Nitric oxide synthesis by hepatic cells is down regulated in endotoxin tolerant rats

Abstract

The administration of a non-lethal dose of lipopolysaccharide (LPS) to experimental animals and human subjects results in a state of hyporesponsiveness to a second lethal challenge. Relatively little is known about the mechanisms of this endotoxin tolerance, especially about the induction of nitric oxide formation after LPS under these condition. Male Sprague-Dawley rats were divided into four groups: 1) rats that received a non-lethal i.v. injection of Escherichia coli LPS (0.5 mg/kg i.v., 'low dose'); 2) rats given a single injection of 'high dose' of LPS (3 mg/kg i.v.); 3) rats administered a low dose of LPS 12-168 h before they were challenged by a second injection of a high dose LPS (0.5 mg/kg followed by 3 mg/kg i.v., 'double injection'); and 4) rats treated with saline instead of LPS (1 ml/kg i.v., 'control'). 6 h after the high dose LPS, the livers were perfused with Krebs Henseleit buffer in a recirculating system at 37°C for 1 h, or hepatic cells were isolated. The isolated hepatocytes, Kupffer and hepatic endothelial cells were incubated in Hank's balanced salt solution (HBSS), containing 1 mM L-arginine, at 37°C for 3 h. The liver perfusate and supernatant from cell incubation were collected for determination of nitrite plus nitrate. Transcripts for inducible nitric oxide synthase (iNOS) were measured by cDNA equalized reverse transcription polymerase chain reaction in freshly isolated hepatic cells. Plasma glucose, lactate, alanine aminotransferase (ALT) and reactive nitrogen intermediates (RNIs) were also determined. High dose LPS alone caused a significant hypoglycemia (from 121.6 ± 3.0 to 84.5 ± 9.2 mg/dl), lactacidemia (from 8.3 ± 0.7 to 40.2 ± 5.7 mg/dl) and increase in plasma ALT (from 20.5 ± 2.8 to 477.8 ± 105.4 u/l). RNI levels in plasma also increased after 3 h and reached the maximum at 24 h after LPS (from 32.0 ± 1.3 to 795.3 ± 121.5 μM). RNI release from the perfused liver 6 h after high dose LPS was increased from 9.0 ± 2.0 to 156.6 ± 24.6 nmoles/g.h. Freshly isolated hepatic cells from control or low dose LPS treated rats released only small amounts of RNI. After high dose LPS administration, however, RNI release by hepatocytes, Kupffer and hepatic endothelial cells was increased 2.5, 14 and 4.5 fold, respectively. The 'high dose' LPS-induced increase of RNI production was associated with upregulation of iNOS mRNA in Kupffer and endothelial cells. After double injection of LPS (group 3), a protective effect was demonstrated by attenuated mortality, glucose changes, lactacidemia, and amino transferase activity, as compared to the high dose group. LPS tolerance with regard to RNI production by the liver was observed by 12 h, reached its maximum at about 72 h and was still evident even 120 h after the first injection of LPS. An attenuated RNI production in the supernatant from isolated hepatic cell cultures was also observed in the double injection group as compared to RNI release following the 'high dose' alone. This was associated with suppression of upregulation of iNOS mRNA induced by high dose LPS in Kupffer and hepatic endothelial cells. In contrast to the attenuated hepatic release of RNI during acute tolerance, RNI levels in plasma did not show hyporesponsiveness.