Early signaling events by endotoxin in PC12 cells: Involvement of tyrosine kinase, constitutive nitric oxide synthase, cGMP-dependent protein kinase, and Ca²⁺ channels

We studied the effects of endotoxin from Escherichia coli (E. coli) on Ca²⁺ channel activity in PC12 cells using the cell-attached patch clamp technique. Endotoxin (1-100 ng/ml) decreased channel availability (n · P(o)) to about one third of control values, an effect that required 3.5 ± 1 min (mean ± SD; n = 13) to reach steady state. The biophysical properties of the channel, including slope conductance (22 pS; 40 mM Ba²⁺), voltage dependence of n · P(o), and open times (τ₁ = 0.78 ms, τ₂ = 8.9 ms) for the two open states at 0 mV, were not altered. The effect of endotoxin was blocked by polymyxin-B, indicating involvement of the lipid-A moiety of lipopolysaccharide, and by the tyrosine kinase (tk) inhibitor, tyrphostin. The effect of endotoxin was mimicked by 8-bromo-cGMP (100 μM), and was blocked by the inhibitor of cGMP-dependent protein kinase (PKG), H-8, suggesting involvement of the cGMP/PKG pathway. The effect of endotoxin also was blocked by the nitric oxide (NO) synthase inhibitor, N(G)-monomethyl-L- arginine monoacetate, suggesting involvement of nitric oxide synthase (NOS). The rapidity of the effect of endotoxin on Ca²⁺ channel activity suggested that constitutive NOS (cNOS) was involved, in accordance with our finding that endotoxin-induced transcriptional induction of NOS, as measured by nitrite production, required >6 hr. We conclude that early signaling events by endotoxin in PC12 cells involve tk, cNOS, cGMP/PKG, and Ca²⁺ channels.