title: Internally applied endotoxin and the activation of BK channels in cerebral artery smooth muscle via a nitric oxide-like pathway.
abstract: 1. In this study the role of nitric oxide synthase (NOS) in the acute activation of large conductance, Ca2+-activated K+ channels (BK channels) by internally applied E. coli lipopolysaccharide (LPS, endotoxin) was examined in vascular smooth muscle cells. 2. Cerebrovascular smooth muscle cells (CVSMCs) were enzymatically dispersed from the middle, posterior communicating and posterior cerebral arteries of adult Wistar rats and maintained at 4 degrees C for 2-4 days before recording with standard patch-clamp techniques. 3. Acute application of LPS (100 microg ml(-1)) to inside-out patches of CVSMC membrane isolated in a cell-free environment rapidly and reversibly increased the open probability, Po of BK channels in these patches by 3.3+/-0.30 fold. 4. Acute application of the nitric oxide (NO) donor sodium nitroprusside (SNP, 100 microM) to inside-out patches of CVSMC membrane, studied in the presence of intact cells, also reversibly increased Po, by some 1.8+/-0.2 fold over control. 5. Kinetic analysis showed that both LPS and SNP increased Po by accelerating the rate of BK channel reopening, rather than by retarding the closure of open channels. 6. Neither LPS nor SNP altered the reversal potential or conductance of BK channels. 7. The NOS substrate L-arginine (1 microM) potentiated the acute activation of BK channels by LPS, while the synthetic enantiomer D-arginine (1 microM) inhibited the action of LPS on BK channels. 8. The acute activation of BK channels by LPS was suppressed by pre-incubation of cells with N(omega)-nitro-L-arginine (50 microM) or N(omega)-nitro-L-arginine methyl ester (1 mM), two competitive antagonists of nitric oxide synthases. N(omega)-nitro-D-arginine (50 microM), a poor inhibitor of NOS in in vitro assays, had no effect on BK channel activation by LPS. 9. These results indicate that excised, inside-out patches of CVSMC membrane exhibit a NOS-like activity which is acutely activated when LPS is present at the cytoplasmic membrane surface. Possible relationships between this novel mechanism and the properties of known isoforms of nitric oxide synthase are discussed.
authors.affiliation: Department of Physiology, University of British Columbia, Vancouver, Canada.
authors.forename: L M
authors.initials: LM
authors.lastname: Hoang
authors.forename: D A
authors.initials: DA
authors.lastname: Mathers
mesh_terms: D000818:Animals; D001920:Bradykinin / Q000378:metabolism*; D002469:Cell Separation; D002536:Cerebral Arteries / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004558:Electric Stimulation; D004731:Endotoxins / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008564:Membrane Potentials / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009599:Nitroprusside / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar
chemical_list: D004731:Endotoxins; D004791:Enzyme Inhibitors; D007473:Ion Channels; D008070:Lipopolysaccharides; D009599:Nitroprusside; D009569:Nitric Oxide; C044101:endotoxin, Escherichia coli; D019001:Nitric Oxide Synthase; D001920:Bradykinin