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 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.
Novartis Respiratory Research Centre, Wimblehurst Road, Horsham, West Sussex.
Parmjit
P
Bahra
Jonathan
J
Mesher
Su
S
Li
Christopher T
CT
Poll
Henry
H
Danahay
D019540:Area Under Curve; D002120:Calcium Channel Agonists / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects*; D020013:Calcium Signaling / Q000187:drug effects*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D007093:Imidazoles / Q000494:pharmacology; D007202:Indicators and Reagents; D028581:Lanthanoid Series Elements / Q000494:pharmacology*; D018048:Receptors, Purinergic P2 / Q000187:drug effects*; D058473:Receptors, Purinergic P2Y2; D020545:Respiratory Mucosa / Q000187:drug effects / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000494:pharmacology
D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D005456:Fluorescent Dyes; D007093:Imidazoles; D007202:Indicators and Reagents; D028581:Lanthanoid Series Elements; C546385:P2RY2 protein, human; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D016257:Fura-2; D014544:Uridine Triphosphate