Modified Ussing Chamber Research Articles

TRANSSCLERAL ALBUMIN DIFFUSION AND SUPRACHOROIDAL ALBUMIN CONCENTRATION IN UVEAL EFFUSION SYNDROME

To test the hypothesis that uveal effusion syndrome is caused by reduced transscleral albumin permeability. Surgical scleral specimens were obtained from a 55-year-old patient with nanophthalmic uveal effusion syndrome. Specimens were clamped in a modified Ussing chamber, and the rate of transscleral diffusion of fluorescein isothiocyanate-albumin was measured over 12 hours, using a spectrophotometer and predetermined standard curves. The diffusion coefficient was determined at 20°C, and then adjusted to body temperature using Einstein's equation. Results in 3 scleral samples were compared with 10 age-matched controls. Albumin and total protein concentration were measured in choroidal fluid and serum. Histologic staining with Alcian blue showed interfibrillary acid mucin deposits. Transmission electron microscopy showed deposits measuring 1 μm to 10 μm and collections of expanded, degenerate collagen fibrils. The mean (±SD) albumin diffusion coefficient was 12% of that in controls (1.22 ± 0.67(-8) × 10 vs. 10.3 ± 7.0 × 10(-8) cm2/second) and below the lower 95% confidence limit of the control group. The diffusion coefficient was calculated to increase 53% to 1.87 ± 1.03 × 10(-8) cm2/second at 37°C. Choroidal albumin concentration was much higher than physiologic levels, measuring 200 g/L (total protein 321 g/L), 5 times the serum albumin concentration of 42 g/L (total protein 70 g/L). Nanophthalmic uveal effusion syndrome can be associated with reduced scleral permeability to albumin, and a very high concentration of retained suprachoroidal albumin. This will lead to an osmotic gradient that retains fluid and may partly explain the pathogenesis of uveal effusion syndrome in some patients.

Abnormal intestinal function related to hypocupremia in a rodent model

Copper deficiency affects the peripheral (PNS) and central (CNS) nervous systems and can lead to neurological deficits in humans. No studies have addressed whether copper deficiency affects the enteric nervous system (ENS). We hypothesized that ENS abnormalities impair intestinal function in copper deficiency. We induced copper deficiency in rats by nutritional deprivation. Once hypocupremia was achieved, we euthanized the animals and harvested the small and large intestine. The longitudinal smooth muscle from the jejunum and colon was suspended in organ baths and contractility in response to electrical field stimulation (EFS) was assessed. Mucosa was also isolated from each region and placed into modified Ussing chambers to determine whether the copper deficiency leads to alterations in epithelial transport measured as a change in short circuit current across the mucosa in response to EFS. A copper deficient diet (CDD) in normal rats for 9 weeks was sufficient to produce hypocupremia. Colonic smooth muscle contractility was significantly decreased in response to EFS in rats fed a CDD compared with controls, however, jejunal smooth muscle contractility in response to EFS in rats fed a CDD rats resembled that observed in controls. No significant changes in secretory function were observed in either region in response to CDD. Dietary copper deficiency produces significant changes in the neural regulation of colonic smooth muscle contractility in a rodent model. Thus, along with CNS and PNS effects in humans, copper deficiency results in abnormal ENS regulation of intestinal function in rats.

Elevated sweat chloride concentration in children without cystic fibrosis who are receiving topiramate therapy

Topiramate, which is used as an anticonvulsant and for migraine prophylaxis in children, causes oligohydrosis as a side-effect, but its effect on sweat chloride concentrations has not been studied systematically. Twenty-one children receiving topiramate and 20 healthy controls with no signs or symptoms of pulmonary or gastrointestinal disease and a negative family history for cystic fibrosis (CF) underwent bilateral pilocarpine iontophoresis and sweat collection via Macroduct® system. Adequate samples (>15 µl volume) were obtained from 17/19 topiramate subjects (89%), and 19/20 (95%) controls. The mean sweat chloride concentration was 37.7 ± 18.8 mmol/L for patients receiving topiramate, and 15.9 ± 6.9 mmol/L for controls (p = 0.0001). The mean sweat volume was 29.1 ± 17.4 µl for patients receiving topiramate, and 41.2 ± 17.5 µl for controls (p = 0.037). Overall 8/17 (47%) of patients on topiramate with a measurable sweat chloride had either an intermediate (>40 mmol/L but <60 mmol/L) or elevated (>60 mmol/L) sweat chloride test result, while 0/19 control subjects had elevated sweat chloride (p = 0.0008). Further analysis of the in vitro activity of topiramate on cultured human bronchial epithelial cells in modified Ussing chambers showed no differences in chloride conductance measured in cells exposed to 10 or 50 µg/ml of topiramate when compared to non-exposed cells. This is the first report of a medication affecting sweat chloride values and shows that topiramate therapy can cause elevated sweat chloride concentrations in the absence of clinical manifestations of CF.

The pathophysiology of early-stage chronic kidney disease–mineral bone disorder (CKD-MBD) and response to phosphate binders in the rat

Chronic kidney disease-mineral bone disorder (CKD-MBD) is a systemic disorder that describes the complex bone and mineral abnormalities that occur in CKD. To understand the pathophysiology of CKD-MBD and determine whether the early use of phosphate binders would alter this physiology, we used a naturally occurring, slowly progressive model of CKD-MBD, the Cy/+ rat. Male Cy/+ rats were compared with their normal littermates at 20 weeks of age after 1 week of no phosphate binder, calcium carbonate, or sevelamer carbonate. The Cy/+ rat had renal function that was 50% of that of normal littermates, elevated parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23), decreased 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] levels, but normal calcium and phosphorus levels. There was a significant positive correlation of blood FGF23 and phosphorus levels and blood FGF23 and urine phosphorus levels. There was an inverse correlation between FGF23 and calcium levels. mRNA from the kidney demonstrated 50% reduction in klotho and Npt2a expression but no difference in CYP27B1. In the intestine, CKD animals had reduced active phosphate absorption in the jejunum using modified Ussing chambers and a reduction in Npt2b expression throughout the small intestine compared with normal littermates. In bone, mRNA expression of FGF23 was reduced (driven by lowering with phosphate binders), and TRAP expression was increased in CKD. By histology, there was increased osteoclast activity and number, and there were reductions in some measures of femoral neck mechanical strength. One week of phosphate binders reduced intestinal phosphate flux, serum phosphorus levels, and urinary phosphate excretion. These results demonstrate marked abnormalities in kidney, intestine, and bone in early CKD-MBD. While phosphate binders were effective in lowering urine phosphorus, they had little effect on end organs after 1 week of administration.

Relationship of Stokes Radius to the Rate of Diffusion across Bruch's Membrane

To determine the effect of Stokes radius (R(S)) on the diffusion of molecules through Bruch's membrane (BM), and to establish a system suitable for the analysis of diffusion through small (<2 mm(2)) samples of BM. Porcine BM/choroid (BM/Ch) was mounted in a modified Ussing chamber. A concentration gradient was simultaneously established for four tracers with R(S) values ranging from <1.0 to 6.15 nm. Samples were collected from both chambers at various time points up to 36 hours and the amount of each tracer was determined using quantitative gel exclusion chromatography. The integrity of samples was determined using scanning electron microscopy. BM/Ch mounted in the chamber exhibited no obvious damage even after 36 hours in the chamber. Flux was significantly (P < 0.05) greater in the BM to Ch direction than that in the Ch to BM direction for only two of the tracers: cytosine and RNase A. Flux also was dependent on R(S); cytosine, the smallest tracer (R(S) < 1 nm), exhibited the greatest flux and ferritin (R(S) = 6.15 nm) the least. Permeability coefficients for each tracer were determined and exhibited a power relationship with R(S). Flux was dependent on the direction of the concentration gradient and the R(S) of the individual tracers. We have successfully demonstrated that quantitative gel exclusion chromatography can be used to follow diffusion of a mixture of tracers across BM/Ch, and that we can measure flux across BM/Ch preparations with an exposed surface area as small as 1.8 mm(2).

Electrogenic transport, oxygen consumption, and sensitivity to acute hypoxia of human colonic epithelium

It is recognized that epithelial ion transport depends on oxygen supply, but this dependence has not been characterized in the human colon in vitro despite its surgical and clinical implications. The aim of this study is to measure the oxygen consumption of colonic epithelium under conditions which preserve vectorial ion transport and to assess the sensitivity of the human colonic epithelium short-circuit current (I (sc)) to acute hypoxia induced in vitro. Isolated mucosa preparations from human sigmoid colon were placed in a modified Ussing chamber which allows simultaneous measurement of short-circuit current (I (sc)) and oxygen consumption (QO(2)). In separate experiments, the sensitivity to acute hypoxia induced in a conventional Ussing chamber was assessed. Basal mean ± SEM values (n = 8) were I (sc) = 3.3 ± 0.5 μEq h(-1) cm(-2) and QO(2) = 8.09 ± 0.55 μmol h(-1) cm(-2). The contribution of the serosal side to the oxygen supply was higher than that of the mucosal side (p = 0.0023). Ouabain reduced I (sc) by 70% (P < 0.0001) and QO(2) by 26% (n = 8; P = 0.0009), suggesting that a large fraction of QO(2) is needed to support ouabain-sensitive electrogenic transport. Induction of hypoxia at both sides of the Ussing chamber caused a rapid decrease in I (sc) after 2 min. I (sc) was also significantly depressed when hypoxia was induced by 5 min in the serosal side (n = 6, P < 0.0001), but was unaffected by hypoxia induced in the mucosal side. The present results allow a better understanding of the clinical consequences of acute hypoxia on intestinal ion transport.

Peroxisome proliferator receptor γ agonists alter electrolyte transport across porcine vas deferens epithelia

Elevated levels of 15_deoxy_Δ12_14_prostaglandin_J2 (15dPGJ2) have been reported in the reproductive tracts of some cases of male infertility. The goal for this project was to determine a possible mechanism by which 15dPGJ2, an endogenous PPARγ ligand, might contribute to male infertility. Vas deferens epithelial cells were isolated from pigs, cultured for 14–21 days and exposed to dexamethasone and/or PPARγ agonists for the final 3–4 days of culture. Cells were mounted in modified Ussing chambers and exposed to amiloride (ENaC blocker), forskolin (adenylyl cyclase activator), and DASU□02 (CFTR blocker). Amiloride sensitive current induced by dexamethasone was potentiated two-fold by concurrent rosiglitazone exposure while there was no effect on baseline, forskolin or DASU-02 responses. Protein and RNA were isolated. Western blots suggest a decrease in CFTR expression and an increase in α, β, and γ ENaC subunits. RT-PCR detected a decrease in RNA coding for CFTR. PPARγ agonist treatment in the PVD9902 cell line attenuated forskolin and DASU-02 responses. These effects were concentration dependent, induced by structurally distinct PPARγ agonists, and blocked by a PPARγ antagonist, T0070907. These outcomes suggest that PPARγ activation by 15dPGJ2 in the reproductive duct could alter luminal electrolytes, which would likely affect sperm viability and function. [NIH R01-HD058398 & P20-RR017686 Core C]

Pancreatoduodenectomy as a source of human small intestine for Ussing chamber investigations and comparative studies with rat tissue

A clear understanding of oral drug absorption is an important aspect of the drug development process. The permeability of drug compounds across intact sections of small intestine from numerous species, including man, has often been investigated using modified Ussing chambers. The maintenance of viable, intact tissue is critical to the success of this technique. This study therefore aimed to assess the viability and integrity of tissue from patients undergoing pancreatoduodenectomy, for use in cross-species Ussing chamber studies. Electrical parameters (potential difference, mV; short-circuit current, µA.cm(-2) ; resistance, Ω.cm(2) ) were monitored over the duration of each experiment, as was the permeability of the paracellular marker atenolol. The permeability values (Papp; cm/s × 10(-6) ) for a training-set of compounds, displaying a broad range of physicochemical properties and known human fraction absorbed values, were determined in both rat and human jejunum, as well as Caco-2 cell monolayers. The results indicate that human jejunum sourced from pancreatoduodenectomy remained viable and intact for the duration of experiments. Permeability values generated in rat and human jejunum correlate well (R(2) = 0.86), however the relationship between permeability in human tissue and Caco-2 cells was comparatively weak (R(2) = 0.58). Relating permeability to known human fraction absorbed (hFabs) values results in a remarkably similar relationship to both rat and human jejunum Papp values. It can be concluded that human jejunum sourced from pancreatoduodenectomy is a suitable source of tissue for Ussing chamber permeability investigations. The relationship between permeability and hFabs is comparable to results reported using alternative test compounds.

Low levels of bile acids increase bacterial uptake in colonic biopsies from patients with collagenous colitis in remission

Patients with collagenous colitis have an impaired mucosal barrier. Moreover, collagenous colitis is associated with bile acid malabsorption. Bile acids can increase bacterial mucosal uptake in humans. Mucosal barrier function was investigated by exposing colonic biopsies to chenodeoxycholic acid (CDCA) or deoxycholic acid (DCA) in Ussing chamber experiments. To find if low levels of bile acids increase bacterial uptake in colonic biopsies from collagenous colitis patients. The study comprised 33 individuals; 25 with collagenous colitis (14 in clinical remission without treatment, 11 with active disease and 10 examined in clinical remission resulting from treatment with 6 mg budesonide); eight healthy individuals undergoing screening colonoscopy served as controls. Endoscopic biopsies from the sigmoid colon were mounted in modified Ussing chambers and assessed for short-circuit current (Isc), potential difference, trans-epithelial resistance and transmucosal passage of Escherichia coli K12 after adding 100 μmol/L CDCA or DCA. When adding 100 μmol/L CDCA or DCA, bacterial uptake increased fourfold in biopsies of patients in remission; CDCA 6.5 units [2.5-9.8] and DCA 6.2 units [2.1-22] (median [IQR]), compared with uptake in biopsies without added bile acids 1.6 units [1.1-3] (P=0.004 and P=0.01 respectively). In active disease and in patients in remission due to budesonide treatment, bile acids did not affect bacterial uptake. Confocal microscopy revealed trans-epithelial passage of E. coli K12 within 30 min. Low concentrations of dihydroxy-bile acids exacerbate mucosal barrier dysfunction in colonic biopsies of patients with collagenous colitis in remission. This allows a substantially increased bacterial uptake, which may contribute to recurrence of inflammation.

Regulation of anion transport across the uterine epithelium ofGallus domesticus

For egg shell production, cations, primarily Ca(2+), and anions, primarily HCO(3)(-), must be secreted across the uterine epithelium. Because alterations of HCO(3)(-) transport influence Ca(2+) secretion, the present study was performed to gain insight into the regulation of anion transport (i.e., chloride and HCO(3)(-) transport) across the egg shell gland of the domestic chicken. To this purpose, unstripped uterus epithelia were mounted in modified Ussing chambers and electrogenic [i.e., short circuit current (I(sc))] and electroneutral anion transport were measured. Stimulating adenylate cyclases by forskolin, thereby enhancing the intracellular cyclic adenosine monophosphate( )(cAMP) concentration, evoked 2 patterns of I(sc) responses. Under HCO(3)(-)-buffered conditions, some of the tissues (3/8) showed an increase of I(sc), whereas in others (5/8) a decrease of I(sc) was observed in the presence of the drug. The I(sc) increase existed in HCO(3)(-) secretion because under HCO(3)(-)-free conditions forskolin stimulated only an I(sc) decrease. In addition, methazolamide, a blocker of carboanhydrases, significantly reduced baseline I(sc). The forskolin-induced decrease of I(sc) presented a Cl(-) absorption. In the absence of HCO(3)(-), forskolin yielded only an I(sc) decrease and the transepithelial flux of (36)Cl(-) was reduced. In the presence of mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid, a nonselective blocker of chloride channels, forskolin-induced I(sc) decrease was inhibited. In addition to cAMP, the regulatory influence of neurons or prostaglandins on baseline I(sc) and anion transport was investigated. Neither tetrodotoxin, a blocker of neuronal Na(+) conductances, electrical field stimulation, nor indomethacin, a blocker of cyclooxygenases, influenced the baseline I(sc) or anion transport. In summary, these data show effects of forskolin (i.e., cAMP) on anion transport across the egg shell gland of the domestic chicken: HCO(3)(-) secretion (supporting Ca(2+) mineralizing of the egg shell) and Cl(-) absorption. Neurons or prostaglandins did not influence the anion transport.

Omeprazole decreases magnesium transport across Caco-2 monolayers

To elucidate the effect and underlying mechanisms of omeprazole action on Mg(2+) transport across the intestinal epithelium. Caco-2 monolayers were cultured in various dose omeprazole-containing media for 14 or 21 d before being inserted into a modified Ussing chamber apparatus to investigate the bi-directional Mg(2+) transport and electrical parameters. Paracellular permeability of the monolayer was also observed by the dilution potential technique and a cation permeability study. An Arrhenius plot was performed to elucidate the activation energy of passive Mg(2+) transport across the Caco-2 monolayers. Both apical to basolateral and basolateral to apical passive Mg(2+) fluxes of omeprazole-treated epithelium were decreased in a dose- and time-dependent manner. Omeprazole also decreased the paracellular cation selectivity and changed the paracellular selective permeability profile of Caco-2 epithelium to Li(+), Na(+), K(+), Rb(+), and Cs(+) from series VII to series VI of the Eisenman sequence. The Arrhenius plot revealed the higher activation energy for passive Mg(2+) transport in omeprazole-treated epithelium than that of control epithelium, indicating that omeprazole affected the paracellular channel of Caco-2 epithelium in such a way that Mg(2+) movement was impeded. Omeprazole decreased paracellular cation permeability and increased the activation energy for passive Mg(2+) transport of Caco-2 monolayers that led to the suppression of passive Mg(2+) absorption.

The Buccal Mucosa as an Alternative Route for the Systemic Delivery of Risperidone

The purpose of this study was to investigate the potential of the buccal mucosa for the systemic delivery of risperidone (RISP), and to determine the impact of Azone® (AZ) on the transport of RISP via this route. The permeability of RISP through porcine buccal mucosa was assessed in modified Ussing chambers at various concentrations to determine the mechanisms involved in transport across the tissue. The effect of AZ was assessed by administering AZ 5% (w/w) to the tissue as a pretreatment or together with RISP in solution or in a mucoadhesive gel formulation. RISP permeated the buccal mucosa via a passive diffusion mechanism and pretreatment or coadministration of AZ 5% did not significantly modify the permeation of RISP. Application of a RISP mucoadhesive gel resulted in a steady state flux of 64.65 ± 8.0 µg/cm2/h, which when extrapolated to the in vivo setting, is predicted to result in RISP plasma concentrations of 11.2–56.1 µg/L for mucosal application areas between 2 and 10 cm2. Given that these predicted concentrations are within the therapeutic range of RISP required in humans, delivery of RISP via the buccal mucosa has the potential to result in therapeutically relevant plasma concentrations for the treatment of schizophrenia. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:4584–4592, 2010

Effect of ethanol on the water permeability of controlled release films composed of ethyl cellulose and hydroxypropyl cellulose

The robustness of controlled release formulations when co-ingested with alcohol is a current concern expressed by regulatory authorities, especially with regard to dose dumping. One such controlled release formulation commonly used is film coating composed of ethyl cellulose (EC) and hydroxypropyl cellulose (HPC). The aim of this study was to investigate how the presence of ethanol in the dissolution medium affects the water permeability of such films. Film samples were prepared in various EC–HPC compositions, and the effect of different ethanol concentrations in the dissolution medium on the permeability was studied using a modified Ussing chamber and tritiated water. It was found that the effect of ethanol on the film permeability varied depending on the composition of the films. The results were interpreted in terms of swelling of the EC in the films, where the swelling increased with increasing ethanol concentration. Thus, for films with low HPC content (non-interconnected pores), the water permeability of the films increased with increasing ethanol concentration as the diffusion through the ethyl cellulose increased due to swelling. However, for films with higher HPC content (having interconnected pores through the films), the permeability decreased, likely due to the swelling of the ethyl cellulose blocking the pores. The interpretation of the results was supported by dynamic mechanic analysis and SEM analysis.

Hymenolepis diminuta: The effects of infection on transepithelial ion transport and tight junctions in rat intestines

In this study, we examine the effect of Hymenolepis diminuta on ion transport in the ileum and on tight junctions in the ileum and colon of rats. We also evaluate the effect of H. diminuta on C-fiber endings in the ileum, the direct habitat of H. diminuta, before and after mechanical stimulation and pharmacological modification by capsaicin (C-fiber irritant).Wistar rats were orally infected with five cysticercoids of H. diminuta. Using a modified Ussing chamber, electrophysiological parameters of the ileum were measured (transepithelial electrical potential difference and transepithelial electrical resistance) as well as the deposition of occludin (a tight junction protein) in the ileum and colon of the rats 8, 16, 25, 35, 40 and 60days post infection.We observed a significant reduction in transepithelial electrical potential difference in the ileum of rats infected with H. diminuta. In both the ileum and colon of rats infected with H. diminuta we also observed a decrease in occludin deposition, which indicates leakage of tight junctions, correlating with the decrease in transepithelial electrical resistance of these tissues. The application of capsaicin confirmed the hypothesis that H. diminuta in rats affects the C-fiber sensory receptors, causing changes in ion transport in the ileum.The results of the performed electrophysiological and immunohistochemical examinations indicate hymenolepidosis-related changes in the active transport of ions and the passive movement of ions.

Ammonia uptake in Manduca sexta midgut is mediated by an amiloride sensitive cation/proton exchanger: Transport studies and mRNA expression analysis of NHE7, 9, NHE8, and V-ATPase (subunit D)

In this study participation of cation/proton exchangers (NHE) in ammonia uptake in the medial midgut of Manduca sexta larvae was investigated employing a modified Ussing chamber. There was a mean inward net ammonia (NH 3 + NH 4 +) flux of 194 ± 17 nmol cm −2 h −1 across the isolated epithelium under conditions of 0.1 mmol L −1 ammonia on both sides of the tissue and a 100-fold inwardly directed P NH3-gradient (pH 8.5 luminal side, pH 6.5 hemolymphal side). Employing a 100-fold NH 4 + gradient amiloride applied to the luminal side inhibited the influx in a dose-dependent manner, with a maximal inhibition of 75% at 20 mmol L −1 and an estimated IC 50 = 2 mmol L −1. Amiloride also caused a dose-dependent but smaller decrease in the short-circuit current ( I sc ). No inhibition by apical or basal applied amiloride was noticed on cellular metabolic ammonia release, of which ca. 1/3 and 2/3 was secreted towards the apical and basal side, respectively. Using molecular methods full and partial sequence information of two putative cation/proton exchangers (MsNHE8, MsNHE7, 9) were obtained, both containing the characteristic amiloride binding motif. An mRNA expression analysis revealed ubiquitous expression patterns for both proteins, with similar expression levels for NHE8 in all tissues investigated and lower mRNA abundances for MsNHE7, 9 in the midgut sections of the caterpillar. In contrast, in this tissue high expression levels of the V-ATPase (D subunit) were detected, likely the sole pump responsible for energizing goblet cell K + excretion, but also involved in columnar cell ammonia uptake.

Enhanced oral absorption of saquinavir with Methyl-Beta-Cyclodextrin—Preparation and in vitro and in vivo evaluation

Saquinavir (SQV) is a weak base compound, whose solubility is strongly influenced by pH variations. Thus, in the present work, we thought it worthy of interest to investigate in-depth the combined effect of pH control and cyclodextrin (CyD) complexation on SQV solubilization. Phase-solubility studies were performed by adding excess drug to buffered (pH from 1.1 to 7.4) aqueous solutions containing increasing concentrations of Methyl-Beta-CyD (M-β-CyD) in order to evaluate the role of the unionized species of SQV in improving solubility by CyD complexation and to be able to select the most suitable conditions for optimizing drug solubilization. Our study reveals that the integrated approach of pH adjustment and CyD complexation can be successfully used for improving the CyD solubilizing power towards an ionizable drug such as SQV, thus allowing a smaller quantity of CyD to solubilize a given amount of drug, offering clear economic and technologic advantages as well. When biopharmaceutics of the optimized cyclodextrin-based formulation of SQV was studied in Wistar rats after intravenous and oral administrations, we found that inclusion of SQV into M-β-CyD could dramatically improve its oral bioavailability and decrease the variation of its oral pharmacokinetics. Compared to the control, the presence of M-β-CyD significantly increased the area under the plasma concentration–time curve (439.7 ± 161.35 to 2312.03 ± 159.53, p < 0.01) and the peak plasma concentration (117.24 ± 35.77 to 1347.88 ± 276.76, p < 0.01) of orally administered SQV. The modulating effect of M-β-CyD on the bidirectional transport of SQV was also investigated using a modified Ussing chamber system. The results demonstrated that the enhancing effect of M-β-CyD on the oral bioavailability of SQV is due not only to its solubilizing effect on SQV but also, at least in part, to the inhibitory effect of M-β-CyD on the P-glycoprotein (P-gp) mediated efflux of SQV in the gastrointestinal tract. The present results suggest that M-β-CyD is particularly useful in designing oral preparations of SQV with an enhanced bioavailability and a reduced variability in absorption.

T1875 Molecular and Functional Evidence for a Predominant Role of Electrogenic Na +/HCO 3− Cotransport (NBCe1) Compared to Electroneutral Na +/HCO 3− Cotransport (NBCn1) in Murine Colon

colonic epithelium from patients with and without neoplasi of the colon studied as short circuit current (SCC) in micro-Ussing-air-suction (MUAS)-chambers (ref 1). Methods: Patients referred to endoscopy were enrolled in this study. All patients were more than 50 years of age. They all gave informed consent and the study was conducted with adherence to the declaration of Helsinki. When endoscopy was performed biopsies were taken app. 30 cm from the anus and at least 10 cm from a tumor if any present. The biopsies were mounted in MUAS-chambers with 10 mL of bicarbonate-Ringer on both sides and treated with (in mM) amiloride (0.1) , teophyllin (0.4), and indomethacin (0.04) prior to serosal cAMP (0.6) application. Tissue viability was controlled with either dibutyryl-cAMP (0.6) or forskolin (0.005). Results: Preliminary data of SCC-signals obtained from human colon biopsies induced by cAMP were 14.4±3.7 and 23.7±11.8 μA*cm-2 for the neoplasi and control group, respectively. Signals for db-cAMP were 105±40 and 138±30 μA*cm-2 for the neoplasi and control group respectively. Data are based on 2 patients and 4 biopsies in each group. No significant difference between the two groups. Conclusions: Serosal application of cAMP produces a secretory response indicating that transport of cAMP can be functionally assessed in MUAS-chambers. The transport of cAMP is likely mediated by an OATP-H transporter, known to transport cAMP. 1. Larsen R, Mertz-Nielsen A, Hansen MB, Poulsen SS, Bindslev N 2001. Novel modified Ussing chamber for the study of absorption and secretion in human endoscopic biopsies. Acta Physiol Scand 173:213-222.

The modulation of intestinal endotoxin transport by different dietary fats

Intestinal derived endotoxin and the subsequent endotoxemia are considered to be major predisposing factors for diseases such as atherosclerosis, sepsis, obesity and diabetes. Therefore, the aim of this study was to assess the effects of dietary fat on intestinal endotoxin transport. We hypothesized that saturated fat (SF) would augment, while n‐3 fatty acids (FA) would attenuate endotoxin transport across the small intestine of pigs. Eleven pigs (three‐four weeks of age) were euthanized and jejunum segments were mounted in modified Ussing chambers. Segments were treated with either no oil (CON), 12.5% (v/v) vegetable oil (VO), DHA (DO), cod liver oil (CLO), coconut oil (CO) or olive oil (OO) in 20 mM bile acid. Ex vivo endotoxin transport was measured by the addition of fluoroscein isothiocyanate labeled lipopolysaccharide (FITC‐LPS, 20 μg/mL) and serial sampling of the mucosal and serosal chambers. Fluorescence was measured in a plate reader and an apparent permeability coefficient (Papp) was calculated using the surface area and transport rate. Compared to the CON, DO and CLO reduced the endotoxin Papp by 50% (P&lt;0.01). However, versus the CON, CO increased the Papp by 60% (P=0.008). OO and VO Papp were not different from the CON. Overall, these data indicate that fat high in n‐3 FA can attenuate intestinal endotoxin transport, while SF augments endotoxin transport. This work was supported by the USDA and NWRC.

Sodium-Dependent Phosphate Uptake in the Jejunum Is Post-Transcriptionally Regulated in Pigs Fed a Low-Phosphorus Diet and Is Independent of Dietary Calcium Concentration

In rodents, severe dietary P restriction increases active phosphate absorption by the intestine. However, it remains unknown if moderate dietary P restriction has a similar effect. Weanling pigs (n = 32; body weight 7.4 ± 0.55 kg) were used in a 2 × 2 factorial design and fed dietary available P (aP) concentrations of 0.23 or 0.40% and Ca concentrations of 0.58 or 1.00% for 14 d. Diets were formulated on an aP basis instead of a total P basis, because pigs are unable to absorb phytate-P present in corn and soybean meal. Jejunal segments were mounted in modified Ussing chambers for determination of Na+-dependent nutrient transport. Intestinal mucosal scrapings were taken for RNA isolation and brush border membrane (BBM) vesicle isolation. Na+-dependent phosphate uptake and gene expression of Na-phosphate cotransporter IIb (NaPi-IIb), SGLT-1 (sodium/glucose cotransporter-1), and calbindin D(9k) and protein expression of NaPi-IIb were evaluated. Na+-dependent phosphate transport increased (P < 0.05) 46% as dietary aP concentration was decreased. However, increased Na+-dependent phosphate uptake was not accompanied by increased NaPi-IIb mRNA expression. Expression of NaPi-IIb protein in the BBM increased (P < 0.01) 84% in pigs fed low-P diets compared with pigs fed adequate-P diets. No dietary Ca effects or aP × Ca interactions were detected for Na-dependent P uptake, mRNA or protein expression of NaPi-IIb, or mRNA expression of calbindin D(9k). These data suggest that restricting dietary aP concentration by only 43% stimulates Na+-dependent phosphate uptake and expression of the NaPi-IIb protein in the BBM of the small intestine and through a post-transcriptional mechanism.

Neutrophils Compromise Retinal Pigment Epithelial Barrier Integrity

We hypothesized that neutrophils and their secreted factors mediate breakdown of the integrity of the outer blood-retina-barrier by degrading the apical tight junctions of the retinal pigment epithelium (RPE). The effect of activated neutrophils or neutrophil cell lysate on apparent permeability of bovine RPE-Choroid explants was evaluated by measuring [3H] mannitol flux in a modified Ussing chamber. The expression of matrix metalloproteinase- (MMP-) 9 in murine peritoneal neutrophils, and the effects of neutrophils on RPE tight-junction protein expression were assessed by confocal microscopy and western blot. Our results revealed that basolateral incubation of explants with neutrophils decreased occludin and ZO-1 expression at 1 and 3 hours and increased the permeability of bovine RPE-Choroid explants by >3-fold (P < .05). Similarly, basolateral incubation of explants with neutrophil lysate decreased ZO-1 expression at 1 and 3 hours (P < .05) and increased permeability of explants by 75%. Further, we found that neutrophils prominently express MMP-9 and that incubation of explants with neutrophils in the presence of anti-MMP-9 antibody inhibited the increase in permeability. These data suggest that neutrophil-derived MMP-9 may play an important role in disrupting the integrity of the outer blood-retina barrier.