Galizia L, Flamenco MP, Rivarola V, Capurro C, Ford P. Role of AQP2 in activation of calcium entry by hypotonicity: implications in cell volume regulation. Am J Physiol Renal Physiol 294: F582-F590, 2008. First published December 19, 2007 doi:10.1152/ajprenal.00427.2007.-We previously reported in a rat cortical collecting duct cell line (RCCD1) that the presence of aquaporin 2 (AQP2) in the cell membrane is critical for the rapid activation of regulatory volume decrease mechanisms (RVD) (Ford et al. Biol Cell 97: 687-697, 2005). The aim of our present work was to investigate the signaling pathway that links AQP2 to this rapid RVD activation. Since it has been previously described that hypotonic conditions induce intracellular calcium ([Ca 2ϩ ]i) increases in different cell types, we tested the hypothesis that AQP2 could have a role in activation of calcium entry by hypotonicity and its implication in cell volume regulation. Using a fluorescent probe technique, we studied [Ca 2ϩ ]i and cell volume changes in response to a hypotonic shock in WT-RCCD1 (not expressing aquaporins) and in AQP2-RCCD1 (transfected with AQP2) cells. We found that after a hypotonic shock only AQP2-RCCD1 cells exhibit a substantial increase in [Ca 2ϩ ]i. This [Ca 2ϩ ]i increase is strongly dependent on extracellular Ca 2ϩ and is partially inhibited by thapsigargin (1 M) indicating that the rise in [Ca 2ϩ ]i reflects both influx from the extracellular medium and release from intracellular stores. Exposure of AQP2-RCCD1 cells to 100 M gadolinium reduced the increase in [Ca 2ϩ ]i suggesting the involvement of a mechanosensitive calcium channel. Furthermore, exposure of cells to all of the above described conditions impaired rapid RVD. We conclude that the expression of AQP2 in the cell membrane is critical to produce the increase in [Ca 2ϩ ]i which is necessary to activate RVD in RCCD1 cells. aquaporin 2; intracellular calcium; renal cells THE KIDNEY COLLECTING DUCT plays an important role in the process of urine concentration through a mechanism regulated by arginine vasopressin. This hormone induces an increase in osmotic water permeability (P f ) by triggering translocation and fusion of intracellular vesicles containing aquaporin 2 (AQP2) to the apical membrane of principal cells (32,33,48). In this condition, two-thirds of the hyposmotic luminal fluid entering the cortical collecting duct (CCD) is reabsorbed. Therefore, CCD cells are faced with both, changes in apical osmolarity and important volume flows which could result in cell volume increases. For this reason, potent volume regulatory mechanisms are needed to maintain cellular homeostasis and epithelial transport (29).Most cells respond to decrease in tonicity first by swelling and second by initiating mechanisms that allow them to recover their original volume (21,35). This complex mechanism, called regulatory volume decrease (RVD), depends on the activation of different ion permeabilities (usually K ϩ , Cl Ϫ , and/or HCO 3 Ϫ ) that reverse the osmotic gradient an...

Arginine-vasopressin (AVP) has been proposed to be involved in the modulation of acid-base transporters; however, the nature of the mechanisms underlying AVP direct action on intracellular pH (pH_i) in the cortical collecting duct (CCD) is not yet clearly defined. The aim of the present study was to elucidate which are the proteins implicated in AVP modulation of pH_i, as well as the receptors involved in these responses using a CCD cell line (RCCD₁); pH_i was monitored with the fluorescent dye BCECF in basal conditions and after stimulation with basolateral 10^-8 M AVP. Specific V1- or V2-receptor antagonists were also used. RT-PCR studies demonstrated that RCCD₁ cells express V1a and V2 receptors. Functional studies showed that while V2-receptor activation induced a biphasic response (alkalinization-acidification), V1-receptor activation resulted in an intracellular acidification. The V2-mediated alkalinization phase involves the activation of basolateral NHE-1 isoform of the Na⁺/H⁺ exchanger while in the acidification phase CFTR is probably implicated. On the other hand, V1-mediated acidification was due to activation of a Cl^-/HCO₃^- exchanger. We conclude that in RCCD₁ cells AVP selectively activates, via a complex of V1 and V2 receptor-mediated actions, different ion transporters linked to pH_i regulation which might have physiological implications.

No abstract

No abstract