P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia

It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were -4.4 +/- 1.3 mV (mean +/- SD, n = 12) and 517.7 +/- 39.4 Omega.cm(2), respectively. The apical membrane potential was -43.9 +/- 1.7 mV, and the ratio of apical to basolateral membrane resistance (R-A/R-B) was 3.5 +/- 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC50 of similar to24 muM (apical) and similar to30 muM (basal). Apical ATP/UTP (100 muM) depolarized apical membrane potential by 17.6 +/- 0.8 mV (n = 7) and decreased R-A/R-B by a factor of approximate to3. The addition of adenosine to either side (100 muM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca2+ concentration (427 +/- 206 nM; 15-25 muMATP, apical; n = 6). This Ca2+ increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 +/- 3 mul.cm(-2).h(-1) (n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y(2) purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca2+ dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.