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At least 1,693 records · Page 94Linked to original sources
PubMed2005

HIV protease inhibitor ritonavir increases endothelial monolayer permeability.

HIV protease inhibitors (PIs) are often associated with metabolic and cardiovascular complications although they are effective anti-HIV drugs. In this study, we determined whether HIV PI ritonavir could increase endothelial permeability, one of the important mechanisms of vascular lesion formation. Human dermal microvascular endothelial cells (HMECs) treated with ritonavir showed a significant increase of endothelial permeability in a dose- and time-dependent manner assayed with a transwell system. Ritonavir significantly reduced the mRNA levels of tight junction proteins zonula occluden-1, occludin, and claudin-1 by 40-60% as compared to controls (P<0.05) by real-time PCR analysis. Protein levels of these tight junction molecules were also substantially reduced in the ritonavir-treated cells. In addition, HMECs treated with ritonavir (7.5, 15, and 30microM) showed a substantial increase of superoxide anion production by 10%, 32%, and 65%, respectively, as compared to controls. Antioxidants (EGCG and SeMet) effectively reduced ritonavir-induced endothelial permeability. Furthermore, ritonavir activated ERK1/2 (phosphorylation), but not P38 and JNK. Specific ERK1/2 inhibitor, PD89059, significantly abolished ritonavir-induced endothelial permeability by 92%. Thus, HIV PI ritonavir increases endothelial permeability, decreases levels of tight junction proteins, and increases superoxide anion production. ERK1/2 activation is involved in the signal transduction pathway of ritonavir-induced endothelial permeability.

Antigens, CD↗
PubMed1999

Alteration of the sodium to potassium ratio in milk and the effect on milk secretion in goats.

Saanen goats were used to determine the effect of the alteration of the intramammary Na to K ratio on milk secretion. Udders were infused via the teat with an isosmotic solution that was high in Na or K to increase or decrease, respectively, the intramammary Na to K ratio. Control glands received an isosmotic sucrose solution. To ensure that the results were not confounded by a decrease in milk secretion as a result of enhanced permeability of mammary tight junctions, the latter was monitored throughout the experiments. An increase in the Na to K ratio caused a significant transient reduction in milk secretion. Therefore, an increase in Na and a decrease in K in milk, commonly observed as a result of the leakiness of tight junctions, may at least partially explain the reduction in milk secretion when the permeability of tight junctions was increased. These experiments further showed that the adverse effects on secretion were not due to a high intracellular concentration per se but were related to a change in the Na to K ratio because a reduction in the ratio also lowered milk secretion. These data support the evidence for activity of Na(+)-K(+)-ATPase in the basolateral secretory cell membranes and passive movement of these ions across the apical cell membranes.

Animals↗
PubMed2002

Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells.

Madin-Darby canine kidney (MDCK) I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical resistance (TER) values in excess of 5,000 Omega. cm(2). When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a >5-fold reduction of TER was observed without changes in the gate function for uncharged solutes or the fence function for apically applied fluorescent lipids. The localization of ZO-1 and occludin was not altered between control and inhibitor-treated cells, indicating that the tight junction was still intact. Furthermore, the complexity of tight junction strands, analyzed by freeze-fracture microscopy, was not reduced. Once the inhibitor was removed and the cells were allowed to synthesize sphingolipids, a gradual recovery of the TER was observed. Interestingly, these inhibitors did not attenuate the TER of MDCK II cells, a cell line that typically exhibits values below 800 omega x cm(2.) These results suggest that glycosphingolipids play a role in regulating the electrical properties of epithelial cells.

Animals↗
PubMedDate not supplied

Blood brain barrier and infection.

The blood brain barrier (BBB) is a highly dynamic structure and consists of endothelial cells, which are characterized by the presence of tight junctions and relative lack of endocytic vesicles. The tight junctions are reinforced by the foot processes of the astrocytes. The BBB functions through these specialised structures, to maintain the environment of the brain in a steady state by regulating the influx and efflux of substances. The protective effect of the BBB is however, lost during bacterial and viral infections. The primary mechanism operative are an increase in the permeability of the BBB and/or direct invasion of the brain by microorganisms. Since the BBB is relatively impermeable to chemotherapeutic agents the treatment of CNS infections is difficult. This paper aims to examine the various mechanisms by which infection spreads to the brain, and suggest measures for successful drug delivery into the brain during infections.

Animals↗
PubMed2001

In vitro assessment of alkylglycosides as permeability enhancers.

series of alkylglycosides has been evaluated on human cell lines to determine its ability to open cellular tight junctions. Alkylglycosides were applied to cell monolayers; the resulting change in resistance was determined by transepithelial electrical resistance measurements. Change in resistance across cell monolayers is an indication of tight junction activation, whereas subsequent increase in resistance signifies monolayer recovery. Of the 13 alkylglycosides tested, 4 caused irreversible solubilization of cell membranes, 5 allowed a partial recovery of the monolayer after a relatively rapid reduction in resistance, and 4 induced a decrease in resistance with more complete cell recovery. Alkylglycosides allowing extensive cell recovery after removal may indicate tight junctions' activity dominance over membrane fluidity. Repeated application of alkylglycosides for 6 hours lowered resistance across cells, which returned to near-normal values after a recovery period of 48 hours. A model dye was transported across the cell monolayer only in the presence of an alkylglycoside, although recovery of cells was incomplete. Activity of the alkylglycosides was unrelated to either the carbon chain length or to the carbohydrate moiety. A direct correlation was established between the concentration of applied alkylglycoside and reduction in resistance over a constant time period. Dodecylmaltoside and octylglucoside were found to be optimal in decreasing resistance at low concentrations and allowing significant recovery of cells. Therefore these 2 alkylglycosides may be useful in facilitating drug transport across biological membranes.

Biological Transport↗
PubMed1983

The intercellular junctions of guinea-pig placental capillaries: a possible structural basis for endothelial solute permeability.

The endothelial cell junction in guinea-pig placental capillaries consists of a continuous ribbon desmosome (zonula adherens) within which lies a particulate tight junction consisting of between one and five anastomosing strands. The intercellular space at these tight junctions is narrowed and is subdivided by junctional bars which are probably continuous with the intramembrane particle rows seen in freeze-fracture replicas of the junctions. Perfusion with lanthanum salts shows the gaps between the junctional bars to be lanthanum-filled and the entire junction to be lanthanum permeable. The estimated size of the spaces between the junctional bars is consistent with the junctional pore size indicated by previous ultrastructural tracer studies. The wider lateral intercellular space of the ribbon desmosome is spanned by more widely spaced "linkers" which may act as a coarser three-dimensional filter in series with size-limiting pores between the tight junctional bars.

Animals↗
PubMed1983

Morphology and permeability of junctional complexes in maturing ameloblasts of rat incisors.

Thin sections of newborn rat incisors were examined by tracer experiments and freeze-fracture replication in order to clarify the morphology and permeability of ameloblast junctional complexes in the maturation stage. Ameloblast junctional complexes consisted of gaps and tight junctions at the proximal and distal ends. Whereas the proximal junctional complexes sealed extracellular spaces incompletely, the distal ones formed complete, belt-like barriers around the cell. Tight junctions of these junctional complexes, however, were composed of both continuous and discontinuous rows of particles with various spaces among them. Intravenously injected horseradish peroxidase (HRP) reached the enamel surface through the extracellular spaces among ameloblasts and was absorbed by ameloblasts of the ruffled borders. Pinocytosis and transcellular migration of HRP could not be demonstrated in maturing ameloblasts except in the ruffled border zone.

Ameloblasts↗
PubMed1994

In vitro and in vivo analysis of the mechanism of absorption enhancement by palmitoylcarnitine.

Long-chain acylcarnitines (12-18 carbon fatty acid esters) dramatically enhance the absorption of hydrophilic drugs across intestinal mucosa without altering the morphology of the epithelium. The mechanism underlying these effects was studied using the colon carcinoma cell line Caco-2. Caco-2 monolayers treated with 0.2 mM palmitoylcarnitine (PCC) show dramatic increases in the transport of hydrophilic markers. This enhanced transport coincides with a rapid drop in transepithelial electrical resistance (TER). The drop in TER is initiated within the first minute after PCC addition and continues for approximately 20 min to a 70 to 85% drop of the initial TER values. This effect is reversible after removing the PCC and does not appear to involve lysis of the apical membrane. Instead PCC's effect appears to be due to loosening of tight junctions as indicated by the accumulation of fluorescent dextrans and the electron dense marker lanthanum nitrate in paracellular spaces. Moreover transmission electron microscopy and freeze fracture electron microscopy indicate that PCC produces significant structural alterations to tight junctions. In contrast to many other tight junction disrupting agents, PCC effects appear to be Ca(++)-independent and PCC does not induce significant disruption of actin filament distribution in Caco-2 cells.

Freeze Fracturing↗
PubMed1982

Alterations in intercellular junctions of the uterine epithelium during the preimplantation phase in the rabbit.

In the rabbit, the pseudopregnant uterus has been used as a model for studying alterations characteristic of the preimplantation phase. Alterations in intercellular junctions of the uterine epithelium were investigated during early pseudopregnancy (day 0 to day 6) by means of the freeze-fracture technique. In the uterine epithelium of oestrous females the zonula occludens belongs to the "tight" type of tight junctions. During pseudopregnancy an impressive proliferation of tight junctional belts can be observed. The basal strands proliferate, forming loops perpendicular to the luminal surface, whereas the more or less parallel arrangement of the luminal strands is maintained. At day 4 of pseudopregnancy macular tight junctions begin to develop on the lower portions of the lateral plasmalemma and are extensive by day 6 post hCG. Small gap junctions are infrequent between cells of the uterine epithelium and show no significant changes during the preimplantation phase. The physiological significance of the present morphological observations is discussed in the light of changes occurring during the preimplantation period.

Animals↗
PubMed1976

Kuhnt intermediary tissue as a barrier between the optic nerve and retina.

Utilizing adult albino rats and lanthanum nitrate as a tracer, electron microscopic studies were done to provide additional information on the blood-optic nerve barrier. Following injection into the cervical artery, lanthanum was observed to fill the intercellular space of the optic nerve parenchyma. Diffusion from the prelaminar optic nerve into the juxta-optic nerve retina was, however, prevented by the Kuhnt tissue at the level of the rod and cone layer. In this region, tight junctions were found between the glial cells of the Kuhnt intermediary tissue and were continuous from the tight junctions of the retinal pigment epithelial cells to the junctions (zonulae adhaerentes) of the outer limiting membrane. From the level of the outer limiting membrane to the inner limiting membrane there was no diffusion of lanthanum into the adjacent retina despite the absence of tight junctions, and the lanthanum which had diffused from the choroid was never observed in the area of the inner limiting membrane of the optic nerve head. It is in this region that the functional barrier may exist.

Animals↗
PubMed1982

A freeze-fracture study of the skate electroreceptor.

The sensory epithelium lining the ampulla of Lorenzini in the skate was examined by the freeze-fracture technique. Anastomosing tight junctions (zonula occludens) completely encircle the apex of each receptor cell joining it to neighbouring support cells. The tight junctions separate two distinctly different regions of the receptor-cell surface. The apical P-face has numerous large particles while just below the tight junctions of the lateral surface have many smaller particles. On its basal surface each receptor cell makes several evaginating ribbon synapses with an afferent nerve. Three regions of the synaptic evagination can be distinguished on the basis of membrane specializations: 1. At the tip of the evagination a regular array of large particles is found on the P-face of the receptor cell directly opposite a similar regular array of large particles on the P-face of the afferent nerve; 2. just above the tip at a narrow constriction, below which vesicles are not found, a population of large particles on the P-face of the receptor cell opposes a well-defined strip of large particles that cleaves with the E-face of the nerve fibre; 3. at the arch of the synaptic evagination randomly occurring dimples are found on the P-face and protrusions on the E-face of the receptor cell. The density of these protrusions increased in skates that were electrically stimulated. We suggest that the co-extensive arrays of particles at the tip of the ribbon synapse is an intercellular junction; that the active zone of the synapse is at or above the constriction; and that membrane retrieval occurs in the synaptic arch region.

Animals↗
PubMed2000

Transepithelial resistance can be regulated by the intestinal brush-border Na(+)/H(+) exchanger NHE3.

Initiation of intestinal Na(+)-glucose cotransport results in transient cell swelling and sustained increases in tight junction permeability. Since Na(+)/H(+) exchange has been implicated in volume regulation after physiological cell swelling, we hypothesized that Na(+)/H(+) exchange might also be required for Na(+)-glucose cotransport-dependent tight junction regulation. In Caco-2 monolayers with active Na(+)-glucose cotransport, inhibition of Na(+)/H(+) exchange with 200 microM 5-(N,N-dimethyl)- amiloride induced 36 +/- 2% increases in transepithelial resistance (TER). Evaluation using multiple Na(+)/H(+) exchange inhibitors showed that inhibition of the Na(+)/H(+) exchanger 3 (NHE3) isoform was most closely related to TER increases. TER increases due to NHE3 inhibition were related to cytoplasmic acidification because cytoplasmic alkalinization with 5 mM NH(4)Cl prevented both cytoplasmic acidification and TER increases. However, NHE3 inhibition did not affect TER when Na(+)-glucose cotransport was inhibited. Myosin II regulatory light chain (MLC) phosphorylation decreased up to 43 +/- 5% after inhibition of Na(+)/H(+) exchange, similar to previous studies that associate decreased MLC phosphorylation with increased TER after inhibition of Na(+)-glucose cotransport. However, NHE3 inhibitors did not diminish Na(+)-glucose cotransport. These data demonstrate that inhibition of NHE3 results in decreased MLC phosphorylation and increased TER and suggest that NHE3 may participate in the signaling pathway of Na(+)-glucose cotransport-dependent tight junction regulation.

Acids↗
PubMed1999

Comparative cytotoxicity of ionic and non-ionic radiocontrast agents on MDCK cell monolayers in vitro.

BACKGROUND: Intravascular radiocontrast agents may cause acute renal failure, particularly in patients with pre-existing renal insufficiency. Direct cytotoxic effects of radiocontrast agents on renal tubular cells may contribute to the pathogenesis of radiocontrast-induced nephropathy. METHODS: We analysed the cytotoxicity of the ionic radiocontrast agents diatrizoate (monomeric) and ioxaglate (dimeric), as well as of the non-ionic radiocontrast agents iohexol (monomeric) and iodixanol (dimeric) on the renal epithelial Madin Darby Canine Kidney (MDCK) cell line grown on permeable supports. The toxicity assays assessed cell viability, transmonolayer resistance and inulin permeability between the apical and basal cell culture compartment. In addition, the distribution of the tight-junction-associated membrane proteins ZO-1 and occludin was analysed using immunofluorescence microscopy. RESULTS: In all assays the high osmolal ionic compound diatrizoate had significant cytotoxic effects that included the partial redistribution of the tight-junction-associated membrane proteins into a cytoplasmic compartment. To a lesser extent this redistribution also occurred with the dimeric ionic compound ioxaglate, but not with the non-ionic radiocontrast agents. With regards to cell viability, transmonolayer resistance and inulin permeability the radiocontrast agents with reduced osmolality were significantly less toxic than diatrizoate, independent of their ionic strength. CONCLUSIONS: Physicochemical factors contribute to the cytotoxicity of radiocontrast agents in vitro. The redistribution of tight-junction-associated membrane proteins by the ionic radiocontrast agents corresponds with the loss of the barrier function of the epithelial cell monolayer, which is a major pathophysiological mechanism in acute renal failure. The radiocontrast agents with reduced osmolality are less cytotoxic than diatrizoate, independent of their ionicity. Hyperosmolality appears to be a more important determinant of the cytotoxicity of diatrizoate than ionic strength.

Animals↗
PubMed2005

Cytoskeleton reorganization and ultrastructural damage induced by gliadin in a three-dimensional in vitro model.

AIM: To evaluate the interplay between gliadin and LoVo cells and the direct effect of gliadin on cytoskeletal patterns. METHODS: We treated LoVo multicellular spheroids with digested bread wheat gliadin in order to investigate their morphology and ultrastructure (by means of light microscopy and scanning electron microscopy), and the effect of gliadin on actin (phalloidin fluorescence) and the tight-junction protein occludin and zonula occluden-1. RESULTS: The treated spheroids had deep holes and surface blebs, whereas the controls were smoothly surfaced ovoids. The incubation of LoVo spheroids with gliadin decreased the number of intracellular actin filaments, impaired and disassembled the integrity of the tight-junction system. CONCLUSION: Our data obtained from an "in vivo-like" polarized culture system confirm the direct noxious effect of gliadin on the cytoskeleton and tight junctions of epithelial cells. Unlike two-dimensional cell culture systems, the use of multicellular spheroids seems to provide a suitable model for studying cell-cell interactions.

Cell Communication↗
PubMed1987

Manganese-bilirubin cholestasis. Further studies in pathogenesis.

To further delineate the mechanism of manganese-bilirubin cholestasis, studies were done to determine: the structural status of the canalicular tight junctions in the model, and the effect of the lack of bilirubin conjugation. The results indicate that canalicular tight junction alterations characteristic of many types of cholestasis develop in this model and that bilirubin conjugation is not a requisite for the cholestatic effect of bilirubin to occur. It is unclear whether the tight junction alterations are the cause of the cholestasis or either secondary effects of the cholestasis or associated noncausal occurrences. The fact that bilirubin does not need to be conjugated in order to effect cholestasis confirms that the cholestasis is due to intrahepatocytic (as opposed to intracanalicular) effects, and may be useful in determining the precise locus and nature of these effects.

Animals↗
PubMedDate not supplied

Formation of junctional complexes in otocysts developed in vitro. A freeze-fracture study.

The thirteenth gestational day inner ear anlage (otocyst) was explanted to an in vitro system and cultured for 8 days, i.e. to a time corresponding to birth. The freeze fracturing technique was used to evaluate morphological differentiation, particularly as regards cell membrane specializations. The epithelial cells were found fully differentiated, e.g. there were regularly arranged stereocilia on the hair cells. The development of tight junctions and gap junctions followed the same pattern as in vivo, but tight junctions did not reach the same degree of regularity and maturation as they did in vivo. There were very few gap junctions in our in vitro specimens. Several tight junctions had an odd appearance, with loss of the normally punctate structure of the strands and areas with considerable thickening. It is possible that this specific morphology can be explained by differentiation in the in vitro system.

Animals↗
PubMed2000

Effect of degree of quaternization of N-trimethyl chitosan chloride for enhanced transport of hydrophilic compounds across intestinal caco-2 cell monolayers.

N-Trimethyl chitosan chloride (TMC) is a permanently quaternized chitosan derivative with improved aqueous solubility compared to native chitosan. TMC is able to open the tight junctions of intestinal epithelia at physiological pH values, where chitosan is insoluble and therefore ineffective. TMCs with degrees of substitution of 40 and 60% were synthesized according to a novel synthesis procedure and their effect on the permeability of the tight junctions of the intestinal Caco-2 monolayers was studied, measuring the transepithelial electrical resistance and the transport of a mainly paracellularly transported compound, [14C]-mannitol. Toxicity studies using nucleic stains were done to establish the transport as a cause of opening of the tight junctions and not of possible cytotoxicity. TMC60 showed higher transport enhancement ratios than TMC40 in all concentrations tested (0.05-1. 0%, w/v). Both derivatives did not affect the viability of the Caco-2 cell monolayers. These results suggest that high charge density is necessary for TMC to substantially improve the paracellular permeability of intestinal epithelia. It is expected that TMC40 and TMC60 will enhance the intestinal permeation of hydrophilic macromolecular drugs such as peptides and proteins.

Biocompatible Materials↗
PubMed1989

Permeability of blood vessels in experimental gliomas: uptake of 99mTc-glucoheptonate and alteration in blood-brain barrier as determined by cytochemistry and electron microscopy.

Experimental gliomas were induced in rats by prenatal exposure to ethyl nitrosourea. Changes in the blood-brain barrier were determined by the uptake of a water-soluble compound, 99mTc-glucoheptonate. Increased uptake of 99mTc-glucoheptonate was measured in intact tumors and in various sectors of dissected tumors. The extent of 99mTc-glucoheptonate uptake greatly varied among different tumors and among different sectors of the same tumor. Ultrastructural and cytochemical analysis of the capillary endothelial wall revealed major alterations in tight junctions, which became permeable to horseradish peroxidase. In brain tissue around the tumors, uptake of 99mTc-glucoheptonate and ultrastructure of tight junctions were comparable to normal brain capillaries. The results of the present study indicate that altered endothelial tight junctions may provide the main route of transport of 99mTc-glucoheptonate through the endothelial wall.

Animals↗