The effects of tolerizing doses of LPS on mRNA and protein levels of three different G protein subunits were investigated to understand the mechanism(s) responsible for the reduction in Gi± protein content in LPS tolerance. Tolerance was induced in rats using Salmonella enteritidis LPS (intraperitoneal route) with a single dose of 100 μg/kg. Peritoneal macrophages were harvested 6 and 24 h later. In some studies, a second dose of LPS 500 μg/kg was given on the following day, and peritoneal macrophages were harvested 5 days after the first injection. Macrophage RNA or a crude membrane fraction was prepared from macrophages, and the mRNA level or the protein content for Gi±3, Gi±2, and Gs± was analyzed using Northern or Western blots, respectively. Compared with the control levels, the message for Gi±3 was reduced (p < .025) at 6 and 24 h and 5 day time periods after LPS treatment. The Gi±2 mRNA was increased relative to the control levels (p < .05) at 6 h and 5 days after LPS treatment, respectively, and Gs± message was not significantly changed. The half-life of Gi±3 mRNA was not significantly different in control versus tolerant macrophages. The Gi±3 mRNA and membrane protein were not significantly changed by incubation with LPS for intervals up to 6 h in vitro. Macrophage membrane Gi±3 and Gi±1 and 2 protein content from tolerant rats were reduced compared with the controls at 6 and 24 h, respectively (p < .05). These studies are consistent with our previous observations of selective changes in macrophage Gi± protein content in LPS tolerance and raise the possibility that this may affect signal transduction events in these cells.
©1999The Shock Society