1 Here we compared the effects of various inhibitors of the activity of
protein tyrosine kinase on (i) the expression of the activity of the inducible
isoform of
nitric oxide (
NO) synthase (iNOS) caused by
endotoxin (
lipopolysaccharide, LPS) in cultured macrophages, (ii) the induction of iNOS and
cyclooxygenase 2 (COX-2)
protein and activity in rats with endotoxaemia, and (iii) the
circulatory failure and organ dysfunction caused by LPS in the anesthetized rat. 2 Activation of murine cultured macrophages with LPS (1 microgram 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
nitrate was attenuated by the
tyrphostins AG126,
AG556,
AG490 or AG1641 or by
genistein in a dose-dependent fashion (IC50: approximately 15 microM). In contrast,
tyrphostin A1 (an analogue of
tyrphostin AG126) or
daidzein (an analogue of
genistein) had no effect on the rise in
nitrite caused by LPS. 3 Administration of LPS (E. coli, 10 mg kg-1, i.v.) caused
hypotension and a reduction of the pressor responses elicited by
noradrenaline (NA, 1 microgram kg-1, i.v.). Pretreatment of rats with the
tyrphostins AG126,
AG490,
AG556, AG1641 or A1 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 affect the
circulatory failure caused by LPS. 4 Endotoxaemia for 360 min resulted in rises in the serum levels of (i)
urea and
creatinine (indicators of
renal failure), (ii)
alanine aminotransferase (ALT),
aspartate aminotransferase (AST),
bilirubin and gamma-glutamyl
transferase (gamma GT) (indicators of liver injury/dysfunction),
lipase (an
indicator of pancreatic injury) as well as
lactate (an
indicator of tissue
hypoxia). None of the
tyrosine kinase inhibitors tested had a significant effect on the rise i the serum levels of
urea, but the
tyrphostins AG126,
AG556 or A1 significantly attenuated the rises in the serum level of
creatinine caused by LPS. In addition, all
tyrphostins and
genistein attenuated the liver injury/failure, the pancreatic injury, the hypoglycaemia and the
lactic acidosis caused by LPS. In contrast,
daidzein did not reduce the organ injury/dysfunction or the
lactic acidosis caused by LPS. 5 Injection of LPS resulted (within 90 min) in a substantial increase in the serum level of
tumor 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 rise 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 and COX-2
protein and activity in the lung, which was attenuated by pretreatment of LPS-rats with the
tyrphostins AG126,
AG556 or
genistein, but not by
daidzein. 6 Thus,
tyrphostins (
AG126,
AG556, AG1641 or A1) and
genistein, but not
daidzein (inactive analogue of
genistein), prevent the (i)
circulatory failure, (ii) the multiple organ
dysfunction (liver and pancreatic dysfunction/injury lactacidosis, hypoglycaemia), as well as (iii) the induction of iNOS and COX-2
protein and activity in rats with endotoxic
shock.