Calu-3 cells respond to basolateral nucleotides with increases in intracellular free calcium ([Ca²⁺]_i) (Clunes et al. 2002). In the present study, we have characterised the receptors underlying these responses in Fura-2-loaded Calu-3 cells grown (2-4 days) on collagen-coated permeable supports (Costar, Transwell Col). Supports were mounted in a specialised chamber, which allowed independent perfusion of the apical and basolateral surfaces (2-3 ml min^-1), and attached to the stage of an inverted microscope equipped with extra long working distance optics. The Ca²⁺ signals evoked by pulses of basolateral nucleotides were quantified (as arbitrary units, means ± S.E.M.) by integrating the increase in Fura-2 fluorescence ratio with respect to time.

Initial experiments confirmed (Clunes et al. 2002) that ATP (EC₅₀ 10.6 ± 1.67 µM, maximal response at 100 µM, n = 6), ADP (EC₅₀ 3.5 ± 0.3 µM, maximal response at 100 µM, n = 5) and UTP (EC₅₀ 53.7 ± 5.6 µM, maximal response at 300 µM, n = 6) all evoked rapid increases in [Ca²⁺]_i. In further experiments cells were exposed to a series of five pulses (30 s) of agonist, delivered at 2 min intervals, in order to evoke autologous desensitisation (> 85 %) to particular nucleotides. We then explored the extent to which these desensitised cells retained sensitivity to other nucleotides. UTP-desensitised cells (300 µM) displayed responses to ADP (100 µM) that were similar to control and ADP-desensitised (100 µM) responded normally to 300 µM UTP (5.86 ± 1.70 arbitrary units). In contrast, the response to ATP was smaller than normal in both groups of cells (P < 0.05 for both, Student’s unpaired t test). Further experiments showed that suramin (10 µM) had no effect upon the concentration- effect curves for UTP (control: EC₅₀ = 30 ± 3 µM, threshold 3 µM, n = 4; suramin-treated: EC₅₀ = 47 ± 4 µM, threshold 3 µM, n = 3) but shifted the curve for ADP to the right (control: EC₅₀ = 0.88 ± 0.33 µM, threshold 30 nM, n = 4; suramin-treated EC₅₀ = 19.4 ± 0.3 µM, threshold 3 µM, n = 6).

ATP thus activates a complex receptor population but UTP and ADP seen to be selective agonists for different receptor subpopulations that can be distinguished on the basis of their sensitivity to suramin. The high affinity for ADP and the sensitivity to suramin suggest that one of these subpopulations corresponds to the well-documented P2Y₁ receptor, but further studies are needed to identify the receptor population that can be activated by UTP but which is insensitive to suramin.

This work was supported by the Wellcome Trust and Tenovus Scotland.