Nitric oxide is the major endothelium-derived relaxing factor (EDRF), and it is thought to relax smooth muscle cells by stimulation of guanylate cyclase, accumulation of its product cyclic GMP, and cGMP-dependent modification of several intracellular processes, including activation of potassium channels through cGMP-dependent protein kinase. Here we present evidence that both exogenous nitric oxide and native EDRF can directly activate single Ca(2+)-dependent K+ channels (K+Ca) in cell-free membrane patches without requiring cGMP. Under conditions when guanylate cyclase was inhibited by methylene blue, considerable relaxation of rabbit aorta to nitric oxide persisted which was blocked by charybdotoxin, a specific inhibitor of K+Ca channels. These studies demonstrate a novel direct action of nitric oxide on K+Ca channels.

Abstract-We previously reported enhanced expression of the p67 phox and gp91 phox components of NAD(P)H oxidase in angiotensin (Ang) II-induced hypertension, suggesting de novo assembly in response to Ang II. To examine the direct involvement of NAD(P)H oxidases in Ang II-induced O 2 Ϫ production, we designed a chimeric peptide that inhibits p47 phox association with gp91 phox in NAD(P)H oxidase (gp91ds-tat). This was achieved by linking a 9-amino acid peptide (aa) derived from HIV-coat protein (tat) to a 9-aa sequence of gp91 phox (known to interact with p47 phox ). As a control, we constructed a chimera containing tat and a scrambled gp91 sequence (scramb-tat). We found that gp91ds-tat decreased O 2 Ϫ levels in aortic rings treated with Ang II (10 pmol/L) but had no effect on either the O 2 Ϫ -generating enzyme xanthine oxidase or potassium superoxide-generated O 2 Ϫ . We infused vehicle, Ang II (0.75 mg · kg, or Ang IIϩscramb-tat intraperitoneally in C57Bl/6 mice and measured systolic blood pressure (SBP) on days 0, 3, 5, and 7 of infusion. SBP increased by day 3 in mice given Ang II and Ang IIϩscramb-tat but was significantly lower with Ang IIϩgp91-tat. On day 7, SBP was still significantly inhibited in mice given Ang IIϩgp91ds-tat, whereas Ang II-induced O 2 Ϫ production was inhibited throughout the aorta as detected by dihydroethidium staining, consistent with the ability of this inhibitor to block the various vascular NAD(P)H oxidase isoforms. These data support the hypothesis that inhibition of the interaction of p47 phox and gp91 phox (or its homologues) can block O 2 Ϫ production and attenuate blood pressure elevation in mice.