Adenosine deaminase 1 and concentrative nucleoside transporters 2 and 3 regulate adenosine on the apical surface of human airway epithelia: implications for inflammatory lung diseases.
Adenosine is a multifaceted signaling molecule mediating key aspects of innate and immune lung defenses. However, abnormally high airway adenosine levels exacerbate inflammatory lung diseases. This study identifies the mechanisms regulating adenosine elimination from the apical surface of human airway epithelia. Experiments conducted on polarized primary cultures of nasal and bronchial epithelial cells showed that extracellular adenosine is eliminated by surface metabolism and cellular uptake. The conversion of adenosine to inosine was completely inhibited by the adenosine deaminase 1 (ADA1) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The reaction exhibited Km and Vmax values of 24 microM and 0.14 nmol x min(-1) x cm(-2). ADA1 (not ADA2) mRNA was detected in human airway epithelia. The adenosine/mannitol permeability coefficient ratio (18/1) indicated a minor contribution of paracellular absorption. Adenosine uptake was Na+-dependent and was inhibited by the concentrative nucleoside transporter (CNT) blocker phloridzin but not by the equilibrative nucleoside transporter (ENT) blocker dipyridamole. Apparent Km and Vmax values were 17 microM and 7.2 nmol x min(-1) x cm(-2), and transport selectivity was adenosine = inosine = uridine > guanosine = cytidine > thymidine. CNT3 mRNA was detected throughout the airways, while CNT2 was restricted to nasal epithelia. Inhibition of adenosine elimination by EHNA or phloridzin raised apical adenosine levels by >3-fold and stimulated IL-13 and MCP-1 secretion by 6-fold. These responses were reproduced by the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and blocked by the adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline (8-SPT). This study shows that adenosine elimination on human airway epithelia is mediated by ADA1, CNT2, and CNT3, which constitute important regulators of adenosine-mediated inflammation.
Hirsh
Andrew J
AJ
Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Stonebraker
Jaclyn R
JR
van Heusden
Catja A
CA
Lazarowski
Eduardo R
ER
Boucher
Richard C
RC
Picher
Maryse
M
D000241:Adenosine / Q000378:metabolism*; D000243:Adenosine Deaminase / Q000378:metabolism*; D002463:Cell Membrane Permeability; D016764:Cell Polarity; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004847:Epithelial Cells / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007249:Inflammation; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008171:Lung Diseases / Q000201:enzymology / Q000473:pathology*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D012137:Respiratory System / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism
D016207:Cytokines; D007527:Isoenzymes; D026901:Membrane Transport Proteins; D018047:Receptors, Purinergic P1; C065171:cif nucleoside transporter; D000243:Adenosine Deaminase; D000241:Adenosine