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

    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 (US)
    Pages (from-to)216-225
    Number of pages10
    JournalJournal of Neuroscience Research
    Volume45
    Issue number3
    DOIs
    StatePublished - 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
    • cGMP
    • endotoxin
    • nitric oxide
    • PC12 cells

    ASJC Scopus subject areas

    • Neuroscience(all)

    Cite this

    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, A.; Nowicki, B.; Chin, L. S.; Singh, S. K.; Perez-Polo, J. R.

    In: Journal of Neuroscience Research, Vol. 45, No. 3, 1996, p. 216-225.

    Research output: Contribution to journalArticle

    Simard, J. Marc ; Tewari, K. ; Kaul, A. ; Nowicki, B. ; Chin, L. S. ; Singh, S. K. ; Perez-Polo, J. R. / Early signaling events by endotoxin in PC12 cells : Involvement of tyrosine kinase, constitutive nitric oxide synthase, cGMP-dependent protein kinase, and Ca2+ channels. In: Journal of Neuroscience Research. 1996 ; Vol. 45, No. 3. pp. 216-225.
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    AU - Kaul, A.

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    AU - Chin, L. S.

    AU - Singh, S. K.

    AU - Perez-Polo, J. R.

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    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.

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