Time course and cellular localization of lipopolysaccharide-induced inducible nitric oxide synthase messenger RNA expression in the rat in vivo.

Objectives: The role of nitric oxide in mediating the early phase of hypotension and cardiovascular hyporeactivity in sepsis, and the cellular sources of inducible nitric oxide synthase expression under in vivo conditions, remain unclear. The objective of this study was to elucidate the time course and cellular location of inducible nitric oxide synthase messenger RNA (mRNA) expression in rats treated with lipopolysaccharide in vivo. Design: Prospective, placebo-controlled, laboratory study. Setting: Experimental laboratory of a postgraduate medical research institution. Subjects: Normal, anesthetized rats. Interventions: Animals were treated with lipopolysaccharide (15 mg/kg ip), saline (1 mL/kg ip), or lipopolysaccharide plus dexamethasone (3 mg/kg ip, 50 mins before lipopolysaccharide administration) in vivo 4 hrs before the removal of tissues. Measurements and Main Results: Total RNA and mRNA were extracted. Total RNA was used for reverse transcription polymerase chain reaction. Poly-(A) + mRNA was isolated for Northern blot analysis. Cryostat sections of heart and lung were prepared for in situ hybridization to elucidate the cellular location of the inducible nitric oxide synthase gene. Hearts, lungs, aortae, and pulmonary arteries from lipopolysaccharide-treated animals expressed inducible nitric oxide synthase mRNA, which was markedly inhibited by pretreatment with dexamethasone (except in aorta). The threshold time point for inducible nitric oxide synthase mRNA induction was between 20 and 40 mins after lipopolysaccharide administration. The quantity of mRNA increased progressively thereafter, reaching a plateau between 4 and 8 hrs and decreasing markedly by 24 hrs. These findings are consistent with the time course of lipopolysaccharide-induced hypotension and cardiovascular hyporeactivity in animal models of septic shock and in man. Inducible nitric oxide synthase mRNA was mainly detected in vascular and airway smooth muscle cells, cardiac myocytes, pneumocytes, and infiltrated inflammatory cells, but was also identified in airway epithelial and vascular endothelial cells. Vascular smooth muscle represented the predominant cell type expressing inducible nitric oxide synthase under these conditions. Conclusions: These results provide further molecular evidence for the involvement of nitric oxide in the early phase of hypotension and cardiovascular hyporeactivity seen in septic shock. They also suggest that nitric oxide derived from vascular smooth muscle cells may contribute significantly to this hypotension and cardiovascular reactivity.