Role of cAMP/PKA signaling cascade in vasopressin-induced trafficking of TRPC3 channels in principal cells of the collecting duct
Role of cAMP/PKA signaling cascade in vasopressin-induced trafficking of TRPC3 channels in principal cells of the collecting duct
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DOI：
10.1152/ajprenal.00586.2009
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发表时间：
2010-04-01
影响因子：
4.2
通讯作者：
Schilling, William P.
中科院分区：
文献类型：
--
作者：
Goel, Monu;Zuo, Cheng-Di;Schilling, William P.
Goel M，Zuo CD，Schilling WP. cAMP/PKA信号级联在加压素诱导的集合管主细胞TRPC 3通道运输中的作用美国肾脏生理学杂志298：F988-F996，2010年。首次发表于2010年1月27日; doi：10.1152/ajprenal.00586.2009。瞬时受体电位通道TRPC 3和TRPC 6在体内和培养的小鼠CD细胞系IMCD-3中与水通道水通道蛋白2（AQP 2）一起沿着表达于集合管（CD）的主细胞中。这些通道主要定位于细胞内囊泡，但在抗利尿激素精氨酸加压素（AVP）刺激下，TRPC 3和AQP 2移位至顶膜。在本研究中，腺苷酸环化酶（AC）/cAMP/PKA信号级联的各种激活剂和抑制剂对通道贩运的影响进行了研究，使用免疫组织化学技术和表面膜蛋白的生物素化。在大鼠肾脏和IMCD-3细胞中，特异性V2受体激动剂[脱氨基-Cys（1），D-Arg（8）]-加压素（dDAVP）可刺激AQP 2和TRPC 3（而非TRPC 6）的转位，特异性V2受体拮抗剂[金刚烷乙酰（1），O-Et-D-Tyr（2），瓦尔（4），氨基丁酰（6），Arg（8，9）]-加压素（AEAVP）可阻断AQP 2和TRPC 3的转位。在IMCD-3细胞中，TRPC 3和AQP 2的易位被AC的直接激活剂forskolin或膜渗透性cAMP类似物dibutyryl-cAMP激活。AC和PKA的特异性抑制剂SQ 22536和H89分别阻断AVP、dDAVP和forskolin诱导的IMCD-3细胞易位。二丁酰cAMP刺激的易位不受AEAVP的影响，但可被H89阻断。AVP和毛喉素诱导的TRPC 3在IMCD-3细胞中的易位也被另外两种PKA抑制剂阻断，特别是Rp-cAMPS和PKA的肉豆蔻酰化抑制剂（m-PKI）。使用表面膜生物素化测定在每个测定条件下对顶CD-3细胞中TRPC 3膜插入的定量证实了通过免疫荧光观察到的易位结果。重要的是，AVP诱导的TRPC 3易位（通过生物素化估计）被H89、Rp-cAMPS或m-PKI平均阻断95.2 +/-1.0%。综上所述，这些结果表明，AVP刺激CD主细胞中的V2受体导致TRPC 3通过刺激AC/cAMP/PKA信号级联转移到顶膜。
Goel M, Zuo CD, Schilling WP. Role of cAMP/PKA signaling cascade in vasopressin-induced trafficking of TRPC3 channels in principal cells of the collecting duct. Am J Physiol Renal Physiol 298: F988-F996, 2010. First published January 27, 2010; doi:10.1152/ajprenal.00586.2009.-Transient receptor potential channels TRPC3 and TRPC6 are expressed in principal cells of the collecting duct (CD) along with the water channel aquaporin-2 (AQP2) both in vivo and in the cultured mouse CD cell line IMCD-3. The channels are primarily localized to intracellular vesicles, but upon stimulation with the antidiuretic hormone arginine vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined using immunohistochemical techniques and by biotinylation of surface membrane proteins. Both in vivo in rat kidney and in IMCD-3 cells, translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino-Cys(1), D-Arg(8)]-vasopressin (dDAVP), a specific V2-receptor agonist, and blocked by [adamantaneacetyl(1), O-Et-D-Tyr(2), Val(4), aminobutyryl(6), Arg(8,9)]-vasopressin (AEAVP), a specific V2-receptor antagonist. In IMCD-3 cells, translocation of TRPC3 and AQP2 was activated by forskolin, a direct activator of AC, or by dibutyryl-cAMP, a membrane-permeable cAMP analog. AVP-, dDAVP-, and forskolin-induced translocation in IMCD-3 cells was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by dibutyryl-cAMP was unaffected by AEAVP but could be blocked by H89. AVP- and forskolin-induced translocation of TRPC3 in IMCD-3 cells was also blocked by two additional inhibitors of PKA, specifically Rp-cAMPS and the myristoylated inhibitor of PKA (m-PKI). Quantification of TRPC3 membrane insertion in IMCD-3 cells under each assay condition using a surface membrane biotinylation assay, confirmed the translocation results observed by immunofluorescence. Importantly, AVP-induced translocation of TRPC3 as estimated by biotinylation was blocked on average 95.2 +/- 1.0% by H89, Rp-cAMPS, or m-PKI. Taken together, these results demonstrate that AVP stimulation of V2 receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade.