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)

Elevation of intracellular cGMP and activation of cGMP-dependent protein kinase (PKG) in vascular smooth-muscle cells produces relaxation, but mechanisms distal to PKG activation are not well understood. Few PKG substrates have been described in smooth muscle that may mediate the action of PKG, including P240, P132 and phospholamban. None of them is a specific PKG substrate, raising the question of whether any specific PKG substrates possibly exist in vascular smooth muscle that may play roles in relaxation. In this study PKG substrates were detected in aortic smooth muscle by adding purified exogenous PKG and [gamma-32P]-ATP. Very few PKG substrates were detectable in whole-tissue homogenates or detergent-solubilized fractions, due to the high basal activity of other protein kinases and the large numbers of other phosphoproteins. Heat or acid treatment of such fractions, to remove any endogenous protein kinase activity and achieve partial protein purification, revealed many potential PKG substrates. Of the 3 substrates identified previously, P240 and P132 were partly heat-stable. Thirty-one new PKG substrates were found: 14 in the initial heat-stable extract and 9 in the heat- and acid-soluble extract, whereas the others were revealed only after chromatography. All of the heat-stable PKG substrates were bound and salt-eluted from a DEAE-cellulose column in 2 major peaks called pool I and II. After sequential application to Q-Sepharose and S-Sepharose columns, 7 PKG substrates were found in pool I, in particular a group of 4 substrates of 40, 33, 28 and 22 kD virtually coeluted through all 3 columns. The former 3 produced similar phosphopeptide maps, suggesting a relationship. All the new substrates from pool I were relatively specific for PKG because they were poorly phosphorylated with exogenous cAMP-dependent protein kinase and not with Ca2+/phospholipid-dependent protein kinase. Further chromatography of the proteins in pool II resulted in an extensive purification of P132 as well as a group of 4 PKG substrates of 33-30 kD. Phosphopeptide mapping of the 132-kD protein revealed a close homology to the 132-kD PKG substrate previously described in rat aortic smooth muscle. These data demonstrate the presence of multiple substrates for PKG in aortic smooth-muscle tissue.
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PMID:Multiple substrates for cGMP-dependent protein kinase from bovine aortic smooth muscle: purification of P132. 863 Mar 52

The large number of covalently bound phosphates on the extracellular phosphoproteins osteopontin (OPN) and bone sialoprotein (BSP) have been implicated in biological functions such as mineral deposition and osteoclast binding. In the present study the state of phosphorylation of BSP and OPN was evaluated by in vitro 32P labeling using a series of protein kinases and quantification. Both the purified bovine BSP and OPN were radiolabeled by [32P]ATP and factor-independent protein kinase. Quantification of 32P radioactivity incorporated on dephosphorylated BSP and OPN provided 6.6 and 8.9 mol of phosphate incorporated/mol, respectively. Native OPN incorporated 1.07 and BSP 2.46 mol of phosphate/mol by factor-independent protein kinase. These data led to calculations that OPN and BSP, respectively, contain 7.83 and 4.14 mol of phosphate/mol in their natural state. Thrombin digests of 32P-labeled BSP showed radioactivity to be associated with fragment of approximately molecular mass values 30 kDa (N-terminal half), with no observable radioactivity associated with the 40-kDa fragment (C-terminal half). Similar experiments with 32P-labeled OPN provided two radiolabeled thrombin fragments, with molecular mass 30 kDa (N-terminal half) and 20 kDa (C-terminal half), both were radioactive. The major phosphorylation was associated with the N-terminal half containing 7.0 mol of phosphate, and 1.9 mol of phosphate were associated with the C-terminal half. Additional experiments of in vitro phosphorylation of OPN and BSP by several other known protein kinases were carried out. cAMP-dependent protein kinase showed no phosphorylation of OPN or BSP, while protein kinase C and cGMP-dependent protein kinase led to minor phosphorylation, each of the latter introduced about 1 mol of phosphate/mol of OPN and BSP molecule.
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PMID:Phosphorylation of purified bovine bone sialoprotein and osteopontin by protein kinases. 866 67

Nitric oxide (NO.) is believed to mediate nitrovasodilators and acetylcholine-induced vasodilatation via increasing intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels. The cellular mechanisms involved in No.-mediated pulmonary vasodilatation are complex and include membrane hyperpolarization. Using the patch-clamp technique in cell-attached and inside-out configurations, we examined the effect of NO. gas, 3-morpholinosydnomimine hydrochloride (SIN-1), and perfusate from ACh-stimulated human pulmonary arterial endothelial cells, or endothelium-derived relaxing factors (EDRF), on the Ca(2+)-dependent K+ (KCa) channels in isolated cultured human pulmonary arterial smooth muscle cells (HPSMC). NO., SIN-1, and EDRF caused similar increases in KCa channel activity. Inhibiting cGMP generation with methylene blue or inhibiting the effect(s) of cGMP with the cGMP antagonist 8-bromoguanosine 3',5'-cyclic monophosphorothioate Rp isomer Rp-cGMPS prevented the NO.- and SIN-1-mediated activation of KCa channels, respectively. Treating the human pulmonary arterial endothelial cells with methylene blue blocked the EDRF-mediated activation of KCa channels in HPSMC. The cGMP analogue 8-bromo-cGMP increased KCa channel activity in intact cells and in excised inside-out HPSMC membrane patches. In the presence of cGMP and ATP, the alpha-isozyme of the cGMP-dependent protein kinase (I alpha-cGMP-PK) significantly increased KCa channel activity, and the channel activation was further increased on addition of the protein phosphatase inhibitors okadaic acid and calyculin A. Furthermore, the cGMP-mediated KCa channel activation was reduced by the cyclic nucleotide-dependent protein kinase inhibitor N-[2-methylamino)ethyl]-5-isoquinlinesulfonamide (H-8). Thus, in HPSMC, the mechanism of NO.- and native EDRF-induced KCa channel activation appears to be mediated via cGMP-I alpha-cGMP-PK phosphorylation of KCa channels.
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PMID:Regulation of Ca(2+)-activated K+ channels in pulmonary vascular smooth muscle cells: role of nitric oxide. 888 62

The effects of intracellular cyclic guanosine monophosphate (cGMP) on L-type calcium current (lCa) and contraction of ventricular myocytes enzymatically isolated from guinea pig hearts were investigated to test the hypothesis that cGMP increases contractions along with ICa in these cells. ICa and contractions, elicited every 15 sec, were recorded simultaneously with a whole-cell voltage-clamp method and a video edge-detector, respectively. Cells were superfused with Tyrode's solution (22 degrees C); the pipette solution contained 120 mM potassium aspartate, 30 mM KCl, 4 mM ATP, 5 mM N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid), 0.01 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and various concentrations of cGMP, which entered the cell interior through the patch electrode. In the presence of 3 nM isoproterenol (ISO) in the bath, ICa was increased 3.2-fold. ICa was further increased by 20% with 30 microM cGMP; cell contractions were also increased by 32%. When ICa was maximal in the presence of 30 nM ISO, cGMP no longer increased ICa or contractions, an indication that the effects of cGMP and ISO were additive. When ICa was increased maximally (4.3-fold) by 100 microM isobutylmethylxanthine, a nonselective phosphodiesterase inhibitor, application of 100 microM cGMP in the pipette decreased ICa by 53% and cell shortening by 64%. Cyclic GMP changed contraction in parallel with ICa in the presence of either ISO or isobutylmethylxanthine. 5'-GMP had no significant effect on ICa or contraction in the presence of ISO or isobutyl-methylxanthine. Cyclic GMP alone, at 30 microM, increased ICa by 25%; this effect on basal ICa was reversed by removal of cGMP from the pipette solution. We conclude that intracellular cGMP had two effects on ICa and contraction, namely, 1) an increase caused by an action on cGMP-inhibited phosphodiesterase and 2) a decrease attributed to activation of cGMP-dependent protein kinase.
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PMID:Biphasic effects of intrapipette cyclic guanosine monophosphate on L-type calcium current and contraction of guinea pig ventricular myocytes. 896 51

In the cortical collecting duct of the rat two Ca(2+)-dependent K+ channels have been described so far. In the luminal membrane a maxi K+ channel with a single channel conductance of 139 +/- 3 pS in excised membrane patches (n = 91) at 0 mV clamp voltage and asymmetrical KCl-concentrations in pipette and bath was found, while in the basolateral membrane an intermediate conductance K+ channel (85 +/- 1 pS, n = 53) and a small K+ channel (28 +/- 2 pS, n = 15) was described. All these K+ channels had similar pharmacological properties since all could be blocked by the K+ channel inhibitors Ba2+, TEA+, and charybdotoxin. Verapamil, known as a L-type Ca2+ channel blocker, was also capable of inhibiting these K+ channels. While the maxi K+ channel from the luminal membrane was upregulated by intracellular Ca2+ (EC50: 5 microM), the small and the intermediate K+ channel from the basolateral membrane were downregulated (IC50: 10 microM). When the cytosolic Ca(2+)-activity was in the physiological range below 1 microM the activity of the maxi K+ channel was low and regulated via intracellular pH and ATP. Furthermore, when CCD cells were strongly depolarized and under hypoosmotic stress, Ca2+ rose and activated this K+ channel, indicating that this channel is involved in volume regulation. Like the maxi K+ channel the intermediate conductance K+ channel from the basolateral membrane was also sensitive to intracellular changes of pH where acidic pH inhibited while alkaline pH activated this channel. But unlike the K+ channels from the luminal membrane the K+ channel from the basolateral membrane is not regulated by ATP up to 5 mM. The activity of the K+ channels from the basolateral membrane decreased steadily after excision of the membrane. This decrease could be prevented by applying cGMP and MgATP to the bath and thus, activating a membrane-bound cGMP-dependent protein kinase (PKG). The activation of the PKG could be reversed by its specific inhibitor KT5823 (1 microM). Due to the opposite regulation via intracellular Ca2+ and the involvement of different protein kinases a specific and independent regulation of K+ secretion and Na+ reabsorption is possible in the CCD of the rat.
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PMID:Ca(2+)-dependent K+ channels in the cortical collecting duct of rat. 926 90

Ca2+ changes induced by nitric oxide (NO.) were investigated in cultured human endothelial cells. Sodium nitroprusside (SNP) (1-100 mumol/L) and S-Nitroso-N-acetylpenicillamine (SNAP) (100 mumol/L) were used as NO. donors. The cytoplasmatic Ca2+ concentration was calculated using ratiometric FURA2 fluorescence measurements. Both NO. donors caused transient oscillatory Ca2+ changes, which were not detectable in the presence of oxyhemoglobin (50 mumol/L). Digital ratio imaging revealed initiation sites within cells where Ca2+ increases started spreading, which indicates that nonuniformly distributed targets might be involved in these reactions. Calcium was released from intracellular stores as indicated by experiments performed in Ca(2+)-free buffer. L-type Ca(2+)-channel blocker diltiazem (100 mumol/L) was not able to block these responses. NO.-induced Ca2+ release from intracellular stores caused capacitative Ca2+ entry. Both thapsigargin (1 mumol/L) and cyclopiazonic acid (10 mumol/L) inhibited the SNP response completely, whereas neither ryanodine (up to 100 mumol/L) nor dantrolene (100 mumol/L) was able to inhibit Ca2+ changes induced by SNP, indicating that primarily inositol 1,4,5-triphosphate (IP3)-dependent stores are released upon stimulation with NO.. A small inhibitory effect of ATP- and SNP-induced peak [Ca2+]i increase was measured in the presence of both caffeine (20 mmol/L) and procaine (1 mmol/L). Evidence is presented that cGMP is not involved in NO.-induced Ca2+ signals, as neither inhibitors of guanylate cyclase (methylene blue and LY 83583) nor cell permeant analogues of cGMP altered or simulated [Ca2+] changes. An inhibitor of cGMP-dependent protein kinase was also ineffective. We therefore propose that endothelial cells have specific targets proximal or at IP3 receptors to induce Ca2+ changes in endothelial cells stimulated with NO..
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PMID:Nitric oxide induces transient Ca2+ changes in endothelial cells independent of cGMP. 928 49

1. The involvement of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKC) in the effects of cyclic AMP-elevating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent-protein kinase activities and ATP-induced calcium signalling ([Ca2+]i was studied in rat aorta. Cyclic AMP-elevating agents used were a beta-adrenoceptor agonist (isoprenaline), a phosphodiesterase 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram). 2. In rat intact aorta, the relaxant effect induced by isoprenaline (0.01-0.03 microM) was decreased by a specific inhibitor of PKA, H-89, whereas a specific inhibitor of PKG, Rp-8-Br-cyclic GMPs, was without effect. NO significant difference in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 microM was used. At the same concentration, isoprenaline did not modify ATP-induced changes in [Ca2+]i in smooth muscle cells. Neither H-89 nor Rp-8-Br-cyclic GMPs modified this response. These findings suggest that PKA is only involved in the relaxant effect induced by low concentrations of isoprenaline (0.01-0.3 microM), whereas for higher concentrations, other mechanisms independent of PKA and PKG were involved. 3. The relaxant effects induced by SK&F 94120 and rolipram were inhibited by Rp-8-Br-cyclic GMPS with no significant effect of H-89. Neither SK&F 94120, nor rolipram at 30 microM significantly modified the activity ratios of PKA and PKG. Rolipram inhibited the ATP-induced transient increase in [Ca2+]i. This decrease was abolished by Rp-8-Br-cyclic GMPS whereas H-89 had no significant effect. These results suggests that PKG is involved in the vascular effects induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross-activation of PKG by cyclic AMP in rat aorta. 4. The combinations of 5 microM SK&F 94120 with rolipram markedly potentiated the relaxant effect of rolipram. This relaxation was decreased by H-89 and not significantly modified by Rp-8-Br-cyclic GMPS. Moreover, the association of the two PDE inhibitors significantly increased the activity ratio of PKA without changing the PKG ratio. The present findings show that PKA rather than PKG is involved in this type of vasorelaxation. The differences in the participation of PKA vs PKG observed when inhibitors of PDE3 and PDE4 were used alone or together could be due to differences in the degree of accumulation of cyclic AMP, resulting in the activation of PKA or PKG which are differently localized in the cell. 5. These findings support for both PKA and PKG in cyclic AMP-mediated relaxation in raT aorta. Their involvement depends on the cellular pathway used to increase the cyclic AMP level.
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PMID:Involvement of cyclic nucleotide-dependent protein kinases in cyclic AMP-mediated vasorelaxation. 929 42

Glutamate, the neurotransmitter released by photoreceptors, excites horizontal cells and OFF-type bipolar cells by activating ionotropic receptors. This study investigated an additional action of glutamate in which it modulates a voltage-gated ion channel in horizontal cells. We find that glutamate and APB (2-amino-4-phosphonobutyrate) produce a delayed and moderately prolonged suppression of an inward rectifier current (IRK+). This effect is proposed to occur via an APB-sensitive metabotropic glutamate receptor (mGluR) because common agonists for the ionotropic or APB-insensitive mGluRs are ineffective and the APB-insensitive receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG) does not block the actions of glutamate or APB. 8-Br-cGMP, 1-methyl-3-isobutylxanthine (IBMX), and atrial natriuretic peptide (ANP) but not 8-Br-cAMP mimic the suppression of IRK+. The effects of glutamate and APB are blocked by protein kinase inhibitors including Rp-8-pCPT-cGMPS, H-8, and H-7 as well as by ATPgammaS. We hypothesize that the APB receptor suppresses IRK+ via upregulation of cGMP and subsequent activation of a cGMP-dependent protein kinase. This pathway is likely regulated by an ATP-dependent phosphorylation. This is a novel signaling pathway for mGluRs and indicates that at least two distinct APB-activated pathways exist in the retina. Functionally, this APB receptor-mediated action found in horizontal cells would provide a means by which spatially restricted changes of glutamate, produced by local illumination of photoreceptors, could regulate IRK+ and consequently the response properties of these neurons. This would serve to adapt selectively retinal regions stimulated by small regions of the visual world.
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PMID:Metabotropic glutamate receptor-mediated suppression of an inward rectifier current is linked via a cGMP cascade. 936 42

Guanosine 3',5'-cyclic monophosphate (cGMP)-binding, cGMP-specific phosphodiesterase (PDE5) is abundant in vascular smooth muscle, and this enzyme is a potent substrate for cGMP-dependent protein kinase (PKG) in vitro. Binding of cGMP to the allosteric sites of PDE5 is required for this phosphorylation to occur. Vascular smooth muscle cells (VSMC) were used to determine if PDE5 is phosphorylated in intact cells when cGMP is increased. With the use of anti-PDE5 antibodies, a phosphorylated 93-kDa protein band was immunoprecipitated from early passaged primary cultures of VSMC that had been preincubated with 32(Pi) to label cellular ATP and then treated with atrial natriuretic factor (ANF). In the absence of ANF, there was no detectable incorporation of radiolabeled phosphate into this band. Phosphorylation of the 93-kDa protein was augmented by pretreating cells with 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) to activate PKG before addition of ANF. 8-BrcGMP, which interacts poorly with the allosteric sites of PDE5, had no effect on PDE5 phosphorylation in the absence of ANF. Phosphorylation of PDE5 in response to treatment of cells with ANF was associated with a two- to fourfold increase in PDE activity in immunoprecipitates. Multiple-passaged VSMC, which are deficient in PKG but retain PDE5, demonstrated no ANF-dependent increase in phosphorylation or catalytic activity of PDE5. However, incubation of immunoprecipitated PDE5 from these cells with purified PKG, cGMP, and a phosphorylation mixture containing [gamma-32P]ATP resulted in 32(Pi) incorporation into PDE5 that was correlated with increased catalytic activity. These studies are the first to demonstrate phosphorylation of PDE5 in intact cells, thus suggesting a physiological role for this enzyme in smooth muscle regulation.
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PMID:ANF elicits phosphorylation of the cGMP phosphodiesterase in vascular smooth muscle cells. 948 47

1. We used patch clamp to study whole-cell K+ currents activated by calcitonin gene-related peptide (CGRP) in smooth muscle cells freshly dissociated from pig coronary arteries. 2. CGRP (50 nM) activated an inward current at -60 mV in symmetrical 140 mM K+ that was blocked by glibenclamide (10 microM), an inhibitor of ATP-sensitive potassium (KATP) channels. CGRP-induced currents were larger in cells dialysed with 0.1 mM ATP than with 3.0 mM ATP. 3. Forskolin (10 microM) activated a glibenclamide-sensitive current, as did intracellular dialysis with cAMP (100 microM). The catalytic subunit of cAMP-dependent protein kinase (protein kinase A, PKA), added to the pipette solution, activated equivalent currents in five out of twelve cells. 4. CGRP-induced currents were reduced by the PKA inhibitors adenosine 3',5'-cyclic monophosphorothioate, RP-isomer, triethylammonium salt (Rp-cAMPS; 100 microM) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulphonamide+ ++ dihydrochloride (H-89; 1 microM), and abolished by inclusion of a PKA inhibitor peptide in the pipette solution. 5. The beta-adrenergic agonist isoprenaline (10 microM) also activated a glibenclamide-sensitive K+ current. 6. CGRP-induced currents were unaffected by the inhibitor of cGMP-dependent protein kinase (PKG) KT5823 (1 microM). Sodium nitroprusside (10 microM) did not activate a glibenclamide-sensitive current in cells held at -60 mV, but did activate an outward current at +60 mV that was abolished by KT5823, or by 100 nM iberiotoxin (an inhibitor of BKCa channels). 7. Our findings suggest that CGRP activates coronary KATP channels through a pathway that involves adenylyl cyclase and PKA, but not PKG.
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PMID:ATP-sensitive K+ channel activation by calcitonin gene-related peptide and protein kinase A in pig coronary arterial smooth muscle. 949 Aug 26


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