ALTERATIONS IN MACROPHAGE G-PROTEINS ARE ASSOCIATED WITH ENDOTOXIN TOLERANCE

Previous studies have suggested that endotoxin tolerance induces macro phage desensitization to endotoxin through altered guanine nucleotide regulatory (G) protein function. In the present study the binding char acteristics of the nonhydrolyzable GTP analogue GTP gamma[S-35] to mac rophage membranes from endotoxin tolerant and control rats were determ ined. Membranes were prepared from peritoneal macrophages harvested fr om rats 72 h after two sequential daily doses of vehicle or Salmonella enteritidis endotoxin (100 mu g/kg on day 1 and 500 mu g/kg on day 2) . GTP gamma[S-35] bound to a single class of sites that were saturable and displaceable in control and endotoxin tolerant macrophage membran es. The maximum specific binding of GTP gamma[S-35] was significantly (P < 0.01) decreased in membranes from tolerant rats compared to contr ol (B-max = 39 +/- 7 pmol/mg protein in control vs, 11 +/- 2 pmol/mg p rotein in endotoxin tolerant; n = 5). There were no significant differ ences in the K-d values, To determine whether the reduced GTP gamma S binding was due to decreases in G proteins, macrophage membrane G prot ein content was determined by western blotting with specific antisera to G(il,2)alpha, G(i3)alpha, G(s) alpha, and the beta subunit of G. Sc anning densitometric analysis demonstrated differential decreases in t olerant macrophage membrane G proteins. G(i3)alpha was reduced the mos t to 48 +/- 8% of controls (n = 3), and this reduction was significant compared to those of other G proteins. G(il,2)alpha and G beta were r educed to 73 +/- 5% (n = 3) and 65 +/- 4% (n = 3) of control values, r espectively. G(s) alpha(L) and G(s) 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 mem brane GTP binding capacity and differential reductions in the content of specific G proteins. The cellular mechanisms leading to such altera tions in G proteins and their functional significance in the acquisiti on of endotoxin tolerance merit further investigation.