Brian G. Kennedy

Rubidium transport in cultured monkey retinal pigment epithelium

Rb+ influx was used to assess Na-K-Cl cotransport and Na,K-ATPase activities in cultured monkey retinal pigment epithelium. Bumetanide-sensitive (Na-K-Cl cotransport-mediated) Rb+ influx exceeds ouabain-sensitive (Na,K-ATPase-mediated) Rb+ influx, with these two transporters accounting for approximately 95% of total Rb+ uptake. Half-maximal inhibition of Rb+ influx by bumetanide is attained at 75 nM bumetanide. The bumetanide-sensitive Rb+ influx depends on both extracellular Na+ and Cl-, and is activated by extracellular Rb+ with a relatively high affinity. Na-K-Cl cotransport activity is stimulated (2.5-fold) by increased extracellular osmolarity. Elevated cAMP content and glycolytic inhibition both depress cotransport activity. Cyanide application, however, had very little effect on Na-K-Cl cotransport activity. Monkey retinal pigment epithelial cells, maintained in culture, provide a system in which the activity and regulation of cation transport mechanisms can be examined.

Immunodetection of an arrestin-like protein in human retinal pigment epithelium

The goal of this study was to determine if an arrestin/S-antigen-like protein is produced by human retinal pigment epithelial (HRPE) cells maintained in tissue culture. Arrestin immunoreactivity was examined in fixed, monolayer cultures of HRPE and on immunoblots of SDS-PAGE separations of whole cell lysates of HRPE using five monoclonal antibodies (mAbs A2G5, A9C6, 3C4.2, 3D1.2 and 5c6.47) that bind to different epitopes in bovine retinal S-antigen. Monolayers of HRPE cells showed immunoreactivity with four of the mAbs though the relative staining intensity varied among mAbs and donors. For example, mAb A2G5 which historically shows very limited crossreactivity among species, reacted strongly with cells from one donor, moderately with cells from a second donor and only weakly with other donor cultures examined. mAb 3D1.2 showed no reactivity with HRPE cells. Immunoblots of SDS-PAGE separations of whole cell lysates of HRPE established from ten different donors confirmed the presence of an arrestin-related polypeptide that comigrated with retinal arrestin. These results demonstrate the presence of an arrestin-like protein in HRPE cells which have been maintained in tissue culture. Though the function of this arrestin homologue in HRPE is not yet established, it could play a role in the downregulation of receptor and/or transport protein activity.