title: Escherichia coli endotoxin inhibits agonist-mediated cytosolic Ca2+ mobilization and nitric oxide biosynthesis in cultured endothelial cells.
abstract: Altered release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) has been proposed as a final common pathway underlying the abnormal vasodilator responses to gram-negative lipopolysaccharide (endotoxin). However, mechanisms responsible for lipopolysaccharide-induced changes in EDRF/NO release from endothelial cells have not been clarified. We evaluated direct effects of Escherichia coli endotoxin on agonist-stimulated cytosolic Ca2+ mobilization and NO biosynthesis in cultured bovine and porcine aortic endothelial cells (ECs). Two methods were used to assay for NO: (1) analysis of NO-induced endothelial levels of cGMP as a biological indicator of NO generation and (2) direct quantitative measurement of NO release (chemiluminescence method). Cytosolic free Ca2+ ([Ca2+]i) was evaluated using fura 2 fluorescence methodology (340/380-nm ratio excitation and 500-nm emission). Incubation of ECs with endotoxin (0.5 microgram/mL, 1 hour plus 1-hour wash) significantly inhibited bradykinin (100 nmol/L)- and ADP (10 mumol/L)-mediated increases in endothelial cell cGMP to 37% and 22% of control responses, respectively. In contrast, endotoxin failed to inhibit the increase in cGMP produced by the non-receptor-dependent Ca2+ ionophore A23187 (1 mumol/L) or sodium nitroprusside (1 mmol/L). Similarly, incubation with endotoxin inhibited ADP-stimulated increases in NO release and EDRF bioactivity to 55% and 56% of control values, respectively, but did not affect A23187-stimulated increases in NO release or EDRF bioactivity. Endotoxin produced significant decreases in both transient and sustained [Ca2+]i responses of ECs to bradykinin and ADP. For example, the initial rapid increase in bovine EC [Ca2+]i in response to bradykinin was reduced to 31% of the initial increases in control cells, and the secondary plateau phase was reduced to only 3% of respective control responses. Concentration-response relation to endotoxin (10(-3)) to 10(0) micrograms/mL) indicated high correlation and similar IC50 values (0.025 and 0.021 micrograms/mL, respectively) for inhibitory effects on cGMP and [Ca2+]i. Endotoxin had no effect on inositol trisphosphate formation ([3H]myo-inositol incorporation) and intracellular Ca2+ release ([Ca2+]i responses in Ca(2+)-free medium) induced by bradykinin. However, agonist-stimulated Mn2+ quenching (index of Ca2+ influx) was significantly attenuated by endotoxin treatment. These studies demonstrate that endotoxin directly decreases agonist (bradykinin and ADP)-mediated biosynthesis and release of EDRF/NO from ECs. These effects can be explained by altered [Ca2+]i mobilization mechanisms, which in turn produce subsequent decreases in activity of the Ca(2+)-calmodulin-dependent constitutive isoform of NO synthase and, ultimately, impairment of agonist-mediated NO release and endothelium-dependent vasodilation.
authors.affiliation: University of Graz, Austria.
authors.forename: W F
authors.initials: WF
authors.lastname: Graier
authors.forename: P R
authors.initials: PR
authors.lastname: Myers
authors.forename: L J
authors.initials: LJ
authors.lastname: Rubin
authors.forename: H R
authors.initials: HR
authors.lastname: Adams
authors.forename: J L
authors.initials: JL
authors.lastname: Parker
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000032:analysis; D003600:Cytosol / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004731:Endotoxins / Q000494:pharmacology*; D004926:Escherichia coli; D008070:Lipopolysaccharides / Q000378:metabolism; D008163:Luminescent Measurements; D009569:Nitric Oxide / Q000032:analysis / Q000096:biosynthesis*; D013552:Swine
chemical_list: D004731:Endotoxins; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D000244:Adenosine Diphosphate; D006152:Cyclic GMP; D001920:Bradykinin; D002118:Calcium