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PubMed1998

Epithelial permeability induced by neutrophil transmigration is potentiated by hypoxia: role of intracellular cAMP.

Mucosal tissues, such as the lung and intestine, are primary targets for ischemic damage. Under these conditions, neutrophil (polymorphonuclear leukocyte; PMN) infiltration into the protective epithelium has been implicated as a pathophysiologic mediator. Because PMN transepithelial migration results in increased paracellular permeability, and because our previous data revealed that epithelial hypoxia enhances PMN transmigration, we hypothesized that macromolecular permeability may be altered in epithelium exposed to hypoxia and reoxygenation (H/R) in the presence of PMNs. Human intestinal epithelia (T84) were grown on permeable supports, exposed to cellular hypoxia (pO2 20 torr) for 0-72 hr, and examined for increases in PMN-evoked permeability by using standard flux assays. Increasing epithelial hypoxia potentiated PMN-induced permeability of labeled paracellular tracers (size range 3-500 kD). Such increases were blocked by monoclonal antibody (mAb) to the PMN integrin CD11b (82 +/- 1% decreased compared with control mAb) and were partially blocked by anti-CD47 mAb (51 +/- 1%). Assessment of barrier recovery revealed that monolayers exposed to H/R were significantly diminished in their ability to reseal following PMN transmigration (recovery of 36 +/- 6% in H/R vs. 94 +/- 2% in normoxic controls). Because intracellular cyclic AMP (cAMP) has been demonstrated to regulate epithelial permeability, and because PMN-derived compound(s), (i.e., 5'-adenosine monophosphate; AMP) elevate epithelial cAMP, we examined the impact of hypoxia on epithelial cAMP responses. These experiments revealed that hypoxic epithelia were diminished in their ability to generate cAMP, and pharmacologic elevation (8-bromo-cAMP) of intracellular cAMP in hypoxic cells normalized both PMN-induced permeability changes and restoration of barrier function. These results support a role for PMN in increased intestinal permeability associated with reperfusion injury and imply a substantial role for cAMP signaling in maintenance of permeability during PMN transmigration.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of cytoskeletal involvement.

Recent studies suggest that an abnormal increase in intestinal tight junction (TJ) permeability may be an important etiologic factor in number of diseases including Crohn's disease, NSAID-associated enteritis, and various infectious diarrheal syndromes. The intracellular processes involved in regulation of intestinal epithelial TJ permeability, however, remain poorly understood. In this study, we used cultured Caco-2 intestinal epithelial cells to examine the intracellular processes involved in extracellular Ca(++) modulation of intestinal epithelial monolayer TJ barrier. Incubation of the filter-grown Caco-2 intestinal monolayers in Ca(++)-free solution (CFS), consisting of modified Krebs-buffer solution containing 0 mM Ca(++) and 1 mM EGTA, resulted in a rapid drop in Caco-2 epithelial resistance and increase in epithelial permeability to paracellular markers mannitol and inulin, indicating an increase in TJ permeability. The increase in Caco-2 TJ permeability was rapidly reversed by the re-introduction of Ca(++) (1.8 mM) into the incubation medium. The CFS-induced increase in Caco-2 TJ permeability was associated with separation of the cytoplasmic and transmembrane TJ proteins, ZO-1 and occludin, and formation of large intercellular openings between the adjoining cells. The CFS-induced modulation of TJ barrier was associated with activation of myosin light chain kinase (MLCK) activity and centripetal retraction of peri-junctional actin and myosin filaments. The inhibition of CFS-induced activation of Caco-2 MLCK with MLCK inhibitor (ML-7) prevented the CFS-induced retraction of actin and myosin filaments and the subsequent alteration of TJ barrier function and structure. Our results suggested that the CFS-induced alteration of TJ proteins and functional increase in TJ permeability was mediated by Caco-2 MLCK activation and the resultant contraction of the peri-junctionally located actin-myosin filaments. Consistent with the role of MLCK in this process, selected inhibitors of Mg(++)-myosin ATPase and metabolic energy, but not protein synthesis inhibitors, also prevented the CFS-induced retraction of actin and myosin filaments and the subsequent increase in TJ permeability. In conclusion, our results indicate that extracellular Ca(++) is crucial for the maintenance of intestinal epithelial TJ barrier function. The removal of extracellular Ca(++) from the incubation medium causes activation of Caco-2 MLCK, which in turn leads to an increase in intestinal monolayer TJ permeability.

Actins↗
PubMed1990

Stimulation of cloned human T lymphocytes via the CD3 or CD28 molecules induces enhancement in vascular endothelial permeability to macromolecules with participation of type-1 and type-2 intercellular adhesion pathways.

Perivascular accumulation of CD29+CD45R0+ memory T lymphocytes at sites of chronic inflammation such as rheumatoid synovium is commonly associated with the localized increase in the endothelial permeability. We have recently demonstrated that a direct interaction between activated CD29+CD45R0+ memory T lymphocytes and vascular endothelial cells (EC) results in the increased permeability of EC. In this report, we have investigated effects on antigen-specific T cell receptor (TcR) alpha/beta+ human T lymphocyte clones on the endothelial permeability to albumin. Our results show that CD29+CD45R0+ cloned human T lymphocytes augment endothelial permeability by a noncytolytic process requiring surface contact between T lymphocytes and EC. Both cytolytic and noncytolytic cloned T lymphocytes were capable of augmenting endothelial permeability and this process did not involve active lysis of EC. Stimulation of T lymphocytes via the CD3/TcR or CD28 molecules resulted in significant enhancement in the ability of T lymphocytes to influence endothelial permeability. Pretreatment of T lymphocytes with monoclonal antibodies directed at either CD11a/CD18 (LFA-1) or CD2 molecules or that of EC with monoclonal antibodies directed at either CD54 (ICAM-1) or CD58 (LFA-3) molecules significantly inhibited T lymphocyte-induced enhancement in endothelial permeability, thus indicating that activated T lymphocytes utilize both type-1 (CD11a/CD18CD54) and type-2 (CD2CD58) intercellular adhesion pathways to augment endothelial permeability and signals received via CD3 or CD28 molecules on T lymphocytes further enhance this process. Furthermore, proinflammatory cytokines interleukin 1 and tumor necrosis factor but not proinflammatory cytokines interleukin 1 and tumor necrosis factor but not interleukin 6 induced resistance in EC to T lymphocyte-mediated effects on their permeability. Collectively, these observations may provide insights into molecular mechanism(s) underlying pathophysiology of localized chronic inflammatory responses in general and more specifically selective accumulation of chronically activated memory T lymphocytes at sites of chronic inflammation.

Antigens, Differentiation↗
PubMed1999

The role of low (< or = 1 mM) phosphate concentrations in regulation of mitochondrial permeability: modulation of matrix free Ca2+ concentration.

Under a variety of conditions, the permeability of the inner mitochondrial membrane to small solutes can be nonselectively increased. A classic mitochondrial permeability transition (MPT) was originally identified based on its dependence on matrix Ca2+ and its extreme sensitivity to cyclosporin A (CsA). It is now clear, however, that several additional and distinct processes can also produce increases in mitochondrial permeability. Both mitochondrial signal peptides (P. M. Sokolove and K. W. Kinnally, 1996, Arch. Biochem. Biophys. 336, 69-76) and butylated hydroxytoluene (BHT) (P. M. Sokolove and L. M. Haley, 1996, J. Bioenerg. Biomembr. 28, 199-206), for example, induce permeability increases that are relatively CsA insensitive and that persist in the presence of EGTA. Inorganic phosphate (Pi) appears to play a key role in each of these permeability increases. High (>1 mM) Pi levels facilitate the classic MPT, while Pi concentrations below 1 mM stimulate the permeability increase induced by signal peptides and inhibit that triggered by BHT. The effect of high Pi concentrations can most probably be explained by exchange of the anion for matrix ADP and the resulting alleviation of ADP-mediated inhibition of the MPT (R. G. Lapidus and P. M. Sokolove, 1994, J. Biol. Chem. 269, 18931-18936). In the experiments reported here, the mechanisms underlying the effects of low Pi concentrations on mitochondrial permeability were investigated, by monitoring mitochondrial volume, with the following results: (1) A hitherto unrecognized ability of Pi (<1 mM) to increase the lag preceding induction of the classic MPT by diamide, phenylarsine oxide, and t-butylhydroperoxide was identified. (2) Data were obtained suggesting that all of the effects of low Pi concentration, stimulation of signal peptide-induced swelling, blockade of BHT-induced swelling, and delay of the classic MPT, can be attributed to the capacity of the anion to complex Ca2+ in the mitochondrial matrix. (3) Differences in the responses of these three systems for enhancing mitochondrial permeability to experimental manipulation indicate that matrix Ca2+ plays more than one role in the regulation of mitochondrial permeability. An additional important finding is the observation that failure of EGTA to alter a mitochondrial process need not mean that the process is Ca2+ independent. In a multicompartment system, absence of EGTA action may instead reflect failure of the chelator to gain access to regulatory Ca2+.

Acetates↗
PubMed1997

Blood-brain barrier permeability to ebiratide and TNF in acute spinal cord injury.

Spinal cord injury (SCI) in mammals has a poor outcome because of a lack of regeneration. Alteration of the local environment after injury may induce regeneration. However, the passage of blood-borne or exogenous neurotrophic substances through the blood-brain barrier (BBB) is not well characterized in either normal or injured states. We investigated the permeability of the BBB in normal and injured states to two markers of permeability (albumin and sucrose), to a peptide (ebiratide), and to a cytokine [tumor necrosis factor-alpha(TNF)]. We found that in normal mice the cervical and lumbar areas of the spinal cord were more permeable than the thoracic area and the brain to all four substances. The penetration of the alpha-MSH/ACTH analogue ebiratide and of TNF, substances that have saturable transport systems across the BBB and may be involved in regenerative processes in the CNS, followed a regional pattern of differential permeability comparable to that of albumin and sucrose. Complete transection at the lumbar level induced a temporal change in the permeability of the BBB. The increased permeability, as measured by the radioactively labeled tracers albumin and sucrose, was most apparent in the lumbar region proximal to the transection. After SCI, the permeability to ebiratide remained unchanged, suggesting that disruption of the BBB did not affect the transport system for ebiratide. By contrast, the increase of permeability to TNF exceeded that detected by the markers albumin and sucrose. This enhanced permeability was inhibited by excess unlabeled TNF in the blood, showing saturability. This suggests that the transport system for TNF may be activated in SCI.

Acute Disease↗
PubMed2001

Epithelial permeability is not increased in rats following small bowel resection.

BACKGROUND: Increased intestinal permeability and translocation of bacteria and/or bacterial products may cause infection and liver dysfunction in patients with the short bowel syndrome. In previous studies, serum from mice undergoing small bowel resection (SBR) enhanced growth of cultured rat intestinal epithelial cells (RIEC-6), implicating a role for a serum factor(s) in the enterocyte response to SBR. These experiments tested the hypothesis that epithelial cell permeability is increased following SBR. MATERIALS AND METHODS: Male Sprague-Dawley rats underwent a 75% SBR or sham operation. Intestinal permeability in the remnant ileum was determined by Ussing chambers on Postoperative Day (POD) 3. Additionally, serum was collected on POD 1, 3, and 7 and mesenteric lymph was harvested on POD 3. Once confluent, RIEC-6 cells were incubated for 3 days in media supplemented with 10% fetal bovine serum (FBS; control), 1% FBS, 1% FBS plus 9% Sham serum, or 1% FBS plus 9% SBR serum or exposed to media with varied concentrations of SBR or Sham lymph. Monolayer permeability was determined by measuring the passage of dextran-rhodamine. RESULTS: Intestinal permeability was reduced in rats undergoing SBR. Sham serum-treated monolayers demonstrated the greatest permeability. Incubation with SBR serum reduced permeability to near control media. There were no permeability differences between SBR and Sham lymph-treated monolayers. CONCLUSION: The early adaptive response of the remnant intestine after SBR is associated with reduced permeability. These results suggest an alternative mechanism for the increased bacterial translocation that has been described following SBR.

Adaptation, Biological↗
PubMed2001

VEGF increases permeability of the endothelial cell monolayer by activation of PKB/akt, endothelial nitric-oxide synthase, and MAP kinase pathways.

VEGF is a key regulator of vascular permeability. However, its signaling pathways are incompletely understood. We tested the hypothesis that VEGF regulates endothelial cell (EC) permeability by activating PKB/akt, NOS, and MAP kinase dependent pathways using human umbilical vein EC (HUVEC). Permeability was measured from FITC-dextran 70-kDa flux across the EC monolayer at baseline and after VEGF at 0.034, 0.068, 1, 10, and 100 nM. VEGF increased HUVEC permeability to FITC-dextran in a dose-dependent manner. VEGF (1 nM) increased permeability from 3.9 x 10(-6) +/- 0.7 x 10(-6) to 14.0 x 10(-6) +/- 1.7 x 10(-6) cm/s (mean +/- SEM; P < 0.001). Permeability changes were also assessed after treatment with 1, 10, and 100 nM wortmannin (PI 3-kinase inhibitor); 0.01, 0.1, and 1.0 nM LY294002 (PI 3-kinase inhibitor); 200 microM l-NMMA (NOS inhibitor); 2.7 microM AG126 (p42/44(MAPK) inhibitor); and 0.006, 0.06, and 0.6 microM SB203580 (p38(MAPK) inhibitor). All inhibitors blocked VEGF-induced permeability changes. Our data demonstrate that (1) VEGF increases permeability of EC monolayers in a dose-dependent fashion, and (2) VEGF-induced permeability is mediated through PI-3 kinase-PKB, NOS, and MAP-kinase signaling cascades. These observations suggest that microvascular hyperpermeability associated with inflammation and vascular disease is mediated by activation of these EC signaling pathways.

Capillary Permeability↗
PubMed2000

Caco-2 cell permeability vs human gastrointestinal absorption: QSPR analysis.

The aim of this study is to elucidate quantitative structure-permeability relationship (QSPR) of various organic molecules through Caco-2 cells, and to ascertain the relationship between gastrointestinal (GI) absorption in humans and Caco-2 cell permeability. Caco-2 cell permeability and human GI absorption data were obtained from the literature. The maximum hydrogen bond-forming capacity corrected for intra-molecular H-bonding (Hbc) and Lien's QSAR model were used in this study. The latest CQSAR software was utilized in calculating the logarithm of partition coefficient in octanol/water (Clog P) and in deriving all regression equations. For 51 compounds, a significant correlation was obtained between Caco-2 cell permeability (log Pcaco-2) and Hbc, octanol/PBS (phosphate buffered saline, pH 7.4) distribution coefficient (log Doct), log MW and an indicator variable (I) for the charge, with a correlation coefficient of 0.797. When these compounds were divided into three subgroups, namely neutral, cationic and anionic compounds, much better correlations (r = 0.968, 0.915 and 0.931, respectively) were obtained using different combinations of various physico-chemical parameters. A plot of human GI absorption vs. Caco-2 cell permeability obtained from different laboratories reveals that Caco-2 cell permeability cannot be used to precisely predict human GI absorption for compounds with Pcaco-2 below 5 x 10(-6) cm/s, due to interlaboratory and experimental variabilities, and the lack of a simple correlation between human GI absorption and Caco-2 cell permeability. Caco-2 cell permeability may be estimated from the structures of drug molecules using the above-mentioned physicochemical parameters. In general, for compounds with Pcaco-2 above 5 x 10(-6) cm/s, human GI absorption ranges from 50 to 100%. This is generally acceptable for development into oral dosage form. For the compounds with Pcaco-2 below 5 x 10(-6) cm/s, careful interpretation of caco-2 cell permeability and use of internal standard for comparison are recommended. Otherwise, good drug candidates may be excluded due to incorrectly predicted poor absorption.

Caco-2 Cells↗
PubMed1992

Combined effects of ozone and cigarette smoke on airway responsiveness and vascular permeability in guinea pigs.

The effects of combined exposure to ozone and cigarette smoke on airway responsiveness and tracheal vascular permeability, compared with those of single exposure were examined in guinea pigs. Airway responsiveness was assessed by measuring the specific airway resistance (sRaw) as a function of increasing concentration of inhaled methacholine aerosol immediately, 5 hr, and 24 hr after exposure. In a parallel study, tracheal vascular permeability was quantified by measuring the tracheal extravasation of intravenously administered Evans blue dye. Neither exposure to 1 ppm ozone for 30 min nor 5 puffs of cigarette smoke increased airway responsiveness or vascular permeability at any time after exposure. Combined exposure to 1 ppm ozone for 30 min and 5 puffs of cigarette smoke caused airway hyper-responsiveness and increased vascular permeability immediately after exposure. Exposure to 1 ppm ozone for 90 min increased both airway responsiveness and vascular permeability immediately after exposure. Exposure to 10 puffs of cigarette smoke increased airway responsiveness but not vascular permeability immediately after exposure. Combined exposure to 1 ppm ozone for 90 min and 10 puffs of cigarette smoke increased both airway responsiveness and vascular permeability immediately after exposure. The combined exposure to ozone and cigarette smoke thus increased both airway responsiveness and tracheal vascular permeability to a greater extent than did exposure to a single agent, suggesting that a combination of air pollutants has a more deleterious effect both on airway responsiveness and on tracheal vascular permeability than does either agent alone in guinea pigs.

Airway Resistance↗
PubMed1988

Comparison of transcellular and transepithelial water osmotic permeabilities (Pos) in the isolated proximal straight tubule (PST) of the rabbit kidney.

Measurements of the water osmotic permeabilities of apical and basolateral membranes of PST cells and of the transepithelial permeability have been carried out using a very fast method with high temporal and spatial resolution. At 25 degrees C the values obtained are: 80.8 +/- 11.9 x 10(-4) cm3/s osmol cm2 of apical (luminal) surface area and 90.1 +/- 13.0 x 10(-4) cm3/s osmol cm2 of basement membrane area (no membrane invaginations taken in account). These values are higher than previously published values due to the use of a faster and more accurate volume measuring and recording system. The transepithelial water osmotic permeability at 25 degrees C is 77 +/- 11 in units of 10(-4) cm3/s osmol cm2 basement membrane area. The transcellular water osmotic permeability is 32 +/- 7 (same units), leaving a paracellular contribution of 45 +/- 10 (same units). In the presence of 2.5 mM parachloromercuribenzenesulfonate (pCMBS) the apical permeability is reduced with an incubation of 10-15 min to 23% of its control value and the basolateral permeability to 8% of its control value (after 25 min) but the transepithelial permeability is only reduced to about 1/2 of the control value. This leaves a transcellular permeability of 6 x 10(-4) cm3/s osmol cm2 of basement membrane area and a paracellular contribution of 33 +/- 6 (same units). These results indicate a significant contribution of the paracellular pathway to the transepithelial water osmotic permeabilities in PST.

4-Chloromercuribenzenesulfonate↗
PubMed1994

Modulation of endothelial cell permeability by lung carcinoma cells: a potential mechanism of malignant pleural effusion formation.

This study examined the hypothesis that tumor cells metastatic to the pleura secrete a soluble factor(s) that directly increases endothelial cell permeability. Nitrocellulose filters were endothelialized with bovine pulmonary artery endothelial cells and exposed to conditioned media from either human lung adenocarcinoma (Calu-3), human lung squamous cell carcinoma (SK-MES-1), or control media for 16 h. The diffusional permeability (Pd x 10(-5) cm/sec) to [14C]albumin was then determined for each monolayer with Ussing-type chambers. Both adenocarcinoma conditioned media (ACCM) and squamous cell carcinoma conditioned media (SCCM) caused a two- to threefold increase in endothelial monolayer permeability. The addition of indomethacin (10 micrograms/ml) blocked the observed permeability increase in ACCM but not in SCCM, suggesting that the increase in permeability by ACCM was secondary to the production of prostaglandins. To confirm this, a variety of prostanoids previously shown to be produced by the Calu-3 cell line were added directly to the endothelial monolayer. Prostaglandin F2 alpha (PGF2 alpha) in both low (10 ng/ml) and high (100 ng/ml) concentrations for 16 h resulted in a three- to fourfold increase in permeability. Prostaglandin E2 (PGE2) resulted in a small increase in [14C]albumin permeability but only at high concentrations (100 ng/ml). PGF2 alpha production by the two tumor cell lines was measured using radioimmunoassay. Baseline adenocarcinoma production of PGF2 alpha was 117.5 pmol/10(6) cells and fell to 24.2 pmol/10(6) cells hours following incubation with indomethacin. The decrease in PGF2 alpha occurred in parallel with the changes in permeability. Concomitant, reversible changes in cell shape and F-actin distribution were detected in endothelial cells exposed to ACCM. No significant production of PGF2 alpha by the squamous cell carcinoma cell line was detected. These results suggest that both adenocarcinoma and squamous cell carcinoma secrete a soluble factor(s) that directly increases endothelial cell permeability to albumin and that in the case of adenocarcinoma this soluble factor may be a prostanoid such as PGF2 alpha.

Adenocarcinoma↗
PubMed1986

Histamine-induced microvascular permeability increases in hamster skin: a response predominantly mediated by H2-receptors.

The pharmacology of histamine-induced increases in cutaneous microvascular permeability was investigated in the hamster by examining the effects of cimetidine and pyrilamine on the increase in microvascular permeability evoked by graded doses of intradermally-injected histamine, and comparing the cutaneous microvascular permeability responses to graded doses of impromidine (0.1-100 micrograms), dimaprit (1-100 micrograms) and beta-histine (0.1-100 micrograms). Pretreatment with pyrilamine (0.1 mg/kg i.v. bolus injection) did not reduce the increase in microvascular permeability produced by any dose of histamine. In contrast, cimetidine (0.5 mg/kg/min i.v. infusion) significantly inhibited the microvascular permeability responses to 10 and 100 micrograms histamine. Although neither cimetidine nor pyrilamine significantly altered the microvascular permeability response to 0.1 and 1 micrograms histamine, inhibition was afforded by a cimetidine-pyrilamine combination. These results suggest a predominantly H2-receptor mediated phenomenon with a minor H1-receptor mediated component. Studies with the H2-receptor agonists impromidine and dimaprit and the H1-receptor agonist beta-histine provide further support for this contention. Dimaprit and impromidine caused a dose-dependent increase in cutaneous microvascular permeability, but betahistine produced only a relatively modest response. In other laboratory species, increased cutaneous microvascular permeability appears to be mediated solely by H1-receptors. Therefore, the hamster skin appears unique with respect to the pronounced H2-receptor involvement in histamine-induced microvascular permeability changes.

Animals↗
PubMed1987

Permeability of ammonia and amines in Rhodobacter sphaeroides and Bacillus firmus.

Permeabilities of uncharged ammonia (NH3), methylamine (CH3NH2), and ethylamine (CH3CH2NH2) in the gram-negative phototrophic bacterium Rhodobacter sphaeroides were measured directly in cells grown heterotrophically under aerobic conditions. The permeability of NH3 was 2.55 +/- 0.73 microns s-1 (n = 20), but the permeabilities of CH3NH2 (MA) and CH3CH2NH2 (EA) were higher, PMA = 17.8 +/- 2.8 microns s-1 (n = 50), PEA = 24.7 +/- 3.9 microns s-1 (n = 44). The relative permeabilities of amines were also determined from their effect on the pH gradient across the cell membrane at alkaline external pH. In aerobically grown R. sphaeroides, both techniques indicated that the permeability of CH3CH2NH2 was about 30% greater than that of CH3NH2 but that the permeability of NH3 was only about 1/5 that of CH3NH2. The relative permeabilities of NH3 (A) and CH3NH2 were different in R. sphaeroides cells grown under three different physiological conditions: (a) cells grown aerobically with ammonium sulfate (PA/PMA about 0.20), (b) cells grown anaerobically with ammonium sulfate as their nitrogen source (PA/PMA about 0.29), and (c) diazotrophic cells (PA/PMA about 0.38). NH3 was also found to be only about 1/3 as permeable as CH3NH2 in the alkalophilic gram-positive bacterium Bacillus firmus. The findings that permeability properties of NH3 and CH3NH2 are very different in different bacteria and vary according to the conditions under which the organism is grown need to be taken into account in the interpretation of experiments where [14C]methylamine is used as an ammonia analog.

Aerobiosis↗
PubMed1983

The anion permeability of vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane.

Band 3 protein was reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 2500:1 phospholipid:protein molar ratio by means of a Triton X-100/beads method. The SO2-4 permeability of the resulting vesicles was measured using an influx assay procedure in which the vesicles were sampled and subsequently eluted over Sephadex columns at appropriate time intervals. The accuracy of the assay was greatly increased by using an internal standard in order to correct for vesicle recovery. In agreement with previous work, it could be demonstrated that incorporation of band 3 in the vesicles caused an increase in SO2-4 permeability, which could be (partially) inhibited by high concentrations of DIDS or a competitive anion such as thiocyanate. However, the magnitude of the increased SO2-4 permeability was highly variable, even when vesicles were reconstituted using band 3 isolated from one batch of ghosts. In addition, the SO2-4 influx curves showed complex kinetics. These results are related to the existence of vesicle heterogeneity with respect to protein content and vesicle size as revealed by stractan density gradient centrifugation and freeze-fracture electron microscopy. Band 3 incorporation also increased the L-glucose permeability of the vesicles which could also be inhibited by DIDS. Glycophorin, which has no known transport function, reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 400:1 phospholipid:protein molar ration increased the bilayer permeability towards SO2-4 as well as towards L-glucose. Surprisingly, the SO2-4 permeability in the vesicles could also be inhibited by DIDS and thiocyanate. It is concluded that the use of DIDS and a competitive anion, thiocyanate, in order to prove that band 3 is functionally reconstituted, is highly questionable. The increased SO2-4 and L-glucose permeability of band 3-lipid as well as glycophorin-lipid vesicles and the inhibitory action of DIDS are discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce the formation of pores. Since the band 3-lipid vesicles are more permeable for SO2-4 than for L-glucose, in contrast to the glycophorin-containing vesicles, it is suggested that some anion specificity of the increased bilayer permeability in the band 3-lipid vesicles is still preserved.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

Control of membrane permeability by external ATP in mammalian cells: isolation of an ATP-resistant variant from Chinese hamster ovary cells.

External ATP causes a great increase in the passive permeability of the plasma membrane for phosphorylated metabolites and other small molecules in cultured mammalian cells. We previously demonstrated that in CHO-K1 cells an ATP-dependent permeability change was induced in the presence of a mitochondrial inhibitor (KCN or rotenone), a cytoskeleton-attacking agent (vinblastine) and a calmodulin antagonist (trifluoperazine). These permeability changes were reversible but long exposure, for 30-60 min, to ATP together with a mitochondrial inhibitor significantly reduced the cell viability of the treated cells. Since this cell lysis was shown to be due to the ATP-dependent permeability change, we could isolate several clones resistant to the action of the external ATP from CHO-K1 cells after repeated treatment with ATP and rotenone. In 9.1 cells, one of the isolated clones, little or no ATP-dependent permeability change was observed in the presence of either a mitochondrial inhibitor, vinblastine or trifluoperazine. This CHO variant could be specifically resistant as to the change in membrane permeability induced by external ATP, since the permeabilities for the 2-deoxyglucose and drugs used in the present studies were similar to those in the case of the parent cells. These results suggest that a specific defect or alteration in the plasma membrane is involved in the ATP-dependent permeability change. It is also reported that Mg2+-dependent ATPase activity was found on the cell surface of both CHO-K1 and 9.1 cells, and this activity was shown to be not involved in the permeability change controlled by external ATP.

Adenosine Triphosphate↗
PubMed1990

Effects of dimethylsulfoxide and mercurial sulfhydryl reagents on water and solute permeability of rat kidney brush border membranes.

The effects of dimethylsulfoxide, DMSO, and mercurial sulfhydryl reagents have been studied on water and small solute permeability of rat renal brush border membrane vesicles. Water and solute permeability was measured by mixing membrane vesicles with hypertonic solutions in a stopped-flow apparatus and following osmotically-induced changes in vesicular volume via changes in scattered light intensity. The rate constant of the fast osmotic shrinkage is proportional to the osmotic water permeability, while the rate constant of the slow reswelling phase is proportional to the solute permeability. Using mannitol as the osmotic agent, the osmotic shrinkage of rat renal brush border membrane vesicles followed a biphasic time course. 80% of the vesicles shrunk with a rate constant of approx. 50 s-1 and 20% with a rate constant of approx. 2 s-1. DMSO decreased dose-dependently the amplitude of the fast osmotic shrinkage, without affecting its rate constant. In contrast to DMSO, HgCl2 decreased the rate constant but not the amplitude of the fast osmotic shrinkage of renal brush border vesicles. Between 40-50 microM HgCl2, the inhibition of the fast osmotic shrinkage was completed. DMSO and HgCl2 increase the activation energy of water permeation in renal membranes from 3 to 12-15 kcal/mol. DMSO and HgCl2 did not affect the rate constant of the slow osmotic shrinkage of renal membrane vesicles and were also without effect on osmotic shrinkage of small intestinal brush border and pure phospholipid vesicles. In renal brush border membranes, HgCl2 at low concentrations (less than 10 microM) increased by 15-fold the permeability to NaCl and urea but not to mannitol, an effect which precedes the inhibition of water permeability at higher HgCl2 concentrations. The increase in small solute permeability was irreversible while the inhibition of water permeability could be reversed with cysteine and dithiothreitol. We conclude that water and small solute pathways in rat renal brush border membranes are completely separate entities, which are effected differently by DMSO and HgCl2. These pathways for water and solutes must be membrane proteins since neither DMSO nor HgCl2 affect the permeability properties of pure phospholipid vesicles.

4-Chloromercuribenzenesulfonate↗
PubMed1989

Intestinal permeability in patients with Crohn's disease and their healthy relatives.

The healthy relatives of patients with Crohn's disease were previously found to have increased intestinal permeability to polyethylene glycol 400. To determine whether the abnormal permeability is uniquely detectable by polyethylene glycol 400, we studied the intestinal permeability of three new probes (lactulose, rhamnose, and mannitol) in 25 patients with Crohn's disease, 41 of their healthy relatives, and 29 normal controls without a family history of inflammatory bowel disease. Patients with Crohn's disease had increased lactulose permeability when compared with relatives or controls. Lactulose absorption by patients with Crohn's disease was 0.41% +/- 0.07% (mean +/- SE), whereas that of their relatives and unrelated controls was 0.28% +/- 0.03% and 0.26% +/- 0.03%, respectively. There was no significant difference between the relatives and controls, but both groups differed from the patients (p less than 0.05 and p less than 0.025, respectively). The patients' lactulose/rhamnose ratio was 70.5% +/- 9.2% vs. 37.2% +/- 3.3% in relatives and 40.6% +/- 5.7% in unrelated controls (p less than 0.0005 and p less than 0.0025, respectively). The two intermediate-sized probes, rhamnose and mannitol, did not detect permeability differences among the three groups. The inability of lactulose, rhamnose, or mannitol to detect permeability abnormalities in healthy relatives of patients with Crohn's disease suggests that these probes penetrate the intestinal barrier by routes or mechanisms that are different from those of polyethylene glycol 400. Lactulose, in particular, detects permeability changes in patients with intestinal inflammation, and polyethylene glycol 400 is able to detect permeability changes in the health relatives of our patients. These data indicate that permeability may be abnormal as a secondary result of inflammation, or as a result of a primary genetic abnormality.

Adult↗
PubMed1992

The role of lipids in ischemia/reperfusion-induced changes in mucosal permeability in developing piglets.

This study determined which nutrient component of formula may be responsible for changes in ischemia/reperfusion-induced mucosal permeability, as quantitated by the plasma-to-lumen clearance of 51Cr-ethylenediaminetetraacetic acid, in newborn piglets. Loops of jejunoileum in 1-day-old and 1-month-old piglets were perfused with predigested and bile acid-solubilized solutions of formula, lipid, protein, carbohydrate, delipidated formula, or fatty acid during 1 hour each of control, ischemia, and reperfusion. Luminal perfusion with formula or lipid led to significantly greater increases in mucosal permeability during reperfusion in newborn intestine than did carbohydrate or protein, whereas mucosal permeability in older animals was not different among solutions. Removal of all lipids from the formula abolished the increased mucosal permeability associated with reperfusion in newborn animals. Perfusion with oleate, a monounsaturated dietary fatty acid, led to still greater increases in reperfusion-associated permeability in newborn but not older intestine. The oleate and lipid perfusions also caused significantly increased mucosal permeability in the absence of ischemia. Thus, it appears that a lipid component of formula, probably a fatty acid, is responsible for the increase in mucosal permeability induced by ischemia/reperfusion in newborn intestine and also leads to increased mucosal permeability in the absence of ischemia/reperfusion. Investigation of the mechanism of these lipid-associated changes in mucosal permeability may provide a rationale for dietary modifications that may decrease the risk of mucosal injury during feeding and ischemic stress in immature intestine.

Animals↗