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 cla mp 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 biophy sical properties of the channel, including slope conductance (22 pS; 4 0 mM Ba2+), voltage dependence of n . P-o, and open times (tau(1) = 0. 78 ms, tau(2) = 8.9 ms) for the two open states at 0 mV, were not alte red. The effect of endotoxin was blocked by polymyxin-B, indicating in volvement of the lipid-A moiety of lipopolysaccharide, and by the tyro sine kinase (tk) inhibitor, tyrphostin. The effect of endotoxin was mi micked by 8-bromo-cGMP (100 mu M), and was blocked by the inhibitor of cGMP-dependent protein kinase (PKG), H-8, suggesting involvement of t he cGMP/PKG pathway. The effect of endotoxin also was blocked by the n itric oxide (NO) synthase inhibitor, N-G-monomethyl-L-arginine monoace tate, suggesting involvement of nitric oxide synthase (NOS), The rapid ity 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 b y nitrite production, required >6 hr. We conclude that early signaling events by endotoxin in PC12 cells involve tk, cNOS, cGMP/PKG, and Ca2 + channels. (C) 1996 Wiley-Liss, Inc.