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The Na+/Mg2+ exchanger represents the main Mg2+ extrusion mechanism operating in mammalian cells including hepatocytes. We have previously reported that this exchanger, located in the basolateral domain of the hepatocyte, promotes the extrusion of intravesicular trapped Mg2+ for extravesicular Na+ with ratio 1. This electrogenic exchange is supported by the accumulation of tetraphenyl-phosphonium within the vesicles at the time when Mg2+ efflux occurs. In this present study, the role of extra- and intra-vesicular Cl− on the Na+/Mg2+ exchange ratio was investigated. The results reported here suggest that Cl− ions are not required for the Na+ to Mg2+ exchange to occur, but the stoichiometry ratio of the exchanger switches from electrogenic (1Na in + :1 Mg out 2+ ) in the presence of intravesicular Cl− to electroneutral (2Na in + :1 Mg out 2+ ) in their absence. In basolateral liver plasma membrane vesicles loaded with MgCl2 labeled with 36Cl−, a small but significant Cl− efflux (~30 nmol Cl−/mg protein/1 min) is observed following addition of NaCl or Na-isethionate to the extravesicular medium. Both Cl− and Mg2+ effluxes are inhibited by imipramine but not by amiloride, DIDS, niflumic acid, bumetanide, or furosemide. In vesicles loaded with Mg-gluconate and stimulated by Na-isethionate, an electroneutral Mg2+ extrusion is observed. Taken together, these results suggest that the Na+/Mg2+ exchanger can operate irrespective of the absence or the presence of Cl− in the extracellular or intracellular environment. Changes in trans-cellular Cl− content, however, can affect the modus operandi of the Na+/Mg2+ exchanger, and consequently impact "cellular" Na+ and Mg2+ homeostasis as well as the hepatocyte membrane potential.

Liver cells from rats chronically fed a Lieber-De Carli diet for 3 wk presented a marked decreased in tissue Mg2+content and an inability to extrude Mg2+into the extracellular compartment upon stimulation with catecholamine, isoproterenol, or cell-permeant cAMP analogs. This defect in Mg2+extrusion was observed in both intact cells and purified liver plasma membrane vesicles. Inhibition of adrenergic or cAMP-mediated Mg2+extrusion was also observed in freshly isolated hepatocytes from control rats incubated acutely in vitro with varying doses of ethanol (EtOH) for 8 min. In this model, however, the defect in Mg2+extrusion was observed in intact cells but not in plasma membrane vesicles. In the chronic model, upon removal of EtOH from the diet hepatic Mg2+content and extrusion required ∼10 days to return to normal level both in isolated cells and plasma membrane vesicles. In hepatocytes acutely treated with EtOH for 8 min, more than 60 min were necessary for Mg2+content and extrusion to recover and return to the level observed in EtOH-untreated cells. Taken together, these data suggest that in the acute model the defect in Mg2+extrusion is the result of a limited refilling of the cellular compartment(s) from which Mg2+is mobilized upon adrenergic stimulation rather than a mere defect in adrenergic cellular signaling. The chronic EtOH model, instead, presents a transient but selective defect of the Mg2+extrusion mechanisms in addition to the limited refilling of the cellular compartments.

Type-I diabetes is associated with a decrease in magnesium content in various tissues, including liver. We have reported that hepatocytes from streptozotocin-injected rats have lost the ability to accumulate Mg2+ following hormonal stimulation. To assess whether the defect is inherent to the Mg2+ transport mechanism located in the hepatocyte cell membrane, plasma membrane vesicles were purified from diabetic livers. Diabetic plasma membranes do not retain intravesicular Mg2+as tightly as vesicles purified from livers of age-matched non-diabetic rats. In addition, the amount of intravesicular Mg2+ these vesicles exchange for extravesicular Na+ or Ca2+ is 2–3-fold larger than in non-diabetic vesicles. The partition of Ca2+/Mg2+ and Na+/Mg2+ exchange mechanisms in the apical and basolateral domains of liver plasma membrane is maintained under diabetic conditions, although the Na+/Mg2+ exchanger in diabetic basolateral membranes has lost the ability to operate in reverse and favor an accumulation of extravesicular Mg2+ within the vesicles in exchange for entrapped Na+. These data indicate the occurrence of a major alteration in Mg2+ transport across the hepatocyte membrane, which can explain, at least in part, the decrease in liver magnesium content observed in diabetic animals and patients. (Mol Cell Biochem 262: 145–154, 2004)

Plasma membrane vesicles purified from livers of 4-week-old streptozotocin-injected diabetic rats present an increased basal and cation-stimulated magnesium (Mg)2+ transport as compared with vesicles purified from age-matched nondiabetic animals. Furthermore, diabetic basolateral membranes are unable to accumulate extravesicular Mg2+ in exchange for intravesicular sodium (Na)+. Loading diabetic vesicles with varying concentrations of D-glucose, in addition to Mg2+, renormalizes basal and Na+- or calcium (Ca)2+-induced Mg2+ extrusion in a dose-dependent manner, but does not restore Na+/Mg2+ exchanger reversibility. A similar effect on Mg2+ extrusion is observed when D-glucose is replaced with 2-deoxy-glucose, amylopectin, or glycogen. The loading with 3-methyl-O-glucose or L-glucose, instead, affects basal and Na+-dependent Mg2+ extrusion, but not Ca2+-dependent Mg2+ fluxes. In contrast, loading the vesicles with hexoses other than glucose or varying extravesicular glucose concentration from 5 to 20 mmol/L does not modify basal or cation-stimulated Mg2+ fluxes. Taken together, these data indicate that basal and cation-stimulated Mg2+ transport across the hepatocyte plasma membrane is altered under diabetic conditions as a result of a decrease in intravesicular (intracellular) glucose.

Serum and tissue Mg(2+) content are markedly decreased in diabetic patients and animals. At the tissue level, Mg(2+) loss progresses over time and affects predominantly heart, liver and skeletal muscles. In the present study, alterations in Mg(2+) homeostasis and transport in diabetic cardiac ventricular myocytes were evaluated. Cardiac tissue and isolated cardiac ventricular myocytes from diabetic animals displayed a decrease in total Mg(2+) content that affected all cellular compartments. This decrease was associated with a marked reduction in cellular protein and ATP content. Diabetic ventricular myocytes were unable to mobilize Mg(2+) following beta-adrenergic receptor stimulation or addition of cell permeant cyclic-AMP. Sarcolemma vesicles purified from diabetic animals, however, transported Mg(2+) normally as compared to vesicles from non-diabetic animals. Treatment of diabetic animals with exogenous insulin for 2 weeks restored ATP and protein levels as well as Mg(2+) homeostasis and transport to levels comparable to those observed in non-diabetic animals. These results suggest that in diabetic cardiac cells Mg(2+) homeostasis and extrusion via beta-adrenergic/cAMP signaling are markedly affected by the concomitant decrease in protein and ATP content. As Mg(2+) regulates numerous cellular enzymes and functions, including protein synthesis, these results provide a new rationale to interpret some aspects of the cardiac dysfunctions observed under diabetic conditions.

Cardiac ventricular myocytes extrude a sizeable amount of their total Mg(2+) content upon stimulation by beta-adrenergic agonists. This extrusion occurs within a few minutes from the application of the agonist, suggesting the operation of rapid and abundantly represented Mg(2+) transport mechanisms in the cardiac sarcolemma. The present study was aimed at characterizing the operation of these transport mechanisms under well defined conditions. Male Sprague-Dawley rats were used to purify a biochemical standardized preparation of sealed rat cardiac sarcolemmal vesicles. This experimental model has the advantage that trans-sarcolemmal cation transport can be studied under specific extra- and intra-vesicular ionic conditions, in the absence of intracellular organelles, and buffering or signaling components. Magnesium ion (Mg(2+)) transport was assessed by atomic absorbance spectrophotometry. The results reported here indicate that: (1) sarcolemma vesicles retained trapped intravesicular Mg(2+) in the absence of extravesicular counter-ions; (2) the addition of Na(+) or Ca(2+) induced a rapid and concentration-dependent Mg(2+) extrusion from the vesicles; (3) co-addition of maximal concentrations of Na(+) and Ca(2+) resulted in an additive Mg(2+) extrusion; (4) Mg(2+ )extrusion was blocked by addition of amiloride or imipramine; (5) pre-treatment of sarcolemma vesicles with alkaline phosphatase at the time of preparation completely abolished Na(+)- but not Ca(2+)-induced Mg(2+) extrusion; (6) Na(+)-dependent Mg(2+) transport could be restored by stimulating vesicles loaded with protein kinase A catalytic subunit and ATP with membrane-permeant cyclic-AMP analog; (7) extra-vesicular Mg(2+) could be accumulated in exchange for intravesicular Na(+) via a mechanism inhibited by amiloride or alkaline phosphatase treatment; (8) Mg(2+) accumulation could be restored via cAMP/protein kinase A protocol. Overall, these data provide compelling evidence for the operation of distinct Na(+)- and Ca(2+)-dependent Mg(2+) extrusion mechanisms in sarcolemma vesicles. The Na(+)-dependent mechanism appears to be specifically activated via protein kinase A/cAMP-dependent phosphorylation process, and can operate in either direction based upon the cation concentration gradient across the sarcolemma. The Ca(2+)-dependent mechanism, instead, only mediates Mg(2+) extrusion in a cAMP-independent manner.

Isolated hepatocytes in physiological [Na(+)]( 0 ) tightly maintain [Mg(2+)]( i ). Upon beta-adrenergic stimulation or in the presence of permeable cAMP, hepatocytes release 5-10% (1-3 mM Mg(2+)) of their total Mg(2+) content. However, isolated basolateral liver plasma membranes (bLPM), release Mg(2+) in the presence of [Na(+)]( o ) even in the absence of catecholamine stimulation. The data indicate that a physiological brake for Mg(2+) efflux is present in the hepatocyte and is removed upon cellular signaling. In contrast, this regulation "brake" is absent in purified bLPM thus rendering them fully active. The present study was carried out to reconstruct the missing regulatory component. Activation of Mg(2+) extrusion in intact cells is consistent with cAMP dependent phosphorylation of the transporter or a regulatory protein. Treatment of bLPM with a non-specific phosphatase such as alkaline phosphatase (AP), decreased Mg(2+) efflux by 70% compared to untreated bLPM. When AP-treated bLPM were loaded with protein kinase A (PKA), and stimulated with permeable cAMP, Mg(2+) transport fully recovered. These data suggest that phosphorylation of the Na(+)/Mg(2+) exchanger or a nearby protein activates the Mg(2+) transport mechanism in hepatocytes.

Isolated hepatocytes release 2-3 nmol Mg2+/mg protein or approximately 10% of the total cellular Mg2+ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg2+ release, a quantitatively similar amount of Ca2+ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg2+:1Ca2+. Calcium induced Mg2+ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg2+:1Ca2+ exchange ratio. The uptake of Ca2+ for the release of Mg2+ occurs in the absence of significant changes in Deltapsi as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP+) electrode or 3H-TPP+. Collapsing the Deltapsi by high concentrations of TPP+ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca2+-induced Mg2+ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca2+ uptake and Mg2+ release. These data demonstrate the operation of an electroneutral Ca2+/Mg2+ exchanger which represents a novel pathway for Ca2+ accumulation in liver cells following adrenergic receptor stimulation.

Activation of PKC signaling induces Mg(2+) accumulation in liver cells. To test the hypothesis that PKC induces Mg(2+) accumulation via MAPKs activation, hepatocytes were incubated in the presence of PD98059 and SB202190 as specific inhibitors of ERK1/2 and p38, respectively, and stimulated for Mg(2+) accumulation by addition of PMA or OAG. Accumulation of Mg(2+) within the cells was measured by atomic absorbance spectrophotometry in the acid extract of cell pellet. The presence of either inhibitor completely abolished Mg(2+) accumulation irrespective of the dose of agonist utilized while having no discernible effect on beta -adrenoceptor mediated Mg(2+) extrusion. A partial inhibition on alpha (1)-adrenoceptor mediated Mg(2+) extrusion was observed only in cells treated with PD98059. To confirm the inhibitory effect of PD98509 and SB202190, total and basolateral liver plasma membrane vesicles were purified in the presence of either MAPK inhibitor during the isolation procedure. Consistent with the data obtained in intact cells, liver plasma membrane vesicles purified in the presence of PD98509 or SB202190 lost the ability to accumulate Mg(2+)in exchange for intra-vesicular entrapped Na(+) while retaining the ability to extrude entrapped Mg(2+) in exchange for extra-vesicular Na(+). These data indicate that ERK1/2 and p38 are involved in mediating Mg(2+) accumulation in liver cells following activation of PKC signaling. The absence of a detectable effect of either inhibitor on beta -adrenoceptor induced, Na(+)-dependent Mg(2+) extrusion in intact cells and in purified plasma membrane vesicles further support the hypothesis that Mg(2+) extrusion and accumulation occur through distinct and differently regulated transport mechanisms.

Acute and chronic ethanol administration results in a decrease in cellular Mg2+ content and an alteration of Mg2+ transport in liver cells. In this study we investigated the extent to which ethanol affects the Mg2+ transport mechanisms in the liver cell membrane. The functionality of these transport mechanisms was assessed in plasma membrane vesicles purified from livers acutely perfused with varying concentrations of alcohol, livers of animals fed with 6% ethanol for 3 weeks, and the respective controls. Acute alcohol administration had little or no effect on the Mg2+ extrusion mechanisms present in the apical and basolateral domains of the hepatocyte but completely impaired the Mg2+ entry mechanism present in the basolateral side of the cell. This effect was already evident at a dose of alcohol as small as 0.01% (approximately 1.5 mM). The chronic administration of ethanol, instead, impaired all the Mg2+ transport mechanisms irrespective of the location and directionality in a time-dependent manner. Taken together, these data indicate a selective sensitivity of the Mg2+ entry mechanism to acute alcohol administration, whereas the Mg2+ extrusion mechanisms are affected only after prolonged exposure to alcohol. These results suggest that the defect in hormone-activated Mg(2+) transport observed in the chronic EtOH model [Young, A., Cefaratti, C., & Romani, A. (2003). Chronic EtOH administration alters liver Mg2+ homeostasis. Am J Physiol 284, G57-G67] depends not only on a reduced cellular Mg2+ content but also on the impaired Mg2+ transport mechanisms present in the hepatocyte plasma membrane, in particular the Mg2+ entry pathway, which prevents the liver cell from restoring cellular Mg2+ homeostasis.

Male Sprague-Dawley rats rendered diabetic by streptozotocin injection presented 10 and 20% decreases in total hepatic Mg2+content at 4 and 8 wk, respectively, following diabetes onset. This decrease was associated with a parallel decrease in K+and ATP content and an increase in Na+level. In diabetic liver cells, the Mg2+extrusion elicited by α1-adrenoceptor stimulation was markedly reduced compared with nondiabetic livers, whereas that induced by β-adrenoceptor stimulation was unaffected. In addition, diabetic hepatocytes did not accumulate Mg2+following stimulation of protein kinase C pathway by vasopressin, diacylglycerol analogs, or phorbol 12-myristate 13-acetate derivates despite the reduced basal content in cellular Mg2+. Experiments performed in purified plasma membrane from diabetic livers located the defect at the level of the bidirectional Na+/Mg2+exchanger operating in the basolateral domain of the hepatocyte cell membrane, which could extrude but not accumulate Mg2+in exchange for Na+. The impairment of Mg2+uptake mechanism, in addition to the decrease in cellular ATP level, can contribute to explaining the decrease in liver Mg2+content observed under diabetic conditions.

Ethanol (EtOH) administration to rats for 4 wk markedly decreased Mg2+content in several tissues, including liver. Total cellular Mg2+accounted for 26.8 ± 2.4 vs. 36.0 ± 1.4 nmol Mg2+/mg protein in hepatocytes from EtOH-fed and control rats, respectively, and paralleled a 13% decrease in cellular ATP content. Stimulation of α1- or β-adrenergic receptor or acute EtOH administration did not elicit an extrusion of Mg2+from liver cells of EtOH-fed rats while releasing 5% of total tissue Mg2+content from hepatocytes of control rats. Despite the 25% decrease in Mg2+content, hepatocytes from EtOH-fed rats did not accumulate Mg2+following stimulation of protein kinase C signaling pathway, whereas control hepatocytes accumulated ∼2 nmol Mg2+· mg protein−1· 4 min−1. Together, these data indicate that Mg2+homeostasis and transport are markedly impaired in liver cells after prolonged exposure to alcohol. The inability of liver cells, and possibly other tissues, to accumulate Mg2+can help explain the reduction in tissue Mg2+content following chronic alcohol consumption.

The plasma membrane of mammalian cells possesses rapid Mg2+transport mechanisms. The identity of Mg2+transporters is unknown, and so are their properties. In this study, Mg2+transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+with negligible leak; 2) the addition of Na+or Ca2+induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+⋅ μm−2⋅ min−1after Na+and Ca2+addition, respectively); 4) coaddition of maximal concentrations of Na+and Ca2+induces an additive Mg2+efflux; 5) both Na+- and Ca2+-stimulated Mg2+effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+channel blockers; 6) extracellular Na+or Ca2+can stimulate Mg2+efflux in the absence of Mg2+gradients; and 7) Mg2+uptake occurs in LPM loaded with Na+but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+transport mechanisms and provide new information on rates of Mg2+transport, specificity of the cotransported ions, and reversibility of the transport.