Roles of [IP.sub.3]R and RyR [Ca.sup.2+] 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 [Ca.sup.2+] 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 (IPsRs) and the ryanodine receptors (RyRs) on the induction of β-cell ER stress and apoptosis. RESEARCH DESIGN AND METHODS--Kinetics of β-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 [Ca.sup.2+] was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)-based probes were used to measure [Ca.sup.2+] in ER and mitochondria. RESULTS--Neither RyR nor [IP.sub.3]R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER [Ca.sup.2+] and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), C/EBP homologous protein (CHOP)-assoeiated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking [IP.sub.3]Rs and RyRs. Conversely, stimulation of ER [Ca.sup.2+] 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 [Ca.sup.2+] channels regulates the susceptibility of β-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in β-cell apoptosis associated with dysfunctional β-cell ER [Ca.sup.2+] homeostasis and ER stress.