Differential relaxing responses to particulate or soluble guanylyl cyclase activation on endothelial cells: a mechanism dependent on PKG-I alpha activation by NO/cGMP.
cGMP is generated in endothelial cells after stimulation of soluble guanylyl cyclase (sGC) by nitric oxide (NO) or of particulate guanylyl cyclase (pGC) by natriuretic peptides (NP). We examined whether localized increases in cytosolic cGMP have distinct regulatory roles on the contraction induced by H2O2 treatment in human umbilical vein endothelial cells. cGMP concentrations and temporal dynamics were different upon NO stimulation of sGC or C-type NP (CNP) activation of pGC and did not correlate with their relaxing effects measured as planar cell surface area after H2O2 challenge. cGMP production due to sGC stimulation was always smaller and more brief than that induced by pGC stimulation with CNP, which was greater and remained elevated longer. The NO effects on cell relaxation were cGMP dependent because they were blocked by sGC inhibition with 1H-(1,2,4)Oxadiazolo(4,3-a)quinoxaline-1-one and mimicked by 8-Br-cGMP. An antagonist of the cGMP-dependent protein kinase type-I (PKG-I) also inhibited the NO-induced effects. The cell contraction induced by H2O2 produces myosin light chain (MLC) phosphorylation and NO prevented it completely, whereas CNP only produced a partial inhibition. Transfection with a dominant negative form of PKG type-I alpha completely reversed the NO-induced effects on MLC phosphorylation, whereas it only partially inhibited the effects due to CNP. Taken together, these results demonstrate that the NO/sGC/cGMP pathway induces endothelial cell relaxation in a more efficient manner than does CNP/pGC/cGMP pathway, an effect that might be related to a selective stimulation of PKG-1 alpha by NO-derived cGMP. Consequently, stimulated PKG-I alpha may phosphorylate important protein targets that are necessary to inhibit the endothelial contractile machinery activated by oxidative stress.
Rivero-Vilches
F J
FJ
Departmento de Fisiología, Universidad de Alcalá, Alcalá de Henares, and Instituto Reina Sofia de Investigacíon Nefrológicas, Madrid, Spain.
de Frutos
S
S
Saura
M
M
Rodriguez-Puyol
D
D
Rodriguez-Puyol
M
M
D002478:Cells, Cultured; D006152:Cyclic GMP / Q000494:pharmacology / Q000502:physiology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D020098:Natriuretic Peptide, C-Type / Q000378:metabolism / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology; D016877:Oxidants / Q000494:pharmacology; D012995:Solubility; D014661:Vasoconstriction / Q000187:drug effects; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology*
D004338:Drug Combinations; D007527:Isoenzymes; D016877:Oxidants; D020098:Natriuretic Peptide, C-Type; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D006152:Cyclic GMP