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


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.
Luciani
Dan S
DS
Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Comlumbia, Canada.
Gwiazda
Kamila S
KS
Yang
Ting-Lin B
TL
Kalynyak
Tatyana B
TB
Bychkivska
Yaryna
Y
Frey
Matthew H Z
MH
Jeffrey
Kristin D
KD
Sampaio
Arthur V
AV
Underhill
T Michael
TM
Johnson
James D
JD
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D015151:Immunoblotting; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D047028:Macrocyclic Compounds / Q000494:pharmacology; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D009942:Organometallic Compounds / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D011419:Propidium / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019284:Thapsigargin / Q000494:pharmacology
D053496:Inositol 1,4,5-Trisphosphate Receptors; D047028:Macrocyclic Compounds; D009942:Organometallic Compounds; D010080:Oxazoles; D019837:Ryanodine Receptor Calcium Release Channel; C110932:tetramethyl rhodamine ethyl ester; C511704:xestospongin C; D012433:Ryanodine; D011419:Propidium; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D019284:Thapsigargin; D053148:Caspase 3; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium