期刊
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
卷 467, 期 10, 页码 2179-2191出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00424-014-1656-2
关键词
(4-6): complement system; Retinal pigment epithelium; RPE; L-type channels; BK channels
类别
资金
- National Institutes of Health (NIH) [R01EY019320]
- Department for Veterans Affairs [RX000444]
- Research to Prevent Blindness (RPB), New York, NY
- Deutsche Forschungsgemeinschaft (DFG) [SK46/2-1, SK46/2-2]
Defective regulation of the alternative pathway of the complement system is believed to contribute to damage of retinal pigment epithelial (RPE) cells in age-related macular degeneration. Thus we investigated the effect of complement activation on the RPE cell membrane by analyzing changes in membrane conductance via patch-clamp techniques and Ca2+ imaging. Exposure of human ARPE-19 cells to complement-sufficient normal human serum (NHS) (25 %) resulted in a biphasic increase in intracellular free Ca2+ ([Ca2+](i)); an initial peak followed by sustained Ca2+ increase. C5- or C7-depleted sera did not fully reproduce the signal generated by NHS. The initial peak of the Ca2+ response was reduced by sarcoplasmic Ca2+-ATPase inhibitor thapsigargin, L-type channel blockers (R)-(+)-BayK8644 and isradipine, transient-receptor-potential (TRP) channel blocker ruthenium-red and ryanodine receptor blocker dantrolene. The sustained phase was carried by Ca(V)1.3 L-type channels via tyrosine-phosphorylation. Changes in [Ca2+](I) were accompanied by an abrupt hyperpolarization, resulting from a transient increase in membrane conductance, which was absent under extracellular Ca2+- or K+-free conditions and blocked by (R)-(+)-BayK8644 or paxilline, a maxiK channel inhibitor. Single-channel recordings confirmed the contribution of maxiK channels. Primary porcine RPE cells responded to NHS in a comparable manner. Pre-incubation with NHS reduced H2O2-induced cell death. In summary, in a concerted manner, C3a, C5a and sC5b-9 increased [Ca2+](i) by ryanodine-receptor-dependent activation of L-type channels in addition to maxi-K channels and TRP channels absent from any insertion of a lytic pore.
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