title: Blockade of ATP binding site of P2 purinoceptors in rat parotid acinar cells by isothiocyanate compounds.
abstract: Extracellular ATP activates a P2Z-type purinergic receptor (purinoceptor) 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 [32P]ATP to intact parotid cells (McMillian et al., Biochem J 255:291-300, 1988) and blocked the activation of the P2Z purinoceptor. The ATP-stimulated 45Ca2+ uptake was blocked by DIDS, H2DIDS (dihydro-DIDS; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid), and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid), but not by DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid), a stilbene disulfonate compound lacking isothiocyanate (SCN-) groups, or by KSCN. The potency of the stilbene disulfonates was related to the number of isothiocyanate groups on each compound. Under the experimental conditions, the IC50 value of DIDS (approximately 35 microM), which has two SCN-groups, was much lower than that of SITS (approximately 125 microM), which has only one SCN-group. The inhibitory effects of DIDS appeared to be much more potent than those of SITS due to the kinetics of their binding to the purinoceptors. Eosin-5-isothiocyanate (EITC) and fluorescein-5-isothiocyanate (FITC), non-stilbene isothiocyanate compounds with single SCN-groups, also blocked the response to ATP and were less potent than DIDS. Trinitrophenyl-ATP (TNP-ATP), an ATP derivative that is not an effective agonist of the parotid P2Z receptor, blocked the covalent binding of DIDS to the plasma membrane, suggesting that 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 the 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 the effectiveness of the stilbene disulfonate compounds in blocking the binding of nucleotide agonists to this purinoceptor.
authors.affiliation: Department of Medicine, Beth Israel Hospital, Boston, MA 02115.
authors.forename: S P
authors.initials: SP
authors.lastname: Soltoff
authors.forename: M K
authors.initials: MK
authors.lastname: McMillian
authors.forename: B R
authors.initials: BR
authors.lastname: Talamo
authors.forename: L C
authors.initials: LC
authors.lastname: Cantley
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D007473:Ion Channels / Q000378:metabolism; D017879:Isothiocyanates; D010306:Parotid Gland / Q000378:metabolism*; D058914:Purinergic Antagonists; D051381:Rats; D013329:Structure-Activity Relationship; D013861:Thiocyanates / Q000737:chemistry; D014227:Triazines / Q000494:pharmacology
chemical_list: D007473:Ion Channels; D017879:Isothiocyanates; D058914:Purinergic Antagonists; D013861:Thiocyanates; D014227:Triazines; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C037152:isothiocyanic acid; C012842:Cibacron Blue F 3GA; D000255:Adenosine Triphosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid