Bolaffi J L, Rodd G G, Ma Y H, Bright D, Grodsky G M
Metabolic Research Unit, University of California, San Francisco 94143.
Endocrinology. 1991 Oct;129(4):2131-8. doi: 10.1210/endo-129-4-2131.
The spontaneous decline of insulin secretion (third phase) that occurs under a variety of secretory conditions is well documented and suggests a general impairment or desensitization of the secretory process. We have examined several aspects of Ca2+ flux as well as regulators of Ca-linked second messenger events in freshly isolated rat islets chronically stimulated with glucose over 24 h, a period that encompasses initial (hour 1), peak (hour 3), and subsequent impaired or desensitized (hour 20-22) secretion. In islets incubated for these periods in HB104 medium with 22 mM glucose, 45Ca2+ uptake did not vary (12.6 +/- 1.6 vs. 10.2 +/- 1.7 vs. 13.2 +/- 3.4 pmol Ca2+/islet.10 min at 1, 3, and 22 h, respectively). Chronic incubation in 2 mM glucose reduced total Ca2+ uptake at each of the time periods, but, again, uptake did not change with desensitization (9.8 +/- 1.4 vs. 6.6 +/- 2.1 vs. 7.8 +/- 2.3 pmol Ca2+/islet.10 min). In 11 mM glucose, the Ca channel antagonist verapamil (1-10 microM) reduced insulin secretion by 55-80% in a dose-dependent manner over 1-3 h; islets continuously exposed to verapamil escaped inhibition by 20 h even at the highest concentration. However, in islets first exposed to 10 microM verapamil only during 20-22 h, hourly insulin secretion was suppressed 25%, 45%, and 33% at 20, 21, and 22 h, respectively, indicating that glucose-desensitized islets were still sensitive to further inhibition of Ca channels. Staurosporine (1 microM), an inhibitor of protein kinase-C activity, progressively inhibited glucose-stimulated insulin secretion from 48% at 1 h to more than 80% by 3 h; again, this inhibitory effect was lost by 20 h of chronic staurosporine. When staurosporine was first administered at 20 h, insulin secretion was modestly suppressed and returned to control values in the next hour. With continuous glucose, the islet response to positive stimulation of endogenous C-kinase activity by carbachol was maintained. The Ca/calmodulin inhibitor trifluoroperazine also inhibited insulin secretion by 75-80% during 1-3 h and continued to exert inhibitory effects through 23 h of continuous administration. We conclude that even though insulin secretion has desensitized to glucose, 1) Ca2+ entry is unchanged and is still regulated by glucose, 2) voltage-dependent Ca channels are still sensitive to blockade by acute verapamil, but can desensitize to chronic verapamil; 3) stimulus-enhanced C-kinase activity may be especially labile during glucose-induced desensitization, while 4) possible Ca/calmodulin potentiation of secretion persists through the three secretory phases.(ABSTRACT TRUNCATED AT 400 WORDS)
在多种分泌条件下发生的胰岛素分泌自发性下降（第三阶段）已有充分记录，这表明分泌过程存在普遍的损伤或脱敏现象。我们研究了原代分离的大鼠胰岛中Ca2+通量的几个方面以及Ca相关第二信使事件的调节因子，这些胰岛在24小时内持续受到葡萄糖刺激，这段时间涵盖了初始（第1小时）、峰值（第3小时）以及随后受损或脱敏（第20 - 22小时）的分泌阶段。在含有22 mM葡萄糖的HB104培养基中孵育这些时间段的胰岛，45Ca2+摄取量没有变化（在第1、3和22小时分别为12.6±1.6、10.2±1.7和13.2±3.4 pmol Ca2+/胰岛·10分钟）。在2 mM葡萄糖中进行慢性孵育会降低每个时间段的总Ca2+摄取量，但同样，摄取量不会随着脱敏而改变（分别为9.8±1.4、6.6±2.1和7.8±2.3 pmol Ca2+/胰岛·10分钟）。在11 mM葡萄糖中，Ca通道拮抗剂维拉帕米（1 - 10 μM）在1 - 3小时内以剂量依赖方式使胰岛素分泌减少55 - 80%；持续暴露于维拉帕米的胰岛即使在最高浓度下到20小时也会逃脱抑制。然而，在仅在第20 - 22小时暴露于10 μM维拉帕米的胰岛中，在第20、21和22小时每小时的胰岛素分泌分别被抑制25%、45%和33%，这表明葡萄糖脱敏的胰岛对Ca通道的进一步抑制仍然敏感。蛋白激酶C活性抑制剂星形孢菌素（1 μM）从第1小时的48%逐渐抑制葡萄糖刺激的胰岛素分泌，到第3小时超过80%；同样，这种抑制作用在慢性星形孢菌素处理20小时后消失。当在第20小时首次给予星形孢菌素时，胰岛素分泌受到适度抑制，并在下一小时恢复到对照值。在持续葡萄糖存在的情况下，胰岛对内源性C激酶活性由卡巴胆碱进行正向刺激的反应得以维持。Ca/钙调蛋白抑制剂三氟拉嗪在1 - 3小时内也将胰岛素分泌抑制75 - 80%，并在持续给药23小时内持续发挥抑制作用。我们得出结论，即使胰岛素分泌对葡萄糖已脱敏，1）Ca2+内流未改变且仍受葡萄糖调节，2）电压依赖性Ca通道对急性维拉帕米的阻断仍敏感，但对慢性维拉帕米会脱敏；3）在葡萄糖诱导的脱敏过程中，刺激增强的C激酶活性可能特别不稳定，而4）分泌过程中可能的Ca/钙调蛋白增强作用在三个分泌阶段持续存在。（摘要截断于400字）
