Endotoxin impairs agonist-induced calcium mobilization in bovine aortic myocytes by a nitric oxide-independent mechanism.

We hypothesized that endotoxin (LPS) would impair vasoconstrictor-agonist-induced 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 coli 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 coincubation with N(omega)-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, coincubation with L-NAME did not ameliorate the Ca2+ mobilization defect (peak Ca2+ increments: control = 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).

Endotoxin impairs agonist-induced calcium mobilization in rat mesangial cells.

We hypothesized that endotoxin would impair agonist-induced calcium (Ca2+) mobilization in rat mesangial cells, owing to the induction of nitric oxide synthase (NOS) and augmented nitric oxide (NO) synthesis. We measured basal and bradykinin-induced peak free cytosolic Ca2+ concentrations through microspectrofluorimetry with fura-2 in confluent mesangial cells, and assayed conditioned medium for nitrite accumulation. Prior to measurement, cells were incubated overnight in serum-supplemented medium, with or without endotoxin, 1-arginine, indomethacin, meclofenamate, or N omega-nitro-L-arginine methyl ester (L-NAME). Endotoxin (1 mg/ml) decreased bradykinin-induced peak Ca2+ responses by 35 to 60% (p < 0.0001) and increased nitrite accumulation > 6-fold (p < 0.01). Arginine supplementation further (> 9-fold, p < 0.0001) increased nitrite accumulation without changing the effect on Ca2+. Inhibition of NOS abolished increments in nitrite concentration but had no effect on impaired Ca2+ responses. Cyclooxygenase (COX) inhibitors, present during incubation with endotoxin, but not afterward, normalized bradykinin-stimulated calcium responses. Thrombin-stimulated Ca2+ responses were similarly affected. We conclude that neither NO nor prostaglandins act directly to impair agonist-induced Ca2+ mobilization following endotoxin exposure; however, this effect may be an indirect effect of COX products, including reactive oxygen intermediates.