Abstract 172: Impact of Pharmacological Inhibition of Neuronal Nitric Oxide Synthase on Brain Microvascular Endothelial Cells
Experimental stroke in animals with deletions of endothelial (eNOS) and neuronal (nNOS) isoforms of nitric oxide synthase have observed greater infarct injury whereas pharmacological inhibition of NOS reported varying impact. nNOS has been identified recently in endothelial cells, however, the functional role of nNOS in brain microvascular endothelial cells (MECs) has never been examined. Our objective was to identify the nNOS in MECs and study its role in the regulation of mitochondrial function and response to anoxic injury.
Methods and Results: Primary brain MECs from humans and rats were used in the studies. Immunohistochemistry identified von Willebrand factor, eNOS, and nNOS in MECs. The nNOS immunoreactivity to three antibodies raised against different sequences of nNOS was observed in the cytoplasm and also in the nucleus when cells were permeabilized. Oxygen consumption rate (OCR) measurements were made from hMECs treated with selective inhibitors of nNOS (N-ω-Propyl-L-arginine; NPA or 7-nitroindazole) and eNOS (L-N5-(1-Iminoethyl)ornithine; NIO) or non-specific inhibitor (NG-Nitro-L-arginine methyl ester; L-NAME) revealed that mitochondrial reserve respiratory capacity was enhanced by nNOS inhibition but diminished by eNOS inhibition or L-NAME (100 mol/L). Drug treatment of hMECs for 3 h followed 24 h later by cell viability measurements showed that nNOS inhibition increased cell proliferation whereas eNOS inhibition diminished cell viability. Exposure to oxygen-glucose deprivation in the presence of nNOS inhibition increased cell viability.
Conclusions: We identified immunoreactive nNOS in brain MECs for the first time. Pharmacological inhibition of nNOS in MECs enhanced mitochondrial capacity, promoted cell proliferation, and afforded protection against anoxic injury. Thus, in MECs, nNOS appears to function distinct from eNOS and may even counteract eNOS actions.
Author Disclosures: V.N.L. Sure: None. A.O. Gordon: None. D. Liu: None. P.V.G. Katakam: None.
- © 2014 by American Heart Association, Inc.