title: Nitrative stress in cerebral endothelium is mediated by mGluR5 in hyperhomocysteinemia.
abstract: 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 1400W. 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.
authors.affiliation: Department of Biological Sciences, Idaho State University, Pocatello, ID 83209, USA.
authors.forename: Jamie N
authors.initials: JN
authors.lastname: Mayo
authors.forename: Richard S
authors.initials: RS
authors.lastname: Beard
authors.forename: Tulin O
authors.initials: TO
authors.lastname: Price
authors.forename: Cheng-Hung
authors.initials: CH
authors.lastname: Chen
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Erickson
authors.forename: Nuran
authors.initials: N
authors.lastname: Ercal
authors.forename: William A
authors.initials: WA
authors.lastname: Banks
authors.forename: Shawn E
authors.initials: SE
authors.lastname: Bearden
mesh_terms: D000578:Amidines / Q000494:pharmacology; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D001921:Brain / Q000098:blood supply / Q000378:metabolism*; D004727:Endothelium / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006710:Homocysteine / Q000235:genetics / Q000378:metabolism; D020138:Hyperhomocysteinemia / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D010648:Phenylacetates / Q000494:pharmacology; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013312:Stress, Physiological; D014443:Tyrosine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism
chemical_list: C107349:2-chloro-5-hydroxyphenylglycine; D000578:Amidines; D001596:Benzylamines; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; C578936:Grm5 protein, mouse; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D010648:Phenylacetates; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; D006710:Homocysteine; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D005998:Glycine