Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells

Chuen Mao Yang, Shue Fen Luo, Wen Bin Wu, Shiow Lin Pan, Yih Jeng Tsai, Chi Tso Chiu, Chuan Chwan Wang

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

1. Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+](i) by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+](i) peak in a concentration-dependent manner. 2. Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 μM) for 30 min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3. The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+](i) were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4α-phorbol 12,13-didecanoate at 1 μM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4. In parallel with the effect of PMA on the BK-induced IPs formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-α, βI, βII, γ, δ, ε, θ and ζ. With PMA treatment of the cells for various times, translocation of PKC-α, PI, βII, γ, δ, ε and θ from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down-regulation of these PKC isozymes. PKC-ζ was not significantly translocated and down-regulated at any of the times tested. 5. Treatment of TECs with 1 μM PMA for either 30 min or 6 h did not significantly change the K(D) and B(max) receptor for BK binding (control: K(D) = 1.7 ± 0.3 nM; B(max) = 50.5 ± 4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. 6. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+](i) increase or inhibit independently both responses to BK. The translocation of pKC-α, βI, βII, δ, ε, γ, and θ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilization in TECs.

Original languageEnglish
Pages (from-to)627-636
Number of pages10
JournalBritish Journal of Pharmacology
Volume125
Issue number4
DOIs
Publication statusPublished - 1998
Externally publishedYes

Fingerprint

Bradykinin
Phorbol Esters
Phosphatidylinositols
Protein Kinase C
Canidae
Hydrolysis
Epithelial Cells
Inositol Phosphates
Bradykinin Receptors
Isoenzymes
Down-Regulation
Staurosporine
Protein C Inhibitor
Protein Kinase Inhibitors
Cell Extracts
Cytosol
Acetates
Western Blotting
Membranes
Antibodies

Keywords

  • Bradykinin
  • Ca
  • Inositol phosphates
  • Phorbol ester
  • Protein kinase C
  • Tracheal epithelial cells

ASJC Scopus subject areas

  • Pharmacology

Cite this

Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells. / Yang, Chuen Mao; Luo, Shue Fen; Wu, Wen Bin; Pan, Shiow Lin; Tsai, Yih Jeng; Chiu, Chi Tso; Wang, Chuan Chwan.

In: British Journal of Pharmacology, Vol. 125, No. 4, 1998, p. 627-636.

Research output: Contribution to journalArticle

Yang, Chuen Mao ; Luo, Shue Fen ; Wu, Wen Bin ; Pan, Shiow Lin ; Tsai, Yih Jeng ; Chiu, Chi Tso ; Wang, Chuan Chwan. / Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells. In: British Journal of Pharmacology. 1998 ; Vol. 125, No. 4. pp. 627-636.
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abstract = "1. Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+](i) by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+](i) peak in a concentration-dependent manner. 2. Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 μM) for 30 min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3. The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+](i) were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4α-phorbol 12,13-didecanoate at 1 μM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4. In parallel with the effect of PMA on the BK-induced IPs formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-α, βI, βII, γ, δ, ε, θ and ζ. With PMA treatment of the cells for various times, translocation of PKC-α, PI, βII, γ, δ, ε and θ from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down-regulation of these PKC isozymes. PKC-ζ was not significantly translocated and down-regulated at any of the times tested. 5. Treatment of TECs with 1 μM PMA for either 30 min or 6 h did not significantly change the K(D) and B(max) receptor for BK binding (control: K(D) = 1.7 ± 0.3 nM; B(max) = 50.5 ± 4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. 6. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+](i) increase or inhibit independently both responses to BK. The translocation of pKC-α, βI, βII, δ, ε, γ, and θ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilization in TECs.",
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TY - JOUR

T1 - Uncoupling of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells

AU - Yang, Chuen Mao

AU - Luo, Shue Fen

AU - Wu, Wen Bin

AU - Pan, Shiow Lin

AU - Tsai, Yih Jeng

AU - Chiu, Chi Tso

AU - Wang, Chuan Chwan

PY - 1998

Y1 - 1998

N2 - 1. Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+](i) by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+](i) peak in a concentration-dependent manner. 2. Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 μM) for 30 min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3. The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+](i) were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4α-phorbol 12,13-didecanoate at 1 μM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4. In parallel with the effect of PMA on the BK-induced IPs formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-α, βI, βII, γ, δ, ε, θ and ζ. With PMA treatment of the cells for various times, translocation of PKC-α, PI, βII, γ, δ, ε and θ from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down-regulation of these PKC isozymes. PKC-ζ was not significantly translocated and down-regulated at any of the times tested. 5. Treatment of TECs with 1 μM PMA for either 30 min or 6 h did not significantly change the K(D) and B(max) receptor for BK binding (control: K(D) = 1.7 ± 0.3 nM; B(max) = 50.5 ± 4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. 6. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+](i) increase or inhibit independently both responses to BK. The translocation of pKC-α, βI, βII, δ, ε, γ, and θ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilization in TECs.

AB - 1. Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+](i) by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Stimulation of TECs by bradykinin (BK) led to IPs formation and caused an initial transient [Ca2+](i) peak in a concentration-dependent manner. 2. Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 μM) for 30 min attenuated the BK-induced IPs formation and Ca2+ mobilization. The maximal inhibition occurred after incubating the cells with PMA for 2 h. 3. The concentrations of PMA that gave half-maximal (pEC50) inhibition of BK-induced IPs accumulation and an increase in [Ca2+](i) were 7.07 M and 7.11 M, respectively. Inactive phorbol ester, 4α-phorbol 12,13-didecanoate at 1 μM, did not inhibit these responses. Prior treatment of TECs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 4. In parallel with the effect of PMA on the BK-induced IPs formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-α, βI, βII, γ, δ, ε, θ and ζ. With PMA treatment of the cells for various times, translocation of PKC-α, PI, βII, γ, δ, ε and θ from cytosol to the membrane was seen after 5 min, 30 min, 2 h, and 4 h treatment. However, 6 h treatment caused a partial down-regulation of these PKC isozymes. PKC-ζ was not significantly translocated and down-regulated at any of the times tested. 5. Treatment of TECs with 1 μM PMA for either 30 min or 6 h did not significantly change the K(D) and B(max) receptor for BK binding (control: K(D) = 1.7 ± 0.3 nM; B(max) = 50.5 ± 4.9 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. 6. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+](i) increase or inhibit independently both responses to BK. The translocation of pKC-α, βI, βII, δ, ε, γ, and θ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilization in TECs.

KW - Bradykinin

KW - Ca

KW - Inositol phosphates

KW - Phorbol ester

KW - Protein kinase C

KW - Tracheal epithelial cells

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