Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

Effects of changing cytosolic free Mg(2+) concentration on L-type Ca(2+) (I(Ca)) and Ba(2+) currents (I(Ba)) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8mM [Mg(2+)] ([Mg(2+)](p)) buffered with 30mM citrate and 10mM ATP. Increasing [Mg(2+)](p) from 0.2 to 1.8mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (I(T)) protocols, respectively. Increasing [Mg(2+)](p) shifted the V(M) for half inactivation by -20mV but dramatically decreased I(Ca) amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I(T) amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg(2+)](p) might also inhibit permeation through open Ca(2+) channels at positive V(M).     

Opiate tolerance-induced modulation of free intracellular calcium in synaptosomes.

Synaptosomes were prepared from morphine-tolerant and non-tolerant mice. Significantly higher levels of basal free intracellular calcium were observed in the synaptosomes from the opiate-tolerant mice compared to synaptosomes from non-tolerant mice (468 nM versus 328 nM, respectively). In addition, morphine (1 microM) failed to attenuate KCl-induced rises in intracellular calcium in the synaptosomes from the tolerant mice. Conversely, morphine produced a concentration-related, naloxone-reversible attenuation of 50 mM KCl-induced rises in intracellular calcium in the synaptosomes from the non-tolerant mice. Omega conotoxin, which blocks both "L" and "N" type calcium channels, attenuated KCl-stimulated rises in intracellular calcium only in synaptosomes from non-tolerant mice. BAY-K 8644, an "L-type" calcium channel agonist, produced nifedipine-reversible increases in intracellular calcium in the synaptosomes from the tolerant animals only. These data suggest that chronic exposure to morphine results in an alteration in either the number of the activation state of calcium channels in the membrane. Changes in intracellular free calcium may be the final common pathway through which neurons adapt to the chronic exposure to morphine.     

Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers

Spontaneous oscillatory fluctuations in membrane potential are often observed in heart cells, but their basis remains controversial. Such activity is enhanced in cardiac Purkinje fibers by exposure to digitalis or K-free solutions. Under these conditions, we find that voltage noise is generated by current fluctuations that persist when membrane potential is voltage clamped. Power spectra of current signals are not made up of single time-constant components, as expected from gating of independent channels, but are dominated by resonant characteristics between 0.5 and 2 HZ. Our evidence suggests that the periodicity arises from oscillatory variations in intracellular free Ca that control ion movements across the surface membrane. The current fluctuations are strongly cross-correlated with oscillatory fluctuations in contractile force, and are inhibited by removing extracellular Ca or exposure to D600. Chelating intracellular Ca with injected EGTA also abolishes the current fluctuations. The oscillatory mechanism may involve cycles of Ca (or Sr) movement between sarcoplasmic reticulum and myoplasm, as previously suggested for skinned cardiac preparations. Our experiments in intact cells indicate that changes in surface membrane potential can modulate cytoplasmic Ca oscillations in frequency and perhaps amplitude as well. A two-way interaction between surface membrane potential and intracellular Ca stores may be a common feature of heart, neuron, and other cell types.     

Selective modulation of L-type calcium current by magnesium lithospermate B in guinea-pig ventricular myocytes

Magnesium lithospermate B (MLB) is the main water-soluble principle of Salviae Miltiorrhizae Radix (also called as 'Danshen' in the traditional Chinese medicine) for the treatment of cardiovascular diseases. MLB was found to possess a variety of pharmacological actions. However, it is unclear whether and how MLB affects the cardiac ion channels. In the present study, the effects of MLB on the voltage-activated ionic currents were investigated in single ventricular myocytes of adult guinea pigs. MLB reversibly inhibited L-type Ca(2+) current (I(Ca,L)). The inhibition was use-dependent and voltage-dependent (the IC(50) value of MLB was 30 microM and 393 microM, respectively, at the holding potential of -50 mV and -100 mV). In the presence of 100 microM MLB, both the activation and steady-state inactivation curves of I(Ca,L) were markedly shifted to hyperpolarizing membrane potentials, whereas the time course of recovery of I(Ca,L) from inactivation was not altered. MLB up to 300 microM had no significant effect on the fast-inactivating Na(+) current (I(Na)), delayed rectifier K(+) current (I(K)) and inward rectifier K(+) current (I(K1)). The results suggest that the voltage-dependent Ca(2+) antagonistic effect of MLB work in concert with its antioxidant action for attenuating heart ischemic injury.     

Block of single cardiac sodium channels by intracellular magnesium

Currents through single cardiac sodium channels have been measured in inside-out patches from guinea pig ventricular cells. To abolish the fast inactivation, Na channels were modified by DPI 201–106. In symmetrical Na solutions, a diminution of outward sodium currents can be observed that depends on the intracellular magnesium concentration and the membrane potential. Inward currents were not altered by the concentrations of magnesium used (between 0 and 22.5 mmol/1). In Mg free solutions a linear current-voltage relation can also be measured in the range of outward Na currents. At +60 mV (symmetrical Na solutions, single channel conductance 24 pS) a half maximal block of cardiac Na channels by intracellular magnesium was found at 2.1 mmol/l. From the analysis of single channel current-voltage relationships the concentration and voltage-dependent block by intracellular magnesium of cardiac sodium channels could be described as binding of Mg at one site with a K _d value of 5.1 mmol/1 at 0 mV. The site is located at an electrical distance of 0.18 from the inside. Offprint requests to: B. Nilius     

Effects of magnesium on inactivation of the voltage-gated calcium current in cardiac myocytes

The effects of changes in intracellular and extracellular free ionized [Mg2+] on inactivation of ICa and IBa in isolated ventricular myocytes of the frog were investigated using the whole-cell configuration of the patch-clamp technique. Intracellular [Mg2+] was varied by internal perfusion with solutions having different calculated free [Mg2+]. Increasing [Mg2+]i from 0.3 mM to 3.0 mM caused a 16% reduction in peak ICa amplitude and a 36% reduction in peak IBa amplitude, shifted the current-voltage relationship and the inactivation curve approximately 10 mV to the left, decreased relief from inactivation, and caused a dramatic increase in the rate of inactivation of IBa. The shifts in the current-voltage and inactivation curves were attributed to screening of internal surface charge by Mg2+. The increased rate of inactivation of IBa was due to an increase in both the steady-state level of inactivation as well as an increase in the rate of inactivation, as measured by two-pulse inactivation protocols. Increasing external [Mg2+] decreased IBa amplitude and shifted the current-voltage and inactivation curves to the right, but, in contrast to the effect of internal Mg2+, had little effect on the inactivation kinetics or the steady-state inactivation of IBa at potentials positive to 0 mV. These observations suggest that the Ca channel can be blocked quite rapidly by external Mg2+, whereas the block by [Mg2+]i is time and voltage dependent. We propose that inactivation of Ca channels can occur by both calcium-dependent and purely voltage-dependent mechanisms, and that a component of voltage-dependent inactivation can be modulated by changes in cytoplasmic Mg2+.     

Tissue-specific modulation of the mitochondrial calcium uniporter by magnesium ions

This paper analyzes the kinetics of the Ca2+ uniporter of mitochondria from rat heart, kidney and liver operating in a range of Ca2+ concentrations near the steady-state value (1-4 microM). Heart mitochondria exhibit the lowest activity, and physiological Mg2+ concentrations inhibit the mitochondrial Ca2+ uniporter by approx. 50% in heart and kidney, and by 20% in liver. At physiological Ca2+ and Mg2+ concentrations the external free Ca2+ maintained by respiring mitochondria in vitro is higher in heart and kidney with respect to liver mitochondria. This behaviour could represent an adaptation of different mitochondria to their specific intracellular environment.     

Cytosolic free calcium spiking affected by intracellular pH change

The characteristics underlying cytosolic free calcium oscillation were evaluated by superfused dual wave-length microspectrofluorometry of fura-2-loaded single acinar cells from rat pancreas. Application of a physiological concentration of cholecystokinin octapeptide (CCK) (20 pM) induced a small basal increase in cytosolic free calcium concentration ([Ca2+]i) averaging 34 nM above the prestimulation level (69 nM) with superimposed repetitive Ca2+ spike oscillation. The oscillation amplitude averaged 121 nM above the basal increase in [Ca2+]i and occurred at a frequency of one pulse every 49 s. Although extracellular Ca2+ was required for maintenance of high frequency and amplitude of the spikes with increase in basal [Ca2+]i, the primary source utilized for oscillation was intracellular. The threshold of the peak [Ca2+]i amplitude for causing synchronized and same-sized oscillations was less than 300 nM. The [Ca2+]i oscillation was sensitive to intracellular pH (pHi) change. This is shown by the fact that the large pHi shift toward acidification (delta pHi decrease, 0.95) led to a basal increase in [Ca2+]i to the spike peak level with inhibiting Ca2+ oscillation. The pHi shift toward alkalinization (delta pHi increase, 0.33) led to a basal decrease in [Ca2+]i to the prestimulation level, possibly due to reuptake of Ca2+ into the Ca2+ stores, with inhibiting Ca2+ oscillation. Whereas extracellular pH (pHo) change had only minimal effects on Ca2+ oscillation (and/or Ca2+ release from intracellular stores), the extra-Ca2+ entry process, which was induced by higher concentrations of CCK, was totally inhibited by decreasing pHo from 7.4 to 6.5. Thus the major regulatory sites by which H+ affects Ca2+ oscillation are accessible from the intracellular space.     

Regulation by magnesium of intracellular calcium movement in skinned muscle fibers

The effect of Mg on Ca movement between the sarcoplasmic reticulum (SR) and myofilament space (MFS) was studied in skinned muscle fibers by using isometric force as an indicator of MFS Ca. In Ca-loaded fibers at 20 degrees C, the large force spike induced by Ca in 1 mM Mg (5 mM ATP) was strongly inhibited in 3 mM Mg, and force development was extremely slow. After a brief Ca stimulus in 1 mM Mg, relaxation in Ca-free solution was significantly faster in 3 mM Mg. These changes were due to altered Ca movements, since the effect of 3 mM Mg on steady force in CaEGTA solutions was small. Changes in Mg alone induced force transients apparently due to altered Ca movement. In relaxed fibers, decreasing the Mg to 0.25 mM caused phasic force development. In contracting fibers in Ca solutions, increasing the Mg caused a large transient relaxation. The effects of increased Mg were antagonized by 0.5 mM Cd, an inhibitor of the SR Ca transport system. The results indicate that active Ca uptake by the SR in situ is stimulated by Mg, and that it can affect local MFS [Ca++] in the presence of a substantial Ca source. These results provide evidence that an increased rate of Ca uptake in 3 mM Mg could account for inhibition of the large force spike associated with Ca-induced Ca release in skinned fibers.     

Model of mitosis based on antagonistic changes of intracellular concentration of free calcium and magnesium ions

Most of structural and metabolic events comprising mitosis can be explained by assumption of changing intracellular ratio of free Ca²⁺/free Mg²⁺ during this process. Evidence is discussed showing that the reason of changes in free divalent cations concentration during mitosis could be the shift induced by spindle protein polymerization in the $\text{SH}\text{SS}$ status of the cellular proteins affecting the membrane ion transport.     

NMR studies of intracellular free calcium, free magnesium and sodium in the guinea pig reticulocyte and mature red cell

During the maturation process reticulocytes lose their intracellular organelles and undergo changes in membrane lipid composition and ion transport properties. While several reports indicate differences in the levels of magnesium, sodium and calcium in reticulocytes and erythrocytes, controversy remains concerning the actual magnitude and direction of ionic alterations during reticulocyte maturation. One problem with all of these studies is that the techniques used are invasive and are limited to measuring only the total cell ion content. We have used 31P, 23Na and 19F nuclear magnetic resonance (NMR) spectroscopy to compare the intracellular free ion and phosphometabolite levels in guinea pig reticulocytes and mature red blood cells. In contrast to a sharply decreased concentration of ATP in erythrocytes in comparison to reticulocytes, the intracellular free magnesium, measured using 31P-NMR, was increased by about 65% upon maturation (150 mumol/l cell water in reticulocytes in comparison to 250 mumol/l cell water in erythrocytes). Sizeable but opposite changes in intracellular sodium (5.5 mumol/ml cells in reticulocytes vs. 8.5 mumol/ml cells in erythrocytes) and intracellular free calcium (99 nM vs. 31 nM in reticulocytes and mature red cells, respectively) were also observed, suggesting that alterations in the kinetics of membrane ion transport systems, accompanying changes in phospholipid and cholesterol content, occur during the process of red cell maturation. However, in contrast to dog red blood cells, there was no evidence for the presence of a Na+/Ca2+ exchanger in guinea pig reticulocytes or erythrocytes.     

Temperature dependence of intracellular free calcium in cardiac myocytes from rat and ground squirrel measured by confocal microscopy

The temperature-dependence of intracellular free calcium ([Ca²⁺]_i) was investigated in indo-1 loaded ventricular myocytes from the rat, a non-hibernator, and from the ground squirrel, a hibernator. The dissociation constant of indo-1 at different temperatures was calibrated both at pH-stat and at α-stat, and the result demonstrated that the α-stat calibration should be preferred. Analysis of the fluorescent image showed a striking increase of [Ca²⁺]_i as well as spontaneous calcium waves in rat cells, indicating an overloaded calcium. In contrast, cardiac myocytes of the ground squirrel were found to keep a constant [Ca²⁺]_i without calcium overload regardless of temperature variation. It is believed that understanding of the mechanisms underlying the intercellular calcium homeostasis of hibemators may lead to solutions of some medical questions.     

Temperature dependence of intracellular free calcium in cardiac myocytes from rat and ground squirrel measured by confocal microscopy

The temperature-dependence of infraeeliular free caleimn (Ca) was investigated in mdo-1 loaded ventricular myocytes from the ral, a non-hibernator, and from the ground squirrel, a hibernator. The dissociation constant of indo-l at different temperatures was calibrated both al pll-tat and at @-stat . and the result demonstrated that the @-stat ralibration should be prettrred . Analysis of the fluoreseent image showed a striking increase of Ca2 as well as spontaneous caleiuni waves in ral cells, indicating an overloaded cakuum. In contrast, cardiac myocytes of the ground sqnirraf were found to keep a constant (Ca2 ) without caleium overload regardless of temperature variation. It is be-lieved that understanding of the mechanisms underlying the interccllular caleima homeostasis of hibrernators may lead to solutions of some medical questions .     

Control of free cytoplasmic calcium by intracellular pH in rat lymphocytes

Activation of Na⁺-H⁺ exchange in rat thymocytes was found to be followed by an increase in free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). We determined whether the change in [Ca²⁺]_i was secondary to the uptake of Na⁺, or to the cytoplasmic alkalinization that result from activation of the antiport. Increasing intracellular [Na⁺] by treating the cells with ouabain or gramicidin failed to affect [Ca²⁺]_i. In contrast, procedures that increased the cytoplasmic pH, such as addition of monensin or NH₃, significantly elevated [Ca²⁺]_i. These results suggest an important role of cytoplasmic pH in the control of [Ca²⁺]_i in lymphocytes.     

On the noninvasive measurement of intracellular free magnesium by 31P NMR spectroscopy

We previously introduced a noninvasive measurement of the concentration of free Mg2+ in intact cells and tissues using 31P NMR. To resolve a controversy in the literature concerning the affinity of Mg2+ for ATP used in our procedure, the apparent dissociation constant of MgATP under simulated intracellular conditions has been determined by three independent magnetic resonance methods, including a newly developed combination procedure for determining this value at intracellular ATP levels. The new combination method, which utilizes 31P NMR to determine the degree of Mg2+ chelation of ATP and the dye antipyrylazo III for optical determination of free Mg2+, yielded a value of (50 +/- 10) microM for this apparent dissociation constant at pH 7.2 in the presence of 0.15 M K+ and 25 degrees C. We further show that hydroxyquinolines are not satisfactory indicators for optical determination of the Mg2+-nucleotide dissociation constant. From our determinations a low value of free Mg2+ (less than 1 mM) is established for all of the tissues studied, including perfused heart muscle, contrary to a recent report in the literature. Saturating human erythrocytes with Mg2+ results in an alpha- and beta-phosphorus resonance separation for intracellular ATP that is indistinguishable from that observed in a noncellular MgATP control under similar conditions, showing that MgATP resonances in this cell are unaffected by the cellular environment.     

Decreased intracellular free magnesium in erythrocytes of spontaneously hypertensive rats

Using 31p-NMR (the phosphorus nuclear magnetic resonance) spectroscopy, we measured intracellular free Mg levels in the erythrocytes of untreated (n = 7) and diltiazem-treated spontaneously hypertensive rats (SHR) (n = 8), and compared them with age-matched Wistar-Kyoto rats (WKY) (n = 10). The intracellular free Mg levels were significantly (p less than 0.01) decreased in untreated SHR compared with those in control WKY. A successful antihypertensive treatment with diltiazem increased the intracellular free Mg levels compared with untreated SHR (p less than 0.05). Furthermore, an inverse correlation was observed between intracellular free Mg levels and blood pressure levels in all groups (r = -0.48, p less than 0.01, n = 25). These observations suggest that abnormalities of intracellular Mg metabolism may be, in part, related to the development or the maintenance of hypertension in SHR.     

Regulation of cation channels in cardiac and smooth muscle cells by intracellular magnesium

Magnesium regulates various ion channels in many tissues, including those of the cardiovascular system. General mechanisms by which intracellular Mg(2+) (Mg(i)(2+)) regulates channels are presented. These involve either a direct interaction with the channel, or an indirect modification of channel function via other proteins, such as enzymes or G proteins, or via membrane surface charges and phospholipids. To provide an insight into the role of Mg(i)(2+) in the cardiovascular system, effects of Mg(i)(2+) on major channels in cardiac and smooth muscle cells and the underlying mechanisms are then reviewed. Although Mg(i)(2+) concentrations are known to be stable, conditions under which they may change exist, such as following stimulation of beta-adrenergic receptors and of insulin receptors, or during pathophysiological conditions such as ischemia, heart failure or hypertension. Modifications of cardiovascular electrical or mechanical function, possibly resulting in arrhythmias or hypertension, may result from such changes of Mg(i)(2+) and their effects on cation channels.