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Apical versus Basolateral P2Y₆ Receptor–Mediated Cl^– Secretion in Immortalized Bronchial Epithelia

¹ Department of Physiology, The Chinese University of Hong Kong, Shatin, Hong Kong

Correspondence and requests for reprints should be addressed to Wing-hung Ko, Ph.D., Rm. 410 D, Basic Medical Sciences Building, Department of Physiology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. E-mail: whko{at}cuhk.edu.hk

Apical and/or basolateral membranes of polarized epithelia express P2Y receptors, which regulate the transport of fluid and electrolytes. In the airway, P2Y receptors modulate Cl^– secretion through the phospholipase C and calcium signaling pathways. Recent evidence suggests that P2Y₆ receptors are expressed in bronchial epithelium and coupled to the cAMP/protein kinase A (PKA) pathways. We examined P2Y receptor subtype expression, including P2Y_6, and the effect of extracellular nucleotides on basal short-circuit current (I_SC) and intracellular calcium concentration ([Ca²⁺]_i) in a human bronchial epithelial cell line (16HBE14o-). Real-time PCR demonstrated P2Y₁, P2Y₂, P2Y₄, and P2Y₆ receptor expression and confirmed that transcript levels were not altered when cells were grown under varied conditions. It was determined that P2Y agonists (ATP, UTP, UDP) stimulated a concomitant increase in I_SC and [Ca²⁺]_i. Apical nucleotides stimulated an increase in [Ca²⁺]_i more efficiently than basolateral nucleotides; however, P2Y agonistic effects on I_SC were greater when applied basolaterally. Since the P2Y₆ receptors differentially regulate apical and basolateral UDP-induced I_SC and [Ca²⁺]_i, we investigated membrane-resident P2Y₆ receptor functions using Cl^– or K⁺ channels blockers. Apical and basolateral UDP activation of I_SC was inhibited by applying DIDS apically or TRAM-34 and clotrimazole basolaterally. Although both apical and basolateral UDP increased PKA activity, only apical UDP-induced I_SC was sensitive to a CFTR inhibitor. These data demonstrate that P2Y agonists stimulate Ca²⁺-dependent Cl^– secretion across human bronchial epithelia and that the cAMP/PKA pathway regulates apical but not basolateral P2Y₆ receptor–coupled ion transport in human bronchial epithelia.

Key Words: P2Y receptors • CI^– secretion • bronchial epithelium, Ca²⁺ • cAMP

CLINICAL RELEVANCE The present study has provided the first evidence of the cellular mechanisms by which P2Y6 receptors differentially regulate apical and basolateral anion secretion in human bronchial epithelia.