Effect of temperature on the occluding junctions of monolayers of epithelioid cells (MDCK)

Summary In previous works it was demonstrated that the monolayer of MDCK cells behaves as a leaky epithelium where the electrical resistance across reflects the sealing capacity of the occluding junction. In the present work we study whether this sealing capacity can be modified by temperature and whether this is accompanied by changes in the structure of the occluding junction. Monolayers were prepared on disks of nylon cloth coated with collagen and mounted as a flat sheet between two Lucite chambers. The changes in resistance elicited by temperature were large (306% between 3 and 37°C), fast (less than 2 sec), and reversible. An Arrhenius plot of conductance versus the inverse of temperature shows a broken curve (between 22 and 31°C), and the activation energies calculated (3.2 and 4.0 kcal·mol^–1) fall within the expected values for processes of simple diffusion. The morphology of the occuluding the number of evaluated in freeze-fracture replicas by counting the number of strands and the width of the band occupied by the junction every 133 nm. In spite of the change by 306% of the electrical resistance and the phase transition, we were unable to detect any appreciable modification of the morphology of the occluding junction. Since the freeze-fracture replicas also show a density of intramembrane particles (IMP) different in the apical from that in the basolateral regions of the plasma membrane, as well as differences between faceE and faceP, we also investigated whether this is modified by temperature. Cold increases the population of IMP, but does not affect their polarization with the incubation time it takes to elicit changes in electrical resistance.     

The permeability of the membranes of experimental secondary cysts of Echinococcus granulosus to [14C]mebendazole

The permeability of secondary E. granulosus cysts to [¹⁴C]mebendazole was studied. The cysts were obtained by transplanting secondary cysts raised in mice into rats. The permeability to [¹⁴C]mebendazole was established by two different experiments: uptake and washout of the drug. The cyst wall permeability to [¹⁴C]mebendazole was found to be 1·33 × 10^?4 cm s^?1, which is of the same order as the diffusion permeability coefficient to water (1·88 × 10^?4 cm s^?1, Rotunno, Kammerer, Perez Esandi & Cereijido, 1974).The drug readily permeates through the cyst wall and experimental data suggest that it moves across the barrier by simple diffusion.     

Ionic fluxes in isolated epithelial cells of the abdominal skin of the frog Leptodactylus ocellatus.

Unidirectional ion fluxes are measured in cells isolated by a trypsination-dissection method from the epithelium of the frog Leptodactylus ocellatus. Potassium seems to be contained in a single cellular compartment. The influx of potassium is 0.0068 mumole min-1 mg-1 of dry weight and is carried by a ouabain-sensitive pump. Sodium seems to be contained in two cellular compartments, one of which does not exchange its Na within the experimental period. The possibility that these compartments reflect the existence of different types of cells is not discarded. 49% of the rate constant for the Na efflux is ouabain-sensitive and 23% is ethacrynic-sensitive. Under control conditions the permeability to potassium (PK), sodium (PNa) and chloride (PC1) are 7.6 X 10(-5), 2.6 X 10(-5) and 2.8 X 10(-5) liters/min mg, respectively. The value of PNa is much higher than predicted by current electrical models of the epithelium. The discrepancy might offer some insight into the nature of the "inner facing barrier" of the skin.     

Occluding junctions of theNecturus gallbladder

Summary The paracellular conducting pathway of theNecturus gallbladder was studied with electrophysiological and electromicroscopic methods. The first one consists of the passage of short (5 msec) and small (32 A cm^–2) current pulses associated with a voltage scanning of the plane of the epithelium at the apical surface with a microelectrode to detect the regions where current flows. The procedure shows that (a) the conductance is evenly distributed along the intercellular regions along the intercellular spaces of the cells where occluding junctions are located; (b) the field above the occluding junctions has the shape of a bell, so that the junction can be sensed at 1–2 m from the region where the intercellular space is visualized by light microscopy; (c) the intersections between three cells, in spite of having 3 half-junctions contributing (instead of two), do not have a higher conductance than the rest of the occluding junction. Scanning electron microscopy shows that (a) cells are densely covered by microvilli which interdigitate above the region of the occluding junctions, and (b) are covered by a surface coat. With transmission electron microscopy, (a) the opening of the occluding junctions at the apical border appears irregular, and most of them oblique; (b) in the last microns the actual mouth of the junction may deviate from the course of the interspace. Freeze-fracture replicas indicate that (a) the occluding junction has a uniform width and little variations in the number of strands around the cell, except (b) at intersections between 3 cells where both, its width and the number of strands, increase toward the basal region.     

The penetration of sodium into the epithelium of the frog skin

The aim of this paper is twofold. First, to describe a method for the measurement of the unidirectional flux of Na from the outer bathing solution into epithelium (J_OT), and second, to describe the use of this method under a variety of experimental conditions in order to obtain some insight into the nature of this flux. The method developed is based on the exposure of a frog skin to a Ringer solution containing ²²Na. The exposure is made so that neighboring points along the surface remain in contact with the ²²Na solution for gradually longer periods, ranging from 0 to 46 sec. Some 8 to 10 samples of the exposed part are used to obtain the time course of the uptake of ²²Na and this time course is used, in turn, to evaluate J_OT. This flux is then studied in skins mounted between two identical Ringer solutions with 115 mM Na (11.25 ± 0.10 [18] µmole·hr^-2 cm^-2), and in skins mounted with Ringer with 1 mM Na on the outside and 115 mM Na on the inside (0.43 ± 0.05 [18] µmole·hr^-1·cm^-2. From the observations that the flux is much larger than the net Na flux across the whole skin, that it is inhibited by K⁺, and is unaffected by ouabain, it is concluded that the penetration of Na⁺ into the epithelium does not occur by simple diffusion and is not directly dependent on an ouabain-sensitive mechanism. In the course of these experiments it was observed that when the skin was crushed between two chambers the uptake of Na in the neighboring exposed areas was decreased.     

Intracellular Electrical Potentials in Frog Skin

The influence of changes in ionic composition of the bathing solutions on intracellular electrical potentials in frog skin has been examined. When the skin bathed in SO₄ Ringer''s solution is penetrated with a microelectrode two approximately equal potential jumps were frequently observed and most experiments were carried out with the electrode located between these steps. Substitution of Cl for SO₄ in the bathing solutions caused a decrease in PD across both the "outer" and "inner" barriers. When the skin was short-circuited an average intracellular potential of -18 mv was found with both Cl and SO₄ Ringer''s. With the skin in SO4 Ringer''s, decrease in Na concentration of the outside solution caused a decrease in PD between the microelectrode and the outside solution which was approximately the same as the decrease in total skin PD. With SO₄ Ringer''s, an increase in K concentration in the inside solution caused a marked decrease in total skin PD. However, only 50 per cent of this change occurred at the inner barrier, between the microelectrode and the inside solution. The remainder of the change occurred at the outer barrier. This observation does not appear to be consistent with the model of the skin proposed by Koefoed-Johnson and Ussing (Acta Physiol. Scand., 1958, 42, 298).     

Structural and functional membrane polarity in cultured monolayers of MDCK cells

Summary MDCK cells form monolayers which have many of the properties usually found in transporting epithelia. The present article is devoted to the study of the structural and functional polarization of MDCK cells, which is one of the central features of transporting epithelia. The results show: (i) that MDCK monolayers transport 2.6 mol hr^–1 cm^–2 of sodium in the apical to basolateral direction; (ii) the passive flux of this ion is relatively large (20.3 mole hr^–1 cm^–2), which is a characteristic of leaky epithelia; (iii) a large fraction of the penetration of sodium into the cells proceeds through an amiloride-sensitive channel, and the exit is operated mainly by a ouabain-sensitive pump; (iv) the net transport of sodium from the apical to the basolateral side agrees with the asymmetric labeling of the pumps with³H-ouabain; (v) this asymmetric labeling agrees, in turn, with a higher concentration of intramembrane particles (IMPs) in freeze-fracture replicas of the basolateral side of the plasma membrane; (vi) the structural polarization of confluent MDCK cells is also revealed by the location of microvilli, occluding junctions, and pinocytotic vesicles; and (vii) the presence of a continuous ring formed by actin microfilaments visualized by immunofluorescence under the lateral aspect of the plasma membrane that may be related to the distribution of the occluding junctions, which act as barriers separating apical from basolateral membrane components.     

The state of water in the outer barrier of the isolated frog skin

Summary The flux of water across the outer barrier of the frog skin is generally regarded as the rate-limiting step in the movement of water across the whole membrane. This paper presents some evidence that, at room temperature, the flux of water across the outer barrier occurs through water in a non-liquid state. The organization of water in a non-liquid state lowers the diffusion coefficient of water through water by several orders of magnitude. The study employs a method recently developed in this laboratory which permits measurement of unidirectional fluxes at the outermost part of an epithelial membrane mounted as a flat sheet. Only above 25°C is the activation energy for the flow of tritiated water (4.3 kcal mole⁻¹) similar to the one observed in free water (4.6 kcal mole⁻¹). At temperatures around 15°C, the energy of activation is 8.5 kcal mole⁻¹. At temperatures near 0°C, at which the frog lives only part of the year, the energy of activation is 16.7 kcal mole⁻¹.