Nitrative Stress in Cerebral Endothelium is Mediated by mGluR5 in Hyperhomocysteinemia

• Published in: Journal of cerebral blood flow and metabolism Vol. 32; no. 5; pp. 825 - 834
• Main Authors: Mayo, Jamie N, Beard, Richard S, Price, Tulin O, Chen, Cheng-Hung, Erickson, Michelle A, Ercal, Nuran, Banks, William A, Bearden, Shawn E
• Format: Journal Article
• Language: English
• Published: London, England SAGE Publications 01.05.2012; Nature Publishing Group; Sage Publications Ltd
• Subjects: Amidines - pharmacology; Animal models; Animals; Benzylamines - pharmacology; Biological and medical sciences; Brain - blood supply; Brain - metabolism; Cerebral blood flow; Cerebral circulation. Blood-brain barrier. Choroid plexus. Cerebrospinal fluid. Circumventricular organ. Meninges; Endothelial cells; Endothelium; Endothelium - metabolism; Enzyme Inhibitors - pharmacology; Excitatory Amino Acid Agonists - pharmacology; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic - drug effects; Gene Expression Regulation, Enzymologic - genetics; Glutamic acid; Glutamic acid receptors (metabotropic); Glycine - analogs & derivatives; Glycine - pharmacology; homocysteine; Homocysteine - genetics; Homocysteine - metabolism; Hyperhomocysteinemia - genetics; Hyperhomocysteinemia - metabolism; Medical sciences; Mice; Mice, Knockout; Microvasculature; Neurology; Nitric oxide; Nitric Oxide - genetics; Nitric Oxide - metabolism; Nitric Oxide Synthase Type II - antagonists & inhibitors; Nitric Oxide Synthase Type II - biosynthesis; Nitric Oxide Synthase Type II - genetics; Nitric-oxide synthase; nitrotyrosine; Original; Pharmacology; Phenylacetates - pharmacology; pyridines; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate - agonists; Receptors, Metabotropic Glutamate - antagonists & inhibitors; Receptors, Metabotropic Glutamate - genetics; Receptors, Metabotropic Glutamate - metabolism; RNA; Stress; Stress, Physiological; Tyrosine - analogs & derivatives; Tyrosine - genetics; Tyrosine - metabolism; Vascular diseases and vascular malformations of the nervous system; Vertebrates: nervous system and sense organs; homocysteine; microvascular; peroxynitrite; blood—brain barrier; oxidative stress; blood-brain barrier; Oxidative stress; Thiol; Nervous system diseases; Blood brain barrier; Cerebral disorder; Encephalon; Endothelium; Sulfur containing aminoacid; Homocystein; Central nervous system disease; Hyperhomocysteinemia; Cerebrovascular disease
• ISSN: 0271-678X; 1559-7016
• DOI: 10.1038/jcbfm.2011.185

Hyperhomocysteinemia (HHcy) disrupts nitric oxide (NO) signaling and increases nitrative stress in cerebral microvascular endothelial cells (CMVECs). This is mediated, in part, by protein nitrotyrosinylation (3-nitrotyrosine; 3-NT) though the mechanisms by which extracellular homocysteine (Hcy) generates intracellular 3-NT are unknown. Using a murine model of mild HHcy (cbs+/– mouse), we show that 3-NT is significantly elevated in cerebral microvessels with concomitant reductions in serum NO bioavailability as compared with wild-type littermate controls (cbs+/+). Directed pharmacology identified a receptor-dependent mechanism for 3-NT formation in CMVECs. Homocysteine increased expression of inducible NO synthase (iNOS) and formation of 3-NT, both of which were blocked by inhibition of metabotropic glutamate receptor-5 (mGluR5) with the specific antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride. Activation of mGluR5 is both sufficient and necessary to drive the nitrative stress because direct activation using the mGluR5-specific agonist (RS)-2-chloro-5-hydroxyphenylglycine also increased iNOS expression and 3-NT formation while knockdown of mGluR5 receptor expression by short hairpin RNA (shRNA) blocked their increase in response to Hcy. Nitric oxide derived from iNOS was required for Hcy-mediated formation of 3-NT because the effect was blocked by 1400 W. These results provide the first evidence for a receptor-dependent process that explains how plasma Hcy levels control intracellular nitrative stress in cerebral microvascular endothelium.