P2 receptor-mediated signal transduction in Ehrlich ascites tumor cells

Abstract

The mechanisms, by which the P2 receptor agonists adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP) evoke an increase in the free cytosolic calcium concentration ([Ca 2+] i) and in intracellular pH (pH i), have been investigated in Ehrlich ascites tumor cells. The increase in [Ca 2+] i evoked by ATP or UTP is abolished after depletion of intracellular Ca 2+ stores with thapsigargin in Ca 2+-free medium, and is inhibited by U73122, an inhibitor of phospholipase C (PLC), indicating that the increase in [Ca 2+] i is primarily due to release from intracellular, Ins(1,4,5)P 3-sensitive Ca 2+ stores. ATP also activates a capacitative Ca 2+-entry pathway. ATP as well as UTP evokes a biphasic change in pH i, consisting of an initial acidification followed by alkalinization. Suramin and 4,4′-diisothiocyano-2,2′-stilbene-disulfonic acid (DIDS) inhibit the biphasic change in pH i, apparently by acting as antagonists at P2 receptors. The alkalinization evoked by the P2 receptor agonists is found to be due to activation of a 5′-( N-ethyl- N-isopropyl)amiloride (EIPA)-sensitive Na +/H + exchanger. ATP and UTP elicit rapid cell shrinkage, presumably due to activation of Ca 2+ sensitive K + and Cl − efflux pathways. Preventing cell shrinkage, either by incubating the cells at high extracellular K + concentration, or by adding the K +-channel blocker, charybdotoxin, does not affect the increase in [Ca 2+] i, but abolishes the activation of the Na +/H + exchanger, indicating that activation of the Na +/H + exchanger is secondary to the Ca 2+-induced cell shrinkage.