Biomedical subjects

R G Contreras

Publications and source records attributed to R G Contreras.

14 recordsLinked to original sources

PubMed1999

Molecular characterization of the tight junction protein ZO-1 in MDCK cells.

Most of the information on the structure and function of the tight junction (TJ) has been obtained in MDCK cells. Accordingly, we have sequenced ZO-1 in this cell type, because this protein is involved in the response of the TJ to changes in Ca2+, phosphorylation, and the cytoskeleton. ZO-1 of MDCK cells comprises 6805 bp with a predicted open reading frame of 1769 amino acids. This sequence is 92 and 87% homologous to human and mouse ZO-1, respectively. Two nuclear sorting signals located at the PDZ1 and GK domains and 17 SH3 putative binding sites at the proline-rich domain were detected. We found two new splicing regions at the proline-rich region: beta had not been reported in human and mouse counterparts, and gamma, which was previously sequenced in human and mouse ZO-1, is now identified as a splicing region. The expression of different beta and gamma isoforms varies according to the tissue tested. With the information provided by the sequence, Southern blot, and PCR experiments we can predict a single genomic copy of MDCK-ZO-1 that is at least 13.16 kb long. MDCK-ZO-1 mRNA is 7.4 kb long. Its expression is regulated by calcium, while the expression of MDCK-ZO-1 protein is not.

Alternative Splicing↗

PubMed1998

Tight junctions and the experimental modifications of lipid content.

Tight junctions (TJs) are cell-to-cell contacts made of strands, which appear as ridges on P faces and complementary furrows on E faces on freeze fracture replicas. Evidences and opinions on whether these strands are composed of either membrane-bound proteins or lipid micelles are somewhat varied. In the present work we alter the lipid composition of Madin-Darby canine kidney monolayers using a novel approach, while studying (i) their transepithelial electrical resistance, a parameter that depends on the degree of sealing of the TJs; (ii) the apical-to-basolateral flux of 4 kD fluorescent dextran (JDEX), that reflects the permeability of the intercellular spaces; (iii) the ability of TJs to restrict apical-to-basolateral diffusion of membrane lipids; and (iv) the pattern of distribution of endogenous and transfected occludin, the sole membrane protein presently known to form part of the TJs. We show that changing the total composition of phospholipids, sphingolipids, cholesterol and the content of fatty acids, does not alter TER nor the structure of the strands. Interestingly, enrichment with linoleic acid increases the JDEX by 631%. The fact that this increase is not reflected in a decrease of TER, suggests that junctional strands do not act as simple resistive elements but may contain mobile translocating mechanisms.

Animals↗

PubMed1998

Role of tight junctions in establishing and maintaining cell polarity.

The tight junction (TJ) is not randomly located on the cell membrane, but occupies a precise position at the outermost edge of the intercellular space and, therefore, is itself considered a polarized structure. This article reviews the most common experimental approaches for studying this relationship. We then discuss three main topics. (a) The mechanisms of polarization that operate regardless of the presence of TJs: We explore a variety of polarization mechanisms that operate at stages of the cell cycle in which TJs may be already established. (b) TJs and polarity as partners in highly dynamic processes: Polarity and TJs are steady state situations that may be drastically changed by a variety of signaling events. (c) Polarized distribution of membrane molecules that depend on TJs: This refers to molecules (mainly lipids) whose polarized distribution, although not the direct result of TJs, depends on these structures to maintain such distribution.

Animals↗

PubMed1996

Morphogenesis of the epithelial cell transporting phenotype: synthesis and distribution of ion channels.

The exchange of substances between higher organisms and the environment takes place across epithelia consisting of one or more cell layers. To perform this function, epithelial cells have two basic differentiated properties: 1) they form tight junctions (TJs) that seal the extracellular space, and 2) they are polarized into an apical and a basolateral domain, with entirely different structural, biochemical and physiological properties. Our understanding of the mechanisms involved in the expression of these properties has been greatly enhanced by the availability of epithelial cell lines that form TJs and polarize in vitro under conditions suitable for experimental control. In this article we summarize our studies on the synthesis and polarized expression of ion channels in epithelial cells. MDCK cells have four types of K+ channels in the apical domain, and a fifth one in the basolateral domain. The basolateral side also has a population of CI- channels. Each type of channel is absolutely polarized. Harvesting with trypsin-EDTA reduces the area of the plasma membrane by 50% and the channel population by 90%. Upon plating, these channels are recovered within a few hours. We describe here the main extracellular and intracellular mechanisms involved in these phenomena.

Animals↗

PubMed1995

Ouabain resistance of the epithelial cell line (Ma104) is not due to lack of affinity of its pumps for the drug.

Na+, K(+)-pumps of most eukaryotic animal cells bind ouabain with high affinity, stop pumping, and consequently loose K+, detach from each other and from the substrate, and die. Lack of affinity for the drug results in ouabain resistance. In this work, we report that Ma104 cells (epithelial from Rhesus monkey kidney) have a novel form of ouabain-resistance: they bind the drug with high affinity (Km about 4 x 10(-8) M), they loose their K+ and stop proliferating but, in spite of these, up to 100% of the cells remain attached in 1.0 microM ouabain, and 53% in 1.0 mM. When 4 days later ouabain is removed from the culture medium, cells regain K+ and resume proliferation. Strophanthidin, a drug that attaches less firmly than ouabain, produces a similar phenomenon, but allows a considerably faster recovery. This reversal may be associated to the fact that, while in ouabain-sensitive MDCK cells Na+, K(+)-ATPases blocked by the drug are retrieved from the plasma membrane, those in Ma104 cells remain at the cell-cell border, as if they were cell-cell attaching molecules. Cycloheximide (10 micrograms/ml) and chloroquine (10 microM) impair this recovery, suggesting that it also depends on the synthesis and insertion of a crucial protein component, that may be different from the pump itself. Therefore ouabain resistance of Ma104 cells is not due to a lack of affinity for the drug, but to a failure of its Na+, K(+)-ATPases to detach from the plasma membrane in spite of being blocked by ouabain.

Animals↗

PubMed1995

A novel type of cell-cell cooperation between epithelial cells.

Ma104 cells (renal, epithelial) have a peculiar way of resisting ouabain: their Na+,K(+)-pumps bind the drug with high affinity, cellular K+ is lost and cell division arrested, but cells do not detach as most cell types do. Then, if up to 4 days later the drug is removed, Ma104 cells recover K+ and resume proliferation (Contreras et al., 1994). In the present work, we investigate whether Ma104 cells are able to protect ouabain-sensitive MDCK cells in co-culture. The main finding is that they do, but in this case protection is not elicited by the usual mechanism of maintaining the K+ content of neighboring cells through cell-cell communications. Ma104 cells treated with ouabain simply remain attached to the substrate and to their MDCK neighbors, and both cells lose K+. This attachment includes tight junctions, because the transepithelial electrical resistance of the monolayers is not abolished by ouabain. Although the beta-subunit of the Na+,K(+)-ATPase is known to possess molecular characteristics of cell-cell attachment molecules, attachment between Ma104-MDCK cells does not seem to be mediated by this enzyme, as immunofluorescence analysis reveals that Na+,K(+)-ATPase is only inserted in the plasma membrane facing a neighboring cell of the same type.

Animals↗

PubMed1995

Expression of potassium channels in epithelial cells depends on calcium-activated cell-cell contacts.

Harvesting MDCK cells with trypsin-EDTA reduces potassium currents (IK) to a mere 10%, presumably by hydrolysis of K+ channels, but replating at confluence restores them in 12-18 hr, through a process that requires transcription, translation and exocytic fusion of intracellular membrane vesicles to the plasma membrane (Ponce & Cereijido, 1991; Ponce et al., 1991a). In the present work we find that this restoration of IK also requires cell-cell contacts and the presence of 1.8 mM Ca2+. The role of extracellular Ca2+ may be substituted by 2.0 microM TRH, 10 nM PMA or 200 micrograms/ml DiC8. drugs that stimulate the system of phospholipase C (PLC) and protein kinase C (PKC). Conversely, the recovery of IK triggered by Ca-dependent contacts can be blocked by 110 microM neomycin, 2.0 microM H7, and 250 nM staurosporine, inhibitors of PLC and PKC. These results suggest that the expression of new K+ channels depends on Ca(2+)-activated contacts with neighboring cells and that the information is conveyed through PLC and PKC, a process in keeping with changes in its enzymatic activity and cellular distribution of PKC. Plasma membrane is also reduced and restored upon harvesting and replating, and depends on Ca(2+)-activated contracts. However, the effects of the chemicals tested on IK differ from the ones they elicit on the recovery of plasma membrane, suggesting that cells can independently regulate their population of K+ channels and the surface of their membrane.

Animals↗

PubMed1993

The making of a tight junction.

MDCK (epithelial cells from the dog kidney) plated at confluence, establish tight junctions in 12-15 hours through a process that requires protein synthesis, formation of a ring of actin filaments in close contact with the lateral membrane of the cells, calmodulin, and a Ca(2+)-dependent exocytic fusion of tight junction (TJ)-associated components. Monolayers incubated in the absence Ca2+ make no TJs. Yet, if Ca2+ is added under these circumstances, TJs are made with a faster kinetics. Ca2+ is needed mainly at a site located on the outer side of the cell membrane, where it activates uvomorulin and triggers the participation of the cellular components mentioned above, via G-proteins associated with phospholipase C and protein kinase C. In principle, the sites of all these molecules and mechanisms involved in junction formation may be where a variety of agents (hormones, drugs, metabolites) act to produce epithelia with a transepithelial electrical resistance (TER) ranging from 10 to 10,000 omega.cm2. This range may be also due to a variety of substances found in serum and in urine, that increase the TER in a reversible and dose-dependent manner.

Actins↗

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

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

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↗