Biomedical subjects

R G Contreras

Publications and source records attributed to R G Contreras.

6 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

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↗