Calcium uptake, release and ryanodine binding in melanosomes from retinal pigment epithelium

High levels of calcium have been reported in pigmented tissues of the vertebrate eye, such as retinal pigment epithelium (RPE). Melanin granules also have high calcium concentrations, suggesting that melanin granules may be a calcium reservoir Here we characterized the uptake and release of calcium in a pure melanosomal fraction obtained from frog RPE. Melanosomes take up (45)Ca by a saturable system with an apparent K(M) of 0.5 mM. About 40% of (45)Ca accumulation was insensitive to low temperature. (45)Ca uptake was not affected by verapamil, nifedipine, dantrolene, vanadate, thapsigargin or cyclopiazonic acid, but it was reduced by 50% by ruthenium red, and increased by the ionophore A23187 and nigericin. Release of (45)Ca-loaded was stimulated by caffeine and inositol 1,4,5 trisphosphate (IP(3)). Caffeine stimulated release of calcium was blocked by either ryanodine or ruthenium red, but calcium released by IP(3) was not affected by heparin. No binding of (3)H-IP(3) was observed. The (3)H-ryanodine binding sites exhibited a K(B) of 1.3 nM and a Bmax of 12.1 fmol/mg protein. Thus, our results suggest that melanosomes may function as intracellular organelles that regulate calcium concentration in RPE. (C) 2000 Harcourt Publishers Ltd.