Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An ATP-regulated inwardly rectifying K(+) channel, whose activity is enhanced by PKA, is present in the plasma membrane of cultured human proximal tubule cells. In this study, we investigated the effects of PKG on this K(+) channel, using the patch-clamp technique. In cell-attached patches, bath application of a membrane-permeant cGMP analog, 8-bromoguanosine 3',5'-monophosphate (8-BrcGMP; 100 microM), stimulated channel activity, whereas application of a PKG-specific inhibitor, KT-5823 (1 microM), reduced the activity. Channel activation induced by 8-BrcGMP was observed even in the presence of a PKA-specific inhibitor, KT-5720 (500 nM), which was abolished by KT-5823. Direct effects of cGMP and PKG were examined with inside-out patches in the presence of 1 mM MgATP. Although cytoplasmic cGMP (100 microM) alone had little effect on channel activity, subsequent addition of PKG (500 U/ml) enhanced it. Furthermore, bath application of atrial natriuretic peptide (ANP; 20 nM) in cell-attached patches stimulated channel activity, which was blocked by KT-5823. In conclusion, cGMP/PKG-dependent processes participate in activating the ATP-regulated K(+) channel and producing the stimulatory effect of ANP on channel activity.
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PMID:Protein kinase G activates inwardly rectifying K(+) channel in cultured human proximal tubule cells. 1221 70

Mitogenic cell proliferation requires a rapid and transient H2O2 generation, which is blocked by catalase or PKA activators. Previously, we observed that anemic HIV(+) individuals expressed acidic pIs of catalase in RBC with significantly high activities [Mol Cell Biochem 165: 77-81, 1996]. These findings led us to hypothesize that cell signaling molecules regulate catalase to control cell mitogenesis. To test the hypothesis, we determined (i) whether RBC counts correlate with their catalase activities, (ii) whether protein kinases and phosphatases alter catalase activity in vitro, and (iii) whether protein kinase activators increase catalase activity to suppress proliferation of cultured cells. The results indicated that RBC counts inversely correlated with RBC catalase activities in both HIV(+) (r: -0.6769, r2: 0.4582, n: 69 male, p < 0.0001) and HIV(-) (r: -0.3827, r2: 0.1464, n: 177 male, p < 0.0001) populations. Catalytic PKA, PKC and Casein Kinase II, but none of PKG, Ca2+/calmodulin kinase II and p34cdc/cyclinB, rapidly elevated catalase activity in vitro by up to 2-fold. Whereas a major CAT subunit (60 kDa) showed immunoreactive phosphoserine and phosphothreonine, the kinases- and gamma-32P-ATP-dependent phosphorylation occurred with a minor component (110 kDa). Among PKC isozymes examined, PKCzeta was the most effective modulator followed by PKCgamma, and protein phosphatase 1gamma and 2A decreased the catalase activity. PKA and PKCzeta activators of forskolin and okadaic acid increased catalase activity and 110 kDa expression in NIH3T3 cells up to 2.4-fold and suppressed the cell growth, showing an inverse correlation of the indices (r: -0.9286, r2: 0.8622, n: 18, p < 0.0001). Taken together, these results suggest for the first time that catalase is under the regulation of cell signaling molecules and capable of modulating mitogenic cell proliferation.
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PMID:Regulation of catalase enzyme activity by cell signaling molecules. 1248 79

In isolated rat pancreatic beta-cells, the nitric oxide (NO) donor NOC-7 at 1 microM reduced the amplitude of the oscillations of cytosolic Ca(2+) concentration ([Ca(2+)](c)) induced by 11.1 mM glucose, and at 10 microM terminated them. In the presence of N(G)-nitro-l-arginine (l-NNA), however, NOC-7 at 0.5 and 1 microM increased the amplitude of the [Ca(2+)](c) oscillations, although the NO donor at 10 microM still suppressed them. Aqueous NO solution also had a dual effect on the [Ca(2+)](c) oscillations. The soluble guanylate cyclase inhibitor LY-83583 and the cGMP-dependent protein kinase inhibitor KT5823 inhibited the stimulatory effect of NO, and 8-bromo-cGMP increased the amplitude of the [Ca(2+)](c) oscillations. Patch-clamp analyses in the perforated configuration showed that 8-bromo-cGMP inhibited whole cell ATP-sensitive K(+) currents in the isolated rat pancreatic beta-cells, suggesting that the inhibition by cGMP of ATP-sensitive K(+) channels is, at least in part, responsible for the stimulatory effect of NO on the [Ca(2+)](c) oscillations. In the presence of l-NNA, the glucose-induced insulin secretion from isolated islets was facilitated by 0.5 microM NOC-7, whereas it was suppressed by 10 microM NOC-7. These results suggest that NO facilitates glucose-induced [Ca(2+)](c) oscillations of beta-cells and insulin secretion at low concentrations, which effects are mediated by cGMP, whereas NO inhibits them in a cGMP-independent manner at high concentrations.
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PMID:Dual effect of nitric oxide on cytosolic Ca2+ concentration and insulin secretion in rat pancreatic beta-cells. 1252 41

We have developed a novel method for quantitating protein phosphorylation by a variety of protein kinases. It can be used with purified kinases and their substrates in vitro or in combination with cell extracts. The method is based on the knowledge that protein kinase C (PKC) adds three phosphates to each molecule of its preferred substrate, myelin basic protein (MBP). A time course is performed in which a kinase is allowed to phosphorylate its preferred substrate or the protein under investigation in the presence of [gamma-32P]ATP. At the same time PKC is allowed to fully phosphorylate MBP. After resolving the products by SDS-PAGE, electrophoretic transfer, and determining the degree of incorporation of 32P by phosphorImager analysis, the data are converted to moles phosphate/mole protein by normalization with phosphorylated MBP. The method is both sensitive and relatively rapid and all the steps are commonly available in the biochemistry laboratory. We have used this method to confirm and extend information on the relationship of MEK1 and MAPK/Erk2 in rat lung fibroblasts exposed to V(2)O(5). A 4-h exposure to V(2)O(5) results in partial phosphorylation of MAPK/Erk2 such that 25% of the potential phosphorylation sites are occupied. We also demonstrate that despite multiple potential phosphorylation sites, recombinant human AP endonuclease is weakly phosphorylated in vitro (4% at best) by PKC, cGMP-dependent protein kinase, casein kinase II, and casein kinase I and not at all phosphorylated by MAPK. Furthermore we are unable to demonstrate phosphorylation in cell extracts from HeLa cells, mouse fibroblasts after oxidative damage with H(2)O(2) or alkylation damage with methylmethane sulfonate, or rat lung fibroblasts after oxidative damage with V(2)O(5).
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PMID:A quantitative method for measuring protein phosphorylation. 1257 52

Hemodynamic shear stress elicits a rise in endothelial [Ca2+]i, which may serve as a key second messenger to regulate many flow-associated physiological and biochemical processes. In the present study, we used Mn2+ quenching of fluorescent dye Fluo3 as an assay to investigate the Ca2+ influx of rat aortic endothelial cells in response to flow. We found that the Ca2+ signaling in response to flow could be greatly influenced by the status of intracellular Ca2+ stores. Depletion of intracellular Ca2+ stores by thapsigargin (4 micromol/L) or cyclopiazonic acid (10 micromol/L) drastically sensitized the Ca2+ influx in response to flow. Ca2+-mobilizing agonist bradykinin (100 nmol/L) or ATP (100 micromol/L) had similar sensitizing effect. The effect of bradykinin or ATP was blocked by Xestospongin C and U73122, suggesting that the sensitization was related to the IP3-mediated store depletion. On the other hand, the Mn2+ quenching in response to flow was greatly reduced by ochratoxin A (100 nmol/L), an agent that could increase the filling state of intracellular Ca2+ stores. In addition, we found that depletion-sensitized Ca2+ influx in response to flow was mediated by a PKG-inhibitable cation channel and that the influx was affected by membrane potential and K+ channel activity. In conclusion, the present study argues for a critical role of intracellular Ca2+ status in determining the Ca2+ signaling in response to flow and it provides a general mechanistic explanation for the stimulatory role of blood-borne agonists on flow-induced Ca2+ influx.
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PMID:Depletion of intracellular Ca2+ stores sensitizes the flow-induced Ca2+ influx in rat endothelial cells. 1259 40

We report the first mutational study of thymidine kinase 1 (TK1) performed in human solid tumors. We sequenced cDNAs representing the complete coding region of TK1 in human breast (n=22) and colorectal (n=26) cancer. Codon 106 near the ATP binding site constantly differed (ATG --> GTG; Met --> Val) from the one deposited by Bradshaw and Deininger in the Genbank database (Accession number NM_003258). Silent polymorphisms at codon 11 (CCC --> CCT; Pro --> Pro) and codon 75 (GCG --> GCA; Ala --> Ala) were frequently detected in tumors as well as in normal tissues. In breast cancer the two polymorphisms were observed in 63.6% of the samples analyzed. No significant association could be found between polymorphisms and TK activity. In colorectal cancer the incidence of the two changes was 73.1% and 69.2%, respectively. Interestingly, one colon cancer with high cytosolic TK activity displayed two missense mutations located in and near the putative phosphorylation site by tyrosine kinase (s) (TAT --> CAT; Tyr --> His) and by cAMP-, cGMP-dependent protein kinase (TAC --> TGC; Tyr --> Cys), respectively; adjacent normal mucosa showed no mutation. This may open new avenues that imply TK1 activity in tumor cell proliferation.
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PMID:Mutation analysis in the coding sequence of thymidine kinase 1 in breast and colorectal cancer. 1269 56

Carbon monoxide (CO) is well known as a relaxing substance in the vasculature, where it is released during the heme oxygenase (HO) reaction. Little is known about the tissue-specific targets of CO in smooth muscles. To date the functional role of CO in the coronary artery remains unclear. The expression of HO-2, the constitutive isoform of HO, but not of HO-1 (inducible HO isoform) was demonstrated by immunohistochemical reaction. Contractile studies, performed under isometrical conditions, showed that CO, as well as hemin (given as a substrate for HO), relax de-endothelized coronary smooth muscle after the blockade of neuronal transmission. The action of hemin was antagonized by preliminary treatment of the vessel with SnPPIX--a competitive inhibitor of HO. The relaxatory effects of hemin were abolished in the presence of guanylyl-cyclase or protein kinase G antagonists. Patch-clamp studies revealed that hemin caused activation of iberiotoxin-blockable K outward current (I(K)) via guanylyl-cyclase and protein-kinase-G-dependent mechanisms. This activation coincided with hyperpolarization of the plasma membrane of single coronary smooth muscle cells by 8+/-3 mV, which was prevented by preliminary exposure of cells to 10 microM SnPPIX. The I(K)-augmenting effect of hemin was not affected by pretreatment of cells with cyclopiazonic acid and/or ryanodine, blockers of phospholipase C or heparin (applied via pipette), but was not observed when ATP was omitted from the dialyzing solution, or in the presence of Na-free, ATP-containing pipette solution. The omission of Ca(2+) from the bath or the replacement of Na with Li in both pipette and bath media also prevented the I(K)-activating effect of hemin. These results suggest that the constitutive HO-2 in coronary artery smooth muscle cells plays role in the modulation of tone. At the level of smooth muscle cells CO and its precursor hemin may cause hyperpolarization of the plasma membrane by activation of iberiotoxin-sensitive I(K) presumably via PKG-dependent activation of the Na/Ca exchanger. This activation is thought to increase the submembrane Ca(2+) concentration in the vicinity of large-conductance, Ca(2+)-sensitive K channels, thus causing voltage-dependent inhibition of Ca(2+) entry and subsequent relaxation of the vessel.
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PMID:Role of constitutively expressed heme oxygenase-2 in the regulation of guinea pig coronary artery tone. 1276 25

(1) Sildenafil (viagra) is a potent PDE5 inhibitor and thus a relaxant drug in corpus carvernosum smooth muscle. In the present work, we evidenced the presence of PDE5 isozyme and investigated the effect of sildenafil on the specific cyclic nucleotide phosphodiesterase (PDE) activity, smooth muscle tone and calcium signaling in the rat main pulmonary artery (MPA). (2) The PDE activity was measured in cytosolic and microsomal fractions. Total cAMP and cGMP-PDE activities were mainly present in the cytosolic fraction. Sildenafil (0.1 micro M) reduced by 72% cGMP-PDE activity, whereas zaprinast (10 micro M), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Sildenafil (0.1 micro M) also inhibited significantly (22%) the cAMP-PDE activity. (3) Western blot analysis revealed the expression of PDE5 mainly in the cytosolic fraction of MPA. Sildenafil concentration-dependently inhibited (IC(50)=3.4 nM) the activity of MPA PDE5 partially purified by HPLC. (4) Sildenafil (0.1 nM-50 micro M) concentration-dependently relaxed MPA rings precontracted with phenylephrine (0.5 micro M). The potency of sildenafil (IC(50)=11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC(50)=600 nM). The vasorelaxant effect of sildenafil was not altered by endothelium removal or in the presence of KT 5823 (1 micro M) and H89 (1 micro M), potent inhibitors of PKG and PKA, respectively. (5) In isolated MPA myocytes, which had been loaded with the calcium fluorophore indo-1, sildenafil (10-100 nM) antagonized ATP- and endothelin-1-induced calcium oscillations but had no effect on the transient caffeine-induced [Ca(2+)](i) response. (6) This study demonstrates the presence of a functional and highly sildenafil-sensitive PDE5 isozyme in rat MPA. Inhibition of this isozyme mainly accounts for the potent pulmonary vasodilator action of sildenafil, which involves alteration in the inositol triphosphate-mediated calcium signaling pathway.
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PMID:Effect of sildenafil on cyclic nucleotide phosphodiesterase activity, vascular tone and calcium signaling in rat pulmonary artery. 1278 11

The involvement of cyclic guanosine 3',5'-monophosphate (cGMP) and cGMP-dependent protein kinase (PKG) and their interaction with the Ca2+-dependent mechanisms in the regulation of ciliary activity are not well understood. To investigate how cGMP regulates ciliary activity, changes in ciliary beat frequency (CBF) and intracellular calcium concentration ([Ca2+]i) of rabbit tracheal ciliated cells in response to 8-bromo-cGMP (Br-cGMP) were simultaneously quantified using digital, high-speed phase-contrast and fluorescence imaging. Br-cGMP induced a response in ciliary activity that could be separated into two parts. Firstly, Br-cGMP induced a concentration-dependent increase in the basal CBF that occurred without increasing the [Ca2+]i. This response was not affected by excessively buffering the [Ca2+]i with BAPTA but was abolished by KT5823, a PKG inhibitor. Secondly, Br-cGMP induced a series of transient increases in CBF that were superimposed on the sustained increases in CBF. These transient increases in CBF correlated with the stimulation of a series of transient increases in [Ca2+]i and were abolished by BAPTA, but were unaffected by KT5823. The magnitude of the transient increases in CBF and [Ca2+]i were not dependent on the concentration of Br-cGMP. The Ca2+-dependent changes in CBF induced by ionomycin or ATP were not affected by KT5823. From these results, we propose that cGMP increases CBF in two ways: firstly through a Ca2+-independent mechanism involving PKG, and secondly through a Ca2+-dependent mechanism following the stimulation of changes in [Ca2+]i. In addition, we suggest that the Ca2+-dependent stimulation of rabbit airway ciliary activity does not initially require PKG activation.
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PMID:The role of cGMP in the regulation of rabbit airway ciliary beat frequency. 1281

The anti-anginal drug nicorandil has been shown to inhibit apoptosis by activating mitochondrial ATP-sensitive potassium (K(ATP)) channels. The possible contribution of the nitrate moiety of this drug to its anti-apoptotic effect has now been investigated in neonatal rat ventricular myocytes subjected to oxidative stress. Exposure of cultured myocytes to 100 micromol/l hydrogen peroxide (H(2)O(2)) increased the number of nuclei stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique as well as induced internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspases-3 and -9, all of which are characteristics of apoptosis. Pretreatment of cells with nicorandil (100 micromol/l) inhibited these effects of H(2)O(2). Both the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoate (5-HD) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, attenuated the anti-apoptotic effect of nicorandil in concentration-dependent manners. Coapplication of ODQ (10 micromol/l) and 5-HD (500 micromol/l) completely abolished nicorandil-induced cytoprotection. The effect of nicorandil was also reduced by an inhibitor of cGMP-dependent protein kinase (KT5823, 1 micromol/l). The nitric oxide donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 50 micromol/l) mimicked the protective effect of nicorandil in a manner sensitive to ODQ but not to 5-HD. A cell-permeable cGMP analog, 8-bromo-cGMP, also reduced H(2)O(2)-induced apoptosis. The inhibition of the H(2)O(2)-induced activation of caspase-3, but not that of caspase-9, by nicorandil in the presence of 5-HD or by SNAP was reversed by the addition of dithiothreitol to the enzyme assay. Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through a nitric oxide/cGMP-dependent mechanism as well as by activating mitochondrial K(ATP) channels.
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PMID:Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through activation of mitochondrial ATP-sensitive potassium channels and a nitrate-like effect. 1465 76


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