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- 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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| 4. |
- Kass, G E, et al.
(författare)
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Receptor-mediated Mn2+ influx in rat hepatocytes : comparison of cells loaded with Fura-2 ester and cells microinjected with Fura-2 salt
- 1994
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 302, s. 5-9
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Tidskriftsartikel (refereegranskat)abstract
- In single Fura-2 ester-loaded hepatocytes, stimulation by vasopressin, but not emptying of the agonist-sensitive Ca2+ store by 2,5-di-(t-butyl)hydroquinone, resulted in an increase in the rate of Fura-2 fluorescence-quenching by Mn2+. Similarly, in cells microinjected with Fura-2 salt, vasopressin stimulated Mn2+ entry while 2,5-di-(t-butyl)hydroquinone or thapsigargin did not. The pattern of Fura-2 quenching by Mn2+ only correlated with the movement of Mn2+ across the plasma membrane.
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| 5. |
- Kass, G E, et al.
(författare)
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Two separate plasma membrane Ca2+ carriers participate in receptor-mediated Ca2+ influx in rat hepatocytes.
- 1994
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434. ; 1223:2, s. 226-33
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Tidskriftsartikel (refereegranskat)abstract
- The plasma membrane Ca2+ carrier system involved in receptor-mediated Ca2+ entry was studied. Using the Ca2+ readdition protocol, the rate of cytosolic free Ca2+ concentration ([Ca2+]i) increase in vasopressin-pretreated hepatocytes was significantly higher than in thapsigargin- or 2,5-di(tert-butyl)hydroquinone-pretreated cells. The addition of Mn2+ to unstimulated hepatocytes resulted in a biphasic quench of fura-2 fluorescence. After an initial phase that was fast in rate but of short duration, the rate of fura-2 quench by Mn2+ became much slower and lasted until all the cellular fura-2 was quenched. Pretreatment of the cells with vasopressin only accelerated the rate of the latter phase but not of the initial one. In agonist-stimulated cells, acidification of the extracellular medium or the presence of ruthenium red, econazole or SK&F 96365 decreased the rates of both [Ca2+]i increase and Mn2+ entry upon addition of the respective cation. By contrast, neomycin and N-tosyl-L-phenylalanine chloromethyl ketone markedly decreased the rate of [Ca2+]i increase upon Ca2+ readdition but had no effect on vasopressin-stimulated Mn2+ entry. None of the treatments affected the ability of vasopressin and thapsigargin to mobilize the internal Ca2+ store. It is concluded that in hepatocytes the two pathways of receptor-mediated Ca2+ entry control two distinct yet pharmacologically related cation carriers.
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