2 DISTINCT CYTOSOLIC CALCIUM RESPONSES TO EXTRACELLULAR ATP IN RAT PAROTID ACINAR-CELLS

1 Increasing concentrations of ATP (0.5 muM-300 muM) produced a biphasic increase in intracellular calcium concentration [Ca]i in rat parotid acinar cells, reflecting two distinct Ca(i) responses to extracellular ATP. 2 In the absence of Mg2+ (with 3 mm CaCl2 in the buffer solution), the more sensitive response was maximal at 3-5 muM and was not further increased by 30 muM ATP. This response to ATP was not well maintained and was blocked by ADP (0.5 mm). A second, much larger increase in Ca(i) was observed on addition of 300 muM ATP. This larger effect, which we have described previously, appears to be mediated by ATP4-, and was selectively reversed by 4,4'-di-isothiocyanato-dihydrostilbene-2,2'-disulphonate as well as by high concentrations of alpha,beta-methylene ATP. 3 Among ATP analogues, only the putative P2Z agonist, 3'-0-(4-benzoyl)benzoyl-ATP distinguished between the two responses. This analogue was at least 10 fold more potent than ATP in stimulating the ATP4--response, but did not evoke the more sensitive response. The agonist potency series for both responses to ATP was identical for other analogues examined (ATP > ATPgammaS = 2-methylthio ATP (a P2y-selective agonist) much greater than ADP, ITP and alpha,beta-methylene ATP (a P2X-selective agonist)). 4 Although the effect of ATP4- could best be characterized as a P2Z-type purinoceptor response, this effect was strongly and selectively blocked by reactive blue 2, a putative P2y-purinoceptor antagonist. Reactive blue 2 may bind to and block P2Z purinoceptors since [gammaP-32]-ATP binding to parotid cells was inhibited by this compound. 5 In contrast to the response to ATP4-, the more sensitive response to ATP was potentiated by reactive blue 2 and was less affected by increases in external Mg2+ and Ca2+. 6 Parasympathetic denervation selectively increased the more sensitive response, suggesting that it may be physiologically regulated.