Roles of IP3R and RyR Ca2+ Channels in Endoplasmic Reticulum Stress and β-Cell Death

OBJECTIVE-Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca2+ release channels in the ER stress-associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP(3)Rs) and the ryanodine receptors (RyRs) on the induction of beta-cell ER stress and apoptosis. RESEARCH DESIGN AND METHODS-Kinetics of beta-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca2+ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)-based probes were used to measure Ca2+ in ER and mitochondria. RESULTS-Neither RyR nor IP3R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca2+ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2 alpha (eIF2 alpha), C/EBP homologous protein (CHOP)-associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP(3)Rs and RyRs. Conversely, stimulation of ER Ca2+ release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization. CONCLUSIONS-This study demonstrates that the activity of ER Ca2+ channels regulates the susceptibility of beta-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in beta-cell apoptosis associated with dysfunctional beta-cell ER Ca2+ homeostasis and ER stress. Diabetes 58:422-432, 2009