P2Y receptors mediate many of the extracellular actions of adenosine 5 '-triphosphate (ATP), uridine 5 '-triphosphate (UTP) and uridine 5 '-diphosphate (UDP). Previously we showed that UTP and UDP both acted via two P2Y receptors to evoke contraction of rat isolated pulmonary arteries (Chootip et al., 2002). For each, one site was insensitive to the antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), whilst the other was inhibited by suramin, but not PPADS. The molecular identity of these receptors is unclear. The aim of the present study was to extend the pharamacological characterisation of these P2Y receptors by recording the calcium-dependent, chloride current (I_Cl,Ca) induced by nucleotides in acutely dissociated pulmonary artery smooth muscle cells (Hartley et al., 1998).

Small (SPA, 200-500 µm i.d.) and large (LPA, 1.0-1.5 mm i.d.) intrapulmonary arteries from male Sprague-Dawley rats (150-250 g) were incubated at 37 °C for 10-15 min with 0.6-0.8 mg.ml^-1 papain, 0.04% BSA and 0.4 mM dithiothreitol. Collagenase (0.6-0.8 mg.ml^-1) was then added and the tissues incubated for a further 5-10 min. Cells were dispersed by mild trituration in enzyme-free solution, then superfused (0.5 ml.min^-1) at room temperature and voltage-clamped at -50 mV in the whole-cell, perforated-patch mode using amphotericin B (150 µg.ml^-1) and an Axopatch 200A amplifier. Drugs were applied using a gravity-feed system, with complete solution exchange in < 2 s. Only one agonist was applied to each cell. Data are expressed as mean ± sem and were compared by Student’s t-test, or one-way ANOVA and Tukey’s comparison as appropriate.

ATP, UTP and UDP (10^-4M) evoked inward currents (peak = 13-727 pA) in 71-93% of SPA (n=118) and LPA (n=117) smooth muscle cells. In most, I_Cl,Ca activated in an oscillating manner. The peak of the first inward current appeared to be larger for ATP (2.4 ± 0.2 pA.pF^-1, n=52) than UTP (1.6 ± 0.2 pA.pF^-1, n=37) or UDP, (1.5 ± 0.4 pA.pF^-1, n=17), but this was only significant in the SPA (P < 0.05). The initial response to ATP in the SPA was also greater than that in the LPA (P < 0.05), but the responses to UTP and UDP were similar in the two vessels. Subsequent currents decreased in amplitude, with a variable time-course, but this was only significant for UTP (P<0.001) and UDP (P<0.05) in the SPA. There was no significant change in the frequency of oscillations (6 - 11.min^-1), apart from an increase to UTP in LPA between the first and second min of agonist application (P<0.05). Suramin (10^-4M) abolished the I_Cl,Ca evoked by ATP in SPA and LPA (n=4,5), but PPADS (10^-4M) had no effect (n=4,5). The currents elicited by UTP or UDP in SPA or LPA were unaffected by suramin and PPADS (n=3-6).

Thus, at least two P2Y subtypes couple to activation of I_Cl,Ca in rat pulmonary artery smooth muscle cells and are likely to play a significant role in nucleotide-induced vasoconstriction. The suramin-sensitive, PPADS-resistant site that is activated by ATP most resembles the P2Y₁₁ receptor, but the suramin- and PPADS-insensitive receptor activated by UTP and UDP does not correspond to any of the known P2Y subtypes.

Chootip et al., (2002). British J. Pharmacol., 137, 637-646.
Hartley et al., (1998). Circ. Res., 83, 940-946.