BACKGROUND: Nitric oxide (NO) has been known to have antimicrobial activity, and the increased NO production in case of sepsis may play as a physiologic defense mechanism. However the increased formation of NO by the inducible nitric oxide synthase (iNOS) has been known to be implicated in pathophysiology of a variety of disease, including circulatory and septic shock. We measured the iNOS activities of mouse macrophage after application of various drugs with lipopolysaccharide (LPS) and gamma-interferon (IFN). The purpose of this study is to evaluate the inhibiting properties of various drugs to iNOS in case of sepsis. METHOD: Thirty ICR (Institue for Cancer Research) mouse weighting 30~40 gm were anesthetized with diethyl ether, and thiogycol broth was injected into peritoneal cavity. Two days later macrophages were collected from peritoneal cavity, and incubated for 24 hours in the CO2 incubator with LPS and IFN mixture and various concentration of dexamethasone, pentoxifylline, aspirin, aprotinin, regular insulin (RI) and neutral protamine hagedorn insulin (NPH). NO concentration were calculating by measuring nitrite concentration which represent the magnitude of NO production. The activities of iNOS in macrophages were measured by analysing iNOS m-RNA expression by Northern blot analysis with autoradiography using polymerase chain reaction (PCR) method. RESULT: The basic NO concentration was 13.0 +/- 8.0 micrometer. With LPS and IFN, NO concentration was increased to 104.4 +/- 31.9 micrometer. The increase in NO production by LPS and IFN was attenuated by dexamethasone (10 (-6) M only), pentoxifylline (above 10 (-10) M), aprotinin, RI, and NPH in dose dependent manner. The addition of LPS and IFN in the culture media caused increase in the iNOS m-RNA production. The aprotinin, RI, and NPH attenuated the increase in iNOS m-RNA production by LPS and IFN. Coclusion: These results suggest that the aprotinin, RI, and NPH prevent the LPS and IFN induced increase of NO production by attenuating the iNOS activity. These properties of aprotinin and insulin may be applied to the treatment of septic shock to block the enhanced formation of NO production.
Animals ; Aprotinin* ; Aspirin ; Autoradiography ; Blotting, Northern ; Culture Media ; Dexamethasone ; Ether ; Incubators ; Insulin* ; Insulin, Isophane ; Interferon-gamma ; Macrophages* ; Mice* ; Nitric Oxide ; Nitric Oxide Synthase Type II* ; Pentoxifylline ; Peritoneal Cavity ; Polymerase Chain Reaction ; Sepsis ; Shock, Septic