Novel Splice Variant of nNOS in Cerebrovascular Endothelial Cells Contributes to Superoxide and Peroxynitrite Generation

Abstract

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 identify nNOS in the brain microvascular endothelial cells (MECs) and characterize its functional role. Primary MECs from humans (hMECs), rats (rMECs), and mice (mMECs) along with cultured primary rat cortical neurons were used in the studies. In addition, rat brain microvessels were isolated by homogenization of cortices and separation by filtration followed by gradient centrifugation. Immunohistochemistry identified von Willebrand factor (endothelial marker), eNOS, and nNOS but stained negative for GFAP (glial marker) and Neu1 (neuronal marker) in rMECs and microvessels. Western blot analysis using antibodies targeting N‐terminal domain of nNOS revealed immunoband of a potential nNOS splice variant at ~130 kD in rMECs as opposed to 160 kD in rat cerebral cortex and cultured neurons. PCR experiments identified the nNOS mRNA in all MECs and rat microvessels. 3′ Rapid Amplification of cDNA Ends method using nNOS specific exon 2 primers revealed a prominent truncated nNOS band in rMECs compared to full length nNOS cDNA (RefSeq NP_434686) found in the brain cortex. Furthermore, siRNA targeting rat nNOS in rMECs was able to knockdown nNOS mRNA. We utilized electron spin resonance spectrometry to measure superoxide (1‐Hydroxy‐3‐methoxycarbonyl‐2,2,5,5‐tetramethyl‐pyrrolidine), peroxynitrite (1‐Hydroxy‐3‐carboxy‐2,2,5,5‐tetramethylpyrrolidine), and NO (colloid Fe(DETC)2). siRNA knockdown or pharmacological inhibition (N‐ω‐Propyl‐L‐arginine and ARL‐17477) of nNOS reduced superoxide levels but increased NO levels in rMECs compared to untreated cells. Inhibition of nNOS also decreased peroxynitrite levels in rMECs. In contrast, eNOS inhibitor (L‐N5‐(1‐Iminoethyl)ornithine) increased superoxide levels but reduced NO levels. Similarly, inhibition of nNOS in neurons increased ROS levels and decreased NO levels. Moreover, co‐treatment with tetrahydrobiopterin, a co‐factor for NOS, reduced the superoxide levels in hMECs. Furthermore, treatment of rMECs with angiotensin II (ATII) and high glucose levels increased superoxide levels. Superoxide generation in MECs at baseline and following the treatment with ATII was sensitive to inhibition by gp‐91‐ds‐tat peptide (NADPH oxidase inhibitor) or ARL‐17477 with combined inhibition greater than individual agent administered alone. Thus, we identified a constitutively active nNOS splice variant in MECs that is distinct from the nNOS isoform expressed in neurons and eNOS and makes significant contribution to superoxide and peroxynitrite generation. In addition, endothelial nNOS contributes to the angiotensin II‐ as well as high glucose‐induced superoxide generation that is comparable to but independent of NADPH oxidase.Support or Funding InformationSupport: PVK: Louisiana BoRSF‐RCS (LEQSF(2014‐17)‐RD‐A‐11) and AHA Scientist Development Grant (14SDG20490359); IR: AHA Postdoctoral Fellowship Grant (15POST23040005); and DWB: NIH grants (HL‐077731and HL093554).