We hypothesized that endotoxin (LPS) would impair vasoconstrictor-agonistinduced calcium (Ca2+) mobilization by a nitric oxide (NO)-dependent mechanism. We Incubated bovine aortic myocytes (passages 16 to 23) for 22 to 24 hours with 0 to 1.0 mg/ml Escherichia coil lipopolysaccharide (LPS). Medium (Dulbecco's modified Eagle's medium [DMEM] + 10% fetal bovine serum [FBS]) was assayed for nitrite (chemiluminescence), and myocytes were loaded with fura-2 acetoxymethyl ester (fura-2AM), after which we assessed basal and thrombin (10 U/ml)-induced peak Ca2+ mobilization by microspectrofluorimetry. LPS (0.01 to 1.0 mg/ml) led to dose-dependent nitrite accumulation, which was blocked by coin- cubation with Nω-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L). LPS also impaired Ca2+ responses in a dose-dependent manner (from -13% at 0.1 mg/ml to -47% at 1.0 mg/ml, n = 8 to 43/dose). However, coincubatlon with L-NAME did not ameliorate the Co mobilization defect (peak Ca2+ increments: confrol = 419 ± 30 nmol/L, vs LPS (1 mg/ml) = 206 ± 18 nmol/L (mean ± SE), n = 15; p < 0.001; control/L-NAME: 417 ± 31 nmol/L vs LPS/L-NAME: 212 ± 19 nmol/L; n = 17 p < 0.001), despite Inhibition of associated nitrite accumulation in the medium (control vs LPS; p < 0.001; control/L-NAME vs LPS/L-NAME: p > 0.05; LPS vs LPS/L-NAME: p < 0.001). Supplemental L-arginine augmented LPS-induced nitrite generation without affecting Ca2+ mobilization. Indomethacin failed to prevent the LPS-induced decrement in thrombin response, but did inhibit LPS-induced myocyte nitrite production, suggesting 'crosstalk' between the NO-synthase and cyclo-oxygenase (COX) systems. These experiments suggest that LPS-Induced vascular contractile Impairment is at least partly mediated by an NO-independent Impairment of agonist-induced myocyte Ca2+ mobilization. This further suggests that any Important contribution of NO synthesis to LPS-induced contractile dysfunction must depend on impairment of the Ca2+ sensitivity of the contractile apparatus (i.e., pharmacomechanical coupling).
ASJC Scopus subject areas
- Pathology and Forensic Medicine