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.

Upon activation of specific cell signaling, hepatocytes rapidly accumulate or release an amount of Mg 2؉ equivalent to 10% of their total Mg 2؉ content. Although it is widely accepted that Mg 2؉ efflux is Na ؉ -dependent, little is known about transporter identity and the overall regulation. Even less is known about the mechanism of cellular Mg 2؉ uptake. Using sealed and right-sided rat liver plasma membrane vesicles representing either the basolateral (bLPM) or apical (aLPM) domain, it was possible to dissect three different Mg 2؉ transport mechanisms based upon specific inhibition, localization within the plasma membrane, and directionality. The bLPM possesses only one Mg 2؉ transporter, which is strictly Na ؉ -dependent, bi-directional, and not inhibited by amiloride. The aLPM possesses two separate Mg 2؉ transporters. One, similar to that in the bLPM because it strictly depends on Na ؉ transport, and it can be differentiated from that of the bLPM because it is unidirectional and fully inhibited by amiloride. The second is a novel Ca 2؉ /Mg 2؉ exchanger that is unidirectional and inhibited by amiloride and imipramine. Hence, the bLPM transporter may be responsible for the exchange of Mg 2؉ between hepatocytes and plasma, and vice versa, shown in livers upon specific metabolic stimulation, whereas the aLPM transporters can only extrude Mg 2؉ into the biliary tract. The dissection of these three distinct pathways and, therefore, the opportunity to study each individually will greatly facilitate further characterization of these transporters and a better understanding of Mg 2؉ homeostasis.Magnesium, the second most abundant cation within mammalian cells, is necessary for a variety of metabolic and cellular functions (1-5). Under resting conditions, cellular-free Mg 2ϩ concentration is held at 0.5-0.8 mM, well below its predicted electrochemical equilibrium, and approximately 5 mM Mg 2ϩ is complexed with ATP and other metabolites (1, 5). In several tissues such as heart and liver, specific hormonal or metabolic stimulation causes, within a few minutes, a massive mobilization of cellular Mg 2ϩ (6 -9). Although cytosolic-free Mg 2ϩ undergoes minimal changes, several intracellular signals leading to a cAMP increase induce a loss of 5-10% of total cellular Mg 2ϩ (6 -9). Conversely, signals activating protein kinase C result in an accumulation of Mg 2ϩ and a consequent increase of total cellular Mg 2ϩ by 5-10% (10). These findings underscore the operation of a very powerful Mg 2ϩ transport machinery within the plasma membranes of mammalian cells. Yet, the Mg 2ϩ transporter(s) has not been isolated, and even the basic kinetic properties of Mg 2ϩ cellular transport remain confusing and contradictory. For example, the dependence of Mg 2ϩ release on extracellular Na ϩ has been established in invertebrate and mammalian cells (11-16) but Na ϩ -independent pathways have also been reported (1,8,9,17,18). Equally contradictory are the findings of variable Na ϩ / Mg 2ϩ stoichiometries (1,19), the role of trans-membrane potenti...

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.

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 Mg(2+) content in several tissues, including liver. Total cellular Mg(2+) accounted for 26.8 +/- 2.4 vs. 36.0 +/- 1.4 nmol Mg(2+)/mg protein in hepatocytes from EtOH-fed and control rats, respectively, and paralleled a 13% decrease in cellular ATP content. Stimulation of alpha(1)- or beta-adrenergic receptor or acute EtOH administration did not elicit an extrusion of Mg(2+) from liver cells of EtOH-fed rats while releasing 5% of total tissue Mg(2+) content from hepatocytes of control rats. Despite the 25% decrease in Mg(2+) content, hepatocytes from EtOH-fed rats did not accumulate Mg(2+) following stimulation of protein kinase C signaling pathway, whereas control hepatocytes accumulated approximately 2 nmol Mg(2+). mg protein(-1). 4 min(-1). Together, these data indicate that Mg(2+) 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 Mg(2+) can help explain the reduction in tissue Mg(2+) content following chronic alcohol consumption.