Effects of membrane potential on Na+ -dependent Mg2+ extrusion from rat ventricular myocytes

To study Mg2+ transport across the cell membrane, the cytoplasmic concentration of Mg2+ ([Mg2+](i)) in rat ventricular myocytes was measured with the fluorescent indicator furaptra (mag-fura-2) under Ca2+ -free conditions (0.1 mM EGTA) at 25 degrees C. The fluorescence ratio signal of furaptra was converted to [Mg2+](i) using calibration parameters previously estimated in myocytes (Watanabe and Konishi, Pflügers Arch 442: 35-40, 2001). After [Mg2+](i) was raised by loading the cells with Mg2+ in a solution containing 93 mM Mg(2+), the cells were voltage-clamped at a holding potential of -80 mV using the perforated patch-clamp technique with amphotericin B. At the holding potential of -80 mV, the reduction of extracellular Mg2+ to 1.0 mM caused a rapid decrease in [Mg2+](i) only in the presence of extracellular Na(+). The rate of the net Mg2+ efflux appeared to be dependent on the initial level of [Mg2+](i); the decrease in [Mg2+](i) was significantly faster in the myocytes markedly loaded with Mg2+. The rate of decrease in [Mg2+](i) was influenced little by membrane depolarization from -80 to -40 mV, but the [Mg2+](i) decrease accelerated significantly at 0 mV by, on average, approximately 40%. Hyperpolarization from -80 to -120 mV slightly but significantly slowed the decrease in [Mg2+](i) by approximately 20%. The results clearly demonstrate an extracellular Na(+)- and intracellular Mg2+ -dependent Mg2+ efflux activity, which is consistent with the Na(+)-Mg2+ exchange, in rat ventricular myocytes. We found that the apparent rate of Mg2+ transport depends slightly on the membrane potential: facilitation by depolarization and inhibition by hyperpolarization with no sign of reversal between -120 and 0 mV.     

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.     

Myoplasmic Mg2+ concentration in Xenopus muscle fibres at rest, during fatigue and during metabolic blockade.

Intracellular free Mg2+ concentration ([Mg2+]i) was measured in isolated single fibres of Xenopus muscle using the fluorescent Mg2+ indicator furaptra. In resting muscle the [Mg2+]i was 1.7 mM in a Mg(2+)-free Ringer solution. There was no significant change in [Mg2+]i over 2 h in Mg(2+)-free Ringer solution. Elevating extracellular [Mg2+] to 40 mM for 5 min caused a small rise (0.13 mM) in [Mg2+]i. There was no detectable rise in [Mg2+]i after 5 min in Na(+)-free Ringer solution. These results suggest that the membrane is relatively impermeable to Mg2+ and that there was no detectable Na(+)-Mg2+ exchange over 5 min. When muscle fibres were fatigued by repeated tetani continued until force declined to about 40% of control, [Mg2+]i showed characteristic changes. During the early period of fatigue when force first showed a small decline and then became almost stable, [Mg2+]i was unchanged; during the final period of fatigue when force declined more rapidly, [Mg2+]i increased by 0.8 mM. Recovery of [Mg2+]i took about 30 min. Recovery of force was complex: tetanic force first declined (post-contractile depression) and then slowly recovered to control. Since the minimum force occurred at about the time when [Mg2+]i had recovered, it seems unlikely that post-contractile depression is caused by elevated [Mg2+]i. Rigor, produced by inhibiting oxidative phosphorylation and glycolysis, was associated with a larger increase (1.6 mM) in [Mg2+]i than fatigue. The rise in [Mg2+]i during fatigue and metabolic blockade could be explained as release of Mg2+ normally bound to ATP. A model of the metabolic changes and the resulting increase in [Mg2+]i explains our results reasonably well.     

Intracellular free magnesium and its regulation, studied in isolated ferret ventricular muscle with ion-selective microelectrodes

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.     

Effects of magnesium on prostacyclin synthesis and intracellular free calcium concentration in vascular cells.

This study investigated the effects of extracellular magnesium concentration ([Mg2+]e; 0.3-3 mM) on intracellular free calcium concentration ([Ca2+]i) and prostacyclin (PGI2) production in cultured human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells from rats (VSMC) under basal and agonist-stimulated conditions. We used histamine as agonist which increases [Ca2+]i and PGI2 production in HUVEC, norepinephrine in VSMC. [Mg2+]e dose-dependently increased basal and agonist-stimulated PGI2 production in both cells. [Mg2+]e dose-dependently reduced basal [Ca2+]i in VSMC, but did not influence in HUVEC. In both cells, increasing [Mg2+]e reduced agonist-stimulated [Ca2+]i responses. Furthermore, [Mg2+]e dose-dependently reduced agonist-stimulated [Ca2+]i in Ca(2+)-free buffer, indicating intracellular Ca2+ release. In VSMC, 10(-6) M diltiazem and 10(-7) M nifedipine, Ca2+ channel blockers, reduced agonist-stimulated [Ca2+]i as well as 3 mM Mg2+, but did not affect PGI2 production. [Mg2+]e amplified dose-dependently arachidonic acid-induced PGI2 production in both cells, suggesting the activation of cyclooxygenase and/or PGI2 synthetase. Our results suggest that [Mg2+]e influences intracellular Ca2+ mobilization of not only vascular smooth muscle cells but also endothelial cells by inhibiting both Ca2+ influx and intracellular Ca2+ release. [Mg2+]e enhances PGI2 production in both types of cells, although the mechanism is likely to be independent from Ca2+ mobilization.     

Total and free Mg2+ contents in erythrocytes: a simple but still undisclosed cell model.

The concentration of intracellular free Mg2+ ([Mg2+]i) in erythrocytes, measured by means of 31P NMR and using a dissociation constant for MgATP of 38-50 microM, amounted to 0.2 mM [Mg2+]i in the erythrocytes of various species, was not significantly different and was independent of their total Mg2+ content. The more probable value of [Mg2+]i using the more realistic KD of Mg ATP or the null-point method may amount to 0.4 mM [Mg2+]i in erythrocytes is lower than the [Mg2+]i in nucleated mammalian cell types. The lower [Mg2+]i may be caused by a different regulation of Mg2+ influx and Mg2+ efflux by intracellular Mg2+ in erythroblasts. Free and reversibly bound Mg2+ represent a Mg2+ buffer. The main Mg2+-binding substances are ATP and 2,3-bisphosphoglycerate (2,3-BPG). Total Mg2+ content in the erythrocytes of various species is correlated to the concentrations of ATP and 2,3-BPG. The changed Mg2+ level in erythrocytes during deoxygenation, maturation, cold storage, in Mg2+ deficiency and in sickle cell anemia was reviewed.     

Extracellular magnesium deficiency induces contraction of arterial muscle: role of PI3-kinases and MAPK signaling pathways

The present study investigated the actions of extracellular Mg2+ ([Mg2+]o) deficiency on isolated rat aortae and rat aortic smooth muscle cells (RASMC). Exposure of isolated, intact rat aortic rings to Mg(2+)-free or Mg(2+)-deficient medium (0.15-0.6 mM) produced endothelium-independent, concentration-dependent contractions: the lower the [Mg2+]o, the stronger the contraction. Pre- or post-incubation of the vessels with low concentrations of U0126, SB-203580, PD-98059, wortmannin, LY-294002, or a SH2 domain inhibitor peptide suppressed [Mg2+]o deficiency-induced contractions significantly. The concentrations of these antagonists required for half-maximal inhibition (IC50) were not very different from the inhibitory constants (Ki) for these drugs. A variety of specific pharmacological antagonists of several known endogenously-formed vasoconstrictors did not inhibit or attenuate the contractions induced by low [Mg2+]o. Mg(2+)-free medium induced a 6- to 7-fold increase in intracellular Ca2+ ([Ca2+]i) in RASMC. Pre- or post-treatment of the cells with U0126, SB-203580, PD-98059, wortmannin, LY-294002, or a SH2 domain inhibitor peptide markedly inhibited the increments in ([Ca2+]i) in RASMC induced by exposure to Mg(2+)-free medium. The present findings suggest that Mg(2+)-deficiency-induced contractions of rat aortae are associated with activation of several cellular signal pathways, such as mitogen-activated protein kinase, phosphatidylinositol-3 (PI3) kinases, and SH2 domain-containing proteins.     

Contribution of Na+/Ca2+ exchanger to glucose-induced [Ca2+]i increase in rat pancreatic islets

The present study was carried out to elucidate the role of the reverse mode of the Na+/Ca2+ exchanger in an increase in intracellular Ca2+ concentration ([Ca2+]i) induced by a stimulatory concentration of glucose in rat pancreatic islets. The effects of KB-R7943, a selective inhibitor of reverse Na+/Ca2+ exchanger, on Na+o removal-induced [Ca2+]i changes were examined by a microfluorimetric method using fura-2 in perifused preparations of isolated rat pancreatic islets. Na+o removal induced a rapid increase in [Ca2+]i under 100 or 5 mM K+ conditions, respectively. The increases in [Ca2+]i induced by Na+o removal were inhibited by KB-R7943. The net amount of the [Ca2+]i increases during Na+o removal (Delta[Ca2+]i), obtained by subtracting the KB-R7943-independent Delta[Ca2+]i in the presence of KB-R7943 from Delta[Ca2+]i in the absence of KB-R7943, was significantly increased when extracellular K+ was raised. Increasing the external glucose concentration from 3 to 20 mM caused a biphasic increase in [Ca2+]i, which exhibited a transient increase (first phase) followed by a sustained increase (second phase) in [Ca2+]i. KB-R7943 (10 microM) partially inhibited the second phase of the [Ca2+]i increase rather than the first phase. These results suggest that the increase in [Ca2+]i induced by Na+o removal may be enhanced when plasma membrane is depolarized, and consequently, Ca2+ influx through the reverse Na+/Ca2+ exchanger may partially contribute to the glucose-induced [Ca2+]i dynamics in rat pancreatic islet cells.     

External calcium sensitivity of low sodium contractures in the control and hypertrophied right ventricle of the ferret.

The existence of possible differences of calcium (Ca2+) fluxes through the sarcolemmal sodium-calcium (Na+/Ca2+) exchanger during hypertrophy has been tested by comparing the characteristics of the contracture--as an indicator of the intracellular Ca2+ concentration--induced by partial or total withdrawal of external sodium (Na+), in the absence of external potassium, in the right ventricular trabeculae of adult ferret hearts. Pressure-overload was induced by pulmonary artery clipping and led to an increase of the right ventricular weight of 60%. At an external Ca2+ concentration ([Ca2+]o) of 3 mM, the dependence of the contractures on extracellular sodium concentration ([Na+]o), the rate of tension development, the time course of spontaneous relaxation and the time course for the repriming of the contracture were unchanged by hypertrophy. However, the relationship between [Ca2+]o and contracture amplitude at various [Na+]o showed that the apparent affinity of the contracture for [Ca2+]o was decreased in hypertrophied preparations. Thus, in 0 mM [Na+]o, half-maximal contracture was induced at a [Ca2+]o of 0.012 +/- 0.016 mM and 0.171 +/- 0.021 mM in control (n = 11) and hypertrophy (n = 12) respectively (P less than 0.001). Although these data may be indicative of a decreased Ca2+ influx through the Na+/Ca2+ exchanger, it cannot be excluded that intracellular buffering mechanism may also be involved in this differential response to [Na+]o withdrawal.     

Concentration, compartmentation and metabolic function of intracellular free Mg2+.

Intracellular total Mg2+ and free Mg2+ are compartmentalized between cell organelles and within the cytosol. Different values of [Mg2+]i in the cytosol of the same cell type were measured by various investigators. A main reason for the differences is the uncertainty of the dissociation constants used for the Mg furaptra complex in the fluorescence method and for MgATP when 31P NMR was employed. The more realistic KD values of Mg furaptra and MgATP measured under in situ conditions are higher than the KDs used by most investigators. The [Mg2+]is obtained and the KDs used by various authors were presented. The role of intracellular Mg2, in metabolic functions and the action of various effectors on [Mg2+]i and [Ca2+] was reviewed. Intracellular Mg2+ may have a permissive role supporting the effector-induced mechanisms that are mediated by Ca2+ as a second messenger.     

Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms.

The roles of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Na(+)-Ca2+ exchange in Ca2+ removal from cytosol were compared in isolated rabbit and rat ventricular myocytes during caffeine contractures and electrically stimulated twitches. Cell shortening and intracellular calcium concentration ([Ca2+]i) were measured in indo-1-loaded cells. Na(+)-Ca2+ exchange was inhibited by replacement of external Na+ by Li+. To avoid net changes in cell or SR Ca2+ load during a twitch in 0 Na+ solution, intracellular Na+ (Na+i) was depleted using a long pre-perfusion with 0 Na+, 0 Ca2+ solution. SR Ca2+ accumulation was inhibited by caffeine or thapsigargin (TG). Relaxation of steady-state twitches was 2-fold faster in rat than in rabbit (before and after Na+i depletion). In contrast, caffeine contractures (where SR Ca2+ accumulation is inhibited), relaxed faster in rabbit cells. Removal of external Na+ increased the half-time for relaxation of caffeine contractures 15- and 5-fold in rabbit and rat myocytes respectively (and increased contracture amplitude in rabbit cells only). The time course of relaxation in 0 Na+, 0 Ca2+ solution was similar in the two species. Inhibition of the Na(+)-Ca2+ exchange during a twitch increased the [Ca2+]i transient amplitude (delta[Ca2+]i) by 50% and the time constant of [Ca2+]i decline (tau) by 45% in rabbit myocytes. A smaller increase in tau (20%) and no change in delta[Ca2+]i were observed in rat cells in 0 Na+ solution. [Ca2+]i transients remained more rapid in rat cells. Inhibition of the SR Ca(2+)-ATPase during a twitch enhanced delta[Ca2+]i by 25% in both species. The increase in tau after TG exposure was greater in rat (9-fold) than in rabbit myocytes (2-fold), which caused [Ca2+]i decline to be 70% slower in rat compared with rabbit cells. The time course of [Ca2+]i decline during twitch in TG-treated cells was similar to that during caffeine application in control cells. Combined inhibition of these Ca2+ transport systems markedly slowed the time course of [Ca2+]i decline, so that tau was virtually the same in both species and comparable to that during caffeine application in 0 Na+, 0 Ca2+ solution. Thus, the combined participation of slow Ca2+ transport mechanisms (mitochondrial Ca2+ uptake and sarcolemmal Ca(2+)-ATPase) is similar in these species. We conclude that during the decline of the [Ca2+]i transient, the Na(+)-Ca2+ exchange is about 2- to 3-fold faster in rabbit than in rat, whereas the SR Ca(2+)-ATPase is 2- to 3-fold faster in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of various intracellular Ca ion concentrations on the calcium current of guinea-pig single ventricular cells

The effects of changing the intracellular Ca concentration ([Ca]i) on the calcium current (iCa) were studied in isolated single ventricular cells of the guinea-pig. [Ca]i was varied by an intracellular perfusion technique using a suction pipette. iCa measured from internally perfused cells at a pCa lower than 9.0 was dependent on the extracellular Ca concentration ([Ca]o). Increasing [Ca]o from 1.8 to 5.4 mM increased the amplitude of iCa, and reduction of [Ca]o from 1.8 to 0.01 mM decreased the amplitude. The inactivation time course of iCa became faster as [Ca]o was increased and slower as [Ca]o was reduced. By decreasing the pCa of the internal perfusate from 9.0 to 6.8, the amplitude of iCa was decreased markedly, but no significant change was observed in its time course. Analysis of the I-V curve led to the conclusion that a change in the driving force was not a major factor in the reduction of iCa. The "steady state inactivation" of iCa was measured by a double-pulse method. The amplitude of iCa elicited by the test pulse was smaller at pCa 7.4 than at pCa 9.0 at potentials of between -27 and +33 mV. By normalizing the iCa amplitude, however, the "steady state inactivation" curve in the control and at high [Ca]i overlapped. Similar results were obtained in Sr-Tyrode solution. The degree of "steady state inactivation" of iCa at the potentials positive to +10 mV was larger in Ca-Tyrode than in Sr-Tyrode solution. It is proposed that the reduction in amplitude of iCa at higher [Ca]i is caused by a reduction of the maximum conductance of iCa (gCa) and that Ca ions passing through iCa channels have a remarkable effect on its inactivation.     

Difference in propagation of Ca2+ release in atrial and ventricular myocytes

Intracellular [Ca2+] ([Ca2+]i) was imaged in atrial and ventricular rat myocytes by means of a high-speed Nipkow confocal microscope. Atrial myocytes with an absent t-tubule system on 8-di- ANEPPS staining showed an initial rise in Ca2+ at the periphery of the cell, which propagated to the interior of the cell. Ventricular myocytes showed a uniform rise in [Ca2+]i after electrical stimulation, consistent with a prominent t-tubular network. In atrial myocytes, there was a much shorter time between the peak of the [Ca2+]i transient and the peak contraction as compared to ventricular myocytes. A regional release of Ca2+ induced by an exposure of one end of the myocyte to caffeine with a rapid solution switcher resulted in a uniform propagation of Ca2+ down the length of the cell in atrial myocytes, but we found no propagation in ventricular myocytes. A staining with rhodamine 123 indicated a much greater density of mitochondria in ventricular myocytes than in atrial myocytes. Thus the atrial myocytes display a lack of "local control" of Ca2+ release, with propagation after the Ca2+ release at the periphery induced by stimulation or at one end of the cell induced by exposure to caffeine. Ventricular myocytes showed the presence of local control, as indicated by an absence of the propagation of a local caffeine-induced Ca2+ transient. We suggest that this finding, as well as a reduced delay between the peak of the [Ca2+]i transient and the peak shortening in atrial myocytes, could be due in part to reduced Ca2+ buffering provided by mitochondria in atrial myocytes as opposed to ventricular myocytes.     

Effects of ouabain on intracellular ion activities of sensory neurons of the leech central nervous system.

1. The intracellular K+, Na+, and Ca2+ of mechanosensory neurons in the central nervous system of the leech Hirudo medicinalis was measured using double-barreled ion-sensitive microelectrodes. 2. After inhibition of the Na(+)-K+ pump with 5 x 10(-4) M ouabain, the intracellular K+ activity (aKi) decreased, while the intracellular Na+ activity (aNai) increased. The input resistance decreased in the presence of ouabain. The intracellular Ca2+ increased more than one order of magnitude after ouabain addition. All changes in intracellular ion activities and membrane resistance were fully reversible. 3. When extracellular Na+ concentration ([Na+]o) was removed [replaced by tris(hydroxymethyl)aminomethane (Tris)], aNai decreased. In the absence of [Na+]o, aKi and aNai remained unchanged after inhibition of the Na(+)-K+ pump by reducing the extracellular K+ concentration ([K+]o) to 0.2 mM. The membrane resistance increased under these conditions. 4. The intracellular Ca2+ decreased or remained constant after removal of [Na+]o. Addition of ouabain in the absence of [Na+]o did not change intracellular Ca2+, which only increased after readdition of [Na+]o. 5. The relative K+ permeability (PK) measured as membrane potential change during a brief increase of the [K+]o from 4 to 10 mM, increased manyfold after addition of ouabain but only little if [Na+]o had been removed before adding ouabain. 6. The results suggest that the intracellular Na+ increase after inhibition of the Na(+)-K+ pump affects the intracellular Ca2+ level by stimulating a Nai(+)-Ca2+ exchange mechanism. The subsequent intracellular Ca2+ activity (aCai) rise may result in an increase of the membrane permeability to K+ ions.     

Effects of gender on intracellular [Ca2+] in rat cardiac myocytes

The present study investigated the effects of gender on intracellular [Ca2+] ([Ca2+]i) in freshly isolated rat cardiac myocytes. Changes in [Ca2+]i in response to varied extracellular [Ca2+], different stimulus frequencies and addition of caffeine and isoprenaline were monitored using fura-2 in both male and female cardiac myocytes. Increasing extracellular [Ca2+] and stimulus frequency resulted in significant increases in peak [Ca2+] and the amplitude of the Ca2+ transient in both male and female cardiac myocytes. However, as extracellular [Ca2+] was raised, peak [Ca2+] and the amplitude of the Ca2+ transient increased significantly more in male than female cardiac myocytes. In addition a significant difference between male and female cells at each stimulus frequency was apparent. The time course of decay of the Ca2+ transient was significantly slower in female cardiac myocytes when compared with male cardiac myocytes, along with significantly slowed times to peak shortening and 50% relaxation, and a reduced extent of shortening. There was no significant difference in the amplitude of caffeine-induced [Ca2+]i responses between male and female cells, however, [Ca2+]i increased more readily in male cells than in female cells when isoprenaline was added. The data demonstrate that, under a variety of conditions, intracellular [Ca2+] rises to higher levels in cardiac myocytes from male as compared to female rats.     

Cytoplasmic free concentrations of Ca2+ and Mg2+ in skeletal muscle fibers at rest and during contraction

This review summarizes estimates for cytoplasmic-free concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) at rest and during contraction of skeletal muscles, from which substantial quantitative information about them has been accumulated. Although the estimates of resting [Ca2+]i in the literature widely differ, which is because of the variety of difficulties related to different methodologies used, recent studies suggest that estimates of resting [Ca2+]i of approximately 0.05-0.1 microM are likely to be correct. Following action potential propagation, the Ca2+ release from the sarcoplasmic reticulum causes a transient rise of [Ca2+]i (Ca2+ transient). The large peak amplitude and brief time course of the Ca2+ transients have been established only recently by studies with low-affinity Ca2+ indicators developed in the past decade. These technical improvements in [Ca2+]i measurements have made it possible to study relationships between [Ca2+]i and force in intact muscle fibers. In the second part of this review, various estimates of [Mg2+]i in the resting muscle are discussed. Relatively recent estimates of the [Mg2+]i level appear to be about 1.0 mM. Using the current knowledge of concentrations and reaction properties of intracellular Ca2+-Mg2+ binding sites, we constructed a model for dynamic Mg2+ movement following Ca2+ transients. The model predicts that with a train of action potentials, the sustained rise of [Ca2+]i produces an elevation of [Mg2+]i of about 200 microM.     

The intracellular concentration of free magnesium in extensor digitorum longus muscles of the rat

Magnesium-sensitive microelectrodes were used to measure the intracellular concentration of free Mg2+, [Mg2+]f, in rat extensor digitorum longus muscles in vitro at 30 degrees C. The intracellular activities of Na+ and K+ were also determined so that allowance could be made for the interference from these ions with the Mg2+ electrode response. The mean value for [Mg2+]f based on twenty-six measurements in twelve muscles was 0.47 mM.     

Mechanisms of Mg2+ influx, efflux and intracellular 'muffling' in leech neurones and glial cells.

Mg2+ is known to influence conductance and gating properties of a multitude of ion channels and is thus able to modulate synaptic transmission. Therefore, a tight regulation of the intracellular free Mg2+ concentration ([Mg2+]i) in neurones and glial cells is crucial for maintaining the functions of central nervous systems. [Mg2+]i is regulated through the balance of Mg2+ influx and Mg2+ efflux, together with heavy damping of [Mg2+]i changes through intracellular buffering and sequestration. To investigate the mechanisms involved in [Mg2+]i regulation, neurones and glial cells from the central nervous system of the leech Hirudo medicinalis proved to be an ideal model system. The present article summarizes the evidence for a Mg2+ influx pathway which is distinct from that for Ca2+, for a dual regulation of Mg2+ efflux (a 1 Na+/1 Mg2+ antiport and a Na(+)-independent Mg2+ efflux mechanism), for pH-dependent Mg2+ buffering through ATP and other intracellular Mg2+ binding components and for the involvement of mitochondria in intracellular Mg2+ sequestration.     

N-methyl-D-aspartate responses in rat cerebellar granule cells are modified by chronic depolarisation in culture.

Following culture in high (25 mM) K+ conditions cerebellar granule cells only respond with a rise in cytosolic free calcium concentration ([Ca2+]i after removal of external Mg2+. When granule cells are grown in low (5 mM) K+ N-methyl-D-aspartate (NMDA) exerts a neurotrophic effect. We show that at the critical time for this effect NMDA will elicit a rise in [Ca2+]i in 5 mM K+ cultures even in the presence of Mg2+ and that growth in 25 mM K+ induces the rapid appearance of a Mg2+ block of NMDA receptors in granule cells. This suggests firstly, that a rise in [Ca2+]i could be involved in the neurotrophic effect of NMDA and secondly, that the characteristics of the NMDA responses in granule cells are modified as a result of growth under depolarising conditions.