Endogenous ATP release inhibits electrogenic Na+ absorption and stimulates Cl− secretion in MDCK cells

Purinergic Signalling

Yi Xie²  James A. Schafer¹ 

[1] University of Alabama at Birmingham$$University of Alabama at Birmingham$$;University of Alabama at Birmingham$$UCLA School of Medicine$$

关键词: Purinergic effects;   

DOI  :  10.1007/s11302-007-9053-3

学科分类：分子生物学，细胞生物学和基因

来源: Springer

【 摘 要 】

Our previous studies with a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) transfected with FLAG-labeled α, β, and γ subunits of epithelial Na⁺ channel (ENaC) showed that, although most of the short-circuit current (I_sc) was amiloride sensitive (AS-I_sc), there was also an amiloride-insensitive component (NS-I_sc) due to Cl⁻ secretion (Morris and Schafer, J Gen Physiol 120:71–85, 2002). In the present studies, we observed a progressive increase in NS-I_sc and a corresponding decrease in AS-I_sc during experiments. There was a significant negative correlation between AS-I_sc and NS-I_sc both in the presence and absence of treatment with cyclic adenosine monophosphate (cAMP). NS-I_sc could be attributed to both cystic fibrosis transmembrane conductance regulator (CFTR) and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive Ca²⁺-activated Cl⁻ channel (CaCC). Continuous perfusion of both sides of the Ussing chamber with fresh rather than recirculated bathing solutions, or addition of hexokinase (6 U/ml), prevented the time-dependent changes and increased AS-I_sc by 40–60%, with a proportional decrease in NS-I_sc. Addition of 100 μM adenosine triphosphate (ATP) in the presence of luminal amiloride produced a transient four-fold increase in NS-I_sc that was followed by a sustained increase of 50–60% above the basal level. ATP release from the monolayers, measured by bioluminescence, was found to occur across the apical but not the basolateral membrane, and the apical release was tripled by cAMP treatment. These data show that constitutive apical ATP release, which occurs under both basal and cAMP-stimulated conditions, underlies the time-dependent rise in Cl⁻ secretion and the proportional fall in ENaC-mediated Na⁺ absorption in FL-MDCK cells. Thus, endogenous ATP release can introduce a significant confounding variable in experiments with this and similar epithelial cells, and it may underlie at least some of the observed interaction between Cl⁻ secretion and Na⁺ absorption.

【 授权许可】

Unknown