Resuscitation of hemorrhagic shock attenuates intrapulmonary nitric oxide formation.

Hemorrhagic shock has been shown to upregulate intrapulmonary inducible nitric oxide (NO) synthase (iNOS) expression. Increased intrapulmonary iNOS expression is reflected by increases in concentrations of NO in the airways. The purpose of this study was to examine the effects of resuscitation on this induction of intrapulmonary NO formation caused by hemorrhage. Eighteen rats were randomized to one of three groups. One group of rats was simply sham-instrumented and monitored. Two other groups experienced hemorrhagic shock (mean systemic blood pressure of 40-45 mmHg) for 60 min. In one of the hemorrhagic shock groups, resuscitation was performed by re-infusing the shed blood and supplementing it with normal saline. Compared with sham-instrumented rats, those exposed to hemorrhagic shock without subsequent resuscitation exhibited a 10-fold increase in exhaled NO concentrations. Additionally, concentrations of both intrapulmonary iNOS protein and mRNA increased. Resuscitation attenuated the hemorrhage-induced upregulation of exhaled NO, iNOS protein and iNOS mRNA. This data suggests that resuscitation attenuates the hemorrhagic shock-induced formation of intrapulmonary NO by downregulating iNOS transcription. We believe that exhaled NO concentrations provide a useful, non-invasive method of monitoring the intrapulmonary inflammatory sequelae of hemorrhagic shock.

Diprivan attenuates the cytotoxicity of nitric oxide in cultured human bronchial epithelial cells.

OBJECTIVE: Excess nitric oxide (NO) and its reactive derivatives cause oxidative reactions that lead to cell death. Propofol, an intravenous anesthetic, exhibits antioxidant properties. Diprivan is a widely used commercial preparation of propofol that is emulsified in 10% intralipids. We sought to test the hypothesis that clinically encountered concentrations of Diprivan attenuate the toxicity of NO in a cell culture model. DESIGN: Prospective, randomized, controlled trial. SETTING: University research laboratory. SUBJECTS: Cultured human bronchial epithelial (IB-3) cells. INTERVENTIONS: Human bronchial epithelial cell cultures were randomly assigned to one of the following six groups: no additives (negative control), NO alone (positive control), NO with either 1 micro M, 10 micro M or 100 micro M Diprivan, and 100 micro M Diprivan alone (Diprivan control). S-nitroso-N-acetylpenicillamine (SNAP) was used to generate NO. MEASUREMENTS AND RESULTS: Hemacytometry with trypan blue staining was used to measure cell survival. To assess direct NO toxicity, immunoblot assays for nitrotyrosine-containing proteins in cell homogenates were performed. Exogenous NO significantly decreased live cell numbers and increased intracellular nitrotyrosine-containing protein concentrations (p<0.001). Diprivan significantly attenuated these changes in a concentration-independent manner (p<0.001). At concentrations as low as 1 micro M, Diprivan exhibited cytoprotective effects. CONCLUSIONS: Diprivan effectively attenuates the cytotoxicity of excessive NO exposure in IB-3 cells at concentrations that are clinically attainable.

Environmental pH regulates LPS-induced nitric oxide formation in murine macrophages.

This study was designed to determine how pH affects nitric oxide (NO) formation induced by lipopolysaccharide (LPS) in cultured murine macrophages (RAW 264.7). The initial pH of LPS-containing culture media was adjusted to one of eight values (6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, and 8.2). After exposure to LPS for eighteen hours, the cultures were harvested for analysis of mRNA, protein, and nitrate/nitrite (stable by-products of NO). Analyses for these substances were performed using semiquantitative RT-PCR, immunoblotting, and colorimetric Griess assays, respectively. We found that acidic culture media favored expression of inducible nitric oxide synthase (iNOS) mRNA. However, alkaline media favored expression of iNOS protein. Our findings suggest that post-transcriptional mechanisms predominate over transcriptional ones in order to regulate pH-mediated effects on NO formation by murine macrophages. The optimal pH for NO formation by iNOS was found in our study to be around 7.2.

Interleukin-10 inhibition of nitric oxide biosynthesis involves suppression of CAT-2 transcription.

Interleukin-10 (IL-10) has been shown to attenuate lipopolysaccharide (LPS) stimulation of inducible nitric oxide synthase (iNOS) in various cell types. Guanosine triphosphate cyclohydrolase I (GTPCH) and type-2 cationic amino acid transporter (CAT-2) are enzymes that regulate iNOS activity. We therefore sought to assess the effects of IL-10 on the expression of these regulatory enzymes in LPS-stimulated macrophages that are known to express iNOS. Five minutes after adding LPS to these macrophage cultures, various doses of recombinant human IL-10 were also added. The samples were harvested for analysis 18 h after exposure to both LPS and IL-10. In LPS-stimulated macrophages, IL-10 attenuated the upregulation of nitric oxide and iNOS protein but not iNOS mRNA. IL-10 also attenuated the LPS-induced upregulation of CAT-2 mRNA. However, IL-10 and LPS had no effect on GTPCH mRNA expression. We therefore conclude that IL-10 inhibits nitric oxide formation in LPS-stimulated macrophages partly by decreasing iNOS protein expression. Moreover, our data suggests that transcriptional control of CAT-2 plays a role in IL-10 mediated influences upon nitric oxide biosynthesis.