Hypercapnia is a symptom of chronic lung disease and is caused by inefficient gas exchange at the alveoli. We have recently shown that high CO2 reduces cAMP stimulated CFTR-dependent HCO3- secretion from polarised cultures of human submucosal gland derived airway epithelial cells (Calu-3) (1). The decrease in HCO3- secretion appears to be due to a specific 5-6 fold reduction in HCO3- accumulation across the basolateral membrane, during cAMP agonist stimulation, via an unidentified Na+-independent/DIDS-sensitive HCO3- transporter. How high CO2 signals a reduction in HCO3- accumulation is not fully understood, but it does involve changes in cAMP and Ca2+ (1). Recent work in rat brain tissue has shown that hypercapnia stimulates ATP release from the medulla oblongata via the opening of connexin channels. The ATP is then thought to signal to local neurones to mediate the adaptive respiratory response to high CO2. The aim of the present work was therefore to investigate if similar paracrine signalling by CO2-induced ATP release was involved in the observed changes in HCO3- transport in Calu-3 cells. Exposing Calu-3 cells to exogenous ATP (100µM) in normocapnia, in the presence of the cAMP agonist forskolin, reduced HCO3- accumulation 5.0 ± 0.86 fold (p<0.01; n = 3) thus mimicking the effect observed in hypercapnia. This effect of ATP is Ca2+-dependent which suggests that increases in extracellular [ATP] ([ATP]e) may underlie the effect of hypercapnia. However, the reported connexin inhibitors cobalt (500µM, n = 4) and proadifen (200µM, n = 4) did not prevent the CO2-mediated decrease in HCO3- accumulation implying that connexins are not involved in any potential CO2-induced ATP release. Furthermore, inhibition of the related gap junction proteins, pannexins, with carbenoxolone (10µM) also had no effect on the CO2-induced change in HCO3- transport. Finally, pre-treating cells with brefeldin A (5µg/ml) did not alter the response to hypercapnia, implying that ATP is not released via exocytosis of ATP-containing vesicles. We next tested whether the effects of ATPe were due to ATP metabolism into adenosine. In control conditions, hypercapnia reduced forskolin-stimulated HCO3- accumulation 5.0 ± 0.44 fold (p<0.05; n = 3) and this was unaffected in cells exposed to an adenosine receptor antagonist CGS-15943 (500nM) in which hypercapnia reduced forskolin-stimulated HCO3- accumulation 4.1 ± 0.05 fold (p<0.05; n = 3). This implies that any role of ATP is likely a direct effect rather than a result of ATP metabolism. In conclusion, hypercapnia may induce ATP release to paracrine signal and thus contribute to changes in cAMP-regulated HCO3- transport, although the mechanism for any putative increases in [ATP]e remains to be determined.