Role of TRPC3 channels in ATP-induced Ca²⁺signaling in principal cells of the inner medullary collecting duct

Abstract

flux across IMCD-3 monolayers is increased by overexpression of TRPC3 and attenuated by a dominant negative TRPC3 construct, suggesting that Ca 2ϩ entry across the apical membrane occurs via TRPC3 channels. To test this hypothesis, we selectively measured the Ca 2ϩ permeability of the apical membrane of fura-2-loaded IMCD-3 cells using the Mn 2ϩ quench technique. Mn 2ϩ influx across the apical membrane was increased 12-to 16-fold by apical ATP and was blocked by the pyrazole derivative BTP2, a known inhibitor of TRPC3 channels, with an IC50 value Ͻ100 nM. In contrast, Mn 2ϩ influx was only increased ϳ2-fold by basolateral ATP. Mn 2ϩ influx was also activated by apical, but not basolateral, 1-stearoyl-2-acetyl-snglycerol (SAG), a known activator of TRPC3 channels. Apical ATPand SAG-induced Mn 2ϩ influx was increased by overexpression of TRPC3 and completely blocked by expression of the dominant negative TRPC3 construct. Mn 2ϩ influx was also stimulated ϳ2-fold by thapsigargin applied to either the apical or basolateral side. Thapsigargin-induced flux was blocked by BTP2 but was unaffected by overexpression of TRPC3 or by dominant negative TRPC3. Apical ATP, but not basolateral ATP, increased transepithelial 45 Ca 2ϩ flux. These results demonstrate that the apical membrane of IMCD-3 cells has two distinct Ca 2ϩ influx pathways: 1) a store-operated channel activated by thapsigargin and basolateral ATP and 2) TRPC3 channels activated by apical ATP. Only activation of TRPC3 leads to net transepithelial apical-to-basolateral Ca 2ϩ flux. Furthermore, these results demonstrate that native TRPC3 is not a store-operated channel in IMCD-3 cells. calcium channels; transient receptor potential channel; fluorescence microscopy; fura 2; manganese quench; inner medullary collecting duct cell line IMCD-3IT IS NOW WELL ESTABLISHED that purinergic receptors of the P2X and P2Y subtype are present in the surface membrane of most cells, including epithelial cells of the renal nephron (1, 32). Purinergic receptors are not only present in the basolateral membrane but also are expressed in the apical membrane of renal epithelial cells, and it is now clear that autocrine purinergic signaling occurs within the tubular lumen throughout the entire nephron (12). Although not fully understood, mechanisms exist for the regulated release of ATP from epithelial cells lining the tubule in response to various agonists or in response to mechanical stimulations such as the shear stress associated with the flow of tubular fluid or changes in membrane stretch associated with hyper-and hypotonicity (12, 28). The enzymes responsible for degradation of ATP, i.e., ectonucleotidases, are also differentially expressed in the apical membrane along the nephron (29). Thus, although the physiological roles of purinergic signaling in the tubular lumen are only slowly coming to light, mechanisms for both the activation and inactivation of this signaling pathway clearly exist within the nephron. There is also increasing evidence that purinergic signaling plays an ...

Flow regulation of endothelin-1 production in the inner medullary collecting duct

“…As shown in Fig. 10, mRNAs for all known P2Y receptors were detected in IMCD3 cells with the exception of P2Y 12 and P2Y 14 . Bands were seen on Western blots for P2Y isoforms (Fig.…”

Evidence of a Role for Fibroblast Transient Receptor Potential Canonical 3 Ca2+ Channel in Renal Fibrosis

“…Recently, the presence of TRPC channels has been depicted in a normal rat kidney fibroblasts cell line and found to control the process of receptor-operated Ca 2+ influx 46,47 ; additionally, TRPC3 was present in the rat kidney and played a key role in the influx of Ca 2+ into principal cells of the collecting duct. 48,[52][53][54] However, the role of such channels in freshly isolated adult renal fibroblasts under native physiologic conditions remains to be elucidated.…”

“…44,45 Few other studies reported the presence of TRPC channels in normal rat kidney cell lines 46,47 and identified their localization in the adult rat and human kidney. [48][49][50][51] TRPC3 is also responsible for transepithelial Ca 2+ flux in principal cells of the renal collecting duct [52][53][54] ; however, a clear functional in vivo role of such channels remains totally unclear, especially in the diseased kidney. Therefore, it is important to study the role of TRPC3 in renal fibroblasts and its role in the pathogenesis of renal fibrosis associated with kidney disease.…”

Effect of proteinS-glutathionylation on Ca2+homeostasis in cultured aortic endothelial cells

“…Time-dependent changes in [Ca 2ϩ ] i were measured in BAEC monolayers as previously described (21). Briefly, BAEC monolayers grown on glass coverslips were loaded with fura-2, mounted in a temperature-controlled perfusion chamber, and placed on the stage of a Leica DMIRE2 inverted microscope.…”