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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

P2Y receptor regulation of sodium transport in human mammary epithelial cells

¹Department of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, Seoul, South Korea; and ²Departments of Physiology and Animal Science, University of Minnesota, St. Paul, Minnesota

Submitted 18 February 2007 ; accepted in final form 9 August 2007

Primary human mammary epithelial (HME) cells were immortalized by stable, constitutive expression of the catalytic subunit of human telomerase. Purinergic receptors were identified by RT-PCR and quantitative RT-PCR from mRNA isolated from primary and immortalized cells grown to confluence on membrane filters. Several subtypes of P2Y receptor mRNA were identified including P2Y₁, P2Y₂, P2Y₄, and P2Y₆ receptors. RT-PCR experiments also revealed expression of A_2b adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current (I_sc) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na⁺ channel (ENaC) -, β-, and -subunits increased approximately threefold compared with that in cells grown without hydrocortisone. In addition, basal benzamil-sensitive Na⁺ transport was nearly twofold greater in hydrocortisone-treated monolayers. Stimulation with UTP, UDP, or adenosine 5'-O-(3-thiotriphosphate) (ATPS) produced increases in intracellular calcium concentration that were significantly reduced following pretreatment with the calcium-chelating agent BAPTA-AM. Concentration-response relationships indicated that the rank order of potency for these agonists was UTP > UDP > ATPS. Basolateral stimulation with UTP produced a rapid but transient increase in I_sc that was significantly reduced if cells were pretreated with BAPTA-AM or benzamil. Moreover, basolateral treatment with either charybdotoxin or clotrimazole significantly inhibited the initial UTP-dependent increase in I_sc and eliminated the sustained current response. These results indicate that human mammary epithelial cells express multiple P2 receptor subtypes and that Ca²⁺ mobilization evoked by P2Y receptor agonists stimulates Na⁺ absorption by increasing the activity of Ca²⁺-activated K⁺ channels located in the basolateral membrane.

adenosine receptors; epithelial sodium channels; KCNN4; SK4; potassium channels