Abstract
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 alpha,beta-methyleneadenosine 5'-diphosphate (alphabetaMeADP), 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. alphabetaMeADP 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. alphabetaMeADP 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.
Highlights
The inhibition of taurine efflux by adenosine receptor antagonists suggested that endogenous adenosine really contributes to swellingdependent volume-sensitive organic anion channel (VSOAC) activation
It has been postulated that hypotonically induced ATP release represents an autocrine stimulus for VSOAC activation [12, 13]
Our results indicate that the hypotonic shock elicits an ATP release in 9HTEoϪ tracheal epithelial cells
Summary
In a previous work performed on a tracheal epithelial cell line, we found that extracellular ATP is a potent modulator of swelling-induced VSOAC activation [11]. This finding led us to test the hypothesis that release of endogenous ATP by the cells could be responsible for VSOAC activation during the hypotonic shock. In contrast with our results, other investigators have found that there is a release of ATP induced by cell swelling [12, 13] To reconcile these conflicting results, we have considered the possibility that ATP efflux in our cells could be masked by the rapid catabolism caused at the extracellular side of the membrane by specific ectoenzymes [14, 15]. Cells respond to osmotic stresses by transporting organic and inorganic osmolytes through the plasma mem-
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