Alterations in macrophage G proteins are associated with endotoxin tolerance

摘要

Previous studies have suggested that endotoxin tolerance induces macrophage desensitization to endotoxin through altered guanine nucleotide regulatory (G) protein function. In the present study the binding characteristics of the nonhydrolyzable GTP analogue GTPytS] to macrophage membranes from endotoxin tolerant and control rats were determined. Membranes were prepared from peritoneal macrophages harvested from rats 72 h after two sequential daily doses of vehicle or Salmonella enteritidis endotoxin (100 /xg/kg on day 1 and 500 jxg/kg on day 2). OTPylS] bound to a single class of sites that were saturable and displaceable in control and endotoxin tolerant macrophage membranes. The maximum specific binding of GTPy[35S] was significantly (P<0.01) decreased in membranes from tolerant rats compared to control (Bmas = 39 + 7 pmol/mg protein in control vs. 11 + 2 pmol/mg protein in endotoxin tolerant; n - 5). There were no significant differences in the Kd values. To determine whether the reduced GTPyS binding was due to decreases in G proteins, macrophage membrane G protein content was determined by western blotting with specific antisera to Gil2&, GBa, Gso and the /3 subunit of G. Scanning densitometric analysis demonstrated differential decreases in tolerant macrophage membrane G proteins. Gi3a was reduced the most to 48 + 8% of controls (n - 3), and this reduction was significant compared to those of other G proteins. Gu 2a and G/3 were reduced to 73 ± 5% (n= 3) and 65 +_ 4% (n = 3) of control values, respectively. GSQ(L) and Gs a(H) were reduced to 61 ± 5% (n = 3) and 68 + 3% (n = 3) of control, respectively. These results demonstrate that endotoxin tolerant macrophages exhibit decreased membrane GTP binding capacity and differential reductions in the content of specific G proteins. The cellular mechanisms leading to such alterations in G proteins and their functional significance in the acquisition of endotoxin tolerance merit further investigation.