Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells

Chuen Mao Yang, Yih Jeng Tsai, Shiow Lin Pan, Chih Chung Lin, Wen Bin Wu, Chuan Chwan Wang, Samuel C M Huang, Chi Tso Chiu

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Abstract

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+](i) by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+](i), were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs 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. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-α, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K(D) and B(max) of the BK receptor for binding (control: K(D) = 1.7 ± 0.2 nM; B(max) = 47.3 ± 4.4 fmolmg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-α, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs. Copyright (C) 1999 Elsevier Science Inc.

Original languageEnglish
Pages (from-to)899-907
Number of pages9
JournalCellular Signalling
Volume11
Issue number12
DOIs
Publication statusPublished - Dec 1999
Externally publishedYes

Fingerprint

Bradykinin
Phorbol Esters
Phosphatidylinositols
Vascular Smooth Muscle
Protein Kinase C
Smooth Muscle Myocytes
Hydrolysis
Isoenzymes
Inositol Phosphates
Down-Regulation
Staurosporine
Protein C Inhibitor
Protein Kinase Inhibitors
Cytosol
Acetates
Western Blotting
Membranes
Antibodies

Keywords

  • Bradykinin
  • Ca
  • Inositol phosphates
  • Phorbol ester
  • Protein kinase C
  • Vascular smooth muscle cells

ASJC Scopus subject areas

  • Cell Biology

Cite this

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells. / Yang, Chuen Mao; Tsai, Yih Jeng; Pan, Shiow Lin; Lin, Chih Chung; Wu, Wen Bin; Wang, Chuan Chwan; Huang, Samuel C M; Chiu, Chi Tso.

In: Cellular Signalling, Vol. 11, No. 12, 12.1999, p. 899-907.

Research output: Contribution to journalArticle

Yang, Chuen Mao ; Tsai, Yih Jeng ; Pan, Shiow Lin ; Lin, Chih Chung ; Wu, Wen Bin ; Wang, Chuan Chwan ; Huang, Samuel C M ; Chiu, Chi Tso. / Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells. In: Cellular Signalling. 1999 ; Vol. 11, No. 12. pp. 899-907.
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abstract = "Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+](i) by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+](i), were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs 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. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-α, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K(D) and B(max) of the BK receptor for binding (control: K(D) = 1.7 ± 0.2 nM; B(max) = 47.3 ± 4.4 fmolmg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-α, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs. Copyright (C) 1999 Elsevier Science Inc.",
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AU - Yang, Chuen Mao

AU - Tsai, Yih Jeng

AU - Pan, Shiow Lin

AU - Lin, Chih Chung

AU - Wu, Wen Bin

AU - Wang, Chuan Chwan

AU - Huang, Samuel C M

AU - Chiu, Chi Tso

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N2 - Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+](i) by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+](i), were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs 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. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-α, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K(D) and B(max) of the BK receptor for binding (control: K(D) = 1.7 ± 0.2 nM; B(max) = 47.3 ± 4.4 fmolmg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-α, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs. Copyright (C) 1999 Elsevier Science Inc.

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KW - Bradykinin

KW - Ca

KW - Inositol phosphates

KW - Phorbol ester

KW - Protein kinase C

KW - Vascular smooth muscle cells

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