The broad, long-term objective of this proposal is to understand on a molecular level the mechanism by which extracellular ATP (adenosine 5'- triphosphate) promotes receptor-mediated alterations of the ion transport events involved in saliva formation (fluid secretion) by the parotid acinar cell. the parotid cell secretes an isotonic primary fluid that is modified by the salivary ductal system, which adds and removes electrolytes. Secretory events are controlled by the release of neurotransmitters, and the parotid receives parasympathetic and sympathetic innervation. The former is believed to regulate fluid secretion, while the latter is involved in the stimulation of exocytosis and amylase release. ATP is costored and coreleased with neurotransmitters, and it may also act as a neurotransmitter. We found that ATP has a similar effect to muscarinic and other phospholipase C- linked agonists in activating the Ca2+-sensitive ion transport mechanisms involved in fluid secretion. However, it does this by a receptor- operated ion channel, and this is different from the mechanism of action of sympathetic and parasympathetic agonists. We found that a specific type of purinergic receptor, P2Z, is present on parotid acinar cells, and propose that when ATP binds to these receptors it activates fluid secretion by a pathway that is separate from other activation pathways in these cells. In this proposal, I will investigate the regulation of the ATP-activated ion current in parotid acinar cells, including determining whether a GTP-dependent protein is involved in its activation. We have identified a number of covalent (4,4'- diisothiocyanatostilbene- 2,2'-disulfonic acid [DIDS]) and noncovalent (Reactive Blue 2, Coomassie Blue) inhibitors of the physiological effects of ATP, including the ATP-stimulated entry of 45Ca2+. Using this assay, I will determine structural features that are important to the binding of these inhibitors to the P2Z receptor, and investigate their competition with ATP to determine if they are P2Z antagonists. In addition, I will use these inhibitors in binding studies using ATPalphaS (an effective P2Z agonist) to define pharmacological criteria for identifying the P2Z receptor. I will also attempt to covalently label the P2Z receptor by cross-linking ATPalpha[35S] to it. Together, these studies will provide a more complete understanding of the pharmacological and biochemical nature of this receptor and its involvement in fluid secretion and saliva formation.