Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells

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Abstract
1 The effect of extracellular nucleotides on the transepithelial ion transport of Madin Darby canine kidney cells (MDCK) was investigated. Cells were grown up to confluency on permeable supports and the short circuit current (Isc) was measured with an Ussing chamber-like mini-perfusion system. 2 Apical ATP stimulated a biphasic Isc increase consisting of a first rapid and transient peak followed by a broader one. 3 The first peak evoked by ATP was reversibly blocked by basilen blue (BB) in a concentration-dependent fashion, with an EC50 of 7.5 μm. 4 The P2Y receptor agonist, 2-methylthioATP (2-MeSATP) caused a single transient Isc increase that was completely blocked by pretreatment with BB. On the contrary, the P2x agonist, α,β-methylene ATP (α,β-meATP) was almost completely ineffective on Isc. UTP essentially induced a monophasic response the time-course of which resembled that of the second peak stimulated by ATP. The agonist potency order was 2-MeSATP ≥ ATP >> UTP, α<β-meATP for the first peak and UTP ≥ ATP > 2-MeSATP > α,β-meATP for the second peak. 5 Monolayer incubation with the membrane permeable calcium chelator [bis-o-aminophenoxy)-ethane-N,N,N′,N′,-tetraacetic acid, tetra(acetoximethyl)-ester] (BAPTA/AM) inhibited the ATP-evoked first peak. 6 The non-hydrolyzable ATP analogue, adenosine-5′-O-(3-thio)-trisphosphate (ATP-γ-S) elicited a biphasic response similar to that of ATP. The P1 receptor agonist, 2-chloroadenosine and CGS-21680, were almost unable to induce an Isc increase. These results rule out the involvement of ATP hydrolysis and P1 receptor activation as responsible for Isc increase. 7 Inhibition of prostaglandins synthesis by indomethacin abolished the second ATP-evoked peak. 8 Chloride replacement with gluconate on both sides of the epithelium completely inhibited the second peak induced by ATP but only reduced the amplitude of the first spike. 9 The results suggest that ATP stimulates Isc increase by two mechanisms. The first one is mediated by a P2Y receptor and by intracellular calcium increase. The second induces prostaglandin synthesis probably through a P2U receptor activation.