833 — Temperature effect on ion transport through the isolated human amnion☆Part I. Electrophysiological studies

In vitro, electrolyte transfer through the human amniotic membrane depends on the temperature of the external solutions. The transepithelial conductance (Gt) is measured with the amnion mounted between two Ussing chambers and the membrane potential

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  383 Bioelectrochemistry and Bioenergetics, 15 1986) 383-394 A section of J. Electroanul. Chem, and constituting Vol. 211 (1986) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ 833 - TEMPERATURE EFFEm ON ION TRANSPORT THROUGH THE ISOLATED HUMAN AMNION PART I. ELECTROPHYSIOLOGICAL STUDIES MICHEL BARA and ANDREE GUIET-BARA Laboratory of Biology of Reproduction, University P.M. Curie, 7 puai Saint-Bernard 75230 Paris Cedex 05 France) (Revised manuscript received October 9th 1985) SUMMARY In vitro, electrolyte transfer through the human amniotic membrane depends on the temperature of the external solutions. The transepithelial conductance (G,) is measured with the amnion mounted between two Ussing chambers and the membrane potential (I&,) and the input resistance Rim) of epithelial-cell membranes are recorded by intracellular microelectrodes. When the temperature increases, G, and R;, increase and the membrane is hyperpolarized. Thus, the paracellular conductance increases and the transcellular conductance decreases. Arrhenius plots of the conductance over the temperature range of 4-4O’C yield apparent activation energies of 4-8 kcal/mole for amnion as a whole and 2-10 kcal/mole for epithelial amniotic-cell membrane. These values agree with the fact that the human amnion is a leaky epithelium. A step discontinuity in the Arrhenius plot with Ca2+ indicates a lipid phase transition near 30°C. Thus, the temperature acts upon’the amnion by changing the fluidity of the membrane lipid charges and the lipid microenvironment of the sites. INTRODUCTION zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Previous studies have indicated and characterized the ionic exchange properties of the human amnion by measuring the ionic conductance for different solutions [l]. The ion selectivity of human amnion is largely controlled by the presence of neutral and negative sites on the membrane of epithelial cells in contact with amniotic fluid and on the membrane of epithelial cells delimiting the intercellular channels [2]. Moreover, human amnion has the physicochemical characteristics of a porous or a sieve-like system [3]; it’s a leaky epithelium, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR .e. the paracellular pathway is more important than the transcellular pathway [4,5]. Presented, in part, at the Bioelectrochemistry Meeting held in Nottingham (Great Britain), September 1983. 0302-4598/86/ 03.50 0 1986 Blsevier Sequoia S.A.  384 To specify the concept of sites on the membrane, and to determine and gain insight into the physical parameters of amnion permeation, the effect of temperature on the transfer of monovalent cations across the isolated human amnion was studied. The apparent activation energies obtained from conductance measurements for permeation through the epithelial amniotic cells were calculated for different cations and for different amniotic areas. This study also intends to provide some indications on the in-uiuo transport of cations between mother and fetus across the amnion. EXPERIMENTAL Membrane preparation Strips of human amnion, isolated from the placental (PZ), ‘reflected (RZ) and umbilical (UpZ: near placenta, UfZ: near fetus) zones of the amniotic sac, were obtained after delivery or cesarean sections and immediately transferred to Hanks’ solution at room temperature (N = 32 placentas were studied). The different areas were studied using two methods: (a) A circular area of amnion (2 cm2) was sampled and mounted between two Ussing chambers filled with saline solutions. To improve mixing and minimize the thickness of the unstirred layers next to the epithelium, the saline solution in each chamber was vigorously agitated by magnetically driven stirring bars or by gas bubbling; (b) A circular area of amnion (1 cm2) was mounted horizontally between two Lucite chambers according to Bara and Guiet-Bara’s device [4]. Solutions Hanks’ solution used contained (mM/dm3): NaCl 150, KC1 6, MgSO, 0.5, MgCl, 0.5, CaCl, 1, glucose 5.5 and was buffered to pH 7.4 with 1 mM NaH,PO,, KH,PO,, NaHCO,. Saline solutions (NaCl, KCl, RbCl, CsCl, LiCll50 mM + 0.25 mM CaCl, and zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDC aCl zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB   mM + 5 mM CaCl, + 5.5 mM glucose) were buffered to pH 7.4 with 1.5 mM Tris. -The different osmolarities and the different composition of the medium had no effect on the structure of the amnion [6,7]. Experimental procedures The saline solutions were recirculated through the chambers and the reservoirs by gas lifts and the temperature of the solutions was controlled by circulating water through the outer jackets of the reservoirs. Constant-temperature circulators were used to control the temperature and pump the water through the water jackets. The temperature of the saline solutions, changed from 4 to 40°C was monitored continuously using a Nododirect telethermometer with a calibrated hypodermic probe.  385 Two experimental procedures were followed: (a) The membranes were either incubated for 10 min at the temperatures studied (4, 10, 15, 25, 30, 37 and 40°C) in Hanks’ solution or saline solutions and the electrical parameters were measured; or (b) the electrical parameters were measured continuously when the temperature was varied between 4 and 40°C (2 degrees per 5 min). The results obtained with the two procedures were significantly identical. Electrical parameters (a) The transamniotic conductance (G,) was measured by observing the transepi- thelial potential difference when a direct current (100 PA) was passed across the whole tissue in the mother-to-fetus direction and back. The potential was recorded with two agar-agar salt bridges (Bactoagar “Difco” 014002 + KC1 3 M) placed 1.3-1.5 mm from each side of the tissue, while current was passed across the tissue by means of Ag ] AgCl electrodes and agar salt bridges and was recorded on a Schlumberger electrometer. The variation of temperature, when the electrodes were in the same bath, does not produce a significant change in residual potential in the electrodes themselves (0.9 + 0.5 mM at 4°C and 1.5 f 0.7 mV at 40°C). (b) Membrane potentials (U,, on the maternal side and U,, on the fetal side) were measured with intracellular glass microelectrodes filled with KC1 3 M inserted into the epithelium under visual control using a binocular microscope and a micromanipulator. The membrane potential, between the microelectrode tip and an Ag ]AgCl indifferent electrode in the bath, was measured with a high-input imped- ance amplifier (Pica-metric amplifier 181, Instr. Lab. Inc.) and an oscilloscope (Tektronix 502A). The input resistance (R,) was measured by injecting a constant- current, square-wave pulse through a double-barrelled microelectrode and analysing the recorded voltage transient. (c) A second microelectrode was inserted into another cell of the same strand and located at a distance varying between 100 and 500 microns away from the site of current injection. Measurements of the space constant (X, in mm) were made by recording the amplitude of the electrotonic potentials generated by a constant pulse of current at different distances from the polarizing electrode. The membrane resistance per unit length (rm) and the intracellular longitudinal resistance per unit length (ri) were given by r, = 2 R,, h and ri = 2 R;,,/h [8]. The electrical coupling factor was calculated by the ratio U,,,/U,,, or U,,/U,,, (membrane potentials in cell 1 and in cell 2 after the passage of electrical current in cell 1). (d) The apparent conductance activation energy E,) for permeation through the epithelium was obtained by the Arrhenius equation: 2.3 log,,G’ Go++-) where G/ = transamniotic conductance at absolute temperature T’  386 G 7' = Transamniotic conductance at absolute temperature T R = gas constant E a = apparent conductance activation energy (kcal/mol) (e) All errors were reported as standard errors of the mean and p was the level of significance. RESULTS The effect of temperature on G Temperature coefficients, expressed by apparent conductance activation energies E a in kcal/mol), were given by Arrhenius curves. Figure 1 shows G,, measured in the fetus (F)-to-mother (M) direction, plotted against 1/T, where the slope is the calculated apparent conductance activation energy for each saline solution. There was a high and significant correlation between G and T (0.78 < r < 0.95, p < 0.01). The different values of E a for each zone of the amnion are given in Table 1. A sharp 10 A---~ EdH÷: 4.9 kcal/Mol o~o £aK÷ 6.5 .... o---~_¢ aN;: 7.2 ,, ~, EaLi*. 7.9 ,, . ~c,~ ~ ~ EaNa*, 5m,¥ Cat+: (1)=2.2 kcal/Mol x~, ±~,..,.,~ (2)=1.6 ,, ,, \x \ \ \\ 40 4 T('C) 0 312 3.13 31.4 315 3.6 10 (T- ) Fig. 1. Arrhenius plots of human-amniotic-membrane (PZ) conductance in the temperature range 4-40°C in various solutions.  387 break in the Arrhenius plot was noted when 5 mM CaCl, were added to NaCl solutions. The break occurs at 30°C, giving two significant apparent activation energies (Table 1). When the fetal-to-mother G, was measured, statistical analysis showed that the E, sequence for the different zones was: RZ = PZ > UpZ > UfZ. When the mother- to-fetus G, was measured, the results indicated a similar trend, although statistical analysis showed the E, sequence to be: RZ > UpZ > PZ > UfZ and confirmed definitely that T, (F-to-M) was lower than G, (M-to-F). The break with Ca2’ was less pronounced and was significant with PZ, Rz and UpZ. Reproducibility of the results was good when the temperature increased (from 4 to 40°C) or decreased (from 40 to 4°C) with a different response latency. The effect of temperature on electrical parameters of epithelial-cell membrane Membrane potential u zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA M Or u,F were measured when the temperature of the saline solutions was varied between 4 and 40°C at a rate of 2°C per 5 min. The results are shown in Fig. 2. The development of U,,, (identical for V,,) for the 4 zones shows a similar trend: a minimum between 31 and 33°C and two maxima, between 4 and 18’C and 38 and 4O’C. There is a relationship between U,, and temperature: it is a curvilinear regression treated by a variable conversion, i.e.: z = log temperature and X= U mM. The correlation coefficients are: -0.78 for PZ, -0.75 for RZ, - 0.75 for UpZ and - 0.76 for UfZ (P < 0.01). These coefficients indicate a high and signifi- cant correlation between U,,, and the temperature. The range between 25 and 35°C was investigated more extensively by varying the temperature at a rate of 1°C per 2 min: there was a minimum between 31 and 33°C (Fig. 3). The range between 31 and 40°C was investigated at the same rate to verify whether the U,, increase was due to an effect of temperature or to an effect of duration of the mounting. Figure 4 and Table 2 indicate that U,, increases with temperature and that the highly S-shaped curve in Fig. 2 becomes linear in Fig. 3 because there was a more rapid warming up. Regression analysis of the data obtained between 30 and 40°C gives a linear plot (regression coefficient r = 0.91 for RZ and PZ, 0.88 for UpZ and 0.86 for UfZ, p < 0.01) with a slope of 1.24 mV/“C for PZ, 1.05 mV/“C for RZ, 0.82 mV/“C for UpZ and 0.63 mV/OC for UfZ. Input resistance Ri,) Fig. 2) Ri, did not vary between 4 and 30°C, but from 30 to 40°C it increased by 74% for PZ, 13% for RZ, 35% for UpZ and 27% for UfZ (Table 2). Apparent activation energies (kcal/mole) were given by Arrhenius curves: 9.6 + 0.3 for PZ; 2.3 + 0.1 for RZ, 5.3 + 0.3 for UpZ and 4.3 f 0.2 for UfZ. * For the criteria used to obtain acceptable results cf. Ref. 21.
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