P2Y receptor regulation of sodium transport in human mammary epithelial cells

Yeong Lee So, Melissa L Palmer, Peter J Maniak, Hwa Jang Soo, Dong Ryu Pan, Scott M O'Grady

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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 A2b adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current (Isc) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na+ channel (ENaC) α-, β-, and γ-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) (ATPγS) 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 > ATPγS. Basolateral stimulation with UTP produced a rapid but transient increase in Isc 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 Isc 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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume293
Issue number5
DOIs
StatePublished - Nov 1 2007

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Uridine Triphosphate
Breast
Epithelial Cells
Sodium
Hydrocortisone
Monolayers
Messenger RNA
Uridine Diphosphate
Polymerase Chain Reaction
Purinergic P2Y2 Receptors
Purinergic P2Y1 Receptors
Cells
Charybdotoxin
Clotrimazole
Membranes
Purinergic Receptors
Calcium-Activated Potassium Channels
Purinergic P1 Receptors
Telomerase
Short circuit currents

Keywords

  • Adenosine receptors
  • Epithelial sodium channels
  • KCNN4
  • Potassium channels
  • SK4

Cite this

P2Y receptor regulation of sodium transport in human mammary epithelial cells. / So, Yeong Lee; Palmer, Melissa L; Maniak, Peter J; Soo, Hwa Jang; Pan, Dong Ryu; O'Grady, Scott M.

In: American Journal of Physiology - Cell Physiology, Vol. 293, No. 5, 01.11.2007.

Research output: Contribution to journalArticle

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abstract = "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 A2b adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current (Isc) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na+ channel (ENaC) α-, β-, and γ-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) (ATPγS) 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 > ATPγS. Basolateral stimulation with UTP produced a rapid but transient increase in Isc 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 Isc 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.",
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AU - Maniak, Peter J

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AU - Pan, Dong Ryu

AU - O'Grady, Scott M

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AB - 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 A2b adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current (Isc) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na+ channel (ENaC) α-, β-, and γ-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) (ATPγS) 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 > ATPγS. Basolateral stimulation with UTP produced a rapid but transient increase in Isc 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 Isc 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.

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