ESCHERICHIA-COLI ENDOTOXIN INHIBITS AGONIST-MEDIATED CYTOSOLIC CA2-OXIDE BIOSYNTHESIS IN CULTURED ENDOTHELIAL-CELLS( MOBILIZATION AND NITRIC)

Altered release of endothelium-derived relaxing factor/nitric oxide (E DRF/NO) has been proposed as a final common pathway underlying the abn ormal vasodilator responses to gram-negative lipopolysaccharide (endot oxin). However, mechanisms responsible for lipopolysaccharide-induced changes in EDRF/NO release from endothelial cells have not been clarif ied. We evaluated direct effects of Escherichia coli endotoxin on agon ist-stimulated cytosolic Ca2+ mobilization and NO biosynthesis in cult ured bovine and porcine aortic endothelial cells (ECs). Two methods we re used to assay for NO: (1) analysis of NO-induced endothelial levels of cGMP as a biological indicator of NO generation and (2) direct qua ntitative measurement of NO release (chemiluminescence method). Cytoso lic free Ca2+ ([Ca2+]i) was evaluated using fura 2 fluorescence method ology (340/380-nm ratio excitation and 500-nm emission). Incubation of ECs with endotoxin (0.5 mu g/mL, 1 hour plus 1-hour wash) significant ly inhibited bradykinin (100 nmol/L)- and ADP (10 mu mol/L)-mediated i ncreases in endothelial cell cGMP to 37% and 22% of control responses, respectively. In contrast, endotoxin failed to inhibit the increase i n cGMP produced by the non-receptor-dependent Ca2+ ionophore A23187 (1 mu mol/L) or sodium nitroprusside (1 mmol/L). Similarly, incubation w ith endotoxin inhibited ADP-stimulated increases in NO release and EDR F bioactivity to 55% and 56% of control values, respectively, but did not affect A23187-stimulated increases in NO release or EDRF bioactivi ty. Endotoxin produced significant decreases in both transient and sus tained [Ca2+](i) responses of ECs to bradykinin and ADP. For example, the initial rapid increase in bovine EC [Ca2+](i) in response to brady kinin was reduced to 31% of the initial increases in control cells, an d the secondary plateau phase was reduced to only 3% of respective con trol responses. Concentration-response relation to endotoxin (10(-3) t o 100 mu g/mL) indicated high correlation and similar IC50 values (0.0 25 and 0.021 mu g/mL, respectively) for inhibitory effects on cGMP and [Ca2+](i). Endotoxin had no effect on inositol trisphosphate formatio n ([H-3]myo-inositol incorporation) and intracellular Ca2+ release ([C a2+](i) responses in Ca2+-free medium) induced by bradykinin. However, agonist-stimulated Mn2+ quenching (index of Ca2+ influx) was signific antly attenuated by endotoxin treatment. These studies demonstrate tha t endotoxin directly decreases agonist (bradykinin and ADP)-mediated b iosynthesis and release of EDRF/NO from ECs. These effects can be expl ained by altered [Ca2+](i) mobilization mechanisms, which in turn prod uce subsequent decreases in activity of the Ca2+-calmodulin-dependent constitutive isoform of NO synthase and, ultimately, impairment of ago nist-mediated NO release and endothelium-dependent vasodilation.