Changes of cytosolic Ca2+ interfere with measurements of cytosolic Mg2+ using mag-fura-2

Abstract

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)

Cytosolic-free calcium increases to greater than 100 micromolar in ATP-depleted proximal tubules.

“…The increases substantially exceeded the values measured in the presence of 20 mM Mg 2ϩ and A23187. Because the timing of these changes in fluorescence was similar to those seen with the Ca 2ϩ indicator, fura-2 (12), and, in view of the reported sensitivity of magfura-2 to Ca 2ϩ (27,28,(30)(31)(32)(33), we then tested the effect of introducing ionomycin during the initial period of superfusion with regular medium containing Ca 2ϩ and Mg…”

“…The affinity of mag-fura-2 for Ca 2 ϩ is substantially greater than its affinity for Mg 2 ϩ , but much less than the affinity of fura-2 for Ca 2 ϩ (27)(28)(29). This property has allowed use of mag-fura-2 in a number of systems to quantify Ca f increases well into the M range (28,(30)(31)(32)(33). Mag-fura-2 has also been used under conditions of ATP depletion to assess release of Mg 2 ϩ associated with hydrolysis of MgATP (33)(34)(35)(36).…”

Regulation by extracellular Na+ of cytosolic Mg2+ concentration in Mg2+‐loaded rat sublingual acini

“…The purpose of this treatment was to prevent the toxic effects of continuous [Ca2+]~ elevation and to avoid the interference of Ca 2+ on [Mg2+]i measurements [24][25][26]. Ca 2+ interference of the [Mg2+]i determination with mag-fura-2 has been reported in several cell systems [24,25] and can become a major concern [25,26]. Hurley et al [25] reported that in exocrine pancreatic and submandibular acini, Ca 2+ significantly interferes with the mag-fura-2 fluorescence.…”

“…Ca 2+ interference of the [Mg2+]i determination with mag-fura-2 has been reported in several cell systems [24,25] and can become a major concern [25,26]. Hurley et al [25] reported that in exocrine pancreatic and submandibular acini, Ca 2+ significantly interferes with the mag-fura-2 fluorescence. Indeed, we tested the contribution of Ca 2+ interference on mag-fura-2 fluorescence and found that when the Mg 2+ concentration is physio- In MDCK cells, Mg 2+ influx is mediated by a unique Mg z+ entry pathway which manifests similar characteristics to voltage-gated Ca 2+ channels [30].…”

Intracellular magnesium transport and regulation in epithelial secretory cells

“…The affinity of Magfura-2 for Ca 2+ is not very different from that for Mg 2+ (29) and several authors have indicated the existence of a clear interference from this source (54,55 (40,41) or single cell studies (46,51). In addition, different approaches can be undertaken, including perfusion of the tissue (28,46) or the employment of static-volume cuvettes (42,43).…”