Daniel Edwin E, Eteraf Tahereh, Sommer Bettina, Cho Woo Jung, Elyazbi Ahmed
Department of Pharmacology, University of Alberta, Edmonton, AB, Canada.
J Cell Mol Med. 2009 Feb;13(2):352-64. doi: 10.1111/j.1582-4934.2008.00667.x. Epub 2008 Jan 23.
In mouse intestine, caveolae and caveolin-1 (Cav-1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L-type Ca(2+) channels, Na(+)-Ca(2+) exchanger type 1 (NCX1), plasma membrane Ca(2+) pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo-sarcoplasmic reticulum (ER-SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L-type Ca (+) channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav-1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium-free media with 100 mM ethylene glycol tetra-acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium-free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L-type Ca(2+) channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav-1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav-1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L-type Ca(2+) channels or an increase in the distance between caveolae and SR affected calcium handling.
在小鼠肠道中，小窝和小窝蛋白 -1（Cav-1）存在于平滑肌（负责执行收缩）和 Cajal 间质细胞（ICC；负责调节收缩节律）中。我们发现许多钙处理/依赖分子与小窝相关，包括 L 型 Ca(2+)通道、1 型钠钙交换体（NCX1）、质膜钙泵和神经元型一氧化氮合酶（nNOS），并且小窝靠近外周肌质网（ER-SR）。此外，我们发现这种组合可能解释了钙从小窝结构域通过 L 型 Ca(+)通道循环至肌质网以维持节律调节和收缩的过程。在此，我们通过比较 Cav-1 基因敲除小鼠（缺乏小窝）及其基因对照小鼠的纵行肌在各种条件下的节律调节和收缩情况，进一步验证这一假设。我们采用了一种方法，即在含有 100 mM 乙二醇四乙酸（EGTA）的无钙培养基中研究起搏频率（指示 ICC 的功能）和收缩幅度（指示平滑肌的功能）。当钙储存最初充足时，ICC 中缺乏小窝会通过减少钙从小窝质膜到肌质网的循环，抑制 ICC 在无钙培养基中维持收缩频率的能力。这种向 ICC 的循环主要涉及 L 型 Ca(2+)通道；即通过打开这些通道可提高起搏频率，而关闭这些通道则会抑制起搏频率。然而，当通过阻断肌质网/内质网 Ca(2+)-ATP 酶（SERCA）泵使这些储存耗尽，或通过卡巴胆碱激活钙释放时，Cav-1 或小窝的缺失几乎没有影响。小窝的缺失对收缩幅度影响很小，这表明钙在平滑肌中循环至肌质网。然而，在某些条件下，ICC 和平滑肌中小窝的缺失都会减缓钙从肌质网的流失速率，这可能反映了与储存操纵性钙通道距离的增加。我们发现有证据表明这些通道与 Cav-1 相关。这些变化均与以下假设一致：肠肌丛 ICC 中小窝相关的细胞外钙减少、L 型 Ca(2+)通道的状态或小窝与肌质网之间距离的增加会影响钙的处理。
