Mechanism that regulates nitric oxide production by lipopolysaccharide-stimulated rat Kupffer cells

Mechanism that regulates nitric oxide (NO) production by stimulated Kupffer cells was studied. Lipopolysaccharide (LPS) stimulated NO production by primary-cultured Kupffer cells in a time- and dose-dependent manner. Twenty-four h after incubation with 1 microgram/ml of LPS, the nitrite concentration in the culture medium increased from 2.87 +/- 1.4 to 73.6 +/- 6.9 nmol/ml. NO production started to increase around 6 h after adding LPS and reached a maximum within 24 h. NO production was inhibited by 0.3 mM of NG-monomethyl-L-arginine and was reversed by adding 3 mM of L-arginine. When incubated with 1 microgram/ml of LPS for 24 h, NO synthase activity in Kupffer cells increased from 0.164 +/- 0.035 to 3.16 +/- 0.11 nmol/min/mg protein. Dexamethasone and prednisolone significantly inhibited the induction of NO synthase and NO production in Kupffer cells. Expression of mRNA for inducible type of NO synthase (iNOS) started to increase around 4 h after adding LPS (1 microgram/ml) and reached a maximum by about 20 h. The enhanced expression of iNOS mRNA was also suppressed by 1 microM dexamethasone. On the other hand, calcium ionophore A23187 significantly enhanced the induction of NO synthase and iNOS mRNA expression in LPS-stimulated Kupffer cells. In addition, a tyrosine kinase inhibitor, genistein (100 microM), significantly inhibited NO production by LPS-stimulated Kupffer cells. Neither phorbol 12-myristate 13-acetate, dibutyryl cAMP, indomethacin nor a protein kinase C inhibitor H-7 affected NO production by Kupffer cells. These results suggested that iNOS mRNA expression, NO synthase activity and NO production increased in LPS-stimulated Kupffer cells by a glucocorticoid-inhibitable mechanism and that Ca2+ and tyrosine phosphorylation might play important roles in LPS-dependent induction of NO synthase.