Human Airway Ecto-adenylate Kinase: A MECHANISM TO PROPAGATE ATP SIGNALING ON AIRWAY SURFACES

Maryse Picher,Richard C Boucher

doi:10.1074/jbc.m208071200

Maryse Picher, Richard C Boucher

Open Access

https://doi.org/10.1074/jbc.m208071200

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Abstract

Mechanically induced ATP release from human airway epithelial cells regulates mucociliary clearance through cell surface nucleotide receptors. Ectoenzymes detected on these cells were recently shown to terminate ATP-mediated responses by sequential dephosphorylation of extracellular ATP into ADP, AMP, and adenosine. We now demonstrate that an ecto-adenylate kinase (ecto-AK) contributes to the metabolism of adenine nucleotides on human airway epithelial surfaces by the reversible reaction: ATP + AMP 2ADP. This phosphotransferase exhibited a bilateral distribution on polarized primary cultures of human bronchial epithelial cells with a 4-fold higher activity on the mucosal surface. Ecto-AK presented an absolute requirement for magnesium and adenine-based nucleotides. UMP, GMP, and CMP could not substitute for AMP as gamma-phosphate acceptor, and UDP could not replace ADP. Apparent K(m) and V(max) values were 23 +/- 5 microM and 1.1 +/- 0.1 nmol x min(-1) x cm(-2) for ATP and 43 +/- 6 microM and 0.5 +/- 0.1 nmol x min(-1) x cm(-2) for ADP. Ecto-AK accounted for 20% of [gamma-(32)P]ATP dephosphorylation, and the impermeant AK inhibitor, diadenosine pentaphosphate, reduced ADPase activity by more than 70% on both epithelial surfaces. Time course experiments on ATP metabolism demonstrated that ecto-AK significantly prolongs effective ATP and ADP concentrations on airway epithelial surfaces for P2 receptor signaling and reduces by 6-fold adenosine production. Our data suggest a role for this nucleotide entrapment cycle in the propagation of purine-mediated mucociliary clearance on human airway epithelial surfaces.

Highlights

Induced ATP release from human airway epithelial cells regulates mucociliary clearance through cell surface nucleotide receptors
We demonstrate that an ecto-adenylate kinase contributes to the metabolism of adenine nucleotides on human airway epithelial surfaces by the reversible reaction: ATP ؉ AMP u 2ADP
Detection of Ectonucleotidase and Ecto-AK Activities on Airway Epithelia—Polarized bronchial epithelial cultures were assayed for nucleotide metabolism

Summary

A MECHANISM TO PROPAGATE ATP SIGNALING ON AIRWAY SURFACES*

Induced ATP release from human airway epithelial cells regulates mucociliary clearance through cell surface nucleotide receptors. Ectoenzymes detected on these cells were recently shown to terminate ATP-mediated responses by sequential dephosphorylation of extracellular ATP into ADP, AMP, and adenosine. We demonstrate that an ecto-adenylate kinase (ecto-AK) contributes to the metabolism of adenine nucleotides on human airway epithelial surfaces by the reversible reaction: ATP ؉ AMP u 2ADP. In a human bronchial cell line lacking P2Y2 receptors (Calu-3 [23]), the basal ion channel activity of the cystic fibrosis transmembrane regulator (CFTR) was inhibited by 8-p-sulfophenyltheophylline and by AMPCP [22], an inhibitor of the cell surface conversion of AMP into adenosine (for a review, see Ref. 24).

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION