Biomedical subjects

M Cereijido

Publications and source records attributed to M Cereijido.

At least 19 recordsLinked to original sources

PubMed1992

Interaction of calcium with plasma membrane of epithelial (MDCK) cells during junction formation.

We have previously shown that upon transferring confluent monolayers of Madin-Darby canine kidney (MDCK) cells from low- to normal-Ca2+ medium, cytosolic Ca2+ increases and tight junctions (TJs) assemble and seal, but the increase in cytosolic Ca2+ does not seem to be necessary for junction formation. In the present work we establish that these are in fact two independent phenomena. We first measured unidirectional Ca2+ fluxes across the plasma membrane of MDCK cells to find suitable inhibitors and tested their effects on the ability of Ca2+ to seal the TJ. Likewise, we studied a variety of multivalent cations. We observed that 1) Ca2+ triggering of junction formation does not depend on its entering the cell, 2) cations like La3+ may impair the influx of Ca2+ without affecting the sealing of TJs, and 3) only Cd2+ is able to block both Ca2+ penetration and junction formation; however, 4) Cd2+ itself cannot trigger junction formation. We interpret that Ca2+ triggers junction formation by acting mainly on an extracellular membrane site and that this site has a higher Ca2+ selectivity than the mechanisms for Ca2+ translocation across the membrane.

Animals↗

PubMed1991

Osmolarity-sensitive release of free amino acids from cultured kidney cells (MDCK).

The amino acid pool of MDCK cells was essentially constituted by alanine, glycine, glutamic acid, serine, taurine, lysine, beta-alanine and glutamine. Upon reductions in osmolarity, free amino acids were rapidly mobilized. In 50% hyposmotic solutions, the intracellular content of free amino acids decreased from 69 to 25 mM. Glutamic acid, taurine and beta-alanine were the most sensitive to hyposmolarity, followed by glycine, alanine and serine, whereas isoleucine, phenylalanine and valine were only weakly reactive. The properties of this osmolarity-sensitive release of amino acids were examined using 3H-taurine. Decreasing osmolarity to 85, 75 or 50% increased taurine efflux from 0.6% per min to 1.6, 3.5 and 5.06 per min, respectively. The time course of 3H-taurine release closely follows that of the regulatory volume decrease in MDCK cells. Taurine release was unaffected by removal of Na+, Cl- or Ca2+, or by treating cells with colchicine or cytochalasin. It was temperature dependent and decreased at low pH. Taurine release was unaffected by bumetanide (an inhibitor of the Na+/K+/2Cl- carrier); it was inhibited 16 and 67 by TEA and quinidine (inhibitors of K+ conductances), unaffected by gadolinium or diphenylamine-2-carboxylate (inhibitors of Cl- channels) and inhibited 50% by DIDS. The inhibitory effects of DIDS and quinidine were additive. Quinidine but not DIDS inhibited taurine uptake by MDCK cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1991

Assembly and sealing of tight junctions: possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin.

The making and sealing of a tight junction (TJ) requires cell-cell contacts and Ca2+, and can be gauged through the development of transepithelial electrical resistance (TER) and the accumulation of ZO-1 peptide at the cell borders. We observe that pertussis toxin increases TER, while AIF3 and carbamil choline (carbachol) inhibit it, and 5-guanylylimidodiphosphate (GTPTs) blocks the development of a cell border pattern of ZO-1, suggesting that G-proteins are involved. Phospholipase C (PLC) and protein kinase C (PKC) probably participate in these processes since (i) activation of PLC by thyrotropin-1 releasing hormone increases TER, and its inhibition by neomycin blocks the development of this resistance; (ii) 1,2-dioctanoylglycerol, an activator of PKC, stimulates TER development, while polymyxin B and 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine dihydrochloride (H7), which inhibit this enzyme, abolish TER. Addition of 3-isobutyl-1-methyl-xanthine, dB-cAMP or forskolin do not enhance the value of TER, but have just the opposite effect. Trifluoperazine and calmidazoline inhibit TER development, suggesting that calmodulin (CaM) also plays a role in junction formation. These results indicate that junction formation may be controlled by a network of reactions where G-proteins, phospholipase C, adenylate cyclase, protein kinase C and CaM are involved.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

Role of calcium in tight junction formation between epithelial cells.

Upon transferring confluent monolayers of Madin-Darby canine kidney (MDCK) cells from a low-Ca2+ medium (1-5 microM) to one with 1.8 mM Ca2+ (Ca switch), tight junctions (TJs) assemble and seal, and transepithelial electrical resistance (TER) develops in 4-5 h, presumably through exocytotic fusion that incorporates junctional components to the surface membrane. In the present work we test this possibility and observe 1) that the Ca switch raises the cytosolic concentration of this ion; 2) that it also increases the membrane area by 22%; 3) that chloroquine, a drug which prevents exocytosis, blocks both the increase of surface membrane and the sealing of TJs; and 4) that if monolayers are not permanently switched to 1.8 mM Ca2+, but are subject to a 15-min pulse, cytosolic free Ca2+ concentration [( Ca2+]c) transiently increases but returns to low values (14 +/- 11 nM) and TER does not develop. Comparisons of the time course of TJ sealing with levels of [Ca2+]c, as well as the relationship between these parameters and extracellular Ca2+ levels, suggest that this ion may act from the extracellular side or in a narrow intracellular domain in the close vicinity of the plasma membrane.

Affinity Labels↗

PubMed1989

Establishment of tight junctions between cells from different animal species and different sealing capacities.

Epithelial cells establish tight junctions (TJs) that offer an ample range of transepithelial electrical resistances (TER), in adjustment to physiological requirements. In the present work, we demonstrate that cells from different animal origins, co-cultured in monolayers, can make sealed TJs, suggesting that this structure has a basic universal structure. TJs cannot be established, however, if one of the partners does not normally express TJs, indicating that each neighbor has to contribute its moiety. Furthermore, we observe that clones of the same cell line, with widely different values of TER, do not differ in the number and length of their junctional strands, suggesting that the difference is due to their ability to express ionic channels traversing their strands. The value of TER achieved in mixed monolayers of cells of the same or different lines is the one that may be expected by taking into account the proportion of each type in the mixture and adding in parallel the electrical resistance that they exhibit in pure monolayers. Therefore, epithelial TJs appear to behave as parallel resistances.

Animals↗

PubMed1989

Development and alteration of polarity.

Overall polarization of the plasma membrane of epithelial cells is the sum of the individual polarizations of its components. These individual polarizations in turn, may vary independently in degree (apical/basolateral ratio) and may be expressed at different stages of the cell cycle. They occur in response to cell contacts, nature of the support, and presence of triggering hormones; once established, polarizations may be subject to disruption and resorting. Epithelial cells transcytose receptors, insert membrane mechanisms during a particular period of the cell cycle, remove and relocate misplaced membrane components, and even completely reverse their polarity in the presence of well established TJs. TJs are not responsible for polarization but, ironically, they should be regarded as a result of the polarization process itself (31). The polarization of single cells, such as neurons and muscle cells mentioned at the beginning of this article, may represent extreme cases of cells that polarize but do not produce TJs. However, if an asymmetrically inserted protein is subsequently released from underlying anchoring structures (e.g. the cytoskeleton) to become free (e.g. 50% of Na,K-ATPase in MDCK cells), then the TJ may play a role in confining the free fraction to the apical or to the basolateral region. But even if TJs fail to completely segregate membrane components, mechanisms can restore polarization as in the case of the Na,K-ATPase trapped on the apical side. Lipid polarization seems to depend on the existence of the fence like character of the TJs and to the best of our knowledge lipid polarization is only found in epithelial cells with well established TJs.

Animals↗

PubMed1989

Repolarization of Na+-K+ pumps during establishment of epithelial monolayers.

Madin-Darby canine kidney (MDCK) cells plated at confluence and incubated for 20 h in low (5 microM) Ca2+ have no tight junctions (TJs), and their Na+-K+-ATPase is randomly distributed over the surface. On transfer to normal Ca2+ levels (1.8 mM) ("Ca2+ switch"), TJs and transepithelial resistance develop quickly, trapping a considerable fraction (35%) of the surface Na+-K+-ATPase on the apical (incorrect) side. This misplaced enzyme is subsequently removed from this region or inactivated, demonstrating that polarization proceeds despite TJs. Simultaneously, the amount of Na+-K+-ATPase on the basolateral side increases in a higher proportion (125%), than could be accounted for by relocation of the misplaced apical enzyme. This incorporation is prevented by cycloheximide, ammonium chloride, primaquine, or chloroquine, suggesting that Na+-K+-ATPase originates in an intracellular pool and that its surface insertion requires synthesis of new enzyme or of a protein factor, since it is carried to the surface membrane through a mechanism of exocytosis. In summary, asymmetric distribution of ion pumps depends 1) on polarized insertion of Na+-K+-ATPase as well as 2) on removal or inactivation of misplaced enzyme.

Animals↗

PubMed1988

Epithelial tight junctions.

Epithelial cells differentiate by polarizing into an apical and a basolateral domain and by forming tight junctions (TJ) that control permeation through the paracellular route. The molecular nature of this structure, as well as the processes of assembly, sealing, and regulation, are not yet fully understood. However, the use of epithelial cell lines cultured as monolayers is helping to elucidate the structure and function of this important cellular feature. Furthermore, the development of specific antibodies that interact directly with junctional components may help to solve the molecular structure of the TJ.

Animals↗

PubMed1987

Voltage and Ca2+-activated K+ channel in cultured epithelial cells (MDCK).

Patch-clamp techniques were used to study a K channel in the cell membrane of MDCK cells. This cell line derives from the kidney of a normal dog, presumably from the distal nephron, a region involved in potassium secretion. The cells were cultured in confluent monolayers and approached from the apical side. The K channel we describe is Ca2+ and voltage activated, has a conductance of 221 +/- 7 pS, and can be inhibited by 10 mM tetraethylammonium and by 1 mM quinidine, but not by 4-aminopyridine, nor by 1 mM Ba2+ added to the outer side. Using the whole-cell configuration, we find that most of the cationic conductance of the membrane is constituted by a K-specific one (maximum K conductance 32.1 +/- 3.9 nS vs. a leak conductance of 1.01 +/- 0.17 nS). Comparisons of the maximum K conductance with that of a single K channel indicates that an MDCK cell has an average of 145 such channels. The membrane capacity is 24.5 +/- 1.4 pF.

Animals↗

PubMed1987

Rescue of a wild-type MDCK cell by a ouabain-resistant mutant.

When wild-type MDCK cells (W-MDCK) were cocultured in mixed monolayers with a ouabain-resistant mutant (R-MDCK), the wild-type cells were protected from the effect of ouabain up to concentrations as high as 100 microM. Rescue depended on the dose of ouabain and on the proportion of each cell type in the coculture. The survival of R-MDCK cells at 1 microM ouabain was not endangered by varying from 1:9 to 9:1 the proportion of W-MDCK cells to be rescued. Ouabain binding revealed two kinds of binding sites in R-MDCK cells, one with high and the other with low affinity. Only the high affinity site was present in W-MDCK cells. Electron probe analysis of individual cells revealed that rescued cells kept a high K and a low Na intracellular contents, similar to control cells. Histograms of intracellular K/Na in cocultured cells treated with ouabain were unimodal. Using microinjection of Lucifer yellow or electrophysiological techniques we estimated that at most 13% of the R-MDCK and W-MDCK cells may be connected at a given time through cell-to-cell junctions. Therefore permanent cell-to-cell communication did not seem to play a central role in the rescue. W-MDCK cells cocultured with R-MDCK cells and subsequently separated, were not rescued. Thus rescue did not seem to depend on the transfer from R-MDCK to W-MDCK cells of either ouabain-resistant Na-K pumps or of information to synthesize them. It is speculated that intercellular communications were sporadic events, so that all cells may become intermittently connected and rescued.

Animals↗

PubMed1986

Microtubules and actin filaments are not critically involved in the biogenesis of epithelial cell surface polarity.

We have studied the role of microtubules and actin filaments in the biogenesis of epithelial cell surface polarity, using influenza hemagglutinin and vesicular stomatitis G protein as model apical and basolateral proteins in infected Madin-Darby canine kidney cells. Addition of colchicine or nocodazole to confluent monolayers at concentrations sufficient to completely disassemble microtubules did not affect the asymmetric budding of influenza or vesicular stomatitis virus and only slightly reduced the typical asymmetric surface distribution of their envelope proteins, despite extensive cytoplasmic redistribution of the Golgi apparatus. Alteration of microtubular function by taxol or dissociation of actin filaments by cytochalasin D also failed to have a significant effect. Furthermore, neither colchicine nor cytochalasin D pretreatment blocked the ability of subconfluent Madin-Darby canine kidney cells to sustain polarized budding of influenza virus a few hours after attachment to the substrate. Our results indicate that domain-specific microtubule or actin filament "tracks" are not responsible for the vectorial delivery of apically or basolaterally directed transport vesicles. In conjunction with currently available evidence, they are compatible with a model in which receptors in the cytoplasmic aspect of apical or basolateral regions provide vectoriality to the transport of vesicles carrying plasma membrane proteins to their final surface localization.

Actin Cytoskeleton↗

PubMed1985

A ouabain resistant epithelial cell that protects the wild type in co-cultures.

MDCK cells (epithelioid of renal origin) can be cultured as monolayers that resemble natural epithelia. A mutant of these cells (R-MDCK) can grow in cultures exposed to high doses of ouabain (e.g. 100 microM) because one half of its population of pumps has a negligible affinity for this drug. It can also protect the wild type for at least 86 h in co-cultures exposed to ouabain. This article reviews several possible mechanisms of protection, and suggests that it is due to exchange of Na+ and K+ taking place through gap junctions. These connections though, do not seem to be continuous features, but the cells couple intermittently so that, in a given moment, only 28% of them communicate.

Animals↗

PubMed1985

Tight junction formation in cultured epithelial cells (MDCK).

Synthesis and assembly of tight junctions are studied in monolayers of MDCK cells plated at a density sufficient for confluence, allowed to attach for 1 hr, and transferred to fresh media without cells containing or not Ca2+. 20 hr later, while monolayers with Ca2+ have fully developed junctions that confer an electrical resistance across of 346 +/- 51 omega cm2, those without Ca2+ have a negligible resistance. If at this time Ca2+ is added, junctions assemble and seal with a fast kinetics, that can be followed through the development of electrical resistance, penetration of ruthenium red, and electron microscopy. Drugs that impair synthesis, maturation and transport of proteins (cycloheximide, tunicamycin, monensin) indicate that protein components are synthesized early upon plating, do not seem to require N-glycosylation, and are stored in the Golgi compartment. Upon addition of Ca2+ they are transferred to the membrane with the participation of microfilaments but not of microtubules. These components seem to insert directly in the position they occupy in the strands, and the cell circles its perimeter with one strand as early as 15 min, even if in some segments it only consists of a row of particles. New strands develop in association with previous ones, and the pattern completes in 4 to 6 hr. Ca2+ is required for the maintenance of the assembly and also for the sealing with neighboring cells. These processes cannot occur below 25 degrees C. Serum is not required. Polarized distribution of intramembrane particles (IMP) in apical and basolateral regions follows the same time course as junction formation, in spite of the fence constituted by those strands that are already assembled. This suggests that IMP do not redistribute by lateral displacements in the plane of the membrane, but by removal and insertion in the apical and basolateral domains.

Animals↗

PubMed1984

Electrical properties of Madin-Darby canine kidney cells.

Madin-Darby canine kidney (MDCK) cells form monolayers with properties resembling natural transporting epithelia. In this paper we first summarize briefly information on the electrical properties of the whole monolayer and on the paracellular permeation route, and then concentrate on the electrical properties of the cells themselves as studied with intracellular microelectrodes of high resistance (approximately 100 M omega). The membrane resistance is -40.5 + 15 (120) mV, yet the shape of the distribution curve shows that a group of artifactually low values is displacing the average, so the actual potential may be close to -50 mV. The electrical resistance is 61.6 +/- 6.3 (92) M omega and the capacity is 45.1 +/- 2.9 (63) pF. The relationship between resistance and capacity was 1981 +/- 177 (61) omega. microF . No electrical coupling was found in 20 pairs of neighboring cells. The current/voltage curve shows a marked asymmetry, and in some cells, voltage becomes dependent on time for large, depolarizing current pulses. This phenomenon can be accentuated by removing Na+ or adding amiloride.

Animals↗

PubMed1984

Changes in paracellular and cellular ionic permeabilities of monolayers of MDCK cells infected with influenza or vesicular stomatitis viruses.

MDCK cells (epithelioid line derived from the kidney of a normal dog) form monolayers which retain the properties of transporting epithelia. In these cells viruses bud asymmetrically: influenza from the apical, and vesicular stomatitis (VSV from the basolateral membrane (E. Rodríguez-Boulán and D.D. Sabatini, Proc. Natl. Acad. Sci. USA 75: 5071-5075, 1978; E. Rodríguez-Boulán and M. Pendergast, Cell 20: 45-54, 1980). In the present study, we analyzed whether these viruses affect specific ion-translocating mechanisms located in the plasma membrane. We studied the effect of infection on membrane and transepithelial conductance, passive and active unidirectional fluxes of Na+ and K+, intracellular potentials, cellular content of Na+ and K+, and formation of blisters which, in these preparations, are due to the vectorial transport of fluid. Two main observations are derived from these studies. First, infection with VSV caused an increase in transepithelial electrical conductance, due to the opening of tight junctions, 5 to 6 hr after the start of infection, coincident with the accumulation of envelope protein in the cell surface and with the rise in the curve of virus budding. Infection with influenza, on the other hand, increased the transepithelial conductance only late in the infection (12 to 14 hr) when virus production has already stopped. Second, viruses did affect membrane permeability. Yet, the changes observed may not be ascribed to a perturbation of the specific translocating mechanisms for Na+ and K+ which operate in the same region of the plasma membrane that the viruses use to penetrate and leave MDCK cells. The methods used in the present study are not suitable to decide whether the nonspecific changes in permeability elicited by the viruses occur over the whole cell membrane or are restricted to a given region.

Animals↗

PubMed1984

Cell-to-cell communication in monolayers of epithelioid cells (MDCK) as a function of the age of the monolayer.

We explore the existence of cell-to-cell communication in monolayers of MDCK cells plated at high densities so that they form a continuous monolayer in a few minutes. Lucifer Yellow CH is injected in the cytoplasm of a given cell by using a glass microelectrode with a fine tip (ca. 100 M omega) and passing square pulses of current of 1.0 nA that last 10 msec, every 20 msec, during 1 to 3 min. We then examine the monolayer with fluorescence microscopy. In 27 out of 111 cells injected during the first 4 to 15 hr after plating, the dye was transferred to neighboring cells. Electron micrographs of freeze-fracture replicas prepared at this time, show that 20 to 25% of the lateral surfaces present the aggregates of intramembrane particles typical of gap junctions. These early hours correspond to the formation of occluding junctions and polarization into an apical and a basolateral domain of the plasma membrane (Cereijido, Meza & Martínez-Palomo, 1981). Cell-to-cell coupling then decreases sharply and, in the period between the 1st and 3rd day (mature monolayers), only 4 out of 49 injected cells were able to transfer the dye to their neighbors in the monolayers. No image of gap junctions was found in freeze-fracture replicas of mature monolayers. The degree of coupling between cells, as well as the number of cells coupled to the injected one, were highly variable. The lack of coupling between cells in mature monolayers observed in this article with Lucifer Yellow CH and electron microscopy is in keeping with the absence of electrical coupling observed in a previous work (Stefani & Cereijido, 1983). The transient existence of communicating junctions observed in monolayers of MDCK cells is similar to that described in the literature for embryo tissues during development.

Animals↗

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↗