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'-tri phosphate (ATP) receptors on the basolateral membranes of frog skin epithel ial cells. Cytosolic calcium ([Ca2+](i)) was measured with fura-2 and Calci um-Green-1 using epifluorescence microscopy and confocal laser scanning mic roscopy respectively. In the presence of Ca2+ in the solutions ATP increase d [Ca2+](i). The increase in [Ca2+](i) was due to the agonist activity of A TP and not to the activity of the potential products of ATP metabolism, i.e . adenosine 5'-diphosphate (ADP): adenosine 5'-monophosphate (AMP) or adeno sine, 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 concentrat ions the initial spike was followed by a plateau phase. In the absence of C a2+ in the extracellular solution ATP caused Ca2+ release from intracellula r stores. This could be inhibited by pre-treatment of the tissue with 1 mu M thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. T he 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 layer s of the epithelium responded to ATP. Our data indicates that serosal ATP a cts on serosal P2Y-type receptors in frog skin epithelium. This is the firs t evidence of a phospholipase C-coupled receptor in this tissue.