P2Y2-receptor-mediated activation of a contralateral, lanthanide-sensitive calcium entry pathway in the human airway epithelium

1 Receptor-mediated calcium entry (RMCE) was examined in well-differentiated cultures of normal human bronchial epithelial cells (HBECs). Changes in intracellular free Ca2+ ([Ca2+](i)) were quantified using fluorescence ratio imaging of Fura-2-loaded cells during perfusion with Ca2+ mobilizing agonists. 2 Initial studies revealed an agonist potency of ATP = uridine triphosphate (UTP) > ADP = uridine diphosphate, consistent with purinergic activation of an apical P2Y(2)-receptor mediating the increase in [Ca2+](i) in HBECs. 3 Apical UTP ( 30 muM) induced a sustained period of elevated [Ca2+](i) between 300 and 600 s following agonist stimulation that extracellular Ca2+ free studies indicated was dominated by Ca2+ influx. 4 RMCE was inhibited by 100 nM La3+ (83 +/- 3%) or Gd3+ (95 +/- 7%)( P<0.005, n = 4 - 11) and was partially attenuated by Ni2+(1 mM)( 58.7 +/- 5.0%, P<0.005, n = 9). 5 RMCE was also partially sensitive ( < 25% inhibition, P<0.01) to the cation channel blockers SKF96365 ( 30 muM) and econazole ( 30 muM), but was insensitive to both verapamil ( 1 muM) and ruthenium red ( 10 muM). 6 Using either a sided Ca2+ readdition protocol or unilateral La3+, established that the RMCE pathway was located exclusively on the basolateral membrane. 7 The pharmacological sensitivity of the P2Y(2)-receptor activated Ca2+ entry pathway in the human airway epithelium is inconsistent with the established profile of TRP channel families and is therefore likely to be of an as-yet uncharacterized molecular identity.