Alterations in macrophage G proteins are associated with endotoxin tolerance

Abstract

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 GTP gamma [35S] 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 micrograms/kg on day 1 and 500 micrograms/kg on day 2). GTP gamma [35S] bound to a single class of sites that were saturable and displaceable in control and endotoxin tolerant macrophage membranes. The maximum specific binding of GTP gamma [35S] was significantly (P < 0.01) decreased in membranes from tolerant rats compared to control (Bmax = 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 GTP gamma S binding was due to decreases in G proteins, macrophage membrane G protein content was determined by western blotting with specific antisera to Gi1,2 alpha, Gi3 alpha, Gs alpha, and the beta subunit of G. Scanning densitometric analysis demonstrated differential decreases in tolerant macrophage membrane G proteins. Gi3 alpha was reduced the most to 48 +/- 8% of controls (n = 3), and this reduction was significant compared to those of other G proteins. Gi1,2 alpha and G beta were reduced to 73 +/- 5% (n = 3) and 65 +/- 4% (n = 3) of control values, respectively. Gs alpha(L) and Gs alpha(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.

Different Modes of IL-10 and TGF-β to Inhibit Cytokine-Dependent IFN-γ Production: Consequences for Reversal of Lipopolysaccharide Desensitization

“…Apart from endogenous IL-10, other intrinsic mechanisms are likely to be involved in LPS desensitization. Described potential mechanisms include alteration of G protein binding (46,47), impaired mitogen-activated protein kinase (48), or Toll-like receptor 4 signaling involving the induction of IL-1R-associated kinase M (49) and suppressor of cytokine signaling 1/3 (50 -52), as well as increased expression of the p50 subunit of NF-B (53, 54). These intrinsic mechanisms might explain why the restoration of TNF-␣ production did not completely correlate to the restored IFN-␥ levels in our experiments.…”

Identification of Gβγ Binding Sites in the Third Intracellular Loop of the M3-muscarinic Receptor and Their Role in Receptor Regulation

“…Antisera to the carboxyl-terminal 10 amino acids of G␤1, which recognize G␤1-4, were generated as described (28). GRK2 was purified from Sf9 insect cells infected with recombinant virus as described (29 ) domains were generated as described previously (21).…”

Selective Interaction of AGS3 with G-proteins and the Influence of AGS3 on the Activation State of G-proteins

“…Immuno Fluore mounting medium was purchased from ICN Biomedicals. Purified bovine brain G-protein and antisera to the COOH-terminal 10 amino acids of G␤1, which recognizes G␤1-4, were kindly provided by Dr. John Hildebrandt (Department of Pharmacology, Medical University of South Carolina, Charleston, SC) (11,12). G␣ i1-3 and G␣ o were purified from Sf9 insect cells infected with recombinant virus as described (13) and kindly provided by Dr. Stephen Graber (West Virginia University School of Medicine, Morgantown, WV).…”