CHANGES OF CYTOSOLIC CA-2+ INTERFERE WITH MEASUREMENTS OF CYTOSOLIC MG-2+ USING MAG-FURA-2

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 (Ca(i)2+) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca(o)2+) but are much less influenced by changes in extracellular Mg2+ (Mg(o)2+). To evaluate interference with measurement of Cytosolic Mg2+ (Mg(i)2+) by changes in Ca(i)2+, we determined the dissociation constant (K(d)) and Hill coefficient (N(H)) of the Ca2+-mag-fura-2 and Mg2+-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 K(d) of the Ca2+-mag-fura-2 complex in acini (determined with ionomycin) was 20.49 +/- 5.20-mu-M, and N(H) was 1.44 +/- 0.16 (n = 24). K(d) of the Mg2+-mag-fura-2 complex in acini was 2.25 +/- 0.98 mM, and N(H) was 1.20 +/- 0.20 (n = 25). Mean K(d) 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 Ca(i)2+ transients. The R value in stimulated acini loaded with BAPTA and mag-fura-2 increased slightly when Mg(o)2+ was increased from <10 nM to 1.2 mM, suggesting that Mg2+ influx contributes to the maintenance of Mg(i)2+ during exposure to carbachol. Under these conditions, pancreatic acinar Mg(i)2+ is 0.53 +/- 0.14 mM (n = 5). In submandibular gland acini, under similar conditions Mg(i)2+ is 0.48 +/- 0.11 mM (n = 5). We conclude that changes in Ca(i)2+ are readily detected in mag-fura-2-loaded cells, especially when Mg(i)2+ is relatively stable. This may limit the conditions under which Mg(i)2+ can be reliably determined using mag-fura-2.