Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Effects of pharmacological modulation of protein kinase A, C and G (PKA, PKC and PKG) were examined on inducible form of nitric oxide synthase (iNOS) expressed in COS cells to elucidate regulatory mechanism of iNOS by protein kinases. Formation of nitric oxide (NO), as an index of NOS activity, was assessed by measurement of nitrite in incubation medium in long term observation and by hemoglobin assay method in kinetic study. In long term observation (18 hours), activation of PKA by 8-Br-cAMP increased NO formation that was inhibited by N-(2-[p-bromocinnamylamino] ethyl)-5-isoquinolinesulfonamide (H89). Though activation of PKC by 12-O-tetradecanoyl phorbol-13-acetate (TPA) decreased NO formation, PKC inhibitor, chelerythrine, failed to inhibit the decrease. Activation of PKG with 8-Br-cGMP and inhibition with KT5823 resulted in no change in NO formation. Western blot analysis revealed that neither 8-Br-cAMP nor TPA affect iNOS expression. In kinetic study (short term perfusion study), no change in NO formation was observed by 8-Br-cAMP and TPA. These results indicate that, in living cells, PKG does not play a regulatory role in iNOS activity and that PKA and PKC do not directly modulate iNOS activity. However, PKA and PKC would possibly modify NOS activity indirectly via cofactors necessary for NO formation.
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PMID:Protein kinase A- and C-dependent modulation of murine inducible nitric oxide synthase. 1164 42

Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.
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PMID:Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. 1172 16

To study the role of cGMP-dependent protein kinase I (cGKI) for cardiac contractility, force of contraction (F(c)) was studied in electrically driven heart muscle from wild-type (WT) mice and from conventional and conditional cGKI knockout mice. Both 8-Br-cGMP and 8-pCPT-cGMP reduced Fc in cardiac muscle from juvenile WT but not from juvenile cGKI-null mutants. Similarly, the cGMP analogues reduced F(c) in forskolin-stimulated ventricular muscle from WT mice but not from cGKI-null mutants. In contrast, carbachol reduced F(c) in both groups of animals. 8-Br-cGMP reduced F(c) also in heart muscle from adult WT mice but not from adult cardiomyocyte-specific cGKI-knockout mice. These results demonstrate that cGKI mediates the negative inotropic effect of cGMP in the myocardium of juvenile and adult mice.
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PMID:cGMP-dependent protein kinase I mediates the negative inotropic effect of cGMP in the murine myocardium. 1178 13

The mechanisms by which the intracellular messenger cGMP can modulate synaptic efficacy remain poorly understood. Here we report that cGMP, acting through cGMP-dependent protein kinase (PKG), has multiple rapid and reversible effects on synaptic transmission in slices and cultures of rodent visual cortex. Extracellular application of the membrane permeable cGMP analog 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP) and the PKG specific activator beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate sp-isomer (Sp-8-Br-PET-cGMPS) reduced stimulus-evoked EPSPs in slices. In cortical cultures, both analogs reduced the frequency of spontaneous EPSCs, but not their amplitude. In both slices and cultures, intracellular perfusion of the postsynaptic neurons with a pseudosubstrate inhibitory peptide specific for PKG had no effect on the reduction in EPSPs and EPSCs, indicating that the inhibition occurred at presynaptic sites. Whole-cell calcium currents in cultured cortical neurons were also reduced by both analogs, which may account for the effect on synaptic release. To determine whether cGMP was also acting at postsynaptic sites, we applied exogenous kainate/AMPA and NMDA to the recorded cells directly. cGMP and its analogs showed little effect on the postsynaptic kainate/AMPA responses but produced a dramatic enhancement of NMDA responses. cGMP-induced NMDA potentiation was prevented by the specific PKG inhibitory peptide infused into the postsynaptic cell. In summary, cGMP, acting through PKG, had depressive presynaptic and facilitatory postsynaptic actions at excitatory synapses in the visual cortex. We suggest that these opposing actions may be useful for altering the balance of synaptic inputs to cortical neurons in ways that enhance signals important for synaptic facilitation and neuronal plasticity.
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PMID:cGMP-induced presynaptic depression and postsynaptic facilitation at glutamatergic synapses in visual cortex. 1181 31

The inhibitory neuromodulator taurine is involved in osmoregulation and cell volume adjustments in the central nervous system. In addition, taurine protects neural cells from excitotoxicity and prevents harmful metabolic events evoked by cell-damaging conditions. The release of taurine in nervous cell preparations is greatly enhanced by glutamate receptor agonists and various cell-damaging conditions. NO-generating compounds also increase taurine release in the mouse hippocampus. The further involvement of the NO/cGMP pathway and protein kinases in preloaded [3H]taurine release from hippocampal slices from adult (3-month-old) and developing (7-day-old) mice in normoxia and in ischemia was now studied using a superfusion system. The release was enhanced by 8-Br-cGMP and the phosphodiesterase inhibitor 2-(2-propyloxyphenyl)-8-azapurin-6-one (zaprinast), particularly in the immature hippocampus, indicating that increased cGMP levels induce taurine release. The release was also increased by the inhibitor of soluble guanylyl cyclase, 1H-(1,2,4)oxadiazolo-(4,3a)quinoxalin-1-one (ODQ) and the protein kinase C activator 4beta-phorbol 12-myristate 13-acetate (PMA), but only in the adult hippocampus. The ischemia-induced release was also enhanced by increased cGMP levels in both adult and developing mice, whereas protein kinase inhibitors had no effects in any conditions. The results demonstrate that cGMP is able to modulate hippocampal taurine release in both adult and developing mice, the rise in cGMP levels evoking taurine release in normoxia and in ischemia. This could be part of the neuroprotective properties of taurine, being thus important particularly in cell-damaging conditions and in preventing excitotoxicity.
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PMID:Taurine release in the developing and adult mouse hippocampus: involvement of cyclic guanosine monophosphate. 1192 68

Holocarboxylase synthetase (HCS) catalyzes the covalent attachment of biotin to five biotin-dependent carboxylases in human cells. Multiple carboxylase deficiency (MCD) is a life-threatening disease characterized by the lack of carboxylase activities because of deficiency of HCS activity. Here, we report the obligatory participation of HCS in the biotin-dependent stimulation of the level of HCS mRNA and those of acetyl-CoA carboxylase and the alpha subunit of propionyl-CoA carboxylase in human cells. Fibroblasts from patients with MCD are unable to increase HCS mRNA in response to biotin unless the vitamin concentration is raised 100-fold, in keeping with mutations that cause a reduced affinity for biotin by the mutant enzyme. The outcome is deficient synthesis of biotinyl-5'-AMP, the active form of the vitamin in the biotinylation reaction. HCS and carboxylase mRNA levels in normal and MCD fibroblasts and HepG2 cells can be restored by the addition of the cGMP analogue, 8-Br-cGMP, and can be abolished by the addition of inhibitors of the soluble form of guanylate cyclase. We propose a regulatory role for biotin in the control of HCS and carboxylase mRNA levels through a signaling cascade that requires HCS, guanylate cyclase, and cGMP-dependent protein kinase.
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PMID:Holocarboxylase synthetase is an obligate participant in biotin-mediated regulation of its own expression and of biotin-dependent carboxylases mRNA levels in human cells. 1195 85

Does cGMP, via protein kinase G, inhibit cAMP-stimulated Ca(2+) current (I(Ca(L))) in mammalian ventricular myocytes by phosphorylating the calcium channel at a site different from that acted on by cAMP or by dephosphorylating the calcium channel through phosphatase(s)? We tested these possibilities in guinea pig ventricular myocytes superfused with Tyrode's solution (35 degrees C) and dialyzed with adenosine 5'-O-(3-thiotriphosphate) ([ATPgammaS](pip)). ATPgammaS is a kinase substrate but thiophosphorylated proteins are not phosphatase substrates. With 5 mM [ATPgammaS](pip), I(Ca(L)) increased gradually over 20 to 25 min and then rapidly in the presence of 3-isobutyl-1-methylxanthine. 8-Bromo-cGMP (8-Br-cGMP; 1 mM) did not inhibit I(Ca(L)) significantly (-3 +/- 11.8%, n = 21) in contrast to results with ATP dialysis (). Similar results were obtained with 0.1 mM carbachol (CCh). I(Ca(L)) increased after longer dialysis (>/=40 min) with ATPgammaS; again, 8-Br-cGMP had no effect. Also, isoproterenol (ISO) did not stimulate and CCh, alone or in the presence of ISO, did not inhibit I(Ca(L)). Block of CCh effect by ATPgammaS, although consistent with cGMP action in muscarinic inhibition, could be explained by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) formation from ATPgammaS via nucleoside diphosphate kinase. GTPgammaS uncouples muscarinic and beta-adrenoceptors from intracellular effectors. Failure of 8-Br-cGMP to reduce I(Ca(L)) irreversibly excludes calcium channel phosphorylation as an inhibitory mechanism. We propose that cGMP inhibits I(Ca(L)) by activating phosphatase(s) in guinea pig ventricular myocytes.
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PMID:On the role of phosphatase in regulation of cardiac L-type calcium current by cyclic GMP. 1196 Oct 49

Nitric oxide (NO) regulates the release of catecholamines from the adrenal medulla but the molecular targets of its action are not yet well identified. Here we show that the NO donor sodium nitroprusside (SNP, 200 microM) causes a marked depression of the single Ca(V)1 L-channel activity in cell-attached patches of bovine chromaffin cells. SNP action was complete within 3-5 min of cell superfusion. In multichannel patches the open probability (NP(o)) decreased by approximately 60 % between 0 and +20 mV. Averaged currents over a number of traces were proportionally reduced and showed no drastic changes to their time course. In single-channel patches the open probability (P(o)) at +10 mV decreased by the same amount as that of multichannel patches (approximately 61 %). Such a reduction was mainly associated with an increased probability of null sweeps and a prolongation of mean shut times, while first latency, mean open time and single-channel conductance were not significantly affected. Addition of the NO scavenger carboxy-PTIO or cell treatment with the guanylate cyclase inhibitor ODQ prevented the SNP-induced inhibition. 8-Bromo-cyclicGMP (8-Br-cGMP; 400 microM) mimicked the action of the NO donor and the protein kinase G blocker KT-5823 prevented this effect. The depressive action of SNP was preserved after blocking the cAMP-dependent up-regulatory pathway with the protein kinase A inhibitor H89. Similarly, the inhibitory action of 8-Br-cGMP proceeded regardless of the elevation of cAMP levels, suggesting that cGMP/PKG and cAMP/PKA act independently on L-channel gating. The inhibitory action of 8-Br-cGMP was also independent of the G protein-induced inhibition of L-channels mediated by purinergic and opiodergic autoreceptors. Since Ca(2+) channels contribute critically to both the local production of NO and catecholamine release, the NO/PKG-mediated inhibition of neuroendocrine L-channels described here may represent an important autocrine signalling mechanism for controlling the rate of neurotransmitter release from adrenal glands.
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PMID:Nitric oxide inhibits neuroendocrine Ca(V)1 L-channel gating via cGMP-dependent protein kinase in cell-attached patches of bovine chromaffin cells. 1204 44

The present study aimed to test the hypothesis that a decrease in preoptic cAMP mediates fever. To this end, body core temperature (T(c)) of unanesthetized, freely moving rats was monitored by biotelemetry before and after pharmacological modulation of the cAMP pathway, and cAMP levels in the anteroventral third ventricular region (AV3V), where the preoptic region (POA) is located, were determined. We observed that intra-POA administration of the cAMP agonist dibutyryl-cAMP (Db-cAMP, 40 microg) reduced T(c). PGE(2) (the proximal mediator of fever, 200 ng) raised T(c) with a concomitant decrease in AV3V cAMP levels from 22.7+/-1.8 to 17.0+/-1.0 fmol/microg protein. Moreover, PGE(2)-induced fever was impaired by the phosphodiesterase inhibitor aminophylline. In order to verify the interaction between the cAMP- and cGMP-dependent pathways in the POA, we then co-injected Db-cAMP and 8-Br-cGMP into the POA. As a result, 8-Br-cGMP augmented the drop in T(c) evoked by Db-cAMP. Lastly, we observed that intra-POA co-microinjection of the protein kinase A inhibitor (Rp-cAMPS, 1 microg) with the protein kinase G inhibitor (Rp-cGMPS, 1 microg), mimicking the effects of reduced production of cAMP and cGMP, respectively, produced a fever-like response. In summary, the present data support that a decrease in the levels of cAMP and cGMP in the POA is associated with the genesis of fever.
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PMID:Role of preoptic second messenger systems (cAMP and cGMP) in the febrile response. 1210 73

1. Regulation of the slowly activating component of delayed rectifier K(+) current (I(Ks)) by intracellular guanosine 3'5' cyclic monophosphate (cGMP) was investigated in guinea-pig sino-atrial (SA) node cells using the whole-cell patch-clamp method. 2. When a cell was dialyzed with pipette solution containing 100 micro M cGMP, I(Ks) started to gradually increase and reached a maximum increase of a factor of 2.37 +/- 0.39 (n = 4) about 10-15 min after rupture of patch membrane. Atrial natriuretic peptide (ANP, 100 nM) also potentiated I(Ks), consistent with intracellular cGMP-induced enhancement of I(Ks). 3. Bath application of a selective blocker of the cGMP-inhibited phosphodiesterase (PDE3) milrinone (100 microM) enhanced I(Ks) by a factor of 1.50 +/- 0.09 (n = 4) but failed to further enhance I(Ks) after a maximum stimulation by intracellular cGMP (100 microM), suggesting that blockade of PDE3 activity is involved in the enhancement of I(Ks). A potent but nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 microM) further increased I(Ks) stimulated by 100 microM milrinone, indicating that PDE subtypes other than PDE3 are also involved in the regulation of basal I(Ks) in guinea-pig SA node cells. 4. Bath application of 100 microM 8-bromoguanosine 3'5' cyclic monophosphate (8-Br-cGMP) increased I(Ks) by a factor of 1.48 +/- 0.11 (n = 5) and this stimulatory effect was totally abolished by cGMP-dependent protein kinase (PKG) inhibitor KT-5823 (500 nM), suggesting that the activation of PKG also mediates cGMP-induced potentiation of I(Ks). 5. These results strongly suggest that intracellular cGMP potentiates I(Ks) not only by blocking PDE3 but also by activating PKG in guinea-pig SA node cells.
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PMID:Potentiation of slow component of delayed rectifier K(+) current by cGMP via two distinct mechanisms: inhibition of phosphodiesterase 3 and activation of protein kinase G. 1218 38


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