Iversen B M, Arendshorst W J
Department of Physiology, University of North Carolina at Chapel Hill 27599-7545, USA.
Am J Physiol. 1998 Mar;274(3):F498-508. doi: 10.1152/ajprenal.1998.274.3.F498.
Calcium signaling mechanisms were examined in vessel segments and dispersed single smooth muscle cells (SMC) of interlobular arteries and afferent arterioles (< 50 microns diameter) from the rat kidney. These resistance vessels were isolated from rat kidneys, using an iron oxide-sieving technique with subsequent collagenase digestion. Individual cells were identified by their characteristic oval appearance and positive staining for smooth muscle-specific alpha-actin and heavy chain myosin SM-1 and SM-2. Cytosolic calcium concentration ([Ca2+]i) was measured using fura 2 ratiometric fluorescence at 340 and 380 nm wavelength with a microscope-based photometer. Angiotensin II (ANG II) and arginine vasopressin (AVP), at concentrations of 10(-10)-10(-6) M, produced dose-dependent increases in [Ca2+]i; maximum increases were 221 +/- 49 nM for ANG II and 237 +/- 49 nM for AVP. The temporal response patterns for both agonists were characterized by a square-shaped, immediate step increase in [Ca2+]i to a near maximum level that was maintained through the recording period of 150-200 s. Responses of individual dispersed SMC and short vessel segments were similar. Losartan antagonized the action of ANG II, indicating mediation by AT1 receptors on preglomerular arteriolar SMC. The V1-selective antagonist [d(CH2)5Tyr(Me)2Tyr(NH2)9]AVP completely inhibited AVP-induced [Ca2+]i changes. The importance of calcium entry in hormone-induced changes in [Ca2+]i was demonstrated by the finding that neither ANG II nor AVP elicited a [Ca2+]i response in media rendered nominally calcium free by addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Calcium entry occurred primarily through L-type, voltage-gated calcium channels as the dihydropyridine, nifedipine, completely prevented or reversed [Ca2+]i changes normally elicited by either hormone. Our results provide new information about the similarity of calcium signaling in single SMC and short segments freshly isolated from renal interlobular arteries and afferent arterioles. The observations indicate that AT1 and V1 receptors are coupled to signal transduction pathways leading to rapid changes in [Ca2+]i. Calcium mobilization appears to play a minor to nonexistent role under the experimental conditions. The predominant mechanism involves calcium entry through dihydropyridine-sensitive, voltage-gated calcium channels in single SMC from these resistance vessels.
研究了大鼠肾脏小叶间动脉和传入小动脉（直径<50微米）血管段及分散的单个平滑肌细胞（SMC）中的钙信号传导机制。使用氧化铁筛分技术并随后进行胶原酶消化，从大鼠肾脏中分离出这些阻力血管。通过其特征性的椭圆形外观以及平滑肌特异性α-肌动蛋白、肌球蛋白重链SM-1和SM-2的阳性染色来鉴定单个细胞。使用基于显微镜的光度计，在340和380nm波长下，采用fura 2比率荧光法测量细胞质钙浓度（[Ca2+]i）。浓度为10（-10）-10（-6）M的血管紧张素II（ANG II）和精氨酸加压素（AVP）使[Ca2+]i呈剂量依赖性增加；ANG II的最大增加量为221±49 nM，AVP为237±49 nM。两种激动剂的时间反应模式的特征是[Ca2+]i呈方形、立即阶跃增加至接近最大水平，并在150-200秒的记录期内保持。单个分散的SMC和短血管段的反应相似。氯沙坦拮抗ANG II的作用，表明其通过肾小球前小动脉SMC上的AT1受体介导。V1选择性拮抗剂[d（CH2）5Tyr（Me）2Tyr（NH2）9]AVP完全抑制AVP诱导的[Ca2+]i变化。通过添加乙二醇双（β-氨基乙基醚）-N，N，N'，N'-四乙酸使培养基名义上无钙，发现ANG II和AVP在其中均未引起[Ca2+]i反应，这证明了钙内流在激素诱导的[Ca2+]i变化中的重要性。钙内流主要通过L型电压门控钙通道发生，因为二氢吡啶硝苯地平完全阻止或逆转了通常由任何一种激素引起的[Ca2+]i变化。我们的结果提供了有关从肾小叶间动脉和传入小动脉新鲜分离的单个SMC和短节段中钙信号传导相似性的新信息。观察结果表明，AT1和V1受体与导致[Ca2+]i快速变化的信号转导途径偶联。在实验条件下，钙动员似乎起很小或不起作用。主要机制涉及通过这些阻力血管单个SMC中对二氢吡啶敏感的电压门控钙通道的钙内流。
