C1C-3 CHLORIDE ION CHANNELS IN VASCULAR SMOOTH MUSCLE

氯（Cl）电流有助于血管平滑肌（VSM）细胞的牵拉和激动剂诱导的去极化。改变Cl梯度、干扰Cl转运或阻断Cl通道都可改变VSM对去甲肾上腺素的收缩反应。 负责这种效应的Cl通道由一氧化氮调节。 这可能代表了一个新的机制来控制VSM的收缩。 肌源性张力也受到C1通道阻滞剂的抑制，并且对C1梯度的变化敏感。 我们已经通过北方印迹显示，在培养的人VSM细胞（主动脉和冠状动脉）中，迄今为止最高表达的C1通道基因是C1 C-3。 C1 C-3在人肺血管中的表达通过原位杂交证实。 最近的研究表明，NIH/3 T3细胞中C1 C-3克隆的表达产生体积激活的氯电流（IC 1vol），其被蛋白激酶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。 我们已经发现，C1 C-3基因在小鼠和大鼠中是可变剪接的，因此外显子2被排除（CIC-3Short），而人类mRNA总是包含该序列（C1 C-3Long）。 这是重要的，因为外显子2含有产生C1 C-3蛋白的符合读码框的ATG，其在氨基末端比先前表达的产生IC 1vol的克隆长58个氨基酸。 这58种氨基酸的功能需要确定。我们建议; 1）采用穿孔膜片钳技术研究钙激活和膨胀激活的氯电流对儿茶酚胺介导的小鼠主动脉VSM细胞去极化的影响。 然后，我们将确定这些电流是否以及如何受一氧化氮调节，2）研究由三种不同细胞类型产生的容积激活氯电流，这些细胞类型要么完全缺乏C1 C-3，（敲除C1 C-3的小鼠胚胎干细胞）或已使其过表达C1 C-3L或C1 C-3S（NIH/3 T3细胞，Fisher大鼠甲状腺上皮细胞），和3）通过使用转基因技术在VSM特异性启动子（SM 22 α）后面过表达C1 C-3来评估C1 C-3在完整鼠血管中的功能。这些研究将定义VSM Cl通道如何被激活和调节，以及C1 C-3如何有助于血管功能和血压的确定。 它们也将进一步加深我们对C1 C-3的基本生物物理特征的理解。对这些问题的理解可能使我们能够设计新的药理学方法来控制血管功能。

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.