[Frontiers in Bioscience 5, d720-734, August 1, 2000]
REGULATION OF CELLULAR MAGNESIUM
Andrea M.P. Romani and Antonio Scarpa
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, 44106-4970
TABLE OF CONTENTS
The abundance of magnesium (Mg2+) within mammalian cells is consistent with its relevant role in regulating tissue and cell functions. At the last count, more than three hundred and fifty enzymes, aside from metabolic cycles, appear to require and be regulated by concentrations of Mg2+ that are well within the physiological range observed in tissues and cells. The absence of detectable major changes in cellular free [Mg2+], and the extremely slow turn-over of the cation across the cell plasma membrane under quiescent condition has supported for more than three decades the assumption that cellular Mg2+ content is kept constant at the level necessary for enzyme and channel function, and that its concentration does not require drastic and rapid changes to form complex with ATP and other phosphonucleotides.
In the last decade, a large body of new experimental observations has significantly reverted this way of thinking. Compelling evidence now suggests that large fluxes of Mg2+ can cross the cell plasma membrane in either direction following a variety of hormonal and non-hormonal stimuli, resulting in major changes in total and, to a lesser extent, free Mg2+ content within tissues, and in a marked variation in the opposite direction of circulating Mg2+ level. The present review will attempt to update our knowledge in this area and provide some insights on how changes in cellular Mg2+ content can result in a modification of the activity rate for several cellular enzymes.
2. Mg 2+ as an intracellular messenger
For many decades the role of Mg2+ in biological systems has been hampered by the difficulty of measuring accurately and selectively Mg2+ in cell and biological system. This has been partly ameliorated by the introduction of atomic absorbance spectrophotometry in the early 1950s and, more recently, by additional analytical methods.
Mg2+ is now recognized being indispensable for enzyme activity and structural modification of phosphometabolites or channels. Yet, the general consensus from a large body of evidence indicates that Mg2+ concentration is relatively stable within the cell and that whilst Mg2+ presence is necessary for cell function, it will not modulate - like Ca2+ - cell function by changing concentrations within the cytsosol. In fact, studies attempting to equate Ca2+ and Mg2+ as signaling molecules for cytosolic enzymes have been disappointing. Ca2+ is a signaling molecule because of the following conditions: a) the free cytosolic concentration is extremely low; b) the concentrations in plasma and cytosolic reservoirs are very high, establishing a large concentration gradient across biological membranes; c) because of the low resting Ca2+ concentration, the movement of a few Ca2+ molecules can increase or decrease the concentration of Ca2+ by several orders of magnitude; d) the increase of Ca2+ concentrations results in specific Ca2+ binding to cytosolic proteins which are modified in their 3-D structure and function upon formation of a complex with Ca2+.
These paradigms of operation are very different for Mg2+. Mg2+ is kept both in the cytosol and in extracellular fluids in the millimolar or submillimolar concentration. Because of this initial large concentration, a total increase or decrease of Mg2+ in the cytosol equivalent to that occurring for Ca2+ will result in negligible free Mg2+ changes. Furthermore, due to the large difference between the radius of hydrated and not hydrated Mg2+, specific coordination to proteins is less likely than that of Ca2+.
For the above reasons, the role of Mg2+ as transient regulator of cytosolic enzymes appears to be intrinsically denied by the coordination chemistry, the concentrations existing in the cytosol, and by experimental evidence. Nevertheless, fluxes of Mg2+ across the cell plasma membrane have been recently measured, leading to massive translocations that increase or decrease total cellular Mg2+ by an equivalent of 1-2 mM (approximately 5-10% of total cell content) within a few minutes. Yet, these fluxes result in minor or no changes in cytosolic free Mg2+ content.
Changes in total cellular Mg2+ in the absence of major changes in cytosolic free Mg2+ can only be explained assuming the fulfillment of the following tenets:
a) When massive total cellular Mg2+ release or uptake occurs, the source or destination of mobilized Mg2+ must be an intracellular compartment or a major binding site;
b) The plasma membrane must possess a sensor (which keeps cytosolic free Mg2+ relatively constant in spite of massive redistribution of the total) and/or a powerful uptake/release mechanism (which extrudes or accumulates the large amount of Mg2+ from the cell to the extracellular fluid, or viceversa, to maintain cytosolic free Mg2+ constant).
Hence, at variance from Ca2+, regulation of cellular functions by Mg2+ should be expected to occur not in the cytosol but within organelles (or binding sites), where Mg2+ is being mobilized, and in the plasma, where Mg2+ concentration can rapidly increase or decrease more than 20%.
In the following pages we will focus mostly on what is known about hormonal modulation of extracellular Mg2+ concentration and fluxes of Mg2+ across the plasma membrane, and on the possible regulation by Mg2+ of metabolic parameters such as respiration, following changes of the cation within organelles.
3. CHANGES IN SERUM Mg2+ LEVEL
Circulating Mg2+ level is 1.5-1.7 mEq/L in humans and in many mammals (1-3). A decrease in serum Mg2+ level has been reported to occur during several chronic diseases, both in humans and in animals (4-6). Yet, there is a remarkable lack of information, or contrasting result, as to whether magnesemia undergoes circadian fluctuations following the release of hormones or physiological stimuli (e.g. fasting or exercise). Studies conducted in conscious humans (7) or ovine (8) infused with catecholamine for a period of time varying between 30 min to 5 hours resulted in a varying level of hypomagnesemia and in a marked increase of Mg2+ excretion in the urine. In contrast, minimal or not changes in serum Mg2+ level were found in rats infused with epinephrine in the presence of alpha-adrenergic receptor blockade (9), or with isoproterenol (10), respectively.
This and other laboratories have reported that the administration of isoproterenol, epinephrine or norepinephrine to perfused rat hearts (11-13) and livers (14,15) results in an extrusion of Mg2+ from the organs into the perfusate. Consistent with this observation, the infusion of increasing doses of isoproterenol to anesthetized rat results in a marked dose- and time-dependent increase in circulating Mg2+ level. The increase is serum Mg2+ is already detectable within 10 min, reaches the maximum within 20 min after the agent administration (10,16), and remains unchanged up to 2 hours even in the absence of the agonist (10). This time course suggests that the increase in serum Mg2+ occurs independently of the hemodinamic changes (i.e. increase in heart rate and decrease in mean arterial pressure) induced by the beta-adrenergic agonist for the limited time of the infusion (16). Additional support to this hypothesis is provided by the inability of sodium nitroprusside to significantly change serum Mg2+ level despite the fact it can mimic the decrease in mean arterial pressure induced by isoproterenol (16). The persistent increase in serum Mg2+ also implies that the stimulation of beta-adrenergic receptor results in the activation of secondary mechanism(s) which account for the long-term persistence of this phenomenon. At its peak, the increase accounts for a net change of ~7 micromol Mg2+/300 g b.w., or 10% above basal level, for an infused dose of 0.1 microgram isoproterenol/kg/min, and ~10 micromol Mg2+/300 g b.w., or 20% above basal level, for a dose of 10 microgram/kg/min. This increase occurs via the specific activation of beta2-adrenergic receptor. It can be mimicked by the administration of the selective beta2-adrenergic agonist salbutamol, and inhibited by the specific beta2-blocker ICI-118551, and not by the beta1-adrenergic agonist prenalterol or the beta1-blocker CGP-20712A, respectively (16). Most likely, this difference is attributable to the larger distribution of beta2 versus beta1 adrenergic receptors present in the all body (17,18), rather than to a different signaling pathway.
Based upon the observed percent change in serum Mg2+, it can be estimated that the circulating level of the cation would increase from 0.75-0.8 mM to ~0.9-1 mM (16) as a result of tissue release into the bloodstream. Yet, the attempt to determine from which tissue(s) Mg2+ is mobilized into the circulation has not provided a conclusive answer (16). Because of the inhibitory effect of carbonic anhydrase inhibitor infused in an anesthetized rat, Gunther and co-workers (10) have proposed that bones may represent the primary source of Mg2+ mobilization following isoproterenol administration. However, as beta2-adrenergic receptors are largely distributed throughout all the organs, it cannot be excluded that other tissues contribute, to a varying extent, to the observed increase in circulating Mg2+ level. It is worth to note that changes in renal excretion do not appear to contribute significantly to determine the initial increase in serum Mg2+ level. Based upon the pre-infusion level of serum Mg2+, the glomerular filtration rate (1.62 mL/min (19)) and the fractional excretion (17% (19)), it can be estimated that only one-third of the increase in serum Mg2+ level observed at 20 min would occur in the case of a total block of the renal fractional excretion (16). More difficult to assess is the extent to which a reduced glomerular filtration, together with a beta-adrenergic stimulated, cAMP-mediated, increase in Mg2+ reabsorption at the level of the thick ascending portion of Henle's loop (20) contribute to the persisting elevated level of circulating Mg2+ up to 2 hours after the end of agonist infusion (10).
As expected, the infusion of epinephrine or norepinephrine to an anesthetized rat results in an increase in circulating Mg2+ of approximately 20%, comparable to that induced by isoproterenol. In this case, however, the increase occurs via activation of both alpha- and beta-adrenergic receptors (21), as it can be prevented by the use of pharmacological inhibitors for these two classes of adrenergic receptor (21). More interesting, however, is the observation that the administration of a bolus of insulin in the absence of glucose clamp induces a marked hypoglycemia which is followed, within 5-10 min, by an increase in serum Mg2+ that resembles, in onset and extent, that induced by catecholamine infusion (21). Because the increase in serum Mg2+ prompted by insulin does not occur in animals pre-treated with alpha- and beta-adrenergic receptor blockers, or with reserpine (21), it can be reasonably concluded that the increase in serum Mg2+ under these experimental conditions is attributable to the release of endogenous catecholamines triggered by hypoglycemic condition.
The discrepancy between the earliest studies, in which changes in serum Mg2+ were minimal or absent, and the most recent observations showing a considerable increase in magnesemia is not easy to explain. Several factors may contribute to the mentioned incongruity. The relative proportion of beta-adrenergic receptor subtypes in different experimental models, the ability of catecholamines and isoproterenol to stimulate with differing hierarchy alpha- and beta-adrenergic receptors or distinct beta-adrenoceptor subtypes, modality, rate and duration of drug infusion are but a few of the possibilities to be considered.
Although the most recent observations suggest that circulating Mg2+ level can increase following the release of endogenous catecholamine or their exogenous infusion, likely through the activation of common mechanism(s), the physiological significance of this increase still remains elusive. Also, it remains unanswered the question whether the increase in serum Mg2+ represents a signal for organs or tissues able to sense this change in circulating Mg2+, and/or for the organs and tissues from which Mg2+ is released.
The hypothesis that the increase in serum Mg2+ has systemic signaling activity implies that organs or tissues have the ability to sense changes in circulating Mg2+ level. Presently, no specific Mg2+ sensing mechanism has been identified. However, the Ca2+ sensing mechanism identified by Brown and collaborators as the physiological regulator of calcemia (22) appears to sense changes in circulating Mg2+ as well, though at higher concentrations than Ca2+ (23), that is in a range that would be consistent with the reported increase in serum Mg2+ level (10,16,21). Whether the Ca2+ sensing mechanism is a bi-functional regulator, or represents the epitome of a new class of sensors still to be identified, is topic for future studies. Regarding this possibility, it has to be mentioned that, in contrast to the original report by Brown et al. (22), Bapty et al have recently observed the operation of a Ca2+-sensing mechanism with comparable sensitivity for extracellular Ca2+ and Mg2+ in mouse distal convoluted tubule cells (MDCT) (24). The activation of this sensor mechanism appears to inhibit the glucagone- or vasopressin-mediated entry of Mg2+ into the cell (25). This observation should explain the clinical and experimental evidence that hypermagnesemia and hypercalcemia can inhibit hormone-stimulated, cAMP-mediated, reabsorption of both Mg2+ and Ca2+ along the different segments of the nephron (26). Also, it may provide distal regulation to restore circulating Mg2+ to a physiological level following the increase observed in anesthetized animals infused with isoproterenol (10,16) or catecholamine (21), and opens an interesting and totally new area of investigation for hormonal regulation of cellular Mg2+ homeostasis.
The absence of showy physiological effects despite the magnitude of the increase in serum Mg2+ is a striking difference with a comparable increase in serum Ca2+, which induces muscle weakness and arrhythmia. Overall, it appears that large variations in magnesemia are well tolerated in vivo. For example, rats infused with boluses of Mg2+ that increase magnesemia by 50% do not manifest significant systemic hemodynamic changes, but present a marked increase in coronary artery flow (27). Baboons that received pharmacological doses of Mg2+ sufficient to prevent cardiac arrhythmias induced by epinephrine presented a significant reduction in the epinephrine-induced increase in mean arterial pressure and systemic vascular resistance (28). Moreover, studies in vitro suggest that Mg2+ can regulate catecholamine release from both peripheral and adrenal sources (29), and that an elevated level of [Mg2+]o has a significant modulatory effect on cardiac contractility (13). Taken together, these observations i) pose for a role of Mg2+ as an endogenous modulator of catecholamine release and activity, and ii) suggest that an increase in circulating Mg2+ following adrenergic stimulation may contribute to improve blood flow and O2 delivery to the heart, and possibly other tissues as well, at a time when an increase in energy production is expected.
4. CHANGES IN CELLULAR Mg2+ CONTENT
Concentrations of total Mg2+ ranging between 17-20 mM have been measured and estimated in the majority of mammalian cell types by a variety of technical approaches (see Table 1 in reference 30). Within the cell, total Mg2+content is distributed almost homogeneously among nucleus, mitochondria and endo-(sarco)-plasmic reticulum (31,32). A considerable amount of Mg2+, approximately 4-5 mM, is present in the cytosol as a complex with adenine trisphosphate and other
Table 1. Panel A. Tissues and cell types in which a cAMP-induced Mg2+ extrusion has been observed
Table 1. Panel B. Tissues and cell types in which an accumulation of Mg2+ has been observed
1Mg2+ accumulation is inferred based upon an observed increase in [Mg2+]i.
phosphometabolites (33). Finally, with the exception of calmodulin (34) and S100 protein (35), no other protein is known to have specific binding sites for Mg2+ within the cell. The techniques for measuring cytosolic free Mg2+ are a far cry from those available to measure Ca2+, H+ or other cations. The approaches commonly used are 31P-NMR, selective Mg2+-electrode, 13C-NMR citrate/isocitrate ratio or the fluorescent indicator Mag-Fura (see Table 1 in ref. 30), and each of them presents significant drawbacks in term of sensitivity and/or selectivity. Using these techniques, cytosolic free Mg2+ has been estimated to range between 0.5-1 mM (i.e. less than 5% of total cellular Mg2+) in the majority of cells analyzed (see Table I in ref. 36). Under resting conditions no major changes in cytosolic free Mg2+ content are observed, and Mg2+ fluxes across the plasma membrane, measured by 28Mg re-distribution (37), appear to be slow. For example, an early report by Page and Polimeni (37) indicates that radioisotope equilibrium is achieved within 72-80 hours in cardiac cells incubated at 37oC, and an even longer period is required when the experimental temperature is reduced to 20oC (37). These premises led to the conclusion that a Mg2+ transport mechanism with a slow rate of activity must be present in the cell plasma membrane (37).
In recent years, this perception has been largely reconsidered following reports by Vormann and Gunther (12,15), Jakob et al (38), Romani and Scarpa (11,14) and Howarth et al (13), which indicate that cardiac (11-13) and liver cells (14,15,38) extrude a considerable amount of cellular Mg2+ within 5-6 min from the administration of an adrenergic agonist.
These first observations have been followed by a large number of similar reports, supporting the notion that different hormonal stimuli can induce significant changes in total and free Mg2+ content in a variety of cells. Based upon these studies, two main paradigms leading to Mg2+ extrusion or accumulation have emerged:
1) a condition in which Mg2+ is extruded from the cell via the stimulation of adrenergic receptors and the increase in cellular cAMP level, or by agents that decrease cellular ATP content, and
2) a condition in which Mg2+ is accumulated into the tissue following a decrease in cAMP level or the stimulation of protein kinase C pathway.
The tissues and organs in which these two processes have been observed are reported in panels A and B of Table 1.
4.1. Mg2+ extrusion following beta-adrenergic receptor stimulation, and role of cAMP
The administration of isoproterenol, epinephrine or norepinephrine results in a marked extrusion of Mg2+ from cardiac (11-13,39) or liver cells (14,15,40-42). This extrusion becomes evident within 1 min from the addition of the agonist to the perfusate, or the incubation system, and reaches the maximum within 5-6 min, irrespective of the dose of agonist administered. Under perfusion conditions, after this period of time Mg2+ extrusion returns towards the basal level, independent of the persistence of the agonist in the perfusate (11,14,42). When a submaximal dose of agonist is added repeatedly to the perfusate, each addition results in an extrusion of Mg2+ progressively smaller than the previous one (11), a result which can be reasonably interpreted as a progressive depletion of intracellular Mg2+ store(s). As for the mechanism involved in the extrusion of Mg2+ from cardiac or liver cells, two distinct considerations can be made, regarding the intracellular signaling pathway and the transport mechanism involved at the plasma membrane, respectively. As for the intracellular signaling pathway, Mg2+ extrusion can be inhibited by the administration of non-selective beta-adrenergic receptor antagonist (propranolol or sotalol) (11,14) or, in the case of cardiac cells, by selective beta1-adrenergic blocking agents (e.g. atenolol) (11). Furthermore, Mg2+ extrusion can be mimicked to a comparable extent by the administration of the cell permeant cyclic AMP analogs di-butyryl-cAMP, 8-Cl-cAMP or 8-Br-cAMP (11,14,40), or forskolin (11,14,40), an agent that activates in an irreversible manner adenylyl cyclase (43). Conversely, it can be inhibited by Rp-cAMPs (44), an isomer of cAMP able to cross the cell plasma membrane and prevent specifically protein kinase A activation by endogenous cAMP (45). These observations suggest that the activation of beta-adrenergic receptors (mainly beta1 subtype in cardiac ventricular myocytes (46)) by catecholamine or isoproterenol increases the cytosolic level of cAMP via adenylyl cyclase, and induces an extrusion of Mg2+ from the organ into the perfusate or the extracellular compartment. As for the mechanism responsible for Mg2+ extrusion across the plasma membrane, it appears to require the presence of a physiological concentration of [Na+]o (39,41) and [Ca2+]o (39,41) (see following section for further details). In fact, the extrusion of Mg2+ induced by isoproterenol or by catecholamine can be inhibited by the removal of any of these two cations from the extracellular milieu (39,41), or by the administration of Na+-transport inhibitors such as amiloride (12), imipramine (47) or quinidine (48), or Ca2+-channel inhibitors such as verapamil (39) or nifedipine (39).
Following the first observations by Gunther and collaborators (12) and by this laboratory (11), the number of reports indicating the operation of the beta-adrenergic mediated Mg2+ extrusion mechanism in tissues or cells other than heart and liver has increased considerably. Erythrocytes (49), lymphocytes (44), Erlich ascites cells (50), sublingual mucous acini (51), thymocytes (52) and HL-60 promyelocytic leukemia cells (53) are just a few examples of tissues or cell types in which Mg2+ extrusion has been reported to occur via an increase in cellular cAMP level, independently of the pathway leading to cAMP increase. Cittadini and collaborators, for example, have observed that prostaglandin PGE1 or PGE2 and arachidonic acid are also able to increase cellular cAMP level and thus induce an extrusion of Mg2+ from freshly isolated spleen lymphocytes (44) or Erlich ascites cells (50,54), respectively. Therefore, a more general signaling pathway responsible for Mg2+ mobilization can be envisioned, whereby the increase in cellular cAMP level triggered by various exogenous stimuli can result in an extrusion of Mg2+ via a Na+-dependent mechanism. Although the extrusion mechanism has not been purified or cloned, the published observations suggest that the increase in cAMP level likely increases the activity rate of the transporter via phosphorylation (52).
4.2. Is there a role for alpha-adrenergic receptors in Mg2+ extrusion?
The reports by Vormann and Gunther (12), Romani and Scarpa (11) and Howarth et al. (13) are consistent with the operation of a beta-adrenergic receptor induced, cAMP-mediated, Na+-dependent Mg2+ extrusion mechanism in both cardiac and liver cells. In contrast, the data by Jakob et al. (38) indicate that Mg2+ can be extruded from liver cells by phenylephrine via activation of alpha1-adrenergic receptor.
A recent report by Keenan et al. (42) further support the presence of two distinct signaling pathways for Mg2+ extrusion in liver cells. In search of hormones that can modulate cellular cAMP level and possibly counteract the effect of the second messenger on Mg2+ homeostasis and extrusion, our laboratory has recently focused its attention on insulin. The results obtained so far indicate that the infusion of insulin into heart (55) or liver (42) prior to isoproterenol or to cell permeant cAMP analogs (8-Br-cAMP or 8-Cl-cAMP) administration completely blocks the extrusion of Mg2+ induced by these agents. The replacement of isoproterenol with epinephrine or norepinephrine results in an extrusion of Mg2+ from liver cells that is minimally reduced (10-15%) by insulin pre-treatment (42). In contrast, insulin pre-treatment does not affect the amplitude of Mg2+ extrusion from livers stimulated by phenylephrine administration (42).
Therefore, it can be hypothesized that both alpha- and beta-adrenergic receptor are able to induce Mg2+ extrusion from a tissue following the stimulation by specific alpha or beta agonists, or by mix agonists such as epinephrine or norepinephrine.
Because catecholamines stimulate both alpha- and beta-adrenergic receptors with a slightly different hierarchy (56), the observation by Jakob et al. (38) and our laboratory (42) pose several questions: Do alpha1- and beta-adrenergic receptors activate the same Mg2+ extrusion mechanism? Which is the signaling mechanism used by alpha1-adrenoceptors? From which intracellular pool(s) is Mg2+ mobilized following alpha1 or beta-adrenergic stimulation? And why the tissue requires a redundancy of signaling and Mg2+ extrusion mechanisms? Are alpha1 and beta-adrenergic receptors activated at the same time by catecholamine administration? Or does one class of receptors operate only when the other is inhibited?
Due to the relative novelty of this observation, no overall satisfactory answer is presently available, though certain observation can be explained. For example, because the stimulation of alpha1-adrenergic receptors increases cytosolic Ca2+ (57), it is reasonable to infer that Ca2+ may activate directly the Mg2+ transporter. Yet, it is not fully defined whether Ca2+ is used to simply activate the transporter, or it is also utilized as a counter-ion for Mg2+ extrusion, or whether Ca2+ activates, in a synergistic or alternative manner, the Na+-dependent mechanism activated by cAMP. Preliminary data from our laboratory would suggest that the administration of phenylephrine to liver cell results in a Mg2+ extrusion via a Ca2+-dependent mechanism (approximately 15-20% of total Mg2+ extrusion) and via an Ca2+-activated Na+-dependent mechanism, that accounts for the majority (80-85%) of Mg2+ mobilization (58). Finally, by comparing the amount of Mg2+ mobilized by epinephrine, isoproterenol or phenylephrine, it appears that epinephrine can mobilize an amount of Mg2+ which is approximately double than the amount of Mg2+ mobilized by the administration of the selective alpha or beta agonist (42,58). In other words, epinephrine appears to mobilize Mg2+ by stimulating concomitantly both alpha and beta-adrenoceptor. Although preliminary and indirect, this observation would indicate that liver cells possess two distinct intracellular pools from where Mg2+ can be mobilized by the selective stimulation of the two classes of adrenergic receptors, respectively.
In Figure 1 the alpha1and beta-adrenergic receptor activated signaling pathways and their modulatory effect on cellular Mg2+ homeostasis are schematically reported.
Figure 1. The intracellular signaling pathways activated following the stimulation of alpha1- and beta- adrenergic receptor are schematically illustrated in the cartoon. The stimulation of beta-adrenergic receptor by proper agonists results in the activation of adenylyl cyclase to increase cytosolic cAMP level. The second messenger may interact at the mitochondrial level with the adenine nucleotide translocase (ANT) and induce an extrusion of Mg2+*ATP from the organelle into the cytosol. Also, cAMP may directly, or indirectly, modulate the operation rate of the Na+/Mg2+ exchanger at the plasma membrane level, determining an extrusion of cellular Mg2+ through this mechanism. The stimulation of alpha1-adrenergic receptor, instead, induces the formation of IP3 and diacyl-glycerol via phospholipase C activation. Inositol-trisphosphate interacts with a specific receptor at the endo-(sarco)-plasmic reticulum level and favors a release of Ca2+ from this pool into the cytosol, which - directly or indirectly - determines an entry of Ca2+ through the plasma membrane (capacitative Ca2+ entry). The activation of this signaling pathway also results in an extrusion of Mg2+ from the cell via the Na+/Mg2+ exchanger and/or via a Na+-independent, Ca2+-modulated (dependent?) extrusion mechanism.
4.3. Other agents that induce Mg2+ extrusion
A marked extrusion of Mg2+ from liver cells (59-61), rat erythrocytes (62), and HL-60 promyelocytic leukemia cells (63) has also been observed following the administration of cyanide (59), FCCP or other mitochondria uncouplers (59,63), fructose (60,62), or ethanol (61). Because all these agents decrease cellular ATP content, it can be reasoned that, by depriving Mg2+ of its most abundant intracellular chelating component, these substances increase cytosolic free Mg2+. This increase, in turn, results in an extrusion of Mg2+ from the cell via a Na+-dependent mechanism. An afterthought of this consideration is how Mg2+ extrusion is activated under these conditions. The determination of cellular cAMP level following acute ethanol administration has failed to evidence an increase in second messenger content (61), thus excluding an involvement of this pathway in the extrusion of Mg2+. Therefore, possibility is there that the increase in cytosolic free Mg2+ subsequent to the decrease in ATP content is sufficient per se to activate the transporter and induce the extrusion of Mg2+. Alternatively, it can be hypothesized that Mg2+ is extruded as a compensatory mechanism for the altered Na+ and K+ distribution within cells depleted of ATP (64). An increase in cytosolic free Mg2+ has been observed by Lemasters and collaborators in cultured hepatocytes treated with cyanide (65), and by Hue and co-workers in liver cells incubated in the presence of fructose (60). However, the increase in free Mg2+ concentration is considerably lower than it would be expected based on the amplitude of ATP decrease in the cell. Although the possibility of a redistribution of Mg2+ among intracellular compartments cannot be excluded, the limited increase in cytosolic Mg2+ would be consistent with a Mg2+ extrusion, at least partial, across the cell plasma membrane, as reported by Dalal et al (59).
4.4. Hormones and stimulatory agents involved in Mg2+ accumulation
While adrenergic agonists stimulate cellular Mg2+ release, other hormones or agonists induce cellular Mg2+ accumulation.
The work by de Rouffignac and collaborators (66-68) and by Quamme and co-workers (reviewed in 2,68,69) suggests that the renal apparatus represent the major site where hormones operate to increase Mg2+ reabsorption and, consequently, decrease its hematic level. To avoid redundancy, we refer to that part of the monograph for a more exhaustive description about the role of the renal apparatus on Mg2+ handling, and limit our interest to hormones that modulate acutely, or within minutes, total and free Mg2+ content at the cell level.
Among the hormones that induce an accumulation of Mg2+ into the tissue or, at least, counteract the Mg2+ mobilizing effect of adrenergic agonists or catecholamine, insulin has a preeminent role. We have briefly mentioned some of the most recent data provided by our laboratory to this regard (see previous paragraph and also ref. 42). By phosphorylating the tyrosyl residues 350 and 354 at the C terminus of beta2-adrenergic receptor, insulin would block the receptor (70), thus preventing its activation by isoproterenol or other agonists. Also, by increasing phosphodiesterase activity (71), insulin enhances the degradation of cAMP to AMP and hampers the signaling by the second messenger inside the cell.
Not only insulin prevents the Mg2+ extrusion induced by isoproterenol or cell permeant cAMP in cardiac (55) and liver cells (42), but also induces a detectable accumulation of Mg2+ in 3T3 fibroblasts (72) and other cell types (73). Vasopressin and angiotensin-II also induce Mg2+ accumulation in cells. Our laboratory has reported that the administration of vasopressin to hepatocytes (40,41) results in an accumulation of Mg2+ in these cells via a Na+-dependent mechanism. Similar results have been observed by Dai et al in renal epithelial cells stimulated by vasopressin (74), and by Touyz and Schiffrin (75) and by Okada et al (76) in smooth muscle cells from mesenteric artery stimulated by vasopressin (75,76) or angiotensin-II (75). The main difference between these data is that we measured changes in total Mg2+ by atomic absorbance spectrophotometry, whereas the other groups detected an increase in cytosolic free Mg2+ by using the fluorescent dye Mag-Fura (74-76). The use of this technique allowed Touyz and Schiffrin (75) to notice that the change in
cytosolic free Mg2+ induced by vasopressin appears to be biphasic. A first, transient, increase in cytosolic free Mg2+ content is rapidly (within 2 min) followed by a decrease. Whether the second phase indicates redistribution into an intracellular compartment, or a belated extrusion across the plasma membrane is undefined. However, because under our experimental conditions (40,41,77) Mg2+ accumulation persists for about 6 min after the drug administration, the former possibility appears to be more likely.
In all the experimental models mentioned above, however, the hormones appear to operate via activation of protein kinase C pathway. In fact, under conditions in which protein kinase C pathway is inhibited by calphostin C (75), or is down regulated by exposure to a supra-maximal dose of phorbol myristate acetate (77), the accumulation of Mg2+ does not occur. On the other hand, the effect of vasopressin can be mimicked, to a comparable extent, by the administration of diacyl-glycerol analogs (e.g. oleoyl-acetyl-glycerol or stearoyl-arachidonoyl-glycerol) or phorbol-myristate acetate analogs (e.g. PMA or PDBU) that permeate the cell plasma membrane and interact directly with protein kinase C (78). Accumulation of Mg2+ by phorbol myristate acetate in thymocytes or S49 lymphoma cell lines has also been reported by Csermely et al (79) and Maguire and Grubbs (80), respectively. Additional agents that induce Mg2+ accumulation are bombesin, which increases cytosolic free Mg2+ concentration in 3T3 fibroblasts (72), and carbachol, which appears to mediate a similar effect in pancreatic islets (73), cardiac ventricular myocytes (11,39), hepatocytes (14,39,77), and sublingual mucosa acini (51), most likely via a decrease in cytosolic cAMP level (81).
As the administration of vasopressin or angiotensin-II induces also changes in cytosolic Ca2+, the possibility that this cation plays a relevant role in modulating Mg2+ uptake cannot be dismissed. Several experimental evidences support this hypothesis. Hepatocytes loaded with BAPTA-AM as an intracellular Ca2+ chelating agent do not accumulate Mg2+ when stimulated by vasopressin or phorbol myristate acetate (41). Also, when hepatocytes are stimulated by vasopressin within two minutes from thapsigargin administration, the amplitude of Mg2+ accumulation declines with time, up to a point in which an extrusion of Mg2+, rather than an accumulation, is observed (41). These observations are difficult to be interpreted because changes in cellular Ca2+ and Mg2+ occur on different time-scales and have different amplitudes (82,83). Cytosolic free Ca2+ can increase several orders of magnitude within seconds or minutes, returning towards basal level thereafter. Cytosolic free Mg2+, which is already in the millimolar range, can increase in absolute amount far more than Ca2+, though the concentration only increases by approximately 10-15% (84). Thus, it is reasonable to postulate that the rapid increase in cytosolic Ca2+ induced by hormones like vasopressin is required to activate an entry of Mg2+ across the cell plasma membrane and its redistribution within intracellular compartments.
An indication that the cell is able to sense the intracellular concentration of Mg2+ and modulate it dynamically is provided by the observation by Quamme and co-workers that an accumulation of Mg2+ occurs in cardiac ventricular myocytes (85), MDKC cells (86), or MDCT cells (87-90) as a consequence of a prolonged exposure (~16 hours) of the cells to 0 mM [Mg2+]o. Under these experimental conditions, the cytosolic free Mg2+ concentration decreases to about half the normal level and remains unaltered as long the cells are maintained in an incubation medium with 0 mM [Mg2+]o. When [Mg2+]o is increased, intracellular free Mg2+ level returns to the normal level (87), at a rate of 170-180 nM/sec, within a time frame that is directly proportional to [Mg2+]o (87). The process occurs within a few minutes and is prevented by the presence of L-type Ca2+-channel inhibitors (i.e. verapamil, nifedipine) or La3+ in the incubation medium (85-87). Because no significant changes in cytosolic [Ca2+]i are observed under these experimental conditions, it appears that these inhibitory agents affect directly the Mg2+ entry mechanism (85-90).
Mg2+ accumulation can also occur as a consequence of ion redistribution. In fact, additional observation from Quamme and collaborators indicates that renal epithelial cells accumulate Mg2+ as a result of phosphate (88) or potassium (89) redistribution across the cell plasma membrane. The latter point is particularly interesting and worth further investigation because it implies that the change in membrane potential determined by a redistribution of K+ across the plasma membrane may result in an activation of Mg2+ transport for charge compensation. Finally, it is interesting to note that the same agents that induce Mg2+ extrusion in other cell types or tissues (e.g. PGE2 (90), isoproterenol (20), cAMP (91)) all induce a Mg2+ uptake at the level of the thick ascending limb of the Henle's loop.
Considering altogether the ability of several tissues to release or accumulate Mg2+ as a result of varying stimulatory conditions, two distinct considerations can be drawn: a) that the cell senses the cytosolic free Mg2+ concentration and adjusts it according to its physiological requirement as a result of changes in energy content or other cations distribution, and b) that the hormones stimulating Mg2+ extrusion from different organs or tissues, thereby increasing plasma Mg2+, also increase Mg2+ reabsorption at the renal level to prevent a net loss of the cation.
5. Mg2+ TRANSPORT ACROSS THE CELL PLASMA MEMBRANE
Heretofore, the Mg2+ transporter(s) in the plasma membrane has(have) not been purified or cloned, and its(their) presence is inferred based upon data provided by Mg2+ fluxes. Not only uncertainty exists about the number of Mg2+ extrusion mechanisms present in the cell plasma membrane but it is also undefined whether Mg2+ release and accumulation are accomplished by the same transporters operating in either direction or by distinct transport mechanism(s).
Gunther and Vormann first reported the operation of a Mg2+ extrusion mechanism in the plasma membrane of chicken and turkey erythrocytes (92,93). The authors observed that in these cells the extrusion mechanism operates as an electroneutral Na+/Mg2+ exchanger (2 Na+in for 1 Mg2+out) (93) inhibited by amiloride (93). Since their report, an increasing number of observations suggests that a similar mechanism operates in the majority of mammalian cells. Table 2 lists the cell types in which the operation of a Na+/Mg2+ exchanger has been observed. Over the years, the list has extended to comprehend a broad variety of normal cells, including cardiac myocytes (12,39,94), smooth muscle cells (95), hepatocytes (15,41,96), erythrocytes (48,92,93,97,100), lymphocytes (44), sublingual mucous acini (101), as well as pathological cell lines (HL-60 promyelocytic leukemia cells (44) or Erlich ascites cells (50)). Nor the list is limited to mammalian cells, as the operation of a similar Mg2+ transport mechanism has also been observed for decades in non-mammalian cell types such as squid axon (102) or barnacle muscle (103), and - more recently - in leech neuron (104).
Table 2. Tissues in which the operation of a Na+/ Mg2+ exchanger has been observed or hypothesized
1Operation of Na+/Mg2+ exchanger evidenced in plasma membrane vesicles from these organs. 2In Paramecium, Na+ and Mg2+ currents have been recorded at the cell plasma membrane.
The modality of activation and operation of this transport mechanism are not fully defined, to the point that its presence in certain cells (e.g. cardiac ventricular myocytes) is questioned (see ref. 105 for more detail). The data available in literature point to an involvement of this Mg2+ transport mechanism in the Mg2+ extrusion induced by beta-adrenergic receptor stimulation, likely through a cAMP-mediated phosphorylation process (52), or by cyanide, fructose or ethanol administration, likely via the increase in cytosolic free Mg2+ content subsequent to the decrease in ATP content determined by these agents (59-63). The inability to measure accurately Mg2+ fluxes across the plasma membrane does not permit to confirm or exclude whether this exchanger is responsible for the slow turnover of cellular Mg2+ observed under basal condition (37). Also, the stoichiometry of operation of the exchanger appears to vary according to the cell type considered. For example, an electroneutral ratio 2:1 has been observed in chicken and turkey erythrocytes (93), but not in hamster (100) or human red blood cells (99,106), in which an exchange of one to three Na+ for one Mg2+ has been documented. An electrogenic operation of the exchanger would imply at least one of the following possibilities: a) that other ions have to be transported across the plasma membrane for charge compensation, or b) that changes in membrane potential would have a marked effect on the Na+/Mg2+ exchanger.
The latter possibility, though it cannot completely dismissed, appears to be the less likely. Attempts to induce modest changes in membrane potential have results in negligible or no change in cellular Mg2+ content (102,107). To this respect, it has to be kept in mind that the results provided by Quamme and collaborators (88-91) have been obtained in Mg2+ deficient cells, as no changes in [Mg2+]i were observed in cells that maintained a physiological level of cytosolic free Mg2+ of ~0.5 mM (74,89). On the other hand, experimental results have been provided by several laboratories about the operation of a Mg2+ extrusion mechanism that does not requires Na+ and therefore termed Na+-independent (41,48, 108,109). In terms of ion counter-transported for Mg2+, the Na+-independent mechanism does not appear to be extremely selective. A Mg2+ extrusion in exchange for extracellular Ca2+ (39,41), Mn2+ (108,110), Cl- (41,109), or HCO3- (96,111), has been described in erythrocytes (108-110), cardiac myocytes (39), hepatocytes (41,96) and other cell types as well. The operation of this transport mechanism is facilitated by the presence of a low concentration of [Na+]o, as increasing concentrations of [Na+]o as well as quinidine, ATP depletion, or cAMP administration (96,108) markedly inhibit its functioning. The extrusion mediated by the Na+-independent mechanism appears to occur on one-to-one ratio, at least when Mn2+ is exchanged for Mg2+ (108), and can also operate in a reverse mode, thus favoring an entry on Mg2+ into the cell (108,112). A particular Na+-independent transport mechanism is the Mg2+/Mg2+ exchanger, whose operation has been hypothesized to occur in erythrocytes (113) and cardiac cells (114), as well as in non-mammalian cell types (115). The data, obtained by monitoring the movement of 24Mg and 28Mg radioisotopes in opposite directions across the plasma membrane of red blood cells (113), indicate a one-to-one transport of Mg2+ across the cell membrane, with not net gain or loss in terms of total cellular Mg2+ content. Kinetic evaluation provides a Km value for the putative Mg2+/Mg2+ exchanger which is not too dissimilar from that obtained for the Na+/Mg2+ exchanger (93), thus rising the question whether these two transport mechanisms are separate entities or two mode of operation of the same transporter, as proposed by Gunther (116).
The possibility that Mg2+ may cross the plasma membrane via a selective channel is appealing but not sufficiently supported by available experimental evidence. Preston and co-workers have provided evidence for the operation of a selective Mg2+-channel in Paramecium (117) that becomes operative following the administration of extracellular GTP (118). Yet, no comparable observation is available for mammalian cells. The possibility that Mg2+ may enter MDCT cells (88-91), cardiac myocytes (85) or MDCK cells (86) via a channel is indirectly supported by the inhibitory effect of Ca2+-channel blockers (verapamil or nifedipine) or La3+ (85-91, 119) on Mg2+ accumulation in these cell types.
Finally, it has to be mentioned the relevant role that intracellular ATP plays at regulating these Mg2+ extrusion pathways. While the results obtained in squid axon (102) indicate that the Na+-dependent Mg2+ efflux requires cellular ATP and is inhibited by procedures that decrease ATP content (102), the data obtained in mammalian cells provided a far more complex scenario. As mentioned previously, the Na+-independent pathway, irrespective of the cation used, appears to be inhibited by the removal of ATP, at least in erythrocytes (109) or hepatocytes (96). As no evidence exists that Mg2+ is extruded from the cell via the operation of an outwardly oriented Mg2+-ATPase, it has been hypothesized that ATP is required to maintain the transporter in a phosphorylated, active state (96-109). On the other hand, under conditions in which ATP level is permanently (chemical hypoxia, (59,65)), or transiently (acute ethanol treatment, (61)) decreased, the extrusion of Mg2+ via a Na+-dependent mechanism inhibited by amiloride appears to be enhanced (59,61). The latest scenarios are more consistent with ATP being the major ligand for Mg2+ in the cytosol of the cell, so that - when ATP content decreases - the consequent increase in free Mg2+ concentration results in an extrusion across the plasma membrane (59,61).
Recently, to better characterize the Mg2+ transport mechanism(s), different research groups have undertaken the more direct approach of purifying plasma membrane vesicle from brush border cells of rabbit ileum (120), of duodenum and jejunum of rat (121,122) and from rat hepatocytes (123).
The transport of Mg2+ in vesicles from rabbit ileum has been investigated by using the fluorescent dye Mag-Fura2 both as cell impermeant and permeant (acetoxymethylester) derivatives, whereas 28Mg or atomic absorbance spectrophometry were used to detect Mg2+ fluxes in plasma membrane from small intestine or liver cells, respectively. The result obtained in ileum and liver plasma membrane support the operation of two Mg2+ transport mechanisms, one of which modulated by an established Na+ gradient across the plasma membrane. Yet, some marked differences can be observed between the two experimental models.
In the case of liver plasma membrane (123), a reversible exchange of Na+ for Mg2+ occurs with a varying stoichiometry. The extrusion mechanism is quite specific, in that Na+ cannot be replaced by other monovalent cations (e.g. Li+ or K+), becomes saturated at a concentration of [Na+]o = 25-30 mM, and is inhibited, though not completely, by amiloride, quinidine or imipramine. In addition, the vesicles possess another Mg2+ transport mechanism that extrudes intravesicular Mg2+ for extravesicular Ca2+. This pathway, which operates only in one direction (Ca2+in:Mg2+out), is activated by micromolar concentrations of Ca2+ or other divalent cations (Ca2+>>Co2+=Mn2+>Sr2+>>Ba2+>Cu2+>>Cd2+), and is also blocked, to a variable extent, by amiloride. Finally, intravesicular ATP does not modulate the activity of these two transporters, though some inhibitory effect by ATP-gamma-S on both mechanisms is observed.
In the case of membrane vesicles from rabbit ileum (120), a saturable Mg2+ uptake mechanism is observed when [Na+]i>[Na+]o, and not when [Na+]i<[Na+]o or [Na+]i=[Na+]o or in the absence of external Na2+. This pathway is not reversible, and is not inhibited by the amiloride analogs DMA or EIPA, or by Ca2+-channel inhibitors. A major effect of intravesicular anions, especially Cl- and SCN-, on Mg2+ transport is observed, and antagonist of anion transport (e.g. H2-DIDS) exerts a stimulatory effect on Mg2+ accumulation into the vesicles. The transport appears to be electroneutral and has a Km for Na+ of 16 mM, a value not dissimilar from the Km that can be calculated from the data of Cefaratti et al (123), or that has been reported by Tashiro and Konishi (95) in smooth muscle cells from guinea pig tenia caecum, and by Gunther and Vormann in chicken erythrocytes (93, 116).
A Mg2+ uptake mechanism has also been described in plasma membrane vesicles from rat duodenum and jejunum (121,122). The vesicles from the duodenum appear to possess a single Mg2+ uptake mechanism with an apparent K(t) of 0.8 mM. Kinetic analysis of Mg2+ uptake in vesicles from jejunum suggest, instead, the presence of two distinct transport mechanisms with a K(t) of 0.15 and 2.4 mM, respectively. The accumulation of Mg2+ is not affected by verapamil, but is reduced by 30-40% by the use of L-leucine or L-phenylalanine as inhibitors of intestinal alkaline phosphatase (121). Under the same experimental conditions 45Ca2+ uptake is unaffected, thus suggesting that Ca2+ and Mg2+ are transported via distinct pathways. Furthermore, the Mg2+ transport mechanisms have an optimal temperature at 28oC and are stimulated by an electronegative potential inside the vesicle (122). When Mg2+ accumulation is performed at 0.1 mM [Mg2+]o, the uptake is stimulated by an alkaline pHo and by a difference in Mg2+ concentration across the plasma membrane, and is inhibited by amiloride or by the presence of other divalent cations (e.g. Co2+, Ni2+ and Ba2+) whereas [Na+]o or alkaline pHi are ineffective (121). When Mg2+ accumulation is performed at 1 mM [Mg2+]o, the uptake is insensitive to changes in pHo or pHi, is not stimulated by a difference in Mg2+ concentration across the plasma membrane, but is still inhibited, though to a lesser extent, by amiloride (121). Under these conditions, the presence of Na+ or K+ in the extravesicular space strongly enhances Mg2+ accumulation. In contrast, the presence of divalent cations inhibits Mg2+ uptake to a varying extent (Co2+>Mn2+>Ca2+>Ni2+>Ba2+>Sr2+) (122).
Figure 2 summarizes the different Mg2+ transport mechanism identified in plasma membrane vesicles from mammalian cells.
Figure 2. The cartoon depicts the Mg2+ transport mechanism recently characterized in plasma membrane vesicles from brush border cells of the small intestine (top part), liver cells and brush border cells of rabbit ileum (bottom part). See section 4 for more detail.
Because of the difference in organs and animals, the diversities in Mg2+ transport cannot be conscribed to a common denominator. Yet, a few general concepts can be defined. Organs like liver appear to utilize Na+ as the primary driving force for Mg2+ transport in either direction across the plasma membrane. Most likely, this transport mechanism is the pathway that different hormones activate to induce Mg2+ extrusion or accumulation. A secondary mechanism, utilizing Ca2+ or other cations, may
play a subsidiary role under more varying situation. Whether this second mechanism is the same transporter activated by alpha1-adrenergic stimulation is, at present, matter of speculation. As for the intestine, due to the absorption function of the organ, it is reasonable to envisage that the Mg2+ transport mechanism(s) here respond(s) primarily to the gradient of Mg2+ present across the brush border of the small intestine and, to a varying extent, to the presence of both mono- and di-valent cations and to changes in pH across the plasma membrane, until a certain level of intracellular accumulation is achieved. The lack of information about which hormones are able to modulate the accumulation through these mechanisms renders more difficult to define under which condition one route of Mg2+ accumulation is preferentially utilized versus the other. However, the operation of two transporters in both liver cells and brush border cells from small intestine with different regulation and sensitivity can be interpreted as the presence of a coarse and a fine tuning mechanism to regulate cellular Mg2+ homeostasis under the most diverse physiological situations.
6. PHYSIOLOGICAL SIGNIFICANCE OF Mg2+ changes within mitochondria
Within the cell mitochondria contain large amount of Mg2+ (40,124), some of which can be rapidly mobilized following the increase in cytosolic cAMP level (39,40). While the overall mechanism affected by cAMP may be far more complex than what initially reported, many conditions leading to cAMP increase are accompanied by an efflux of Mg2+ from the mitochondria (39,40,51). Little is known as to whether changes in matrix Mg2+ can affect the rate of mitochondrial dehydrogenases and therefore the rate of respiration.
There is significant evidence that changes in Ca2+ matrix can control respiration (125, 126). Those observations, however, have been questioned by other studies showing:
a) that several mitochondrial dehydrogenases can increase activity within minutes in the absence of an increase in mitochondrial Ca2+ (127,128);
b) that mitochondrial Mg2+ changes during state 3 to 4 transition (129) and in the presence of cAMP (39,40);
c) that the activity of the isolated alpha-ketoglutarate dehydrogenase is regulated in vitro by both Ca2+ and Mg2+ (130).
The role of matrix Mg2+ as regulator of dehydrogenases and overall mitochondrial respiration has been recently investigated by measuring the activity of several dehydrogenases in mitochondria whose matrix Ca2+ and/or Mg2+ varied. From the data, it appears that alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase are not regulated by changes in mitochondrial Mg2+. By contrast, removal of Mg2+ stimulates several fold succinate and glutamate dehydrogenases (131). This and other experimental evidence clearly indicate that changes in matrix Mg2+ (in addition to those of Ca2+) could control respiration and metabolic pathways regulated by changes in concentration of matrix substrates.
Hence, in principle, the effect of catecholamine on increasing respiration could be explained as the decrease in mitochondrial Mg2+ resulting from an increase in cellular cAMP. This in turn will stimulate some dehydrogenases directly and render others more susceptible to the concentrations of Ca2+ present in the mitochondrial matrix.
It is clear that the understanding of cell and body Mg2+ homeostasis is presently at a primordial stage, and it is a far cry with respect to that existing in the literature for other ions such as Ca2+, H+, K+ or Na+. The list of questions is far greater than the list of answers:
a) What is the effect of increasing plasma Mg2+ concentrations on body functions? While there is a large body of evidence, often contradictory on several physiological responses, a common denominator (a Rosetta's stone) to classify these observations in a logical framework is still missing.
b) What is the role of various distinct organs and tissues (i.e. bone, muscle and kidney) in redistributing plasma and parenchymal Mg2+ in response to stimuli?
c) What is the nature, or even the operation and regulation, of the plasma membrane transporter(s)?
d) How is Mg2+ redistributed among cytosolic organelles or structures?
e) What is the role of Mg2+ changes within organelles on their function and metabolism?
For Mg2+ research it is clearly a new frontier, interesting and challenging!
Supported by NIH HL 18708
1. G H Mudge: Agents affecting volume and composition of body fluids. In: The Pharmacological Basis of Therapeutics. Eds.: Goodman Gilman A, Godman LS, Rall TW, Murad F, MacMillian, NY (1989)
2. G.A Quamme: Magnesium Homeostasis and renal magnesium handling. Miner Electrolyte Metab 19, 218-225 (1993)
3. Geigy Scientific Tables. Ed.: Lentner C., Ciba-Geigy, Basel, Switzerland (1984)
4. R. Taylor, L Agius: The biochemistry of diabetes. Biochem J 250, 625-640 (1988)
5. L M Resnick: Ionic basis of hypertension, insulin resistance, vascular disease, and related disorders. The mechanism of "syndrome X". Am J Hypertens 6, 123S-134S (1993)
6. S Wallach, R L Verch: Tissue magnesium content in diabetic rats. Magnesium 6, 302-306 (1987)
7. H Joborn, G Akerstrom, S Ljunghall: Effects of exogenous catecholamines and exercise on plasma magnesium concentrations. Clin Endocrinol 23:219-226 (1985)
8. Y Rayssiguier: Hypomagnesemia resulting from adrenaline infusion in ewes: its relation to lipolysis. Horm Metab Res 9:309-314 (1977)
9. G Guideri: Catecholamine modulation of Mg2+ plasma levels in the rat. Arch Intern Pharmacodyn Therap 14, 122-125 (1992)
10. T Gunther, J Vormann: Mechanism of beta-agonist-induced hypermagnesemia. Magnes Bull 14, 122-125 (1992)
11. A Romani, A Scarpa: Hormonal control of Mg2+ in the heart. Nature 346, 841-844 (1990)
12. J Vormann, T Gunther: Amiloride sensitive net Mg2+ efflux from isolated perfused rat hearts. Magnesium 6, 220-224 (1987)
13. F C Howarth, J Waring, B I Hustler, J Singh: Effects of extracellular magnesium and beta adrenergic stimulation on contractile force and magnesium mobilization in the isolated rat heart. Magnes Res 7, 187-197 (1994)
14. A Romani, A Scarpa: Norepinephrine evokes a marked Mg2+ efflux from liver cells. FEBS Lett 269, 37-40 (1990)
15. T Gunther, J Vormann, V Hollriegl: Noradrenaline-induced Na+-dependent Mg2+ efflux from rat liver. Magnes Bull 13, 122-124 (1991)
16. D Keenan, A Romani, A Scarpa: Differential regulation of circulating Mg2+ in the rat by beta1- and beta2-adrenergic receptor stimulation. Circ Res 77, 973-983 (1995)
17. P B Molinoff: Alpha- and beta-adrenergic receptor subtypes, properties, distribution and regulation. Drugs 28, 1-14 (1984)
18. P J Barnes: Beta-adrenergic receptors and their regulation. Am J Respir Crit Care Med 152, 838-860 (1995)
19. I M Shafik, G A Quamme: Early adaptation of renal Mg2+ reabsorption in response to magnesium restriction. Am J Phyiol 257, F974-F977 (1989)
20. C Bailly, M Imbert-Teboul, N Roinel, C Amiel: Isoproterenol increases Ca, Mg, and NaCl reabsorption in mouse thick ascending limb. Am J Physiol 258, F1224-F1231 (1990)
21. A K Konstantakos, A Romani, A Scarpa: Catecholamine-induced hypermagnesemia: Effect of epinephrine in pharmacological and physiological conditions. Am J Physiol, submitted (1999)
22. E M Brown, G Gamba, D Riccardi, M Lombardi, R Butters, O Kifor, A Sun, M A Hediger, J Lytton, S C Herbert: Cloning and characterization of an extracellular Ca2+-sensing from bovine parathyroid. Nature 366, 575-580 (1993)
23. E F Nemeth, A Scarpa: Rapid mobilization of cellular Ca2+ in bovine parathyroid cells by external divalent cations. J Biol Chem 262, 5188-5196 (1987)
24. B W Bapty, L J Dai, G Ritchie, F Jirik, L Canaff, G N Hendy, G A Quamme: Extracellular Mg2+- and Ca2+-sensing in mouse distal convoluted tubule cells. Kidney Intern 53, 583-592 (1998)
25. B W Bapty, L J Dai, G Ritchie, L Canaff, G N Hendy, G A Quamme: Mg2+/Ca2+ sensing inhibits hormone-stimulated uptake in mouse distal convolute tubule cells. Am J Physiol 275, F353-F360 (1998)
26. G A Quamme, J H Dirks: Effect of intraluminal and contraluminal magnesium on magnesium and calcium transfer in the rat nephron. Am J Physiol 238, F187-F198 (1980)
27. D J Dipette, K Simpson, J Guntupalli: Systemic and regional hemodynamic effect of acute magnesium administration in the normotensive and hypertensive state. Magnesium 6, 136-149 (1987)
28. M F M James, R C Cork, G M Harlen, J F White: Interaction of adrenaline and magnesium in the cardiovascular system of the baboon. Magnesium 7, 37-43 (1988)
29. J B Stanbury: The blocking action of magnesium on sympathetic ganglia. J Pharmacol Exp Ther 93, 52-62 (1948)
30. A Romani, C Marfella, A Scarpa: Mg2+ homeostasis in cardiac ventricular myocytes. In: Biochemistry of Cell Membranes: a compendium of selected topics. Eds.: Papa S, Tager J M, Birkhauser-Verlag, Base, Switzerland (1995)
31. T Gunther: Functional compartmentation of intracellular magnesium. Magnesium 5, 53-59 (1986)
32. R L Griswold, N Pace: The intracellular distribution of metal ions in rat liver. Exp Cell Res 11, 362-367 (1956)
33. L Garfinkel, R A Altschuld, D Garfinkel: Magnesium in cardiac energy metabolism. J Mol Cell Cardiol 18, 1003-1013 (1986)
34. S Oki, M Ikura, M Zhang: Identification of Mg2+ binding sites and role of Mg2+ on target recognition by calmodulin. Biochemistry 36, 4309-4316 (1997)
35. Y Ogoma, H Kobayashi, T Fujii, Y Kondo, A Hachimori, T Shimizu, M Hatano: Binding study of metal ions to S100 protein: 43Ca, 25Mg, 67Zn and 39K N.M.R.. Int J Biol Macromol 14, 279-286 (1992)
36. A Romani, A Scarpa: Regulation of cell magnesium. Arch Biochem Biophys 298, 1-12 (1992)
37. P I Polimeni, E Page. In: Recent Advances in study in cardiac cells and metabolism. Ed.: Dhalle N S, vol IV, pp. 217-232, University Park Press, Baltimore (1974)
38. A Jakob, J Becker, G Schottli, G Fritzsch: alpha1-adrenergic stimulation causes Mg2+ release from perfused rat liver. FEBS Lett 246, 127-130 (1989)
39. A Romani, C Marfella, A Scarpa: Regulation of magnesium uptake and release in the heart and in isolated ventricular myocytes. Circ Res 72, 1139-1148 (1993)
40. A Romani, E Dowell, A Scarpa: Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria. J Biol Chem 266, 24376-24384 (1991)
41. A Romani, C Marfella, A Scarpa: Hormonal regulation of Mg2+ uptake in hepatocytes. J Biol Chem 268, 15489-15495 (1993)
42. D Keenan, A Romani, A Scarpa: Regulation of Mg2+ homeostasis by insulin in perfused rat livers and isolated hepatocytes. FEBS Lett 395, 241-244 (1996)
43. R D Huang, M F Smith, W L Zahler: Inhibition of forskolin-activated adenylate cyclase by ethanol and other solvents. J Cyclic Nucleot Res 8, 385-394 (1982)
44. F I Wolf, A Di Francesco, V Covacci, A Cittadini: Regulation of magnesium from rat spleen lymphocytes. Arch Biochem Biophys 344, 397-403 (1997)
45. J D Rothermel, L H Parker Botelho: A mechanistic and kinetic analysis of the interactions of the diastereisomers of adenosine 3',5'-(cyclic)phosphorothioate with purified cyclic AMP-dependent protein kinase. Biochem J 251, 757-762 (1988)
46. P Stemmer, P L Wisler, A M Watanabe: Isolated myocytes in experimental cardiology. In: The heart and cardiovascular system. Scientific Foundations. Eds. Fozzard H A Haber E, Jennings R B, Katz A M, Morgan H E, Raven Press, New York (1992)
47. J-C Feray, R Garay: Demonstration of a Na+:Mg2+ exchange in human red cells by its sensitivity to tricyclic antidepressant drugs. Naunyn-Schmied Arch Pharmacol 338, 332-337(1988)
48. J-C Feray, R Garay: An Na+-stimulated Mg2+ transport system in human red blood cells. Biochim Biophys Acta 856, 76-84 (1986)
49. T Matsuura, Y Kanayama, T Inoue, T Takeda, I Morishima: cAMP-induced changes of intra-cellular Mg2+ levels in human erythrocytes. Biochim Biophys Acta 1220, 31-36 (1993)
50. F I Wolf, A Di Francesco, V Covacci, A Cittadini: cAMP activates magnesium efflux via the Na/Mg antiporter in ascite cells. Biochem Biophys Res Commun 202, 1209-1214 (1994)
51. G H Zhang, J E Melvin: Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini. J Biol Chem 267, 20721-20727 (1992)
52. T Gunther, J Vormann: Activation of Na+/Mg2+ antiport in thymocytes by cAMP. FEBS Lett 297, 132-134 (1992)
53. F I Wolf, V Covacci, N Bruzzese, A Di Francesco, A Sachets, D Cord, A Cittadini: Differentiation of HL-60 promyelocytic leukemia cells is accompanied by a modification of magnesium homeostasis. J Cell Biochem 71, 441-448 (1998)
54. F I Wolf, A Di Francesco, V Covacci, D Corda, A Cittadini: Regulation of intracellular magnesium in ascites cells: Involvement of different regulatory pathways. Arch Biochem Biophys 331, 194-200 (1996)
55. A Romani, V Matthews, A Scarpa: Synergism for Mg2+ and glucose transport in heart following insulin administration. Biophys J 72, W210 (1997)
56. G Milligan, D Svoboda, C Brown: Why there are so many adrenoceptor types? Biochem Pharmacol 48, 1059-1071 (1994)
57. K P Minneman: Alpha1-adrenergic receptor subtypes, inositol phosphates, and sources of cell Ca2+. Pharmacol Rev 40, 87-119 (1988)
58. T E Fagan, A Romani: Activation of a Na+- and a Ca2+-dependent Mg2+ extrusion mechanism by alpha- and beta-adrenergic agonists in perfused rat liver. FASEB J 13(4), (1999)
59. P Dalal, A Romani, A Scarpa: Redistribution of Mg2+ in isolated rat hepatocytes upon chemical hypoxia. Biophys J 74, A193 (1998)
60. V Gaussin, P Gailly, J-M Gillis, L Hue: Fructose-induced increase in intracellular free Mg2+ ion concentration in rat hepatocytes: relation with the enzymes of glycogen metabolism. Biochem J 326, 823-827 (1997)
61. P A Tessman, A Romani: Acute effect of EtOH on Mg2+ homeostasis in liver cells: Evidence for the activation of an Na+/Mg2+ exchanger. Am J Physiol 275, G1106-G1116 (1998)
62. J Hwa, A Romani, C Marfella, A Scarpa: Mg2+ transport in erythrocytes: Regulation by intra-cellular pH or cAMP. Biophys J 63, A307 (1993)
63. F I Wolf, A Di Francesco, A Cittadini: Characterization of magnesium efflux from Erlich ascites tumor cells. Arch Biochem Biophys 308, 335-341 (1994)
64. A Gasbarrini, A B Borle, H Farghali, C Bender, A Francavilla, D van Thiel: Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes. J Biol Chem 267, 6654-6663 (1992)
65. A W Harman, A-L Nieminen, J J Lemasters, B Herman: Cytosolic free magnesium, ATP, and blebbing during chemical hypoxya in cultured rat hepatocytes. Biochem Biophys Res Commun 170, 477-483 (1990)
66. A Di Stefano, M Wittner, R Nitchke, R Braitsch, R Greger, C Bailly, C Amiel, N Roniel, C De Rouffignac: Effects of parathyroid hormone and calcitonin on Na+, Cl-, K+, Mg2+ and Ca2+ transport in cortical and medullary thick ascending limbs of mouse kidney. Pflugers Arch 417, 161-167 (1990)
67. C De Rouffignac, B Mandon, M Wittner, A Di Stefano: Hormonal control of renal magnesium handling. Miner Electrolyte Metab 19, 226-231 (1993)
68. C De Rouffignac, G A Quamme: Renal magnesium handling and its hormonal control. Physiol Rev 74, 305-322 (1994)
69. G A Quamme: Renal magnesium handling: New insights in understanding old problems. Kidney Inter 52, 1180-1195 (1997)
70. V Karoor, K Baltensperger, H Paul, M C Czech, C C Malbon: Phosphorylation of tyrosyl residues 350/354 of the beta-adrenergic receptor is obligatory for counterregulatory effects of insulin. J Biol Chem 270, 25305-25308 (1995)
71. J A Smoake, G-M M Moy, B Fang, S S Solomon: calmodulin-dependent cyclic AMP phospho-diesterase in liver plasma membranes: Stimulated by insulin. Arch Biochem Biophys 323, 223-232 (1995)
72. S Ishijima, M Tatibana: Rapid mobilization of intracellular Mg2+ by bombesin in Swiss 3T3 cells: Mobilization through external Ca2+- and tyrosine kinase-dependent mechanism. J Biochem 115, 730-737 (1994)
73. E Gylfe: Insulin secretagogues induce Ca2+-like changes in cytoplasmic Mg2+ in pancreatic beta cells. Biochim Biophys Acta 1055, 82-86, (1990)
74. L-J Dai, B Bapty, G Ritchie, G A Quamme: Glucagone and arginine vasopressin stimulate Mg2+ uptake in mouse distal convoluted tubule cells. Am J Physiol 274, F328-F335 (1998)
75. R M Touyz, E L Schiffrin: Angiotensin II and vasopressin modulate intracellular free magnesium in vascular smooth muscle cells through Na+-dependent protein kinase C pathways. J Biol Chem 271, 24353-24358 (1996)
76. K Okada, S-E Ishikawa, T Saito: Cellular mechanisms of vasopressin and endothelin to mobilize [Mg2+]i in vascular smooth muscle cells. Am J Physiol 263, C873-C878 (1992)
77. A Romani, C Marfella, A Scarpa: Regulation of Mg2+ uptake in isolated rat myocytes and hepatocytes by protein kinase C. FEBS Lett 296, 135-140 (1992)
78. M Castagna, Y Takai, K Kabuchi, K Sano, U Kikkawa, Y Nishizuka: Direct activation of calcium-activated phospholipid-dependent protein kinase by tumor promoting phorbol esters. J Biol Chem 257, 7848-7851 (1982)
79. P Csermely, P Fodor, J Somogyi: The tumor promoter tetradecanoylphorbol-13-acetate elicits the redistribution of heavy metal in subcellular fractions of rabbit thymocytes as measured by plasma emission spectroscopy. Carcinogenesis 8,1663-1666 (1987)
80. R D Grubbs, M E Maguire: Regulation of magnesium but not calcium by phorbol esters. J Biol Chem 261, 12550-12554 (1986)
81. K H Jakobs, K Aktories, G Schultz: GTP-dependent inhibition of cardiac adenylate cyclase by muscarinic cholinergic agonists. Naunyn-Schmiedeberg's Arch Pharmacol 310, 113-119 (1979)
82. X Zhu and L Birnbaumer: Calcium channels formed by mammalian Trp homologues. News in Physiol Sci 13, 211-217 (1998)
83. R D Grubbs, M E Maguire: Magnesium as a regulatory cation: Criteria and evaluation. Magnesium 6, 113-127 (1987)
84. M M Fatholahi, A Scarpa: Cytosolic free magnesium changes in isolated rat cardiomyocytes and smooth muscle cell line A7r5. Biophys J 76, A305
85. G A Quamme, S W Rabkin: Cytosolic free magnesium in cardiac myocytes: Identification of a Mg2+ influx pathway. Biochem Biophys Res Commun 167, 1406-1412 (1990)
86. G A Quamme, L-S Dai: Presence of a novel influx pathway for Mg2+ in MDCK cells. Am J Physiol 259, C521-C525 (1990)
87. L-J Dai, G A Quamme: Intracellular Mg2+ and magnesium depletion in isolated renal thick ascending limb cells. J Clin Invest 88, 1255-1264 (1991)
88. L-J Dai, P A Friedman, G A Quamme: Phosphate depletion diminishes Mg2+ uptake in mouse distal convoluted tubule cells. Kidney Inter 51, 1710-1718 (1997)
89. L-J Dai, P A Friedman, G A Quamme: Cellular Mechanism of chlorothiazide and cellular potassium depletion on Mg2+ uptake in mouse distal convoluted tubule cells. Kidney Inter 51, 1008-1017 (1991)
90. L-J Dai, B Bapty, G Ritchie, G A Quamme: PGE2 stimulates Mg2+ uptake in mouse distal convoluted tubule cells. Am J Physiol 275, F833-F839 (1998)
91. L-J Dai, G A Quamme: Cyclic nucleotides alter intracellular free Mg2+ in renal epithelial cells. Am J Physiol 262, F1100-F1104 (1992)
92. T Gunther, J Vormann, R Forster: Regulation of intracellular magnesium by Mg2+ efflux. Biochem Biophys Res Commun 119, 124-131 (1984)
93. T Gunther, J Vormann: Mg2+ efflux is accomplished by an amiloride-sensitive Na+/Mg2+ antiport. Biochem Biophys Res Commun 130, 540-545 (1985)
94. R D Handy, I F Gow, D Ellis, P W Flatman: Na-dependent regulation of intracellular free magnesium concentration in isolated rat ventricular myocytes. J Mol Cell Cardiol 28, 1641-1651 (1996)
95. M Tashiro, M Konishi: Na+ gradient-dependent Mg2+ transport in smooth muscle cells of guinea pig tenia cecum. Biophys J 73, 3371-3384 (1997)
96. T Gunther, V Hollriegl: Na+- and anion-dependent Mg2+ influx in isolated hepatocytes. Biochim Biophys Acta 114949-54 (1993)
97. T Gunther, J Vormann, V Hollriegl: Characterization of Na+-dependent Mg2+ efflux from Mg2+-loaded rat erythrocytes. Biochim Biophys Acta 1023, 455-461 (1990)
98. P W Flatman, L M Smith: Magnesium transport in ferret red cells. J Physiol 431, 11-25 (1990)
99. H Ludi, H J Schatzmann: Some properties of a system for sodium-dependent outward movement of magnesium from metabolizing human red blood cells. J Physiol 390, 367-382 (1987)
100. W Xu, J S Willis: Sodium transport through the amiloride-sensitive Na-Mg pathway of hamster red cells. J Membr Biol 141, 277-287 (1994)
101. G H Zhang, J E Melvin: Na+-dependent release of Mg2+ from an intracellular pool in rat sublingual mucous acini. J Biol Chem 271, 29067-29072 (1996)
102. R Di Polo, L Beauge': An ATP-dependent Na+/Mg2+ countertransport is the only mechanism for Mg2+ extrusion in squid axons. Biochim Biophys Acta 946, 424-428 (1988)
103. P F Baker, A C Crawford: Mobility and transport of magnesium in squid giant axons. J Physiol 227, 855-874 (1972)
104. D Gunzel, W-R Schlue: Sodium-magnesium antiport in Retzius neurons of the leech Hirudo medicinalis. J Physiol 491 595-608 (1996)
105. E Murphy, C C Freudenrich, M Lieberman: Cellular magnesium and Na/mg exchange in heart cells. Annu Rev Physiol 53, 273-287 (1991)
106. H J Schatzmann: Asymmetry of the magnesium sodium exchange across the human red cell membrane. Biochim Biophys Acta 1148, 15-18 (1993)
107. P W Flatman: Mechanisms of magnesium transport. Annu Rev Physiol 53, 259-271 (1991)
108. J-C Feray, R Garay: One-to-one Mg2+:Mn2+ exchange in rat erythrocytes. J Biol Chem 262, 5763-5768 (1987)
109. T Gunther, J Vormann: Characterization of Na+-independent Mg2+ efflux from erythrocytes. FEBS Lett 271, 149-151 (1990)
110. T Gunther, J Vormann E J Cragoe Jr: Species-specific Mn2+/Mg2+ antiport from Mg2+-loaded erythrocytes. FEBS Lett 261, 47-51 (1990)
111. T Gunther: Mechanisms and regulation of Mg2+ efflux and Mg2+ influx. Miner Electrolyte Metab 19, 250-265 (1993)
112. T Gunther, J Vormann: Induction of Mn2+/H+ antiport in chicken erythrocytes by intracellular Mg2+ and Mn2+. FEBS Lett 265, 55-58 (1990)
113. T Gunther, J Vormann: Characterization of Na+/Mg2+ antiport by simultaneous 28Mg2+ influx. Biochem Biophys Res Commun 148, 1069-1074 (1987)
114. M E Maguire, R Crome, D J Hearse, A S Mannings: Efflux of 28Mg from isolated rat heart: Effects of ischaemia, reperfusion, temperature and extracellular magnesium concentration. J Mol Cell Cardiol 16, 34 (1984)
115. F J Brinley: Calcium and magnesium transport in single cells. Fed Proc 32, 1735-1739 (1973)
116. T Gunther: Putative mechanism of Mg2+/Mg2+ exchange and Na+/Mg2+ antiport. Magnes Bull 18, 2-6 (1996)
117. R R Preston: A magnesium current in Paramecium. Science 250, 285-288 (1990)
118. K D Clark, R R Preston: Extracellular GTP causes membrane-potential oscillations through the parallel activation of Mg2+ and Na+ currents in Paramecium tetraurelia. J Membr Biol 157, 159-167 (1997)
119. L-J Dai, P A Friedman, G A Quamme: Acid base changes alter Mg2+ uptake in mouse distal convolute tubule cells. Am J Physiol 272, F759-F766 (1997)
120. R Juttner, H Ebel: Characterization of Mg2+ transport in brush border membrane vesicles of rabbit ileum studied with mag-fura-2. Biochim Biophys Acta 1370, 51-63 (1998)
121. M Baillien, H Wang, M Cogneau: Uptake of 28Mg by duodenal and jejunal brush border membrane vesicle in the rat. Magnesium 8, 315-329 (1995)
122. M Baillien, M Cogneau: Characterization of two mechanisms of 28Mg uptake in rat jejunal brush border membrane vesicles. Magnesium 8, 331-339 (1995)
123. C Cefaratti, A Romani, A Scarpa: Characterization of two Mg2+ transporters in sealed plasma membrane vesicles from rat liver. Am J Physiol 275, C995-C1008 (1998)
124. P W Flatman: Magnesium transport across cell membrane. J Membr Biol 80, 1-14 (1984)
125. J G McCormack, A Halestrap, R M Denton: Role of calcium ions in regulation of mammalian intramitochondrial metabolism. Physiol Rev 70, 391-425 (1990)
126. R G Hansford: Physiological role of mitochondrial Ca2+ transport. J Bioenerg Biomembr 26, 495-508 (1994)
127. C S Moravec, M Bond: Calcium is released from the junctional sarcoplasmic reticulum during cardiac muscle contraction. Am J Physiol 260, H989-H997 (1991)
128. C S Moravec, M Bond: Effect of inotropic stimulation on mitochondrial calcium in cardiac muscle. J Biol Chem 267, 5310-5316 (1992)
129. G P Brierley, M Davis, D W Jung: Respiration-dependent uptake and extrusion of Mg2+ by isolated heart mitochondria. Arch Biochem Biophys 253, 322-332 (1987)
130. A Panov, A Scarpa: Independent modulation of the activity of alpha-ketoglutarate dehydrogenase complex by Ca2+ and Mg2+. Biochemistry 35, 427-432 (1996)
131. A Panov, A Scarpa: Mg2+ control of respiration in isolated rat liver mitochondria. Biochemistry 35, 12849-12856 (1996)