Rembold C M, Kendall J M, Campbell A K
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Cell Calcium. 1997 Jan;21(1):69-79. doi: 10.1016/s0143-4160(97)90098-1.
The physiologic relevance of Ca2+ release from the sarcoplasmic reticulum in arterial smooth muscle contraction is controversial. Therefore, we sought to measure changes in sarcoplasmic reticulum free [Ca2+] (i.e. [Ca2+]sr) in the intact rat tail artery. We exploited a novel technique to measure [Ca2+]sr with genetically targeted apoaequorin acting as a pseudo-luciferase rather than as classic aequorin. Intact rat tail arteries were infected with a replication deficient adenoviral vector (RAdER) containing the apoaequorin gene targeted to the sarcoplasmic reticulum. Addition of apoaequorin's substrate, coelenterazine, to the perfusate increased light production in a [Ca2+] dependent manner, consistent with apoaequorin action on coelenterazine. Within the limits of the photon counting system, imaging of infected rat tail artery segments revealed light production from the whole thickness of the vascular wall. Phenylephrine stimulation decreased apoaequorin generated light and induced a contraction. Washout of phenylephrine relaxed the tissues and increased light indicating refilling of the sarcoplasmic reticulum with Ca2+. Incubation in 10 microM cyclopiazonic acid, a SERCA inhibitor, did not alter apoaequorin generated light or induce a contraction. In the presence of cyclopiazonic acid, phenylephrine contractions were enhanced and apoaequorin generated light decreased further than that observed in the absence of cyclopiazonic acid. Cyclopiazonic acid also prevented the increase in apoaequorin generated light upon washout of phenylephrine, consistent with its inhibition of sarcoplasmic reticulum refilling. These results suggest that light production from targeted apoaequorin, delivered by a replication deficient adenovirus, is a valid measure of changes in [Ca2+]sr in the intact arterial wall. There appeared to be a correlation between Ca2+ release and contraction in these lightly loaded arteries.
肌浆网释放Ca2+在动脉平滑肌收缩中的生理相关性存在争议。因此，我们试图测量完整大鼠尾动脉中肌浆网游离Ca2+浓度（即[Ca2+]sr）的变化。我们采用了一种新技术，利用基因靶向的脱辅基水母发光蛋白作为假荧光素酶而非经典的水母发光蛋白来测量[Ca2+]sr。将完整的大鼠尾动脉用携带靶向肌浆网的脱辅基水母发光蛋白基因的复制缺陷型腺病毒载体（RAdER）进行感染。向灌注液中添加脱辅基水母发光蛋白的底物腔肠素，会以Ca2+依赖的方式增加发光，这与脱辅基水母发光蛋白对腔肠素的作用一致。在光子计数系统的测量范围内，对感染的大鼠尾动脉节段进行成像显示，血管壁全层均有发光现象。去氧肾上腺素刺激会降低脱辅基水母发光蛋白产生的光，并诱导收缩。冲洗掉去氧肾上腺素后，组织松弛且发光增加，表明肌浆网重新充满了Ca2+。在10微摩尔/升的环匹阿尼酸（一种肌浆网Ca2+-ATP酶抑制剂）中孵育，并未改变脱辅基水母发光蛋白产生的光，也未诱导收缩。在环匹阿尼酸存在的情况下，去氧肾上腺素引起的收缩增强，且脱辅基水母发光蛋白产生的光比不存在环匹阿尼酸时下降得更多。环匹阿尼酸还阻止了冲洗去氧肾上腺素后脱辅基水母发光蛋白产生的光的增加，这与其对肌浆网再充盈的抑制作用一致。这些结果表明，由复制缺陷型腺病毒递送的靶向脱辅基水母发光蛋白产生的光是完整动脉壁中[Ca2+]sr变化的有效测量指标。在这些轻度负荷的动脉中，Ca2+释放与收缩之间似乎存在相关性。
