Biochemical evidence for an ecto alkaline phosphodiesterase I in human airways

Because dinucleotides are signaling molecules that can interact with cell s urface receptors and regulate the rate of mucociliary clearance in lungs, w e studied their metabolism by using human airway epithelial cells. A membra ne-bound enzyme was detected on the mucosal surface of polarized epithelia that metabolized dinucleotides with a broad substrate specificity (diadenos ine polyphosphates and diuridine polyphosphates [Up(n)U], n = 2 to 6). The enzymatic reaction yielded nucleoside monophosphates (NMP) and Npn-1 (N = A or U), and was inhibited by nucleoside 5'-triphosphates (alpha,beta met ad enosine triphosphate [ATP] > ATP greater than or equal to uridine triphosph ate > guanidine triphosphate > cytidine triphosphate). The apparent Michael is constant (K-m,K-app) and apparent maximal velocity (V-max,V-app) for [H- 3]Up(4)U were 22 +/- 4 mu M and 0.24 +/- 0.05 nmoles.min(-1).cm(-2), respec tively. Thymidine 5'-monophosphate p-nitrophenyl ester and adenosine diphos phate (ADP)ribose, substrates of ecto alkaline phosphodiesterase 1 (PDE I) activities, were also hydrolyzed by the apical surface of airway epithelia. ADP-ribose competed with [H-3]Up,U, with a K-i of 23 +/- 3 mu M. The metab olism of ADP-ribose and Ap(4)A was not affected by inhibitors of cyclic nuc leotide phosphodiesterases (3-isobutyl-1-methylxanthine, Ro 20-1724, and 1, 3-dipropyl-8-p-sulfophenylxanthine), but similarly inhibited by fluoride an d N-ethylmaleimide. These results suggest that a PDE I is responsible for t he hydrolysis of extracellular dinucleotides in human airways. The wide sub strate specificity of PDE 1 suggests that it may be involved in several sig naling events on the luminal surface of airway epithelia, including purinoc eptor activation and cell surface protein ribosylation.