Comparison and characterization of retinal pericytes and retinal pigment epithelial cells on subcellular...

A comparative study of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization in bovine retinal capillary pericytes (BRCP) and bovine retinal pigment epithelial cells (BRPE) was carried out. Both cells were permeabilized with saponin. The two cell types had similar basal levels of [Ca2+]i (130 nM for BRCP, 132 nM for BRPE) and responded to IP3 in a dose-dependent manner. However, when stimulated by various concentrations of IP3 (1-10 microM), the increase in [Ca2+]i of BRCP was always two- to threefold higher than that in BRPE. Subcellular-fractionation studies showed that a single population of IP3 binding site with a high affinity and high specificity of IP3 mainly localized to plasma membrane in these two cell types. Although the dissociation constant of specific [32P]-IP3 binding sites (Kd 1.9-2.8 nM) was similar, the profile of maximal binding capacity (Bmax) of each fraction was markedly different. In comparison, plasma membrane fractions of BRCP were with Bmax of 165 fmol/mg protein versus 90 fmol/mg protein for BRPE membranes. The ATP-dependent Ca2+ uptake and IP3-dependent Ca2+ release were observed in the both plasma membrane fractions. With quantitative correlation, the membrane fraction (2 mg) of BRCP released 0.2 nmol Ca2+ whereas BRPE only released 0.07 nmol Ca2+ with the same dose of IP3 (5 microM). The selectively higher density of IP3 binding sites in coupling to the larger Ca(2+)-release in the membrane of BRCP suggests that the quantity of Ca2+ mobilized is determined by the spatially preferential distribution of membrane-associated IP3 binding sites. These findings may provide an explanation for the differences observed between BRCP and BRPE in IP3-induced DNA replication.