Calcium-Sensing Receptor (CaSR) Activity in Human Retinal Pigment Epithelium

Abstract

Purpose: : The RPE is a transporting epithelial monolayer that controls hydration and composition of the subretinal space (SRS). The calcium-sensing receptor (CaSR) is a G-protein-coupled plasma membrane receptor whose ligand is extracellular calcium. Calcium concentration in SRS varies with light/dark transitions, and the CaSR, if expressed in the RPE, could modulate RPE transport in response to these changes. We previously demonstrated expression of CaSR in native RPE. The present study investigated CaSR activity in cultured RPE.

Methods: : CaSR activity was assessed in RPE monolayers by measuring changes in intracellular calcium ([Ca²⁺]_i) as a function of step-wise changes in extracellular calcium ([Ca²⁺]_o) with- and without addition of R-568 a known allosteric modulator of CaSR. Relative changes in [Ca²⁺]_i in were monitored by single cell digital fluorescence imaging in cells loaded with fluo-3. Experiments were performed using primary cultures established from three adult human RPE donor cultures (Caucasian, age range 47 - 82 years; passage numbers ranging from 17 to 73). The control incubation solution contained (in mM) NaCl (140), KCl (2), CaCl₂ (0.5), MgCl₂ (1.6) glucose (5), and HEPES (15), adjusted to pH 7.4 with NaOH. Concentration-response curves were obtained for calcium alone (CaCl₂ @ 0.5-, 2-, 3- and 5 mM) and for calcium plus 2 µM, 4 µM or 8 µM R-568.

Results: : In RPE, changes in extracellular Ca²⁺ were mirrored by changes in intracellular [Ca²⁺]_. Intracellular [Ca²⁺] increased monotonically with step-wise increases in extracellular [Ca²⁺] ranging from 0.5 mM Ca²⁺ to 5 mM Ca²⁺. The rise in [Ca²⁺]_i was rapid and sustained for the duration of exposure to each solution. The hypothesis that [Ca²⁺]_o-induced changes in [Ca²⁺]_i were mediated by CaSR-receptor activation was tested pharmacologically by repeating the extracellular Ca²⁺ dose-response with the addition of R-568. In the presence of drug, the [Ca²⁺]_o-evoked [Ca²⁺]_i response was reduced.

Conclusions: : CaSR is active in RPE and mediates [Ca²⁺]_o-evoked changes in [Ca²⁺]_i. R-568, a known allosteric modulator of CaSR, appeared to decrease the affinity of the RPE CaSR for its ligand, Ca²⁺. This is in contrast to the reported effect of R-568 on the parathyroid CaSR. The demonstration of CaSR activity in RPE together with our earlier immunohistochemical data showing the presence of CaSR on the apical RPE surface supports the hypothesis that SRS calcium can act as a first messenger in changing RPE transport activity.

Keywords: retinal pigment epithelium • calcium • ion transporters