Unperturbed islet α-cell function examined in mouse pancreas tissue slices
Unperturbed islet α-cell function examined in mouse pancreas tissue slices
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DOI：
10.1113/jphysiol.2010.200345
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发表时间：
2011-01-15
影响因子：
5.5
通讯作者：
Gaisano, Herbert Y.
中科院分区：
文献类型：
--
作者：
Huang, Ya-Chi;Rupnik, Marjan;Gaisano, Herbert Y.
由于胰岛分离和分散成单细胞的技术困难，对健康和糖尿病中胰岛α细胞生物学的关键研究很少且不一致。我们通过胰腺切片的制备，规避了这些困难。我们在原位天然状态下对α细胞进行功能表征（电生理），然后将被测细胞加载生物细胞素染料，随后通过免疫细胞化学确认细胞身份。我们发现了大量的α细胞，它们在几个离子通道的电生理参数（atp敏感的K+、Na+和Ca2+电流）和作为胰高血糖素颗粒胞吐测量的电容变化中表现出广泛的分布。这可以解释以前关于α细胞的报告明显不一致，由于α细胞采样不足，无意中显示了扭曲的数据。我们的创新方法将使未来的研究能够阐明糖尿病中的α细胞失调。由于采用孤立的胰岛和分散的单细胞的传统策略存在技术困难，对健康和糖尿病中的α细胞生物学的关键研究很少，有时也不一致。急性胰腺切片制备是为了克服传统胰岛细胞分离程序中固有的酶和机械扰动而开发的。这种制备保留了胰岛内细胞通讯和胰岛结构在原位的天然状态。采用膜片移液管直接检测组织切片内的α -细胞，并进行电生理表征。生物细胞素染色标记和免疫细胞化学证实了斑块细胞的身份。小鼠胰腺切片中的α -细胞表现出良好的I-Na（在生理膜电位下可兴奋）、I-KATP、小细胞尺寸、低静息膜电导和可诱导的低电压和高压激活的I-Ca的特征，后者与通过电容测量确定的胞外分泌有关。与之前的报道相反，我们对α -细胞进行了大量无偏采样，揭示了所有这些参数的广泛分布，包括K-ATP电导的数量，Na+和Ca2+电流的振幅，以及由一系列去极化脉冲引起的电容变化。所提出的胰腺切片制备与标准贴片钳夹技术相结合，可以对α细胞进行大采样和快速评估，从而揭示α细胞离子通道特性的广泛分布。这一特殊的特征解释了先前关于这些α细胞离子通道性质的报道的明显不一致。我们的创新方法将使未来的研究能够阐明糖尿病期间发生的胰岛α细胞失调。
Non-technical summaryCritical investigation into pancreatic islet alpha-cell biology in health and diabetes has been sparse and inconsistent because of technical difficulties in islet isolation and dispersion into single cells. We have circumvented these difficulties by employing the pancreas slice preparation. We functionally characterized (electrophysiologically) the alpha-cells in their in situ native state, then loaded the tested cells with biocytin dye to subsequently confirm the cell identities by immunocytochemistry. We characterized a very large number of alpha-cells, which showed a wide-range distribution in the electrophysiological parameters of several ion channels (ATP-sensitive K+, Na+ and Ca2+ currents) and capacitance changes as measure of exocytosis of glucagon granules. This could explain the apparent inconsistency of previous reports on alpha-cells that inadvertently showed skewed data due to insufficient sampling of alpha-cells. Our innovative approaches will enable future studies into elucidating alpha-cell dysregulation in diabetes.Critical investigation into alpha-cell biology in health and diabetes has been sparse and at times inconsistent because of the technical difficulties with employing conventional strategies of isolated islets and dispersed single cells. An acute pancreas slice preparation was developed to overcome the enzymatic and mechanical perturbations inherent in conventional islet cell isolation procedures. This preparation preserves intra-islet cellular communication and islet architecture in their in situ native state. alpha-Cells within tissue slices were directly assessed by patch pipette and electrophysiologically characterized. The identity of the patched cells was confirmed by biocytin dye labelling and immunocytochemistry. alpha-Cells in mouse pancreas slices exhibited well-described features of I-Na (excitable at physiological membrane potential), I-KATP, small cell size, low resting membrane conductance, and inducible low and high voltage-activated I-Ca, the latter correlating with exocytosis determined by capacitance measurements. In contrast to previous reports, our large unbiased sampling of alpha-cells revealed a wide range distribution of all of these parameters, including the amount of K-ATP conductance, Na+ and Ca2+ current amplitudes, and capacitance changes induced by a train of depolarization pulses. The proposed pancreas slice preparation in combination with standard patch-clamping technique allowed large sampling and rapid assessment of alpha-cells, which revealed a wide distribution in alpha-cell ion channel properties. This specific feature explains the apparent inconsistency of previous reports on these alpha-cell ion channel properties. Our innovative approach will enable future studies into elucidating islet alpha-cell dysregulation occurring during diabetes.