Apical Methyl-β-cyclodextrin (mβCd) Treatment In A6 Renal Cells Does Not Affect Basolateral PGE2-induced Cl- Secretion , But Stimulates An Early Cl- Peak Current, Activated By Hypotonic Shock

Abstract

We explore in this study whether cholesterol content of the apical membrane of distal kidney cells from Xenopus laevis (A6) affects the behavior of two types of anion channels, recently described by Bao et al. (AJP,2008,295,G234). Voltage clamp technique in an Ussing-type chamber, enabling the recording of short-circuit current, was applied on polarized monolayers of A6 cells, grown on permeable filter supports for 13 - 31 days. To create a gradient for Cl- secretion, the apical solution was Cl- and Na+ free, whereas the basolateral solution contained normal NaCl. Twice basolateral addition of PGE2 (1 μM) during 15 minutes with an interval of 60 minutes produced a first transient peak increase in Isc of 7.25 ± 0.98 μAcm-2 (N=6), followed by a plateau of 2.77 ± 0.50 μAcm-2. The second PGE2 stimulation elicited a peak current of 6.63 ± 1.20 μAcm-2. When the apical membrane was treated for at least 1h with 10 mM mβCD, PGE2 evoked Cl- secretion was not different from control. A hypotonic shock was induced twice with an interval of 60 minutes on the basolateral side by a sudden reduction of solution osmolality from 260 to 140 mOsm/kg H2O. In control a transient increase in Isc of 0.27 ±0.11 μAcm-2 (N=3) was observed during the second stimulation only. After 60 minutes treatment with 10 mM mβCD the hypotonic shock evoked an augmented transient Isc of 1.82 ± 0.08 μAcm-2 (N=5). These results suggest that apical Cl- channels in A6 cells, activated either by PGE2 or by cell swelling, are regulated differently by the membrane cholesterol content. This work was supported by the grants FWO Flanders G.0270.07 and BOF 03B0BF.