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Early signaling events by endotoxin in PC12 cells: Involvement of tyrosine kinase, constitutive nitric oxide synthase, cGMP‐dependent protein kinase, and Ca 2+ channels
Author(s) -
Simard J.M.,
Tewari K.,
Kaul A.,
Nowicki B.,
Chin L.S.,
Singh S.K.,
PerezPolo J.R.
Publication year - 1996
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/(sici)1097-4547(19960801)45:3<216::aid-jnr3>3.0.co;2-g
Subject(s) - nitric oxide , nitric oxide synthase , cgmp dependent protein kinase , protein kinase a , lipopolysaccharide , kinase , chemistry , tyrosine kinase , biochemistry , microbiology and biotechnology , signal transduction , biology , endocrinology , mitogen activated protein kinase kinase
We studied the effects of endotoxin from Escherichia coli (E. coli) on Ca 2+ channel activity in PC12 cells using the cell‐attached patch clamp technique. Endotoxin (1–100 ng/ml) decreased channel availability (n · P 0 ) 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 Ba 2+ ), voltage dependence of n · P 0 , and open times (τ 1 = 0.78 ms, τ 2 = 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 Ca 2+ 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 Ca 2+ channels. © 1996 Wiley‐Liss, Inc.