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

J. Marc Simard, K. Tewari, A. Kaul, B. Nowicki, L. S. Chin, S. K. Singh, J. R. Perez-Polo

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Abstract

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.

Original languageEnglish
Pages (from-to)216-225
Number of pages10
JournalJournal of Neuroscience Research
Volume45
Issue number3
DOIs
StatePublished - 7 Oct 1996

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Keywords

  • Ca channels
  • PC12 cells
  • cGMP
  • endotoxin
  • nitric oxide

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