The Ca2+-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca2+ or Mg2+ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca2+ (Ca2+(i)) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G(q/11) or G(i/o) signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca2+(i) mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists.

The aminoglycoside antibiotic gentamicin elicits proximal tubular toxicity and cell death. In calcium-sensing receptor (CaR)-transfected HEK-293 (CaR-HEK) cells and CaR-expressing proximal tubule-derived opossum kidney (OK) cells, chronic gentamicin treatment elicits dose-dependent, caspase-mediated apoptotic cell death. Here we investigated whether the renal cell toxicity of the CaR agonist gentamicin could be prevented by CaR antagonism or by lithium cotreatment which may interfere with receptor-mediated signalling. Chronic treatment of OK and CaR-HEK cells with low concentrations of gentamicin elicited cell death, an effect that was ameliorated by cotreatment with the CaR negative allosteric modulator (calcilytic) NPS-89636. This calcilytic also attenuated CaR agonist-induced ERK activation in these cells. In addition, 1 mM LiCl, equivalent to its therapeutic plasma concentration, also inhibited gentamicin-induced toxicity in both cell types. This protective effect of lithium was not due to the disruption of phosphatidylinositol-mediated gentamicin uptake as the cellular entry of Texas red-conjugated gentamicin into OK and CaR-HEK cells was unchanged by lithium treatment. However, the protective effect of lithium was mimicked by glycogen synthase 3beta inhibition. Together, these data implicate CaR activation and a lithium-inhibitable signalling pathway in the induction of cell death by gentamicin in renal epithelial cells in culture.

The calcium-sensing receptor (CaR) is expressed on intestinal epithelial serosal membrane and in Caco-2 cells. In renal epithelium, CaR expressed on the basolateral membrane acts to limit excess tubular Ca2+ reabsorption. Therefore, here we investigated whether extracellular calcium (Ca(o)2+) can regulate active or passive 45Ca2+ transport across differentiated Caco-2 monolayers via CaR-dependent or CaR-independent mechanisms. Raising the Ca(o)2+ concentration from 0.8 to 1.6 mM increased transepithelial electrical resistance (TER) and decreased passive Ca2+ permeability but failed to alter active Ca2+ transport. The Ca(o)2+ effect on TER was rapid, sustained and concentration-dependent. Increasing basolateral Mg2+ concentration increased TER and inhibited both passive and active Ca2+ transport, whereas spermine and the CaR-selective calcimimetic NPS R-467 were without effect. We conclude that small increases in divalent cation concentration elicit CaR-independent increases in TER and inhibit passive Ca2+ transport across Caco-2 monolayers, most probably through a direct effect on tight junction permeability. Whilst it is known that the complete removal of Ca(o)2+ lowers TER, here we show that Ca(o)2+ addition actually increases TER in a concentration-dependent manner. Therefore, such Ca(o)2+-sensitivity could modulate intestinal solute transport including the limiting of excess Ca2+ absorption.