Amount Of Cyclic GMP Research Articles

Melatonin Modulates the Effects of Gastric Injury in Rats: Role of Prostaglandins and Nitric Oxide

Experimental evidence has been presented connecting melatonin with the prevention or treatment of gastrointestinal disorders either by the scavenging properties of active oxygen or by receptor-mediated stimulation of gene expression of neutralizing enzymes. Prostaglandins and nitric oxide are important neuroimmunomodulators in digestive physiology and different studies have indicated that the protective properties of melatonin may be explained by prostaglandin and/or nitric oxide mechanisms. The aim of the present study was to examine the effect of intraperitoneal administration of melatonin on in vivo changes in PGE₂, generated in gastric mucosal lesions by ischemia-reperfusion. Cyclic GMP nucleotide was also studied as an index of the principal enzymatic activity involved in the metabolism of nitric oxide, the nitric oxide synthase. The different immunological tests showed that the intraperitoneal administration of melatonin prevents the postischemic decrease in prostaglandins. The concentration of this eicosanoid in the rat mucosa treated with 20 mg·kg^–1 of melatonin was significantly higher (p < 0.05) than that in the control rats. The amount of cyclic GMP in the stomach decreased because of ischemia-reperfusion. In treated animals however, a marked increase occurred in concentrations of GMP, but the difference was not statistically significant. The results suggest that the mechanism of protection afforded by melatonin against lesions induced by gastric ischemia-reperfusion may be due to stimulation of the synthesis of eicosanoid protectors during the ischemic process.

Role of endothelium in biphasic hypoxic response of the isolated pulmonary artery in the rat.

We investigated the roles of the endothelium in the hypoxic responses of the isolated main pulmonary artery (PA) in the rat. Hypoxia was induced by gassing an organ chamber with 95% N2 + 5% CO2 (PO2 = 34.6 +/- 3.1 Torr) instead of 16% O2 + 5% CO2 + balance N2 (PO2 = 92.8 +/- 3.0 Torr). Vascular rings were precontracted with 2 x 10(-8) M phenylephrine. A transient hypoxic contraction and a subsequent relaxation were observed in the endothelium-intact rings. The hypoxic contraction was reduced in the endothelium-denuded rings. In contrast, there were no significant differences between the hypoxic relaxation in the endothelium-intact and endothelium-denuded rings. Inhibitors of endothelium-derived relaxing factor (EDRF) activity, 2 x 10(-6) M NG-monomethyl-L-arginine (L-NMMA) and 10(-6) M methylene blue, produced 53% and 66% reductions in hypoxic contraction, respectively, Furthermore, the amount of cyclic GMP in the endothelium-intact PA rings which had been precontracted with phenylephrine decreased from 2.10 +/- 0.45 pmol/mg protein during normoxia to 0.90 +/- 0.18 pmol/mg protein during hypoxia. Indomethacin and OKY-046 did not influence hypoxic contraction or relaxation. These results suggest that hypoxic contraction of the isolated pulmonary artery in the rat is partially induced by inhibition of the release of EDRF.

Release of prostacyclin, endothelium-derived relaxing factor and endothelin by freshly harvested cells attached to microcarrier beads.

Cultured endothelial cells have been used in the past as a source of endothelium-derived relaxing factor (EDRF) and of prostacyclin (PGI2). Although cell cultures are essential for observation of prolonged exposure to media or when there is delayed response, they are time consuming and sterile conditions are essential. In the present study, we report that endothelial cells, freshly harvested from bovine aortas, readily attached themselves to cytodex-3 microcarrier beads and released an endothelium-derived relaxing factor (EDRF), prostacyclin (PGI2) and increased the amount of cyclic GMP in vascular smooth muscle. Attachment to microcarrier beads was essential since it increased the surface area and the number of attached cells and permitted collection of cell free filtrates because of the formation of dense networks of cells and beads. As a result superfusion of cells and beads on the filter did not dislodge bound cells which remain on the filter. Conditioned filtrates from freshly harvested endothelial cells attached to microcarrier beads caused marked relaxation of endothelium-deprived bovine pulmonary artery strips. The degree of relaxation depended on the number of cells; maximal relaxation occurred with 50 million cells at ED50 of 14 million. High values of cyclic GMP were found in vascular smooth muscle exposed to conditioned filtrate. The calcium ionophore A23187 further increased the amount of cyclic GMP. Large amounts of PGI2 were released by freshly harvested endothelial cells particularly after stimulation with the calcium ionophore. In contrast, endothelin production by freshly harvested cells attached to microcarrier beads was barely detectable after 30 min incubation and was beyond the limit of detection by bioassay procedures. Freshly harvested endothelial cells attached to microcarrier beads appear to be a useful adjunct to tissue cultures under specific experimental conditions.

Cyclic nucleotides and the activity of glia maturation factor in the hypoplastic cerebellum of developing jaundiced Gunn rats.

Developmental changes of cyclic nucleotides were studied in the hypoplastic cerebellum of jaundiced Gunn rats over the period of postnatal days 8 to 30. The mitogenic activity of glia maturation factor was also measured at day 15. In jaundiced homozygotes (jj), the amount of cyclic GMP on a protein basis was not significantly different from that in control heterozygotes (j+) at either day 8 or 15, but at day 30 it was reduced to about 19% of the control. On the other hand, a lowered nucleotide level on a wet weight basis in jj rats was already statistically significant at day 15. In contrast to cyclic GMP, the rates of increase of cyclic AMP on a wet weight basis were almost the same in the two groups of rats, but the nucleotide levels on a protein basis at days 15 and 30 were a little, but significantly, higher in jj rats than in j+ rats. The activity of glia maturation factor in jj rats was found to be 1.5-3 times as high as that in j+ rats. Possible implications of the present results are discussed.

Receptor-mediated phosphorylation of spermatozoan proteins.

These studies are the first to report egg peptide-mediated stimulation of protein phosphorylation in spermatozoa. Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) or resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2) stimulated the incorporation of 32P into various proteins of isolated spermatozoan membranes in the presence, but not absence, of GTP. The Mr of three of the phosphorylated proteins were 52,000, 75,000, and 100,000. GTP gamma S (guanosine 5'-O-(3-thiotriphosphate] but not GDP beta S (guanosine 5'-O-(2-thiodiphosphate] or GMP-PNP (guanylyl imidodiphosphate) also supported the peptide-mediated stimulation of protein phosphorylation. The peptides markedly stimulated guanylate cyclase activity, and GTP gamma S or GTP but not GMP-PNP served as effective substrates for the enzyme. The accumulation of cyclic AMP was not stimulated by the peptides. Subsequently, it was shown that added cyclic GMP or cyclic AMP increased 32P incorporation into the same membrane proteins as those observed in the presence of peptide and GTP. The amount of cyclic GMP (up to 3 microM) formed by membranes in the presence of peptide and 100 microM GTP equated with the amount of added cyclic GMP required to increase the 32P content of a Mr 75,000 protein selected for further study. 32P-Peptide maps of the Mr 75,000 protein indicated that the same domains were phosphorylated in response to cyclic nucleotides or to egg peptide and GTP. Intact cells were subsequently incubated with 32P to determine if the radiolabeled proteins observed in isolated membranes also would be obtained in intact cells. The 32P contents of proteins of Mr 52,000, 75,000, and 100,000 were significantly increased by the addition of resact. Peptide maps confirmed that the increased 32P incorporation obtained in a Mr 75,000 protein of isolated membranes occurred on the same protein domains as the 32P found on the Mr 75,000 protein of intact cells. These results suggest that a GTP or GTP gamma S requirement for peptide-mediated protein phosphorylation in spermatozoan membranes is mainly due to the enhanced formation of cyclic GMP, and it therefore is likely that peptide-induced elevations of cyclic nucleotide concentrations in spermatozoa are responsible for the specific increases in 32P associated with at least three sperm proteins, all apparently localized on the plasma membrane.

Bacterial growth and the concentrations of cyclic nucleotides inLegionella pneumophila cultures

The growth kinetics ofLegionella pneumophila, and the rates of DNA and RNA synthesis in this organism, are qualitatively similar to that of other gram negative organisms of clinical importance. After a brief stationary phase, culture viability is rapidly lost. This loss of viability is not associated with detectable cell lysis or with significant breakdown of cellular DNA; it is, however, preceded by the rapid breakdown of approximately 50% of the cumulatively labeled RNA. During logarithmic growth in buffered yeast extract broth, bacterial cells secreted into the medium large amounts of cyclic GMP at both 37°C and room temperature, while those grown in a buffered synthetic broth did not. Cyclic AMP increased moderately during stationary phase at 37°C and room temperature in both media. Neither added cyclic AMP nor added cyclic GMP influenced the rate of growth ofL. pneumophila in either broth, with or without 1-methyl-3-isobutyl-xanthine, a phosphodiestease inhibitor. Dibutyrul cyclic AMP and 8-bromo cyclic GMP individually or in combination also had no effect on growth.

Urinary Excretion of Adenosine 3′,5′-Monophosphate and Guanosine 3′,5′-Monophosphate in Normal Children and Those with Cystic Fibrosis

The urinary excretion of adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic GMP) was examined in 98 normal children and 46 children with cystic fibrosis between the ages of 9 months and 18 yr. Diurnal variations in cyclic AMP and cyclic GMP excretion were observed in subjects from either group, and peak levels of cyclic nucleotide excretion were generally observed during the period of 0700 to 2100 h. Excretion rates (mumol/day) of cyclic AMP and cyclic GMP increased significantly with age. When cyclic AMP and cyclic GMP excretion rates were normalized for urinary creatine, or body weight, the values declined significantly with age in both groups of patients. Cyclic GMP excretion normalized for body surface area also decreased with age, while the value for cyclic AMP (2.86 plus or minus 0.08 mumol/day/m2, mean plus or minus SE) was constant with age in both normals and cystic fibrosis children. With some comparisons of age groups there were significant differences in cyclic nucleotide excretion between normal subjects and children with cystic fibrosis. The differences noted were dependent upon the methods used to normalize excretion rates (urinary creatine, body weight, surface area, and the ratio of cyclic AMP to cyclic GMP excreated). In general patients with cystic fibrosis excreted greater amounts of cyclic GMP than did normals. The most striking comparison was the ratio of cyclic AMP to cyclic GMP excreted which was 9.09 plus or minus 0.50 in all normal children and 4.41 plus or minus 0.32 in children with cystic fibrosis (P smaller than 0.001).