Effect of external magnesium on intracellular free sodium: Na+ flux via Na+/Mg2+ antiport is masked by other Na+ transport systems in rat cardiac myocytes.

Mg2+ efflux from heart cells on a Na+/Mg2+ antiport has been postulated, but the Na+ flux component of the antiport has not been demonstrated. The study aimed to establish if the Na+ flux component could be measured by following changes in [Na+]i with SBFI during conditions known to reverse the antiport (5 mmol/L Mg2+(o), Na+(o)- & Ca2+(o)-free): and after minimising the activity of other Na+ transport pathways. Resting [Na+]i was 8 +/- 0.7 mmol/L (mean +/- S.E., n = 39 cells) in normal Tyrode's solution. [Na+]i decreased below the normal level in all cells (a decline of 4-5 mmol/L, n = 21) during perfusion with 5 mmol/L Mg2+(o) (Na+(o)- & Ca2+(o)-free). Controls using 1 mmol/L Mg2+(o) showed similar declines in [Na+]i, but the fall was greatest when Na+(o) was replaced by K+(o) (decline of 6 mmol/L) rather than the tetramethylammonium ion (TMA+). The rate of decrease in [Na+]i during perfusion with 5 mmol/L Mg2+(o) (Na+(o)- & Ca2+(o)-free) was slowed by 20 microM ouabain (n = 5) or by elevation of pHo to pH 9 (n = 7) so that [Na+]i remained close to the initial value. The decrease of [Na+]i was not affected by 10 microM imipramine (n = 15). These data suggest that the Na+ efflux component of the Na+/Mg2+ antiport is masked in Na+(o)- and Ca2+(o)-free conditions by other Na+(i) efflux pathways.     

Na+/Mg2+ antiport in erythrocytes of spontaneously hypertensive rats: role of Mg2+ in the pathogenesis of hypertension.

Total Mg2+ content in plasma and erythrocytes did not significantly differ between WKY and SHR. Mg2+ efflux via Na+/Mg2+ antiport was 10% lower in non Mg(2+)-loaded erythrocytes of SHR than in WKY, and 16% lower in Mg(2+)-loaded erythrocytes of SHR. The activation of Na+/Mg2+ antiport in erythrocytes by Cl-, as tested by substitution of Cl- with SCN-, and the regulation of Na+/Mg2+ antiport by protein kinases, as tested by PMA and staurosporine, showed no differences between WKY and SHR. The reduction of Na+/Mg2+ antiport was explained by a reduction in the number of Na+/Mg2+ antiporter molecules in SHR erythrocytes. Mg2+ efflux in KCl medium by K+/Mg2+ antiport via the unspecific choline exchanger was not significantly reduced in SHR and was equally affected by PMA and staurosporine in WKY and SHR. An explanation for some controversial results, unchanged or reduced concentration of Mg2+ in serum, total Mg2+ and free Mg2+ in erythrocytes of SHR and patients with essential hypertension was proposed. The role of Na+/Mg2+ antiport and [Mg2+]i in the pathogenesis of experimental and clinical hypertension was discussed.     

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.     

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.     

Stimulation of choline/Mg2+ antiport in rat erythrocytes by mefloquine.

In non Mg(2+)-loaded and non malaria-infected rat erythrocytes, mefloquine (100 micromol x l (-1)) stimulated choline/Mg2+ antiport without affecting the Na+/Mg2+ antiport. The stimulation of the choline/Mg2+ antiport by mefloquine, found in this study, and by trifluoperazine and fluvoxamine, reported previously [Ebel et al. Biochim Biophys Acta 2004; 1167: 132-40], was associated with CF3 groups attached to the quinoline or benzene ring. The effect of mefloquine on choline/Mg2+ antiport in vitro was not related to the antimalarial action of mefloquine in vivo. In rat erythrocytes, the choline/Mg2+ antiport can be differentiated from the Na+/Mg2+ antiport through the use of cinchonine that inhibited the choline/Mg2+ antiport [Ebel et al. Biochim Biophys Acta 2002; 1559: 135-44], and mefloquine that stimulated the choline/Mg2+ antiport, whereby the Na+/Mg2+ antiport was not affected by either drug at proper concentrations. The Na+/Mg2+ antiport and choline/Mg2+ antiports behave as different molecular entities.     

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.     

Expression and functional activity of the Na/Mg exchanger, TRPM7 and MagT1 are changed to regulate Mg homeostasis and transport in rumen epithelial cells

The present study was performed to show the molecular identity of functionally characterized Mg transport pathways in rumen epithelial cells (REC) and to investigate the effects of extracellular [Mg] changes on their expression and activity. By using RT- PCR, Western blot, flow cytometry and immunocytochemistry, TRPM7, MagT1 and a Na+/Mg2+ exchanger were found in REC. Compared with control conditions ([Mg]e = 1.2 mM), a decreased or increased MagT1 (-30%; 20%) and Na+/Mg2+ exchanger (-25%; 40%) protein abundance was observed after a 24-h incubation of REC in low (0.12 mM)- and high (5 mM)-Mg medium, respectively. To determine the Mg transport capacity, [Mg2+]i changes were measured by use of mag-fura 2. The basal [Mg2]i (0.43 +/- 0.03 mM) was not influenced by the [Mg] of the pre-incubation medium. However, compared to control cells, REC incubated in low- or high-Mg medium showed significantly reduced (59%) and elevated (57%) Mg extrusion rates, respectively. In addition, they were characterized by an increased influx capacity (30-40%). In low-Mg cells the latter results mainly from a strong TRPM7 related transport component whereas in high-Mg cells the imipramine-sensitive, the Na+/Mg2+ exchanger-mediated transport component causes this effect. In conclusion, TRPM7, MagT1 and a Na+/Mg2+ exchanger are shown to be the main Mg transport proteins in REC and their expression and functional activity is influenced by the cellular Mg status. The latter responses permit adaptation of epithelial Mg absorption and enable REC to maintain a physiological [Mg2+]i which is a prerequisite for various cell functions.     

Mechanisms, regulation and pathologic significance of Mg2+ efflux from erythrocytes.

Mg2+ efflux from erythrocytes can be performed by the Na+/Mg2+ antiport and by Na+-independent Mg2+ efflux. Na+-independent Mg2+ efflux functions via the unspecific choline exchanger as choline/Mg2+ or K+/Mg2+ antiport and as Mg2+ efflux accompanied by intracellular Cl- for charge compensation, as found for example in sucrose medium. Na+/Mg2+ antiport in erythrocytes exchanges 2 extracellular Na+ for 1 intracellular Mg2+. Driving forces are the Na+ and Mg2+ gradients. By reversing these gradients, the Na+/Mg2+ antiporter can mediate Mg2+ influx. The Na+/Mg2+ antiporter can exchange 24Mg2+ for 28Mg2+ and other divalent cations for intracellular Mg2+. In the exchange mechanism, extra- and intracellular Na+ can compete with Mg2+. Na+/Mg2+ antiport is inhibited by amiloride, quinidine and imipramine. Na+/Mg2+ antiport is drastically activated by intracellular Mg2+ due to an allosteric transition. The affinity of intracellular Mg2+ to the Na+/Mg2+ antiporter is dependent on intracellular ATP due to phosphorylation. Besides this mechanism, in non Mg2+-loaded erythrocytes, the activity of Na+/Mg2+ antiport is regulated by phosphorylation-dephosphorylation and by intracellular Cl-. The drastically Mg2+-activated Na+/Mg2+ antiporter is not further stimulated by phosphorylation and intracellular Cl-. Na+-independent Mg2+ efflux via the choline exchanger is also inhibited by amiloride, quinidine and imipramine, and can also be regulated by phosphorylation-dephosphorylation. Na+/Mg2+ antiport of erythrocytes is altered in various pathologic conditions.     

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.     

Differential roles of the sodium-calcium exchanger in renin secretion and renal vascular resistance

The intracellular free calcium concentration ([Ca2+]i) is a central regulator of renin secretion and the contractility of vascular smooth muscle cells. As [Ca2+]i results from calcium influx and calcium extrusion, we were interested in the role of the Na+/Ca2+-exchanger as an important calcium-extrusion pathway in the regulation of renin secretion. Therefore, we investigated the effects of inhibiting the Na+/Ca2+-exchanger, either by reducing the extracellular sodium concentration ([Na+]e) or using pharmacological tools, on renin secretion and vascular resistance in the isolated perfused rat kidney model. Stepwise reductions of [Na+]e led to progressive (up to sevenfold) increases in renal vascular resistance ([Na+]e 7 mM) whilst renin secretion rates were not altered significantly. Similarly, pharmacological blockade of the Na+/Ca2+-exchanger by benzamil (100 microM) or KB-R7943 (30 microM) resulted in significant vasoconstrictions without altering basal renin secretion rates. Also renin secretion that was pre-stimulated by isoproterenol (10 nM), blockade of macula densa salt transport by bumetanide (100 microM) or lowering the perfusion pressure to 40 mmHg was not attenuated by Na+/Ca2+-exchanger inhibition, although the vascular resistance increased significantly. In contrast, angiotensin II (100 pM) reduced pre-stimulated renin secretion values by 50%. The subsequent lowering of the [Na+]e however did not augment the inhibition of renin secretion, although the renal vascular resistance increased markedly. We conclude that the Na+/Ca2+-exchanger has no functional role in the regulation of [Ca2+]i in juxtaglomerular cells controlling renin secretion, whereas it markedly affects the preglomerular vascular smooth muscle cells of the renal vasculature.     

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.     

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.     

A genistein-sensitive Na+/Ca2+ exchange is responsible for the resting [Ca2+]i and most of the Ca2+ plasma membrane fluxes in stimulated rat cerebellar type 1 astrocytes

The differential role of Na+/Ca2+ exchange in the regulation of intracellular ionized calcium ([Ca2+]i) in immunological and pharmacologically identified type 1 astrocytes and Purkinje cells was studied in rat cerebellar culture, using Ca2+ (Fluo-3, Fura-2) and Na+ (SBFI) fluorescence measurements. The mean resting [Ca2+]i was significantly higher (191 +/- 8 nM, n=25) in type 1 astrocytes than in Purkinje cells (92 +/- 2.5 nM, n=35). In contrast to Purkinje cells, in unstimulated cerebellar type 1 astrocytes, forward and reverse Na+/Ca2+ modes operate under resting physiological conditions, being responsible for most of the total Ca2+ transplasma membrane fluxes. Four observations support this hypothesis: (1) under resting conditions of temperature and ionic composition, Na+o removal causes a remarkable increase in [Ca2+]i, being inhibited by 2',4' dichlorobenzamil (DCB), and 2-[2-[4-(nitrobenzilloxiphenyl ethyl] isothiourea metanesulfonate (KB-R7943); (2) Ca2+o removal in the presence of Na+o causes an important drop in [Ca2+]i, which is absent in Li+o or NMG+o (N-methyl-D-glucamine) containing medium; (3) the reverse mode exchange inhibitor KB-R7943 mimics the removal of Ca2+o only in the presence of Na+o; and (4) under loaded [Na+]i conditions (ouabain or the activation of taurine-Na+-cotransport), reverse mode exchange increases in both astrocytes and Purkinje cells. In type 1 astrocytes stimulated with endothelin-3 (ET-3), the recovery of the Ca2+i signal occurs largely through the Na+/Ca2+ exchanger. Genistein, a tyrosine kinase inhibitor, completely and reversibly blocks all exchange activity, but not its inactive analogue daidzein, thus suggesting that the Na+/Ca2+ exchanger of cerebellar type 1 astrocytes may be modulated by phosphorylation. Our main conclusion is that in rat cerebellar type 1 astrocytes under resting physiological conditions, most of the total transplasma membrane Ca2+ fluxes take place through the Na+/Ca2+ exchanger, thus accounting for the resting [Ca(2+)]i.     

Magnesium net fluxes and distribution in rabbit myocardium in irreversible contracture

Distribution of magnesium (Mg) in heart muscle was studied by measuring fluxes of Mg and transmembrane potentials as a function of perfusate [Mg2+] after a massive increase in permeability of the sarcolemma was induced in the Langendorff prepared heart from the Nembutal-anesthetized rabbit. After onset of 0 mM [Ca2+] perfusion which produced excitation-contraction (E-C) uncoupling and mechanical arrest, action potentials recorded from subepicardial cells showed an increase in duration and decrease in amplitude, which progressed until no transmembrane potentials could be observed. Restoration of physiological salt solution perfusion after 15 min of [Ca2+]-free perfusion caused an irreversible contracture that was associated with 1) efflux of potassium (K) and myoglobin, 2) perfusate [Mg2+]-dependent flux of Mg, and 3) transmembrane potentials of 0 mV. The magnitude of net efflux of K and myoglobin during contracture was unaffected by perfusate [Mg2+]. During the first 2 min of contracture, net efflux of Mg (mumoles per gram wet muscle +/- SE) was 1.37 +/- 0.09 and 0.48 +/- 0.19 during 0 mM and 2.5 mM [Mg2+] perfusion, respectively; but a net influx of 0.56 +/- 0.23 occurred during 5 mM [Mg2+] perfusion. Total sarcoplasmic [Mg] may correspond to perfusate [Mg2+] of 3.6 mM, which was found by interpolation to prevent any net flux of Mg during contracture. 3.6 mM may, therefore, represent the upper limit of the intracellular free-ionized Mg concentration in rabbit heart.     

Subcellular localization of glutamate-stimulated intracellular magnesium concentration changes in cultured rat forebrain neurons using confocal microscopy

Glutamate can stimulate increases in intracellular magnesium concentration ([Mg2+]i) and induce neurotoxicity, both independent of Ca2+ changes. Although Mg2+ is essential within the cell, very little is known about how it is regulated, especially in neurons. Therefore we used the fluorescent indicator, magindo-1 and confocal microscopy to examine possible intracellular pools of Mg2+ in cultured neurons that can be dynamically regulated by glutamate. The magindo-1 fluorescence signal was present throughout the cell body and extends into the neuronal processes. The magindo-1 405 nm/490 nm ratio signal was similar in the cytoplasm and nucleus, suggesting that resting [Mg2+]i is uniform across the neuron. The addition of 100 microM glutamate/10 microM glycine in an extracellular Ca2+- and Na+-free buffer stimulated an increase in [Mg2+]i in both the nuclear and cytoplasmic regions of similar magnitude and duration. This glutamate exposure also stimulated a [Mg2+]i increase in neuronal processes which was inhibited by the N-methyl-D-aspartate receptor antagonist, MK-801 (10 microM). The glutamate-stimulated [Mg2+]i increase in both the cell body and neuronal processes was dependent on the extracellular Mg2+ concentration. These findings suggest glutamate-stimulated [Mg2+]i changes may not only impact cytoplasmic processes, but also directly trigger nuclear events involved, for example, in neuronal injury.     

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.     

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries.

The contribution of the Na+/Ca2+ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (ASK) with pronounced myogenic tone and mesenteric small arteries (AMS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na+/Ca2+ exchanger, the extracellular Na+ concentration ([Na+]o) was lowered from 143 to 1.2 mM by substituting choline-Cl for NaCl. The ASK developed significant myogenic tone and constricted further in low [Na+]o. Nifedipine (1 microM) reduced both myogenic tone and low [Na+]o-induced contraction. Because the membrane potential of ASK was not changed by low [Na+]o (-35 +/- 2 mV at 143 mM [Na+]o, -37 +/- 3 mV at 1.2 mM [Na+]o), depolarization-induced Ca2+ influx was not a cause of the low [Na+]o-induced contraction. The AMS did not develop significant myogenic tone. Although low [Na+]o also constricted AMS, the magnitude of constriction was significantly weaker than that in ASK (17 +/- 4 vs. 47 +/- 6%, P < 0.01, at 58 mM Na+). With Bay K 8644, AMS developed myogenic tone, and low [Na+]o-induced constriction was significantly increased. In conclusion, Na+/Ca2+ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca2+-extrusion.     

Influence of extracellular Na+ removal on cytosolic Ca2+ concentration in smooth muscle cells of rabbit cerebral artery.

There are some controversies over the contribution of Na+/Ca2+ exchanger (NCX) to the regulation of cytosolic Ca2+ concentration ([Ca2+]c) in smooth muscle. To prove the functional role of Na+/Ca2+ exchanger, we examined whether the removal of extracelluar Na+ could affect [Ca2+]c of rabbit cerebral arterial smooth muscle. The fluorescence ratio of fura-2 (R(340/380)) was measured in the single myocyte of rabbit middle cerebral artery and Na+ was substituted with the same concentration of NMDG+ or Li+. In 21 out of 230 cells tested, Na+ removal increased R(340,380) (deltaR(340/380)) by 115 +/- 16.5% of the deltaR(340/380) induced by 10 mM caffeine in the same cell. The Na+ removal-induced deltaR(340/380) was blocked by a selective inhibitor of cardiac type NCX exchanger (KB-R7943, (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea, 10 microM). In those cells where the Na+ removal by itself did not increase R(340/380), the caffeine-induced deltaR(340/380) was increased by Na+-removal (130 +/- 9.8% of control response, n=30). Under the whole-cell patch clamp condition, short application of caffeine induced transient increase of outward current (I(K,Ca)-transient) which reflect the change of subsarcolemmal [Ca2+]. The application of KB-R7943 increased the amplitude of I(K,Ca)-transient (n=4). These results suggest the functional existence of NCX in rabbit cerebral artery smooth muscle.