We have recently shown that polarised cultures of human airway epithelial cells (Calu-3) express a DIDS-sensitive basolateral Cl–HCO3- anion exchanger (BlAE), which is inhibited via a cAMP-dependent, but PKA-independent, mechanism (1). The aim of this study was to investigate the downstream targets of cAMP responsible for BlAE regulation. The activity of the exchanger was assessed by measuring Cl–dependent changes in intracellular pH using BCECF-loaded Calu-3 cells grown on Transwell supports. In the absence of cAMP agonists, exposing Calu-3 cells to a Cl–free solution (0Cl-) in the basolateral perfusate produced an alkalinisation of 0.27 ± 0.03 pH units (mean ± SEM.; P<0.05 n=10). Restoring Cl- caused pH to recover at a rate of 0.62 ± 0.07 pH units min-1. The response to 0Cl- was completely abolished if cells were pre-treated with the adenylyl cyclase (tmAC) activators, forskolin or adenosine, the membrane-permeable cAMP analogue, dibutyryl cAMP or the phosphodiesterase inhibitor, IBMX (n=3-10), clearly implicating a rise in cAMP as a key event in the inhibition of the BlAE. The guanine-nucleotide exchange protein directly activated by cAMP (Epac) is another cAMP target. However, incubating Calu-3 cells with the Epac agonist, 8CPT-2Me-cAMP-AM, had no effect on BlAE activity, nor did it prevent forskolin inhibiting the exchanger (n=4). To investigate if a rise in cAMP was linked to a change in cytosolic Ca2+, Calu-3 cells were acutely exposed to carbachol (n=7) or thapsigargin (n=3), but neither agent affected resting BlAE activity, nor the subsequent inhibition by forskolin. However, pre-loading cells with the Ca2+ buffer, BAPTA-AM, did reduce BlAE activity by 63.3±5.8% suggesting some dependence of exchanger function on cytosolic Ca2+ levels. To investigate if the actin cytoskeleton was important in cAMP-regulation of BlAE, Calu-3 cells were pre-treated with cytochalasin D or lantruculin B for 60 mins. Both agents reduced transepithelial resistance (TER) by ~ 40 % after 20 mins but TER remained stable thereafter (n=3). In cytochalasin D treated cells, resting BlAE activity was similar to untreated cells but there was a marked reduction in the ability of adenosine to inhibit the BlAE (n=4, P<0.05). Interestingly, this was not observed for forskolin (n=4), suggesting that actin disruption specifically reduced the ability of adenosine receptors to couple to cAMP production via tmAC, thereby leading to higher BlAE activity. In contrast, lantruculin B reduced resting BlAE activity, but did not prevent cAMP inhibition. In conclusion, our results provide clear evidence that a rise in cAMP inhibits BlAE activity, through a downstream mechanism that does not involve Epac, nor a rise in cytosolic calcium, but does appear to require an intact actin cytoskeleton. Work supported by an Iraqi Government Studentship to SI