Ryanodine receptor‐operated activation of TRP‐like channels can trigger critical Ca²⁺signaling events in pancreatic β‐cells

Abstract

There is little information available concerning the link between the ryanodine (RY) receptors and the downstream Ca(2+) signaling events in beta-cells. In fura-2 loaded INS-1E cells, activation of RY receptors by 9-methyl 5,7-dibromoeudistomin D (MBED) caused a rapid rise of [Ca(2+)]i followed by a plateau and repetitive [Ca(2+)]i spikes on the plateau. The [Ca(2+)]i plateau was abolished by omission of extracellular Ca(2+) and by SKF 96365. In the presence of SKF 96365, MBED produced a transient increase of [Ca(2+)]i, which was abolished by thapsigargin. Activation of RY receptors caused Ca(2+) entry even when the ER Ca(2+) pool was depleted by thapsigargin. The [Ca(2+)]i plateau was not inhibited by nimodipine or ruthenium red, but was inhibited by membrane depolarization, La(3+), Gd(3+), niflumic acid, and 2-aminoethoxydiphenyl borate, agents that inhibit the transient receptor potential channels. The [Ca(2+)]i spikes were inhibited by nimodipine and ryanodine, indicating that they were due to Ca(2+) influx through the voltage-gated Ca(2+) channels and Ca(2+)-induced Ca(2+) release (CICR). Activation of RY receptors depolarized membrane potential as measured by patch clamp. Thus, activation of RY receptors leads to coherent changes in Ca(2+) signaling, which includes activation of TRP-like channels, membrane depolarization, activation of the voltage-gated Ca(2+) channels and CICR.

Functional Ryanodine Receptors in the Plasma Membrane of RINm5F Pancreatic β-Cells

“…We conclude that depletion of intracellular Ca 2ϩ stores fails to evoke significant SOCE in RINm5F cells. The lack of detectable SOCE is consistent with reports from insulinoma (44) and ␤-cells (20,27,(45)(46)(47) where SOCE was either very small or undetectable.…”

“…The results so far establish that agonists of RyR evoke Ca 2ϩ (or Mn 2ϩ ) entry that is independent of SOCE or voltage-gated Ca 2ϩ channels. Another study also showed that a RyR-selective agonist stimulates Ca 2ϩ entry via a SOCE-independent pathway in ␤-cells, although it speculated that it resulted from RyRmediated release of Ca 2ϩ from a non-ER store causing activation of Ca 2ϩ -permeable TRP (transient receptor potential) channels in the PM (20). Our subsequent experiments show that the Ca 2ϩ entry is mediated directly by RyR in the PM.…”

“…In each case, the density of the PM channels was very low, typically no more than ϳ10 channels/cell, and often just 2-3 channels/cell. None of these reports established the molecular identity of the channel and several were reluctant to conclude that it was a RyR (20,(61)(62)(63), but together they provide evidence that small numbers of RyR may be expressed in the PM of several different electrically excitable cells. Our results extend these observations to RINm5F cells and pancreatic ␤-cells and, more importantly, they unequivocally establish that RyR2 expressed in the PM can directly mediate Ca 2ϩ entry.…”

“…Glucose rapidly equilibrates across the plasma membrane (PM) of ␤-cells and its oxidative metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing K ATP channels to close (19). This allows an unidentified leak current to depolarize the PM (20) and activate voltage-gated Ca 2ϩ channels, predominantly L-type Ca 2ϩ channels (21). The resulting Ca 2ϩ entry is amplified by Ca 2ϩ -induced Ca 2ϩ release from intracellular stores (7), triggering exocytotic release of insulin-containing dense-core vesicles (22).…”

Possible Involvement of Transient Receptor Potential Channels in Electrophile-Induced Insulin Secretion from RINm5F Cells

“…These results suggest that the stimulation of TRPA1 eventually induces VDCC activation, and thereby increases in Ca 2+ influx. An association between TRP channels and VDCC has been reported in diverse systems, [38][39][40][41] including β-cells, 42) which showed that lowering the extracellular Na + concentration inhibited an islet amyloid polypeptide-stimulated TRPV4-mediated increase in [Ca 2+ ] i in cultured MIN6 β-cells. As TRPV4 is permeable to Ca 2+ and Na + to a similar extent, 43) the authors of that study concluded that islet amyloid polypeptide induces Na + entry via TRPV4, which thus stimulates membrane depolarization and Ca 2+ entry through VDCC.…”