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J Biol Chem, Vol. 274, Issue 17, 11701-11707, April 23, 1999

Autocrine Regulation of Volume-sensitive Anion Channels in Airway Epithelial Cells by Adenosine

Luciana Musante, Olga Zegarra-Moran, Paolo G. Montaldo^§, Mirco Ponzoni^§, and Luis J. V. Galietta

From the  Laboratory of Molecular Genetics and ^§ Laboratory of Oncology, Gaslini Institute, 16148 Genova, Italy

The activity of volume-sensitive Cl channels was studied in human tracheal epithelial cells (9HTEo) by taurine efflux experiments. The efflux elicited by a hypotonic shock was partially inhibited by adenosine receptor antagonists, by ,-methyleneadenosine 5'-diphosphate (MeADP), an inhibitor of the 5'-ectonucleotidase, and by adenosine deaminase. On the other hand, dipyridamole, a nucleoside transporter inhibitor, increased the swelling-induced taurine efflux. Extracellular ATP and adenosine increased taurine efflux by potentiating the effect of hypotonic shock. MeADP strongly inhibited the effect of extracellular ATP but not that of adenosine. These results suggest that anion channel activation involves the release of intracellular ATP, which is then degraded to adenosine by specific ectoenzymes. Adenosine then binds to purinergic receptors, causing the activation of the channels. To directly demonstrate ATP efflux, cells were loaded with [³H]AMP, and the release of radiolabeled molecules was analyzed by high performance liquid chromatography. During hypotonic shock, cell supernatants showed the presence of ATP, ADP, and adenosine. MeADP inhibited adenosine formation and caused the appearance of AMP. Under hypotonic conditions, elevation of intracellular Ca²⁺ by ionomycin caused an increase of ATP and adenosine in the extracellular solution. Our results demonstrate that volume-sensitive anion channels are regulated with an autocrine mechanism involving swelling-induced ATP release and then hydrolysis to adenosine.

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