ENDOTOXIN-MEDIATED SYNTHESIS OF NITRIC-OXIDE IS DEPENDENT ON G(Q) PROTEIN SIGNAL-TRANSDUCTION

Background. Nitric oxide (NO) is a ubiquitous multifunctional free rad ical produced during sepsis, shock, reperfusion injury, and allograft rejection. Many studies are presently evaluating the functional roles of NO production in these settings. However, the signal transduction m echanisms underlying initiation of NO production are largely unknown. This study defines the cell surface receptor proteins that mediate end otoxin-induced NO synthesis in, ANA-1 murine macrophages. Methods. End otoxin (LPS, 10 mu g/ml) was added to ANA-1 macrophages to induce NO s ynthesis. In selected instances guanosine 5'-O-(2-thiodiphosphate)-tri lithium salt (GOTP), pertussis toxin, cholera toxin, or suramin were a dded as inhibitors of specific subclasses of heterotrimeric G proteins . Calphostin was added as a protein kinase C inhibitor, and ET-OCH3 wa s added as a phospholipase C-P inhibitor NO release was quantified by measurement of the NO metabolite, nitrite. Membrane guanosine triphosp hatase (GTPase) activity was also analyzed. Steady-state levels of ind ucible nitric oxide synthase (iNOS) mRNA were determined by using reve rse transcription-polymerase chain reaction analysis. Results. Inhibit ion of G protein function by suramin of GOTP significantly decreased s ynthesis of NO and expression of iNOS mRNA. Pertussis and cholera toxi n did trot alter NO synthesis, suggesting that the G(i) and G(s) class es are not involved. Inhibition of protein kinase C or upstream phosph olipase C-beta activity decreased NO synthesis, implicating the G(q) c lass of heterotrimeric proteins. Conclusions. In ANA-1 macrophages, en dotoxin-mediated NO synthesis is dependent on heterotrimeric G(q) prot ein-phospholipase C-beta-protein kinase C signal transduction.