BLOCKADE OF ATP BINDING-SITE OF P(2) PURINOCEPTORS IN RAT PAROTID ACINAR-CELLS BY ISOTHIOCYANATE COMPOUNDS

Extracellular ATP activates a P2Z-type purinergic receptor (purinocept or) in rat parotid acinar cells that increases the intracellular free Ca2+ concentration via the entry of extracellular Ca2+ through an ATP- sensitive cation channel (Soltoff et al., Am J Physiol 262: C934-C940, 1992). To learn more about the ATP binding site of the purinoceptor, we examined the effects of several stilbene isothiocyanate analogs of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), which block the binding of [P-32]ATP to intact parotid cells (McMillian et al., B iochem J 255: 291-300, 1988) and blocked the activation of the P2Z pur inoceptor. The ATP-stimulated Ca-45(2+) uptake was blocked by DIDS, H2 DIDS (dihydro-DIDS; 4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid), and SIT'S cetamido-4'-isothiocyanatostilbene-2,2'-disulfonic a cid), but not by DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid), a s tilbene disulfonate compound lacking isothiocyanate (SCN-) groups, or by KSCN. The potency of the stilbene disulfonates was related to the n umber of isothiocyanate groups on each compound. Under the experimenta l conditions, the IC50 value of DIDS (approximately 35 muM), which has two SCN-groups, was much lower than that of SITS (approximately 125 m uM), which has only one SCN- group. The inhibitory effects of DIDS app eared to be much more potent than those of SITS due to the kinetics of their binding to the purinoceptors. Eosin-5-isothiocyanate (EITC) and fluoroscein-5-isothiocyanate (FITC), non-stilbene isothiocyanate comp ounds with single SCN- groups, also blocked the response to ATP and we re less potent than DIDS. Trinitrophenyl-ATP (TNP-ATP), an ATP derivat ive that is not an effective agonist of the parotid P2Z receptor, bloc ked the covalent binding of DIDS to the plasma membrane, suggesting th at ATP and DIDS bind to the same site. Reactive Blue 2 (Cibacron Blue 3GA), an anthraquinone-sulfonic acid derivative that is a noncovalent purinergic antagonist, also blocked the covalent binding of DIDS to th e plasma membrane. These results suggest that isothiocyanate compounds interact with the ATP binding site of this P2 purinoceptor, and that isothiocyanate groups make an important contribution in determining th e effectiveness of the stilbene disulfonate compounds in blocking the binding of nucleotide agonists to this purinoceptor.