Erne P, Hermsmeyer K
Cardiovascular Research Laboratory, Earle A. Chiles Research Institute, Providence Medical Center, Portland, OR 97213.
Naunyn Schmiedebergs Arch Pharmacol. 1991 Dec;344(6):706-15. doi: 10.1007/BF00174755.
We investigated two putative K+ channel openers, pinacidil and BRL34915 (cromakalim), and demonstrated their vasorelaxant effectiveness on rat artery contractions induced by K+, tetraethylammonium (TEA), or norepinephrine. The K+ channel opener-induced decrease in tension was rapid, even when tension was stimulated by 100 mmol/l K+. Measurements of intracellular free Ca++ (activity) by ultra-high sensitivity digital imaging microscopy was carried out by briefly loaded fura 2 (fluorescence ratio) quantitation in isolated, contracting cells of rat azygos vein. Submicron resolution was achieved by measuring cytoplasmic Ca(++)-sensitive fluorescence at each pixel, and size and intensity of areas with high Ca++ concentrations, called hot spots, were determined by a computer-generated, 3 lambda algorithm. Hot spots, which most likely represent the sites of Ca++ release and re-uptake by Ca(++)-regulatory organelles, increased in size and intensity upon addition of K+ or norepinephrine, reaching an early peak prior to the whole cell average peak in cytoplasmic Ca++ activity. Both norepinephrine and K(+)-induced stimulation resulted in Ca++ activity increases that were primarily due to Ca++ release from storage sites. Reduction of free Ca++ activity to resting or lower levels occurred upon addition of pinacidil or cromakalim. Intracellular Ca++ decreases due to K+ channel openers appeared abruptly beginning at the central portions of the cells, resulting in a pronounced early drop in central Ca++ activity while elevated Ca++ levels persisted at the periphery. While this late stage residual of peripheral Ca++ appears to be a significant step in the vascular muscle relaxant action of both K+ channel opener drugs, the level of Ca++ at peripheral sites was greater in response to pinacidil than to cromakalim. The results of this study suggest that in addition to increasing K+ conductance, pinacidil and cromakalim cause 1) decreased Ca++ activity in central regions of the myocytes, and 2) a shift in Ca++ distribution to primarily subsarcolemmal sites. These observations lead us to hypothesize separate control of peripheral and central Ca++ activity within a vascular muscle cell, with Ca++ redistribution that can be altered by vasorelaxants. We suggest that intracellular Ca++ redistribution may contribute the membrane potential-independent part of the vasorelaxant action of the K+ channel openers.
我们研究了两种假定的钾通道开放剂，匹那地尔和BRL34915（克罗卡林），并证明了它们对由钾离子、四乙铵（TEA）或去甲肾上腺素诱导的大鼠动脉收缩具有血管舒张作用。即使张力由100 mmol/l钾离子刺激，钾通道开放剂引起的张力降低也是迅速的。通过在大鼠奇静脉的分离收缩细胞中短暂加载fura 2（荧光比率）定量，采用超高灵敏度数字成像显微镜测量细胞内游离钙离子（活性）。通过测量每个像素处的细胞质钙敏感荧光实现亚微米分辨率，通过计算机生成的3λ算法确定高钙离子浓度区域（称为热点）的大小和强度。热点很可能代表钙离子调节细胞器释放和重新摄取钙离子的部位，在加入钾离子或去甲肾上腺素后，其大小和强度增加，在细胞质钙离子活性的全细胞平均峰值之前达到早期峰值。去甲肾上腺素和钾离子诱导的刺激均导致钙离子活性增加，这主要是由于钙离子从储存部位释放。加入匹那地尔或克罗卡林后，游离钙离子活性降低至静息水平或更低水平。钾通道开放剂引起的细胞内钙离子减少从细胞中央部分突然开始，导致中央钙离子活性明显早期下降，而外周钙离子水平持续升高。虽然外周钙离子的这个后期残留似乎是两种钾通道开放剂药物血管平滑肌舒张作用的一个重要步骤，但匹那地尔引起的外周部位钙离子水平高于克罗卡林。本研究结果表明，除了增加钾离子电导外，匹那地尔和克罗卡林还导致1）心肌细胞中央区域钙离子活性降低，以及2）钙离子分布转移至主要位于肌膜下的部位。这些观察结果使我们推测血管平滑肌细胞内外周和中央钙离子活性存在独立调控，钙离子再分布可被血管舒张剂改变。我们认为细胞内钙离子再分布可能促成了钾通道开放剂血管舒张作用中与膜电位无关的部分。
