Changes in vascular responsiveness to a thromboxane mimetic in endotoxin tolerance

Our previous studies have demonstrated that peritoneal macrophages obtained from endotoxin-tolerant rats exhibit altered cellular activation by endoto xin, possibly involving changes In guanine nucleotide regulatory (G) protei n-coupled signal transduction pathways. Endotoxin-tolerant rats also exhibi t cross tolerance and altered hemodynamic responses to thromboxane (Tx)A(2) mimetics, suggesting potential changes In vascular responsiveness. We test ed the hypothesis that endotoxin tolerance results In vascular hyporesponsi veness to a TxA(2) mimetic via alterations in the TxA(2) receptor, G protei n function, and/or second messenger production. Rats were rendered endotoxi n tolerant by Increasing sublethal consecutive doses of Salmonella enteriti dis endotoxin (100 to 5000 mug/kg, ip) for 4 days. The animals were sacrifi ced 2 days after the final dose of endotoxin for removal of aortas. Contrac tile responses of aortic rings to U46619, a TxA(2) agonist, were assessed i n control and tolerant rats. The EC50 values for U46619 were 14.8 +/-6.6 nM and 32.3 +/-3.1 nM (n=5-7), (P<0.05) for control and tolerant rats, respec tively. Crude membranes were prepared from aortas of control and tolerant r ats, and binding of I-BOP TxA(2)/PGH2 receptor agonist, [1S-(1 alpha, 2 bet a (5Z), 3 alpha (1E,3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-bu tenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), a TxA(2) agonist, was assessed by Scatchard analysis. I-BOP binding to the TxA(2) re ceptor was saturable and revealed a single class of TxA(2) receptors for bo th groups. There was no significant difference in control (n=7) compared wi th tolerant (n=5) K-d values (2.1<plus/minus>0.2 vs. 2.4 +/-0.9 nM, respect ively), or B-max (31 +/-6 vs. 28 +/- 12 fmol/mg protein, respectively). To assess potential changes in G protein function, aortic membrane GTpase acti vity was determined. GTPase activity in tolerant membranes was significantl y reduced (P<0.05) compared with control membranes (309<plus/minus>23 (n=5) vs. 440 +/- 32 (n=7) pmol/mg/protein/min, respectively). However, U46619-s timulated phosphoinositide production was similar in vascular tissue from c ontrol and tolerant rats. These observations suggest that the decreased con tractile response to TxA(2) mimetics In endotoxin tolerance does not result from a change in receptor number, affinity of TxA(2) receptors, or changes in phosphatidylinositol metabolism but is associated with decreased vascul ar G protein function.