Calcimimetic R-568 and its enantiomer S-568 increase nitric oxide release in human endothelial cells

Abstract

Background: Calcimimetics, such as R-568, are thought to activate G protein-linked Ca(2+)-sensing receptor (CaSR) by allosterically increasing the affinity of the receptor for Ca(2+) allowing for efficient control of uremic hyperparathyroidism. Several recent studies suggest they possess additional vascular actions. Although it has been postulated that calcimimetics may have a direct effect on CaSR in the blood vessels, further studies are needed to elucidate their vascular CaSR-dependent versus CaSR-independent effects.

Methodology/principal findings: Focusing on human umbilical vein endothelial cells (HUVECs), we studied the CaSR expression and distribution by Immunofluorescence and Western Blot analysis. CaSR function was evaluated by measuring the potential effect of calcimimetic R-568 and its enantiomer S-568 upon the modulation of intracellular Ca(2+) levels (using a single cell approach and FURA-2AM), in the presence or absence of Calhex-231, a negative modulator of CaSR. To address their potential vascular functions, we also evaluated R- and S-568-stimulated enzymatic release of Nitric Oxide (NO) by DAF-2DA, by Nitric Oxide Synthase (NOS) radiometric assay (both in HUVECs and in Human Aortic Endothelial Cells) and by measuring eNOS-ser1177 phosphorylation levels (Immunoblotting). We show that, although the CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. In addition, regardless of the presence or absence of Calhex-231, both R- and S-568 significantly increased intracellular Ca(2+) levels by mobilization of Ca(2+) from intracellular stores, which in turn augmented NO release by a time- and Ca(2+)-dependent increase in eNOS-ser1177 phosphorylation levels.

Conclusions/significance: Taken together, these data indicate that in human endothelium there is no stereoselectivity in the responses to calcimimetics and that CaSR is probably not involved in the action of R- and S-568. This suggests an additional mechanism in support of the CaSR-independent role of calcimimetics as vasculotrope agents.

Figure 1. CaSR protein expression in HUVECs by Immunofluorescence Confocal Microscopy and Western Blot.

Immunofluorescent localization of CaSR in HUVECs with specific antibody and negative control after fixation and permeabilization protocol (A and B), or after fixation but not membrane permeabilization (C and D). Representative Western Blot of CaSR protein levels in HAoVSMC, HAEC and HUVEC total lysate, and in HUVEC membrane and cytoplasm extracts (E).

Figure 2. Effects of different doses of R-568 or S-568 on HUVECs [Ca²⁺]_i.

Traces (mean value ± SE) representing [Ca²⁺]i variations in FURA-2AM-loaded HUVECs stimulated with R-568 (A–C) or S-568 (D–F) at 1 µM (A and D), 10 µM (B and E) or 100 µM (C and F) concentrations. Representative traces after stimulation with R-568 100 µM + Calhex 10 µM (inset C) or S-568 100 µM + Calhex 10 µM (inset F).

Figure 3. Effects of R-568 or S-568 on HUVECs [Ca²⁺]_i without extra or intracellular Ca²⁺.

Representative Ca²⁺-variations in FURA-2AM-loaded HUVECs stimulated with 100 µM R-568 or 100 µM S-568 in Ca²⁺ free medium (A and B) or after depletion of intracellular Ca²⁺ stores by thapsigargin (tg) (C and D).

Figure 4. Effects of R-568 or S-568 +/- Calhex (10 µM) on HUVECs NO production.

NO levels (mean value ± SE) in DAF-2DA-loaded HUVEC populations treated with R-568 or S-568 (1–100 µM) +/- Calhex 10 µM (A). eNOS-ser1177 phosphorylation in HUVECs stimulated with R-568 (B) or S-568 (C) (0.1–100 µM) +/- Calhex (10 µM). Representative immunoblot of eNOS-ser1177 phosphorylation (Upper Panel, B and C). Representative data from three experiments (means ± SD, Lower Panel, B and C). Phospho-eNOS (p-eNOS) expression was normalized vs total eNOS expression.

Figure 5. Effects of 100 µM R-568 and S-568 with and without extra and intracellular Ca²⁺ on NO production.

Intracellular NO levels (mean value ± SE) in DAF-2DA-loaded HUVECs (A–C). Time effect of R-568 or S-568 100 µM on eNOS-ser1177 phosphorylation (D). Representative immunoblot of eNOS-ser1177 phosphorylation (Upper Panel). Representative data from three experiments (means ± SD, Lower Panel, p<0.003 vs 0′). Phospho-eNOS (p-eNOS) was normalized vs eNOS total.