Fig. 4.
NO inhibits NSF regulation of platelet granule exocytosis. (A) NSF is a target of exogenous NO: dose–response. Murine platelets were harvested, and equal numbers were incubated with control or DEA-NONOate, lysed, immunoprecipitated with antibody to nitrosocysteine, and immunoblotted with antibody to NSF. NSF is nitrosylated in a dose-dependent manner. (Each step was repeated three times with similar results.) (B) NSF is a target of endogenous NO. Murine platelets were harvested, preincubated with control or l-NAME 5 mM, treated with control or 1 μM Ca2+ ionophore, lysed, immunoprecipitated with antibody to nitrosocysteine, and immunoblotted with antibody to NSF. (C) NO inhibits NSF separation from syntaxin-4 in vitro. Recombinant His6-NSF was pretreated or not pretreated with DEA-NONOate and incubated with a-SNAP and GST-SNARE fusion polypeptides expressed in platelets. ATP or ATP-gS was added, and the mixture was precipitated with glutathione-Sepharose. Precipitated proteins were immunoblotted with antibody to the NSF tag. (D) NSF is a target of NO. Platelets were permeabilized, exposed to 1 mM DEA-NONOate, and then incubated with recombinant NSF or nitrosylated NSF. Platelets were then stimulated or not stimulated with TRAP and Ca2+, and exocytosis was measured by FACS. (n = 3 ± SD, *, P < 0.05 for NO plus NSF vs. NO plus NSF-NO.)