STIMULATION OF THE KATP CHANNEL BY ADP AND DIAZOXIDE REQUIRES NUCLEOTIDE HYDROLYSIS IN MOUSE PANCREATIC BETA-CELLS
STIMULATION OF THE KATP CHANNEL BY ADP AND DIAZOXIDE REQUIRES NUCLEOTIDE HYDROLYSIS IN MOUSE PANCREATIC BETA-CELLS
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DOI：
10.1113/jphysiol.1993.sp019598
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发表时间：
1993-04-01
影响因子：
5.5
通讯作者：
RORSMAN, P
中科院分区：
文献类型：
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作者：
LARSSON, O;AMMALA, C;RORSMAN, P
1. 使用从组织培养物中维持的小鼠胰腺β细胞中分离出的由内向外的贴片，研究了ADP和高血糖化合物二氮嗪刺激ATP调节的K+通道（K(ATP)通道）活性的机制。2．二氮嗪和ADP增加K(ATP)通道活性的能力在贴片切除后随着时间的推移而下降，并且在15-40分钟后没有观察到刺激。3。 ADP 激活 K(ATP) 通道需要细胞内 Mg2+ 的存在。 AMP 可以模仿 ADP 的刺激作用，但仅限于 ATP 存在的情况下。用不可水解的类似物 β,γ-亚甲基 ATP 替代 ATP 不会干扰 ADP 刺激 K(ATP) 通道活性的能力。相比之下，K(ATP) 通道活性的增强主要依赖于可水解的ADP，并且在用α,β-亚甲基ADP 替代标准ADP 后没有观察到刺激。4。二氮嗪增强 K(ATP) 通道活性的能力取决于内部 Mg2+ 和 ATP 的存在。用 β,γ-亚甲基 ATP 替代 ATP 后，未观察到二氮嗪对 K(ATP) 通道活性的刺激。然而，在 ADP 存在的情况下，在其本身没有刺激作用的浓度（10 μM）下，二氮嗪在稳定的 ATP 类似物的存在下也具有刺激作用。5。在 ATP 存在的情况下，细胞内 ADP 显着增强二氮嗪对 K(ATP) 通道活性的刺激作用。 ADP 的这种增强作用不能通过稳定的类似物 α,β-亚甲基 ADP 重现，并且以细胞内 Mg2+ 的存在为条件。 AMP (0.1 mm) 也观察到通道活性的类似增强。在没有 ATP 的情况下，如果存在 ADP，二氮嗪仍然能够刺激通道活性。 AMP.6 未重现此效果。在无核苷酸溶液和存在 0.1 mm ATP 的情况下，K(ATP) 通道开放时间的分布由时间常数几乎等于 20 ms 的单指数描述。添加 ADP 或二氮嗪导致出现时间常数大于 100 ms 的第二种成分，占事件总数的 40-70%。在后一种实验条件下，通道的开放概率相对于单独存在ATP时观察到的增加了五倍以上。7．我们认为，ADP 和二氮嗪诱导的 K(ATP) 通道活性刺激反映了相同的基础机制，并且涉及不属于 K(ATP) 通道本身一部分的可扩散细胞质调节成分。二氮嗪的作用取决于 Mg-ADP 的存在，可能是由于 ADP 作用放大所致，最终诱导一种以长持续时间开放为特征的新型动力学状态。
1. The mechanisms by which ADP and the hyperglycaemic compound diazoxide stimulate the activity of the ATP-regulated K+ channel (K(ATP) channel) were studied using inside-out patches isolated from mouse pancreatic beta-cells maintained in tissue culture.2. The ability of diazoxide and ADP to increase K(ATP) channel activity declined with time following patch excision and no stimulation was observed after 15-40 min.3. Activation of K(ATP) channels by ADP required the presence of intracellular Mg2+. The stimulatory effect of ADP was mimicked by AMP but only in the presence of ATP. Replacement of ATP with the non-hydrolysable analogue beta,gamma-methylene ATP did not interfere with the ability of ADP to stimulate K(ATP) channel activity. By contrast, enhancement of K(ATP) channel activity was critically dependent on hydrolysable ADP and no stimulation was observed after substitution of alpha,beta-methylene ADP for standard ADP.4. The ability of diazoxide to enhance K(ATP) channel activity was dependent on the presence of both internal Mg2+ and ATP. Diazoxide stimulation of K(ATP) channel activity was not observed after substitution of beta,gamma-methylene ATP for ATP. However, in the presence of ADP, at a concentration which in itself had no stimulatory action (10 muM), diazoxide was stimulatory also in the presence of the stable ATP analogue.5. The stimulatory action of diazoxide on K(ATP) channel activity in the presence of ATP was markedly enhanced by intracellular ADP. This potentiating effect of ADP was not reproduced by the stable analogue alpha,beta-methylene ADP and was conditional on the presence of intracellular Mg2+. A similar enhancement of channel activity was also observed with AMP (0.1 mm). In the absence of ATP, diazoxide was still capable of stimulating channel activity provided ADP was present. This effect was not reproduced by AMP.6. In both nucleotide-free solution and in the presence of 0.1 mm ATP, the distribution of the K(ATP) channel open times were described by a single exponential with a time constant of almost-equal-to 20 ms. Addition of ADP or diazoxide resulted in the appearance of a second component with a time constant of > 100 ms which comprised 40-70% of the total number of events. Under the latter experimental conditions, the open probability of the channel increased more than fivefold relative to that observed in the presence of ATP alone.7. We propose that ADP- and diazoxide-induced stimulation of K(ATP) channel activity reflects the same basal mechanism and involves a diffusible cytoplasmic regulatory component which is not part of the K(ATP) channel itself The effect of diazoxide is dependent on the presence of Mg-ADP and may result from amplification of the ADP action, culminating in the induction of a novel kinetic state characterized by openings of long duration.