Upregulation of inducible nitric oxide synthase and nitric oxide occurs later than the onset of the hyperdynamic response during sepsis

Polymicrobial sepsis is characterized by an early, hyperdynamic phase (i.e., 2-10 h after cecal ligation and puncture [CLP]) followed by a late, hypodynamic phase (16 h after CLP or later). Although nitric oxide (NO) plays an important role in the pathophysiologic response during sepsis, it remains unknown how early NO is upregulated after the onset of sepsis and which organs are responsible for producing the increased amount of NO. To study this, male rats were subjected to sepsis by CLP followed by fluid resuscitation. Blood samples were then taken at 2, 5, 10, or 20 h after CLP or sham operation. In additional groups of animals, the kidneys, small intestine, heart, liver, and lungs were harvested at 5 or 10 h after CLP. Plasma and tissue levels of nitrate and nitrite (NO3-/NO2-, stable products of NO) were determined by using a colorimetric assay. Inducible NO synthase (iNOS) mRNA was examined in various tissues harvested at 10 h after CLP by reverse transcription-polymerase chain reaction (RT-PCR) technique. The results indicate that plasma levels of NO3-/NO2- (mainly reflecting INOS activity) did not increase at 2-5 h but were significantly elevated at 10-20 h after CLP. Tissue levels of NO3-/NO2- increased significantly in the kidneys, small intestines, heart, and liver at 10 h but not at 5 h after CLP. Similarly, INOS gene expression was upregulated in the kidneys, small intestines, and liver. Thus, the above organs appear to be important sites responsible for producing the increased NO during sepsis. Because we previously showed that the hyperdynamic response occurs as early as 2 h after CLP and because iNOS-derived NO production is not upregulated earlier than 10 h after the onset of sepsis, it appears that factors other than NO are responsible for producing the hyperdynamic response during sepsis.