Mechanisms for monovalent cation-dependent depletion of intracellular Mg2+:Na(+)-independent Mg2+ pathways in guinea-pig smooth muscle

Abstract

It has been suggested that magnesium deficiency is correlated with many diseases. 31P NMR experiments were carried out in order to investigate the effects of Na+ substitution on Mg2+ depletion in smooth muscle under divalent cation-free conditions. In the taenia of guinea-pig caeci, the intracellular free Mg2+ concentration ([Mg2+]i) was estimated from the chemical shifts of (1) the beta-ATP peak alone and (2) beta- and gamma-ATP peaks. Both estimations indicated that [Mg2+]i decreased only very slowly in Mg(2+)-free, Ca(2+)-free solutions in which Na+ was substituted with large cations such as NMDG (N-methyl-D-glucamine) and choline. Furthermore, the measurements of tension development supported the suggestion of preservation of intracellular Mg2+ with NMDG substitution. Substituting extracellular Na+ with the small cation, Li+, also shifted the beta-ATP peak towards a lower frequency, but the frequency shift was significantly less than that seen upon Na+ substitution with K+. The estimated [Mg2+]i depletion was, however, comparable with that seen after Na+ substitution with K+ using the titration curves of metal-free and Mg(2+)-bound ATP obtained in Li(+)-based model solutions. It was concluded that Mg2+ rapidly decreases only when small cations were the major electrolyte of the extracellular medium. Na+ substitutions with NMDG, choline or Li+ had little effect on intracellular ATP concentration after 100 min treatment.

Figure 1. Titration data for metal free- (filled square) and Mg²⁺-bound ATP (filled circle)

The data points are fitted by sigmoid curves (eqn (8)). The fitting parameters are shown in Table 1. A Na⁺-based solution was used in A. In B, Li⁺ was used instead of Na⁺.

Figure 2. Effects of removal and reapplication of extracellular cations on spontaneous mechanical activity

The extracellular divalent cations and Na⁺ were iso-osmotically replaced with K⁺ in (A) or with NMDG in (B). After 100 min exposure to divalent cation-free, Na⁺-free solutions, 2.4 m M Ca²⁺ was added (in both A and B). A, Mg²⁺ (1.2 mM) was added 100 min after the reapplication of Ca²⁺. Subsequently, after 100 min the Na⁺ concentration was returned to the initial concentration. The histogram in C shows averaged magnitude of mechanical response to the reapplication of extracellular cations (n = 6 for each experiment). The magnitude was normalized by taking the control mechanical activity (in ‘normal’ solution) as 100 %. * Significant difference (unpaired t-test, P < 0.05) compared with the mechanical response to Ca²⁺ reapplication in Mg²⁺-free, Ca²⁺-free solution (K⁺ substitution) at 100 min.

Figure 3. ³¹p NMR spectra obtained in divalent cation-free, Na⁺- free solutions

After observing control ³¹P NMR spectra (Aa and Ba), extracellular divalent cations and Na⁺ were iso-osmotically substituted with K⁺ for 100 min (A) or with NMDG for 200 min (B). Vertical dashed lines represent the initial chemical shifts of γ- and β-ATP. Each spectrum was obtained from the sum of 2500 NMR signals over 25 min. The ³¹P NMR spectra (Ab, Bb and Bc) were obtained during exposures to divalent cation-free, Na⁺-free solutions for 75–100 and 175–200 min, respectively.

Figure 4. The time course of changes in [Mg²⁺]_i during exposure to divalent cation-free, Na⁺-free solutions

Changes in [Mg²⁺]_i induced by substituting extracellular divalent cations and Na⁺ with K⁺ (filled square)and NMDG(filled circle). Each point represents the negative logarithm of the mean [Mg²⁺]_i value (p[Mg²⁺]_i). [Mg²⁺]_i was estimated from the chemical shifts of the β-ATP and PME-1 peaks using eqn (6). The pH dependence of K_D,MgATP described by Zhang et al. (1997) (K_{D,Zhang,MgATP}(pH)) was used in A, while that described by Bock et al. (1984) (K_D,Bock,MgATP(pH)) was used in B.

Figure 5. ³¹p NMR spectra obtained in a divalent cation-free, Na⁺-free solution which was made by iso-osmotically substituting with Li⁺

Vertical dashed lines represent the initial chemical shifts of γ- and β-ATP. The spectrum (a) shows control; (b) and (c) are ³¹P NMR spectra obtained during exposures to divalent cation-free, Na⁺-free solutions for 25–50 and 75–100 min, respectively.