The Na/Ca exchange system was investigated in rapidly perfused small trabeculae (diameter around 250 micron) from ferret ventricle. In these preparations tension and Em were recorded, aiNa, aiK and aiMg were measured using ion-selective... more
The Na/Ca exchange system was investigated in rapidly perfused small trabeculae (diameter around 250 micron) from ferret ventricle. In these preparations tension and Em were recorded, aiNa, aiK and aiMg were measured using ion-selective microelectrodes. The mean aiNa at room temperature (22-24 degrees C) was 10.9 mmol/l +/- S.D. 4.2 mmol/l. When [Na]o was reduced from 155 mmol/l to 1.5 mmol/l there was a rapid decrease in aiNa (half time 30 s), which was associated with a small increase in resting tension. The decrease in aiNa was not influenced by altering pHo, blocking the Na/K pump and was not passive, for during the decrease the driving force for Na ions was inward. The aiNa decreased if [Ca]o was increased or [Na]o was decreased or vice versa. On the basis of these findings it was concluded that the decrease was due to Na/Ca exchange. Since Ca uptake does occur under Na free perfusion and since [Ca]i hardly changes it was proposed that the mitochondria take up Ca and so prevent an excessive rise in cytoplasmic calcium. The Ca uptake mechanism of the mitochondria is inhibited by [Mg]i, so [Mg]i was measured using ion selective microelectrodes. An essential preliminary step was the determination of aiK since the Mg microelectrodes react not only to Mg but also to Na and K. The mean aiK was 104.4 mmol/l +/- S.D. 9.0 mmol/l. Based on the measured aiNa and aiK, the [Mg]i was 0.4 mmol/l, a level not high enough to markedly inhibit the Ca uptake mechanism of the mitochondria. Criteria for judging the impalement quality when using ion selective microelectrodes are discussed.
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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... more
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.
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1. The effect of [K]0 on the current-voltage relationship is described. In the negative potential range the curves cross over as [K]0 is increased. At positive potentials the curves re-cross so that in low [K]0 there is more outward... more
1. The effect of [K]0 on the current-voltage relationship is described. In the negative potential range the curves cross over as [K]0 is increased. At positive potentials the curves re-cross so that in low [K]0 there is more outward current than in high [K]0. 2. Chord conductance has been calculated from the current-voltage relationship and this is taken as a measure of gK. It is shown gK is a function of both the potassium driving force (EM--EK) and [K]0. 3. Current-voltage relationships obtained by the voltage clamp technique have been compared to net current-voltage relationships obtained by phase plane analysis of the action potential. [K]0 is shown to have similar effects on both. 4. The effect of [K]0 at positive potentials suggests that delayed outward current during large depolarizing voltage clamp steps is due to an activation of a time-dependent outward current and not to potassium accumulation. An analysis of current tails also suggests the presence of a time-dependent outward current. 5. In contrast delayed outward current changes during small depolarizing voltage clamp steps are probably due to potassium accumulation. 6. Evidence is presented which indicates that inward current tails following depolarizing voltage clamp steps are due to potassium accumulation.
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ABSTRACT
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... more
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.
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ABSTRACT
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The apparent dissociation constants (Kapp) and total ligand concentrations ([Ligand]T) from extensive published and unpublished macroelectrode measurements for Mg2+/ATP, Mg2+/EDTA and Ca2+/EGTA buffers have been recalculated. These... more
The apparent dissociation constants (Kapp) and total ligand concentrations ([Ligand]T) from extensive published and unpublished macroelectrode measurements for Mg2+/ATP, Mg2+/EDTA and Ca2+/EGTA buffers have been recalculated. These calculations were made feasible by the introduction of an Excel program which reduced the time of calculation for Kapp and [Ligand]T from over an hour to under five minutes. These estimations of Kapp and [Ligand]T allowed, not only a comparison between measured and calculated ionised magnesium and calcium concentrations ([Mg2+] and [Ca2+]) for Mg2+/ATP, Mg2+/EDTA and Ca2+/EGTA buffers but also a comparison amongst calculated values. Calculated [X2]1 values always differed from measured, and calculated values differed amongst themselves by factors of at least 2. These variations cast doubts on the published absolute values for intracellular [Mg2+] estimated by 31P-NMR and the resting values for [Ca2+] in cells. The allowable range for [X2+] in the buffers ...
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1. A new perfused preparation of frog's ventricle is described, whose main advantage is that there are short diffusion distances between the cells and the washing fluid.2. This preparation responds within a few seconds to alterations... more
1. A new perfused preparation of frog's ventricle is described, whose main advantage is that there are short diffusion distances between the cells and the washing fluid.2. This preparation responds within a few seconds to alterations in sodium, potassium or calcium concentration, or alteration in the osmotic pressure of the bathing fluid. The speed of these alterations is consistent with the diffusional distances involved measured histologically.3. On depolarizing the preparation with Ringer + excess potassium, tension development starts at about -25 mV and is still increasing at -8 mV (the lowest voltage studied). As in earlier experimetns in frog ventricle (Niedergerke, 1956b) and in skeletal muscle (Hodgkin & Horowicz, 1960) the relation between potential and tension is very steep.4. Sodium free contractures (Lüttgau & Niedergerke, 1958) are very rapid in onset with this preparation. To maintain the tension developed, a rapid rate of washing is necessary for 1-2 min. After th...
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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... more
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 hyperventi...
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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... more
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 concent...
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A method for mounting and rapidly perfusing small ventricular trabeculae (diameter around 250 micron) from either ferret or guinea-pig is described. Tension, membrane potential (Em) and intracellular Na activity (aiNa) were measured. aiNa... more
A method for mounting and rapidly perfusing small ventricular trabeculae (diameter around 250 micron) from either ferret or guinea-pig is described. Tension, membrane potential (Em) and intracellular Na activity (aiNa) were measured. aiNa was measured using Na-sensitive micro-electrodes. At room temperature (22-26 degrees C), [Na]o 155 mmol/l and [Ca]o 5.4 mmol/l, aiNa was 10.9 mmol/l +/- S.D. 4.2 mmol/l (n = 148). When [Na]o was reduced from 155 to 1.5 mmol/l contractures developed. These were about twitch height in guinea-pig but less than the twitch height in ferret. Associated with the development of the contracture there was a decrease in aiNa. The aiNa halved within 30 s. The decrease in aiNa was not influenced by changing pHo from 7.4 to 9.5, K-free solution or strophanthidin 50 mumol/l and was not passive since, even when the [Na]o was 1.5 mmol/l, the driving force for Na ions remained inward. The aiNa decreased if [Ca]o was increased and [Na]o decreased or vice versa. On th...
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1. The exchanges of potassium and various other substances have been measured in beating frog's ventricles, using both superfused and distended preparations. In both preparations the high fluid flow rates used (1 ml./sec) cleared the... more
1. The exchanges of potassium and various other substances have been measured in beating frog's ventricles, using both superfused and distended preparations. In both preparations the high fluid flow rates used (1 ml./sec) cleared the ventricular cavity with a half-time (T(1/2)) of about 130 msec.2. Histological sections show that the modal strand radius in the relaxed or contracted distended ventricle is 17.5 mu, and in the relaxed and contracted superfused ventricle is 17.5 and 27.5 mu respectively.3. In quiescent ventricles the resting potassium influx and efflux are approximately equal at about 16 p-mole/cm(2).sec. This figure is computed from Niedergerke's (1963b) estimate of a cell size of 3.5 mu taken from electron-micrographs. If the older figure of 9.2 mu from single isolated cells is used (Skramlik, 1921) then the fluxes are about 44 p-mole/cm(2).sec. To allow for some cell damage in these preparations a further increase in flux of about 30% may be necessary.4. Cont...
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1. A double sucrose gap method to clamp small bundles (diameter 0.8-1.2 mm) of sheep or calf ventricular fibres is described.2. Comparison between micro-electrode recordings from the central gap and the externally recorded potentials... more
1. A double sucrose gap method to clamp small bundles (diameter 0.8-1.2 mm) of sheep or calf ventricular fibres is described.2. Comparison between micro-electrode recordings from the central gap and the externally recorded potentials showed good agreement between the time course and amplitude of the action potentials. The rapid sodium inward current was not controlled on depolarizing clamp steps. On repolarization, control was obtained within 20 msec. The method is regarded as only suitable for a study of slow currents.3. During clamps of several seconds duration slow changes in outward current can be demonstrated. The potential, where the instantaneous current-voltage relationship crosses the voltage axis, shifted in a positive direction as the clamp duration was increased (clamp amplitude constant), and did not alter much if the clamp amplitude was increased while the duration remained constant. For these reasons it is concluded that K ions accumulate round the cells.4. A comparis...
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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... more
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.
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We have considered the manner in which data obtained with ion-selective electrodes should be evaluated. The potential difference recorded by such electrodes, with respect to a stable reference, is converted to a concentration by a... more
We have considered the manner in which data obtained with ion-selective electrodes should be evaluated. The potential difference recorded by such electrodes, with respect to a stable reference, is converted to a concentration by a non-linear transformation--the Nernst or Nikolsky equation. The mean and standard deviation of such estimations of concentration are then usually presented, which assumes that the latter variable is normally distributed. If, however, the recorded potential difference (PD) is the normally distributed variable and the mean value calculated, then a different value of mean concentration will be obtained. We show here that the recorded PD is indeed the normally distributed variable using data from a variety of ion-selective electrode measurements and conclude that the mean values of quoted ion concentrations have been overestimated by 6-43%.
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In sheep and calf ventricular bundles, increasing the internal calcium by increasing the frequency of voltage-clamping to plateau range potentials increased the time-independent outward current. This effect was more marked with higher... more
In sheep and calf ventricular bundles, increasing the internal calcium by increasing the frequency of voltage-clamping to plateau range potentials increased the time-independent outward current. This effect was more marked with higher [Ca]o, and was reduced if the Ca current blockers Verapamil or D 600 were used. 2. If the internal Ca was increased by the addition of cyanide and reduction of external sodium the outward current was also increased. The frequency-dependent increase in outward current also occurred in this Na-poor (12 mM) solution. 3. Tension measurement on the ventricular bundles showed that a Na-free solution with cyanide did not cause a contracture. On changing from Tyrode to a Na-free solution containing cyanide, and on changing back to Tyrode there was a potentiation of the twitch. 4. In Na-poor solution with cyanide, although no contracture was found, ECa was less positive, suggesting that under these circumstances Ca accumulates at the inner side of the membrane,...
Research Interests: Calcium, Biological Sciences, Sheep, Animals, The, and 6 moreCattle, Sodium, Cyanides, Action Potentials, Heart Ventricles, and Verapamil
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Research Interests: Kidney diseases, Humans, Magnesium, Female, Plasma, and 3 moreMedical Physiology, Clinical Sciences, and Ions
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... more
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.
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THESE experiments were started to test the flux predictions made by Noble1 from his equations used to describe the action potential of Purkinje fibres. The experiments were done on frog ventricle, as this is the most suitable tissue... more
THESE experiments were started to test the flux predictions made by Noble1 from his equations used to describe the action potential of Purkinje fibres. The experiments were done on frog ventricle, as this is the most suitable tissue histologically. Although the results are in general agreement with these equations, they suggest a considerably greater diminution in the potassium permeability during the plateau of the cardiac action potential than that used for the equations.
Research Interests: Multidisciplinary, Nature, Animals, Heart, Anura, and 3 morePotassium, Perfusion, and Action potential
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ABSTRACT
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In measurements of the intracellular free calcium concentration ([Ca2+]) using either microelectrodes or fluorescent probes, calibration is normally carried out in EGTA calcium buffer solutions. In the first part of the article the... more
In measurements of the intracellular free calcium concentration ([Ca2+]) using either microelectrodes or fluorescent probes, calibration is normally carried out in EGTA calcium buffer solutions. In the first part of the article the general properties of calcium buffer solutions are discussed, the equations used to calculate the apparent calcium binding constant (Kapp) are derived, and the difficulties in the calculation are discussed. The effects of the purity of EGTA as well as the influence of calcium contamination on the buffer solutions are explained. Because of the difficulties in calculating Kapp, and the importance of EGTA purity and calcium contamination, it is suggested that it is easier to measure all three under the appropriate experimental conditions using the method of Bers (1982). In the second part a do-it-yourself guide to the preparation of EGTA calcium buffer solutions is given. An experimental example is provided using the Bers method to measure purity, contamination, and Kapp. It is concluded that unless all three factors are known it is not possible to prepare accurate EGTA calcium buffer solutions.
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The effect of pH changes on Ca2+ transport by isolated heart mitochondria was measured. Two components of Ca2+ transport were identified, an accumulation dependent on mitochondrial respiration and a Na+-dependent efflux. A decrease of pH... more
The effect of pH changes on Ca2+ transport by isolated heart mitochondria was measured. Two components of Ca2+ transport were identified, an accumulation dependent on mitochondrial respiration and a Na+-dependent efflux. A decrease of pH over the range 7.7-6.7 reduced the initial rate and the total amount of respiration dependent Ca2+ accumulation. At pH 7.2 the [Na+] required to activate half-maximal efflux, k1/2, was 7.5 +/- 1.1 mM. Decreasing the pH over the range 7.7 to 6.9 increased the k1/2 from 3.6 to 11.6. The effect of acidosis was more profound on the respiration dependent Ca2+ uptake than the Na+-dependent efflux.
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ABSTRACT