| 1. |
- Arkhammar, P., et al.
(författare)
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Protein kinase C modulates the insulin secretory process by maintaining a proper function of the beta-cell voltage-activated Ca2+ channels
- 1994
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Ingår i: Journal of Biological Chemistry. - : Baishideng Publishers. - 0021-9258 .- 1083-351X. ; 269:4, s. 2743-2749
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Tidskriftsartikel (refereegranskat)abstract
- In the present study an attempt was made to further elucidate the molecular mechanisms whereby protein kinase C (PKC) modulates the beta-cell stimulus-secretion coupling. Regulation of Ca2+ channel activity, [Ca2+]i, and insulin release were investigated in both normal pancreatic mouse beta-cells and in similar beta-cells deprived of PKC activity. [Ca2+]i was measured with the intracellular fluorescent Ca2+ indicator fura-2 and the Ca2+ channel activity was estimated by the whole cell configuration of the patch-clamp technique. To reveal the various isoenzymes of PKC present in the mouse beta-cell, proteins were separated by one-dimensional gel electrophoresis and Western blotting was performed. The production of inositol phosphates was measured by ion-exchange chromatography and insulin release was measured radioimmunologically. Acute stimulation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate resulted in suppression of both the carbamylcholine-induced increase in [Ca2+]i and production of inositol 1,4,5-trisphosphate. Under these conditions the increase in [Ca2+]i in response to glucose was similar to that found in control cells. When beta-cells were deprived of PKC, by exposure to 200 nM 12-O-tetradecanoylphorbol-13-acetate for 24-48 h, there was an enhanced response to carbamylcholine. This response constituted increases in both the [Ca2+]i signal and production of inositol 1,4,5-trisphosphate. Interestingly, cells with down-regulated PKC activity responded more slowly to glucose stimulation, when comparing the initial increase in [Ca2+]i, than control cells. On the other hand, the maximal increase in [Ca2+]i was similar whether or not PKC was present. Moreover, PKC down-regulated cells exhibited a significant reduction of maximal whole cell Ca2+ currents, a finding that may explain the altered kinetics with regard to the [Ca2+]i increase in response to the sugar. Both the alpha and beta 1 forms of the PKC isoenzymes were present in the mouse beta-cell and were also subjected to PKC down-regulation. Hence, either of these isoenzymes or both may be involved in the modulation of phospholipase C and Ca2+ channel activity. Since insulin release under physiological conditions is critically dependent on Ca(2+)-influx through the voltage-gated L-type Ca2+ channels, the kinetics of hormone release was expected to demonstrate a similar delay as that of the [Ca2+]i increase. Although not as pronounced, such a delay was indeed also observed in the onset of insulin release. There was, however, no effect on the total amounts of hormone released. There was,h owever, no effect on thet otal amounts of hormone released. The present study con- firms that PKC has multiple roles and thereby interacta at different sites in the complex series of events consti- tuting the #?-cell signal-transduction pathway. It is sug- gested that PKC may be tonically active and effective in the maintenance of the phosphorylation state of the voltage-gated L-type Ca2+ channel, enabling an appro- priate function of this channel in the insulin secretory process.
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| 2. |
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| 3. |
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| 4. |
- Haby, Christelle, et al.
(författare)
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Inhibition of serine/threonine protein phosphatases promotes opening of voltage-activated L-type Ca2+ channels in insulin-secreting cells
- 1994
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 298:Pt 2, s. 341-346
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Tidskriftsartikel (refereegranskat)abstract
- The biological activity of many proteins, including voltage-sensitive ion channels, is controlled by their state of phosphorylation. Ca2+ influx through voltage-activated L-type Ca2+ channels serves as the major stimulatory signal in insulin-secreting cells. We have now investigated the extent to which Ca2+ handling in clonal insulin-secreting RiNm5F cells was affected by okadaic acid, an inhibitor of various serine/threonine protein phosphatases. Whole-cell patch-clamp experiments showed that okadaic acid generated an increase in membrane current, suggesting that it promotes Ca2+ influx through L-type voltage-gated Ca2+ channels probably by modifying their phosphorylation state. Okadaic acid was found to provoke a transient rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i) but had no further effect on the K(+)-induced increase. The Ca2+ transient induced by okadaic acid was dependent on the presence of extracellular Ca2+ and was abolished by D600, a blocker of voltage-activated L-type Ca2+ channels. Concomitant with the rise in [Ca2+]i, okadaic acid induced insulin secretion, a phenomenon that was also dependent on extracellular Ca2+. It is proposed that hyperphosphorylation of voltage-activated L-type Ca2+ channels in insulin-secreting cells lowers the threshold potential for their activation.
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| 5. |
- Islam, M. Shahidul, et al.
(författare)
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Ca(2+)-induced Ca2+ release in insulin-secreting cells
- 1992
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 296:3, s. 287-291
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Tidskriftsartikel (refereegranskat)abstract
- The sulphydryl reagent thimerosal (50 microM) released Ca2+ from a non-mitochondrial intracellular Ca2+ pool in a dose-dependent manner in permeabilized insulin-secreting RINm5F cells. This release was reversed after addition of the reducing agent dithiothreitol. Ca2+ was released from an Ins(1,4,5)P3-insensitive pool, since release was observed even after depletion of the Ins(1,4,5)P3-sensitive pool by a supramaximal dose of Ins(2,4,5)P3 or thapsigargin. The Ins(1,4,5)P3-sensitive pool remained essentially unaltered by thimerosal. Thimerosal-induced Ca2+ release was potentiated by caffeine. These findings suggest the existence of Ca(2+)-induced Ca2+ release also in insulin-secreting cells.
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| 6. |
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| 7. |
- Islam, M. Shahidul, et al.
(författare)
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Cyclic ADP-ribose in beta cells
- 1993
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Ingår i: Science. - 0036-8075 .- 1095-9203. ; 262:5133, s. 584-586
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Tidskriftsartikel (refereegranskat)
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| 8. |
- Islam, M. Shahidul, et al.
(författare)
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Interaction with the inositol 1,4,5-trisphosphate receptor promotes Ca2+ sequestration in permeabilised insulin-secreting cells
- 1991
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Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 288:1-2, s. 27-29
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Tidskriftsartikel (refereegranskat)abstract
- Electropermeabilised insulin-secreting RINm5F cells sequestered Ca2+, resulting in a steady-state level of the ambient free Ca2+ concentration corresponding to 723 +/- 127 nM (mean +/- SEM, n = 10), as monitored by a Ca(2+)-selective minielectrode. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) promoted a rapid and pronounced release of Ca2+. This Ca2+ was resequestered and a new steady-state Ca2+ level was attained, which was always lower (460 +/- 102 nM, n = 10, P less than 0.001) than the steady-state Ca2+ level maintained before the addition of Ins(1,4,5)P3. Whereas the initial reuptake of Ca2+ subsequent to Ins(1,4,5)P3 stimulation was relatively slow, the later part of reuptake was fast as compared to the reuptake phases of a pulse addition of extraneous Ca2+. In the latter case the uptake of Ca2+ resulted in a steady-state level similar to that found in the absence of Ins(1,4,5)P3. Addition of Ins(1,4,5)P3 under this condition resulted in a further Ca2+ uptake and thus a lower steady-state Ca2+ level. Heparin, which binds to the Ins(1,4,5)P3 receptor, also lowered the steady-state free Ca2+ concentration. In contrast to Ins(1,4,5)P3, inositol 1,3,4,5-tetrakisphosphate was without effect on Ca2+ sequestration. These findings are consistent with the presence of a high-affinity Ins(1,4,5)P3 receptor promoting continuous release of Ca2+ under basal conditions and/or the Ins(1,4,5)P3 receptor being actively involved in Ca2+ sequestration.
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| 9. |
- 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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| 10. |
- 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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