Purinergic stimulation induces Ca2+-dependent activation of Na+-K+-2Cl- cotransporter in human nasal epithelia.
Increasing evidence suggests that P2 receptors (P2Rs) in airway epithelial cells perform critical functions in auto- or paracrine regulation of fluid and mucus secretion. In the present study, we characterized the effects of P2R stimulation on Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity in normal human nasal epithelial (NHNE) cells. [Ca(2+)](i) and pH(i) were measured in primary cultures of NHNE cells using a double perfusion chamber, which enabled us to analyze membrane-specific transporter activities. NKCC activities were estimated by the pH(i) reduction due to Na(+)-dependent and bumetanide-sensitive intracellular uptake of NH(4)(+). NKCC activities were observed in the basolateral membrane, but not in the luminal membrane, of NHNE cells. Interestingly, P2Rs were expressed in both membranes, and the stimulation of either luminal or basolateral P2R increased NKCC activity. Blockades of luminal Cl(-) channels, basolateral K(+) channels, or protein kinase C did not affect the activation of NKCC by basolateral P2R stimulation. The effects of luminal P2R stimulation were partially reduced by Cl(-) channel blockers. However, chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) treatment completely blocked the stimulatory effects of luminal and basolateral P2Rs on NKCC. In addition, increasing [Ca(2+)](i) by treatment with ionomycin-stimulated NKCC activity. These results provide evidence that stimulation of P2Rs directly activates basolateral NKCC by Ca(2+)-dependent pathways in NHNE cells, which is an important aspect of the purinergic regulation of ion and fluid secretions in human airway epithelia under physiologic and pathologic conditions.
Shin
Ji-Hyun
JH
Department of Otorhinolaryngology, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.
Namkung
Wan
W
Choi
Jae Young
JY
Yoon
Joo-Heon
JH
Lee
Min Goo
MG
D002118:Calcium / Q000032:analysis / Q000502:physiology*; D016764:Cell Polarity; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels; D009666:Nose / Q000166:cytology*; D011493:Protein Kinase C; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*
D007473:Ion Channels; D018048:Receptors, Purinergic P2; D028021:Sodium-Potassium-Chloride Symporters; D011493:Protein Kinase C; D002118:Calcium