Background/Aims: The extracellular calcium-sensing receptor (CaR) is expressed in pancreatic β-cells where it is thought to facilitate cell-to-cell communication and augment insulin secretion. However, it is unknown how CaR activation improves β-cell function. Methods: Immunocytochemistry and western blotting confirmed the expression of CaR in MIN6 β-cell line. The calcimimetic R568 (1µM) was used to increase the affinity of the CaR and specifically activate the receptor at a physiologically appropriate extracellular calcium concentration. Incorporation of 5-bromo-2’-deoxyuridine (BrdU) was used to measure cell proliferation, whilst changes in non-nutrient-evoked cytosolic calcium were assessed using fura-2-microfluorimetry. AFM-single-cell-force spectroscopy related CaR-evoked changes in epithelial (E)-cadherin expression to improved functional tethering between coupled cells. Results: Activation of the CaR over 48hr doubled the expression of E-cadherin (206±41%) and increased L-type voltage-dependent calcium channel expression by 70% compared to control. These changes produced a 30% increase in cell-cell tethering and elevated the basal-to-peak amplitude of ATP (50µM) and tolbutamide (100µM)-evoked changes in cytosolic calcium. Activation of the receptor also increased PD98059 (1-100µM) and SU1498 (1-100µM)-dependent β-cell proliferation. Conclusion: Our data suggest that activation of the CaR increases E-cadherin mediated functional tethering between β-cells and increases expression of L-type VDCC and secretagogue-evoked changes in [Ca²⁺]_i. These findings could explain how local changes in calcium, co-released with insulin, activate the CaR on neighbouring cells to help ensure efficient and appropriate secretory function.

a b s t r a c tIn this study we use a novel approach to quantitatively investigate mechanical and interfacial properties of clonal b-cells using AFM-Single Cell Force Spectroscopy (SCFS). MIN6 cells were incubated for 48 h with 0.5 mM Ca 2+ ± the calcimimetic R568 (1 lM). AFM-SCFS adhesion and indentation experiments were performed by using modified tipless cantilevers. Hertz contact model was applied to analyse force-displacement (F-d) curves for determining elastic or Young's modulus (E). Our results show CaSR-evoked increases in cell-to-cell adhesion parameters and E modulus of single cells, demonstrating that cytomechanics have profound effects on cell adhesion characterization.

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Background/Aims: Evidence supports a role for the extracellular calcium-sensing receptor (CaR) in regulating proliferation in a variety of cell types, including pancreatic β-cells. The present study examines the mechanism by which CaR activation augments β-cell proliferation and determines the extent by which cell-cell contact modifies this response. Methods: Western blot analysis and immunocytochemistry confirmed CaR expression within MIN6 and primary mouse β-cells. DNA synthesis was used as a marker of cell proliferation by measuring the incorporation of ALEXA-tagged-5-bromo-2'-deoxyuridine (BrdU) in individual cells. Results: Expression of CaR-protein increased in MIN6 monolayer cells compared to passage-matched 3dimensional pseudoislets. Activation of CaR using calcimimetic R568 (1μM) enhanced proliferation in MIN6 monolayers at physiologically appropriate concentrations of extracellular calcium (0.5mM). Pseudoislet formation reduces CaR-evoked proliferation compared with monolayers counterpart (P<0.001). CaR-evoked proliferation was significantly inhibited by PD98059 and SU1498 (1-100μM), suggesting that CaR-stimulated β-cell turnover was dependent both on the MAPK/ERK pathway. Cell proliferation was also inhibited in cells lacking E-cadherin ligation. Proliferation was recovered by addition of R568 to ECAD-neutralized cells (P<0.01, n=3) but was unable to fully regain the R568-evoked effect when applied to cells that retain Ecadherin ligation. Conclusion: These studies support a role of the CaR in p42/44 MAPK-mediated proliferation within pancreatic β-cells. The extent of CaR-activated proliferation depends upon the degree of cell-to-cell contact.