Abstract
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.
Highlights
Magnesium, the second most abundant cation within mammalian cells, is necessary for a variety of metabolic and cellular functions [1,2,3,4,5]
One intriguing possibility is the presence in hepatocytes of a physiological “break” on Mg2ϩ transport, which could be transiently removed as the result of activation of cellular pathways
Plasma Membrane Loading—5-ml aliquots of aLPM or bLPM vesicles were resuspended in 25 ml of incubation medium (1:5 v/v) in the presence of 20 mM MgCl2 or, when specified, Naϩ or Ca2ϩ and loading was empirically determined by four passes in a Thomas C Potter with a tight fitting pestle at 4 °C as described previously [21]
Summary
Alkaline phosphatase is expressed as mol of p-nitrophenyl hydrolyzed/mg of proteinϪ1/minϪ1, whereas all the other data are expressed as mol Pi/mg of proteinϪ1/minϪ1. All experimental protocols were performed as previously described [21]. All data are reported as mean Ϯ S.E.; n ϭ 15, 8, 8, and 10 for 5Ј-nucleotidase, MgATPase, Naϩ/Kϩ-ATPase, and alkaline phosphatase in homogenate and total LPM, respectively. N ϭ 10, 6, 6, and 10 for 5Ј-nucleotidase, MgATPase, Naϩ/Kϩ-ATPase, and alkaline phosphatase in aLPM and bLPM, respectively All data are reported as mean Ϯ S.E.; n ϭ 15, 8, 8, and 10 for 5Ј-nucleotidase, MgATPase, Naϩ/Kϩ-ATPase, and alkaline phosphatase in homogenate and total LPM, respectively. n ϭ 10, 6, 6, and 10 for 5Ј-nucleotidase, MgATPase, Naϩ/Kϩ-ATPase, and alkaline phosphatase in aLPM and bLPM, respectively
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