Calcium influx induced by activation of receptor tyrosine kinases in SV40-transfected human corneal endothelial cells.
This study was undertaken to investigate electrophysiological properties of immortalized SV40-transfected human corneal endothelial cells (HCEC-SV40) combined with the analysis of intracellular Ca(2+) responses mediated by ligands for receptor tyrosine kinases (RTK). In addition, the effects of several tyrosine kinase inhibitors were tested on Ca(2+) inflow mediated by induction of capacitative calcium entry (CCE). Patch-clamp techniques and measurements of the intracellular free Ca(2+) ([Ca(2+)](i)) by fura-2 were performed using HCEC-SV40. Stimulation of fibroblast growth factor receptors (FGFR) (e.g. by basic-FGF) (10 ng ml(-1)) elicited activation of Ca(2+) permeable channels and a subsequent increase of cytosolic free Ca(2+) in HCEC-SV40. This effect could be disrupted by the L-type Ca(2+) channel blocker nifedipine (5 microM). In addition, nifedipine significantly reduced the magnitude of CCE. Inhibition of protein tyrosine kinases (PTKs) by genistein, lavendustin A, or tyrphostin 51 (all 5 microM) also led to a reduction of CCE in HCEC-SV40. This study demonstrates for the first time that L-type Ca(2+) channel activity in HCEC-SV40 is linked to the activity of FGF receptor tyrosine kinases. These data regarding Ca(2+) inflow through Ca(2+) channels could be useful for investigation of culture and vitality conditions of HCEC.
Mergler
Stefan
S
Medizinische Klinik m. S. Hepatologie und Gastroenterologie, Universitätsklinikum Charité der Humboldt-Universität zu Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany. stefan.mergler@charite.de
Dannowski
Haike
H
Bednarz
Jürgen
J
Engelmann
Katrin
K
Hartmann
Christian
C
Pleyer
Uwe
U
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D025802:Electric Capacitance; D004727:Endothelium / Q000201:enzymology / Q000378:metabolism; D016220:Fibroblast Growth Factor 1 / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D006801:Humans; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection / Q000379:methods
D015220:Calcium Channels; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D002118:Calcium