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Using Mg2+ macroelectrodes based on the sensor ETH 7025 and accurate Mg2+-EDTA buffer solutions, the apparent Mg2+-ATP dissociation constant (Kapp) was measured at 25 and 37 degrees C in background solutions mimicking the cationic intracellular milieu of muscle cells. The mean +/- SD (in microM) at 25 degrees C was 157.0 +/- 13 (n = 4), 127.5 +/- 12.0 (n = 11), 101.0 +/- 9.0 (n = 4) and at 37 degrees C was 106.6 +/- 9.6 (n = 4), 87.4 +/- 4.9 (n = 4), 78.1 +/- 2.0 (n = 4) at pH values of 6.7, 7.2, and 7.7, respectively. The dependence of Kapp at 25 degrees C on the ionic strength was also measured, the mean +/- SD (microM) being 61.9 +/- 2.2 (n = 3), 127.5 +/- 12 (n = 11), and 243.0 +/- 11.8 (n = 3) at ionic strengths of 0. 087, 0.156 (normal background), and 0.3 m, respectively. These values are larger than the Kapp values most commonly used in the literature (87.4 microM compared to 38 microM at pH 7.2 and 37 degrees C) to estimate the [Mg2+]i in 31P NMR experiments, attributed to the difficulties in setting the [Mg2+]i without the use of Mg2+ buffer solutions. If these new values are used, the literature values for [Mg2+]i estimated by 31P NMR increase by a factor of around 1.5, making them similar to values obtained by direct Mg2+ microelectrode measurements., (Copyright 1997 Academic Press.)

Using Mg(2+)-selective macroelectrodes based on the neutral carriers ETH 7025 and ETH 5506, methods were developed to determine accurately the apparent binding constant (Kapp) and purity, and hence the ionized magnesium concentration ([Mg2+]), in Ca(2+)-free, Mg2+ buffer solutions manufactured with either CDTA (trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid monohydrate) or EDTA. In nominally Ca(2+)-free solutions, calibration of the macroelectrodes was possible down to 1 mumol l-1 in both intracellular (ETH 7025)- and extracellular (ETH 5506)-like physiological solutions. The measured [Mg2+] in the buffer solutions overlapped with the [Mg2+] set by dilution alone, suggesting that the method was reliable. These buffer solutions could then be used to manufacture standard Mg2+ solution containing known [Mg2+] at a set calcium concentration. Ca2+ sensitivity limits the use of ETH 7025 and, for extracellular measurements in Ca(2+)-containing solutions, Mg(2+)-selective macroelectrodes manufactured with ETH 5506 were the electrodes of choice. In 0.5-0.9 mmol l-1 Ca+, measurement of [Mg2+] was possible down to 10 mumol l-1. At [Mg2+] greater than 0.25 mmol l-1 there was no interference from Ca2+ (0.5-1.5 mmol l-1). With CDTA and/or EDTA buffer solutions there was a wide variation between the calculated values for the [Mg2+] and the measured [Mg2+] (the calculated values differed by a factor of up to 4.5 and 3, respectively). At present, measurement of the Kapp and ligand purity in the appropriate solution at the desired pH and temperature would seem to be the best strategy to adopt rather than attempting to calculate the constant. Since no recognized international standard exists for [Mg2+] at the micromolar level, values in the literature for Kd, etc. in this range can only be regarded as approximate.

1. Respiratory alkalosis accompanies the clinical syndrome of tetany, precipitates cardiac arrhythmias and predisposes to coronary vasoconstriction. Magnesium plays a critical role in the maintenance of membrane function, and magnesium depletion is often associated with cardiac arrhythmias or vasoconstriction. 2. As technology for detecting circulating ionized magnesium (the most interesting form with respect to physiological and biological properties) is now available in the form of new magnesium-selective electrodes, the effect of respiratory alkalosis induced by voluntary overbreathing for 30 min on circulating ionized magnesium was studied in eight healthy subjects. 3. The total plasma magnesium concentration was not modified by hyperventilation. On the contrary, hyperventilation was associated with a significant reduction in the ionized magnesium concentration of 0.05 (0.02-0.15) mmol/l (median and range) and in the free magnesium fraction of 0.06 (0.01-0.19). During hyperventilation the relative intravascular magnesium mass, calculated from changes in total plasma magnesium concentration and haematocrit, decreased significantly. 4. It is concluded that acute overbreathing reduces the circulating ionized magnesium concentration and the intravascular magnesium mass. It is therefore conceivable that extracellular magnesium deficiency is at least a subsidiary cause of the syndrome of tetany and the cardiac complications that are precipitated by hyperventilation.

Human erythrocytes either untreated or Mg2+(-)loaded by means of A23187 in the presence of 3 or 12 mM MgCl2 were haemolysed by freezing and thawing. In the haemolysates the concentration of total Mg2+ (by atomic absorption spectrophotometry), free Mg2+ (by a Mg2+(-)sensitive electrode) and ATP were determined. The increase in the free Mg2+ concentration was also measured when titrating the haemolysates with MgCl2. The experiments yielded a total Mg2+ buffer capacity of 4.85mM with a Ka of 0.9 mM-1, indicating that 2,3 bisphosphoglycerate was the main Mg2+ buffer in haemolysates of erythrocytes which had been ATP-depleted during preparation. The activity coefficient of free Mg2+ in erythrocytes was the same as in the chloride-based calibration solutions.

The polyamines spermine, spermidine and putrescine interact with Mg(2+)-sensitive macroelectrodes based on the neutral carrier ETH 7025; the interaction in decreasing order being spermine, spermidine and putrescine. The effect is small and dependent on the ionized magnesium concentration ([Mg2+]free), only resulting in a significant increase in the measured [Mg2+]free with spermine and spermidine at [Mg2+]free less than 0.5 mM. The polyamines compete with Mg2+ for common binding sites on Mg2+ buffers such as ATP, ADP and citrate, releasing bound Mg2+ and increasing the [Mg2+]free. This effect is most prominent with Mg ATP and the action of the polyamines in decreasing order is spermine, spermidine and putrescine. Increases in polyamines which occur in cell proliferation and cancer could thus secondarily increase the [Mg2+]free resulting in an activation of Mg(2+)-dependent metabolic pathways.

Because of the reported presence of a Na(+)-Mg2+ exchanger in guinea-pig but not in ferret myocardium, the Mg2+ extrusion mechanism in guinea-pig myocardium has been reinvestigated using Mg(2+)- and Na(+)- selective microelectrodes and the fluorochromes mag-fura-2 and -5. The mean [Mg2+]i measured with microelectrodes in trabeculae or papillary muscles was 0.72 mmol/l (n = 22, thirteen experiments; range 0.42-1.23 mmol/l). Increasing [Mg2+]o from 0.5 mmol/l to either 10.5 or 20 mmol/l caused small increases in [Mg2+]i. Decreasing [Na+]o by 50% had no effect on the [Mg2+]i and there was no change in [Na+]i on increasing [Mg2+]o from 0.5 to 10.5 mmol/l. Varying pHo or changing pHi with NH4Cl did not influence the [Mg2+]i. In vitro calibration of mag-fura-2 and -5 using the ratio method gave values for K'd (experimentally determined dissociation constant) of 22.2 +/- 2.7 (mean +/- S.D., n = 7) and 25.7 +/- 1.3 (n = 4) mmol/l respectively. Mag-fura-2 reacted to physiological concentrations of Ca2+ and mag-fura-5 to changes in pH. In isolated myocytes, Na+ removal gave an apparent increase of [Mg2+]i with mag-fura-2 but not with mag-fura-5. However, when the pHi was altered with NH4Cl mag-fura-5 showed an apparent decrease in [Mg2+]i on application and an apparent increase on removal, with a time course similar to the pHi changes. It is concluded that Mg2+ extrusion in guinea-pig myocardium is not via a Na(+)-Mg2+ exchanger. The use of mag-fura-2 and -5 are limited in their application because of Ca2+ and H+ sensitivity respectively.

The new fluorescent indicator, mag-fura-5, was evaluated for its ability to measure accurately physiological changes in cytosolic free magnesium. The apparent dissociation constants (Kd) of the fluorochrome for Mg2+, Mg2+/EGTA and Ca2+/EGTA solutions were 14.7 mM, 15.4 mM, and 1.8 mM respectively. The calculated difference in the fluorescence ratios and in the resulting pMg between the standards with low-Ca2+ or low H+ backgrounds and the corresponding samples with approximately physiological levels were not significant. In contrast, the changes due to an increased Ca2+ or H+ content were statistically significant, with mean pMg differences of 0.10 +/- 0.09 (P < 0.02) and 0.33 +/- 0.26 (P < 0.01) respectively. Repetitive measurements on 3 consecutive days yielded comparable data with differences not exceeding 4%. Because of the good reproducibility, it is suggested that the new fluorescent probe may be suitable for free cytosolic magnesium determinations in isolated cells.

Ion-sensitive microelectrodes (ISEs) have been used to measure intracellular [Mg2+] ([Mg2+]i) in cardiac muscle, although most measurements have tended to overestimate the value due to the poor selectivity of the Mg2+ ionophore in the sarcoplasm and to inaccurate collation of individual ISE measurements. This paper highlights the correct method for analysis of data from multiple ISE experiments. Since [Mg2+]i is constrained at a lower concentration than would be expected by passive distribution of the ion, some of the possible mechanisms underlying Mg2+ extrusion from ferret ventricular myocardium were investigated. During elevation of the extracellular [Mg], mean [Mg2+]i rose from 1.61 to 1.91 mM. The same intervention had no significant effect on membrane potential, intracellular [Na+] or pH measured with ISEs, and there was no change in resting [Ca2+], as assessed from fura-2 fluorescence. The data are not consistent with a simple mechanism for Na(+)-Mg2+ exchange as the primary mode of Mg2+ regulation in cardiac muscle or with an Mg2+ extrusion mechanism involving steady-state ion exchange.

Intracellular free magnesium ([Mg2+]i) was measured in isolated ferret papillary muscles using ion-selective microelectrodes filled with the new magnesium sensor ETH 5214. This new sensor, unlike its predecessor ETH 1117, does not react to marked changes in K+, Na+ or pH. Reducing Ca2+ from 20 microM to around 10 nM also did not affect the response so these electrodes are ideally suited to study intracellular Mg2+ and its regulation. The mean value for the [Mg2+]i from thirty-two experiments (forty-two impalements) was 0.85 mM, confirming previous estimates from this laboratory. Intracellular Mg2+ is not passively distributed and the possibility that Mg2+ is transported out of the cell by a Na(+)-Mg2+ exchanger was investigated. An increase in [Mg2+]o caused an increase in [Mg2+]i, as did stepwise reduction in the [Na+]o. However, this increase in [Mg2+]i on Na+ reduction also occurred in Mg2(+)-free solution suggesting that the increase in [Mg2+]i was due to the increase in intracellular Ca2+ on Na+ reduction. Moreover, increasing [Na+]i by strophanthidin did not change the [Mg2+]i and on increasing [Mg2+]o there was no reduction in the [Na+]i. Blocking ATP production lead to small increases in the [Mg2+]i. These results are not consistent with a Na(+)-Mg2+ exchanger as being the main outward transport mechanism for Mg2+ in this tissue.

The free Mg concentration ([Mg]i) was measured in ferret ventricular muscle using single-barrelled Mg-sensitive micro-electrodes. The electrodes were calibrated in solutions containing the [K]i and [Na]i measured in this tissue. In thirteen measurements from seven experiments conforming to strict criteria for calibration and penetration the estimated mean [Mg]i was 0.4 mmol/l. This is to be regarded as an upper limit since it was estimated by linear extrapolation of the calibration curve.

Determination of intracellular free magnesium [( Mg]i) with ion-selective Mg microelectrodes filled with the neutral carrier ETH 1117 is critically dependent on the values taken for K and Na in the calibrating solutions, for intracellularly the Mg microelectrodes also respond to these ions. This study was designed to test the influence of K and Na on the measured [Mg]i in ferret ventricular muscle. To estimate the effect of these ions, two methods were used: calculation based on the Nicolsky-Eisenman equation and calibration of the electrodes in solutions containing various concentrations of K and Na. Selectivity coefficients were measured for K and Na by the fixed interference method. While the Nicolsky-Eisenman equation could describe the behavior of an electrode in a given calibrating solution, the effects of changes in either the concentration of K and/or Na could not be predicted by the equation. Comparison between calibrating solutions containing the mean values for K and Na and calibrating solutions containing the lowest measured values for K and Na showed that the mean [Mg]i in ferret ventricle would increase from 0.4 to 0.9 mmol/l. A critical appraisal of the literature values for [Mg]i in muscle tissue suggests that values over 2 mmol/l were either unphysiological or too high for methodological reasons.