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 x 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 x Po, and open times (tau 1 = 0.78 ms, tau 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 microM), 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, 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.
Simard
J M
JM
Division of Neurological Surgery, University of Maryland School of Medicine, Baltimore, USA.
Tewari
K
K
Kaul
A
A
Nowicki
B
B
Chin
L S
LS
Singh
S K
SK
Perez-Polo
J R
JR
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004731:Endotoxins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D008050:Lipid A / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D015290:Second Messenger Systems / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004731:Endotoxins; D004791:Enzyme Inhibitors; D008050:Lipid A; D008070:Lipopolysaccharides; C044101:endotoxin, Escherichia coli; D019001:Nitric Oxide Synthase; D011505:Protein-Tyrosine Kinases; D017869:Cyclic GMP-Dependent Protein Kinases