title: Endotoxin impairs agonist-stimulated intracellular free calcium (Ca(i)) responses in freshly dispersed aortic endothelial cells.
abstract: Impairment in endothelial cell intracellular free calcium (Ca(i)) 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 Ca(i) 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 Ca(i) (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 micromol/L) produced a biphasic increase in Ca(i) that was markedly decreased in cells from LPS-treated animals (P < 0.0001). Peak ADP-stimulated Ca(i) 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 micromol/L) produced sustained increases in Ca(i) (R = 1.4 +/- 0.13) in CON cells; however, LPS abolished Ca(i) responses to ACh. Exposure of endothelial cells to substance P (100 nmol/L) produced a biphasic increase in Ca(i) that was not different between groups. In the absence of extracellular Ca2+ (plus 10 micromol/L EGTA), exposure to ADP (10 micromol/L) produced transient increases in Ca(i) (Ca2+ release) that were decreased in cells from LPS-treated versus CON animals. Exposure to ACh in zero Ca2+ (10 micromol/L) produced smaller increases in Ca(i) (peak R = 1.3 +/- 0.12) in CON cells (when compared to ADP); however, Ca(i) responses to ACh remained absent in cells from LPS-treated animals. Re-exposure to Ca2+ produced sustained ACh-induced Ca(i) responses (Ca2+ influx) in cells from CON, but not LPS-treated animals; LPS markedly impaired (P< 0.05) ADP-induced sustained Ca(i) responses. Our data demonstrate that in vivo LPS exposure elicits decreased agonist-stimulated endothelial Ca(i) responses primarily involving impaired Ca2+ influx mechanisms. Known dependence of endothelial agonist-stimulated NO synthesis on Ca(i) suggests that defects in cell Ca2+ mobilization may contribute to LPS-induced impaired NO biosynthesis and decreased endothelium-dependent relaxation.
authors.affiliation: Dalton Cardiovascular Research Center and Department of Physiology, University of Missouri, Columbia 65211, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Jones
authors.forename: M
authors.initials: M
authors.lastname: Sturek
authors.forename: H R
authors.initials: HR
authors.lastname: Adams
authors.forename: J L
authors.initials: JL
authors.lastname: Parker
mesh_terms: D000818:Animals; D001011:Aorta / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism* / Q000494:pharmacology; D017136:Ion Transport / Q000187:drug effects; D013552:Swine
chemical_list: D004731:Endotoxins; D002118:Calcium