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cGMP-dependent Protein Kinase (cgmp-dependent + protein_kinase)
Selected AbstractsEndocannabinoids mediate muscarine-induced synaptic depression at the vertebrate neuromuscular junctionEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007Zachary Newman Abstract Endocannabinoids (eCBs) inhibit neurotransmitter release throughout the central nervous system. Using the Ceratomandibularis muscle from the lizard Anolis carolinensis we asked whether eCBs play a similar role at the vertebrate neuromuscular junction. We report here that the CB1 cannabinoid receptor is concentrated on motor terminals and that eCBs mediate the inhibition of neurotransmitter release induced by the activation of M3 muscarinic acetylcholine (ACh) receptors. N -(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, a CB1 antagonist, prevents muscarine from inhibiting release and arachidonylcyclopropylamide (ACPA), a CB1 receptor agonist, mimics M3 activation and occludes the effect of muscarine. As for its mechanism of action, ACPA reduces the action-potential-evoked calcium transient in the nerve terminal and this decrease is more than sufficient to account for the observed inhibition of neurotransmitter release. Similar to muscarine, the inhibition of synaptic transmission by ACPA requires nitric oxide, acting via the synthesis of cGMP and the activation of cGMP-dependent protein kinase. 2-Arachidonoylglycerol (2-AG) is responsible for the majority of the effects of eCB as inhibitors of phospholipase C and diacylglycerol lipase, two enzymes responsible for synthesis of 2-AG, significantly limit muscarine-induced inhibition of neurotransmitter release. Lastly, the injection of (5Z,8Z,11Z,14Z)- N -(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (an inhibitor of eCB transport) into the muscle prevents muscarine, but not ACPA, from inhibiting ACh release. These results collectively lead to a model of the vertebrate neuromuscular junction whereby 2-AG mediates the muscarine-induced inhibition of ACh release. To demonstrate the physiological relevance of this model we show that the CB1 antagonist N -(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide prevents synaptic inhibition induced by 20 min of 1-Hz stimulation. [source] The hinge region operates as a stability switch in cGMP-dependent protein kinase I,FEBS JOURNAL, Issue 9 2007Arjen Scholten The molecular mechanism of cGMP-dependent protein kinase activation by its allosteric regulator cyclic-3,,5,-guanosine monophosphate (cGMP) has been intensely studied. However, the structural as well as thermodynamic changes upon binding of cGMP to type I cGMP-dependent protein kinase are not fully understood. Here we report a cGMP-induced shift of Gibbs free enthalpy (,,GD) of 2.5 kJ·mol,1 as determined from changes in tryptophan fluorescence using urea-induced unfolding for bovine PKG I,. However, this apparent increase in overall stability specifically excluded the N-terminal region of the kinase. Analyses of tryptic cleavage patterns using liquid chromatography-coupled ESI-TOF mass spectrometry and SDS/PAGE revealed that cGMP binding destabilizes the N-terminus at the hinge region, centered around residue 77, while the C-terminus was protected from degradation. Furthermore, two recombinantly expressed mutants: the deletion fragment ,1-77 and the trypsin resistant mutant Arg77Leu (R77L) revealed that the labile nature of the N-terminus is primarily associated with the hinge region. The R77L mutation not only stabilized the N-terminus but extended a stabilizing effect on the remaining domains of the enzyme as well. These findings support the concept that the hinge region of PKG acts as a stability switch. [source] Phosphorylation of phosphodiesterase-5 by cyclic nucleotide-dependent protein kinase alters its catalytic and allosteric cGMP-binding activitiesFEBS JOURNAL, Issue 9 2000Jackie D. Corbin In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem.265, 14971,14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50,70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2,0.5 ,m PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the Km value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 ,m. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed. [source] G-substrate gene promoter SNP (,1323T>C) modifies plasma total cholesterol and triglyceride phenotype in familial hypercholesterolemia: Intra-familial association study in an eight-generation hyperlipidemic kindredGERIATRICS & GERONTOLOGY INTERNATIONAL, Issue 2 2004Yukiko Nobe Background: Plasma lipid and lipoprotein generally reflect the complex influences of multiple genetic loci, for instance, even familial hypercholesterolemia (FH), a representative example of monogenic hyperlipidemia, often presents with phenotypic heterogeneity. Methods: In the course of investigating familial coronary artery disease in Utah, we studied 160 members of an eight-generation extended family of FH, to examine possible genetic modification of lipoprotein phenotype by ,modifier locus'. G-substrate (GSBS) is an endogenous substrate for cGMP-dependent protein kinase. We carried out an intrafamilial correlation analysis of modifier effect of ,1323T>C substitution in the GSBS gene among 85 LDLR-mutation carriers and 75 non-carriers. Results: In the LDLR - mutation carriers, the plasma cholesterol levels were highest among ,1323C homozygotes (mean ± SD = 454 ± 101 mg/dL), lowest among ,1323T homozygotes (mean ± SD, 307 ± 72 mg/dL) and intermediate among ,1323T/C heterozygotes (mean ± SD, 314 ± 62 mg/dL; P = 0.015). Similarly, in the LDLR-mutation carriers, the plasma triglyceride levels were highest among ,1323C homozygotes (mean ± SD, 371 ± 381 mg/dL), lowest among ,323T homozygotes (mean ± SD, 171 ± 94 mg/dL), and intermediate among ,1323T/C heterozygotes (mean ± SD, 218 ± 130 mg/dL; P = 0.003). No such gene-interactive effect was observed among non-carriers of the LDLR-mutation. Conclusion: These results indicate a significant modification of the phenotype of FH with defective LDLR allele, by GSBS-1323C allele in the kindred studied. [source] Putative Role of Carbon Monoxide Signaling Pathway in Penile Erectile FunctionTHE JOURNAL OF SEXUAL MEDICINE, Issue 1 2009Mohamed T. Abdel Aziz MD ABSTRACT Introduction., Erectile response depends on nitric oxide (NO) generated by NO synthase (NOS) enzyme of the nerves and vascular endothelium in the cavernous tissue. NO activates soluble guanylate cyclase (sGC), leading to the production of cyclic guanosine monophosphate (cGMP). cGMP activates cGMP-dependent protein kinase that activates Ca2+/ATPase pump that activates Ca2+/K efflux pump extruding Ca2+ across the plasma membrane with consequent smooth muscle cell relaxation. A role similar to that of NOS/NO signaling has been postulated for carbon monoxide (CO) produced in mammals from heme catabolism by heme oxygenase (HO) enzyme. Aim., To assess CO signaling pathway for erectile function by reviewing published studies. Methods., A systematic review of published studies on this affair based on Pubmed and Medical Subject Heading databases, with search for all concerned articles. Main Outcome Measures., Documentation of positive as well as negative criteria of CO/HO signaling focused on penile tissue. Results., The concept that HO-derived CO could play a role in mediating erectile function acting in synergism with, or as a potentiator for, NOS/NO signaling pathway is gaining momentum. CO/HO signaling pathway has been shown to partially mediate the actions of oral phosphodiesterase type 5 inhibitors. In addition, it was shown that the use of CO releasing molecules potentiated cavernous cGMP levels. However, increased CO production or release was reported to be associated, in some studies, with vasoconstriction. Conclusion., This review sheds a light on the significance of cavernous tissue CO signaling pathway that may pave the way for creation of therapeutic modalities based on this pathway. Abdel Aziz MT, Mostafa T, Atta H, Wassef MA, Fouad HH, Rashed LA, and Sabry D. Putative role of carbon monoxide signaling pathway in penile erectile function. J Sex Med 2009;6:49,60. [source] The locust foraging geneARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2010C. Lucas Abstract Our knowledge of how genes act on the nervous system in response to the environment to generate behavioral plasticity is limited. A number of recent advancements in this area concern food-related behaviors and a specific gene family called foraging (for), which encodes a cGMP-dependent protein kinase (PKG). The desert locust (Schistocerca gregaria) is notorious for its destructive feeding and long-term migratory behavior. Locust phase polyphenism is an extreme example of environmentally induced behavioral plasticity. In response to changes in population density, locusts dramatically alter their behavior, from solitary and relatively sedentary behavior to active aggregation and swarming. Very little is known about the molecular and genetic basis of this striking behavioral phenomenon. Here we initiated studies into the locust for gene by identifying, cloning, and studying expression of the gene in the locust brain. We determined the phylogenetic relationships between the locust PKG and other known PKG proteins in insects. FOR expression was found to be confined to neurons of the anterior midline of the brain, the pars intercerebralis. Our results suggest that differences in PKG enzyme activity are correlated to well-established phase-related behavioral differences. These results lay the groundwork for functional studies of the locust for gene and its possible relations to locust phase polyphenism. © 2010 Wiley Periodicals, Inc. [source] KMUP-1 activates BKCa channels in basilar artery myocytes via cyclic nucleotide-dependent protein kinasesBRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2005Bin-Nan Wu This study investigated whether KMUP-1, a synthetic xanthine-based derivative, augments the delayed-rectifier potassium (KDR)- or large-conductance Ca2+ -activated potassium (BKCa) channel activity in rat basilar arteries through protein kinase-dependent and -independent mechanisms. Cerebral smooth muscle cells were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K+ - and Ca2+ channel activities. KMUP-1 (1 ,M) had no effect on the KDR current but dramatically enhanced BKCa channel activity. This increased BKCa current activity was abolished by charybdotoxin (100 nM) and iberiotoxin (100 nM). Like KMUP-1, the membrane-permeable analogs of cGMP (8-Br-cGMP) and cAMP (8-Br-cAMP) enhanced the BKCa current. BKCa current activation by KMUP-1 was markedly inhibited by a soluble guanylate cyclase inhibitor (ODQ 10 ,M), an adenylate cyclase inhibitor (SQ 22536 10 ,M), competitive antagonists of cGMP and cAMP (Rp-cGMP, 100 ,M and Rp-cAMP, 100 ,M), and cGMP- and cAMP-dependent protein kinase inhibitors (KT5823, 300 nM and KT5720, 300 nM). Voltage-dependent L-type Ca2+ current was significantly suppressed by KMUP-1 (1 ,M), and nearly abolished by a calcium channel blocker (nifedipine, 1 ,M). In conclusion, KMUP-1 stimulates BKCa currents by enhancing the activity of cGMP-dependent protein kinase, and in part this is due to increasing cAMP-dependent protein kinase. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and the relaxation of cerebral arteries. British Journal of Pharmacology (2005) 146, 862,871. doi:10.1038/sj.bjp.0706387 [source] Poly- l -proline type II peptide mimics as probes of the active site occupancy requirements of cGMP-dependent protein kinaseCHEMICAL BIOLOGY & DRUG DESIGN, Issue 4 2005R. Zhang Abstract:, Based on the X-ray crystal structure of cAMP-dependent protein kinase (PKA) with the endogenous inhibitor PKI and the X-ray crystal structure of cyclin-dependent kinase 2 (CDK2) with a substrate peptide, a proposal is put forth that some protein kinases bind peptide substrates in their active sites in the poly- l -proline type II (PPII) conformation. In this work, PPII peptide mimics are evaluated as pseudosubstrate inhibitors of cGMP-dependent protein kinase (PKG) to explore if PKG also binds peptide substrates in the PPII conformation. Inhibition data of our PPII mimetics provide evidence that the P , 1, P , 2, and P , 3 residues of substrate peptides bind in the PPII conformation (, approximately ,75°, , approximately 145°). In addition, the inhibition data also suggest that the P , 1, P , 2, and P , 3 residues in substrate peptides bind with a gauche(,) ,1 angle. [source] | ||||||||||||||||