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Diabetes. 2009 Feb;58(2):422-32. doi: 10.2337/db07-1762. Epub 2008 Nov 25.

Roles of IP3R and RyR Ca2+ channels in endoplasmic reticulum stress and beta-cell death.

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  • 1Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Comlumbia, Canada.

Abstract

OBJECTIVE:

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca(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 (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 Ca(2+) was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)-based probes were used to measure Ca(2+) in ER and mitochondria.

RESULTS:

Neither RyR nor IP(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(2+) and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha), 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 Ca(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(2+) 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 Ca(2+) homeostasis and ER stress.

PMID:
19033399
[PubMed - indexed for MEDLINE]
PMCID:
PMC2628616
Free PMC Article
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