Antisense evidence for two functionally active forms of nitric oxide synthase in brain microvascular endothelium

Other workers have identified two constitutive forms of NOS in endothelial cells: endothelial or eNOS and neuronal or nNOS. The present study tests the functional significance of these NOS in brain surface arterioles of mice. Antisense oligodeoxynucleotides (ODN) were injected into the cerebral ventricles. Anti eNOS and anti nNOS were tested separately and in combination. Each antisense reduced the dilation produced by topical acetylcholine (ACh) or by tetrahydrobiopterin (THBP). These are endothelium dependent, NOS dependent dilators, with the THBP being a cofactor for NOS. The endothelium derived mediator actually causing the dilation is EDRF(ACh). When both antisenses were given together there was an additive effect which approached 100% inhibition. Neither sense nor mismatched (scrambled) ODN inhibited either ACh or THBP. Moreover, anti eNOS did not inhibit dilation by bradykinin (endothelium dependent but not NOS dependent) or by sodium nitroprusside (endothelium independent). The data strongly support the conclusion that both eNOS and nNOS are functionally important in the endothelium of mouse pial arterioles. Each isoform of NOS appears to contribute significantly to the synthesis of basally released (THBP triggered) and agonist (ACh) released EDRFACh.