RESUMO
Epithelia in multicellular organisms constitute the frontier that separates the individual from the environment. Epithelia are sites of exchange as well as barriers, for the transit of ions and molecules from and into the organism. Therapeutic agents, in order to reach their target, frequently need to cross epithelial and endothelial sheets. Two routes are available for such purpose: the transcellular and the paracellular pathways. The former is employed by lipophilic drugs and by molecules selectively transported by channels, pumps and carriers present in the plasma membrane. Hydrophilic molecules cannot cross biological membranes, therefore their transepithelial transport could be significantly enhanced if they moved through the paracellular pathway. Transit through this route is regulated by tight junctions (TJs). The discovery in recent years of the molecular mechanisms of the TJ has allowed the design of different procedures to open the paracellular route in a reversible manner. These strategies could be used to enhance drug delivery across epithelial and endothelial barriers. The procedures employed include the use of peptides homologous to external loops of integral TJ proteins, silencing the expression of TJ proteins with antisense oligonucleotides and siRNAs as well as the use of toxins and proteins derived from microorganisms that target TJ proteins.
Assuntos
Sistemas de Liberação de Medicamentos , Endotélio/citologia , Endotélio/fisiologia , Células Epiteliais/fisiologia , Junções Íntimas/fisiologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Endotélio/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Junções Íntimas/efeitos dos fármacosRESUMO
A fundamental function of epithelia and endothelia is to separate different compartments within the organism and to regulate the exchange of substances between them. The tight junction (TJ) constitutes the barrier both to the passage of ions and molecules through the paracellular pathway and to the movement of proteins and lipids between the apical and the basolateral domains of the plasma membrane. In recent years more than 40 different proteins have been discovered to be located at the TJs of epithelia, endothelia and myelinated cells. This unprecedented expansion of information has changed our view of TJs from merely a paracellular barrier to a complex structure involved in signaling cascades that control cell growth and differentiation. Both cortical and transmembrane proteins integrate TJs. Among the former are scaffolding proteins containing PDZ domains, tumor suppressors, transcription factors and proteins involved in vesicle transport. To date two components of the TJ filaments have been identified: occludin and claudin. The latter is a protein family with more than 20 members. Both occludin and claudins are integral proteins capable of interacting adhesively with complementary molecules on adjacent cells and of co-polymerizing laterally. These advancements in the knowledge of the molecular structure of TJ support previous physiological models that exhibited TJ as dynamic structures that present distinct permeability and morphological characteristics in different tissues and in response to changing natural, pathological or experimental conditions.
Assuntos
Células Epiteliais/fisiologia , Junções Íntimas , Animais , Humanos , Imunoglobulinas/química , Lipídeos , Bainha de Mielina/fisiologia , Estrutura Terciária de Proteína , Xenopus/embriologiaRESUMO
ZO-1, ZO-2 and ZO-3 are tight junction (TJ)-associated proteins that belong to the MAGUK family. In addition to the presence of the characteristic MAGUK modules (PDZ, SH3 and GK), ZOs have a distinctive carboxyl terminal with splicing domains, acidic- and proline-rich regions. The modular organization of these proteins allows them to function as scaffolds, which associate to transmembrane TJ proteins, the cytoskeleton and signal transduction molecules. ZOs shuttle between the TJ and the nucleus, where they may regulate gene expression.
Assuntos
Núcleosídeo-Fosfato Quinase/química , Núcleosídeo-Fosfato Quinase/fisiologia , Junções Íntimas/fisiologia , Animais , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Conexina 43/fisiologia , Citoesqueleto/fisiologia , Guanilato Quinases , Humanos , Substâncias Macromoleculares , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Ocludina , Fosfoproteínas/química , Fosfoproteínas/fisiologia , Fosforilação , Estrutura Terciária de Proteína , Proteínas da Zônula de Oclusão , Proteína da Zônula de Oclusão-1 , Proteína da Zônula de Oclusão-2 , Domínios de Homologia de srcRESUMO
BACKGROUND: Tight junctions play a critical role in tubular function. In mammalian kidney, the transepithelial electrical resistance and the complexity of the tight junction increase from the proximal to the collecting tubule. The differential expression of three tight junction proteins, ZO-1, ZO-2, and occludin, along isolated rabbit renal tubules is examined in this article. METHODS: Microdissected rabbit renal tubules were processed for immunofluorescence detection of ZO-1, ZO-2, and occludin. The quantitation of these proteins was done by Western blot determinations in Percoll isolated tubules. RESULTS: ZO-1 stained cell boundaries independently of the identity of the tubule. However, the amount found in distal segments was significantly higher than that expressed in proximal regions. ZO-2 in the proximal region was found diffusely distributed in the cytoplasm, with faint staining at cell borders, while a clear signal at cell perimeters was detectable from the Henle's loop to collecting tubules. Nuclear staining of ZO-2 was found along the whole nephron. The presence of occludin at the proximal region was faint and discontinuous, while its expression in the more distant portions was conspicuous. The quantity of ZO-2 and occludin present at the distal region was significantly higher compared with the proximal segment. CONCLUSIONS: The distribution of ZO-1, ZO-2, and occludin follows the increase in junction complexity encountered in renal tubules. The amount of the three proteins found in proximal and distal segments is significantly higher in the latter.
Assuntos
Túbulos Renais/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Junções Íntimas/metabolismo , Animais , Western Blotting , Núcleo Celular/metabolismo , Imunofluorescência , Secções Congeladas , Técnicas In Vitro , Masculino , Ocludina , Coelhos , Distribuição Tecidual , Proteína da Zônula de Oclusão-1 , Proteína da Zônula de Oclusão-2RESUMO
The establishment of the junctional complex in epithelial cells requires the presence of extracellular calcium, and is controlled by a network of reactions involving G-proteins, phospholipase C and protein kinase C. Since potential candidates for phosphorylation are the tight junction associated proteins ZO1, ZO2 and ZO3, in a previous work we specifically explored these molecules but found no alteration in their phosphorylation pattern. To continue the search for the target of protein kinase C, in the present work we have studied the subcellular distribution and phosphorylation of vinculin and alpha-actinin, two actin binding proteins of the adherent junctions. We found that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in the phosphorylation of vinculin, alpha-actinin remains unchanged. The increased phosphorylation of vinculin is due to changes in phosphoserine and phosphothreonine content and seems to be regulated by protein kinase C, since: (1) DiC8 (a kinase C stimulator) added to monolayers cultured without calcium significantly increases the vinculin phosphorylation level; (2) H7 and calphostin C (both protein kinase C inhibitors) completely abolish this increase during a calcium switch; (3) inhibition of phosphorylation during a calcium switch blocks the subcellular redistribution of vinculin and alpha-actinin. These results therefore suggest that vinculin phosphorylation by protein kinase C is a crucial step in the correct assembly of the epithelial junctional complex.
Assuntos
Actinina/metabolismo , Cálcio/farmacologia , Proteína Quinase C/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Vinculina/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Cães , Microscopia de Fluorescência , Fosforilação , Serina/metabolismo , Frações Subcelulares/metabolismo , Treonina/metabolismoRESUMO
The variable clinical course of human giardiasis may be due in part to differences in the virulence of various strains of Giardia lamblia. To address this issue, the in vitro cytopathic effect of isolates obtained from human symptomatic or asymptomatic infections was assessed by ultrastructural and electrophysiological methods. Axenic trophozoites of 10 strains of G. lamblia isolated from children with infections in Mexico City were cultured for 12 to 24 hr on live MDCK epithelial cells. No decrease in transepithelial resistance of MDCK monolayers mounted in Ussing chambers was detected with any of the isolates analyzed. On the contrary, trophozoites or media in which the isolates grew produced up to a twofold increase in transepithelial resistance. Transmission and scanning electron microscopy revealed that all isolates of G. lamblia, irrespective of their origin, gave rise to focal regions of microvilli depletion. These modifications were induced by the close attachment of the ventrolateral flange of the parasite adhesive disk to the apical surface of MDCK cells. The circular imprints evolved progressively to larger areas devoid of microvilli. In conclusion, under in vitro conditions, isolates of G. lamblia trophozoites derived from symptomatic or asymptomatic human infections damage epithelial cultured cells mainly by depleting their microvilli. None of the isolates showed evidence of an invasive effect.
Assuntos
Portador Sadio/parasitologia , Giardia lamblia/patogenicidade , Giardíase/parasitologia , Animais , Linhagem Celular , Criança , Eletrofisiologia , Epitélio/parasitologia , Epitélio/ultraestrutura , Giardia lamblia/ultraestrutura , Humanos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Microvilosidades/parasitologia , Microvilosidades/ultraestrutura , VirulênciaRESUMO
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.
Assuntos
Calmodulina/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Junções Intercelulares/fisiologia , Proteína Quinase C/fisiologia , Fosfolipases Tipo C/fisiologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Toxina Adenilato Ciclase , Animais , Cálcio/fisiologia , Carbacol/farmacologia , Células Cultivadas , Condutividade Elétrica/efeitos dos fármacos , Condutividade Elétrica/fisiologia , Células Epiteliais , Epitélio/metabolismo , Epitélio/fisiologia , Junções Intercelulares/metabolismo , Isoquinolinas/farmacologia , Rim/citologia , Rim/metabolismo , Rim/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neomicina/farmacologia , Toxina Pertussis , Piperazinas/farmacologia , Polimixina B/farmacologia , Proteína Quinase C/antagonistas & inibidores , Trifluoperazina/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
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.
Assuntos
Cálcio/fisiologia , Junções Intercelulares/fisiologia , Marcadores de Afinidade/farmacologia , Animais , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Cloroquina/farmacologia , Citosol/metabolismo , Ácido Egtázico/farmacologia , Condutividade Elétrica , Epitélio/efeitos dos fármacos , Epitélio/fisiologia , Epitélio/ultraestrutura , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , Indóis , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/ultraestrutura , Cinética , Manitol/metabolismo , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Verapamil/farmacologiaRESUMO
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.