P2Y-mediated Ca²⁺ signals are well documented in cultured epithelial cells but most such studies have been undertaken using cells grown on impermeable supports, which often fail to become polarised. As cell polarity can be an important determinant of P2Y receptor expression (Clunes et al. 1998; Wilson et al. 1998), we now explore the effects of nucleotides upon intracellular free Ca²⁺ ([Ca²⁺]_i) in Calu-3 cells grown to confluence (3-4 days) on collagen-coated, permeable membranes (Costar Transwell-Col). These cells were loaded with fura-2 by incubation in Hepes-buffered physiological saline containing 3 mM fura-2 AM and 2.5 mM probenecid, and mounted in a chamber that allowed the apical and basolateral sides of the cultured epithelium to be superfused independently (2-3 ml min, 37 °C). Fura-2 fluorescence ratios were recorded using an inverted microscope equipped with extra long working distance optics. All numbers are means ± S.E.M.

Apical ATP (100 mM, n = 6) and UTP (300 mM, n = 6) failed to evoke discernible responses but were effective basolaterally. Experiments (n > 4) in which increasing concentrations of P2Y receptor agonists (0.1-300 mM) were delivered to the basolateral membrane as 30 s pulses revealed a rank order of potency (EC₅₀) of ADP-β-S (8.4 ± 1.1 mM) ▓Dgr│ ATP (10.7 ± 1.9 mM) > UTP (53.7 ± 6.0 mM). The maximal response to ADP-β-S was only ~50 % of that evoked by ATP, suggesting that this P2Y₁ receptor agonist acts as a partial agonist. ATP-stimulated cells (100 mM) became essentially insensitive (6 ± 1 % of control sensitivity, n = 4) to UTP, whereas cells exposed to a maximally effective concentration of UTP (300 mM) retained 30 ± 10 % of their sensitivity to ATP. Calu-3 cells thus seem to express a complex population of P2Y receptors in the basolateral, but not the apical membrane. This is surprising as functional studies have shown that apical nucleotides do evoke increased ion transport in these cells (Chambers et al. 2002). Moreover our findings also contrast with earlier data from cells grown on impermeable substrates which indicated that UTP-sensitive receptors are not expressed by Calu-3 cells (Communi et al. 1999).

The authors thank The Wellcome Trust and Tenovus for their financial support.