Human airway ecto-adenylate kinase. A mechanism to propagate ATP signaling on airway surfaces.
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.
Picher
Maryse
M
Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA. pichm@med.unc.edu
Boucher
Richard C
RC
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000263:Adenylate Kinase; D001980:Bronchi / Q000166:cytology / Q000201:enzymology*; D002412:Cations; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003550:Cystic Fibrosis / Q000201:enzymology; D004847:Epithelial Cells / Q000201:enzymology; D006801:Humans; D015398:Signal Transduction; D013379:Substrate Specificity
D002412:Cations; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase