Search PubMed⌕ Search

Biomedical subjects

M Cereijido

Publications and source records attributed to M Cereijido.

At least 55 records · Page 3Linked to original sources
PubMed1984

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

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 degrees 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 degrees C), and the activation energies calculated (3.2 and 4.0 kcal X mol-1) fall within the expected values for processes of simple diffusion. The morphology of the occluding junction was 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 face E and face P, 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.

Animals↗
PubMed1983

Electrical properties of cultured epithelioid cells (MDCK).

This is a study of the intracellular electrical potential, membrane resistance, and capacity of MDCK cells (epithelioid of renal origin) cultured in monolayers on a collagen couch. These monolayers have a transepithelial resistance of 256 +/- 12 (22) ohm cm2 (mean +/- standard error, and number of observations), and the cells have 61.6 +/- 6.3 (92) M omega across their plasma membrane. The electrical capacity of the cells is 45.1 +/- 2.9 (63) pF and is much higher than expected for a cell of its size (diameter 14 micrometers, height 5 micrometers) and cannot be attributed to intercellular coupling, as no evidence of this type of connection was found in 20 pairs of neighboring cells. On the contrary, the high capacity is in keeping with previous studies using electron microscopy showing microvilli and a high degree of lateral infolding. The relationship between resistance and capacity was 1981 +/- 177 (61) omega . microF. The cells have an intracellular potential of -40.5 +/- 15 (120) mV. Yet the shape of the distribution curve suggests that the actual value may be somewhat higher (some -50 mV). The current/voltage curve of the distribution curve suggests that the actual value may be somewhat higher (some -50 mV). The current/voltage curve shows a marked asymmetry, and in some cells the voltage becomes time-dependent for large, depolarizing current pulses.

Animals↗
PubMed1983

Occluding junctions and paracellular pathways studied in monolayers of MDCK cells.

MDCK cells (epithelioid, derived from the kidney of a normal dog) cultured in monolayers on a permeable support, exhibit properties of natural transporting epithelia. Comparisons of the electrical resistance across the plasma membrane of MDCK cells (as studied with microelectrodes) and the resistance across the whole monolayer, (mounted as a flat sheet between two chambers) indicate that most of the current flows through an extracellular pathway. Scanning of the electrical field over the apical surface shows that this pathway is located at the intercellular space. Yet conductance is not evenly distributed along the intercellular space as in leaky epithelia, but is restricted to sites scattered irregularly along the intercellular space. Studies of freeze fracture electron microscopy indicate that the number of strands of the junctions is also distributed irregularly, varying from 1 to 10 in a few nanometers. This suggests that regions with few strands would correspond to spots with high conductance and vice versa. However, in this preparation the sealing property of the junction bears little relationship to its structure. Thus by changing the temperature from 37 to 3 degrees C and back, the electrical resistance increases reversibly by 306%, while the number and arrangement of the strands show no significant modification. The resistance of the monolayer varies also with the age of the cells, suggesting that sealing and ion-permeating components of the junction may be dynamic entities that are not permanently installed, but can be accommodated to the requirements of the tissue.

Animals↗
PubMed1982

Occluding junctions in MDCK cells: modulation of transepithelial permeability by the cytoskeleton.

In MDCK cell monolayers the opening and resealing of occluding junctions can be induced by removal and restoration of calcium to the external medium. The overall changes in permeability of the occluding junctions in the monolayer can be monitored by the drop and recovery of the total transepithelial electrical resistance. We have investigated the effects of cytochalasin B (CB) on this process. When CB is added to sealed monolayers there is a gradual drop in the electrical resistance across the monolayer. This drop is accompanied by a slow disorganization of the microfilament pattern of these cells, including a disturbance of a ring of cortical microfilaments that is normally associated with the junctions. Cells in open monolayers treated with CB will not reseal and have an altered filament distribution. These cells do not have a continuous cortical ring. We have used a voltage scanning technique that uses a microelectrode to measure the resistance at selected points along the junction which surrounds a single cell. In untreated, closed monolayers, the junction is heterogeneous with alternating points of high and low conductance. In closed monolayers treated with CB, although there are low conductance points, we have observed an increased frequency of high conductance points that correlates with the change in the overall conductance. The frequency of high conductance points along the junction and the overall conductance both increase with time of exposure to CB. In an effort to understand the molecular basis for the permeability changes induced by EGTA and CB, we have looked for differences in the protein components of the cell membranes of open, closed, and CB-treated MDCK monolayers. This was done by radioiodinating the surface membrane proteins under control and experimental conditions that bring about permeability changes. No significant differences in the labeled protein patterns were found under these conditions. These results suggest that the permeability changes involve only a structural rearrangement of membrane components. In additions we have observed that about 36% of the surface label remains bound to the insoluble cytoskeletons obtained from cells in control and experimental conditions that alter the permeability of the tight junctions. The iodinated proteins attached to the CS include polypeptides with Mr of greater than or equal to 120K daltons as well as peptides with Mr = 56K, 50K, 36K, and 18K daltons.

Animals↗
PubMed1982

Occluding junctions of the Necturus gallbladder.

The paracellular conducting pathway of the Necturus gallbladder was studied with electrophysiological and electromicroscopic methods. The first one consists of the passage of short (5 msec) and small (32 microA 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 micron 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.

Animals↗
PubMed1981

Fluxes, junctions, and blisters in cultured monolayers of epithelioid cells (MDCK).

The formation of blisters is a transient phenomenon that led Dr. Leighton to describe its observation with time-lapse photography in the MDCK monolayer to a "gently boiling oatmeal." One may ask why is it transient and why most areas are momentarily not blistering. The observations discussed in this article indicate that (a) at the blister, junctions are really tight; (b) when transported fluid is allowed to escape through a permeable support, junctions are also tight, but (c) when the support is impermeable the junctions allow ouabain and peroxidase through. This suggests that, if the attachment of the monolayer is strong enough, the accumulation of fluid (Figure 7) bursts the junctions. If, on the contrary, junctions withstand the pressure, factor 3 of Figure 7 prevails over the others, and a blister is formed. In all the rest of the monolayer junctions seem to be open. This is in keeping with the observation by Rabito et al.,27 that if the monolayers are prepared on weakened supports, blisters are much bigger than under control conditions. It also allows the measurement of ionic fluxes and labeling of pumping sites at the basolateral membrane. As a corollary, one may say that some of the factors known to affect blistering 26 may very well act through modifications in the occluding junctions.

Animals↗
PubMed1981

Occluding junctions in cultured epithelial monolayers.

When MDCK cells are cultured in monolayers, they synthesize, assemble, and seal occluding junctions that limit the paracellular route. These processes may be impaired by inhibitors of the protein synthesis but not by inhibitors of the synthesis of RNA. Once established, the occluding junctions confer to the monolayer an overall electrical resistance of 80-600 omega . cm2. At the microscopical level, the resistance of individual junctions have large variations along the perimeter of a given cell. This agrees with the images of freeze-fracture electron microscopy where the network of the junction varies abruptly from 1 to 10 strands. The junctions are impermeable to macromolecular tracers, have a 9 to 1 Na+/Cl- discrimination, and a cation selectivity following the order: K+ greater than Na+ greater than Rb+ greater than Cs+ greater than Li+. Sealing requires extracellular Ca2+, but the junctions open when the concentration of Ca2+ in the cytoplasm increases. The structural components of the cytoskeleton (microtubules and microfilaments) seem to be involved in the junctional events as revealed by staining with immunofluorescent specific antibodies. If the cells are treated with cytochalasin B, actin microfilaments disorganize, the junctions open, and the electrical resistance across the monolayers falls. The resealing of the tight junction is inhibited by this drug.

Animals↗
PubMed1980

Occluding junctions in a cultured transporting epithelium: structural and functional heterogeneity.

MDCK cells (epithelioid of renal origin) form monolayers which are structurally and functionally similar to transporting epithelia. One of these similarities is the ability to form occluding junctions and act as permeability barriers. This article studies the junctions of MDCK monolayers formed on a permeable and transparent support (a disk of nylon cloth coated with collagen) by combining two different approaches: (i) Scanning of the electric field: the disk is mounted as a flat sheet between two Lucite chambers and pulses of 20--50 microA cm-2 are passed across. The apical surface of the monolayer is then scanned with a microelectrode to detect those points where the current is flowing. This shows that the occluding junctions of this preparation are not homogeneous, but contain long segments of high resistance, intercalated with sites of high conductance. (ii) Freeze fracture electron microscopy: the junctions are composed of regions of eight to ten strands intercalated with others where the strands are reduced to one or two ridges. The sites of high conductance may correspond to those segments where the number of junctional strands is reduced to 1 or 2. It is concluded that the occluding junctions of MDCK monolayers are functionally and morphologically heterogeneous, with "tight" regions intermixed with "leaky" ones.

Animals↗
PubMed1980

Structural and functional membrane polarity in cultured monolayers of MDCK cells.

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 mumol 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 with 3H-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.

Amiloride↗
PubMed1980

Experimental modulation of occluding junctions in a cultured transporting epithelium.

The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy. As in natural epithelia, the function of occluding junctions as permeability barriers specifically depends on extracellular Ca++ concentration and fails if this ion is replaced by Mg++ or Ba++. The removal of Ca++ and the addition of EGTA to the bathing medium opened the junctions and reduced the transepithelial resistance. Resealing was achieved within 10-15 min by restoring Ca++. Quantitative freeze-fracture electron microscopy showed that junctional opening, caused by lack of Ca++, was accompanied by simplification of the pattern of the membrane strands of the occluding junction without disassembly or displacement of the junctional components. Resealing of the cellular contacts involved the gradual return to a normal junctional pattern estimated as the average number of strands constituting the junction. The occluding junctions were also opened by the addition of the ionophore A23187, suggesting that the sealing of the contacts requires high Ca++ on the extracellular side and low Ca++ concentration of the cytoplasmic compartment. The opening process could be blocked by low temperature (7.5 degrees C). Resealing did not depend on serum factors and did not require protein synthesis; therefore, it seems to be caused by reassembly of preexisting membrane junctional components. The restoration of the junctions occurred simultaneously with the establishment of ion-selective channels; the Na+/Cl- and the cation/cation selectivity were recovered with the same time-course as the electrical resistance. The role of the cytoskeleton in the process of junctional reassembly is reported in the companion article.

Animals↗
PubMed1980

Occluding junctions and cytoskeletal components in a cultured transporting epithelium.

MDCK cells form uninterrupted monolayers and make occluding junctions similar to those of natural epithelia. This aricle explores the relationship between these junctions and the cytoskeleton by combining studies on the distribution of microfilaments and microtubules with the effect of drugs, such as colchicines and cytochalasin B, on the degree of tightness of the occluding junctions. To study the degree of tightness, monolayers were prepared by plating MDCK cells on mylon disks coated with collagen. Disks were mounted as flat sheets between two Lucite chambers, and the sealing capacity of the junctions was evaluated by measuring the electrical resistance across the monolayers. Equivalent monolayers on coverslips were used to study the distribution of microtubules and microfilaments by indirect immunofluorescence staining with antibodies against tubulin and actin. This was done both on complete cells and on cytoskeleton preparations in which the cell membranes had been solubilized before fixation. Staining with antiactin shows a reticular pattern of very fine filaments that spread radially toward the periphery where they form a continuous cortical ring underlying the plasma membrane. Staining with antitubulin depicts fibers that extend radially to form a network that occupies the cytoplasm up to the edges of the cell. Colchicine causes a profound disruption of microtubules but only a 27 percent decrease in the electrical resistance of the resting monolayers. Cytochalasin B, when present for prolonged periods, disrupts the cytoplasmic microfilaments and abolishes the electrical resistance. The cortical ring of filaments remains in place but appears fragmented with time. We find that removal of extracellular Ca(++), which causes the tight junctions to open, also causes the microfilaments and microtubules to retract toward the center of the cells. The process of junction opening and fiber retraction is reversed by the restoration of Ca(++). Colchicine has no effect on either the opening or reversal processes, but cytochalasin B inhibits the resealing of the junctions by disorganizing the filaments in the ring and at the apical border of the cells. These cytochalasin B effects are fully reversible. The correlation among cell shape, cytoskeletal patterns, and electrical resistance in the EGTA-opened and resealed monolayers suggests that microfilaments, through their association with plasma membrane components, play a role in positioning the junctional strands and influence the degree of sealing of the occluding junctions.

Animals↗
PubMed1978

Studies on chloride permeability of the skin of Leptodactylus ocellatus: I. Na+ and Cl- effect on passive movements of Cl-.

The outflux of chloride through the isolated skin (JCl31) of the South American frog Leptodactylus ocellatus (L.) is carried by a mechanism that saturates at high concentration of chloride on the inside, and is stimulated by the presence of Cl- in the outer solution (trans side). The presence of Na+ on the outside, by itself, does not increase JCl31. However, when JCl31 is already increased by chloride on the trans side, the addition of Na+ produces a significant further increase. At low concentration of Cl- on the outside JCl31 is carried by an exchange diffusion mechanism. At high concentrations of Cl- outside, JCl31 proceeds through a route which involves changes in electrical parameters. The results suggest that both mechanisms are located on the cell membranes and, therefore, that the fluxes would cross through the cytoplasm of the cells. Na+ stimulates the second mechanism only.

Animals↗
PubMed1978

Studies on chloride permeability of the skin of Leptodactylus ocellatus: II. Na+ and Cl- effect of inward movements of Cl-.

At low concentration (1 mM) of Cl- in the outer solution, the influx of chloride through the isolated skin (JCl13) of the South American frog Leptodactylus ocellatus (L.) seems to be carried by two mechanisms: (i) a passive one that exhibits the characteristics of an exchange diffusion process, and (ii) an active penetration. Studies of the influx and efflux of chloride (JCl13 and JCl31) indicate that the presence of a high (107 mM) concentration of Cl- in the outer solution activates the translocation of this ion through the cells. Studies of the unidirectional flux of Cl- across the outer barrier (JCl12) indicate that Na+ out stimulates the penetration of Cl- at this level. Cl- out, in turn, stimulates the JNa12, but this effect is only detected at low concentrations of Na+ out.

Animals↗
PubMed1978

Studies on chloride permeability of the skin of Leptodactylus ocellatus: III. Na+ and Cl- effect on electrical phenomena.

During their flux through the skin of the frog Leptodactylus ocellatus, Na+ and Cl- interact with each other. This interaction gives rise to electrical phenomena which are studied in the present paper. The skin is mounted in Na2SO4 Ringer's with 115 mM Na+ on the inside, and a variety of outer solutions. The osmolarity of all solutions is kept constant at 237.8 mosmol by adding sucrose. When the main anion used on the outside is SO=4 the electrical potential difference (deltapsi) rises steadily with the concentration of sodium (Na+)0 up to 87 mV, which is reached at about 20 mM. Thereafter deltapsi remains constant. When the main anion is Cl- it is observed that deltapsi rises steadily with (NaCl)0 with a slope similar to the curve obtained with SO=4 (37 mV per decade), but with a lower intercept attributed to an inward Cl pumping which is characteristic of this frog species. At 2--9 mM (NaCl)0 a Cl-specific channel is activated. Further increases of (NaCl)0 produce a decrease of deltapsi. The specificity of the activation of this site by monovalent cations and its use by monovalent anions is also studied.

Animals↗
PubMed1978

Amiloride and calcium effect on the outer barrier of the frog skin.

Amiloride (0.1 mM) as well as Ca++ (10 mM) inhibit Na+ transport across frog skin by blocking Na+ entrance across the outer barrier of the epithelium. The inhibition produced by amiloride consists of an "early" and a "late" phase which together account for almost a total inhibition of the short-circuit current (SCC). The analysis of the time course indicates that the two phases are due to the inhibition of superficially and deeply located Na sites, respectively, Ca++, instead, only blocks a fraction of the SCC, and this fraction seems to correspond to the inhibition of the same population of Na sites blocked by the "late" phase of amiloride effect. The location of the two populations of Na sites as well as the possible relationship between them are discussed in terms of maturation of the outermost cell layer.

Amiloride↗
PubMed1978

Polarized monolayers formed by epithelial cells on a permeable and translucent support.

An epithelial cell line (MDCK) was used to prepare monolayers which, in vitro, develop properties of transporting epithelia. Monolayers were formed by plating cells at high densities (10(6) cells/cm2) on collagen-coated nylon cloth disks to saturate the area available for attachment, thus avoiding the need for cell division. An electrical resistance developed within 4-6 h after plating and achieved a steady-state value of 104 +/- 1.8 omega-cm2 after 24 h. Mature monolayers were morphologically and functionally polarized. They contained junctional complexes composed of desmosomes and tight junctions with properties similar to those of "leaky" epithelia. Monolayers were capable of maintaining a spontaneous electrical potential sensitive to amiloride, produced a net water flux from the apical to basal side, and discriminated between Na+ and Cl- ions. The MDCK permeability barrier behaves as a "thin" membrane with negatively charged sites. It has: (a) a linear conductance/concentration relationship; (b) an asymmetric instantaneous current/voltage relationship; (c) a reduced ability to discriminate between Na+ and Cl- caused by lowering the pH; and (d) a characteristic pattern of ionic selectivity which suggests that the negatively charged sites are highly hydrates and of medium field strength. Measurements of Na+ permeability of electrical and tracer methods ruled out exchange diffusion as a mechanism for ion permeation and the lack of current saturation in the I/deltapsi curves does not support the involvement of carriers. The discrimination between Na+ and Cl- was severely but reversibly decreased at low pH, suggesting that Na+-specific channels which exclude Cl- contain acidic groups dissociated at neutral pH. Bound Ca++ ions are involved in maintaining the integrity of the junctions in MDCK monolayers as was shown by a reversible drop of resistance and opening of the junctions in Ca++-free medium containing EGTA. Several other epithelial cell lines are capable of developing a significant resistance under the conditions used to obtain MDCK monolayers.

Animals↗
PubMed1975

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.

Abdomen↗