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ATP stimulates Cl^- secretion and reduces amiloride-sensitive Na⁺ absorption in M-1 mouse cortical collecting duct cells

John E. Cuffe*, Andreas Bielfeld-Ackermann*, Jörg Thomas¹, Jens Leipziger¹ and Christoph Korbmacher*

1. Using equivalent short circuit current (I_SC) measurements we examined the effect of extracellular ATP on transepithelial ion transport in M-1 mouse cortical collecting duct cells. Apical addition of ATP produced a rapid transient peak increase in I_SC. This was followed by a fall below basal I_SC due to a reduction in the amiloride-sensitive I_SC component.

2. The ATP-induced I_SC increase was preserved in the presence of apical amiloride while it was reduced in the absence of extracellular Cl^- and in the presence of the apical Cl^- channel blockers diphenylamine-2-carboxylic acid (DPC, 1 mM), DIDS (300 µM) and niflumic acid (100 µM).

3. The stimulatory effect of apical ATP on I_SC was concentration dependent with an EC₅₀ of about 0·6 µM. Basolateral ATP elicited a similar I_SC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors.

4. ATP and UTP applied to either the apical or the basolateral bath equi-potently stimulated I_SC while 'purified' ADP and UDP had no effect consistent with P2Y₂ purinoceptors, the expression of which was confirmed using RT-PCR.

5. Intracellular calcium concentration ([Ca²⁺]_i) measurements using fura-2 demonstrated that ATP and UTP elicited a rise in [Ca²⁺]_i with EC₅₀ values of 1·1 and 0·6 µM, respectively. The shape and time course of the calcium response were similar to those of the I_SC response. The peak I_SC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre-incubated with the calcium chelator BAPTA AM.

6. We conclude that in M-1 cells extracellular ATP reduces amiloride-sensitive Na⁺ absorption and stimulates Cl^- secretion via calcium-activated Cl^- channels through activation of P2Y₂ purinoreceptors located in the apical and basolateral membrane.

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