Cardiovascular Research

Cardiovascular Research 2002 53(2):290-291; doi:10.1016/S0008-6363(01)00561-2
© 2002 by European Society of Cardiology

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Copyright © 2001, European Society of Cardiology

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

Tatsuto Kiyosue^*

Department of Physiology, Oita Medical University, 1-1 Idai-ga-oka, Hasama, Oita 879-5593, Japan

kiyosue{at}oita-med.ac.jp

^* Tel.: +81-975-865-651; fax: +81-975-496-046

Received 4 December 2001; accepted 4 December 2001

See article by Wei et al. [1] (pages 334–340) in this issue.

	1. Effects of intracellular Mg2+ and ATP on NCX

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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.

	2. Similarities to the regulation of cardiac L-type Ca2+ channel by Mg2+i

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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, I_CaL [6,7]. Combination of forskolin and IBMX did not increase the I_CaL 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 I_CaL augmentation produced by low Mg²⁺_i. Thus, the channel phosphorylation is not related to I_CaL stimulation in frog ventricular myocytes.

Pelzer et al. [8] confirmed the augmentation of I_CaL 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 I_CaL. 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.

	3. Mg2+i under normal and pathophysiological conditions

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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.

	References

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1. Wei S.-K., Quigley J.F., Hanlon S.U., O'Rourke B., Haigney M.C.P. Cytosolic free magnesium modulates Na/Ca exchange currents in pig myocytes. Cardiovasc Res (2002) 53:334–340.[Abstract/Free Full Text]
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