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

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Abstract
1. Receptor-mediated calcium entry (RMCE) was examined in well-differentiated cultures of normal human bronchial epithelial cells (HBECs). Changes in intracellular free Ca(2+) ([Ca(2+)](i)) were quantified using fluorescence ratio imaging of Fura-2-loaded cells during perfusion with Ca(2+) 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 [Ca(2+)](i) in HBECs. 3. Apical UTP (30 microm) induced a sustained period of elevated [Ca(2+)](i) between 300 and 600 s following agonist stimulation that extracellular Ca(2+) free studies indicated was dominated by Ca(2+) influx. 4. RMCE was inhibited by 100 nm La(3+) (83+/-3%) or Gd(3+) (95+/-7%) (P<0.005, n=4-11) and was partially attenuated by Ni(2+) (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 microm) and econazole (30 microm), but was insensitive to both verapamil (1 microm) and ruthenium red (10 microm). 6. Using either a sided Ca(2+) readdition protocol or unilateral La(3+), established that the RMCE pathway was located exclusively on the basolateral membrane. 7. The pharmacological sensitivity of the P2Y(2)-receptor activated Ca(2+) 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.