cyclic-gmp and dimethylamine

Impaired nitric oxide (NO) bioavailability and low levels of circulating endothelial progenitor cells (EPC) are correlated to an increased risk for development of cardiovascular diseases. We investigated whether improved systemic NO bioavailability and increased levels of EPC after GH treatment are related and mediated by the IGF-I.. Healthy middle-aged volunteers (n = 16) were treated for 10 d with recombinant human GH. Before and after GH treatment, we analyzed markers of NO bioavailability and EPC levels. GH treatment was responded by significant increases in plasma IGF-I levels. Urinary cGMP levels were increased and diastolic blood pressure reduced after GH treatment (P < 0.05). Likewise, plasma nitrate and nitrite levels were increased, whereas the NO synthase inhibitor asymmetric dimethylarginine was reduced. Correspondingly, IGF-I treatment increased expression of the asymmetric dimethylarginine-metabolizing enzyme dimethylarginie dimethylaminohydrolase-1 and dimethylarginie dimethylaminohydrolase-2 in cultured human endothelial cells. IGF-I levels correlated with cGMP concentrations (r = 0.51; P < 0.05). EPC numbers were increased after GH treatment and correlated with markers for NO bioavailability. These findings were also observed in mice treated with GH for 7 d. GH treatment additionally increased aortic endothelial NO synthase expression of mice. Importantly, blocking of the IGF-I receptor in vivo abolished the GH-mediated effects on markers of increased NO bioavailability.. GH treatment induced markers of increased NO bioavailability and enhanced circulating EPC numbers in healthy volunteers. Animal data demonstrate increased NO availability to be mediated via an increase in IGF-I plasma levels. Thus, GH treatment enhances systemic NO bioavailability via IGF-I and may be beneficial in certain cardiovascular diseases.

Topics: Aged; Amidohydrolases; Animals; Biomarkers; Cells, Cultured; Cyclic GMP; Dimethylamines; Dinoprost; Endothelial Cells; Female; Glyceraldehyde-3-Phosphate Dehydrogenases; Growth Hormone; Human Growth Hormone; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Male; Mice; Middle Aged; Nitrates; Nitric Oxide; Nitrites; Stem Cells; Vascular Endothelial Growth Factor A

The selectivity of Ca2+ over Na+ is approximately 3.3-fold larger in cGMP-gated channels of cone photoreceptors than in those of rods when measured under saturating cGMP concentrations, where the probability of channel opening is 85-90%. Under physiological conditions, however, the probability of opening of the cGMP-gated channels ranges from its largest value in darkness of 1-5% to essentially zero under continuous, bright illumination. We investigated the ion selectivity of cGMP-gated channels as a function of cyclic nucleotide concentration in membrane patches detached from the outer segments of rod and cone photoreceptors and have found that ion selectivity is linked to gating. We determined ion selectivity relative to Na+ (PX/PNa) from the value of reversal potentials measured under ion concentration gradients. The selectivity for Ca2+ over Na+ increases continuously as the probability of channel opening rises. The dependence of PCa/PNa on cGMP concentration, in both rods and cones, is well described by the same Hill function that describes the cGMP dependence of current amplitude. At the cytoplasmic cGMP concentrations expected in dark-adapted intact photoreceptors, PCa/PNa in cone channels is approximately 7.4-fold greater than that in rods. The linkage between selectivity and gating is specific for divalent cations. The selectivity of Ca2+ and Sr2+ changes with cGMP concentration, but the selectivity of inorganic monovalent cations, Cs+ and NH4+, and organic cations, methylammonium+ and dimethylammonium+, is invariant with cGMP. Cyclic nucleotide-gated channels in rod photoreceptors are heteromeric assemblies of alpha and beta subunits. The maximal PCa/PNa of channels formed from alpha subunits of bovine rod channels is less than that of heteromeric channels formed from alpha and beta subunits. In addition, Ca2+ is a more effective blocker of channels formed by alpha subunits than of channels formed by alpha and beta subunits. The cGMP-dependent shift in divalent cation selectivity is a property of alphabeta channels and not of channels formed from alpha subunits alone.

Topics: Ambystoma; Animals; Bass; Calcium; Calcium Channel Blockers; Calcium Channels; Cations, Divalent; Cattle; Cesium; Cyclic GMP; Diltiazem; Dimethylamines; Dose-Response Relationship, Drug; Electrophysiology; Ion Channel Gating; Ligands; Membrane Potentials; Methylamines; Mutagenesis; Oocytes; Quaternary Ammonium Compounds; Recombinant Proteins; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Sodium; Xenopus

1. Amino acids with a charged or a polar residue in the putative pore region, between lysine 346 and glutamate 372 of the alpha-subunit of the cGMP-gated channel from bovine rods were mutated to a different amino acid. The mRNA encoding for the wild-type, i.e. the alpha-subunit, or mutant channels was injected in Xenopus laevis oocytes. 2. When glutamate 363 was mutated to asparagine, serine or alanine, the current activated by a steady cGMP concentration declined in mutant channels. No current decline was observed when glutamate 363 was mutated to aspartate, glutamine or glycine, when theronine 359, 360 and 364 were mutated to alanine or when other charged residues in the pore region were neutralized. 3. The amount of current decline and its time course were significantly voltage dependent. In mutant E363A the current decline developed within about 1.5 s at -100 mV, but in about 6 s at +100 mV. In the same mutant, the current declined to about 55% of its initial level at +100 mV and to about 10% at -100 mV. 4. The current decline in mutants E363A, E363S and E363N was only moderately dependent on the cGMP concentration (from 10 to 1000 microM) and was not caused by a reduced affinity of the mutant channels for cGMP. Analysis of current fluctuations at a single-channel level indicated that current decline was primarily caused by a decrease of the open probability. 5. The wild-type channel was not permeable to dimethylammonium. When glutamate 363 was replaced by a smaller residue such as serine, mutant channels became permeable to dimethylammonium. 6. The current decline observed in mutant channels is reminiscent of desensitization of ligand-gated channels and of inactivation of voltage-gated channels. These results suggest also that gating and permeation through the cGMP-gated channel from bovine rods are intrinsically coupled and that glutamate 363 is part of the molecular structure controlling both the gating and the narrowest region of the pore.

Topics: Amino Acid Sequence; Animals; Cattle; Cyclic GMP; Dimethylamines; Electrophysiology; Glutamic Acid; Ion Channel Gating; Lysine; Molecular Sequence Data; Mutagenesis; Oocytes; Point Mutation; Time Factors; Xenopus laevis