Christie Cefaratti

Differential Localization and Operation of Distinct Mg2+ Transporters in Apical and Basolateral Sides of Rat Liver Plasma Membrane

Upon activation of specific cell signaling, hepatocytes rapidly accumulate or release an amount of Mg2+ equivalent to 10% of their total Mg2+ content. Although it is widely accepted that Mg2+ efflux is Na+-dependent, little is known about transporter identity and the overall regulation. Even less is known about the mechanism of cellular Mg2+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 Mg2+ transport mechanisms based upon specific inhibition, localization within the plasma membrane, and directionality. The bLPM possesses only one Mg2+ transporter, which is strictly Na+-dependent, bi-directional, and not inhibited by amiloride. The aLPM possesses two separate Mg2+ 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 Ca2+/Mg2+ exchanger that is unidirectional and inhibited by amiloride and imipramine. Hence, the bLPM transporter may be responsible for the exchange of Mg2+ between hepatocytes and plasma, and vice versa, shown in livers upon specific metabolic stimulation, whereas the aLPM transporters can only extrude Mg2+ 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 Mg2+homeostasis.

Characterization of two Mg2+ transporters in sealed plasma membrane vesicles from rat liver.

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+ . micrometer -2 . min-1 after 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.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.

Functional characterization of two distinct Mg2+ extrusion mechanisms in cardiac sarcolemmal vesicles

Cardiac ventricular myocytes extrude a sizeable amount of their total Mg2+ content upon stimulation by β-adrenergic agonists. This extrusion occurs within a few minutes from the application of the agonist, suggesting the operation of rapid and abundantly represented Mg2+ 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 (Mg2+) transport was assessed by atomic absorbance spectrophotometry. The results reported here indicate that: (1) sarcolemma vesicles retained trapped intravesicular Mg2+ in the absence of extravesicular counter-ions; (2) the addition of Na+ or Ca2+ induced a rapid and concentration-dependent Mg2+ extrusion from the vesicles; (3) co-addition of maximal concentrations of Na+ and Ca2+ resulted in an additive Mg2+ extrusion; (4) Mg2+ 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 Ca2+-induced Mg2+ extrusion; (6) Na+-dependent Mg2+ 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 Mg2+ could be accumulated in exchange for intravesicular Na+ via a mechanism inhibited by amiloride or alkaline phosphatase treatment; (8) Mg2+ accumulation could be restored via cAMP/protein kinase A protocol. Overall, these data provide compelling evidence for the operation of distinct Na+- and Ca2+-dependent Mg2+ 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 Ca2+-dependent mechanism, instead, only mediates Mg2+ extrusion in a cAMP-independent manner.

Involvement of ERK1/2 and p38 in Mg2+ accumulation in liver cells

Activation of PKC signaling induces Mg2+ accumulation in liver cells. To test the hypothesis that PKC induces Mg2+ 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 Mg2+ accumulation by addition of PMA or OAG. Accumulation of Mg2+ within the cells was measured by atomic absorbance spectrophotometry in the acid extract of cell pellet. The presence of either inhibitor completely abolished Mg2+ accumulation irrespective of the dose of agonist utilized while having no discernible effect on β -adrenoceptor mediated Mg2+ extrusion. A partial inhibition on α 1-adrenoceptor mediated Mg2+ 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 Mg2+in exchange for intra-vesicular entrapped Na+ while retaining the ability to extrude entrapped Mg2+ in exchange for extra-vesicular Na+. These data indicate that ERK1/2 and p38 are involved in mediating Mg2+ accumulation in liver cells following activation of PKC signaling. The absence of a detectable effect of either inhibitor on β -adrenoceptor induced, Na+-dependent Mg2+ extrusion in intact cells and in purified plasma membrane vesicles further support the hypothesis that Mg2+ extrusion and accumulation occur through distinct and differently regulated transport mechanisms.

Lack of insulin impairs Mg2+ homeostasis and transport in cardiac cells of streptozotocin-injected diabetic rats.

Serum and tissue Mg2+ content are markedly decreased in diabetic patients and animals. At the tissue level, Mg2+ loss progresses over time and affects predominantly heart, liver and skeletal muscles. In the present study, alterations in Mg2+ 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 Mg2+ 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 Mg2+ following β‐adrenergic receptor stimulation or addition of cell permeant cyclic‐AMP. Sarcolemma vesicles purified from diabetic animals, however, transported Mg2+ 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 Mg2+ homeostasis and transport to levels comparable to those observed in non‐diabetic animals. These results suggest that in diabetic cardiac cells Mg2+ homeostasis and extrusion via β‐adrenergic/cAMP signaling are markedly affected by the concomitant decrease in protein and ATP content. As Mg2+ 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. J. Cell. Biochem. 104: 1034–1053, 2008.

Altered Mg2+ transport across liver plasma membrane from streptozotocin-treated rats

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)

Intravesicular glucose modulates magnesium2+ transport in liver plasma membrane from streptozotocin-treated rats

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.

Delayed restoration of Mg2+ content and transport in liver cells following ethanol withdrawal

Liver cells from rats chronically fed a Lieber-De Carli diet for 3 wk presented a marked decreased in tissue Mg(2+) content and an inability to extrude Mg(2+) into the extracellular compartment upon stimulation with catecholamine, isoproterenol, or cell-permeant cAMP analogs. This defect in Mg(2+) extrusion was observed in both intact cells and purified liver plasma membrane vesicles. Inhibition of adrenergic or cAMP-mediated Mg(2+) 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 Mg(2+) extrusion was observed in intact cells but not in plasma membrane vesicles. In the chronic model, upon removal of EtOH from the diet hepatic Mg(2+) content and extrusion required approximately 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 Mg(2+) 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 Mg(2+) extrusion is the result of a limited refilling of the cellular compartment(s) from which Mg(2+) 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 Mg(2+) extrusion mechanisms in addition to the limited refilling of the cellular compartments.