Early signaling events by endotoxin in PC12 cells: Involvement of tyrosine kinase, constitutive nitric oxide synthase, cGMP-dependent protein kinase, and Ca2+ channels
✍ Scribed by J.M. Simard; K. Tewari; A. Kaul; B. Nowicki; L.S. Chin; S.K. Singh; J.R. Perez-Polo
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 871 KB
- Volume
- 45
- Category
- Article
- ISSN
- 0360-4012
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✦ Synopsis
We studied the effects of endotoxin from Escherichiu coli (E. coli) on Ca2+ channel activity in PC12 cells using the cell-attached patch clamp technique. Endotoxin (1-100 nglml) decreased channel availability (n -Po) 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 . Po, and open times ( T ~ = 0.78 ms, T~ = 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 FM), and was blocked by the inhibitor of cGMPdependent 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, NG-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.