Elemental maps in human allantochorial placental vessels cells: 2. MgCl ² and MgSO ⁴ effects

ARTICLE

Auteur(s) : Claire Michelet-Habchi^a, Philippe Barberet^a, Raj Kumar Dutta^a, Philippe Moretto^a, Andrée Guiet-Bara^b, Michel Bara^b

^a Centre d'Études Nucléaires de Bordeaux-Gradignan, Interface Physique-Biologie, Le Haut-Vigneau, BP 120, 33175 Gradignan Cedex, France. 
^b Laboratoire de Physiologie et Physiopathologie, Université P. et M. Curie, Bât. A, 4 Place Jussieu, 75252 Paris Cedex 05, France.

Introduction

Alterations in extracellular and intracellular magnesium in numerous tissues have been shown to alter ion channel currents. The magnesium regulation of various ionic channels interferes with cellular functions. Magnesium ions are known to block the current through K⁺ channels (inwardly rectifier, delayed rectifier, Ca-dependent, ATP-dependent), Na⁺ channels and Ca²⁺ voltage-gated channels and to activate the current through chloride channels [1]. Magnesium ions interfere with numerous functions, and particularly extracellular magnesium ions which are implicated in the blood vessel smooth muscle tone and reactivity, regulated by membrane potential [2]. The membrane potential of vascular cells is regulated by ionic channels which induce depolarization or hyperpolarization. Previous studies [3-5] have demonstrated the influence of magnesium salts on the membrane potential of vascular smooth muscle cells (VSMCs) and of vascular endothelial cells (VECs) of human allantochorial placental vessels which are representative of the small arteries involved in the control of fetal and maternal utero-placental resistance, in contrast to the usually studied umbilical arteries [3]. The membrane potential of these cells has been investigated and is modulated by several channels: voltage-sensitive potassium channels (K_df), Ca-activated (K_Ca) and ATP-sensitive (K_ATP) potassium channels and voltage-sensitive calcium channels in VSMCs [6]; K_df, K_Ca and voltage-sensitive calcium channels in VECs [7]. Previous studies [8] have investigated the possibility of applying nuclear microprobe analysis to human vascular allantochorial placental cells in order to reveal the structure of arteries and to generate maps of the different inorganic ions across the walls of arteries.

The aim of this study was to investigate the influence of two magnesium salts (physiological magnesium chloride and magnesium sulfate, frequently used in the case of pre-eclampsia and/or eclampsia) [9] added to a survival medium (Hanks's solution) on the elemental ion distribution (Na, K, Cl, P, Ca, Mg) in VSMCs and in VECs to investigate the possible relationship with the ionic movement across the membrane channels.

Material and methods

Sample preparation

Short segments of allantochorial arteries (results obtained with veins are identical) [3] were sampled in human placenta collected after normal delivery at term. Samples were immediately incubated in different media: Hanks' solution, Hanks' solution + 2 mM MgCl₂, and Hanks' solution + 2 mM MgSO₄ (the concentration of 2 mM of Mg corresponds to a middle pharmacological magneso-therapy and to the dose inducing a measurable effect on the ionic channels³). Then the samples were cryofixed using the following method: the samples were quench-frozen in isopentane chilled with liquid nitrogen and stored in liquid nitrogen until sectioning.

The preparation of placental vessels was carried out according to a classical scheme: arteries were cut using a cryo-microtome equipped with a tungsten carbide blade and placed in a cryostat at – 25°C. The sections (20 µm thick) were directly collected from the blade using fresh Formvar^R films (50 µg/cm² areal mass) stretched on pure aluminium sample holders. The section were left overnight in the cryostat until complete freeze-drying.

Nuclear microprobe analysis

All sections were analyzed using the CENBG nuclear microprobe. This experimental setup has been previously presented [10]. The microanalysis was carried out using a 1.5 MeV proton beam, focused down to a small (2 µm in diameter) spot on the sample. A beam current of 150 pA was employed. Particle Induced X-ray Emission (PIXE) and Rutherford Backscattering Spectometry (RBS) were employed simultaneously in order to determine both the mineral content and the organic mass of the analyzed tissues. For each sample, two analyses were performed: a large scan ( ~ 1 mm²) in order to identify the different strata obtained from the elemental maps of minerals, then a small scan ( ~ – 160 × 150 µm²) was made on the lumen border to locate the monolayer of endothelial cells.

Data reduction

In order to measure the ionic content of smooth muscle cells and endothelial cells in arteries, the strata were delimited on the elemental maps (figure 1). In each lamina, a large region was then chosen for its homogeneity and local spectra were extracted using a specific computer treatment of list data files. The endothelial cells on the lumen border maps were also identified, cell by cell, and local spectra were also extracted from small regions.

Statistical comparisons between various specimens were carried out with Student's test. The values of the significance level (p) of 0.05 and less were considered as significant.

Results

Vascular smooth muscle cells (VSMC)

The ionic concentrations measured in VSMC are presented in figure 2. The histograms compare, for each ion, the artery walls incubated in Hanks' solution + 2 mM MgCl₂ and those incubated in Hanks' solution + 2 mM MgSO₄ to those incubated in Hanks' solution taken as a reference. Whatever the medium, the ionic concentrations remained constant (0.06 < p < 0.9) except a significant increase of Na (p < 0.05) with MgCl₂ and of Mg (p < 0.005) concentrations in Hanks' solution + MgCl₂ and in Hanks' solution + MgSO₄.

Vascular Endothelial cells (VEC)

The data obtained in VEC are presented in figure 3. Results are expressed in terms of concentration ratios against sulphur taken as a reference. Measurements indicated a significant increase of the Na (p < 0.01) and Mg (p < 0.01) concentration in Hanks' solution + MgCl₂ and a significant increase of Mg (p < 0.01) concentration in Hanks' solution + MgSO₄ compared to Hanks' solution alone. The other elemental ion concentrations, whatever the medium, remained constant (0.09 < p < 0.9).

Discussion

In human allantochorial placental vessels, the previous electrophysiological studies [3, 4] have indicated that MgCl₂ and MgSO₄ depolarize VSMCs with different thresholds, indicating that MgCl₂ influences cell membrane potential directly while MgSO₄ interferes first with the endothelial cells, which may act as an intermedary between Mg²⁺ ions and VSMCs. This effect has been confirmed on VECs: MgCl₂ and MgSO₄ induce depolarization and the influence of MgCl₂ is higher and more direct than that of MgSO₄.

The membrane potential of VSMCs of human allantochorial placental vessels is the major factor which explains the excitation-contraction coupling. Indeed, the smooth muscle cells of these vessels may be considered as tonic because they only respond to excitatory stimuli with graded depolarization. This depolarization may be a consequence of various factors, particularly the secretion of endothelium-derived depolarized factors (EDDFs).

In 1997 [13], a scheme was proposed to explain the effects of Mg salts on the membrane potential of human allantochorial placental vessel cells: 1- a direct action of external Mg²⁺ ions on the membrane potential by secretion of EDDFs which depolarize the membrane; 2- an indirect action on the membrane potential by: * a regulation of K⁺ and Ca²⁺ channels; * an action on the internal Mg²⁺ concentration; * an interaction with internal Na⁺ and Ca²⁺ concentrations; and * a regulation of the Na⁺/Ca²⁺ exchanger.

Previous electrophysiological studies [5, 14] have confirmed the regulation of K⁺ and Ca²⁺ channels by external Mg salts. Indeed, in VSMCs incubated in a physiological medium+MgCl₂, K_df, K_Ca and K_ATP channels are blocked (K_df are open at low MgCl₂ concentration), while in VECs, K_df and K_Ca are blocked. In a physiological medium+MgSO₄, the same observations occur, except the K_df open at low concentrations. This observation also occurs to Ca²⁺ — voltage gated channels which are blocked [14, 15].

The present microanalysis study indicates that in VSMCs and in VECs, the two Mg salts have no effect on the concentrations of K, Cl, P and Ca, but that MgCl₂ significantly increases Na and Mg concentrations while MgSO₄ increases only Mg concentration.

These results confirm the blockage of the potassium and calcium channels by Mg salts (the influx and the outflux of K and Ca ions remain constant and their concentrations have not modified). Moreover, the data indicate an interaction between Mg salts and internal Na concentration in VSMCs and internal Mg concentration in VSMCs and in VECs, corroborating the hypothesis scheme [13]. Internal Ca concentration is not modified, therefore it is difficult to consider a relationship between Mg ions and Na⁺/Ca²⁺ exchanger, but it seems possible to consider the existence of an Na⁺/Mg²⁺ exchanger, demonstrated in numerous cell membranes [16], exchanger which might be the major element of the regulation of Na and Mg distribution in VSMCs and in VECs.

Conclusion

The results of the present study indicate the importance of Mg²⁺ in the regulation of intracellular ion concentrations in VSMCs and in VECs of allantochorial placental vessel cells, indicating the relationship between Mg²⁺ ions and ionic channels, Na and Mg internal concentrations and with a possible Na⁺/Mg²⁺ exchanger. Further research should be undertaken to establish whether the effects of Mg on the intracellular ion concentrations is dependent on the Mg-salt dose.

References

1. Bara M., Ibrahim B., Guiet-Bara A. (2001): Magnesium and ionic membrane channels, In: Advances in Magnesium Research: Nutrition and Health, Rayssiguier Y., Mazur A., Durlach J. eds, John Libbey & Comp., 65-72.

2. Daut J., Standen N.B., Nelson M.T. The role of membrane potential of endothelial and smooth muscle cells in the regulation of coronary blood flow. J Cardiovasc Electrophysiol 1994; 5, 154-81.

3. Ibrahim B., Guiet-Bara A., Leveteau J., Challier J.C., Vervelle C., Bara M. Membrane potential of smooth muscle cells of human placental chorionic vessels. Comparative effects of MgCl₂ and MgSO₄. Magnesium Res 1995; 8, 127-35.

4. Ibrahim B., Leveteau J., Guiet-Bara A., Bara M. Influence of magnesium salts on the membrane potential of human endothelial placental vessel cells. Magnesium Res 1995; 8, 233-6.

5. Guiet-Bara A., Bara M. (2001): Magnesium sulphate and magnesium chloride effects on K(df), K(Ca) and K(ATP) channels of smooth muscle and endothelial cells of allantochorial human placental vessels. In: Advances in Magnesium Research: Nutrition and Health, Rayssiguier Y., Mazur A., Durlach J. eds, John Libbey, Comp., 465-7.

6. Guiet-Bara A., Ibrahim B., Leveteau J., Bara M. Calcium channels, potassium channels and membrane potential of smooth muscle cells of human allantochorial placental vessels. Bioelectrochem Bioenerg 1999; 48, 407-13.

7. Guiet-Bara A., Bara M. Evidence of K and Ca channels in endothelial cells of human allantochorial placental vessels. Cell Mol Biol 2002; 48, OL137-OL142.

8. Moretto Ph., Bara M., Guiet-Bara A., Michelet C. Influence of an external medium on the ionic distribution in human allantochorial placental vessels. Nucl Instr Meth B 1999; 158, 380-5.

9. Durlach J., Bara M. (2000): Le magnésium en biologie et médecine, 403 pp., Ed. Med. Int. Cachan (France).

10. Moretto Ph. Nuclear microprobe: a microanalytical technique in biology. Cell Mol Biol 1996; 42, 1-16.

11. Maxwell J.A., Campbell J.L., Teesdale W.S. The Guelph PIXE software package. Nucl Inst Meth B 1989; 43, 218-22.

12. Moretto Ph., Razafindrabe L. Stimulation of RBS spectra for quantitative mapping of inhomogeneous biological tissue. Nucl Inst Meth B 1994; 104, 171-5.

13. Bara M., Ibrahim B., Leveteau J., Guiet-Bara A. Implication of external Mg²⁺ ions in the depolarization of smooth muscle cell membrane of the human chorionic placental vessels: an hypothesis. Magnesium Res 1997; 10, 3-10.

14. Bara M., Guiet-Bara A. Magnesium regulation of Ca²⁺ channels in smooth muscle and endothelial cells of human allantochorial placental vessels. Magnesium Res 2001; 14, 11-18.

15. Schwinger R.H., Frank K., Hoischen S., Muller-Ehmsen J., Brixius K. Effect of changed extracellular K⁺ and Mg²⁺ — concentration on intracellular Ca²⁺ homeostasis, contraction coupling and force-frequency relations in the human myocardium. Herz 1997; 22 (Suppl. 1), 18-27.

16. Bara M., Guiet-Bara A., Durlach J. Regulation of sodium and potassium pathways by magnesium in cell membranes. Magnesium Res 1993; 6, 167-77.