The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation

Géza Szücs, Stephan Heinke, Christine De Greef, Luc Raeymaekers, Jan Eggermont, Guy Droogmans, Bernd Nilius

Research output: Contribution to journalArticle

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Abstract

We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (ICl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation of ICl,vol occurred at a hypotonicity of 27.5 ± 1.2%. Run-down of the current upon repetitive activation was less than 15% within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (-)-indolactam did not affect ICl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 μmol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19-31 pseudosubstrate peptide, did not influence ICl,vol. Trifluoperazine and tamoxifen fully blocked IcCl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 μmol/1 respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca2+] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 μol/1) and genistein (100 μol/1) did not affect ICl,vol Exposing CPAE cells to lysophosphatidic acid (1μmol/1), an activator of p42 MAPkinase and the focal adhesion kinase p125FAK in endothelial cells, neither evoked a Cl- current nor affected ICl,vol Neither wortmannin (10 μmol/1), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/1), which interferes with the p70S6 kinase pathway, affected ICl,vol Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl- current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl- current induced by hypotonicity.

Original languageEnglish
Pages (from-to)540-548
Number of pages9
JournalPflugers Archiv European Journal of Physiology
Volume431
Issue number4
DOIs
Publication statusPublished - Feb 1996

Fingerprint

Phosphorylation
Endothelial cells
Pulmonary Artery
Chlorides
Endothelial Cells
Chemical activation
Induced currents
Calmodulin
Protein Kinase C
Acetates
MAP Kinase Kinase 3
Hypotonic Solutions
Trifluoperazine
Focal Adhesion Protein-Tyrosine Kinases
Genistein
Clamping devices
Patch-Clamp Techniques
Sirolimus
Tamoxifen
Phosphatidylinositol 3-Kinases

Keywords

  • Endothelium
  • Patch clamp Volume-activated Cl currents
  • Phosphorylation

ASJC Scopus subject areas

  • Physiology

Cite this

The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation. / Szücs, Géza; Heinke, Stephan; De Greef, Christine; Raeymaekers, Luc; Eggermont, Jan; Droogmans, Guy; Nilius, Bernd.

In: Pflugers Archiv European Journal of Physiology, Vol. 431, No. 4, 02.1996, p. 540-548.

Research output: Contribution to journalArticle

Szücs, Géza ; Heinke, Stephan ; De Greef, Christine ; Raeymaekers, Luc ; Eggermont, Jan ; Droogmans, Guy ; Nilius, Bernd. / The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation. In: Pflugers Archiv European Journal of Physiology. 1996 ; Vol. 431, No. 4. pp. 540-548.
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abstract = "We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (ICl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation of ICl,vol occurred at a hypotonicity of 27.5 ± 1.2{\%}. Run-down of the current upon repetitive activation was less than 15{\%} within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (-)-indolactam did not affect ICl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 μmol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19-31 pseudosubstrate peptide, did not influence ICl,vol. Trifluoperazine and tamoxifen fully blocked IcCl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 μmol/1 respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca2+] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 μol/1) and genistein (100 μol/1) did not affect ICl,vol Exposing CPAE cells to lysophosphatidic acid (1μmol/1), an activator of p42 MAPkinase and the focal adhesion kinase p125FAK in endothelial cells, neither evoked a Cl- current nor affected ICl,vol Neither wortmannin (10 μmol/1), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/1), which interferes with the p70S6 kinase pathway, affected ICl,vol Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl- current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl- current induced by hypotonicity.",
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AU - Szücs, Géza

AU - Heinke, Stephan

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AU - Raeymaekers, Luc

AU - Eggermont, Jan

AU - Droogmans, Guy

AU - Nilius, Bernd

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N2 - We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (ICl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation of ICl,vol occurred at a hypotonicity of 27.5 ± 1.2%. Run-down of the current upon repetitive activation was less than 15% within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (-)-indolactam did not affect ICl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 μmol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19-31 pseudosubstrate peptide, did not influence ICl,vol. Trifluoperazine and tamoxifen fully blocked IcCl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 μmol/1 respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca2+] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 μol/1) and genistein (100 μol/1) did not affect ICl,vol Exposing CPAE cells to lysophosphatidic acid (1μmol/1), an activator of p42 MAPkinase and the focal adhesion kinase p125FAK in endothelial cells, neither evoked a Cl- current nor affected ICl,vol Neither wortmannin (10 μmol/1), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/1), which interferes with the p70S6 kinase pathway, affected ICl,vol Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl- current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl- current induced by hypotonicity.

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