Mathematical Model of Nucleotide Regulation on Airway Epithelia

Abstract

In the airways, adenine nucleotides support a complex signaling network mediating host defenses. Released by the epithelium into the airway surface liquid (ASL) layer, they regulate mucus clearance through P2 (ATP) receptors, and following surface metabolism through P1 (adenosine; Ado) receptors. The complexity of ASL nucleotide regulation provides an ideal subject for biochemical network modeling. A mathematical model was developed to integrate nucleotide release, the ectoenzymes supporting the dephosphorylation of ATP into Ado, Ado deamination into inosine (Ino), and nucleoside uptake. The model also includes ecto-adenylate kinase activity and feed-forward inhibition of Ado production by ATP and ADP. The parameters were optimized by fitting the model to experimental data for the steady-state and transient concentration profiles generated by adding ATP to polarized primary cultures of human bronchial epithelial (HBE) cells. The model captures major aspects of ATP and Ado regulation, including their >4-fold increase in concentration induced by mechanical stress mimicking normal breathing. The model also confirmed the independence of steady-state nucleotide concentrations on the ASL volume, an important regulator of airway clearance. An interactive approach between simulations and assays revealed that feedforward inhibition is mediated by selective inhibition of ecto-5-nucleotidase. Importantly, the model identifies ecto-adenylate kinase as a key regulator of ASL ATP and proposes novel strategies for the treatment of airway diseases characterized by impaired nucleotide-mediated clearance. These new insights into the biochemical processes supporting ASL nucleotide regulation illustrate the potential of this mathematical model for fundamental and clinical research. Mucociliary clearance (MCC)4 constitutes the first line of defense against airway infection (1). Inhaled pathogens are trapped by a mucus layer, positioned above ciliated epithelia by a periciliary (PCL) layer. Together, mucus and PCL form the ASL layer, which is continuously transported cephalad by coordinated ciliary beating. Through their interactions with P2 receptors, adenine nucleotides regulate all major epithelial functions supporting MCC, including Cl Ϫ /liquid secretion via the Ca 2ϩ -activated Cl Ϫ channel and the cystic fibrosis transmembrane regulator (CFTR) (2), mucin secretion (3, 4), and ciliary beat activity (5, 6). Nucleotides are released by the epithelium into the ASL layer under basal conditions (7,8), and their release rate increases in response to mechanical stress imparted by tidal breathing (8 -12). Nucleotide-mediated epithelial responses are modified by surface enzymes (ectonucleotidases) that convert a fraction of the ATP into Ado (13-15). This nucleoside activates airway epithelial signaling pathways through P1 receptors, typically the A 2B receptor, which stimulates ciliary beat activity (16) and Cl Ϫ /liquid secretion by CFTR (17). In healthy airways, the purinergic regulation of MCC is mediated by both P1 and P2 receptor-m...

UDP-glucose promotes neutrophil recruitment in the lung

“…Differences in rates of extracellular metabolism account for this apparent discrepancy. For example, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) sequentially dephosphorylate ATP to ADP and AMP (ATP → ADP → AMP + 2 phosphates) and are abundantly expressed on the surface of most cell types [49], including airway epithelial cells [39,50] and neutrophils [51]. Alkaline phosphatase, which is expressed on airway epithelial cells [52], sequentially dephosphorylates ATP, ADP, and AMP.…”

Cystic fibrosis remodels the regulation of purinergic signaling by NTPDase1 (CD39) and NTPDase3

“…We recently developed a mathematical model for the multi-enzyme network regulating extracellular nucleotides on airway epithelia (80). The simulations and experimental validations showed that the ectonucleotidases interact via substrate competition.…”

“…Given the extensive ramifications of purinergic signaling in airway defenses, anomalies in the regulation of ATP, in CF, may contribute to the chronic airway obstruction and inflammation. The mechanisms regulating ATP on airway epithelial surfaces were recently summarized in a mathematical model, which combines several ATP-hydrolyzing enzymes: nonspecific alkaline phosphatase (NSAP), nucleotide pyrophosphatase/phosphodiesterases (NPPs), and nucleoside triphosphate diphosphohydrolases (NTPDases) (80). These ectonucleotidases convert ATP into ADP and/or AMP; then, AMP is converted into ADO by NSAP and ecto 5=-nucleotidase (CD73).…”

“…The rhythmic mechanical stress caused by breathing induces ATP release from the epithelia (10), which binds P2Y 2 receptors (P2Y 2 Rs) to induce fluid secretion via Ca 2ϩ -activated Cl Ϫ channels, and mucin secretion to shield the surface and entrap the pathogens. Surface enzymes, named ectonucleotidases, convert a fraction of the ATP into adenosine (ADO) (80), which binds A 2B receptors (A 2B Rs) to stimulate cilia beating and CFTR activity (4). Purinergic signaling also regulates inflammatory responses, as ATP induces cytokine secretion from the epithelium (16,64) and regulates the activities of inflammatory cells (32).…”

Maintained tubuloglomerular feedback responses during acute inhibition of P2 purinergic receptors in mice

“…ATP may also directly activate P2 receptors on the afferent arteriole, with P2X1 receptors being the most likely receptor subtype since its presence in afferent arterioles has been well documented (8). Alternatively, ATP may be dephosphorylated to AMP and adenosine by ecto-ATPases and ecto-5= nucleotidase, a paradigm that has been shown to underlie the effects of ATP in a number of tissues (10,18,34). Such a mode of action of ATP is supported by the consistent and complete inhibition of TGF responses in vivo by inhibitors of A 1 adenosine receptors (A1AR) and by the absence of TGF responses in mice with targeted deletion of A1AR (5,6,29,32).…”