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D. Krizaj, D.R. Copenhagen; Ryanodine Receptors, PMCAs and Mitochondria Interact in Inner Segments of Salamander Rods and Cones . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1065.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the contribution of intracellular Ca stores, transporters and mitochondria to Ca homeostasis in rod and cone ISs. Methods: Enzymatically isolated photoreceptors from tiger salamander retina were loaded with Fura-2 AM or Fluo-4 AM. Changes in intracellular calcium [Ca2+]i were monitored with a CCD camera or confocal microscope. For immunofluorescence, isolated cells or slices were fixed in 4% paraformaldehyde, incubated overnight with the primary antibody and for 2 hours in a secondary FITC- and/or TxRed conjugated IgG. Results: Ryanodine -sensitive stores were stimulated with caffeine. 10 mM caffeine evoked large [Ca2+]i increases in rod, but not cone, ISs. Caffeine-evoked Ca responses in cone inner segments were unmasked in the presence of inhibitors of the plasma membrane Ca ATPases (PMCAs) and mitochondrial Ca sequestration. Caffeine-evoked responses were blocked by ryanodine, a selective blocker of Ca release, and by cyclopiazonic acid, a blocker of Ca sequestration by SERCAs into the endoplasmic reticulum. These two inhibitors also substantially reduced the amplitude of depolarization-evoked [Ca2+]i increases, providing evidence for Calcium-Induced Calcium Release (CICR) in rods and cones. The magnitude and kinetics of caffeine-evoked Ca elevation depended on the basal [Ca2+]i, PMCA activity and on mitochondrial function. Immunocytochemistry revealed localization of ryanodine receptors and SERCA2b (but not SERCA1 or 3) pumps to synaptic terminals, perikarya and ellipsoids of rod and cone ISs. Conclusions: These results reveal roles for the endoplasmic reticulum, voltage-gated Ca channels, PMCAs and mitochondrial Ca stores in regulating Ca in photoreceptor inner segments and suggest a role for CICR in regulation of synaptic transmission. Acknowledgements: Supported by NIH (DK and DRC) and Research to Prevent Blindness (DRC).
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