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Electrophysiological Investigation of Cultured Human Corneal Endothelial Cells

1Mergler S., 2Dannowski H., 3Bednarz J., 3Engelmann K., 2Hartmann C., 2Pleyer U.,
1Universitätsklinikum Charité, Campus Virchow, Med. Klinik m.S. Hepatologie & Gastroenterologie (Berlin)
2Humboldt-Universität zu Berlin, Charité Campus Virchow-Klinikum, Augenklinik (Berlin)
3Universitätsklinikum Hamburg-Eppendorf, Klinik und Poliklinik für Augenheilkunde (Hamburg)

Background: Apart from oxygen free radicals, Ca2+ plays an important role concerning apoptotic-induced proceedings. In addition, Ca2+ is decisively involved in the regulation of different cellular functions and is a second messenger for several signaling pathways. So far, there are no investigations in human corneal endothelium about Ca2+ channels. Putative Ca2+ channel activities and Ca2+ responses should be measured, which were mediated by ligands for protein tyrosine kinases (PTKs) coupled receptors such as fibroblast growth factor receptor 1 (FGFR1).
Methods: Primary cultures from human corneal endothelium as well as the permanent cell line HCEC-SV-40 were established (Exp Eye Res. 1999; 69:547-553). The electrophysiological studies were performed by patch-clamp techniques (Pflügers Arch. 1981; 391:85-100) and a fluorimetric measuring method (J. Biol. Chem. 1985; 260: 3440-3450).
Results: Depolarization in HCEC-SV40 led to Ba2+ inward currents similar to L-type Ca2+ channel currents. Extracellular application of b-FGF (basic fibroblast growth factor) (10 ng/ml) led to an increase in intracellular Ca2+ concentration (from 115 ± 3 nM; n = 5 to 153 ± 3 nM; n = 6). This effect was at higher levels in HCEC-SV40 (from 108 ± 3 nM to 177 ± 21 nM; both n = 4). In addition, a-FGF (acidic-FGF) (10 ng/ml) led to a similar effect in HCEC-SV40, which was disrupted by the specific L-type channel blocker nifedipine (5 µM). In contrast, inhibition of FGF receptors by tyrosine kinase inhibitors led to a decreased magnitude of capacitative Ca2+ entry (CCE).
Conclusions: We could demonstrate L-type channel activity by nifedipine for the first time. Presumably, the prevailing tyrosine kinase-sensitive Ca2+ permeable channel activity could be associated with a high density of FGF receptor tyrosine kinases. In addition, FGF receptors in HCE-SV40 cells could be expressed at higher levels than in HCEC cells. These data about Ca2+ conductance could be used for check of culture and preservation of HCEC. Supported in part: DFG (PL 150/10-1)