Flatt P R, Abrahamsson H, Arkhammar P, Berggren P O, Rorsman P, Swanston-Flatt S K
Department of Biochemistry, University of Surrey, Guildford, UK.
Br J Cancer. 1988 Jul;58(1):22-9. doi: 10.1038/bjc.1988.154.
Regulation of insulin release, membrane potential, transmembrane 45Ca fluxes and cytoplasmic free Ca2+ concentration, [Ca2+]i, was examined using suspensions of transplantable NEDH rat insulinoma cells previously cultured for 2-3 days to eliminate necrotic tumour cells and counter prior hypoglycaemia. Insulinoma cells displayed a resting [Ca2+]i of 94 +/- 8 nM (n = 17) and released 104 +/- 15 ng insulin 10(-6) cells (n = 7) during 60 min incubations with uptake of 2.7 +/- 0.2 nmol 45Ca 10(-6) cells (n = 7). High concentrations of glucose did not affect membrane potential, transmembrane 45Ca fluxes, [Ca2+]i or insulin release by insulinoma cells. K+ at 25 mM depolarised the plasma membrane, induced a small increase in 45Ca efflux and increased [Ca2+]i by 65%. This modest action was not associated with demonstrable effects on 45Ca uptake and insulin release. The effect of 25 mMK+ on [Ca2+]i was counteracted by D-600, but this blocker of voltage-activated Ca2+ channels and verapamil lacked effects on transmembrane 45Ca fluxes and insulin release. The Ca2+-calmodulin antagonist, trifluoroperazine, was also without effect on 45Ca fluxes and insulin release. Ca2+ ionophore ionomycin increased [Ca2+]i, whereas A23187 and X537A did not affect transmembrane 45Ca fluxes. Moreover, insulin release was independent of extracellular Ca2+ over the range 0-20.4 mM despite marked affects on transmembrane 45Ca fluxes and a greater than 4-fold change of [Ca2+]i. Dibutyryl cyclic AMP increased insulin release by 55% without affecting transmembrane 45Ca fluxes or [Ca2+]i. The phosphodiesterase inhibitor, theophylline, also enhanced insulin release by 10-36% with no change of 45Ca uptake. The effectiveness of theophylline was independent of extracellular Ca2+ over the range 0-10.2 mM. These results indicate that inappropriate Ca2+ regulation is a key pathogenic feature underlying the inappropriate insulin secretion of rat insulinoma cells.
利用可移植的NEDH大鼠胰岛素瘤细胞悬液，对胰岛素释放、膜电位、跨膜45Ca通量和细胞质游离Ca2+浓度[Ca2+]i的调节进行了研究。这些细胞先前培养2 - 3天以消除坏死肿瘤细胞并应对先前的低血糖。胰岛素瘤细胞的静息[Ca2+]i为94±8 nM（n = 17），在60分钟孵育期间，每10(-6)个细胞释放104±15 ng胰岛素（n = 7），同时每10(-6)个细胞摄取2.7±0.2 nmol 45Ca（n = 7）。高浓度葡萄糖对胰岛素瘤细胞的膜电位、跨膜45Ca通量、[Ca2+]i或胰岛素释放没有影响。25 mM的K+使质膜去极化，诱导45Ca外流略有增加，并使[Ca2+]i增加65%。这种适度的作用与对45Ca摄取和胰岛素释放的明显影响无关。25 mM K+对[Ca2+]i的作用被D - 600抵消，但这种电压激活Ca2+通道阻滞剂和维拉帕米对跨膜45Ca通量和胰岛素释放没有影响。Ca2+ - 钙调蛋白拮抗剂三氟拉嗪对45Ca通量和胰岛素释放也没有作用。Ca2+离子载体离子霉素增加了[Ca2+]i，而A23187和X537A对跨膜45Ca通量没有影响。此外，尽管对跨膜45Ca通量有显著影响且[Ca2+]i有超过4倍的变化，但在0 - 20.4 mM范围内，胰岛素释放与细胞外Ca2+无关。二丁酰环磷腺苷使胰岛素释放增加55%，而不影响跨膜45Ca通量或[Ca2+]i。磷酸二酯酶抑制剂茶碱也使胰岛素释放增加了10 - 36%，而45Ca摄取没有变化。在0 - 10.2 mM范围内，茶碱的有效性与细胞外Ca2+无关。这些结果表明，不适当的Ca2+调节是大鼠胰岛素瘤细胞不适当胰岛素分泌的关键致病特征。
