Garcia-Perez A, Smith W L
J Clin Invest. 1984 Jul;74(1):63-74. doi: 10.1172/JCI111419.
The studies reported here were designed to determine if there is an apical-basolateral asymmetry to the release of prostaglandins by or to the biochemical effects of prostaglandins on the renal collecting tubule. Canine cortical collecting tubule (CCCT) cells were isolated by immunodissection and seeded at supraconfluent densities on Millipore filters. The resulting confluent monolayer of CCCT cells: (a) developed and maintained a transcellular potential difference of 1 mV (apical side negative); (b) exhibited a permeability to inulin that was the same as that obtained with similar monolayers of Madin-Darby canine kidney (MDCK) cells; and (c) released adenosine 3',5'-cyclicmonophosphate (cAMP) in response to arginine vasopressin (AVP) added to the basolateral but not the apical surface of the monolayer. These results indicate that confluent monolayers of CCCT cells on Millipore filters have characteristics of asymmetry that are seen with intact collecting tubules. Moreover, PGE2 added to either side of the CCCT cell monolayer crossed the monolayer at the same slow rate as inulin, which demonstrated the feasibility of examining the sidedness of the effects of and the release of PGE2. Although AVP caused cAMP release only when added to the basolateral side of CCCT cells, AVP caused the release of PGE2 when added to either the apical or basolateral surface. This result implies that there are at least two AVP receptor systems, one coupled to cAMP synthesis and one to PGE2 formation. In contrast to the results observed with AVP, bradykinin caused PGE2 release only when added to the apical surface of CCCT cells, which suggested that urinary but not blood borne kinins elicit PGE2 formation by the canine collecting tubule. PGE2 was released in comparable amounts on each side of the monolayer in response both to AVP and to bradykinin. High concentrations (greater than or equal to 10(-8) M) of PGE2 added to either side of the monolayer caused the release of cAMP. However, at concentrations (10(-10) - 10(-12) M) at which PGE2 had no independent effect on cAMP release, PGE2 inhibited the release of cAMP, which normally occurred in response to AVP. This inhibition occurred with PGE2 added to either the apical or basolateral surface of the CCCT cell monolayer. PGE2 (10(-11) M) also inhibited the AVP-induced accumulation of intracellular cAMP by CCCT cells seeded on culture dishes. This inhibition was only observed when the cells were preincubated with PGE2 for greater than or equal to 20 min. Our results are consistent with the concept that inhibiton by prostaglandins of the hydroosmotic effect of AVP is due to inhibition of AVP-induced cAMP production. This inhibition does not appear to involve a direct physical interaction of PGE2 with the AVP receptor which is coupled to adenylate cyclase, since CCCT cells must be preincubated with PGE2 for 20 min for the inhibition to be observed, and since PGE2 added to the apical surface of CCCT cells inhibits cAMP release in response to AVP acting from the basolateral surface.
本文报道的研究旨在确定前列腺素的释放是否存在顶端 - 基底外侧不对称性，或者前列腺素对肾集合管的生化效应是否存在这种不对称性。通过免疫解剖分离犬肾皮质集合管（CCCT）细胞，并以超汇合密度接种于密理博滤膜上。由此形成的CCCT细胞汇合单层：（a）产生并维持1 mV的跨细胞电位差（顶端侧为负）；（b）对菊粉的通透性与用类似的麦迪逊 - 达比犬肾（MDCK）细胞单层所获得的通透性相同；（c）响应添加到单层基底外侧而非顶端表面的精氨酸加压素（AVP）而释放腺苷3',5'-环磷酸（cAMP）。这些结果表明，密理博滤膜上的CCCT细胞汇合单层具有与完整集合管相同的不对称特征。此外，添加到CCCT细胞单层任一侧的前列腺素E2（PGE2）穿过单层的速率与菊粉相同，这证明了研究PGE2效应的方向性和释放的可行性。尽管AVP仅在添加到CCCT细胞基底外侧时才导致cAMP释放，但AVP添加到顶端或基底外侧表面时均会导致PGE2释放。该结果表明至少存在两种AVP受体系统，一种与cAMP合成偶联，另一种与PGE2形成偶联。与AVP的结果相反，缓激肽仅在添加到CCCT细胞顶端表面时才导致PGE2释放，这表明尿中而非血源性激肽可引发犬集合管产生PGE2。响应AVP和缓激肽，单层两侧释放的PGE2量相当。添加到单层任一侧的高浓度（大于或等于10(-8) M）PGE2导致cAMP释放。然而，在PGE2对cAMP释放无独立作用的浓度（10(-10) - 10(-12) M）下，PGE2抑制了通常由AVP引起的cAMP释放。这种抑制在PGE2添加到CCCT细胞单层的顶端或基底外侧表面时均会发生。PGE2（10(-11) M）也抑制接种于培养皿上的CCCT细胞中AVP诱导的细胞内cAMP积累。仅当细胞与PGE2预孵育大于或等于20分钟时才观察到这种抑制作用。我们的结果与以下概念一致，即前列腺素对AVP水渗透效应的抑制是由于抑制了AVP诱导的cAMP产生。这种抑制似乎不涉及PGE2与偶联腺苷酸环化酶的AVP受体的直接物理相互作用，因为CCCT细胞必须与PGE2预孵育20分钟才能观察到抑制作用，并且因为添加到CCCT细胞顶端表面的PGE2抑制了从基底外侧表面作用的AVP引起的cAMP释放。
