INHIBITION OF NITRIC-OXIDE SYNTHASE GENE-EXPRESSION IN-VIVO AND IN-VITRO BY REPEATED DOSES OF ENDOTOXIN

We have examined the effects of repeated endotoxin administration in v ivo and in vitro on the induction of nitric oxide synthase (NOS). In v ivo, hepatic NOS activity and mRNA were increased markedly by the admi nistration of Escherichia coli lipopolysaccharide (LPS). The change in hepatic NOS activity coincided with a marked accumulation of hepatic citrulline. Both enzyme activity and citrulline concentration returned to normal by 12 h after LPS administration. At this time, a subsequen t administration of endotoxin caused no change in either NOS mRNA, NOS activity, or citrulline concentration, and thus an endotoxin-refracto ry state for nitric oxide (NO) synthesis was established. Normal sensi tivity was reestablished by 24 h after the initial dose. In vitro stud ies using both a macrophage cell line (HD11) and primary macrophages i ndicated that LPS pretreatment caused cells in culture to become compl etely refractory to subsequent stimulation by LPS. Finally, we tested the hypothesis that NO may be involved in the development of the refra ctory state. Various inhibitors blocked the initial synthesis of NO by >90% but failed to influence the development of the refractory state. Our study demonstrates both in vivo and in vitro that NO synthesis is completely blocked after repeated exposure to endotoxin by a mechanis m that appears to be pretranslational. This model of early endotoxin t olerance may provide insight into the molecular mechanisms that regula te expression of the NOS gene.