EFFECTS OF TYRPHOSTINS AND GENISTEIN ON THE CIRCULATORY FAILURE AND ORGAN DYSFUNCTION CAUSED BY ENDOTOXIN IN THE RAT - A POSSIBLE ROLE FOR PROTEIN-TYROSINE KINASE

1 Here we compared the effects of various inhibitors of the activity o f protein tyrosine kinase on (i) the expression of the activity of the inducible isoform of nitric oxide (NO) synthase (iNOS) caused by endo toxin (lipopolysaccharide, LPS) in cultured macrophages, (ii) the indu ction of iNOS and cyclo-oxygenase 2 (COX-2) protein and activity in ra ts with endotoxaemia, and (iii) the circulatory failure and organ dysf unction caused by LPS in the anaesthetized rat. 2 Activation of murine cultured macrophages with LPS (1 mu g ml(-1)) resulted, within 24 h, in a significant increase in nitrite (an indicator of the formation of NO) in the cell supernatant. This increase in nitrite was attenuated by the tyrphostins AG126, AG556, AG490 or AG1641 or by genistein in a dose-dependent fashion (IC50:similar to 15 mu M). In contrast, tyrphos tin Al (an analogue of tyrphostin AC126) or daidzein (an analogue of g enistein) had no effect on the rise in nitrite caused by LPS. 3 Admini stration of LPS (E. coli, 10 mg kg(-1), i.v.) caused hypotension and a reduction of the presser responses elicited by noradrenaline (NA, 1 m u g kg(-1), i.v.). Pretreatment of rats with the tyrphostins AG126, AG 490, AG556, AG1641 or Al attenuated the circulatory failure caused by LPS. Although genistein attenuated the vascular hyporeactivity to NA, it did not affect the hypotension caused by LPS. Daidzein did not affe ct the circulatory failure caused by LPS. 4 Endotoxaemia for 360 min r esulted in rises in the serum levels of (i) urea and creatinine (indic ators of renal failure), (ii) alanine aminotransferase (ALT), aspartat e aminotransferase (AST), bilirubin and gamma-glutamyl transferase (ga mma GT) (indicators of liver injury/dysfunction), lipase (an indicator of pancreatic injury) as well as lactate (an indicator of tissue hypo xia). None of the tyrosine kinase inhibitors tested had a significant effect on the rise in the serum levels of urea, but the tyrphostins AG 126, AG556 or Al significantly attenuated the rises in the serum level s of creatinine caused by LPS. In addition, all tyrphostins and genist ein attenuated the liver injury/failure, the pancreatic injury, the hy poglycaemia and the lactic acidosis caused by LPS. In contrast, daidze in did not reduce the organ injury/dysfunction or the lactic acidosis caused by LPS. 5 Injection of LPS resulted (within 90 min) in a substa ntial increase in the serum level of tumour necrosis factor alpha (TNF alpha), which was attenuated by pretreatment of LPS-rats with any of the tyrphostins used. Genistein, but not daidzein, also reduced the ri se in the serum levels of TNF alpha caused by LPS. Endotoxaemia for 6 h also resulted in a substantial increase in the expression of iNOS an d COX-2 protein and activity in the lung, which was attenuated by pret reatment of LPS-rats with the tyrphostins AG126, AG556 or genistein, b ut not by daidzein. 6 Thus, tyrphostins (AG126, AG490, AG556, AG1641 o r Al) and genistein, but not daidzein (inactive analogue of genistein) , prevent the (i) circulatory failure, (ii) the multiple organ dysfunc tion (liver and pancreatic dysfunction/injury, lactacidosis, hypoglyca emia), as well as (iii) the induction of iNOS and COX-2 protein and ac tivity in rats with endotoxic shock.