Tolerance to LPS decreases macrophage G protein content.
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 Gialpha protein content in LPS tolerance. Tolerance was induced in rats using Salmonella enteritidis LPS (intraperitoneal route) with a single dose of 100 microg/kg. Peritoneal macrophages were harvested 6 and 24 h later. In some studies, a second dose of LPS 500 microg/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 Gialpha3, Gialpha2, and Gsalpha was analyzed using Northern or Western blots, respectively. Compared with the control levels, the message for Gialpha3 was reduced (p < .025) at 6 and 24 h and 5 day time periods after LPS treatment. The Gialpha2 mRNA was increased relative to the control levels (p < .05) at 6 h and 5 days after LPS treatment, respectively, and Gsalpha message was not significantly changed. The half-life of Gialpha3 mRNA was not significantly different in control versus tolerant macrophages. The Gialpha3 mRNA and membrane protein were not significantly changed by incubation with LPS for intervals up to 6 h in vitro. Macrophage membrane Gialpha3 and Gialpha1 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 Gialpha protein content in LPS tolerance and raise the possibility that this may affect signal transduction events in these cells.
Fernando
L P
LP
Department of Physiology, Medical University of South Carolina, Charleston 29425, USA.
Makhlouf
M
M
Fernando
A N
AN
Ashton
S
S
Halushka
P V
PV
Cook
J A
JA
D000818:Animals; D018076:DNA, Complementary; D004361:Drug Tolerance; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000378:metabolism*; D008297:Male; D051381:Rats; D020318:Rats, Long-Evans
D018076:DNA, Complementary; D008070:Lipopolysaccharides; D019204:GTP-Binding Proteins