Abstract
In human airways, extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection. Thus, defining the mechanisms of adenosine generation is critical for elucidating the role of this nucleoside in airway homeostasis. In this study, we identified the source of adenosine on the mucosal surface of human airway epithelia. Polarized primary cultures of human nasal or bronchial epithelial cells were assayed for transepithelial transport, cytosolic and cell surface adenosine production. Ussing chamber experiments indicated that serosal 1 microM [(3)H]adenosine was not transported to the mucosal compartment. Messenger RNA for the cytosolic AMP-specific 5'-nucleotidase (CN-I) was not detected in human bronchial epithelial cells, suggesting that mucosal adenosine did not originate from intracellular pools. In contrast, extracellular 0.1 mm ATP was rapidly dephosphorylated into adenosine on the mucosal epithelial surface. We identified two ectonucleotidases that mediated the conversion of AMP to adenosine: ecto 5'-nucleotidase (ecto 5'-NT, CD73) and alkaline phosphatase (AP). Both mucosal and serosal epithelial surfaces displayed ecto 5'-NT activity (K(m) = 14 microM, V(max) = 0.5 nmol x min(-1) x cm(-2)), whereas AP activity was restricted to the mucosal surface (K(m,)(high) = 36 microM, V(max) = 1.2 nmol x min(-1) x cm(-2); K(m,)(low) = 717 microM, V(max) = 2.8 nmol x min(-1) x cm(-2)). In bronchial cultures and tissues, ecto 5'-NT accounted for >80% of total activity toward 0.01 mm AMP, compared with <15% for 5 mm AMP. The proximal airway AP isoform was identified as nonspecific AP (NS AP) by levamisole sensitivity and mRNA expression. The two ectoenzymes presented opposite airway distributions, ecto 5'-NT and NS AP mRNA dominating in higher and lower airways, respectively. Collectively, these experiments support a major role for extracellular nucleotide catalysis and for ecto 5'-NT and NS AP in the regulation of adenosine concentrations on airway surfaces.
Highlights
In human airways, extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection
Adenosine Accumulation on the Apical Surface of Human Airway Epithelia—First, we explored the possibility that serosal adenosine could reach the mucosal surface
Despite numerous studies demonstrating that ATP is released from the mucosal surface of human airway epithelia (20 –28), the origin of extracellular adenosine has not been established
Summary
Extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection. The two ectoenzymes presented opposite airway distributions, ecto 5-NT and NS AP mRNA dominating in higher and lower airways, respectively These experiments support a major role for extracellular nucleotide catalysis and for ecto 5-NT and NS AP in the regulation of adenosine concentrations on airway surfaces. Two members of the P2X receptor subfamily were recently identified in human airway epithelial cultures: P2X4 and P2X5 [8] These ATP-gated cationic channels increased Ca2ϩdependent ClϪ secretion [8] and CBF [9] in airway epithelia. The importance of adenosine receptor-mediated regulation of MCC remains unclear because of the lack of information on endogenous sources of extracellular adenosine on the mucosal surface of airway epithelia. The pharmacological manipulation of endogenous adenosine levels by targeting these ectoenzymes remains an unexplored area
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