L-type Ca2+ Channel Activity in Cultured Human Corneal Endothelial Cells

Abstract

Abstract: : Purpose: Little is known about the role of Ca²⁺ channels in human corneal endothelial cells (HCEC). This study was designed to investigate the electrophysiological properties of putative voltage-dependent Ca²⁺ channels in these cells. In addition, the effect of the L-type channel blocker nifedipine was tested on Ca²⁺ influx in unstimulated HCEC. Methods: Primary cultivated HCEC (prHCEC) as well as permanent SV40-immortalized HCEC (HCEC-SV40) were investigated by the patch-clamp technique in conjunction with measurements of intracellular free Ca²⁺ ([Ca²⁺]_i) using the fluorescence dye fura-2 (Grynkiewicz et al., 1985, J. Biol. Chem. 260: 3440-3450). Results: Extracellular application of nifedipine (5 µM) led to a reduction in [Ca²⁺]_i to 76 ± 20% of control (set to 100%) (n = 4) and 85 ± 14% of control (n = 6) in prHCEC and HCEC-SV40 respectively. Inversely, extracellular application of the L-type channel opener Bay K 8644 (5 µM) led to an increase of Ca²⁺ channel current density in HCEC-SV40 from 1.24 ± 0.24 pApF^-1 (n = 9) (without drug) to 3.11 ± 0.54 pApF^-1 (n = 9) (recovery occurred to 1.60 ± 0.71 pApF^-1; n = 3). [Ca²⁺]_i was reversibly increased, as well. In addition, intracellular application of inositol-1,4,5-trisphosphate (InsP3; 10 µM) via the patch-pipette led to an increase of Ca²⁺ channel amplitudes in HCEC-SV-40 during whole-cell configuration. Conclusions: HCEC-SV40 as well as prHCEC express voltage-dependent L-type channels. Active store depletion by IP3 induces activation of Ca²⁺ channels which are involved in the IP3 linked Ca²⁺ signaling. These observations could be important for the evaluation of the quality and constitution of immortalized HCEC cultures, e.g. for clinical corneal transplantats. CR: none

Keywords: cornea: endothelium • calcium • electrophysiology: non-clinical