Impairment in endothelial cell intracellular free calcium (Cai) mobilization mechanisms may contribute to decreased nitric oxide (NO) biosynthesis and impaired vasorelaxation responses of endotoxemic guinea pigs to endothelium-dependent vasodilators. We tested this hypothesis using fura-2 microfluorometry to compare agonist-stimulated Cai responses of aortic endothelial cells freshly dispersed from guinea pigs 16 h after intraperitoneal injection of Escherichia coli endotoxin (lipopolysaccharide, LPS; 4 mg/kg) or saline (CON). In the presence of normal extracellular Ca2+ (2 mmol/L), basal (non-stimulated) endothelial Cai (340/380 nm fluorescence ratio, R) was not different between CON and LPS cells (1.1 ± 0.03 and 1.1 ± 0.03, respectively). However, exposure to ADP (10 μmol/L) produced a biphasic increase in Cai that was markedly decreased in cells from LPS-treated animals (P < 0.0001). Peak ADP-stimulated Cai responses averaged 2.2 ± 0.21 in CON cells and 1.5 ± 0.11 (P < 0.01) in cells dispersed from LPS-treated animals. Exposure to acetylcholine (ACh; 10 μmol/L) produced sustained increases in Cai (R = 1.4 ± 0.13) in CON cells; however, LPS abolished Cai responses to ACh. Exposure of endothelial cells to substance P (100 nmol/L) produced a biphasic increase in Cai that was not different between groups. In the absence of extracellular Ca2+ (plus 10 μmol/L EGTA), exposure to ADP (10 μmol/L) produced transient increases in Cai (Ca2+ release) that were decreased in cells from LPS-treated versus CON animals. Exposure to ACh in zero Ca2+ (10 μmol/L) produced smaller increases in Cai (peak R = 1.3 ± 0.12) in CON cells (when compared to ADP); however, Cai responses to ACh remained absent in cells from LPS-treated animals. Re-exposure to Ca2+ produced sustained ACh-induced Cai responses (Ca2+ influx) in cells from CON, but not LPS-treated animals; LPS markedly impaired (P < 0.05) ADP-induced sustained Cai responses. Our data demonstrate that in vivo LPS exposure elicits decreased agonist-stimulated endothelial Cai responses primarily involving impaired Ca2+ influx mechanisms. Known dependence of endothelial agonist-stimulated NO synthesis on Cai suggests that defects in cell Ca2+ mobilization may contribute to LPS-induced impaired NO biosynthesis and decreased endothelium-dependent relaxation.
- Bacterial lipopolysaccharide
ASJC Scopus subject areas
- Emergency Medicine
- Critical Care and Intensive Care Medicine