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)

This report describes the fractional separation of microvessels from human brain for establishment of segmentally derived endothelial cell (EC) cultures. The investigation comprised evaluation of media constituents and purity of the cell culture and focused on functional biochemical characterization of endothelium derived from large microvessels (EC) Cells contained endothelial marker factor VIII (von Willebrand antigen), secreted endothelin-1 (ET-1) and prostaglandins, and took up 86Rb+ as a measure of K+. Exogenous ET-1 stimulated phosphatidylinositol hydrolysis and K+ uptake; BQ-123 (selective ETA receptor antagonist) but not IRL-1038 or BQ-788 (selective ETB receptor antagonists) inhibited both. Ouabain (inhibitor of Na(+)-K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (74-80 and 20-40%, respectively) the ET-1-stimulated K+ uptake. Staurosporine [protein kinase C (PKC) inhibitor] selectively reduced Na(+)-K(+)-Cl- cotransport, whereas verapamil but not nifedipine (L-type voltage-dependent Ca2+ channel blockers) decreased Na(+)-K(+)-ATPase activity induced by ET-1. Phorbol 12-myristate 13-acetate (PMA; activator of PKC) stimulated K+ uptake, which was only decreased with bumetanide. N-ethylisopropylamiloride (inhibitor of Na+/H+ exchange) reduced the ET-1-stimulated but not the PMA-induced K+ uptake. Results indicate that phosphatidylinositol hydrolysis and ion transport systems in large microvascular EC are stimulated by ET-1 through activation of ETA receptors. The findings also suggest that the ET-1-stimulated Na(+)-K(+)-ATPase activity, in contrast to Na(+)-K(+)-Cl- cotransport, is not mediated by PKC. In addition, the data suggest a linkage between Na(+)-K(+)-ATPase activity and Na+/H+ exchange.
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PMID:Functional properties of cultured endothelial cells derived from large microvessels of human brain. 903 29

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 microM-3 mM) produced concentration-dependent, well-sustained contraction. Its -logEC50 was 3.74 +/- 0.05 (mean +/- s.e.mean) and its maximal effect was equivalent to 97.5 +/- 4.2% of the response to acetylcholine (ACh, 1 mM). 2. Vanadate (200 microM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 microM), zileuton (10 microM), a mixture of atropine, mepyramine and phentolamine (each at 1 microM), or by mast cell degranulation with compound 48/80. 3. Vanadate (200 microM)-induced contraction was unaltered by tissue exposure to verapamil or nifedipine (each 1 microM) or to a Ca2+-free, EGTA (0.1 mM)-containing physiological salt solution (PSS). However, tissue incubation with ryanodine (10 microM) in Ca2+-free, EGTA (0.1 mM)-containing PSS reduced vanadate-induced contraction. A series of vanadate challenges was made in tissues exposed to Ca2+-free EGTA (0.1 mM)-containing PSS with the object of depleting intracellular Ca2+ stores. In such tissues cyclopiazonic acid (CPA; 10 microM) prevented Ca2+-induced recovery of vanadate-induced contraction. 4. Tissue incubation in K+-rich (80 mM) PSS, K+-free PSS, or PSS containing ouabain (10 microM) did not alter vanadate (200 microM)-induced contraction. Ouabain (10 microM) abolished the K+-induced relaxation of human bronchus bathed in K+-free PSS. This action was not shared by vanadate (200 microM). The tissue content of Na+ was increased and the tissue content of K+ was decreased by ouabain (10 microM). In contrast, vanadate (200 microM) did not alter the tissue content of these ions. Tissue incubation in a Na+-deficient (25 mM) PSS or in PSS containing amiloride (0.1 mM) markedly inhibited the spasmogenic effect of vanadate (200 microM). 5. Vanadate (200 microM)-induced contractions were markedly reduced by tissue treatment with each of the protein kinase C (PKC) inhibitors H-7 (10 microM), staurosporine (1 microM) and calphostin C (1 microM). Genistein (100 microM), an inhibitor of protein tyrosine kinase, also reduced the response to vanadate. 6 Vanadate (0.1-3 mM) and ACh (1 microM- 3 mM) each increased inositol phosphate accumulation in bronchus. Such responses were unaffected by a Ca2+-free medium either alone or in combination with ryanodine (10 microM). 7. In human cultured tracheal smooth muscle cells, histamine (100 microM) and vanadate (200 microM) each produced a transient increase in intracellular Ca2+ concentration ([Ca2+]i). 8. Intracellular microelectrode recording showed that the contractile effect of vanadate (200 microM) in human bronchus was associated with cellular depolarization. 9. It is concluded that vanadate acts directly on human bronchial smooth muscle, promoting the release of Ca2+ from an intracellular store. The Ca2+ release mechanism involves both the production of inositol phosphate second messengers and inhibition of Ca-ATPase. The activation of PKC plays an important role in mediating vanadate-induced contraction at values of [Ca2+]i that are close to basal.
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PMID:The spasmogenic effects of vanadate in human isolated bronchus. 925 12

We showed before that partial inhibition of Na/K-ATPase by non-toxic concentrations of ouabain caused hypertrophic growth of neonatal rat cardiac myocytes, and induced several early- and late-response genes that are markers of cardiac hypertrophy. The aim of this study was to determine if the genes of the alpha-subunit isoforms of Na/K-ATPase were among those regulated by ouabain; and if so, to begin the characterization of the pathways regulating these genes. When neonatal myocytes, expressing alpha1- and alpha3-isoform messages, were exposed to 5-100 micro M ouabain, alpha1 mRNA was not affected, but alpha3 mRNA was decreased in a dose- and time-dependent manner. Ouabain-induced down-regulation of alpha3 mRNA was accompanied by a decrease in alpha3-protein content in these myocytes. There was a significant correlation between ouabain effects on alpha3-repression and skeletal alpha-actin induction; also, ouabain's transcriptional effects on both genes were antagonised by retinoic acid. These findings suggested the association of alpha3 repression with ouabain-induced hypertrophy. Phenylephrine and a phorbol ester, two hypertrophic stimuli that do not inhibit Na/K-ATPase, also down-regulated alpha3 mRNA without affecting alpha1 mRNA, suggesting that alpha3-repression is a common feature of the hypertrophic phenotype in these myocytes. Ouabain-induced repression of alpha3 required the influx of extracellular Ca2+, and was antagonized by inhibitors of protein kinase C, Ca2+-calmodulin kinase, and mitogen-activated protein kinase but not by inhibition of protein kinase A. These data, and prior findings on the mechanisms of hypertrophic effects of phenylephrine and phorbol esters, suggest that transcriptional repression of alpha3 by ouabain and other hypertrophic stimuli involves a common step regulated by a mitogen-activated protein kinase.
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PMID:Differential regulation of Na/K-ATPase alpha-subunit isoform gene expressions in cardiac myocytes by ouabain and other hypertrophic stimuli. 940 89

1. This study was designed to investigate the mechanism(s) of the negative inotropic effects of alpha1-adrenoceptor agonists observed in rat isolated left atria after exposure to free radicals. 2. Ouabain and calphostin C were used in contraction experiments to block the sodium pump and protein kinase C. Methoxamine-induced phospholipase C and Na+/K+ ATPase activities were measured. 3. Methoxamine (300 microM) increased contractile force by 1.6 +/- 0.2 mN in control atria but decreased contractile force in electrolysis-treated atria by 2.0 +/- 0.1 mN (P < 0.05), as determined 10 min after methoxamine addition. In contrast, the positive inotropic effects of endothelin-1 (30 nM) and isoprenaline (10 microM) were reduced from 2.6 +/- 0.3 to 1.3 +/- 0.1 mN and from 2.6 +/- 0.3 to 1.7 +/- 0.2 mN, respectively, by electrolysis treatment (P < 0.05), but not converted into a negative inotropic action. 4. In an inositol phosphate assay we observed that the stimulation of phospholipase C by methoxamine was attenuated by electrolysis when the (electrolyzed) medium from the organ bath was used, but the phospholipase C responses were restored by the use of fresh medium. However, fresh medium did not counteract the negative inotropic effect of methoxamine. Accordingly, the negative inotropic effect of methoxamine is not directly related to the impaired phospholipase C responses seen in atria subjected to electrolysis. 5. Ouabain (10 microM) and the protein kinase C inhibitor calphostin C (50 nM), completely prevented the negative inotropic effect of 300 microM methoxamine in electrolysis-treated atria. 6. Measurement of the Na+/K+ ATPase activity, revealed that in control atria, alpha1-adrenoceptor stimulation with 300 microM methoxamine, decreased the Na+/K+ ATPase activity by 14.4 +/- 7.7%. In contrast, methoxamine increased the Na+/K+ ATPase activity by 48.8 +/- 8.9% (P < 0.05) in electrolysis-treated atria. Interestingly, this increase in Na+/K+ ATPase activity was completely counteracted by calphostin C (1.4 +/- 0.1% over basal). 7. These results indicate that the negative inotropic effects of alpha1-adrenoceptor agonists, observed in rat isolated left atria exposed to free radicals, are likely to be caused by protein kinase C-mediated phosphorylation and subsequent activation of the Na+/K+ ATPase.
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PMID:Possible mechanism of the negative inotropic effect of alpha1-adrenoceptor agonists in rat isolated left atria after exposure to free radicals. 953 25

The present experiments investigated the release of [3H]acetylcholine ([3H]ACh) from the guinea pig myenteric plexus treated with 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a drug that impairs ACh accumulation by synaptic vesicles. Ouabain, an Na+-K+ ATPase inhibitor, released [3H]ACh synthesised in the presence of (-)-vesamicol, while electrical field stimulation or KCl depolarisation were not effective to release the transmitter in this condition. The effect of ouabain was Ca2+-dependent and in the presence of (-)-vesamicol it was blocked by calphostin C, an inhibitor of protein kinase C (PKC). In addition, stimulation of kinase C activity by a phorbol ester, but not by its inactive isomer, prevented (-)-vesamicol from interfering with the release of [3H]ACh in electrically-stimulated myenteric plexus, similar to the effect of ouabain. We conclude that release of [3H]ACh induced by ouabain in the presence of (-)-vesamicol depends on PKC activation.
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PMID:Role of protein kinase C in the release of [3H]acetylcholine from myenteric plexus treated with vesamicol. 957 99

We showed before that in neonatal rat cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophic growth and transcriptional regulations of genes that are markers of cardiac hypertrophy. In view of the suggested roles of Ras and p42/44 mitogen-activated protein kinases (MAPKs) as key mediators of cardiac hypertrophy, the aim of this work was to explore their roles in ouabain-initiated signal pathways regulating four growth-related genes of these myocytes, i.e. those for c-Fos, skeletal alpha-actin, atrial natriuretic factor, and the alpha3-subunit of Na+/K+-ATPase. Ouabain caused rapid activations of Ras and p42/44 MAPKs; the latter was sustained longer than 90 min. Using high efficiency adenoviral-mediated expression of a dominant-negative Ras mutant, and a specific inhibitor of MAPK kinase (MEK), activation of Ras-Raf-MEK-p42/44 MAPK cascade by ouabain was shown. The effects of the mutant Ras, an inhibitor of Ras farnesylation, and the MEK inhibitor on ouabain-induced changes in mRNAs of the four genes indicated that (a) skeletal alpha-actin induction was dependent on Ras but not on p42/44 MAPKs, (b) alpha3 repression was dependent on the Ras-p42/44 MAPK cascade, and (c) induction of c-fos or atrial natriuretic factor gene occurred partly through the Ras-p42/44 MAPK cascade, and partly through pathways independent of Ras and p42/44 MAPKs. All ouabain effects required extracellular Ca2+, and were attenuated by a Ca2+/calmodulin antagonist or a protein kinase C inhibitor. The findings show that (a) signal pathways linked to sarcolemmal Na+/K+-ATPase share early segments involving Ca2+ and protein kinase C, but diverge into multiple branches only some of which involve Ras, or p42/44 MAPKs, or both; and (b) there are significant differences between this network and the related gene regulatory pathways activated by other hypertrophic stimuli, including those whose responses involve increases in intracellular free Ca2+ through different mechanisms.
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PMID:Multiple signal transduction pathways link Na+/K+-ATPase to growth-related genes in cardiac myocytes. The roles of Ras and mitogen-activated protein kinases. 961 40

Ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured in digitonin-permeabilized monolayers of cultured cells derived from rabbit non-pigmented ciliary epithelium. Diminished Na,K-ATPase activity was observed in cells that had been pre-treated 10 min with the protein kinase C activator, PDBu, as well as in cells that had been cooled to 4 degrees C for 4 h then rewarmed to 37 degrees C for 30 min (cool-rewarm manoeuvre). In the intact cells, ouabain binding was not decreased either by PDBu treatment or the cool-rewarm manoeuvre. However, both PDBu and the cool-rewarm manoeuvre increased the rate of ouabain-sensitive potassium (86Rb) uptake measured in intact cells. Cell ATP content was diminished in PDBu-treated cells and cells subjected to the cool-rewarm manoeuvre. We suggest that an episode of ATP depletion might initiate a mechanism which causes lasting, partial inhibition of Na,K-ATPase activity. In keeping with this suggestion, diminished Na,K-ATPase activity was observed in cells that had been pre-treated 20 min with the metabolic inhibitors CCCP or rotenone and in cells pre-treated 2.5 h in dextrose-free medium. This study illustrates that Na,K-ATPase activity measured in the permeabilized cell is a complex parameter which is not necessarily a reliable indicator of sodium pump responses in the intact cell.
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PMID:Partial inhibition of Na,K-ATPase activity in cultured rabbit non-pigmented ciliary epithelium following an episode of cytoplasmic ATP depletion. 977 20

The Na+/K+-ATPase is a target protein for protein kinase C (PKC). The PKC-mediated phosphorylation of the rat alpha1 subunit at Ser-23 results in the inhibition of its transport function. To understand the molecular basis of the inhibition by PKC, the Ser-23 in the rat alpha1 subunit has been replaced by negatively (Asp, Glu) or positively (Lys) charged, or uncharged (Gln, Ala) residues, and the mutants were expressed in Xenopus oocytes. Ouabain-specific 86Rb uptake and pump-generated current as well as sensitivity to ouabain and to external K+ have been investigated. When Ser-23 was replaced by the negatively charged residues, transport function was inhibited, and simultaneously synthesis of the alpha subunits was enhanced. In addition, if Ser-23 was substituted by Glu, the K(I) value for inhibition of transport by ouabain was drastically increased from 46.5 microM to 1.05 mM. The data suggest that insertion of a negative charge within the N-terminus of alpha subunit of the Na+/K+-ATPase due to phosphorylation of Ser-23 plays an important role in the PKC-mediated inhibition of transport function.
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PMID:Mutations of Ser-23 of the alpha1 subunit of the rat Na+/K+-ATPase to negatively charged amino acid residues mimic the functional effect of PKC-mediated phosphorylation. 1042 61

Partial inhibition of Na/K-ATPase by ouabain causes hypertrophic growth and regulates several early and late response genes, including that of Na/K-ATPase alpha3 subunit, in cultured neonatal rat cardiac myocytes. The aim of this work was to determine whether ouabain and other hypertrophic stimuli affect Na/K-ATPase beta1 subunit gene expression. When myocytes were exposed to non-toxic concentrations of ouabain, ouabain increased beta1 subunit mRNA in a dose- and time-dependent manner. Like the alpha3 gene, beta1 mRNA was also regulated by several other well-known hypertrophic stimuli including phenylephrine, a phorbol ester, endothelin-1, and insulin-like growth factor, suggesting involvement of growth signals in regulation of beta1 expression. Ouabain failed to increase beta1 subunit mRNA in the presence of actinomycin D. Using a luciferase reporter gene that is directed by the 5'-flanking region of the beta1 subunit gene, transient transfection assay showed that ouabain augmented the expression of luciferase. These data support the proposition that ouabain regulates the beta1 subunit through a transcriptional mechanism. The effect of ouabain on beta1 subunit induction, like that on alpha3 repression, was dependent on extracellular Ca2+ and on calmodulin. Inhibitions of PKC, Ras, and MEK, however, had different quantitive effects on ouabain-induced regulations of beta1 and alpha3 subunits. The findings show that partial inhibition of Na/K-ATPase activates multiple signaling pathways that regulate growth-related genes, including those of two subunit isoforms of Na/K-ATPase, in a gene-specific manner.
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PMID:Regulation of Na/K-ATPase beta1-subunit gene expression by ouabain and other hypertrophic stimuli in neonatal rat cardiac myocytes. 1120 57

Riboflavin (vitamin B2) is essential for fetal development and must be acquired from maternal sources. The uptake mechanism of riboflavin and the major regulatory pathways involved were characterized in a model for the placental barrier, the human choriocarcinoma cell line, BeWo. Uptake of [3H]riboflavin was saturable (Kt = 1.32 +/- 0.68 nM, Jmax = 266.63 +/- 26.89 fmol/mg of protein/20 min), and was significantly reduced at low temperature and in the presence of metabolic inhibitors (azide, 2-deoxyglucose) or structural analogs. Ouabain, amiloride, sodium-free buffers, and medium with pH values ranging from 3 to 8 did not affect uptake of [3H]riboflavin. In contrast, substitution of chloride with other monovalent anions significantly inhibited its uptake. Induced differentiation of BeWo cells into syncytiotrophoblasts by forskolin or 8-bromo-cyclic adenosine monophosphate introduced a time-dependent decrease of riboflavin uptake. Preincubation with activators of cyclic nucleotide-dependent protein kinase pathways (3-isobutyl-1-methylxanthine and p-chlorophenylthio-cyclic guanosine monophosphate) and calmodulin antagonists (calmidazolium and W-13) resulted in a concentration-dependent reduction of [3H]riboflavin uptake, whereas specific modulators of protein kinase C pathways did not have significant effects. 3-Isobutyl-1-methylxanthine exerted its regulatory effect on riboflavin uptake via decreasing both Kt and Jmax of the riboflavin uptake process (Kt = 6.32 +/- 1.29 nM, Jmax = 135.57 +/- 10.42 fmol/mg of protein/20 min). In summary, we report the presence of high- affinity riboflavin transporter(s) on the microvillous membrane of BeWo cells that appears to be modulated by cellular cyclic nucleotide levels and calmodulin.
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PMID:Riboflavin uptake in human trophoblast-derived BeWo cell monolayers: cellular translocation and regulatory mechanisms. 1140 51


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