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, Anil Kaul, B. Nowicki, L. S. Chin, S. K. Singh, J. R. Perez-Polo

Research output: Contribution to journalArticle

13 Citations (Scopus)

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

Fingerprint

Cyclic GMP-Dependent Protein Kinases
PC12 Cells
Endotoxins
Nitric Oxide Synthase
Protein-Tyrosine Kinases
Tyrphostins
Polymyxin B
Lipid A
Patch-Clamp Techniques
Nitrites
Lipopolysaccharides
Arginine

Keywords

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

Cite this

@article{564302f1c88b4d8cbe4b2ac40048c9a9,
title = "Early signaling events by endotoxin in PC12 cells: Involvement of tyrosine kinase, constitutive nitric oxide synthase, cGMP-dependent protein kinase, and Ca2+ channels",
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.",
keywords = "Ca channels, PC12 cells, cGMP, endotoxin, nitric oxide",
author = "Simard, {J. Marc} and K. Tewari and Anil Kaul and B. Nowicki and Chin, {L. S.} and Singh, {S. K.} and Perez-Polo, {J. R.}",
year = "1996",
month = "10",
day = "7",
doi = "10.1002/(SICI)1097-4547(19960801)45:3<216::AID-JNR3>3.0.CO;2-G",
language = "English",
volume = "45",
pages = "216--225",
journal = "Journal of Neuroscience Research",
issn = "0360-4012",
publisher = "Wiley-Liss Inc.",
number = "3",

}

Early signaling events by endotoxin in PC12 cells : Involvement of tyrosine kinase, constitutive nitric oxide synthase, cGMP-dependent protein kinase, and Ca2+ channels. / Simard, J. Marc; Tewari, K.; Kaul, Anil; Nowicki, B.; Chin, L. S.; Singh, S. K.; Perez-Polo, J. R.

In: Journal of Neuroscience Research, Vol. 45, No. 3, 07.10.1996, p. 216-225.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Early signaling events by endotoxin in PC12 cells

T2 - Involvement of tyrosine kinase, constitutive nitric oxide synthase, cGMP-dependent protein kinase, and Ca2+ channels

AU - Simard, J. Marc

AU - Tewari, K.

AU - Kaul, Anil

AU - Nowicki, B.

AU - Chin, L. S.

AU - Singh, S. K.

AU - Perez-Polo, J. R.

PY - 1996/10/7

Y1 - 1996/10/7

N2 - 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.

AB - 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.

KW - Ca channels

KW - PC12 cells

KW - cGMP

KW - endotoxin

KW - nitric oxide

UR - http://www.scopus.com/inward/record.url?scp=0029743373&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1097-4547(19960801)45:3<216::AID-JNR3>3.0.CO;2-G

DO - 10.1002/(SICI)1097-4547(19960801)45:3<216::AID-JNR3>3.0.CO;2-G

M3 - Article

C2 - 8841982

AN - SCOPUS:0029743373

VL - 45

SP - 216

EP - 225

JO - Journal of Neuroscience Research

JF - Journal of Neuroscience Research

SN - 0360-4012

IS - 3

ER -