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Role of inducible nitric oxide synthase in the acute activation of murine vascular smooth muscle BK channels by internally applied lipopolysaccharide Yakubovich, Natalia

Abstract

The role of inducible nitric oxide synthase (iNOS) in the acute activation of large conductance, Ca²⁺-activated K⁺ channels (BK channels) by the internally applied Escherichia coli lipopolysaccharide (LPS) was studied in murine vascular smooth muscle cells. Primary cell culture, immunocytochemistry, and patch clamp recording techniques were utilized. Immunocytochemical studies showed that rat cerebrovascular arteries fixed promptly upon donor rat sacrifice stained negative for iNOS. However, within 1.5 h of brain removal, iNOS-like immunoreactivity could be detected in cerebrovascular smooth muscle cells (CVSMCs) enzymatically dispersed from rat cerebral arteries, suggesting rapid induction of this protein during cell isolation. Confocal microscopy demonstrated localization of iNOS-like immunoreactivity in the cytoplasm and under the sarcolemma of rat CVSMCs. LPS was then applied to the cytoplasmic face of inside-out membrane patches excised from rat CVSMCs within 2.5-8 h of donor rat sacrifice. It was found that 50 μg/ml LPS rapidly and reversibly increased the open probability of B K channels in these patches, leaving the single channel conductance unaltered. Kinetic analysis showed that LPS activated B K channels by shortening long-duration channel closures, while having little effect on the average duration of channel openings. Importantly, the acute activation of B K channels by LPS was not altered in the presence of the non-isoform specific NOS inhibitor N[sup ω]-nitro-L-arginine (L-NNA, 100 uM). Inside-out patch clamp recordings obtained from wild-type mouse aortic smooth muscle cells (ASMCs) revealed BK channels which had large conductance, were activated by elevation in intracellular calcium and upon membrane depolarization, and were blocked by intracellular tetraethylammonium (25 mM). The effects of LPS on these channels were next compared in wild-type and iNOS knockout (iNOS-KO) mice. Cytoplasmic.application of 50 μg/ml LPS acutely activated B K channels in inside-out patches of ASMC membrane derived from iNOSKO mice, with a degree of potentiation not significantly different from that observed in wildtype mice. These studies establish that internally applied LPS can activate murine vascular smooth muscle B K channels independently of iNOS expression or activity. The mechanism which underlies this novel LPS response remains to be elucidated.

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