studied. alpha-Thrombin (0.1 nM) induced biphasic changes in pHi which consisted in a temporary decrease in pH with its subsequent steady increase due to the Na/H exchange activation which was inhibited by EIPA and controlled by extracellular Na+. The biphasic changes in pHi induced by DIP-alpha-thrombin (0.1 pM-1 nM), a catalytically inactive form with an intact recognition site, were similar to those of alpha-thrombin, whereas beta/gamma-thrombin (10-1000 pM), a catalytically active form characterized by structural disturbances in the recognition site, was able to induce only the initial phase of acidification. The thrombin recognition site modulators, alpha 1-thymosin and heparin, blocked the ability of the enzyme to induce the alkalinization of pHi. Nigericin stimulated the Na/H-exchange in mast cells. The rate of the Na/H-exchange activation determined with nigericin, decreased with an increase in the alpha-thrombin dose from 0.1 pM up to 10 nM. Activation of protein kinase C (PKC) in mast cells by PMA used at 1 nM and 10 nM led to the alkalinization of the cytoplasm as a result of the Na/H-exchange activation blocked by EIPA. The PKC inhibitor, H-7, suppressed the pHi increase induced by both PMA and alpha-thrombin. The alpha-thrombin-induced acidification of the cytoplasm was completely blocked by SITS in Ca(2+)-free media, whereas in media with Ca2+ SITS inhibited the pHi decline. Acidification of the cytoplasm by thrombin seems to be due to both Ca2+ influx and activation of Cl- fluxes. It is concluded that the observed activation of the Na/H-exchange by thrombin is induced by a cascade of intracellular reactions involving PKC.
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PMID:[Activation of Na/H exchange by thrombin in peritoneal mast cells]. 142 May 91

1. Glomerular epithelial cells (GEC) were cultured from human kidneys and immunologically characterized. 2. The effect of extracellular nucleotides on the cytosolic free calcium activity [Ca2+]i was investigated with the fura-2 microfluorescence method. Extracellular UTP, UDP, UMP, ATP, adenosine 5'-O-(3-thio)-trisphosphate (ATP-gamma-S), inosine-triphosphate (ITP), guanyltriphosphate (GTP), 2-methylthio-ATP, AMP, alpha,beta-methylene-ATP and adenosine led to a rapid, transient, concentration-dependent increase of [Ca2+]i, followed by a plateau above the baseline level. 3. In a calcium-free extracellular solution, the rapid increase of [Ca2+]i was still present, whereas the plateau level was abolished. 4. ATP and UTP (ED50 both: 10(-5) M) stimulated inositol trisphosphate (InsP3) formation in GEC. 5. The order of potency for the purine nucleotides in stimulating InsP3 formation was ATP = ATP-gamma-S greater than ADP greater than 2-methylthio-ATP greater than AMP = a,beta methylene-ATP = adenosine. 6. The increase of InsP3 induced by ATP (10(-5) M) could be inhibited by the P2 receptor blocker suramin (greater than 10(-4) M). Reactive blue 2 exhibited a weak stimulating effect on the InsP3 formation and only a weak inhibitory effect at a concentration of 10(-3) M was observed. 7. Protein kinase C activation by preincubation of GEC with phorbol 12-myristate 13-acetate (PMA, 100 ng ml-1, 15 min) abolished the effect of ATP (10(-5) M) on InsP3 formation. Downregulation of protein kinase C by long term incubation (18 h) with PMA had no significant effect on the phosphoinositol turnover induced by ATP.8. The results indicate that an increase of [Ca2+]i and inositol phosphate breakdown can be mediated via activation of a P2 receptor in human GEC.
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PMID:Effect of nucleotides on the cytosolic free calcium activity and inositol phosphate formation in human glomerular epithelial cells. 142 72

Endotoxin induces insulin hypersecretion in vivo, which results in hyperinsulinemia and glucose dyshomeostasis. Polymyxin-B (PMX-B), an inhibitor of protein kinase C (PKC), has been shown to ameliorate the consequences of endotoxin-induced hyperinsulinemia in vivo. To explore the mechanism for this effect in vitro, this study determined whether PMX-B could alter endotoxin-induced insulin hypersecretion in isolated pancreatic islets of Langerhans. Pancreases were obtained from fasted, male, Sprague-Dawley rats treated with either saline (control) or endotoxin (S. enteritidis B, 16.7 mg/kg, i.v.). Three hours after the experimental treatment, islets were isolated by collagenase digestion and then incubated for 1 hr in Krebs Ringer bicarbonate buffer containing 0.5% bovine albumin, 10 mM HEPES, 300 mg/dl D-glucose, phorbol 12-myristate 13-acetate (PMA, 1 microM when present), and PMX-B (1 or 10 mM when present). In the absence of PMA and PMX-B, "endotoxic" islets hypersecreted immunoreactive insulin (IRI) relative to control islets. PMA, the prototypical PKC activator, significantly increased IRI secretion from both control and "endotoxic" islets. The additional inclusion of PMX-B (1 mM or 10 mM) in the incubation media significantly reduced insulin secretion from both control and "endotoxic" islets and suppressed the insulin hypersecretion observed in "endotoxic" islets. Since insulin secretion occurs at least partially through mechanisms dependent on PKC activation, the ability of PMX-B to suppress insulin hypersecretion in "endotoxic" islets suggests that activation of PKC within pancreatic beta-cells may play a role in the excess insulin secretion and hyperinsulinemia associated with endotoxicosis.
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PMID:Polymyxin-B suppresses endotoxin-induced insulin hypersecretion in pancreatic islets. 142 25

Lysophosphatidylcholine (lysoPC) transferred from oxidatively modified low density lipoprotein (Ox-LDL) to the endothelial surface membrane has been shown to produce a selective unresponsiveness to cell surface receptor-regulated endothelium-dependent relaxation (EDR) in the rabbit aorta. To determine its mechanism we examined the effects of lysoPC on endothelial surface receptor-mediated transmembrane signals. Incubation for 1 minute with palmitoyl lysoPC (5-10 microM) decreased thrombin (Th, 2 units/ml)- or histamine (His, 0.1 mM)-stimulated inositol 1,4,5-trisphosphate (IP3) production in primary cultures of human umbilical vein endothelial cells (HUVECs). LysoPC also decreased Th- or His-induced intracellular calcium ([Ca2+]i, fura 2) elevation. Pretreatment with protein kinase C (PKC) inhibitors staurosporine (100 nM) or H-7 (50 microM) prevented the inhibitory actions of lysoPC, but HA-1004 had no effect. Incubation for 5 minutes with phorbol 12-myristate 13-acetate (PMA, 100 nM) produced the inhibitory actions on the Th- or His-induced intracellular signals, which closely mimic those exhibited by lysoPC. However, the inhibitory effect of lysoPC was lost in cells that were depleted of PKC by pretreatment for 24 hours with 100 nM PMA. Furthermore, incubation of the cells for 1 minute with lysoPC stimulated PKC activity in the membrane fraction. In organ chamber experiments with porcine coronary artery rings, pretreatment with staurosporine (20 nM) attenuated lysoPC-induced impairment of EDR in response to Th. These results indicate that lysoPC, which accumulates in Ox-LDL and atherosclerotic arterial walls, inhibits the early transmembrane signaling pathway in endothelial cells, and PKC activation could at least partially be involved in the negative regulation by lysoPC.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lysophosphatidylcholine inhibits surface receptor-mediated intracellular signals in endothelial cells by a pathway involving protein kinase C activation. 142 37

We have shown previously that angiotensin-II (A-II) controls proto-oncogene (c-fos, jun-B and c-jun) mRNA accumulation in bovine adrenal fasciculata cells (BAC). Since BAC contain both subtypes (AT-1 and AT-2) of the A-II receptor, we have investigated which subtype was involved in the effect of A-II on proto-oncogene mRNA by using a selective antagonist for AT-1 (DUP 753) and for AT-2 (CGP 42112A). DUP 753, but not CGP 42112A, inhibited the stimulatory effect of A-II on proto-oncogene mRNA, with ID50s of 4 x 10(-7) M, 7 x 10(-7) M and 2 x 10(-6) M for c-fos, jun-B and c-jun, respectively. Neither of the two antagonists by themselves had a direct effect on proto-oncogene mRNA. As the A-II AT-1 receptors are coupled to the phospholipase C system in BAC, we have investigated whether the A-II effects on the proto-oncogenes were mediated by protein kinase C (PKC) or by Ca2+ calmodulin. First, activation of PKC by the phorbol ester, PMA, increased the level of three proto-oncogene mRNAs, whereas calcium ionophore had no effect. Second, staurosporine, a specific inhibitor of PKC, reduced the stimulatory action of A-II on proto-oncogene mRNA by 80-90%, whereas trifluoroperazine, an inhibitor of calmodulin, had no significant effect. These results demonstrate that the effects of A-II on proto-oncogene mRNA are mediated by AT1 receptor subtypes, mainly through activation of the PKC pathway.
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PMID:Angiotensin-II-induced expression of proto-oncogene (c-fos, jun-B and c-jun) mRNA in bovine adrenocortical fasciculata cells (BAC) is mediated by AT-1 receptors. 142 67

Homotypic aggregation of B-lymphocytes, B-cell lines and class-II-positive T cells via HLA class II molecules was examined. Signaling via DR antigens induced rapid aggregation in a dose- and time-dependent manner, maximum and stable aggregation was induced within 20 minutes. On the contrary, rapid signaling via DP or DQ required prestimulation with either PMA or anti-sIg. Aggregation was temperature and energy dependent. [Ca2+] and [Mg2+] concentrations and an intact cytoskeleton were required while neither mRNA or protein synthesis were required. Furthermore, FACS analysis revealed that aggregation was not directly correlated with cell surface expression of HLA class II molecules. Our results demonstrate that aggregation was mediated through a protein tyrosine kinase (PTK)-dependent pathway that preceded activation of protein kinase C (PKC) and failure to generate either the PTK signal or the PKC signal prevented aggregation. The contribution of a tyrosine kinase was further demonstrated by the total inhibition of aggregation following treatment with an anti-CD45 mAb.
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PMID:HLA class-II-mediated homotypic aggregation: involvement of a protein tyrosine kinase and protein kinase C. 142 32

The binding of natural killer (NK) cells to either susceptible tumor cells or antibody-coated targets results in rapid activation of phospholipase C (PLC) in NK cells. PLC activation generates inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers, which, in turn, increase intracellular free calcium concentrations ([Ca2+]i) and protein kinase C (PKC) activity, respectively. These proximal signals initiate a cascade of as yet undefined biochemical events, leading eventually to the exocytosis of preformed cytotoxic granules. To investigate the signal transduction pathways involved in granule exocytosis, we utilized streptolysin-O-permeabilized human NK cells as our experimental model. Our initial studies indicated that the separate activation of either PKC (using the phorbol ester, PMA) or G protein-dependent pathways (using guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S)) stimulated granule exocytosis in a time-, concentration-, and Ca(2+)-dependent manner. PMA-stimulated exocytosis was inhibited by staurosporine or a PKC pseudosubstrate antagonist peptide, but was not affected by GDP. In contrast, GTP gamma S-stimulated exocytosis was effectively inhibited by GDP, but not by staurosporine or the PKC pseudosubstrate antagonist. These observations suggest that NK cell exocytosis can be stimulated by at least two separate pathways; one involving PKC and the other involving a G protein. However, co-stimulation with PMA and GTP gamma S synergistically enhanced exocytosis, suggesting that even though the two exocytotic pathways were biochemically distinct, cross-talk between the two pathways may potently influence the exocytotic process. These results define a regulatory role for PKC- and G protein-dependent pathways during granule exocytosis from NK cells.
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PMID:Interaction between protein kinase C-dependent and G protein-dependent pathways in the regulation of natural killer cell granule exocytosis. 142 33

Apoptosis is induced in immature thymocytes and T cell hybridomas upon stimulation via the TCR/CD3 complex. This phenomenon appears to be related to negative selection of T cell clones in the thymus. In T cell hybridomas, it has been shown that glucocorticoids inhibit TCR/CD3-mediated apoptosis, whereas glucocorticoids alone induce apoptosis. All-trans-retinoic acid (RA) at 0.1 to 10 microM also inhibited TCR/CD3-mediated apoptosis assessed by DNA fragmentation and cytolysis, but RA alone hardly induced apoptosis. RA enhanced the effects of glucocorticoids to induce apoptosis and to inhibit TCR/CD3-mediated apoptosis. TCR/CD3-mediated stimulation can be mimicked by the combination of ionomycin, a calcium ionophore, and PMA, an activator of protein kinase C, and the combination-induced DNA fragmentation was also inhibited by RA. RA, however, failed to inhibit the combination-induced increase in intracellular Ca2+ concentration or the combination-induced translocation of protein kinase C from the cytosolic fraction to the particulate fraction. Time course studies of RA addition into the culture indicated that a 3- to 6-h delay in the addition of RA did not reduce its inhibitory effect on anti-CD3-induced DNA fragmentation. These results suggest that RA interferes with the apoptotic process at some point after its initiation stage. It has been suggested that negative selection involves not only TCR/CD3-mediated signals but also LFA-1-mediated signals. RA at 0.01 to 1 microM significantly inhibited the induction of thymocyte apoptosis by co-immobilized mAb to CD3 and LFA-1 molecules. RA by itself hardly induced apoptosis, but enhanced glucocorticoid-induced apoptosis. The results suggest that thymic selection might be influenced by RA at near-physiologic concentrations. The receptors of glucocorticoids and RA belong to the erbA oncogene-related steroid hormone receptor superfamily. Thyroid hormones and 1 alpha,25-dihydroxy vitamin D3, whose receptors also belong to the superfamily, failed to modulate apoptosis in both T cell hybridomas and thymocytes.
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PMID:Retinoic acids inhibit activation-induced apoptosis in T cell hybridomas and thymocytes. 143 Nov 7

The proliferative responses of Peyer's patch (PP) T cells from aged BALB/c mice to concanavalin A (Con A) are considerably reduced, as compared to those of the young (P < 0.001). This reduced reactivity of aged T cells could be partly, but not entirely, corrected by interleukin 2 (IL-2) (P < 0.001). PP T cells from aged mice responded synergistically to a protein kinase C (PKC) activator, phorbol myristate acetate (PHA), plus a calcium ionophore, ionomycin, at much lower concentrations than to Con A (P < 0.001); however, the maximal proliferative response still remained nearly at 8/10th of the young (P < 0.01) and higher levels of PMA (but not of ionomycin) were required (P < 0.001). Addition of IL-2 restored the diminished response to the levels of the young T cells (P < 0.05), but that of Con A did not (P > 0.05). The proliferative responses of PP B cells to lipopolysaccharide (LPS) do not differ from those of the young (P > 0.05), but the spontaneous proliferation of aged (unstimulated) B cells is enhanced nearly twofold versus that of the young (P < 0.001). Like the PP T cells, PP B cells from aged mice also responded synergistically to PMA plus ionomycin but to a lesser degree than those of the young under the same stimulation (P < 0.01). Their maximal proliferation required higher levels of PMA, but not of ionomycin and was also diminished (P < 0.01), compared to that of the young. B cell stimulatory co-factors, IL-4 and IL-6, failed to affect the response of aged and young B cells to PMA plus ionomycin (P > 0.05), whereas LPS remediates the reduced response of aged B cells to PMA plus ionomycin. Thus, T and B cells from senescent PP demonstrate an impaired proliferative responsiveness via the Ca-dependent PKC pathway. A T cell mitogen and B cell stimulatory cytokines did not alter this activation pathway, once optimally stimulated. Whereas, T cell stimulatory cytokine IL-2 and B cell mitogen LPS could restore the age-associated decline of the corresponding lymphocyte subsets, T and B cells, in activation of the Ca-dependent pathway. The altered transmembrane signal transduction appears to be intrinsically defective in these aged PP T and B cells.
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PMID:Effects of phorbol myristate and ionomycin on in vitro growth of aged Peyer's patch T and B cells. 143 53

Experiments were performed to immunologically identify protein kinase C (PKC) in cultured IEC-6 cells. Polyclonal antibodies specific to PKC revealed an immunoreactive band of approximately 84 kDa in both cytosolic and solubilized particulate fractions. Treatment with phorbol 12-myristate 13-acetate (PMA; 10 nM x 60 min) increased the intensity of the 84-kDa band by 25% in the solubilized particulate fraction while decreasing it by 36% in the cytosolic fraction. Prolonged 24-h treatment with 300 nM PMA completely abolished the 84-kDa band in both fractions. Isoform-specific antisera demonstrated that alpha- and epsilon-isoforms of PKC were expressed in IEC-6 cells. Treatment of quiescent cultures with PMA induced a maximal 400% increase in ornithine decarboxylase (ODC) activity. Similarly, addition of exogenous phospholipase C (PLC) to quiescent cells stimulated ODC activity. Downregulation of PKC with 300 nM PMA x 24 h inhibited basal, serum, and PLC-stimulated ODC activity by 70%. Northern analysis revealed that PKC downregulation was correlated with a marked reduction in ODC mRNA levels, suggesting regulation of ODC enzyme at this level. Despite their ability to modulate ODC activity in quiescent cultures, neither PMA nor PLC induced [3H]thymidine incorporation at 24 h. Furthermore, downregulation of PKC did not attenuate thymidine incorporation. However, chronic PMA treatment caused the cells to contact-inhibit at a 30% lower cell density, 3.16 x 10(6) vs. 2.1 x 10(6) cells/35-mm plate, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein kinase C regulation of IEC-6 cell ornithine decarboxylase. 144 49


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