The sulphydryl reagent thimerosal (50/~M) released Ca '+ from a non-mitoehondrial intraeellalar Ca:" pool in a dose.dependent manner in pgrmeabilizoa insalin-~ceretin 8 RINmSF cells. Thi~ release was reversed after addition of the reducing agent dithiothreitol. Ca:" was releas,,M from an Ins(l.4,5)P;-insensitive pool, since release was observed even after depletion of the lns(I,4,5)P~-sensitive pool by a supramaximal dose of lns(2.4,5)P~ or thapsigargin. The lns(I.4,5)P~-sensitive pool remained essentially unaltered by thimerosal. Thimero~al.induced Ca :~ release was potentiated by caffeine. These findings suggest the existence of Ca"*-induced Ca:" release also in insulin-secreting cells.

Type 2 melastatin-related transient receptor potential channel (TRPM2), a member of the melastatin-related TRP (transient receptor potential) subfamily is a Ca2+-permeable channel activated by hydrogen peroxide (H2O2). We have investigated the role of TRPM2 channels in mediating the H2O2-induced increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in insulin-secreting cells. In fura-2 loaded INS-1E cells, a widely used model of β-cells, and in human β-cells, H2O2 increased [Ca2+]i, in the presence of 3 mM glucose, by inducing Ca2+ influx across the plasma membrane. H2O2-induced Ca2+ influx was not blocked by nimodipine, a blocker of the L-type voltage-gated Ca2+ channels nor by 2-aminoethoxydiphenyl borate, a blocker of several TRP channels and store-operated channels, but it was completely blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA), a potent inhibitor of TRPM2. Adenosine diphosphate phosphate ribose, a specific activator of TRPM2 channel and H2O2, induced inward cation currents that were blocked by ACA. Western blot using antibodies directed to the epitopes on the N-terminal and on the C-terminal parts of TRPM2 identified the full length TRPM2 (TRPM2-L), and the C-terminally truncated TRPM2 (TRPM2-S) in human islets. We conclude that functional TRPM2 channels mediate H2O2-induced Ca2+ entry in β-cells, a process potently inhibited by ACA.

The ryanodine (RY) receptors in beta-cells amplify signals by Ca2+-induced Ca2+ release (CICR). The role of CICR in insulin secretion remains unclear in spite of the fact that caffeine is known to stimulate secretion. This effect of caffeine is attributed solely to the inhibition of cAMP-phosphodiesterases (cAMP-PDEs). We demonstrate that stimulation of insulin secretion by caffeine is due to a sensitization of the RY receptors. The dose-response relationship of caffeine-induced inhibition of cAMP-PDEs was not correlated with the stimulation of insulin secretion. Sensitization of the RY receptors stimulated insulin secretion in a context-dependent manner, that is, only in the presence of a high concentration of glucose. This effect of caffeine depended on an increase in [Ca2+]i. Confocal images of beta-cells demonstrated an increase in [Ca2+]i induced by caffeine but not by forskolin. 9-Methyl-7-bromoeudistomin D (MBED), which sensitizes RY receptors, did not inhibit cAMP-PDEs, but it stimulated secretion in a glucose-dependent manner. The stimulation of secretion by caffeine and MBED involved both the first and the second phases of secretion. We conclude that the RY receptors of beta-cells mediate a distinct glucose-dependent signal for insulin secretion and may be a target for developing drugs that will stimulate insulin secretion only in a glucose-dependent manner.

Effects of sulfhydryl modification on the ATP regulated K' channel (K,,, channel) in the pancreatic B-cell were studied, using the patch clamp technique. Application of the sulfhydryl oxidizing agents thimerosal and [ 18,191.