Water and ions coupling in an absorving-secreting "aquaporin-lacking" epithelial model: the RCCD1 cells.

CAPURRO, CLAUDIA; RIVAROLA, VALERIA; KIERBEL, ARLINET; ESCOUBET, BRIGITTE; FARMAN, NICOLETTE; BLOT-CHABAUD, MARCEL; PARISI, MARIO

Buenos Aires - Argentina

Congreso; XV International Congress of Nephrology; 1999

The purposc of this work was to study water and ions coupling in a new cell-line established from the rat cortical collecting duct (RCCD1). These cells exhibit high transepithelial resistance, an impotant potential difference and associated short-circuit current (IK). Na+ absorption, and K+ and HCO3- secretion have also been described. In this study the minute by minute recordings of the transcpithelial net water fluxcs (Jw, ul.min-1.cm-2) were associated, in different experimental conditions, to the measurerneni of electrophysiological parameters. Intracellular pH and mannitol permeability were also studied. Three different aquaporins are present in the native cortical collecüng duct: AQP 2, 3 and 4. Our RNAse protection studies showed no exprcssion of these aquaporins in RCCD1 cells. Nevertheless, we repont a simultaneous net water secretion (Jw: -0.162 ± 0.016) observed in the absence of any osmotic or chemical gradient and moving even against a hydrostatic pressure gradient. This secretor Jw was inhibited by: 5 x 10"3 M apical BaCl2 (control: -0.304 ± 0.015 vs BaCI2: -0,153 ± 0.028, p < 0.01, n=7); 10-4 M apical glybenclamide (control: -0.16! ±: 0,022' vs glybenclamide: -0.059 ± 0.017, p < 0.01, n=7) and 5 x 10-3 M serosal bumetanide (control: -0.069 ± 0.009 vs bumetanide: -0.029 ± 0.01 1, p < 0.05, n=6). lt was also observed, in substitution experiments, that replacement of both Cl and HCO3- strongly affected the secretor mechanism. It was previously reported that vasopressin (AVP) stímulates cyclic AMP production and sodium reabsorption in RCCD1 cells.lt was now observed that AVP (and cyclic AMP) induced water reabsorption reducíng the spontaneous secretor flux. The time course of Isc and Jw responses to AVP were quite in parallel. On the other side, AVP did not modify the observed osmotic permeability, while inducing amiloride sensitive (10-3 M) intracellular alkalinization. Paracellular permeabity (14C-mannitol tested) was not affected. The spontaneously observed secretor jw was reduced by amiloride ( 10-5 M), while the responso to AVP was abolished. These results led us to the following conclusions: 1) Important regulated transepithelial and at least partially transcellular water movements were observed in RCCD1 cells, in the absence of expressed aquaporins. 2)!These water movements, absorptive or secretor ones, are coupled to different ionic transfers and 3) The lipid bilayer or water-ion-coupled transporters would be the molecular palhways involved in the here observed transcellular water movements.