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M Karl, K Engelmann, O Strauss; Involvement of Ca2+-Channels and Src Tyrosine Kinases in the Regulation of Photoreceptor Outer Segment (ROS) Phagocytosis By Retinal Pigment Epithelial (RPE) Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4578.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Investigation of the role of L-type Ca2+ channels in the regulation of ROS phagocytosis by normal RPE and dystrophic RCS rat RPE cells. Since L-type channels are activated by cytosolic subtype of tyrosine kinase pp60c-src (Strauss et al. 2000, BBRC 270:806) the influence of this tyrosine kinase was studied. Method: Before measurements of phagocytosis rate, cells were maintained FCS-free for 24 h. To assay phagocytic activity, cells were incubated with SNAFL® - 2 labelled pig ROS and phagocytosis rate (PR) was measured after 5 h incubation using a cytofluorometer assay (distinguishing binding (PRbnd) and ingestion (PRing)) and in addition using FACScan for verification. SV40 transfected human RPE, unpassaged primary human RPE, normal rat RPE (congenic control of RCS) and dystrophic rat RPE (RCS) were investigated. To test the involvement of L-type channels the specific blocker nifedipine (NIF) was applied 5 min before incubation with labeled ROS. To test the involvement of tyrosine kinase, cells were incubated in herbimycin A (HA, 1µM) for 12 h before ROS incubation. Results: SV40 RPE in presence of 5% FCS showed an increased PRing to 188 7% S.E.M. (n = 3) as shown by other groups. Doubleing extracellular calcium to 2 mmol decreased PRing to 85 ± 10% S.E.M. (n = 2) in SV40 RPE incubated FCS-free. Blocking Ca2+ channels by NIF led to an inhibition of PR in all normal RPE. In SV40 RPE PRing was reduced to 66 ± 10% S.E.M. (n = 8). The dystrophic RPE showed the known bFGF stimulation (24 h preincubation, PRing 169 16% S.E.M., n = 3) and no FCS-stimulation (PRing 89 18% S.E.M., n = 3). NIF application to dystrophic RPE led to a recovery in phagocytic capacity. Here, phagocytosis is stimulated to PRing 165 3% S.E.M. and PRbnd 256 2% S.E.M. (n = 3) by 1 µM NIF in FCS-free conditions. HA inhibited PRing of SV40 RPE to 74% ± 5% S.E.M. (n = 3) in FCS-free condition and to 81% ± 9% S.E.M. (n = 3) and PRbnd 110% ± 7% S.E.M. (n = 3) under 5% FCS-stimulation. Comparable results were found on RCS RPE in absence (PRing 77 ± 9% S.E.M., PRbnd 72 ± 14% S.E.M., n = 4) and presence of FCS (PRing 70 ± 11% S.E.M., PRbnd 37 ± 13%S.E.M., n = 3), human primary and normal rat RPE. Conclusion: Influx of Ca2+ through L-type channels is required to maintain phagocytosis activity of ROS by the RPE, but not to initiate this process. Phagocytosis depends on the activity of a src tyrosine kinase. In RCS RPE, which display a changed regulation of L-type channels, inhibition of L-type channels increases phagocytosis rate of ROS by the RPE.
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