Functional characterization of two distinct Mg(2+) extrusion mechanisms in cardiac sarcolemmal vesicles

Abstract

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.