Islam M S, Larsson O, Nilsson T, Berggren P O
Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden.
Biochem J. 1995 Mar 15;306 ( Pt 3)(Pt 3):679-86. doi: 10.1042/bj3060679.
In the pancreatic beta-cell, an increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) by caffeine is believed to indicate mobilization of Ca2+ from intracellular stores, through activation of a ryanodine receptor-like channel. It is not known whether other mechanisms, as well, underlie caffeine-induced changes in [Ca2+]i. We studied the effects of caffeine on [Ca2+]i by using dual-wavelength excitation microfluorimetry in fura-2-loaded beta-cells. In the presence of a non-stimulatory concentration of glucose, caffeine (10-50 mM) consistently increased [Ca2+]i. The effect was completely blocked by omission of extracellular Ca2+ and by blockers of the L-type voltage-gated Ca2+ channel, such as D-600 or nifedipine. Depletion of agonist-sensitive intracellular Ca2+ pools by thapsigargin did not inhibit the stimulatory effect of caffeine on [Ca2+]i. Moreover, this effect of caffeine was not due to an increase in cyclic AMP, since forskolin and 3-isobutyl-1-methylxanthine (IBMX) failed to raise [Ca2+]i in unstimulated beta-cells. In beta-cells, glucose and sulphonylureas increase [Ca2+]i by causing closure of ATP-sensitive K+ channels (KATP channels). Caffeine also caused inhibition of KATP channel activity, as measured in excised inside-out patches. Accordingly, caffeine (> 10 mM) induced insulin release from beta-cells in the presence of a non-stimulatory concentration of glucose (3 mM). Hence, membrane depolarization and opening of voltage-gated L-type Ca2+ channels were the underlying mechanisms whereby the xanthine drug increased [Ca2+]i and induced insulin release. Paradoxically, in glucose-stimulated beta-cells, caffeine (> 10 mM) lowered [Ca2+]i. This effect was due to the fact that caffeine reduced depolarization-induced whole-cell Ca2+ current through the L-type voltage-gated Ca2+ channel in a dose-dependent manner. Lower concentrations of caffeine (2.5-5.0 mM), when added after glucose-stimulated increase in [Ca2+]i, induced fast oscillations in [Ca2+]i. The latter effect was likely to be attributable to the cyclic AMP-elevating action of caffeine, leading to phosphorylation of voltage-gated Ca2+ channels. Hence, in beta-cells, caffeine-induced changes in [Ca2+]i are not due to any interaction with intracellular Ca2+ pools. In these cells, a direct interference with KATP channel- and L-type voltage-gated Ca(2+)-channel activity is the underlying mechanism by which caffeine increases or decreases [Ca2+]i.
在胰腺β细胞中，咖啡因使细胞质游离Ca2+浓度（[Ca2+]i）升高，这被认为是通过激活一种类兰尼碱受体通道，促使Ca2+从细胞内储存库中释放出来。目前尚不清楚是否还有其他机制也参与了咖啡因诱导的[Ca2+]i变化。我们通过在装载了fura-2的β细胞中使用双波长激发显微荧光测定法，研究了咖啡因对[Ca2+]i的影响。在存在非刺激浓度葡萄糖的情况下，咖啡因（10 - 50 mM）持续升高[Ca2+]i。去除细胞外Ca2+以及使用L型电压门控Ca2+通道阻滞剂（如D - 600或硝苯地平）可完全阻断该效应。毒胡萝卜素耗尽激动剂敏感的细胞内Ca2+池并不会抑制咖啡因对[Ca2+]i的刺激作用。此外，咖啡因的这种作用并非由于环磷酸腺苷（cAMP）增加，因为福斯可林和3 - 异丁基 - 1 - 甲基黄嘌呤（IBMX）未能在未受刺激的β细胞中升高[Ca2+]i。在β细胞中，葡萄糖和磺脲类药物通过使ATP敏感性钾通道（KATP通道）关闭来升高[Ca2+]i。咖啡因也会抑制KATP通道活性，这在切除的内向外膜片中得到了测量。因此，在存在非刺激浓度葡萄糖（3 mM）的情况下，咖啡因（> 10 mM）可诱导β细胞释放胰岛素。所以，膜去极化和电压门控L型Ca2+通道开放是这种黄嘌呤药物升高[Ca2+]i并诱导胰岛素释放的潜在机制。矛盾的是，在葡萄糖刺激的β细胞中，咖啡因（> 10 mM）会降低[Ca2+]i。这种效应是由于咖啡因以剂量依赖的方式降低了通过L型电压门控Ca2+通道的去极化诱导的全细胞Ca2+电流。较低浓度的咖啡因（2.5 - 5.0 mM）在葡萄糖刺激使[Ca2+]i升高后加入，会诱导[Ca2+]i快速振荡。后一种效应可能归因于咖啡因升高cAMP的作用，导致电压门控Ca2+通道磷酸化。因此，在β细胞中，咖啡因诱导的[Ca2+]i变化并非由于与细胞内Ca2+池的任何相互作用。在这些细胞中，对KATP通道和L型电压门控Ca(2+)通道活性的直接干扰是咖啡因升高或降低[Ca2+]i的潜在机制。
