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

We have examined the regulation of neuronal nitric oxide synthase (NOS) by phosphorylation with cyclic-GMP (PKG) and cyclic-AMP-dependent (PKA) protein kinases. In vitro phosphorylation studies indicate that both PKG and PKA phosphorylate NOS on a single site. Phosphoamino-acid analysis and peptide mapping demonstrate that phosphorylation by either cyclic-nucleotide kinase occurs on a similar serine residue. Phosphorylation of purified NOS by either PKG or PKA diminishes catalytic activity. Stimulation by 8-Br-cGMP of HEK-293 cells stably transfected with the cDNA for neuronal NOS (293.NOS cells) results in phosphorylation of immunoprecipitated NOS. Incubation of 293-NOS cells with 8-bromo-cGMP or dibutyryl-cAMP reduces nitrite release in response to stimulation with calcium ionophore A23187. Phosphorylation-induced decreases in NOS activity may counterbalance and modulate NOS activating signals.
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PMID:Cyclic nucleotide dependent phosphorylation of neuronal nitric oxide synthase inhibits catalytic activity. 753 10

Cisplatin resistance, induced in murine fibrosarcoma cells (SSK) in vitro or in vivo by low-dose irradiation, can be overcome by activation of the cyclic GMP(cGMP)-dependent transduction pathway. This is mediated either by stimulating cGMP formation with sodium nitroprusside or by replacing cGMP with a selective activator of the cGMP-dependent protein kinase, 8-bromo-cGMP. The cyclic AMP-dependent transduction pathway is not involved in cisplatin resistance. Instead, activation of cAMP sensitises both parental and resistant SSK cells equally to the action of cisplatin. There is a 1.8 to 2.5-fold increase in drug toxicity, depending on the activating agent. Enhancement of cisplatin sensitivity is induced by specific inhibition of cAMP hydrolysis, increase in cAMP formation or by increasing the activation potential to cAMP-dependent protein kinase by specific cAMP analogues. Cells that have lost cisplatin resistance respond to cGMP- or cAMP-elevating agents in the same way as the parental SSK cells. The radiation sensitivity is unchanged in all cell lines, even after activation of cAMP or cGMP. These results suggest that specific DNA repair pathways are altered by radiation but affected only in cisplatin damage repair, which is regulated by cGMP. Although there is ample cooperativity and interaction between the cAMP- and the cGMP-dependent transduction pathways, specific substrate binding by cGMP appears to play an important role in radiation-induced cisplatin resistance.
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PMID:Reversal of radiation-induced cisplatin resistance in murine fibrosarcoma cells by selective modulation of the cyclic GMP-dependent transduction pathway. 764 Feb 7

Natriuretic peptides inhibit the release and action of many hormones through cyclic guanosine monophosphate (cGMP), but the mechanism of cGMP action is unclear. In frog ventricular muscle and guinea-pig hippocampal neurons, cGMP inhibits voltage-activated Ca2+ currents by stimulating phosphodiesterase activity and reducing intracellular cyclic AMP; however, this mechanism is not involved in the action of cGMP on other channels or on Ca2+ channels in other cells. Natriuretic peptide receptors in the rat pituitary also stimulate guanylyl cyclase activity but inhibit secretion by increasing membrane conductance to potassium. In an electrophysiological study on rat pituitary tumour cells, we identified the large-conductance, calcium- and voltage-activated potassium channels (BK) as the primary target of another inhibitory neuropeptide, somatostatin. Here we report that atrial natriuretic peptide also stimulates BK channel activity in GH4C1 cells through protein dephosphorylation. Unlike somatostatin, however, the effect of atrial natriuretic peptide on BK channel activity is preceded by a rapid and potent stimulation of cGMP production and requires cGMP-dependent protein kinase activity. Protein phosphatase activation by cGMP-dependent kinase could explain the inhibitory effects of natriuretic peptides on electrical excitability and the antagonism of cGMP and cAMP in many systems.
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PMID:Potassium channel stimulation by natriuretic peptides through cGMP-dependent dephosphorylation. 767 99

The Alzheimer's disease (AD) beta-amyloid precursor proteins (beta APPs) are large membrane-spanning proteins that give rise to the beta A4 peptide deposited in AD amyloid plaques. beta APPs can also yield soluble forms (APPss) that are potently neuroprotective against glucose deprivation and glutamate toxicity, perhaps through their ability to lower the intraneuronal calcium concentration ([Ca2+]i). We have investigated the mechanism through which APPss exert these effects on cultured hippocampal neurons. The ability of APPss to lower rapidly [Ca2+]i was mimicked by membrane-permeable analogues of cyclic AMP (cAMP) and cyclic GMP (cGMP), as well as agents that elevate endogenous levels of these cyclic nucleotides. However, only cGMP content was increased by APPs treatment, and specific inhibition of cGMP-dependent protein kinase (but not cAMP-dependent kinase) blocked the activity of APPss. A membrane-permeable analogue of cGMP (8-bromo-cGMP) also mimicked the ability of APPss to attenuate the elevation of [Ca2+]i by glutamate, apparently through inhibition of NMDA receptor activity. In addition, 8-bromo-cGMP afforded protection against glucose deprivation and glutamate toxicity, and the protection by APPss against glucose deprivation was blocked by an inhibitor of cGMP-dependent kinase. Together, these data suggest that APPss mediate their [Ca2+]i-lowering and excitoprotective effects on target neurons through increases in cGMP levels.
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PMID:Role of cyclic GMP in the regulation of neuronal calcium and survival by secreted forms of beta-amyloid precursor. 772 92

Prior studies indicate that the natriuretic effects of atrial natriuretic peptide (ANP) are due, in part, to an inhibition of the passive movement of sodium ions from tubular lumen through apical cation channels into renal tubular epithelium. The present work demonstrates that ANP also exerts a potent inhibitory effect on the active pumping of sodium ions by renal tubular sodium and potassium-activated adenosine triphosphatase (Na, K-ATPase). This action of ANP is relatively long lasting, is due to a change in enzyme Vmax and is specific for ouabain-sensitive activity. Enzyme modulation occurs with an EC50 for ANP of 0.1 nM, is independent of intracellular [Na+] and is associated with an increase in tissue cyclic GMP (cGMP), but not cyclic AMP (cAMP). Modulation of Na, K-ATPase by ANP is mimicked by 8-bromo-cGMP and okadaic acid (OA) and is blocked by KT 5823, a selective inhibitor of cGMP-dependent protein kinase (PKG), but not by KT 5720, a selective inhibitor of cyclic AMP-dependent protein kinase (PKA), which suggests that the action of ANP on the sodium pump involves cGMP-mediated changes in protein phosphorylation. Regulation of renal Na, K-ATPase activity also occurs with nitric oxide-generating compounds, such as nitroglycerin and sodium nitroprusside (SNP). However, the ability of ANP to modulate Na, K-ATPase does not appear to involve this latter pathway because the effects of ANP on the sodium pump cannot be blocked by either N omega-nitro-L-arginine, an inhibitor of NO synthase, or hemoglobin, which blocks NO through binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Atrial natriuretic peptide modulates sodium and potassium-activated adenosine triphosphatase through a mechanism involving cyclic GMP and cyclic GMP-dependent protein kinase. 789 13

Ultraviolet irradiation of human platelet cytosol in the presence of 32P-labelled cyclic GMP (cGMP) can specifically label 110, 80, 55, 49 and 38 kDa proteins; the 110 kDa species is the subunit of cGMP-inhibited phosphodiesterase (PDE III) and the 80 kDa species that of cGMP-dependent protein kinase (Tang et al., 1993, Biochem. J. 294, 329). We have now shown that although photolabelling of platelet PDE III was inhibited by unlabelled cGMP, 8-bromo-cGMP and cyclic AMP (cAMP), it was not affected by phosphorothioate analogues of these cyclic nucleotides. Specific concentration-dependent inhibitions of the photolabelling of PDE III were observed with the following PDE inhibitors: trequinsin (IC50 = 13 +/- 2 nM), lixazinone (IC50 = 22 +/- 4 nM), milrinone (IC50 = 56 +/- 12 nM), cilostamide (IC50 = 70 +/- 9 nM), siguazodan (IC50 = 117 +/- 29 nM) and 3-isobutyl 1-methylxanthine (IBMX) (IC50 = 3950 +/- 22 nM). Thus, measurements of the inhibitory effects of compounds on the photolabelling of platelet PDE III provide a simple quantitative means of investigating their actions at a molecular level that avoids the need to purify the enzyme. Photolabelling of rat platelet lysate or rat heart homogenate by [32P]cGMP showed that the 110 kDa PDE III present in human material was replaced by a 115 kDa protein, labelling of which was also blocked by PDE III inhibitors. Heart and other rat tissues contained much less of this putative 115 kDa PDE III than rat platelets. In contrast, the 80 kDa protein was labelled much less in platelets than in many other rat tissue homogenates (e.g., heart, aorta, uterus and lung). Thus, comparison of the relative amounts of specific photolabelled proteins in different cells may provide an indication of different patterns of cyclic nucleotide action. We compared the abilities of phosphodiesterase inhibitors to block the photolabelling of PDE III in human platelet cytosol and to increase the iloprost-stimulated accumulation of cAMP in intact platelets. Whereas trequinsin (EC50 = 19 +/- 3 nM), lixazinone (EC50 = 122 +/- 8 nM), milrinone (EC50 = 5320 +/- 970 nM) and siguazodan (EC50 = 18880 +/- 3110 nM) all increased platelet cAMP to the same maximum extent, cilostamide and IBMX increased cAMP further, indicating that they inhibited a PDE isozyme in addition to PDE III.
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PMID:Photoaffinity labelling of cyclic GMP-inhibited phosphodiesterase (PDE III) in human and rat platelets and rat tissues: effects of phosphodiesterase inhibitors. 792 8

1 The effects of two 8-substituted analogues of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were compared with those of forskolin and isoprenaline on [3H]-noradrenaline release and vasoconstriction induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery, in the absence and in the presence of protein kinase inhibitors. 2 8-Bromo-adenosine 3':5'-cyclic monophosphate (8-bromo-cyclic AMP, 10-300 microM), 8-(4-chlorophenyl-thio)-adenosine 3':5' cyclic monophosphate (8-pCPT-cyclic AMP, 3-300 microM), forskolin (0.3-10 microM) and isoprenaline (1 nM-1 microM) all concentration-dependently enhanced stimulation-induced [3H]-noradrenaline release. The effect of cyclic AMP analogues was larger (2.5 fold at 300 microM) than those of cyclic AMP elevating drugs (1.6 fold at 10 microM for forskolin and 1.5 fold at 30 nM for isoprenaline). 3 At concentrations active at the prejunctional level, the four drugs had differential effects on stimulation-induced vasoconstriction, which was enhanced by the two cyclic AMP analogues, decreased by forskolin and not significantly altered by isoprenaline. 4 The [3H]-noradrenaline release-enhancing effects of 8-bromo-cyclic AMP, forskolin and isoprenaline were significantly decreased by the cyclic AMP-dependent protein kinase (PKA) inhibitor (N-[2-((3-(4-bromophenyl)-2-propenyl)-amino)-ethyl]-5- isoquinolinesulphonamide, di-hydrochloride) (H-89; 100 nM). By contrast they were unaffected by the cyclic GMP-dependent protein kinase (PKG) inhibitor, 8-bromo-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-bromo-cyclic GMPS; 10 microM). By contrast they were unaffected by the cyclic GMP-dependent protein kinase (PKG) inhibitor,8-bromo-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-bromo-cyclic GMPS; 10 MicroM).At the same concentrations the PKA inhibitor attenuated only the nerve-induced vasoconstrictor responses obtained in the presence of 8-bromo-cyclic AMP, whereas the PKG inhibitor did not modify that obtained in the presence of 8-bromo-cycic AMP or forskolin.5. Exposure to the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (1 MicroM) enhanced nerve-evoked [3H]-noradrenaline release, and this effect was decreased by the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(-indol-3-yl)-maleimide (GF 109203X; 100 nM). However, the latter drug did not modify the enhancing effect of 8-bromo-cyclic AMP on [3H]-noradrenaline release.6. It is concluded that activation of cyclic AMP-dependent protein kinase is involved in the enhancing effect of cyclic AMP-elevating compounds on prejunctional release of noradrenaline. In addition the results provide no clear-cut evidence for a vasodilator role of PKA.
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PMID:Effects of cyclic AMP and analogues on neurogenic transmission in the rat tail artery. 800 6

The photoaffinity labelling of platelet cyclic GMP (cGMP)-binding proteins by [32P]cGMP was studied; at least five labelled proteins (110, 80, 55, 49 and 38 kDa) were detected in platelet cytosol and four (80, 65, 49 and 38 kDa) in platelet membranes. The 110 kDa species was identified as cGMP-inhibited cyclic AMP (cAMP) phosphodiesterase (PDE III) by immunoprecipitation and by the inhibition of photolabelling by specific inhibitors of this enzyme. Similarly, the 80 kDa species was identified as cGMP-dependent protein kinase by immunoprecipitation and by the effects of cGMP analogues on photolabelling. Addition of cAMP greatly enhanced the labelling of this 80 kDa protein, implying the existence of a potentially important interaction between the effects of cGMP and cAMP. The 65 kDa photolabelled protein appears to be a novel platelet cyclic-nucleotide-binding protein. In contrast, the 49 and 55 kDa photolabelled species are probably the RI and RII regulatory subunits of cAMP-dependent protein kinase, and the 38 kDa protein(s) may be proteolytic fragment(s) of RI and/or RII.
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PMID:Photoaffinity labelling of cyclic GMP-binding proteins in human platelets. 839 9

1. The modulation of the guanosine 3':5'-cyclic monophosphate (cyclic GMP)- and adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase activities by the diastereomers of 8-bromo-beta phenyl-1, N2-ethenoguanosine 3':5'-cyclic monophosphorothioate, ((Rp)- and (Sp)-8-bromo-PET-cyclic GMPS) was investigated by use of purified protein kinases. In addition, the effects of (Rp)-8-bromo-PET-cyclic GMPS on protein phosphorylation in intact human platelets and on [3H]-noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries were also studied. 2. Kinetic analysis with purified cyclic GMP-dependent protein kinase (PKG) type I alpha and I beta, which are expressed in the rat tail artery, revealed that (Rp)-8-bromo-PET-cyclic GMPS is a competitive inhibitor with an apparent Ki of 0.03 microM. The activation of purified cyclic AMP-dependent protein kinase (PKA) type II was antagonized with an apparent Ki of 10 microM. 3. In human platelets, (Rp)-8-bromo-PET-cyclic GMPS (0.1 mM) antagonized the activation of the PKG by the selective activator 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate (8-pCPT-cyclic GMP; 0.2 mM) without affecting the activation of PKA by (Sp)-5, 6-dichloro-1-beta-D-ribofurano-sylbenzimidazole- 3':5'-cyclic monophosphorothioate ((Sp)-5,6-DCl-cyclic BiMPS; 0.1 mM). 4. (Rp)-8-bromo-PET-cyclic GMPS was not hydrolysed by the cyclic GMP specific phosphodiesterase (PDE) type V from bovine aorta but potently inhibited this PDE. 5. The corresponding sulphur free cyclic nucleotide of the two studied phosphorothioate derivatives, 8-bromo-beta-phenyl-1, N2-ethenoguanosine-3':5'-cyclic monophosphate (8-bromo-PET-cyclic GMP), had no effect on electrically-induced [3H]-noradrenaline release but concentration-dependently decreased the stimulation-induced vasoconstriction. (Rp)-8-bromo-PET-cyclic GMPS (3 microM) shifted the vasoconstriction response to the right without affecting stimulation evoked tritium overflow. 6. The NO donor, 3-morpholinosydnonimine (SIN-1) relaxed rat tail arteries precontracted with phenylephrine (1 microM). The SIN-1 concentration-relaxation curve was shifted in a parallel manner to the right by (Rp)-8-bromo-PET-cyclic GMPS, suggesting that the relaxation was mediated by a cyclic GMP/PKG-dependent mechanism. 7. The [3H]-noradrenaline release-enhancing effect and stimulation-induced decrease in vasoconstriction of forskolin were unaffected by (Rp)-8-bromo-PET-cyclic GMPS. Moreover, the forskolin concentration-relaxation curve was not changed in the presence of the PKG inhibitor, suggesting a high selectivity in intact cells for PKG- over PKA-mediated effects. 8. The results obtained indicate that (Rp)-8-bromo-PET-cyclic GMPS presently is the most potent and selective inhibitor of PKG and is helpful in distinguishing between cyclic GMP and cyclic AMP messenger pathways activation. Therefore, this phosphorothioate stereomer may be a useful tool for studying the role of cyclic GMP in vitro.
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PMID:Inhibition of cyclic GMP-dependent protein kinase-mediated effects by (Rp)-8-bromo-PET-cyclic GMPS. 871 84

In the goldfish, it has been proposed that gonadotropin (GTH) release induced by GTH-releasing hormone (GnRH) involves Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC), protein kinase C (PKC) activation, and arachidonic acid (AA) metabolism, but not cyclic AMP (cAMP) action. However, cAMP appears to mediate GnRH action in other teleosts. In this study, the relative importance of PKC and cAMP in mediating GnRH action in goldfish was studied using primary cultures of dispersed pituitary cells. Consistent with an involvement of PKC in GnRH action, the GTH responses to the PKC activating tetradecanoyl phorbol acetate (TPA), salmon (s)GnRH, and chicken (c)GnRH-II were inhibited by two selective PKC inhibitors, calphostin C, and staurosporine. Furthermore, GTH release responses induced by sGnRH or cGnRH-II were not additive to responses stimulated by the PKC-activating diglyceride DiC8, in either long-term static incubation or acute perifusion experiments. In static incubation studies, the GTH responses to sGnRH and DiC8 were potentiated by the VSCC agonist Bay K 8644, suggesting that VSCC participates in both PKC and GnRH action. Concentrations of K+ < 100 mM did not elicit GTH secretion when tested alone, but were effective in stimulating GTH release in the presence of subthreshold doses of DiC8 or TPA. This suggests that minimal activation of PKC greatly enhances the effectiveness of Ca2+ influx to increase GTH secretion. Taken together, these results indicate that PKC is an important mediator of GnRH-induced, VSCC-dependent GTH release. In contrast to the involvement of PKG, cAMP-dependent mechanisms showed no evidence of direct participation in GnRH-induced GTH release in goldfish. In static incubation studies, the GTH responses to sGnRH and cGnRH-II were not affected by H89, a cAMP-dependent protein kinase (PKA) inhibitor. Furthermore, the GTH release stimulated by cAMP was additive to the response to sGnRH, cGnRH-II, DiC8, TPA, or AA. However, compared to the response to forskolin or TPA alone, combinations of forskolin and TPA resulted in a potentiated increase in GTH release. The acute GTH response to forskolin was also enhanced by DiC8. Thus, cAMP-dependent mechanisms may constitute an independent pathway that interacts positively with GnRH-dependent mechanisms in the regulation of GTH release.
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PMID:Interactions between signaling pathways in mediating GnRH-stimulated GTH release from goldfish pituitary cells: protein kinase C, but not cyclic AMP is an important mediator of GnRH-stimulated gonadotropin secretion in goldfish. 880 63


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