In rat pancreatic and submandibular gland acini during exposure to carbachol, changes in the fluorescence emission intensity ratio (R) of acini loaded with mag-fura-2 resemble changes in cytosolic Ca2+ concentration (Ca2+i) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca2+o) but are much less influenced by changes in extracellular Mg2+ (Mg2+o). To evaluate interference with measurement of cytosolic Mg2+ (Mg2+i) by changes in Ca2+i, we determined the dissociation constant (Kd) and Hill coefficient (NH) of the Ca(2+)-mag-fura-2 and Mg(2+)-mag-fura-2 complexes in standard solutions, in intact acini after loading with the acetoxymethyl ester of mag-fura-2 (mag-fura-2/AM), and in lysates derived from acini loaded with mag-fura-2/AM. The Kd of the Ca(2+)-mag-fura-2 complex in acini (determined with ionomycin) was 20.49 +/- 5.20 microM, and NH was 1.44 +/- 0.16 (n = 24). Kd of the Mg(2+)-mag-fura-2 complex in acini was 2.25 +/- 0.98 mM, and NH was 1.20 +/- 0.20 (n = 25). Mean Kd values were slightly lower in acinar lysates and in solutions of standard mag-fura-2. In acini from either gland, 1,2-bis(2-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (BAPTA) suppressed carbachol-induced Ca2+i transients. The R value in stimulated acini loaded with BAPTA and mag-fura-2 increased slightly when Mg2+o was increased from less than 10 nM to 1.2 mM, suggesting that Mg2+ influx contributes to the maintenance of Mg2+i during exposure to carbachol. Under these conditions, pancreatic acinar Mg2+i is 0.53 +/- 0.14 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)
In dose-dependent fashion, extracellular ATP reduces the increase in cytosolic Ca2+ concentration ([Ca2+]o) due to mobilization of cellular Ca2+ stores by both epinephrine [half-maximal inhibitory concentration (IC50) = 35.7 +/- 12.9 microM; Hill coefficient (NH) = -2.0 +/- 0.7, n = 8] and by carbachol (IC50 = 27.0 +/- 7.0 microM, NH = -2.3 +/- 0.7, n = 9). Inhibition is due to ATP4- but does not result from any emptying or inaccessibility of Ca2+ stores, which are readily mobilized by thapsigargin in the presence of ATP4-. Reduction of Ca2+ mobilization is rapid but is not due to direct interference by ATP with the interaction of carbachol or epinephrine with their respective cell surface receptors. A benzoyl derivative of ATP, 3'-O-(4-benzoyl) adenosine 5'-triphosphate (BZATP) is more potent than ATP in reducing [Ca2+]i due to mobilization of stored Ca2+ by either carbachol or epinephrine (IC50 for carbachol = 3.9 +/- 0.4 microM, NH = -3.2 +/- 0.5; IC50 for epinephrine = 3.8 +/- 0.2, NH = -2.6 +/- 0.7, n = 3) but GTP, UTP, ADP, and adenosine do not inhibit mobilization of stored Ca2+ by either carbachol or epinephrine. Neither ATP nor BZATP prevents the influx of extracellular Ca2+ stimulated by carbachol or epinephrine. These results suggest that ATP inhibits Ca2+ mobilization by autonomic neurotransmitters after occupation of P2Z purinoceptors.
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