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PubMed1970

The water and nonelectrolyte permeability induced in thin lipid membranes by the polyene antibiotics nystatin and amphotericin B.

Nystatin and amphotericin B increase the permeability of thin (<100 A) lipid membranes to ions, water, and nonelectrolytes. Water and nonelectrolyte permeability increase linearly with membrane conductance (i.e., ion permeability). In the unmodified membrane, the osmotic permeability coefficient, P(f), is equal to the tagged water permeability coefficient, (P(d))(w); in the nystatin- or amphotericin B-treated membrane, P(f)/(P(d))(w) approximately 3. The unmodified membrane is virtually impermeable to small hydrophilic solutes, such as urea, ethylene glycol, and glycerol; the nystatin- or amphotericin B-treated membrane displays a graded permeability to these solutes on the basis of size. This graded permeability is manifest both in the tracer permeabilities, P(d), and in the reflection coefficients, sigma (Table I). The "cutoff" in permeability occurs with molecules about the size of glucose (Stokes-Einstein radius approximate, equals 4 A). We conclude that nystatin and amphotericin B create aqueous pores in thin lipid membranes; the effective radius of these pores is approximately 4 A. There is a marked similarity between the permeability of a nystatin- or amphotericin B-treated membrane to water and small hydrophilic solutes and the permeability of the human red cell membrane to these same molecules.

Amphotericin B↗
PubMed1996

Mechanisms of cigarette smoke induced increased airspace permeability.

BACKGROUND: Increased epithelial permeability of the airspaces occurs commonly in the lungs of cigarette smokers. It is likely to be important in augmenting the inflammatory response in the airspaces and hence may have a role in the pathogenesis of emphysema. It has previously been shown that intratracheal instillation of cigarette smoke condensate induces increased epithelial permeability in vivo in rats and in vitro in epithelial cell monolayers, associated with a disturbance in the lung antioxidant, glutathione (GSH). The aim of this study was to assess the role of neutrophils, GSH, and tumour necrosis factor (TNF) in the increased epithelial permeability following intratracheal instillation of cigarette smoke condensate. METHODS: Epithelial permeability of the airspaces was measured in rat lungs as the passage of intratracheally instilled 125-iodine labelled bovine serum albumin (BSA) into the blood. The permeability of a monolayer of human type II alveolar epithelial cells to 125I-BSA was also measured. RESULTS: Cigarette smoke condensate produced a 59.7% increase in epithelial permeability over control values peaking six hours after instillation and returning to control values by 24 hours. Depletion of neutrophils and, to a lesser extent, macrophages by an intraperitoneal injection of antineutrophil antibody did not influence the increased epithelial permeability induced by cigarette smoke condensate. Although instillation of human recombinant TNF alpha produced an increase in epithelial permeability in the rat lung from 0.62 (0.61)% to 1.27 (0.08)%, only a trivial amount of TNF alpha was detected in bronchoalveolar lavage (BAL) fluid in vivo or in culture medium from BAL leucocytes obtained from animals treated with cigarette smoke condensate (94.9 (28.8) units/ml). Furthermore, antiTNF antibody did not abolish the increased epithelial permeability produced by cigarette smoke condensate. The role of GSH was assessed by measuring the changes in both the reduced (GSH) and oxidised form (GSSG) in lung tissue and in BAL fluid. One hour after instillation of cigarette smoke condensate there was a marked fall in the GSH content in the lung (from 809.8 (31.8) to 501.7 (40.5) nmol/g) in association with increased GSSG levels (from 89.8 (2.7) to 148.7 (48.8) nmol/g). This was followed by a return of GSH levels to control values, with a concomitant decrease in GSSG levels six hours after instillation. GSH levels in BAL fluid fell dramatically following cigarette smoke condensate (from 2.56 (0.30) to 0.31 (0.21) nmol/ml) and this fall was sustained up to six hours after instillation of cigarette smoke condensate. CONCLUSIONS: These studies suggest that neutrophils and TNF do not have a major role in the increased epithelial permeability induced by cigarette smoke condensate. However, the data support a role for the depletion of the antioxidant glutathione in the increased epithelial permeability caused by cigarette smoke condensate.

Animals↗
PubMed1994

Nitric oxide synthesis inhibition increases epithelial permeability via mast cells.

In this study, we assessed the involvement of mast cells and mast cell-derived mediators in the enhanced epithelial permeability associated with nitric oxide synthesis inhibition. Permeability of the small bowel was assessed by measuring the clearance of a small marker (51Cr-labeled EDTA) from blood to lumen in the presence of the nitric oxide synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). L-NAME caused a very rapid (10 min) increase in epithelial permeability, reaching peak values (sixfold increase) within 20 min. Two mast cell stabilizers, doxantrazole and lodoxamide, greatly attenuated the rise in mucosal permeability. Rat mast cell protease II activity (marker of mucosal mast cell degranulation) was increased significantly only in the plasma of L-NAME-treated animals. Chronic dexamethasone administration depleted rats of mucosal mast cells and also prevented the L-NAME-induced rise in mucosal permeability. The increase in epithelial permeability was mediated by a number of mediators: platelet-activating factor caused the early rise in epithelial permeability, and histamine caused the later increase in epithelial permeability. Superoxide dismutase attenuated the L-NAME-induced rise in epithelial permeability, suggesting an important and continuous role for superoxide. Transepithelial flux of 51Cr-EDTA across rat intestinal epithelial cell monolayers did not increase in the presence of L-NAME, suggesting that inhibition of nitric oxide does not directly cause epithelial permeability alterations, whereas the in vivo data implicate a potential role for the mast cell. In conclusion, nitric oxide synthesis inhibition activates mast cells in the mucosa and consequently increases epithelial permeability.

Animals↗
PubMed2003

Intermittent high-permeability hemofiltration modulates inflammatory response in septic patients with multiorgan failure.

BACKGROUND/AIM: Continuous venovenous hemofiltration with high-permeability hemofilters is a novel approach in the adjuvant therapy of septic patients. High-permeability hemofilters are characterized by an increased pore size which facilitates the filtration of inflammatory mediators. The present study examines whether intermittent high-permeability hemofiltration has an immunomodulatory effect on polymorphonuclear leukocytes and mononuclear cells. METHODS: Twenty-eight septic patients with acute renal failure were randomly allocated to either receive intermittent high-permeability or conventional hemofiltration. Intermittent high-permeability hemofiltration consisted of a daily 12-hour course of high-permeability hemofiltration alternated by conventional hemofiltration. For high-permeability hemofiltration, a newly developed high-flux polyamide membrane (P2SH) with a nominal cutoff point of 60 kD was used. For conventional hemofiltration a high-flux polyamide hemofilter (Polyflux 11S, cutoff point 30 kD) was used. RESULTS: The polymorphonuclear leukocyte phagocytosis activity before starting hemofiltration was almost double the rate of healthy controls in both groups (p < 0.001). The phagocytosis rate decreased significantly during the course of intermittent high-permeability hemofiltration (p < 0.05), whereas the values remained unchanged in the conventional hemofiltration group. Incubation of high-permeability filtrates with blood from healthy donors resulted in a significant induction of phagocytosis (p < 0.001), whereas conventional filtrates had no phagocytosis-stimulating effects. In addition, incubation of healthy-donor mononuclear cells with high-permeability but not conventional filtrates resulted in a significant tumor necrosis factor alpha release (p < 0.001). CONCLUSIONS: Intermittent high-permeability hemofiltration is a novel extracorporeal elimination modality which exhibits immunomodulatory effects on leukocytes, attenuating polymorphonuclear neutrophil phagocytosis. Further studies are necessary to elucidate whether these effects translate in a clinical improvement in patients suffering from sepsis.

Acute Kidney Injury↗
PubMed1995

Effect of neutrophil mediators on epithelial permeability.

Inflammatory lung disease is associated with increased epithelial permeability, but it is unclear how inflammatory cells alter epithelial permeability. Neutrophils have azurophilic granules containing elastase, cathepsin G, and defensins which are released at sites of inflammation. Experiments using whole animals and cultured cells suggest that neutrophil elastase contributes to increased epithelial permeability. Using Madin-Darby canine kidney epithelial (MDCK) monolayers, a well-described epithelial model, we asked whether neutrophil elastase directly affects epithelial permeability independent of cell death or cell detachment from the substratum. We measured permeability using 3H-mannitol. We found that neutrophil elastase increased epithelial permeability in a time- and concentration-dependent fashion. Increased permeability required prolonged (> or = 6 h) exposure to elastase, but was not associated with cytolytic injury or cell detachment. These findings are potentially relevant to the lung because we found a similar time- and concentration-dependent effect when we added elastase to cultured human bronchial epithelial cells. In MDCK cells, permeability increased without alterations in cell actin at the light microscopic level. Interestingly, elastase-induced permeability was both prevented and reversed by serum, but not by serum albumin. Complete reversal occurred if serum was added up to 16 h after adding elastase. Proteolytic activity is important in HNE-induced epithelial permeability because soy bean trypsin inhibitor completely blocks the effect and alpha 1 proteinase inhibitor (alpha 1 PI) partially blocks the effect. Charge interactions also appear to be important because the polyanions heparin and sulfated dextran completely blocked increased permeability following elastase but only partially blocked elastolytic activity in isotonic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Paracellular and transcellular pathways facilitate insulin permeability in rat gut.

The aim of this study was to conduct a systematic investigation of the absorption of insulin in the rat intestine in the presence of permeation enhancers and protease inhibitors. An in-situ perfused rat gut model was used for the co-perfusion of insulin and PEG 4000 in the presence or absence of bile salts, bile salt:fatty acid surfactant systems and protease inhibitors. Perfusion experiments were conducted for 180 min with perfusate and blood collection at regular intervals. Permeability coefficients for insulin were calculated from plasma insulin and PEG 4000 permeability coefficients were calculated from lumenal disappearance data. In the absence of enzyme inhibitors, insulin permeability was consistently lower than PEG 4000, but increased in proportion to PEG 4000 permeability. Large increases in insulin permeability were obtained for mixed micellar systems and protease inhibitors. In the presence of protease inhibitors and simple micelle systems, PEG 4000 permeability was three-fold greater than insulin permeability. In the presence of absorption enhancers, PEG 4000 permeability increased up to a maximum value of 3.63 x 10(-6)cm s(-1), a value five-fold less than that of the estimated aqueous boundary layer permeability for PEG 4000. This suggests that PEG 4000 permeability is primarily membrane controlled. Insulin permeability is enhanced to a maximum value of 9.17 x 10(-6)cm s(-1), suggesting that paracellular transport routes do not account exclusively for insulin permeation across the intestinal epithelium. The results add support to suggestions that routes other than the paracellular route may contribute to insulin absorption in rat gut.

Animals↗
PubMed1988

The effect of oxygen free radicals on calcium permeability and calcium loading at steady state in cardiac sarcoplasmic reticulum.

It has been proposed that oxygen free radical production is an important mediator of the myocardial dysfunction during the course of acute ischemia. We tested this hypothesis by characterizing the pathway of calcium efflux across sarcoplasmic reticulum (SR) membranes affected by oxygen free radicals. The effect of oxygen free radicals on the steady state calcium load, calcium permeability, and Ca,Mg-ATPase activity of isolated canine cardiac SR vesicles was investigated at pH 7.0. In vitro generation of oxygen free radicals by xanthine oxidase (0.09 units/ml), acting on xanthine in doses up to 50 microM as a substrate, increased the permeability of the SR vesicles to calcium, determined by measuring net efflux of calcium after stopping pump-mediated fluxes, and decreased total intravesicular calcium and free intravesicular calcium with no effect on Ca,Mg-ATPase activity. The effect of oxygen free radicals on calcium permeability was calcium gradient-dependent. Xanthine alone or xanthine plus denatured xanthine oxidase had no effect on this system. Superoxide dismutase (SOD, 56 units/ml), but not denatured SOD, significantly inhibited the effect of xanthine-xanthine oxidase reaction. The calcium permeability of the SR membrane decreased with decreasing calcium load. In addition, inasmuch as extravesicular calcium exerts only a slight effect on calcium permeability, the decrease in the permeability with calcium load is specifically related to the calcium load. Oxygen free radical-induced increase in calcium permeability was unaffected by Mg concentration between 2.1 and 21 mM. In summary, our data reveal that .O2- can produce a diminished level of accumulated calcium, which is reflected by the decreased calcium load and an increase in passive calcium permeability, and that the decreased calcium accumulation in the presence of the xanthine-xanthine oxidase system may not be mainly due to an inhibited calcium pump but due to an increased calcium permeability. Our results also suggest that increased SR membrane passive calcium permeability induced by oxygen free radicals is not carrier mediated. It is postulated that, with the oxygen free radical-mediated progressive increase in calcium permeability, free cytosolic calcium concentrations would increase in ischemic myocardium.

Animals↗
PubMed1984

Permeability of latex and polyvinyl chloride gloves to 20 antineoplastic drugs.

Permeability of latex and polyvinyl chloride (PVC) gloves to 20 injectable antineoplastic drugs was studied. Four types of gloves were evaluated: latex surgical gloves, latex examination gloves, and PVC gloves in two thicknesses. Each glove material was exposed to each drug for 90 minutes, and permeation was tested using a mutagenicity assay. Individual fingertips of thin PVC gloves and latex surgical gloves were tested for permeability at five time points (2-30 minutes) using a doxorubicin coloration assay. All drugs permeated the thin PVC gloves. Latex surgical gloves were definitely permeable to two drugs (carmustine and thiotepa) and exhibited borderline permeability to mechlorethamine hydrochloride. The thick PVC gloves were definitely permeable to four drugs (carmustine, thiotepa, mechlorethamine hydrochloride, and daunorubicin hydrochloride) and exhibited borderline permeability to two drugs (doxorubicin and mercaptopurine). The latex examination gloves were permeable to carmustine, thiotepa, mechlorethamine hydrochloride, and cyclophosphamide. Doxorubicin permeation of individual fingertips of thin PVC gloves varied in time and amount. Doxorubicin did not permeate the latex surgical glove material, but testing with thiotepa showed that individual fingertips of this material also varied in permeability. Glove thickness was a major determinant of permeability; latex surgical gloves were the least permeable and thin PVC gloves the most permeable to the antineoplastic drugs tested. Within individual gloves and glove types, time and amount of permeation were not uniform.

Antineoplastic Agents↗
PubMed1997

The mitochondrial permeability transition in toxic, hypoxic and reperfusion injury.

Opening of a non-specific, high conductance permeability transition pore or megachannel in the inner mitochondrial membrane causes onset of the mitochondrial permeability transition, which is characterized by mitochondrial swelling, depolarization and uncoupling. Inducers of the permeability transition include Ca2+, oxidant stress and a permissive pH greater than 7.0. Blockers include cyclosporin A, trifluoperazine and pH < 7. Using laser scanning confocal microscopy, we developed techniques to visualize onset of the mitochondrial permeability transition in situ in living cells. In untreated cells, the permeability transition pore is continuously closed and does not 'flicker' open. By contrast, the pore opens in liver and heart cells after exposure to oxidant chemicals, calcium ionophore, hypoxia and ischemia/reperfusion, causing mitochondrial uncoupling and aggravation of ATP depletion. In injury to hepatocytes from tert-butylhydroperoxide, an analog of lipid hydroperoxides generated during oxidative stress, onset of the mitochondrial permeability transition is preceded by oxidation of mitochondrial pyridine nucleotides, mitochondrial generation of oxygen radicals and an increase of mitochondrial Ca2+, all inducers of the mitochondrial permeability transition. In ischemia, the acidosis of anaerobic metabolism protects strongly against cell death. During reperfusion, recovery of pH to normal levels is a stress that actually precipitates cell killing. Onset of the mitochondrial permeability transition may be responsible, in part, for this pH-dependent injury, or pH paradox. The mitochondrial permeability transition may also be responsible for a variety of pathological phenomena. In particular, the mitochondrial permeability transition may underlie Reye's syndrome and Reye's-like drug toxicities. In conclusion, multiple mechanisms contribute to cell injury after hypoxia, ischemia/reperfusion and toxic chemicals, but a common final pathway leading to acute cellular necrosis may be ATP depletion after mitochondrial failure. One important mechanism causing mitochondrial failure is the mitochondrial permeability transition, which both uncouples oxidative phosphorylation and accelerates ATP hydrolysis. Interventions that block this pH-dependent phenomenon protect against onset of cell death.

Animals↗
PubMed2000

Inflammatory mediators and modulation of blood-brain barrier permeability.

1. Unlike some interfaces between the blood and the nervous system (e.g., nerve perineurium), the brain endothelium forming the blood-brain barrier can be modulated by a range of inflammatory mediators. The mechanisms underlying this modulation are reviewed, and the implications for therapy of the brain discussed. 2. Methods for measuring blood-brain barrier permeability in situ include the use of radiolabeled tracers in parenchymal vessels and measurements of transendothelial resistance and rate of loss of fluorescent dye in single pial microvessels. In vitro studies on culture models provide details of the signal transduction mechanisms involved. 3. Routes for penetration of polar solutes across the brain endothelium include the paracellular tight junctional pathway (usually very tight) and vesicular mechanisms. Inflammatory mediators have been reported to influence both pathways, but the clearest evidence is for modulation of tight junctions. 4. In addition to the brain endothelium, cell types involved in inflammatory reactions include several closely associated cells including pericytes, astrocytes, smooth muscle, microglia, mast cells, and neurons. In situ it is often difficult to identify the site of action of a vasoactive agent. In vitro models of brain endothelium are experimentally simpler but may also lack important features generated in situ by cell:cell interaction (e.g. induction, signaling). 5. Many inflammatory agents increase both endothelial permeability and vessel diameter, together contributing to significant leak across the blood-brain barrier and cerebral edema. This review concentrates on changes in endothelial permeability by focusing on studies in which changes in vessel diameter are minimized. 6. Bradykinin (Bk) increases blood-brain barrier permeability by acting on B2 receptors. The downstream events reported include elevation of [Ca2+]i, activation of phospholipase A2, release of arachidonic acid, and production of free radicals, with evidence that IL-1 beta potentiates the actions of Bk in ischemia. 7. Serotonin (5HT) has been reported to increase blood-brain barrier permeability in some but not all studies. Where barrier opening was seen, there was evidence for activation of 5-HT2 receptors and a calcium-dependent permeability increase. 8. Histamine is one of the few central nervous system neurotransmitters found to cause consistent blood-brain barrier opening. The earlier literature was unclear, but studies of pial vessels and cultured endothelium reveal increased permeability mediated by H2 receptors and elevation of [Ca2+]i and an H1 receptor-mediated reduction in permeability coupled to an elevation of cAMP. 9. Brain endothelial cells express nucleotide receptors for ATP, UTP, and ADP, with activation causing increased blood-brain barrier permeability. The effects are mediated predominantly via a P2U (P2Y2) G-protein-coupled receptor causing an elevation of [Ca2+]i; a P2Y1 receptor acting via inhibition of adenyl cyclase has been reported in some in vitro preparations. 10. Arachidonic acid is elevated in some neural pathologies and causes gross opening of the blood-brain barrier to large molecules including proteins. There is evidence that arachidonic acid acts via generation of free radicals in the course of its metabolism by cyclooxygenase and lipoxygenase pathways. 11. The mechanisms described reveal a range of interrelated pathways by which influences from the brain side or the blood side can modulate blood-brain barrier permeability. Knowledge of the mechanisms is already being exploited for deliberate opening of the blood-brain barrier for drug delivery to the brain, and the pathways capable of reducing permeability hold promise for therapeutic treatment of inflammation and cerebral edema.

Animals↗
PubMed1994

Differential role of two Ca(2+)-permeable non-NMDA glutamate channels in rat retinal ganglion cells: kainate-induced cytoplasmic and nuclear Ca2+ signals.

1. The permeability of non-N-methyl-D-aspartate (non-NMDA) glutamate channels to divalent cations and specifically the entry of Ca2+ and subsequent elevations in cytoplasmic and nuclear Ca2+ signals were investigated in cultured neonatal rat retinal ganglion cells using the whole cell patch-clamp technique and Ca2+ imaging with confocal microscopy. In addition, divalent-permeable non-NMDA receptor channels were studied in retinal slices using a Co2+ staining technique. 2. Using Ca2+ (2.5 mM) as the only permeable cation in the external solution, stimulation with 100 microM kainate produced nondesensitizing, nonselective cation currents with either low or high Ca2+ permeability. Both currents were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Neurons with the low divalent-permeable currents (type 1) had reversal potentials of -41.5 +/- 4.4 mV (mean +/- SD), and neurons with the high divalent-permeable currents (type 2) had reversal potentials of -22.6 +/- 5.5 mV. The permeability ratio PCa/PCs was 3.3 for the type 1 currents and 8.5 for the type 2 currents, indicating a 2.5-fold greater permeability to Ca2+ for the type 2 non-NMDA glutamate channels. 3. Both types of non-NMDA glutamate channels showed relatively little selectivity between Ca2+ and Co2+. The type 1 neurons had a slightly higher permeability to Co2+ than to Ca2+, whereas the type 2 neurons were equally permeable to both divalent cations. The type 2 neurons had a much higher permeability for both divalent cations compared with the type 1 neurons. 4. Staining for Co2+ uptake through kainate-stimulated non-NMDA glutamate channels in retinal slices provided additional evidence for the presence of the two ganglion cell populations. Activation of the neurons by kainate in conditions isolating the non-NMDA glutamate channel caused differential uptake of Co2+. In contrast, depolarization in the presence of the non-NMDA antagonist CNQX failed to cause Co2+ influx. 5. Imaging experiments using confocal microscopy showed that kainate stimulation induced an increase in intracellular Ca2+ in both types of retinal ganglion cells, but only the type 2 neurons showed a substantial increase in cytoplasmic and nuclear Ca2+ signals. Kainate-induced Ca2+ signals in the type 2 neurons were almost nine times greater than those of the type 1 neurons. 6. When intracellular Ca2+ stores were depleted by brief treatment with thapsigargin, kainate-induced Ca2+ signals in the type 1 neurons were unchanged. However, in the type 2 neurons kainate no longer induced large Ca2+ signals in the cytoplasm and nucleus, despite normal influx of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

[Glomerular permeability for serum proteins in different morphological types of primary chronic glomerulopathy].

Membranous glomerulonephritis (MGN), mesangiocapillary (MCGN), membranoproliferative glomerulonephritis (MPGN) and focal segmental sclerosis or hyalinosis (FSSH) were studied for glomerular filter permeability to serum albumins, IgA and IgG. In MGN the permeability for large-molecular globulins is not dependent on that for albumin, permeabilities for the globulins appeared correlated. In MPGN permeability for IgG depends on albumins permeability, correlations between that for IgA and IgG are similar to relevant findings in MGN. In MCGN glomerular permeability for IgA depends on that for albumins, and IgG depends on IgA permeability. In FSSH better albumin permeability implies increased permeability for both globulins, while enhanced permeability for IgA entails the same trend for albumins and IgG. Variable permeability of the glomerular filter for serum proteins in different morphological forms of chronic glomerulopathy may result from dissimilar defects in diverse layers of the filter and in interaction of basal membrane structures with cells responsible for glomerular impermeability for serum proteins.

Adolescent↗
PubMed2005

Maturation of rat proximal tubule chloride permeability.

We have previously shown that neonate rabbit tubules have a lower chloride permeability but comparable mannitol permeability compared with adult proximal tubules. The surprising finding of lower chloride permeability in neonate proximals compared with adults impacts net chloride transport in this segment, which reabsorbs 60% of the filtered chloride in adults. However, this maturational difference in chloride permeability may not be applicable to other species. The present in vitro microperfusion study directly examined the chloride and mannitol permeability using in vitro perfused rat proximal tubules during postnatal maturation. Whereas there was no maturational change in mannitol permeability, chloride permeability was 6.3 +/- 1.3 x 10(-5) cm/s in neonate rat proximal convoluted tubule and 16.1 +/- 2.3 x 10(-5) cm/s in adult rat proximal convoluted tubule (P < 0.01). There was also a maturational increase in chloride permeability in the rat proximal straight tubule (5.1 +/- 0.6 x 10(-5) cm/s vs. 9.3 +/- 0.6 x 10(-5) cm/s, P < 0.01). There was no maturational change in bicarbonate-to-chloride permeabilities (P(HCO3)/P(Cl)) in the rat proximal straight tubules (PST) and proximal convoluted tubules (PCT) or in the sodium-to-chloride permeability (P(Na)/P(Cl)) in the proximal straight tubule; however, there was a significant maturational decrease in proximal convoluted tubule P(Na)/P(Cl) with postnatal development (1.31 +/- 0.12 in neonates vs. 0.75 +/- 0.06 in adults, P < 0.001). There was no difference in the transepithelial resistance measured by current injection and cable analysis in the PCT, but there was a maturational decrease in the PST (7.2 +/- 0.8 vs. 4.6 +/- 0.1 ohms x cm2, P < 0.05). These studies demonstrate there are maturational changes in the rat paracellular pathway that impact net NaCl transport during development.

Aging↗
PubMed2000

Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.

PURPOSE: The objective of this study was a systematic characterization and evaluation of cell culture models based on mixtures of Caco-2/HT29-MTX co-cultures for their use in screening for drug absorption and intestinal permeability in comparison to the properties of the respective mono-cultures. METHODS: Co-cultures of Caco-2 cells (absorptive-type) and HT29-MTX cells (goblet-type) were set up. Three different co-cultures (initial seeding ratios Caco-2/HT29-MTX: 90/10, 70/30, and 50/50) were grown on permeable filter supports, and monolayers were used for permeability studies with model compounds for paracellular absorption (atenolol, furosemide, H334/75, mannitol, terbutaline), transcellular absorption (antipyrine, ketoprofen, metoprolol, piroxicam), carrier-mediated absorption (D-glucose, Gly-Pro, and L-phenylalanine) as well as substrates for carrier-mediated secretion via P-glycoprotein (cimetidine and talinolol). Electrophysiological and microscopic controls were performed to characterize the cell cultures. RESULTS: For compounds undergoing passive intestinal absorption permeabilities were generally higher in co-cultures than in Caco-2 monolayers, yielding highest values in pure HT29-MTX monolayers. This difference was most obvious for compounds transported via the paracellular pathway, where HT29-MTX cells may be up to 30 times more permeable than Caco-2 cells, whereas for lipophilic and highly permeable compounds, the difference in permeability values was less obvious. For drugs undergoing intestinal secretion mediated by P-glycoprotein, co-cultivation of Caco-2 cells with HT29-MTX cells led to increased apical to basolateral permeability which was decreased in the opposite direction, consistent with the fact that HT29-MTX cells do not express P-glycoprotein. When a carrier-mediated absorption mechanism is involved, the permeabilities observed were lower than the values reported for human small intestine and co-cultivation of HT29-MTX cells with Caco-2 cells resulted in even lower values as compared to the plain Caco-2 cultures. CONCLUSIONS: Co-cultures of HT29-MTX and Caco-2 cells offer the opportunity of modifying the permeability barrier of the cell monolayers both with respect to paracellular resistance and secretory transport via P-gp. Thus, in special cases, they allow more flexibility in adapting the in vitro system to the in vivo situation as compared to the monocultures. Another advantage is the obvious robustness of the method with respect to the reproducibility of the results. A problem remaining, however, is the quantitative expression of carriers involved in intestinal uptake of many nutrients and drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Contribution of the paracellular route to the pH-dependent epithelial permeability to cationic drugs.

The aim of this work was to investigate the contribution of the paracellular route to the pH-dependent permeability to cationic drugs in three models expressing different drug permeabilities: hexadecane membranes (HDMs), Caco-2, and 2/4/A1 cell monolayers. The high- and low-permeability drugs alfentanil and cimetidine were used as model drugs. The paracellular permeability was calculated: 1. from the assumption that the ionized form (P(mi)) permeates a cell monolayer only by the paracellular route, and 2. on basis of the pore-restricted diffusion. For both drugs, sigmoidal relationships between membrane permeability and pH were observed in all models. The P(mi) was in excellent agreement with the paracellular permeability of cimetidine in the two cell models, whereas no significant P(mi) of the drugs could be observed in HDM. The results showed that the paracellular route has a significant role in the permeability of small basic hydrophilic drugs, such as cimetidine in leaky, small intestinal-like epithelia such as 2/4/A1. By contrast, in tighter epithelia such as Caco-2 and in artificial membranes such as HDM, the permeability of the ionized forms of the drugs and the paracellular permeability are lower or insignificant, respectively. These findings will have implications in the experimental design and data interpretation of pH-dependent drug transport experiments in cell culture models as well as in artificial membrane models such as HDM and parallel artificial membrane permeability assay (PAMPA).

Administration, Cutaneous↗
PubMed1996

A quantitative spatial analysis of the blood-spinal cord barrier. I. Permeability changes after experimental spinal contusion injury.

Blood-spinal cord barrier (BSB) permeability was measured using quantitative autoradiography following contusion injury to the rat spinal cord. Permeability was assessed by calculating blood-to-tissue transfer constants (Ki values) for the vascular tracer [14C]-alpha-aminoisobutyric acid (AIB) in injured (3, 7, 14, and 28 days postinjury), laminectomy control, and uninjured control animals. Permeability was quantitated using four separate imaging techniques in gray and white matter throughout the rostro-caudal extents of the forming lesion. Away from the epicenter, gray matter permeability was further differentiated within discrete spinal lamina using computerized templates. Regardless of the type of analysis used, increased AIB permeability (Ki values) was noted at all survival times in all tissue regions with respect to both uninjured and laminectomy control groups. The data indicate a large increase in individual Ki values throughout the dorsoventral axis of the spinal cord at 3 days postinjury (approximately 6-9 ml/kg/min). By 7 days, Ki values were quantitatively smaller (approximately 4-5 ml/kg/min) in all regions compared with 3-day tissues. Despite further attenuation of AIB uptake in the gray matter at 14 and 28 days postinjury, circumferential white matter tracts showed a secondary increase in permeability compared to 7-day tissue. Permeability in the white matter at 14-28 days postinjury (approximately 5-6 ml/kg/min) was comparable to that at 3 days postinjury (6-7 ml/kg/min). Measurements of the axial distribution of AIB permeability indicate increased BSB permeability over several segments rostral and caudal to the lesion epicenter (approximately 3 cm in both directions). Secondary elevations of AIB transfer in the spinal white matter between 14 and 28 days were colocalized with zones of immunohistochemically defined microglial clusters. The known plasticity of this cell type in response to changes in the extracellular microenvironment suggests that the spinal white matter at later survival times (14-28 days postinjury) is an area of dynamic vascular and/or axonal reconstruction. The implications of increased permeability to both tissue injury and neural regeneration are discussed.

Aminoisobutyric Acids↗
PubMed2001

Synergism between leukocyte adherence and shear determines venular permeability in the presence of nitric oxide.

The effects of nitric oxide (NO) on vascular permeability tend to be controversial. While many studies indicate that NO plays a protective role in several models of inflammation by reducing leukocyte-endothelial cell adhesion, in vitro studies have shown that endothelial permeability to macromolecules increases with increasing shear forces via a NO-dependent mechanism. Most investigations of the role of shear on endothelial permeability have been performed either in vitro or in cannulated vessels, but whether NO and leukocyte adherence (WBC-adh) mediate shear-induced control of albumin leakage in autoperfused venules has yet to be determined. By measuring permeability under regular conditions of blood flow and WBC-adh, we recently found that venular albumin permeability of the rat mesentery correlates strongly with basal levels of both shear rate and WBC-adh. Using the same model in the present study, we were able to further elucidate some of the mechanisms and mediators of venular permeability. In one set of experiments, a role for NO in shear-mediated permeability was confirmed. In additional experiments, a permeability-enhancing collaboration between shear and WBC-adh was revealed: shear-induced permeability was found to be dependent on the presence of adherent leukocytes, and similarly, leukocyte-mediated permeability was found to be dependent on shear. This synergism was present both under basal conditions and following the inflammatory stimulus of ischemia-reperfusion.

Animals↗
PubMed1992

Role of nitric oxide in modulating permeability of hamster cheek pouch in response to adenosine 5'-diphosphate and bradykinin.

The goal of this study was to determine the role of the synthesis and release of nitric oxide in modulating alterations in microvascular permeability of the hamster cheek pouch in response to adenosine 5'-diphosphate and bradykinin. We used intra-vital fluorescent microscopy to examine the permeability of the hamster cheek pouch to agonists before and following application of enzymatic inhibitors of nitric oxide, NG-monomethyl-L-arginine (L-NMMA; 0.01, 0.1, and 1.0 microM) and NW-nitro-L-arginine methyl ester (L-NAME; 0.01, 0.1, and 1.0 microM). Increases in permeability of the hamster cheek pouch were quantitated by the formation of microvascular leaky sites. ADP and bradykinin produced an increase in the number of venular leaky sites, and superfusion of L-NMMA and L-NAME significantly decreased ADP- and bradykinin-induced increases in microvascular permeability. To determine the specificity of nitric oxide blockade on microvascular permeability, we examined changes in permeability in response to adenosine, and examined the effects of D-NMMA on microvascular permeability. Adenosine-induced increases in permeability were not altered by treatment with L-NMMA, and D-NMMA did not inhibit ADP-induced increases in microvascular permeability. Thus, these findings suggest that production of nitric oxide, in response to application of ADP and bradykinin, has a role in modulating macromolecular permeability of the hamster cheek pouch in vivo.

Adenosine Diphosphate↗