Jackson W F, Huebner J M, Rusch N J
Department of Biological Sciences, College of Arts and Sciences, Western Michigan University, Kalamazoo 49008, USA.
Microcirculation. 1997 Mar;4(1):35-50. doi: 10.3109/10739689709148316.
The goal of the present study was to develop a method to isolate viable arteriolar muscle cells from single cremasteric arterioles, which retain the contractile and electrophysiological phenotype of the donor microvessels.
Arterioles were hand-dissected from rat and hamster cremaster muscles and dissociated by incubation in papain and dithioerythritol for 35 min followed by incubation in collagenase, elastase, and soybean trypsin inhibitor for 10 to 25 min in solutions containing 100 microM Ca2+. 10 microM sodium nitroprusside, and 1 mg/ml albumin at 37 degrees C.
Populations of single smooth muscle cells enzymatically isolated from cremasteric arterioles showed elongated fusiform morphology and intact plasmalemmal membranes as indicated by retention of calcein, by exclusion of ethidium homodimer-1 and by high membrane resistances (11 +/- 0.8 C omega, n = 36 for rat cells; 8 +/- 0.6 C omega, n = 21 for hamster cells: p < 0.05). Muscle cells contracted in a concentration-dependent fashion in response to pipette application of norepinephrine (10 nM-100 microM). Cell shortening in response to 1 microM norepinephrine was inhibited by 10 microM phentolamine, 1 microM sodium nitroprusside, and 1 microM nifedipine or nominally Ca(2+)-free media. Resting membrane potential recorded in patch-clamped cells by perforated patch methods was -48 +/- 1 mV (n = 47) for rat cells and -44 +/- 2.8 mV (n = 14) for hamster cells (p > 0.05). Families of voltage-dependent K+ currents were observed during stepwise depolarizing pulses from -60 mV to more positive potentials. Blockers of voltage-gated and ATP-sensitive K+ channels (4-aminopyridine [3 mM] and glibenclamide [1 microM], respectively) inhibited membrane K+ conductance, increased membrane resistance, and depolarized cells by 20 +/- 4 mV (n = 8) and 14 +/- 3 mV (n = 6), respectively.
The present method permits isolation of smooth muscle cells from a single cremasteric arteriole. These cells seem to retain the contractile phenotype, alpha-adrenergic signaling cascade, membrane potential, and K+ conductances described for the donor arteriole. Correlating the functional and electrophysiological properties of these smooth muscle cells to in situ and in vitro studies of their donor arterioles should provide a useful extension for understanding the physiology, pathophysiology, biophysics, and cell biology of the microcirculation in skeletal muscle.
本研究的目的是开发一种从单个提睾肌小动脉中分离出有活力的小动脉肌细胞的方法，这些细胞保留供体微血管的收缩和电生理表型。
从小鼠和大鼠的提睾肌中手工分离出小动脉，在含有100微摩尔/升钙离子、10微摩尔/升硝普钠和1毫克/毫升白蛋白的溶液中，于37℃下，先在木瓜蛋白酶和二硫苏糖醇中孵育35分钟，然后在胶原酶、弹性蛋白酶和大豆胰蛋白酶抑制剂中孵育10至25分钟，使其解离。
从提睾肌小动脉中酶解分离出的单个平滑肌细胞群体呈现出细长的梭形形态，质膜完整，这通过钙黄绿素的保留、乙锭同二聚体-1的排除以及高膜电阻得以证明（大鼠细胞的膜电阻为11±0.8千兆欧，n = 36；仓鼠细胞的膜电阻为8±0.6千兆欧，n = 21：p < 0.05）。通过移液管施加去甲肾上腺素（10纳摩尔至100微摩尔），肌细胞以浓度依赖的方式收缩。10微摩尔酚妥拉明、1微摩尔硝普钠、1微摩尔硝苯地平或无钙培养基可抑制细胞对1微摩尔去甲肾上腺素的缩短反应。通过穿孔膜片钳方法记录的膜片钳细胞的静息膜电位，大鼠细胞为-48±1毫伏（n = 47），仓鼠细胞为-44±2.8毫伏（n = 14）（p > 0.05）。在从-60毫伏到更正电位的逐步去极化脉冲期间，观察到电压依赖性钾电流家族。电压门控钾通道和ATP敏感性钾通道的阻滞剂（分别为3毫摩尔4-氨基吡啶和1微摩尔格列本脲）抑制膜钾电导，增加膜电阻，并使细胞分别去极化20±4毫伏（n = 8）和14±3毫伏（n = 6）。
本方法允许从单个提睾肌小动脉中分离出平滑肌细胞。这些细胞似乎保留了供体小动脉所描述的收缩表型、α-肾上腺素能信号级联、膜电位和钾电导。将这些平滑肌细胞的功能和电生理特性与其供体小动脉的原位和体外研究相关联，应该为理解骨骼肌微循环的生理学、病理生理学、生物物理学和细胞生物学提供一个有用的扩展。
