| 4. |
- Islam, M. Shahidul, et al.
(författare)
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Mobilization of Ca2+ by thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone in permeabilized insulin-secreting RINm5F cells : evidence for separate uptake and release compartments in inositol 1,4,5-trisphosphate-sensitive Ca2+ pool
- 1993
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 293:Pt 2, s. 423-429
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Tidskriftsartikel (refereegranskat)abstract
- We characterized and directly compared the Ca(2+)-releasing actions of two inhibitors of endoplasmic-reticulum (ER) Ca(2+)-ATPase, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ), in electropermeabilized insulin-secreting RINm5F cells. Ambient free calcium concentration ([Ca2+]) was monitored by Ca(2+)-selective mini-electrodes. After ATP-dependent Ca2+ uptake, thapsigargin and tBuBHQ released Ca2+ with and EC50 of approximately 37 nM and approximately 2 microM respectively. Both agents mobilized Ca2+ predominantly from the Ins(1,4,5)P3-sensitive Ca2+ pool, and in this respect thapsigargin was more specific than tBuBHQ. The total increase in [Ca2+] obtained with thapsigargin and Ins(1,4,5)P3 was, on the average, only 7% greater than that with Ins(1,4,5)P3 alone. In contrast, the total increase in [Ca2+] obtained with tBuBHQ and Ins(1,4,5)P3 was 33% greater than that obtained with only InsP3 (P < 0.05). Although Ca2+ was rapidly mobilized by thapsigargin and tBuBHQ, complete depletion of the Ins(1,4,5)P3-sensitive Ca2+ pool was difficult to achieve. After the release by thapsigargin or tBuBHQ, Ins(1,4,5)P3 induced additional Ca2+ release. The additional Ins(1,4,5)P3-induced Ca2+ release was not altered by supramaximal concentrations of thapsigargin and tBuBHQ, or by Bafilomycin A1, an inhibitor of V-type ATPases, but was decreased by prolonged treatment with the ER Ca(2+)-ATPase inhibitors. These results suggest the existence of distinct uptake and release compartments within the Ins(1,4,5)P3-sensitive Ca2+ pool. When treated with the inhibitors, the two compartments became distinguishable on the basis of their Ca2+ permeability. Apparently, thapsigargin and tBuBHQ readily mobilized Ca2+ from the uptake compartment, whereas Ca2+ from the release compartment could be mobilized only very slowly, in the absence of Ins(1,4,5)P3.
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| 5. |
- Islam, M. Shahidul, et al.
(författare)
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Sulfhydryl oxidation induces rapid and reversible closure of the ATP-regulated K+ channel in the pancreatic beta-cell
- 1993
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Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 319:1-2, s. 128-132
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Tidskriftsartikel (refereegranskat)abstract
- Effects of sulfhydryl modification on the ATP regulated K+ channel (KATP channel) in the pancreatic beta-cell were studied, using the patch clamp technique. Application of the sulfhydryl oxidizing agents thimerosal and 2,2'-dithio-bis(5-nitropyridine) (DTBNP), in micromolar concentrations, caused complete inhibition of the KATP channel, in inside-out patches. The inhibition was rapid and was reversed by the disulfide reducing agents dithiothreitol and cysteine. Thimerosal, which is poorly membrane permeable, inhibited channel activity, only when applied to the intracellular face of the plasma membrane. In contrast, DTBNP, which is highly lipophilic, caused closure of the KATP channel and consequent depolarization of the membrane potential, also when applied extracellularly. Our results indicate the presence of accessible free SH groups on the cytoplasmic side of the KATP channel in the pancreatic beta-cell. These SH groups are essential for channel function and it is possible that thiol-dependent redox mechanisms can modulate KATP channel activity.
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