Regulation of transcervical permeability by two distinct P2 purinergic receptor mechanisms

Micromolar concentrations ofATP stimulate biphasic change in transepithelial conductance acrossCaSki cultures, an acute increase (phase I response) followed by aslower decrease (phase II response). Phase I andphase II responses involve two distinct calcium-dependentpathways, calcium mobilization and calcium influx. To test thehypothesis that phase I and phase II responsesare mediated by distinct P2 purinergic receptors, changes inpermeability were uncoupled by blocking calcium mobilization with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(BAPTA) or by lowering extracellular calcium, respectively. Under theseconditions ATP EC₅₀ was 25 µM for phase Iresponse and 2 µM for phase II response. The respectiveagonist profiles were ATP > UTP > adenosine5'-O-(3-thiotriphosphate) (ATP-S) = N⁶-(6-aminohexyl]carbamoylmethyl)adenosine5'-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-S. Suramin blocked phase Iresponse and ATP-induced calcium mobilization, whereas pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid (PPADS) blocked phase II response and ATP-augmented calcium influx. ATP time course andpharmacological profiles for phase II response and augmented calcium influx were similar, with a time constant of 2 min and asaturable concentration-dependent effect (EC₅₀ of 2-3µM). RT-PCR experiments revealed expression of mRNA for both theP2Y₂ and P2X₄ receptors. These results suggestthat the ATP-induced phase I and phase IIresponses are mediated by distinct P2 purinergic receptor mechanisms.