Effects of airway surface liquid height on the kinetics of extracellular nucleotides in airway epithelia

Experimental techniques aimed at measuring the concentration of signaling molecules in the airway surface liquid (ASL) often require an unrealistically large ASL volume to facilitate sampling. This experimental limitation, prompted by the difficulty of pipetting liquid from a very shallow layer (~15μm), leads to dilution and the under-prediction of physiologic concentrations of signaling molecules that are vital to the regulation of mucociliary clearance. Here, we use a computational model to describe the effect of liquid height on the kinetics of extracellular nucleotides in the airway surface liquid coating respiratory epithelia. The model consists of a reaction–diffusion equation with boundary conditions that represent the enzymatic reactions occurring on the epithelial surface. The simulations reproduce successfully the kinetics of extracellular ATP following hypotonic challenge for ASL volumes ranging from 25μl to 500μl in a 12-mm diameter cell culture. The model reveals that [ATP] and [ADO] reach 1200nM and 2200nM at the epithelial surface, respectively, while their volumetric averages remain less than 200nM at all times in experiments with a large ASL volume (500μl). These findings imply that activation of P2Y2 and A2B receptors is robust after hypotonic challenge, in contrast to what could be concluded based on experimental measurements of volumetric concentrations in large ASL volumes. Finally, given the central role that ATP and ADO play in regulating mucociliary clearance, we investigated which enzymes, when inhibited, provide the greatest increase in ATP and ADO concentrations. Our findings suggest that inhibition of NTPDase1/highTNAP would cause the greatest increase in [ATP] after hypotonic challenge, while inhibition of the transporter CNT3 would provide the greatest increase in [ADO]. •A mathematical model is proposed for the biochemical network of extracellular nucleotides in airway epithelia, which includes diffusion of nucleotides in the airway surface liquid.•The model predicts the kinetics of ATP, ADP, AMP, ADO and INO after hypotonic challenge.•Due to a sharp concentration gradient, nucleotide concentrations near the epithelial surface are much higher than volume-averaged concentrations.•CNT3 inhibition leads to the greatest availability of ADO for A2B activation.•NTPDase1/highTNAP inhibition leads to the greatest availability of ATP for P2Y2 activation.