Evidence for P₂Y-type ATP receptors on the serosal membrane of frog skin epithelium

Abstract

The present study presents the first evidence for P₂Y-type adenosine 5'-triphosphate (ATP) receptors on the basolateral membranes of frog skin epithelial cells. Cytosolic calcium ([Ca²⁺]_i) was measured with fura-2 and Calcium-Green-1 using epifluorescence microscopy and confocal laser scanning microscopy respectively. In the presence of Ca²⁺ in the solutions ATP increased [Ca²⁺]_i. The increase in [Ca²⁺]_i was due to the agonist activity of ATP and not to the activity of the potential products of ATP metabolism, i.e. adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) or adenosine, as shown by a comparison of the magnitude of the increases in [Ca²⁺]_i caused by the various compounds. The rise in [Ca²⁺]_i was predominantly monophasic at low ATP concentrations (below 100 µM). At higher concentrations the initial spike was followed by a plateau phase. In the absence of Ca²⁺ in the extracellular solution ATP caused Ca²⁺ release from intracellular stores. This could be inhibited by pre-treatment of the tissue with 1 µM thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. The nucleotide uridine 5'-triphosphate (UTP) had similar effects on [Ca²⁺]_i although the plateau level of the [Ca²⁺]_i response was higher with this P₂Y agonist. Confocal laser scanning microscopy showed that all cell layers of the epithelium responded to ATP. Our data indicates that serosal ATP acts on serosal P₂Y-type receptors in frog skin epithelium. This is the first evidence of a phospholipase C-coupled receptor in this tissue.