Heat-sensitive TRPV channels in retinal pigment epithelial cells: regulation of VEGF-A secretion.
OBJECTIVE
Choroidal neovascularization in age-related macular degeneration is caused, to a large extent, by increased secretion of vascular endothelial growth factor (VEGF)-A by the retinal pigment epithelium (RPE). The purpose of the study was to identify pathways that lead to increased VEGF secretion by the RPE.


METHODS
Ca(2+) signaling was studied in ARPE-19 and human RPE cells in primary culture by means of Ca(2+) imaging. Membrane conductance was measured in the whole-cell configuration of the patch-clamp technique. VEGF-A secretion was measured by using ELISA.


RESULTS
Freshly isolated RPE cells or ARPE-19 cells were shown to express TRPV1, -2, -3, and -4 channels. Increasing the temperature or stimulation by IGF-1 increased the VEGF-A secretion rate in both cell types. These effects were both sensitive to the TRPV channel blocker ruthenium red (20 μM). The heat-inducible Ca(2+) signals were blocked by the TRPV channel blockers La(3+) and ruthenium red by 68% and 52%, respectively. In contrast, high concentrations of 2-APB (3 mM) increased [Ca(2+)](i), whereas the TRPV1 channel opener capsaicin and the TRPV3 channel opener camphor had no effect. Reduction of TRPV2 expression by siRNA attenuated the heat-evoked Ca(2+) response. In addition, a heat-activated inwardly rectifying current was measured that was completely blocked by ruthenium red. IGF-1 also increased whole-cell current with a corresponding increase in [Ca(2+)](i), which was blocked by the PI3-kinase blocker LY294002.


CONCLUSIONS
The data strongly suggest that TRPV2 channels expressed by the RPE are involved in the Ca(2+) signaling that mediates both heat-dependent and IGF-1 (via PI3-kinase activation)-induced VEGF secretion.
Cordeiro
Sönke
S
Experimentelle Ophthalmologie, Klinik und Poliklinik für Augenheilkunde, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.
Seyler
Sebastian
S
Stindl
Julia
J
Milenkovic
Vladimir M
VM
Strauss
Olaf
O
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006358:Hot Temperature; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012430:Ruthenium Red / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D050916:TRPV Cation Channels; C400769:TRPV2 protein, human; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D012430:Ruthenium Red; D007334:Insulin-Like Growth Factor I; D007811:Lanthanum; D002118:Calcium