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cGMP Concentrations (cgmp + concentration)
Selected AbstractsInhibition of carbachol-evoked oscillatory currents by the NO donor sodium nitroprusside in guinea-pig ileal myocytesEXPERIMENTAL PHYSIOLOGY, Issue 4 2005Seung-Soo Chung The effect of sodium nitroprusside (SNP) on carbachol (CCh)-evoked inward cationic current (Icat) oscillations in guinea-pig ileal longitudinal myocytes was investigated using the whole-cell patch-clamp technique and permeabilized longitudinal muscle strips. SNP (10 ,m) completely inhibited Icat oscillations evoked by 1 ,m CCh. 1H-(1,2,4) Oxadiazole [4,3-a] quinoxaline-1-one (ODQ; 1 ,m) almost completely prevented the inhibitory effect of SNP on Icat oscillations. 8-Bromo-guanosine 3,,5,-cyclic monophosphate (8-Br-cGMP; 30 ,m) in the pipette solution completely abolished Icat oscillations. However, a pipette solution containing Rp-8-Br-cGMP (30 ,m) almost completely abolished the inhibitory effect of SNP on Icat oscillations. When the intracellular calcium concentration ([Ca2+]i) was held at a resting level using BAPTA (10 mm) and Ca2+ (4.6 ,m) in the pipette solution, CCh (1 ,m) evoked only the sustained component of Icat without any oscillations and SNP did not affect the current. A high concentration of inositol 1,4,5-trisphosphate (IP3; 30 ,m) in the patch pipette solutions significantly reduced the inhibitory effect of SNP (10 ,m) on Icat oscillations. SNP significantly inhibited the Ca2+ release evoked by either CCh or IP3 but not by caffeine in permeabilized preparations of longitudinal muscle strips. These results suggest that the inhibitory effects of SNP on Icat oscillations are mediated, in part, by functional modulation of the IP3 receptor, and not by the inhibition of cationic channels themselves or by muscarinic receptors in the plasma membrane. This inhibition seems to be mediated by an increased cGMP concentration in a protein kinase G-dependent manner. [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] YC-1 increases cyclo-oxygenase-2 expression through protein kinase G- and p44/42 mitogen-activated protein kinase-dependent pathways in A549 cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2002Ming-Shyan Chang YC-1, an activator of soluble guanylate cyclase (sGC), has been shown to increase the intracellular cGMP concentration. This study was designed to investigate the signaling pathway involved in the YC-1-induced COX-2 expression in A549 cells. YC-1 caused a concentration- and time-dependent increase in COX activity and COX-2 expression in A549 cells. Pretreatment of the cells with the sGC inhibitor (ODQ), the protein kinase G (PKG) inhibitor (KT-5823), and the PKC inhibitors (Go 6976 and GF10923X), attenuated the YC-1-induced increase in COX activity and COX-2 expression. Exposure of A549 cells to YC-1 caused an increase in PKC activity; this effect was inhibited by ODQ, KT-5823 or Go 6976. Western blot analyses showed that PKC-,, -,, -,, -, and -, isoforms were detected in A549 cells. Treatment of A549 cells with YC-1 or PMA caused a translocation of PKC-,, but not other isoforms, from the cytosol to the membrane fraction. Long-term (24 h) treatment of A549 cells with PMA down-regulated the PKC-,. The MEK inhibitor, PD 98059 (10,50 ,M), concentration-dependently attenuated the YC-1-induced increases in COX activity and COX-2 expression. Treatment of A549 cells with YC-1 caused an activation of p44/42 MAPK; this effect was inhibited by KT-5823, Go 6976, long-term (24 h) PMA treatment or PD98059, but not the p38 MAPK inhibitor, SB 203580. These results indicate that in human pulmonary epithelial cells, YC-1 might activate PKG through an upstream sGC/cGMP pathway to elicit PKC-, activation, which in turn, initiates p44/42 MAPK activation, and finally induces COX-2 expression. British Journal of Pharmacology (2002) 136, 558,567; doi:10.1038/sj.bjp.0704777 [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] Suppression of cyclic GMP-specific phosphodiesterase 5 promotes apoptosis and inhibits growth in HT29 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2005Bing Zhu Abstract Phosphodiesterase 5 (PDE5) is a major isoform of cGMP phosphodiesterase in a variety of human tumor cell lines and plays a key role in regulating intracellular cGMP concentrations ([cGMP]i). Here, we demonstrate that suppression of PDE5 gene expression by antisense pZeoSV2/ASP5 plasmid transfection results in a sustained increase in [cGMP]i, growth inhibition, and apoptosis in human colon tumor HT29 cells. With stable transfection, antisense transcripts exhibited a specific suppression in PDE5 activity, mRNA levels, and a 93 kDa hPDE5A1 protein. In cloned antisense cells, prolongation of the cell growth doubling times correlate positively with suppressed PDE5 activity and increased [cGMP]i. The growth inhibition in PDE5 antisense clones is due to an increased apoptotic rate and delayed cell-cycle progression. These results corroborate previous findings with the PDE5 inhibitor exisulind and its derivatives showing that sustained [cGMP]i induces apoptosis and growth inhibition in tumor cells. Furthermore, an inducible mitotic inhibitor p21WAF1/CIP1 has been found to account for the delay of cell-cycle progression in PDE5 antisense clones at G2/M phase. A proteolytic cleavage of p21WAF1/CIP1 in the antisense clones is also increased at the later stage of serum stimulation. The protein kinase G (PKG) inhibitor, KT5823, can prevent the cleavage of p21WAF1/CIP. These data substantiate a pivotal role for PDE5 as a modulator of apoptosis and cell-cycle progression for human carcinoma via a mechanism involving the activation of [cGMP]i/PKG signaling pathways. © 2004 Wiley-Liss, Inc. [source] Modulation of the cGMP signaling pathway by melatonin in pancreatic , -cellsJOURNAL OF PINEAL RESEARCH, Issue 2 2009Ina Stumpf Abstract:, Melatonin influences the second messenger cyclic guanosine 3,,5,-monophosphate (cGMP) signaling pathway in pancreatic , -cells via a receptor-mediated mechanism. In the present study, it was determined how the regulation of cGMP concentrations by melatonin proceeds. The results provide evidence that melatonin acts via the soluble guanylate cyclase (sGC), as molecular investigations demonstrated that long-term incubation with melatonin significantly reduced the expression levels of the sGC mRNA in rat insulinoma , -cells (INS1) cells, whereas mRNA expression of membrane guanylate cyclases was unaffected. Incubation with melatonin abolished the S-nitrosoacetyl penicillamine-induced increase of cGMP concentrations in INS1 cells. In addition, the cGMP-inhibitory effect of melatonin was reversed by preincubation with the sGC inhibitors 1H-(1,2,4)oxadiazolo(4,3- ,)quinoxalin-1-one and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one. Nitric oxide (NO) production was not influenced after 1 hr of melatonin application, but was influenced after a 4 hr incubation period. Preincubation of INS1 cells with the NO synthase inhibitor NG -monomethyl- l -arginine did not abolish the cGMP-inhibitory effect of melatonin. Transcripts of cyclic nucleotide-gated (CNG) channels were significantly reduced after melatonin treatment in a dose-dependent manner, indicating the involvement of these channels in mediating the melatonin effect in INS1 cells. The results of this study demonstrate that melatonin mediates its inhibitory effect on cGMP concentrations in pancreatic , -cells by inhibiting the sGC, but does not influence NO concentration or NO synthase activity in short-term incubation experiments. In addition, it was demonstrated that melatonin is involved in modulation of CNG channel mRNA. [source] Insulin-Like Growth Factor-1 Restores Erectile Function in Aged Rats: Modulation the Integrity of Smooth Muscle and Nitric Oxide-Cyclic Guanosine Monophosphate Signaling ActivityTHE JOURNAL OF SEXUAL MEDICINE, Issue 6 2008Xiao-Yong Pu MD ABSTRACT Introduction., Insulin-like growth factor-1 (IGF-1) is one of the growth factors that have a wide range of biologic effects. We have confirmed that gene transfer of IGF-1 to the penis could improve erectile capacity. However, there are some limitations in gene therapies, such as toxicity or a risk of insertional mutagenesis. Protein treatment may be another choice for decreasing these risks. Aim., To investigate whether intracavernosal injection of IGF-1 protein can restore erectile function in the aging rat. Main Outcome Measures., Erectile responses, morphological changes, and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling pathways-related marker were determined. Methods., Ten young (4 months) and 30 old (24 months) Sprague-Dawley male rats were enrolled in this study. The old rats were divided into three groups: vehicle-only (N = 10), IGF-1 1 µg/kg (N = 10) and IGF-1 10 µg/kg treatment group (N = 10). After 4 and 8 weeks of single IGF-1 injection treatment, intracavernous pressure (ICP) responses with electrical stimulation to the cavernous nerve were evaluated. The percent of smooth muscle in corpus cavernosum tissue, the expression of mRNA and protein of endothelial nitric oxide synthase (eNOS) were also evaluated. The activity of nitric oxide synthase (NOS) and concentration of guanosine 3,,5,-cyclic-monophosphate (cGMP) that act upon the major NO-cGMP signaling pathways in penile tissue were also analyzed. Results., After IGF-1 treatment, the ICP responses was significantly increased as the young control group in both the IGF-1 1 µg/kg and the IGF-1 10 µg/kg group compared with the vehicle-only group at 4 and 8 weeks (P < 0.05). Masson's trichrom staining showed the percentage of cavernosal smooth muscle was increased in IGF-1 treatment group. IGF-1 increased e-NOS expression. NOS activities and cGMP concentrations were also significantly increased in IGF-1 treatment rats. Conclusions., IGF-1 improved erectile function in aged rats via restoration the integrity of smooth muscle of corpus cavernosum and modulation of NO-cGMP pathways. Pu, X-Y, Wang X-H, Gao W-C, Yang Z-H, and Li S-L. Insulin-like growth factor-1 restores erectile function in aged rats: Modulation the integrity of smooth muscle and nitric oxide-cyclic guanosine monophosphate signaling activity. J Sex Med 2008;5:1345,1354. [source] | |||||||||||||