Mg2+ buffering in cultured chick ventricular myocytes: quantitation and modulation by Ca2+

Abstract

To characterize the Mg2+ buffering of cultured chick ventricular myocytes, cytosolic Mg2+ was increased by liberating Mg2+ normally chelated by ATP upon total depletion of ATP content. Because the total Mg content and cell volume remained constant during this time, the difference between the amount of Mg2+ liberated (2.7 mM) and the 0.9 mM increase in cytosolic Mg2+ activity measured fluorometrically with mag-fura-2 indicates a sizable Mg2+ buffering. A new term, the Mg2+ buffer coefficient (BMg), was derived to quantify this buffering. We also determined that cytosolic Mg2+ activity increased by only 0.6 mM in cells acutely exposed to zero external Ca2+ during ATP depletion. In the absence of extracellular Ca2+, the basal cytosolic Ca2+ activity (alpha Ca2+i) was reduced by 72%, whereas the increase in alpha Ca2+i induced by ATP depletion was substantially blunted; no difference in either the time course of adenine nucleotide changes or the Ca and Mg content was observed. The BMg value calculated for these cells indicates that Mg2+ buffering is substantially greater in the absence of extracellular Ca2+ (2.5) than when extracellular Ca2+ is present (1.4), indicating that alpha Ca2+i affects cytosolic Mg2+ activity in ventricular myocytes. Therefore the Mg2+ buffering of ventricular myocytes appears to be comprised of at least two components: 1) a Ca(2+)-insensitive adenine nucleotide pool and 2) a Ca(2+)-sensitive nonadenine nucleotide pool.

Mechanism of oxidative stress‐induced intracellular acidosis in rat cerebellar astrocytes and C6 glioma cells

“…from 2 to 4 mÒ) seen following treatment with IAA and HµOµ is probably due to the existence of intracellular Mg¥ buffers, e.g. phopholipids and proteins (Koss, Putnam & Grubbs, 1993). Contamination of the mag-fura_2 signal by changes in [Ca¥]é can be ruled out since only a very slight change in [Ca¥]é was seen on addition of 100 ìÒ HµOµ (Fig.…”

“…This is further strong evidence that HµOµ may cause partial depletion of glycolytic ATP, resulting in hydrolysis-induced acid production. Assuming the resting [ATP]é and [ADP]é to be •4 and 0·4 mÒ, respectively (Table 1 in Koss et al 1993), hydrolysis of 50% of the cellular content of adenine nucleotide will produce a proton concentration of 4·4 mÒ (assuming 1 H¤ is produced per phosphate hydrolysed and hydrolysis of 2 mÒ ATP and 0·4 mÒ ADP to AMP and phosphate). At pHé 6·8 (i.e.…”

Mg‐Atp Binding: Its Modification by Spermine, the Relevance to Cytosolic Mg2+ Buffering, Changes in the Intracellular Ionized Mg2+ Concentration and the Estimation of Mg2+ by 31P‐NMR

“…Grubbs and colleagues (Koss et al 1993;Grubbs & Walter, 1994) have attempted to estimate the muffling coefficient by decreasing the cellular ATP to increase the intracellular ---------------------------------------------------------------------- Schwiening & Thomas (1996) - …”

“…This is illustrated in Fig. 4A, which is a plot of the magnesium titration curves for 10 mmol l¢ ATP (normal value in heart ;Geisbuhler et al 1984) and also, for comparison, for 20 mmol l¢ ATP, at 25°C and a pH of 7·3, using the measured Kapp ) and other intracellular binding sites (Koss et al 1993), this rise will be the upper limit. Decreasing Each of these will now be considered in turn.…”

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

“…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).…”