Bourdeau J E, Lau K
Department of Medicine, Michael Reese Hospital and Medical Center, Chicago, Illinois 60616.
J Clin Invest. 1989 Feb;83(2):373-9. doi: 10.1172/JCI113894.
PTH stimulates active Ca reabsorption in isolated perfused rabbit kidney connecting tubules (CNTs). The existence of PTH-sensitive adenylate cyclase and the reproduction of increased epithelial Ca transport by dibutyryl-cAMP suggest that cAMP is the mediator. Accordingly, we studied the effects of PTH and 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP) on cytosolic free calcium concentration [( Ca2+]i) in individual rabbit CNTs. [Ca2+]i was estimated by continuous epifluorescence microscopy of single fura-2-loaded tubules during dual wave-length excitation. In nonperfused controls at 37 degrees C, [Ca2+]i decreased with time. In contrast to vehicle controls, synthetic bovine (1-34) PTH (0.1 nM) increased [Ca2+]i within 4 min, produced a maximal effect in 7.2 min, and sustained its effect for at least 2 min after washout. 8-Br-cAMP (1 mM) mimicked the effect of PTH, but with an earlier onset of action. To test the hypothesis that lumen Ca is the predominant source of the rise in [Ca2+]i, we studied singly perfused CNTs. In the absence of bath and lumen Ca, PTH elicited no rise in [Ca2+]i, implying that intracellular Ca stores are not the major source. In contrast, there was a rise when Ca was replenished in both media. In the continuous presence of bath Ca, lumen Ca was estimated to contribute 65% of the total rise in [Ca2+]i in response to PTH when it was first deleted and then replenished. However, when the sequence of lumen Ca manipulation was reversed, the contributions by lumen and bath Ca were found to be essentially equal. We conclude (a) at a physiologic concentration, PTH increases [Ca2+]i in rabbit CNTs, (b) 8-Br-cAMP mimics this action, implicating cAMP as a second messenger, and (c) the PTH-stimulated rise in [Ca2+]i depends importantly on both bath and tubular luminal fluid Ca.
甲状旁腺激素（PTH）可刺激离体灌注兔肾连接小管（CNT）对钙的主动重吸收。PTH敏感的腺苷酸环化酶的存在以及二丁酰环磷腺苷（dibutyryl-cAMP）可重现上皮细胞钙转运增加的现象表明，环磷腺苷（cAMP）是介导物。因此，我们研究了PTH和8-溴腺苷3',5'-环磷腺苷（8-Br-cAMP）对单个兔CNT细胞溶质游离钙浓度[Ca2+]i的影响。通过在双波长激发期间对单个负载fura-2的小管进行连续落射荧光显微镜观察来估计[Ca2+]i。在37℃的非灌注对照中，[Ca2+]i随时间下降。与载体对照相比，合成牛（1-34）PTH（0.1 nM）在4分钟内使[Ca2+]i升高，在7.2分钟时产生最大效应，并在冲洗后至少持续2分钟。8-Br-cAMP（1 mM）模拟了PTH的作用，但起效更早。为了检验管腔钙是[Ca2+]i升高的主要来源这一假设，我们研究了单根灌注的CNT。在浴液和管腔中均无钙的情况下，PTH不会引起[Ca2+]i升高，这意味着细胞内钙储存不是主要来源。相反，当两种介质中都补充钙时，[Ca2+]i会升高。在持续存在浴液钙的情况下，当首先去除然后补充管腔钙时，估计管腔钙对PTH诱导的[Ca2+]i总升高的贡献为65%。然而，当管腔钙操作顺序颠倒时，发现管腔钙和浴液钙的贡献基本相等。我们得出结论：（a）在生理浓度下，PTH可升高兔CNT中的[Ca2+]i；（b）8-Br-cAMP模拟了这一作用，表明cAMP是第二信使；（c）PTH刺激引起的[Ca2+]i升高主要依赖于浴液钙和肾小管腔液钙。
