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

摘要

class="enumerated" style="list-style-type:decimal">Using equivalent short circuit current (ISC) 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 ISC. This was followed by a fall below basal ISC due to a reduction in the amiloride-sensitive ISC component.The ATP-induced ISC 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).The stimulatory effect of apical ATP on ISC was concentration dependent with an EC50 of about 0.6 μM. Basolateral ATP elicited a similar ISC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors.ATP and UTP applied to either the apical or the basolateral bath equi-potently stimulated ISC while ‘purified’ ADP and UDP had no effect consistent with P2Y2 purinoceptors, the expression of which was confirmed using RT-PCR.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 ISC response. The peak ISC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre-incubated with the calcium chelator BAPTA AM.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.