Figure 3.
The A2B ADO-R is the main ADO-R subtype expressed in human bronchial epithelial cells (HBECs). (A) Banding patterns obtained after RT-PCR of cDNAs from normal HBECs using primers against the ADO-Rs shown. Lanes 1–3 show bands from three different donors against ADO-Rs as described. Lane 4 is a positive control (A1, whole brain; A2A, A2B, A3, whole lung) and lane 5 shows the respective primers run in water. All ladders are 100 bp. The identity of the A2B bands were confirmed by sequencing. (B) Dose-dependent increase in transepithelial potential difference (Vt) after mucosal ADO addition. HBECs were mounted in Ussing chambers and Vt was measured under open circuit conditions. All measurements were made in the presence of 10−4 M amiloride and with mucosal Cl- being replaced with gluconate to maximize the gradient for Cl- efflux. Under these conditions, the baseline PD was −5.9 ± 2.1 mV. All n = 6. (C) Changes in airway surface liquid (ASL) height measured by XZ confocal microscopy 10 min post-addition of ADO and its analogs as dry powders suspended in perfluorocarbon (final concentration ∼ 200 μM). All n = 6. (D) Left, changes in ASL height 10 minutes after ADO addition in the presence of DPCPX, ZM241385, alloxazine, or MRS1191 (all pre-added at 10−6 M), caffeine (3 × 10−5 M), and ALT801 (2 × 10−7 M). All n = 6–8. Right, changes in ASL height 10 minutes after ADO addition in the presence of glibenclamide or DIDS (both added at 2 × 10−4 M). All n = 6. (N.B., for C and D, all cultures were pretreated with 1 U/ml aprotinin to inhibit ENaC before P1 agonist addition.) Data shown as mean ± SEM. *P < 0.05 as compared with ADO alone.