Removal of intracellular Mg2+ activates cardiac Na+/Ca2+ exchanger

See article by Wei et al. [1] (pages 334–340) in this issue. The Na+/Ca2+ exchanger (NCX) expels Ca2+ from myocytes in exchange for extracellular Na+ and balances Ca2+ influx through L-type Ca2+ channels during cardiac excitation. In this issue of Cardiovascular Research, Wei et al. [1] report that cardiac NCX is regulated by intracellular Mg2+ (Mg2+i). Their results suggest some similarities of mammalian cardiac NCX to that of squid nerves (NCX-SQ) [2]. In squid nerve, rapid removal of Mg2+i produced marked activation of the exchanger. Further removal of ATP did not suppress the activated NCX-SQ, but readmission of Mg2+i abolished this NCX-SQ stimulation. Phosphatidylinositols, which are known to modulate the activity of cardiac NCX [3], were not involved in the stimulation of NCX-SQ by Mg2+i removal [2]. In pig ventricular myocytes [1], when Mg2+i was reduced by cell dialysis with a low Mg2+ internal solution (0.18 mM), whole cell NCX current was augmented regardless of the levels of free Ca2+, ATP and MgATP. These findings are quite comparable to those in squid nerves: removal of ATP in the absence of Mg2+i kept the NCX-SQ in a highly stimulated state. Among the results by Wei et al. [1], the absence of the effect of ATP on stimulated NCX by Mg2+i removal is rather unexpected (Fig. 2 in their report). In many preparations including cardiac [4,5], intracellular ATP directly or indirectly regulates the activity of NCX. As most cytosolic enzymes use MgATP as a substrate, simple reduction of Mg salt of the internal solution may result in reduction of MgATP. Depletion of MgATP, in turn, may reduce phosphorylation of the protein and the content of phosphatidylinositol 4,5-bisphosphate (PIP2), both of which regulate the activity of NCXs [5]. However, in pig ventricular cells [1] and also in squid nerves [2], the decreases in ATP and/or MgATP did not affect the NCXs upregulated by Mg2+i removal. DiPolo et al. [2] hypothesized that removal of intracellular Mg2+ latches the NCX-SQ in a highly activated state. The authors indicated that possible suppression of Mg2+-dependent phosphatase (protein phosphatase 2C type) under the Mg2+ depleted condition has kept the NCX-SQ in the phosphorylated state. The above story on NCXs has interesting similarities to the effects of intracellular Mg2+ on the cardiac L-type Ca2+ channel. In frog ventricular cells, decrease in Mg2+i to 10−6 M/l profoundly increased the amplitude of the L-type Ca current, ICaL[6,7]. Combination of forskolin and IBMX did not increase the ICaL already enhanced by Mg2+ depletion. It was proposed that Mg2+ as well as Ca2+ competitively inhibit the L-type Ca2+ channel. Under physiological condition, a large portion of the channels is bound to Mg2+ and is in a non-available state. Either decrease in Mg2+i or channel phosphorylation puts the channel in an available state. A combination of a non-hydrolysable ATP analogue, AMP-PCP, an inhibitor of glycolysis, 2-deoxyglucose, and an uncoupler of mitochondrial ATP synthesis, cardoxylcyanide-M-chlorophenyl-hydrazone (CCCP) did not inhibit the ICaL augmentation produced by low Mg2+i. Thus, the channel phosphorylation is not related to ICaL stimulation in frog ventricular myocytes. Pelzer et al. [8] confirmed the augmentation of ICaL by low Mg2+i in ventricular myocytes from guinea-pig. Unlike frog ventricular cells, an inhibitor of a wide variety of protein kinases, K252a, completely abolished the low Mg2+i-induced increase in ICaL. They proposed possible involvement of the channel phosphorylation by cyclicAMP-dependent protein kinase (PKA), since degradation of cyclicAMP by phosphodiesterases requires Mg2+. Removal of Mg2+i may also slow down the dephosphorylation of the channels by protein phosphatase 2C as was suggested in case of the NCX-SQ. The cardiac NCX is stimulated by phospholipase C-activating agonists, such as phenylephrine, endothelin 1, and angiotensin II. Phorbol esters can mimic the effects of these agonists and inhibitors of protein kinase C (PKC) block the stimulatory effects. Therefore, phosphorylation of the NCX via PKC is involved in this regulation [5,9]. However, data are lacking about the contribution of protein phosphorylation in the activation of the cardiac NCX by low Mg2+i. In squid nerve vesicles, application of alkaline phosphatase concentration-dependently suppressed the NCX-SQ [2]. However, the same alkaline phosphatase failed to suppress mammalian cardiac NCX. It was suggested that NCX-SQ is regulated mainly by a phosphorylation/dephosphorylation process, and cardiac NCX by lipid metabolism. In this context, a further study is needed to exclude possible contribution of phosphatidylinositols using mammalian myocytes. Anyhow, it is of great interest that depletion of Mg2+i activates both NCXs (from squid nerve and mammalian heart) and the cardiac L-type Ca2+ channel. It is of crucial importance to determine whether the channel phosphorylation/dephosphorylation is the common process or not. Magnesium is the second or third abundant metal atom in the cell interior, its total cellular content being estimated to be around 10 mM/l [10]. However, over 90% of the intracellular Mg is bound or sequenced. Intracellular free Mg2+ concentration measured using 31P-nuclear magnetic resonance (NMR) spectrometry, Mg2+-sensitive microelectrodes or Mg2+-sensitive dye, is reported to be 0.5 to 1 mM/l (1 to 2 mequiv./l). Assuming an extracellular Mg2+ concentration of 1 mM, the equilibrium potential for Mg2+ would be −30 to +10 mV. This means that there is a considerable inward electrochemical driving force for Mg2+ at normal resting potential (−80 mV). To balance passive influx of Mg2+, the cardiac cells must have active Mg2+ extrusion system(s). Like many other cell types, Na+/Mg2+ exchanger would be the most probable mechanism in the heart [11]. The [Mg2+]i is thought to be almost stable under normal physiological conditions but there is evidence that supports considerable elevation and reduction in cellular Mg content or [Mg2+]i under different physiological and pathological conditions [10]. For example, β-agonists cause large increases in efflux of Mg2+, while activation of protein kinase C increases Mg2+ uptake [12]. A three- to fivefold increase in Mg2+i is reported at the onset of myocardial ischaemia, possibly due to rapid break down of MgATP [13]. On the other hand, a reduction of cardiac total Mg content occurred in patients with congestive heart failure, myocardial infarction, and patients undergoing cardiac surgery [14,15]. In a canine model of pacing-induced heart failure, a 50% reduction of [Mg2+]i was reported [16]. Unfortunately, quantitative experimental data correlating Mg2+i and the extent of NCX stimulation are still lacking. At present, it is too early to say that NCX activation by decreased Mg2+i has any significant role in Ca2+ handling in the cells under pathological conditions.