(1995) 8, 3, 207-214
Summary: The effect of the addition of MgCl2 on the Na+, K+, and Cl- concentration and distribution in epithelial and compact layers of the human amniotic membrane was investigated using the Bordeaux nuclear microprobe. Particle-induced X-ray emission and Rutherford backscattering spectrometry techniques were used to provide quantitative measurements. In physiological medium (Hanks' solution), the monovalent ion concentrations were identical in both layers. The addition of Mg2+ ions in Hanks' solution induced a decrease of, K+, and Cl- concentration in both layers and Na+ concentration in the compact layer. The results obtained from nuclear microanalysis might be explained from electrophysiological data which indicate that the addition of Mg2+ ions results in an increase in the cellular, paracellular and exchanger ion pathways.
Keywords: Chlorine, human amniotic membrane, ionic exchange, magnesium, nuclear microanalysis, potassium, sodium.
In the human amniotic membrane, a 'leaky membrane', the total transmembrane conductance is principally due to monovalent ions (Na+, K+, Cl-)1. The conductance is controlled by several components: paracellular and cellular (ionic channels, Na/K-ATPase, antiports and exchangers). Among the multiple magnesium properties, the stabilization of cell membranes and potentiating effects of Na/K-ATPase have been reported previously2. It is generally assumed that in magnesium deficit, the permeability of plasma membranes increases. Mono and divalent cations, such as Na+ and Ca2+, accumulate in the cell, whereas K+ and phosphorus efflux are promoted. This effect is amplified by disregulation of the ATPase-dependent active transport of Na+, K+ and Ca2+. The direct consequence is cell membrane depolarization. In the human amnion, Mg2+ ion interfere with monovalent ions transfer. Previous studies3 have shown that Mg2+ ions (2 mM) increase all components of the transamniotic conductance.
This effect depends on the nature of the magnesium salts present in the physiological medium4-6. Ultrastructural studies7 have indicated that MgCl2, at low concentrations (2 mM) decreases the volume of intercellular space and of the podocytes and has no effect on the volume of microvilli vs the cell volume. These studies do not provide information on the amniotic mapping of the ions present in the physiological fluid. MicroPIXE is one of the few methods of microanalysis which permits a simultaneous detection of most minerals acting in cellular pathways. It provides unique possibilities for revealing directly the distribution of these elements at cell level. Previous studies8-11 have indicated the first step in the development of this technique which has been to check the consequences of a simple incubation in a widely used physiological fluid. These studies have shown the distribution of Na+, K+ and Cl- in the epithelial and compact layers of the human amnion. The aim of this study was to observe the effects of magnesium supplementation on the monovalent ion distribution in the layers of the amnion and to elucidate the previous electrophysiological data.
Specimens of human amnion, isolated from the placental zone of the amniotic sac, were obtained after 10 normal deliveries at term. For each specimen, three strips were collected, the first one being immediately quench-frozen in isopentane cooled with liquid nitrogen, without any rinsing procedure. This sample was considered as a control sample. The second strip was transferred into Hanks' solution, a physiological fluid used in electrophysiological studies (composition in mM/litre: NaCl 150, KCl 6, MgSO4 0.5, MgCl2 0.5, CaCl2 1, glucose 5.5, NaH2PO4NaHCO3 1) at 37°C and pH 7.4. For the third one, magnesium (MgCl2, 2 mM) was added to normal Hanks' solution. After lh incubation, the two incubated strips were cryofixed as previously. The three strips were kept in liquid nitrogen until sectioned. Sectioning was performed at -30°C using a cryomicrotome (Reichert-Jung frigocut 2800). Thin frozen sections, in the thickness range 20-30 µg/cm2 , were collected on the knife, placed directly on thin formvarR foils of about 20 µg/CM2 and kept in the cryostat for several hours until complete freeze-drying. The slides were then stored in a dessicator over silica-gel before analysis. The morphology of the amnion sections was elucidated using light microscopy of adjoining sections mounted on glass slides and stained with haematoxylin and eosin.
Microanalysis and data processing
The analysis were performed using the CENBG microprobe facility in Bordeaux12. Well defined parts of amnion slides were chosen including epithelial cells and compact lamina. These regions were irradiated with a 1 Mev proton beam focused down to a 2 µM spot diameter. The beam current, measured on the target, was 150 pA and the total collected charge was 0.5 µC. The extension of the scan ranging, according to the sample structure, was chosen between 50 x 50 µm2 and 100 x 100 µm2. PIXE and RBS (Rutherford backscattering spectrometry) analysis were carried out simultaneously in order to ensure the mass standardization.
Sodium, potassium and chlorine (PIXE) were determined as well as carbon, nitrogen and oxygen (RBS). X-rays were detected using a 80 mm2 Si(Li) solid state detector (Link system) fitted with a thin beryllium window (8 µm), which allowed us to measure the NaKα line with low attenuation. The backscattered particles were detected at 135°C of the beam with an Si 20 mm2 detector thus allowing the measurement of the organic mass and beam current monitoring. Elemental mapping of Na, K and Cl revealed a high compartmentalization of ionic species thus allowing a precise delimitation of the epithelial (EL) and compact layers (CL) over the whole scanned area. An off-line specific treatment of data permitted us to extract X-rays and partial spectra of backscattered particles issuing from the previously deflned subregions (EL and CL)10.
Quantitative results expressed in term of dry mass were obtained using the following scheme. All PIXE spectra were fitted with GUPIX software13. RBS data were treated using an extension of the RUMP code 14 , a program developed in our group15 taking into account the autoabsorption of low energy x-rays. Unfortunately, this program was not available at the beginning of this study. In order to include all experimental data in the reported results, we therefore expressed quantitative values using elemental rations (Na/S, K/S and Cl/S). Sulphur was considered to be the best reference element because of its unvarying concentration in Epithelial Layers and Compact Layers, whether the amnion was incubated or not. This point was checked using the mass normalization procedure described above. The related data are displayed in Table 1.
The statistical comparisons between reference and incubated specimens were carried out with Student's t test.
It has previously11, been shown that the EL is clearly defined by higher phosphorus level (Fig. 1). The distribution of Na+, K+ and Cl- in a sample incubated in Hanks' solution + 2 mM MgCl2 is given in Fig. 2. The delineated subregions used for the determination of the quantitative values and the RBS energy spectra are shown in Fig. 3. The related PIXE spectra are presented in Fig. 4.
The resulting concentrations of Na+, K+ and Cl- are displayed as a Na/S, K/S and Cl/S ratios because sulfur was the only element with sufficient stability, even after incubation, to be used as a simultaneous reference for EL and CL (Table 1).
Sodium (Fig. 5)
In unwashed control samples, the Na+ concentration was identical in EL and CL (P = 0.07). Concentrations were strongly increased in EL (x3) and CL (x4) (P < 0.001) after incubation in Hanks' solution, without modifications between EL and CL. The addition of MgCl2 had no significant effect on the Na+ concentration in EL (P = 0.096), but decreased it significantly (P < 0.05) in CL.
Potassium (Fig. 6)
In unwashed control samples, the K+ concentration was identical in EL and CL (P = 0.57). Incubation in Hanks' solution had no significant effect on the K+ concentration in EL and CL. However, the addition of MgCl2 induced a significant decreased of the K+ concentration in EL (P < 0.05) and in CL (P < 0.01).
Chlorine (Fig. 7)
In unwashed control samples, the Cl concentration was identical in EL and CL (P = 0.13). After incubation in Hanks' solution, the Cl concentration was increased significantly in EL and CL (P< 0. 001), without modifications between the repartition in these two layers. The addition of MgCl2 in Hanks' solution induced a significant decrease of the Cl- concentration in EL and Cl (P < 0.01).
The monovalent ions (Na+, K+, Cl-) are localized in the 'exchanging' layer (EL) and in the 'supporting' layer (CL). There are distributed in the same way in the two layers. This data confirms previous observations10,11 and indicates the great importance of the compact layer in the human amniotic membrane.
In incubated samples, the Na+ and Cl- concentrations are substantially higher than in control samples in both layers, while the K+ concentration remains constant. These data are consistent with the fact that the Na+ and Cl- concentrations in Hanks' solution are the more important. The increased Na+ and Cl- concentrations in compact layer imply that this layer acts as a buffer which can fix minerals.
The addition of MgCl2 in the Hanks' solution induces a decrease of K+ and Cl- concentrations in both layers, and of Na+ concentration in the compact layer. Electrophysiological studies3,16 have indicated that the addition of MgCl2 increases the movement of monovalent ions across the epithelial cells: cellular and paracellular conductances are increased.
In the EL, after addition of Mg2+ ions, the Na+ concentration remains constant. Mg2+ ions initiate a movement of Na+ ions (leak of Na+ ions from epithelial cells) and the concentration decrease induces increased activity of various exchangersl7 to maintain the internal Na+ concentration, drawing from CL. Consequently, the Na+ concentration remains identical in EL and decreases in CL.
Previous studiesl8-20 may explain the results of nuclear microanalysis on the K+ concentration: decrease in EL and CL. Indeed, Mg2+ ions act as an activator of the K+ transfer on the maternal side (that is, from CL to EL via the podocytes) and in the intercellular space (from the interior of the epithelial cells to the intercellular space)1. Moreover, Mg2+ ions are competitive inhibitors of K+ ions on the fetal side (in microvilli), that is, Mg2+ ions remove K+ ions from external fixed sites and hinder the uptake of K+ in the epithelial cells. The possible mechanism is: Mg2+ ions remove K+ ions from CL (decrease in CL); K+ ions enter in EL (Mg2+ activator of K+ transfer in podocytes): Mg2+ ions displace K+ from the epithelial cells to the intercellular space (increase of total conductance) and then to the external medium: the competitive inhibition effect of Mg2+ ions on the microvilli hinders the re-entry of K+ from the external medium to the epithelial cells: the K+ concentration decreases in EL.
A decrease of Cl- concentration is observed in EL and CL after addition of MgCl2 in Hanks' solution. Previous electrophysilogical studies3 have indicated that Mg2+ ions induces an opening of the Cl- channels and an effect on the ionic exchangers16, Cl/HCO3 particularly. This antiport system is associated with Na/H which has been activated by the leak of Na+ ions from the epithelial cells. The decrease by the Cl- concentration in EL induces an intervention of CL, which acts as a buffer and stocks the ions. CL participates in the regulation of the internal medium and Cl- ions enter the EL from the CL. In this case, the concentration of Cl- ions in the CL decreases. Then, the Cl- concntration in the EL increases and Mg2+ ions again remove Cl- ions. Consequently, the Cl- concentration is decreased in both EL and CL.
Nuclear microanalysis processing provides information on the distribution of monovalent ions in the epithelial and compact layers of the human amniotic membrane and on the importance of the compact layer. The variation of the ion concentrations after addition of Mg2+ ions may be explained by the electrophysiological data. A correlation has been shown between two techniques of studying the effects of Mg2+ ions on the transamniotic ionic transfer.
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M. Bara, A. Guiet-Bara, P. Moretto, L. Razafindrabe, Y. Llabador, M. Simonoff et J. Durlach (Paris, Bordeaux-Gradignan, France)
Résumé: L'effet de l'addition de MgCl2 sur la concentration en Na+, K+, Cl- dans les couches épithéliale et compacte de la membrane amniotique humaine isolée a été étudie en utilisant la microsonde nucléaire de Bordeaux. Les techniques PIXE et RBS ont été utilisées pour obtenir des mesures quantitatives. Dans un milieu physiologique (solution de Hanks), la concentration des ions monovalents est identique dans les deux couches. L'addition des ions Mg2+ dans la solution de Hanks entraine une diminution de la concentration en Na+, K+, Cl dans les deux couches, mise a part la concentration de Na+ dans la couche épithéliale qui reste constante. Les résultats obtenus avec la microanalyse nucleaire sont expliqués à partir des données fournies par les études életrophysiologiques qui montrent que l'addition de MgCl2 induit une augmentation des échanges cellulaires et paracellulaires des ions monovalents.
Mots clés: Chlore, échanges ioniques, magnésium, membrane amniotique humaine, microanalyse nucléaire, potassium, sodium.
Address for correspondence: Dr. Micheal Bara, Physiopathology of Development Laboratory, Cellular Interactions Group, University P.M. Curie, 4 Place Jussieu, 75252, Paris, Cedex 05, France. Tel: +33-1-44-27-35-06 Fax: +33-1-44-07-15-85.
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