REGULATION OF OSCILLATORY OXYGEN CONSUMPTION IN SINGLE CLONAL PANCREATIC?? CELLS

单克隆胰腺振荡耗氧量的调节？

• 批准号: 6120184
• 负责人: BARBARA E. CORKEY
• 金额: --
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120184
• 关键词: Mammalia; aging; animal tissue; bioengineering /biomedical engineering; biomedical equipment development; biomedical resource; child (0-11); clinical research; female; growth /development; male; mother /infant health care; reproductive system; technology /technique; women's health

The molecular mechanism by which glucose and other fuels stimulate insulin release is still uncertain. ?-Cells possess a unique stimulus-response coupling system which requires that the fuel stimulus be metabolized to initiate membrane electrical events then secretion. Exposure of pancreatic ? cells to stimulatory concentrations of glucose is known to decrease the activity of the KATP-channels. This results in depolarization of the membrane potential, Ca2+-dependent bursting electrical activity and insulin secretion. It is clear that fuel-induced secretion, likeother secretory processes, is mediated in part by elevations in cytosolic free Ca2+. Although it is hypothesized that changes in metabolism precede changes in Ca2+, this has never been documented at the single cell level. Single cell Ca2+ studies have been published extensively and it is well-established that oscillations in Ca2+ occur in response to glucose stimulation. However, the pattern observed in single cells (mostly oscillatory) is quite different from the pattern obtained in cell suspensions (monophasic). The present studies were undertaken to evaluate whether the pattern of O2 consumption in single ?-cells supported a model in which metabolic oscillations preceded and caused oscillatory changes in Ca2+. A second goal was to determine the percentage of responding cells and whether increasing glucose concentrations increased O2 consumption in each cell or recruited previously unresponsive cells, as has been hypothesized for Ca2+. The results of these studies showed tha t oscillations in O2 consumption occurred in the basal state and increased in amplitude in response to stimulation by glucose (15 mM). These oscillations were blocked by uncoupling respiration with the chemical uncoupler, FCCP, which stimulated O2 consumption. They were also blocked and respiration totally inhibited by antimycin A, a bc1 complex inhibitor. The question of whether Ca2+ modulated the oscillations in O2 and, indeed, whether the O2 oscillations were independent of Ca2+, was addressed by monitoring O2 in the absence of added Ca2+. Oscillations in O2 occurred inthe absence of Ca2+, increased in response to stimulatory glucose and increased still further in response to restoration of a normal extracellular Ca2+ level. In the presence of normal extracellular Ca2+, basal O2 oscillations were greater and increased still further in response to glucose. These studies indicate that the O2 oscillations did not require Ca2+ but that the amplitude of the O2 oscillations were much greater under conditions where insulin secretion was stimulated. When studying the response of cell to maximal stimulation by glucose most cells exhibited basal oscillations in O2 consumption and that about half of the cells responded with a marked increase in the amplitude of oscillations while the remainder did not respond at all. The data obtained also al lowed analysis of the amplitude and period of oscillations in O2 consumption from two different clonal ?-cell lines. In order to determine how the cells responded to substimulatory concentrations of glucose experiments were performed in which O2 consumption was monitored in multiple cells following addition of two consecutive submaximal additions of glucose. It was determined that cells exhibited and all or none response with no evidence of a concentration dependence.

葡萄糖和其他燃料刺激的分子机制 胰岛素释放仍不确定。 ?-细胞具有独特的 刺激-反应耦合系统，要求燃料 然后刺激被代谢以启动膜电事件 分泌。 暴露胰腺 ?细胞刺激 已知葡萄糖浓度会降低 KATP 通道。 这导致膜去极化 电位、Ca2+依赖性爆发电活动和胰岛素 分泌。 很明显，燃料引起的分泌，像其他分泌一样 分泌过程部分是由细胞质的升高介导的 游离Ca2+。 尽管推测新陈代谢的变化 先于 Ca2+ 的变化，这从未在单一记录中记录过 细胞水平。 单细胞 Ca2+ 研究已广泛发表 众所周知，Ca2+ 的振荡是作为响应而发生的 葡萄糖刺激。 然而，在单细胞中观察到的模式 （主要是振荡）与中获得的模式有很大不同 细胞悬浮液（单相）。 目前的研究是为了 评估单个 ?-细胞的 O2 消耗模式是否 支持代谢振荡先于并导致的模型 Ca2+ 的振荡变化。 第二个目标是确定 反应细胞的百分比以及是否增加葡萄糖 浓度增加每个细胞或招募的 O2 消耗 先前无反应的细胞，正如 Ca2+ 的假设一样。 这 这些研究的结果表明，氧气消耗量的波动 发生在基础状态并响应于振幅增加 葡萄糖（15 mM）刺激。 这些振荡被阻止 使用化学解偶联剂 FCCP 解偶联呼吸作用， 刺激 O2 消耗。 他们的呼吸也受到阻碍 完全被 bc1 复合物抑制剂抗霉素 A 抑制。 这 Ca2+ 是否调节 O2 振荡的问题，事实上， O2 振荡是否独立于 Ca2+，已解决 在不添加 Ca2+ 的情况下监测 O2。 O2 中的振荡 发生在缺乏 Ca2+ 的情况下，响应刺激而增加 葡萄糖并进一步增加以响应恢复 正常细胞外 Ca2+ 水平。 在正常情况下 细胞外Ca2+、基础O2振荡更大且增加 更进一步地响应葡萄糖。 这些研究表明 O2 振荡不需要 Ca2+，但需要 O2 的振幅 在胰岛素条件下，振荡要大得多 分泌受到刺激。 当研究细胞的反应时 葡萄糖的最大刺激大多数细胞表现出基础振荡 氧气消耗量，大约一半的细胞做出反应 振荡幅度明显增加，而其余部分 根本没有反应。 获得的数据还可以分析 两个 O2 消耗量的振荡幅度和周期 不同的克隆β细胞系。 为了确定细胞如何 对亚刺激浓度的葡萄糖实验作出反应 监测多个单元的 O2 消耗量 连续两次次最大添加葡萄糖后。 确定细胞表现出全部或没有反应 没有证据表明存在浓度依赖性。