ABSTRACT
RATIONALE: Nitric oxide, the classic endothelium-derived relaxing factor (EDRF), acts through cyclic GMP and calcium without notably affecting membrane potential. A major component of EDRF activity derives from hyperpolarization and is termed endothelium-derived hyperpolarizing factor (EDHF). Hydrogen sulfide (H(2)S) is a prominent EDRF, since mice lacking its biosynthetic enzyme, cystathionine γ-lyase (CSE), display pronounced hypertension with deficient vasorelaxant responses to acetylcholine. OBJECTIVE: The purpose of this study was to determine if H(2)S is a major physiological EDHF. METHODS AND RESULTS: We now show that H(2)S is a major EDHF because in blood vessels of CSE-deleted mice, hyperpolarization is virtually abolished. H(2)S acts by covalently modifying (sulfhydrating) the ATP-sensitive potassium channel, as mutating the site of sulfhydration prevents H(2)S-elicited hyperpolarization. The endothelial intermediate conductance (IK(Ca)) and small conductance (SK(Ca)) potassium channels mediate in part the effects of H(2)S, as selective IK(Ca) and SK(Ca) channel inhibitors, charybdotoxin and apamin, inhibit glibenclamide-insensitive, H(2)S-induced vasorelaxation. CONCLUSIONS: H(2)S is a major EDHF that causes vascular endothelial and smooth muscle cell hyperpolarization and vasorelaxation by activating the ATP-sensitive, intermediate conductance and small conductance potassium channels through cysteine S-sulfhydration. Because EDHF activity is a principal determinant of vasorelaxation in numerous vascular beds, drugs influencing H(2)S biosynthesis offer therapeutic potential.
Subject(s)
Endothelium, Vascular/metabolism , Hydrogen Sulfide/metabolism , KATP Channels/metabolism , Vasodilation/physiology , Acetylcholine/pharmacology , Animals , Aorta/cytology , Aorta/metabolism , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Charybdotoxin/pharmacology , Cystathionine gamma-Lyase/deficiency , Cystathionine gamma-Lyase/genetics , Endothelium-Dependent Relaxing Factors/metabolism , Female , Glyburide/pharmacology , Hypertension/metabolism , Male , Membrane Potentials/drug effects , Mesenteric Arteries/injuries , Mesenteric Arteries/metabolism , Mesenteric Arteries/pathology , Mice , Mice, Inbred C57BL , Phenylephrine/pharmacology , Potassium Chloride/pharmacology , Rats , Rats, Wistar , Vasodilation/drug effectsABSTRACT
D-Serine, formed from L-serine by serine racemase (SR), is a physiologic coagonist at NMDA receptors. Using mice with targeted deletion of SR, we demonstrate a role for D-serine in NMDA receptor-mediated neurotoxicity and stroke. Brain cultures of SR-deleted mice display markedly diminished nitric oxide (NO) formation and neurotoxicity. In intact SR knock-out mice, NO formation and nitrosylation of NO targets are substantially reduced. Infarct volume following middle cerebral artery occlusion is dramatically diminished in several regions of the brains of SR mutant mice despite evidence of increased NMDA receptor number and sensitivity.
Subject(s)
Brain Ischemia/enzymology , Brain Ischemia/genetics , Cytoprotection/genetics , Neurotoxins/metabolism , Racemases and Epimerases/genetics , Serine/metabolism , Animals , Brain/blood supply , Brain/enzymology , Brain/physiopathology , Brain Infarction/enzymology , Brain Infarction/genetics , Brain Infarction/therapy , Brain Ischemia/therapy , Cells, Cultured , Disease Models, Animal , Down-Regulation/genetics , Gene Deletion , Gene Expression Regulation, Enzymologic/genetics , Genetic Therapy/methods , Infarction, Middle Cerebral Artery/enzymology , Infarction, Middle Cerebral Artery/genetics , Infarction, Middle Cerebral Artery/therapy , Isomerism , Male , Mice , Mice, Knockout , Nitric Oxide/metabolism , Nitric Oxide Synthase Type I/genetics , Nitro Compounds/metabolism , Receptors, N-Methyl-D-Aspartate/agonists , Receptors, N-Methyl-D-Aspartate/metabolismABSTRACT
Hydrogen sulfide (H2S), a messenger molecule generated by cystathionine gamma-lyase, acts as a physiologic vasorelaxant. Mechanisms whereby H2S signals have been elusive. We now show that H2S physiologically modifies cysteines in a large number of proteins by S-sulfhydration. About 10 to 25% of many liver proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are sulfhydrated under physiological conditions. Sulfhydration augments GAPDH activity and enhances actin polymerization. Sulfhydration thus appears to be a physiologic posttranslational modification for proteins.
Subject(s)
Hydrogen Sulfide/metabolism , Signal Transduction , Sulfhydryl Compounds/metabolism , Actins/metabolism , Animals , Biopolymers/metabolism , Chromatography, High Pressure Liquid , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Mice , Protein Processing, Post-Translational , Tandem Mass Spectrometry , Tubulin/metabolismABSTRACT
D-serine is a physiologic coagonist with glutamate at NMDA-subtype glutamate receptors. As D-serine is localized in glia, synaptically released glutamate presumably stimulates the glia to form and release D-serine, enabling glutamate/D-serine cotransmission. We show that serine racemase (SR), which generates D-serine from L-serine, is physiologically inhibited by phosphatidylinositol (4,5)-bisphosphate (PIP2) presence in membranes where SR is localized. Activation of metabotropic glutamate receptors (mGluR5) on glia leads to phospholipase C-mediated degradation of PIP2, relieving SR inhibition. Thus mutants of SR that cannot bind PIP2 lose their membrane localizations and display a 4-fold enhancement of catalytic activity. Moreover, mGluR5 activation of SR activity is abolished by inhibiting phospholipase C.
