Abstract
Human mammary epithelial (HME) cells express several P2Y receptor subtypes located in both apical and basolateral membranes. Apical UTP (10 μM) or ATP-γ-S (10 μM) stimulation of monolayers mounted in Ussing chambers evoked a rapid, but transient decrease in short circuit current (Isc), consistent with activation of an apical K conductance. In contrast, basolateral stimulation with UTP (10 μM) produced a longer duration increase in Isc that was blocked by 5 μM benzamil. Inhibition of Ca2+ mobilization using BAPTA-AM abolished these changes in Isc. Moreover, apical pretreatment with charybdotoxin (200 nM), blocked the effect on Isc by >80% and a similar degree of inhibition was observed with clotrimazole (10 μM) and TRAM-34 (0.5 μM). Silencing expression of KCa3.1 by transfection of HME cells with an shRNA containing lentivirus construct targeting the channel produced ~80% inhibition of mRNA expression and 71% inhibition of the apical Isc response. In addition, silencing the P2Y2 receptor by expression of an shRNA containing lentivirus construct reduced the level of P2Y2 mRNA by 75% and inhibited the apical effects of 10 μM ATP-γ-S by 65%. These results suggest that P2Y2 receptors mediate the effects of purinoceptor agonists on K secretion by regulating the activity of KCa3.1 channels expressed in the apical membrane of HME cells.
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