Raychowdhury Malay K, Ramos Arnolt J, Zhang Peng, McLaughin Margaret, Dai Xiao-Qing, Chen Xing-Zhen, Montalbetti Nicolás, Del Rocío Cantero María, Ausiello Dennis A, Cantiello Horacio F
Nephrology Div. and Electrophysiology Core, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129, USA.
Am J Physiol Renal Physiol. 2009 Jan;296(1):F87-97. doi: 10.1152/ajprenal.90509.2008. Epub 2008 Oct 22.
The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK(1) renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R-gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK(1) cells stained with anti-V2R antibodies and in LLC-PK(1) cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 microM AVP (trans) or forskolin (cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.
肾上皮细胞的初级纤毛是一种非运动性的感觉细胞器，参与机械感觉转导信号。我们实验室最近的研究表明，肾上皮初级纤毛表现出丰富的通道活性；然而，该细胞器中特定膜受体的存在及其功能作用迄今尚不清楚。在这里，我们确定了肾上皮细胞初级纤毛中与2型血管加压素受体（V2R）相关的功能性信号通路。除了正常定位于基底外侧膜外，V2R还在LLC-PK(1)肾上皮细胞的初级纤毛中表达。通过V2R-gfp嵌合体的自发荧光确定初级纤毛中V2R的存在，并通过用抗V2R抗体染色的野生型LLC-PK(1)细胞以及过表达V2R-Flag的LLC-PK(1)细胞用抗Flag抗体进行免疫细胞化学分析来证实。在所有测试的细胞类型中，纤毛V2R与V/VI型腺苷酸环化酶（AC）共定位。通过测量分离纤毛中的cAMP产生以及通过在重构脂质双层中或进行膜片钳记录测试AVP诱导的阳离子选择性通道活性，证实了受体与AC的功能偶联。在存在但不是不存在ATP（1 mM，顺式）的情况下添加10 microM AVP（反式）或福斯可林（顺式）刺激了纤毛膜中的阳离子选择性通道活性。添加PKA抑制剂PKI可降低这种通道活性。这些数据首次证明了V2R在肾上皮细胞初级纤毛中的存在，以及一条功能性cAMP信号通路，该通路靶向纤毛通道功能并可能有助于控制初级纤毛的感觉功能。
