PURINERGIC RECEPTOR ACTIVATION OF CL- SECRETION IN T84 CELLS

The regulation by ATP of Cl- secretion in T84 cells grown on filters was investigated by measuring short-circuit current (I(sc) = net Cl- secretion). ATP (greater-than-or-equal-to 10-mu-M) added to the basolateral side markedly stimulated I(sc) both in the presence and absence of forskolin-activated I(sc). Fluorescence microscopy of cells loaded with the Ca2+ indicator fura-2 showed that ATP stimulated a transient increase in intracellular free Ca2+ concentration [Ca2+]i. The augmentation of forskolin-stimulated I(sc) by ATP was at least partly caused by mobilization of Ca2+ from an internal store because prior depletion of the store using ionomycin prevented the response. The activity sequence for stimulation of I(sc) in the presence of forskolin was adenosine 5'-O-(3-thiotriphosphate) = 5'-adenylylimidodiphosphate (AMP-PNP) > ATP > ADP > AMP, suggesting the presence of a P2 purinergic receptor. Neither beta,gamma-methyleneadenosine 5'-triphosphate nor alpha,beta-methyleneadenosine 5'-triphosphate increased the I(sc). Stimulation of I(sc) by ATP in the absence of forskolin was at least partly due to the breakdown of ATP to AMP and adenosine, which act at P1 receptors to stimulate I(sc), since 1) inhibition of the ecto-phosphohydrolase 5'-nucleotidase by alpha,beta-methylene-ADP partially inhibited stimulation of I(sc) by ATP, 2) the adenosine receptor antagonists caffeine and 8-phenyltheophylline markedly inhibited the ATP-stimulated I(sc), and 3) AMP-PNP, a weakly hydrolyzable analogue of ATP, caused a much smaller increase in I(sc) compared with ATP. Adenosine had no effect on [Ca2+]i. Measurement by high-performance liquid chromatography analysis of nucleotides in the extracellular medium bathing the basolateral surface showed directly that ATP was hydrolyzed to ADP, AMP, and adenosine. Therefore, at least two different signal transduction pathways are involved in the action of ATP. It is concluded that T84 cells possess both P1 and P2 purinergic receptors and that ATP may activate both by directly binding to the P2 receptor and indirectly by its conversion to AMP and adenosine which bind to the P1 receptor.