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

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 mu M) 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 mu M 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.