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The effects of E. Coli endotoxin on BK Channels in rat cerebrovascular smooth muscle cell membranes Hoang, Linda Mong Nguyen

Abstract

Patch clamp recordings were used to study the effects of E. coli bacterial endotoxin c on the properties of large-conductance, Ca²⁺ dependent K⁺ channels (BK channels) in the membrane of enzymatically dispersed rat cerebrovascular smooth muscle cells (CVSMCs). Acute cytoplasmic application of LPS (10-100 µg/ml) to inside-out patches of CVSMC membrane isolated in a cell-free environment rapidly and reversibly increased the open probability of BK channels, leaving the conductance of these channels unaltered. The magnitude of this effect decreased as the concentration of free Ca²⁺ at the cytoplasmic membrane face was lowered, but was little affected by changes in membrane potential. LPS activation of B K channels persisted in membrane patches which had been isolated from their parent cells for one hour. Kinetic analysis showed that LPS accelerated reopening of BK channels, while having little effect on mean channel open time. A detoxified LPS from which the fatty acid chains of Lipid A had been largely removed was about half as effective as the parent endotoxin molecule in activating B K channels. A purified E. coli Lipid A had negligible effects on the function of B K channels at concentrations up to 20 µg/ml. The possible role of nitric oxide synthase (NOS) in mediating the acute activation of B K channels by LPS was next investigated. It was found that the NOS substrate L-arginine (1 µM) potentiated the effect of LPS on BK channels, while the synthetic enantiomer D-arginine inhibited the action of LPS on these channels. Activation of BK channels by LPS was also suppressed by preincubation of CVSMCs with the competitive NOS inhibitor N^nitro-L-arginine (50 µM) while the D-isomer of this agent was ineffective. A second competitive inhibitor of NOS, N°-nitro-Larginine methyl ester (1 mM) also inhibited the action of LPS on BK channels. These studies have shown that application of LPS to the cytoplasmic face of the CVSMC membrane acutely activates B K channels in these membranes. The possibility that this novel effect was mediated by a constitutive, NOS-like enzyme present in the CVSMC membrane is discussed. The significance of these results for future research in the areas of bacterial pathogenesis and vascular smooth muscle physiology are briefly considered.

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