C1C-3 CHLORIDE ION CHANNELS IN VASCULAR SMOOTH MUSCLE

氯电流在牵张和激动剂诱导的血管平滑肌细胞去极化过程中起重要作用。改变氯离子梯度、干扰氯离子转运或阻断氯离子通道都会改变VSM对去甲肾上腺素的收缩反应。负责这一作用的氯通道受一氧化氮的调节。这可能代表了一种新的VSM收缩控制机制。肌源性张力也被C1通道阻滞剂抑制，并对C1梯度的变化敏感。Northern blotting表明，在培养的人VSM细胞(主动脉和冠状动脉)中，到目前为止表达最高的C1通道基因是C1C-3。原位杂交证实C1C-3在人肺中有血管表达。最近的研究表明，C1C-3克隆在NIH/3T3细胞中的表达产生一种体积激活的氯电流(IC1VOL)，该电流被蛋白激酶C的激活剂抑制。目前尚不清楚对肿胀的反应是否转化为对机械拉伸的敏感性，或者这些通道是否可以被收缩剂激活。我们发现C1C-3基因在小鼠和大鼠中交替剪接，因此排除了外显子2(CIC-3Short)，而人的mRNA总是包括这个序列(C1C-3Long)。这一点很重要，因为外显子2包含一个框内ATG，它产生的C1C-3蛋白的氨基末端比之前表达的产生IC1vol.的克隆长58个氨基酸。这58个氨基酸的功能还有待确定。我们建议：1)使用穿孔膜片钳记录来确定钙激活和肿胀激活的氯电流对儿茶酚胺介导的小鼠主动脉VSM细胞去极化的贡献。然后，我们将确定这些电流是否以及如何受一氧化氮的调节；2)研究三种不同类型的细胞产生的容量激活氯电流，这些细胞要么完全缺乏C1C-3(小鼠胚胎干细胞，C1C-3缺失)，要么过表达C1C-3L或C1C-3S(NIH/3T3细胞，Fisher大鼠甲状腺上皮细胞)，以及3)通过使用转基因技术在VSM特异性启动子(SM22pha)后面过表达C1C-3，来评估C1C-3在完整小鼠血管中的功能。这些研究将确定VSM Cl通道是如何激活和调节的，以及C1C-3如何对血管功能和血压测定做出贡献。它们还将加深我们对C1C-3的基本生物物理特性的理解。对这些问题的了解可能会让我们设计出控制血管功能的新的药理学方法。

Chloride (Cl) currents contribute to both stretch and agonist- induced depolarization of vascular smooth muscle (VSM) cells. Changing the Cl gradient, interfering with C1 transport, or blocking Cl channels all alter VSM contractile responses to norepinephrine. The Cl channels responsible for this effect are regulated by nitric oxide. This may represent a new mechanism for the control of VSM contractility. Myogenic tone is also inhibited by C1 channel blockers and sensitive to changes in the C1 gradient. We have shown by Northern blotting that in cultured human VSM cells (aortic and coronary), by far the most highly expressed C1 channel gene is C1C-3. Vascular expression of C1C-3 was confirmed by in situ hybridization in human lung. It has recently been shown that expression of a C1C-3 clone in NIH/3T3 cells produces a volume-activated chloride current (IC1vol) which is inhibited by activators of protein kinase C. It is not known if a response to swelling translates to sensitivity to mechanical stretch or if these channels can be activated by contractile agents. We have found that the C1C-3 gene is alternatively spliced in mice and rats so that exon 2 is excluded (CIC-3Short) while human mRNA always includes this sequence (C1C-3Long). This is important because exon 2 contains an in frame ATG producing a C1C-3 protein which is 58 amino acids longer at the amino terminus than the previously expressed clone which produced IC1vol. The function of these 58 amino acids needs to be determined. We propose to; 1) use perforated patch-clamp recording to define the contribution of the calcium-activated and swelling-activated chloride currents to catecholamine-mediated depolarization of mouse aortic VSM cells. We will then determine if, and how, these currents are regulated by nitric oxide, 2) study the volume-activated chloride currents produced by three different cell types which either completely lack C1C-3 (mouse embryonic stem cells with C1C-3 knocked out) or have been made to overexpress C1C-3L or C1C-3S (NIH/3T3 cells, Fisher Rat Thyroid epithelial cells), and 3) assess the function of C1C-3 in intact murine blood vessels by using transgenic technology to overexpress C1C-3 behind a VSM-specific promoter (SM22alpha). These studies will define how VSM Cl channels are activated and regulated and how C1C-3 contributes to blood vessel function and determination of blood pressure. They will also further our understanding of the basic biophysical characteristics of C1C-3. An understanding of these issues may allow us to design new pharmacological approaches to the control of vascular function.