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Identification and Functional Characterization of Neuronal Nitric Oxide Synthase in Primary Brain Microvascular Endothelial Cells
Author(s) -
Peterson Nicholas,
Gordon Angellica,
Sure Venkata,
Rajaprabhakaran Gowthamram,
Liu Dan,
Dutta Somhrita,
Rutkai Ibolya,
Busija David,
Katakam Prasad
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.795.6
Subject(s) - enos , superoxide , nitric oxide , chemistry , nitric oxide synthase , endothelial nos , endothelial stem cell , citrulline , microbiology and biotechnology , arginine , biochemistry , biology , enzyme , in vitro , organic chemistry , amino acid
Experimental stroke in animals with gene deletions of endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase isoforms have shown opposite effects on infarct injury. nNOS has been identified recently in endothelial cells, however, its functional significance is unclear. Our objective was to localize nNOS in brain microvascular endothelial cells (MECs) and characterize its functional role. Primary MECs from humans (hMECs) and rats (rMECs) were used in the studies. Immunohistochemistry identified von Willebrand factor, eNOS, and nNOS (total and phospho S1417, S847) in MECs. Immunoblot analysis showed nNOS immunobands at ~130 kD in MECs as opposed to 160 kD in brain. Oxygen consumption rate measurements by Seahorse Analyzer in hMECs treated with selective inhibitors of nNOS (50 nmol/L or 1 µmol/L N‐ω‐Propyl‐L‐arginine; NPA) and eNOS (1 µmol/L L‐N 5 ‐(1‐Iminoethyl)ornithine; NIO) or non‐specific inhibitor (100 µmol/L N G ‐Nitro‐L‐arginine methyl ester; L‐NAME) revealed that maximal mitochondrial respiration was enhanced by NPA but diminished by NIO or L‐NAME. Drug treatment of hMECs for 3 h followed 24 h later by cell viability measurements showed that NPA increased whereas NIO diminished cell proliferation. Measurements of superoxide in MECs by electron spin resonance spectroscopy showed that NPA reduced but NIO increased superoxide generation. Thus, we identified a constitutively active nNOS splice variant in MECs. nNOS appears to produce superoxide and negatively regulates mitochondrial reserve capacity and cell proliferation. We conclude that in brain MECs, nNOS effects are distinctly opposite of eNOS.