Rabbit Intestinal Mucosa Research Articles

Design and evaluation of propranolol hydrochloride loaded thiolated Zein/PEO electrospun fibrous matrix for transmucosal drug delivery.

Thiolated polymers have garnered wide attention from researchers on mucoadhesive drug delivery. This work explores the thiolation of zein protein using cysteine amino acid via the EDC crosslinker. The optimization of thiolation and purification have been done and confirmed using Ellman's assay and Raman spectra. The thiolated Zein/PEO polymer blend has been appraised for electrospinning to fabricate fibrous matrices. The extent of thiol modification augmented the mechanical properties and adhesion in rabbit intestinal mucosa. In vitro cytotoxicity evaluations such as direct contact assay, MTT assay, and live dead assay performed in RPMI 2650 cells corroborated the non-cytotoxicity of the fabricated matrices with and without propranolol hydrochloride (PL). Detailed drug release studies were conducted in PBS. Drug release in PBS followed the Korsmeyer Peppas model of release. On treating RPMI 2650 cells with the matrix, F-actin and adherens junctional proteins retained integrity, and consequently, drug permeation would proceed through the transcellular transport mechanism. Transepithelial electrical resistance (TEER) measurement of the RPMI 2650 cell monolayer also supported the transcellular transport mechanism. Ex vivo permeation study through porcine buccal mucosa showed 41.26 ± 0.56% PL permeation within 24 h of study. It validated the competence of the electrospun thiolated Zein/PEO matrix for transmucosal drug delivery.

Ethosomal gel for rectal transmucosal delivery of domperidone: design of experiment, in vitro, and in vivo evaluation

Despite high efficiency of domperidone (DOM) in prophylaxis of emesis accompanied with radiotherapy and chemotherapy, it still can bother cancer patients by its powerful side effects and difficulty of its oral administration. The study was designed to develop and optimize DOM loaded ethosomal gel for rectal transmucosal delivery. Ethosomal formulations were prepared using a 21, 51 full-factorial design where the impact of lecithin concentration and additives were investigated. The optimum ethosomal vesicles were subsequently incorporated in Carbopol gel base where rheological behavior, spreadability, mucoadhesion, and in vivo pharmacokinetic parameters were studied. Based on Design Expert® software (Stat Ease, Inc., Minneapolis, MN), the optimum formulation illustrated entrapment efficiency of 70.02%±5.52%, and vesicular size of 112 ± 3.3 nm, polydispersity index of 0.32 ± 0.01, zeta potential of −59 ± 0.28 mV, and % drug released after 6 h of 76.30%±2.45%. Moreover, ex vivo permeation through rabbit intestinal mucosa increased four times compared to free DOM suspension. The gel loaded with ethosomes showed excellent mucoadhesion to rectal mucosa. DOM ethosomal gel showed a raise in C max and AUC0–48 of DOM by twofolds compared to free DOM gel. The study suggested that ethosomes incorporated in gels could be an efficient candidate for rectal transmucosal delivery of DOM.

In Vivo Evaluation of a Novel Chitosan-Polycaprolactone Based Mucoadhesive Gastro-Retentive Sustained Release Drug Delivery System for Milnacipran HCl.

The study was aimed to develop a gastro-retentive mucoadhesive sustained release matrix formulation for milnacipran HCl (MCN) by using the design of experiment (DoE). The gastro-retentive swellable mucoadhesive matrix tablets were prepared by modified solvent-based wet granulation through mixing milnacipran (MCN), chitosan low molecular weight (CH-LM), chitosan medium molecular weight (CH-MM), and polycaprolactone (PCL). Optimization of the formulation was carried out via DoE. Formulations were characterized by DSC, FTIR, and in vitro drug release testing. In vitro mucoadhesive studies were performed on rabbit's intestinal mucosa. In vivo drug release studies were performed on dogs. Optimized matrix formulations showed no significant interaction among the polymers and MCN, confirmed by DSC and FTIR, and were characterized as swellable controlled release matrix systems. The optimized formulations MOPT3 and MOPT4 showed significantly improved adhesion time of 12h on the gastric mucosa. Based on the in vivo analysis, the elimination half-life of MCN was increased that proved the matrix formulation to be sustained release DDS. The Tmax was extended from 2 to 12 ± 1.63h for MOPT4. Cmax of matrix was reduced to 121.60 ± 9.496ng/ml as compared to 149.22 ± 9.942ng/ml of solution. The bioavailability of the matrix formulation was significantly improved as compared to the MCN solution by 272.20 ± 48.11%. The controlled drug release and strong mucoadhesive properties of the gastro-retentive matrix formulations suggested the potential application of the formulations for the extended oral delivery of MCN.

Enhancing the Intestinal Permeation of the Chondroprotective Nutraceuticals Glucosamine Sulphate and Chondroitin Sulphate Using Conventional and Modified Liposomes.

Liposomes are promising systems for the delivery of macromolecules and poorly absorbed drugs, owing to their ability to compartmentalize drugs, their biodegradability and biocompatibility. The aim of the present study was to formulate and evaluate conventional and modified glucosamine sulphate (GluS) and chondroitin sulphate (CS) liposomal formulations, to enhance their oral permeation for the treatment of osteoarthritis (OA). Liposomal formulations were prepared by the thin-film hydration method using two types of phospholipids; Epikuron 200© and Epikuron 200© SH, and three permeation enhancers; poloxamer 407, cetylpyridinium chloride, and sodium deoxycholate. In-vitro characterization of liposomal formulations was conducted in terms of entrapment efficiency, particle size, zeta potential, viscosity, physical stability and mucoadhesive strength. Surface morphology and vesicle shape, ex-vivo intestinal permeation, and histopathological studies were further carried out on the selected formulation. Results showed that the liposomal formulation containing sodium deoxycholate was the most optimum formula, showing high entrapment efficiency (60.11% for GluS and 64.10% for CS) with a particle size of 4.40 µm, zeta potential of -17.2 mV and viscosity of 2.50 cP. The aforementioned formula displayed the highest cumulative % permeated of GluS and CS through rabbit intestinal mucosa compared to the solution of drugs and other liposomal formulations (64.20% for GluS and 78.21% for CS) after 2 hours. There were no histopathological alterations in the intestinal tissue, suggesting the safety of the utilized liposomal formulation. In light of the above, liposomes can be considered promising oral permeation-enhancer system for GluS and CS, which is worthy of future bioavailability experimentation.

Lipid nanocarriers for tamoxifen citrate/coenzyme Q10 dual delivery

Nanotechnology based combinatorial therapy has emerged as an effective strategy for cancer treatment due to its synergistic activity, suppression of multi-drug resistance and successful delivery to target site. The objective of this work was to develop and characterize tamoxifen citrate (TC) and coenzyme Q10 (CoQ10) lipid nanocarriers for oral breast cancer chemotherapy. Stearic acid (2%w/v) and poloxamer188 (3%w/v) were selected as optimal lipid matrix and surfactant for development of solid lipid nanocarriers (SLNs). Incorporation of lecithin into lipid matrix (SLN9) significantly reduced particle size to 180 nm and increased %EE of CoQ10 (45%). Nanostructured lipid carriers containing 10% Labrafac oil showed further decrease in particle size reaching only 81 nm and increased %EE up to 94% and 56% for TC and CoQ10, respectively. Lipid nanocapsules showed more prominent effect on decreasing particle size (36 nm). Lipid nanocarriers offered controlled drug release profiles. The study showed that lipid carriers significantly improved drugs permeation through rabbit intestinal mucosa and suggested them as potential delivery systems for improving the bioavailability of TC/CoQ10 therapeutic molecules. Subsequent studies will be performed in order to elucidate the cytotoxicity and genotoxicity of selected lipid nanocarriers formulas on MCF-7 (adenocarcinoma breast cancer cells) versus normal cells (WISH cell line).

Influence of 5-tridecylpyrazole-3-carboxylic acid, a new hypolipidaemic agent, on cholesteryl ester formation in rabbit intestinal mucosa.

The comparative effects of 5-tridecylpyrazole-3-carboxylic acid (TDPC), beta-sitosterol and melinamide on the esterification of cholesterol (CH) have been investigated in rabbit intestinal microsomes and cytosol in-vitro. The three agents did not show an effect on cholesteryl ester formation by cholesterol esterase (CEase). TDPC and beta-sitosterol did not affect cholesteryl oleate formation from oleoyl CoA by microsomal acyl CoA:cholesterol acyltransferase (ACAT), whereas melinamide significantly inhibited cholesteryl oleate formation. TDPC significantly inhibited the incorporation of oleic acid into cholesteryl oleate, which is associated with acyl CoA synthetase (ACS) plus ACAT in mucosal microsomes, at a concentration of 20-100 microM. On the other hand, 5-tridecylpyrazole-3-carbinol (TDPC-OH) a congener of TDPC, and beta-sitosterol did not show any effect. From these results, it is demonstrated that carboxylic moiety of TDPC is necessary to inhibit ACS in-vitro. According to the kinetic analytical results, it is suggested that TDPC acts as a competitive inhibitor of ACS. These results suggest that the inhibitory effect of TDPC on cholesteryl ester formation may be mediated by an inhibition of ACS activity. It is apparent from the data presented that there are substantial differences between TDPC, beta-sitosterol and melinamide with respect to their action on cholesteryl ester formation in rabbit intestinal mucosa.

Preparation and Evaluation of Buccal Bioadhesive Films Containing Clotrimazole

Buccal bioadhesive films, releasing topical drugs in the oral cavity at a slow and predetermined rate, provide distinct advantages over traditional dosage forms. The aim of present study was to prepare and evaluate buccal bioadhesive films of clotrimazole for oral candidiasis. The film was designed to release the drug at a concentration above the minimum inhibitory concentration for a prolonged period of time so as to reduce the frequency of administration of the available conventional dosage forms. The different proportions of sodium carboxymethylcellulose and carbopol 974P (CP 974P) were used for the preparation of films. Carbopol was used to incorporate the desired bioadhesiveness in the films. The films were prepared by solvent casting method and evaluated for bioadhesion, in vitro drug release and effectiveness against Candida albicans. In vitro drug release from the film was determined using a modified Franz diffusion cell while bioadhesiveness was evaluated with a modified two-arm balance using rabbit intestinal mucosa as a model tissue. Films containing 5% CP 974P of the total polymer were found to be the best with moderate swelling along with favorable bioadhesion force, residence time and in vitro drug release. The microbiological studies revealed that drug released from the film could inhibit the growth of C. albicans for 6 h. The drug release mechanism was found to follow non-Fickian diffusion.

Alpha and Omega of Carotenoid Cleavage

In early 1900s, based on indirect evidence, Steenbock and Morton independently predicted that beta-carotene could be the biological precursor of vitamin A, although this notion was contested by others. In the 1930s, Thomas Moore showed the in vivo formation of vitamin A from beta-carotene. But it was not until Jim Olson and DeWitt Goodman independently showed in 1965 the formation of retinal, the aldehyde form of vitamin A from beta-carotene in cell-free extracts of liver and intestine, that this vital pathway of beta-carotene was recognized. Despite compelling evidence in several experimental systems for the central cleavage of beta-carotene to retinal by many investigators, there were some careful independent studies by Glover et al., Ganguly et al., Hansen and Meret and Krinsky et al. showing the eccentric cleavage of beta-carotene resulting in the formation of apocarotenoids both in vivo and in vitro. In an attempt to resolve this controversial issue, we revisited this problem in 1989 and showed beyond doubt the formation of retinal as the sole enzymatic product of a cytosolic enzyme from rabbit and rat intestinal mucosa by mass spectrometry and tracer analysis of the crystallized product. This was confirmed in 1996 by Nagao using the pig intestinal extract. Yeum et al. confirmed in 2000 that retinal is the sole product of beta-carotene cleavage in the presence of alpha-tocopherol, and that the observed formation of apocarotenoids occurs only in the absence of an antioxidant like alpha-tocopherol. In the same year, Barua and Olson also concluded from their in vivo studies in rats that central cleavage is by far the major pathway for the formation of vitamin A from beta-carotene. Beta, beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) is the key enzyme that cleaves beta-carotene into two molecules of retinal. It is a cytosolic enzyme primarily localized in the duodenal mucosa although it has been found in liver. It is a 66 kDa sulfhydryl protein, requires molecular oxygen and is activated by ferrous ions. It is highly specific for 15:15' ethylenic bond of carotenoids although it has fairly broad specificity towards a number of carotenoids with at least one intact beta-ionone ring. The dioxygenase was recently cloned from Drosophila melanogaster and from the chicken intestine. The recombinant protein was found to form retinal as the sole cleavage product of beta-carotene. No apo-carotenoids were formed. Therefore, it is unequivocally proven that the major, if not the sole, pathway of beta-carotene cleavage to vitamin A is by oxidative cleavage of the central ethylenic bond of beta-carotene to yield two molecules of retinal. Most recently, human dioxygenase has also been cloned. Thus, the wisdom, vision and epoch-making mission of Jim Olson in the science of beta-carotene metabolism have been accomplished. I have no doubt that the impact of his original discovery of the dioxygenase and its importance in vitamin A nutriture should be forthcoming in the near future.

Histopathological study of rabbit intestinal mucosa infected with a hybrid strain of Shigella dysenteriae 1 carrying LPS biosynthesis genes of Salmonella enterica serovar typhimurium

The rfb gene cluster and the rfc gene of Salmonella enterica were introduced earlier into an invasive Shigella dysenteriae 1 strain by triparental cross. Antiserum was raised in rabbit against lipopolysaccharide isolated from the hybrid strain. Both the hybrid and the invasive S. dysenteriae 1 strain were found to have a titer of 1:2560 while for S. enterica, it was 1:640. Ligated ileal loops were prepared in rabbit, which were inoculated with 10 8 CFU ml −1 each of the hybrid strain, and invasive S. dysenteriae 1 strain used as positive control. Escherichia coli K12 was also used as a negative control. After 18 h, the fluid accumulation ratios were 0.2 and 1.6 for hybrid and invasive strains of S. dysenteriae 1, respectively. Rabbit intestinal mucosa infected with hybrid S. dysenteriae 1 strain showed the presence of intact villus tips and unruptured intestinal mucosa whereas total necrosis of intestinal mucosa and villi was observed in the S. dysenteriae 1-infected region.

Free radical lipid peroxidation inhibits enzymatic conversion of beta-carotene into vitamin A.

Free radical oxidation of arachidonic acid with soybean lipoxygenase was accompanied by inhibition of retinal synthesis from beta-carotene catalyzed by enzyme preparation from rabbit intestinal mucosa. Lipoxygenase inhibitor salicylhydroxamic acid and antioxidants suppressing free radical reactions (ethyl gallate, alpha-tocopherol, astaxanthine, and quercetin) promoted conversion of beta-carotene into retinal catalyzed by beta-carotene-15,15'-dioxygenase. These results indicate that lipoperoxides and/or products of their homolysis attenuate enzymatic conversion of beta-carotene and confirm the important role of natural antioxidants in the maintenance of stable vitamin A content in mammals.

DRA expression and brush border membrane anion exchange characteristics in rat and rabbit duodenum

Backround and Aims: Duodenal bicarbonate secretion in the resting state is in part mediated by an apical anion exchanger whose molecular nature is unknown. Recent data suggest that the dra gene, which is defective in congenital chlorid diarrhea, encodes an anion exchanger which, in oocytes, mediates sulfate as well as Cl uptake. We now investigated if the DRA gene product may be the apical anion exchanger in the duodenum. Methods: To test this hypothesis, we cloned rat and rabbit DRA cDNA fragments and studied the relative expression levels of DRA and NHE3 (Na+/H+ exchanger isoform 3) in the different segments of the rat and rabbit intestine by Northern analysis and semiquantitative PCR. We then isolated rat and rabbit duodenal and jejunal brush border membrane vesicles and studied 36Clund 35S0~uptake into Cl, HCOj, SO~and gluconate-loaded vesicles in the absence and presence of a pH-gradient (to distinguish SO~from HS04 transport) and the absence and presence of DIDS. We also examined HC03secretion in stripped rat and rabbit intestinal mucosa in the Ussing-chamber. Results: DRA was highly expressed in rat and rabbit intestinal tract, with the expression pattern colon>duodenum>ileum>jejunum in the rat and ileumc-colon> duodenum in the rabbit, and clearly differed from the expression pattern for NHE3 which was colon>ileum> jejunum>duodenum in both species. This excess DRA over NHE3 expression in the small intestine but not the proximal colon may explain why the resting state (absorbing) small intestine in the Ussing-chamber displays electroneutral HC03secretion whereas the proximal colon acidifies the luminal perfusate under resting conditions. Both rat and rabbit BBMvs displayed DIDS-sensitive CIICI-, HC03-/Ct, SO~-/Cl, and CIISO~exchange, findings compatible with DRA characteristics. Anion affinity for the internal and external anion binding sites differed somewhat between rat and rabbit but were Cl >HCOj in both species. Summary and Conclusions: The high duodenal DRA expression levels and the fact that the transport characteristics of rat and rabbit BBM anion exchange corresponds with those found in oocytes and Hek293 cells suggests that DRA is the likely transport protein which mediates electroneutral HCOj secretion in the duodenum.

Evaluation of a novel, natural oligosaccharide gum as a sustained-release and mucoadhesive component of calcitonin buccal tablets

The objective of this study was to evaluate the gum from Hakea gibbosa (hakea) as a sustained-release and mucoadhesive component in buccal tablets for a model peptide, namely, salmon calcitonin. Flat-faced core tablets containing either 12 or 32 mg of hakea and 40 µg (200 IU) of salmon calcitonin (sCT) per tablet were formulated using a direct compression technique and were coated with Cutina on all but one face. The in vitro release profiles were sigmoidal in nature and according to a mathematical model indicated super Case II transport as the primary mechanism of release. The resulting plasma sCT and calcium concentrations were determined following both intravenous administration and buccal application of mucoadhesive tablets in rabbits. Following intravenous administration, the mean values determined for t1/2 (α), t1/2 (β), Vd, and CL for sCT were 0.76 ± 0.06 min, 67 ± 18 min, 1484 ± 454 mL/kg, and 19 ± 2 mL/min·kg, respectively. Following the application of the mucoadhesive buccal tablets which contained 40 µg of sCT and either 12 or 32 mg of hakea, the calculated apparent bioavailability (F) and clearance (CL) were 37 ± 6% and 19 ± 3.3 mL/min·kg and 16 ± 8% and 18 ± 0.4 mL/min·kg, respectively. Serum calcium concentrations indicated that biologically active sCT was delivered across the rabbit buccal mucosa. The strength of mucoadhesion of the tablets was also quantitated in terms of the force of detachment as a function of time. The force of detachment for the mucoadhesive buccal tablets containing either 12 or 32 mg of hakea and 40 µg of sCT increased from 4.47 ± 0.68 to 8.41 ± 1.0 N and 8.23 ± 1.62 to 14.98 ± 1.63 N, respectively, from 5 to 90 min following application to excised rabbit intestinal mucosa. These results demonstrate that the novel, natural gum from Hakea gibbosa may be used to sustain the release of sCT from a unidirectional-release buccal tablet. The mechanism of in vitro release is likely to involve peptide diffusion/polymer dissolution. The mucoadhesive strength, as measured by the force of detachment, can be modulated by altering the amount of hakea in the tablet. The mucoadhesive buccal tablets described in this paper represent an improved transbuccal delivery system for therapeutic polypeptides.

Prevalence, in-vitro secretory activity, and cytotoxicity of Aeromonas species associated with childhood gastroenteritis in Chennai (Madras), India.

An investigation on the prevalence of Aeromonas in gastrointestinal illnesses of pediatric inpatients 1 month to 3 years of age was conducted from February 1997 through January 1998 in Madras. Sixteen Aeromonas spp. were isolated from 11 male and five female children among the 341 pediatric inpatients suffering from acute diarrhoea. A. caviae, which was isolated from nine cases, was found to be the most predominant isolate, followed by A. veronii biovar sobria, isolated from six cases, and A. hydrophila, isolated from one case. Shigella flexneri was recovered along with Aeromonas veronii biotype sobria serotype 035 from one 5-month-old female child. We did not notice any seasonal pattern in the association between Aeromonas and childhood gastroenteritis. None of the 147 stool samples obtained from age-matched non-diarrhoeic control children yielded Aeromonas spp. Isolation of Aeromonas spp. from patients suffering from gastroenteritis was found to be significant (chi 2 = 7.1312; P = 0.008, < 0.01). Among the 16 Aeromonas isolates, seven isolates of A. caviae and two isolates of A. veronii biovar sobria induced a secretory response in rabbit intestinal mucosa mounted in Ussing chambers as demonstrated by a significant increase in the short circuit current. Nine of the 16 Aeromonas isolates, including three isolates of A. caviae, five isolates of A. veronii biovar sobria, and the solitary isolate of A. hydrophila were also cytotoxic to CHO cells. Five of the six isolates of A. veronii biovar sobria and the A. hydrophila isolate produced hemolysin. The results of this study indicate that Aeromonas species are important causative agents of diarrhoea in childhood gastroenteritis and are prevalent throughout the year in Madras.

Vibrio mimicus attaches to the intestinal mucosa by outer membrane hemagglutinins specific to polypeptide moieties of glycoproteins.

Vibrio mimicus is the closest organism to Vibrio cholerae. V. mimicus E-33, which is a highly adhesive and enteropathogenic strain, is known to produce three types of hemagglutinins (HAs), i.e., a 31-kDa exocellular metalloprotease (Vm-HA/protease), lipopolysaccharide (Vm-LPSHA), and a 39-kDa major outer membrane protein (Vm-OMPHA). Hemagglutination induced by Vm-LPSHA and Vm-OMPHA was inhibited by glycoproteins, including mucin, fetuin, and asialofetuin, but not by monosaccharides, disaccharides, or N-acetylated saccharides. The inhibitory potential of each glycoprotein for Vm-OMPHA was greatly augmented by treatment with a glycolytic enzyme such as beta-D-galactosidase or beta-D-glucosidase, while pronase treatment achieved complete abolition of the inhibitory potential. The inhibitory ability of the glycoproteins for Vm-LPSHA was also abolished by pronase treatment; however, glycolytic enzyme treatment showed no effect. Hence, the polypeptide portion of glycoproteins may directly associate with Vm-OMPHA and Vm-LPSHA, but the sugar moiety may act as a barrier to interaction with Vm-OMPHA. The glycoproteins as well as Fab antibodies against Vm-OMPHA and Vm-LPSHA eliminated the ability of E-33 cells to agglutinate rabbit erythrocytes and to attach to rabbit intestinal mucosa. Additionally, expression of the hemagglutinating ability by the bacterial cells was accompanied by efficient bacterial adherence to the intestinal mucosa. Finally, the hemagglutinating activity of Vm-OMPHA was markedly increased by incubation with Vm-HA/protease. These results indicate that all three HAs may have significant roles in the glycoprotein-mediated intestinal adherence of V. mimicus E-33.

Expression of virulence-related properties by, and intestinal adhesiveness of, Vibrio mimicus strains isolated from aquatic environments.

A study of the major pathogenic characteristics of Vibrio mimicus was carried out with 77 strains isolated from aquatic environments in Okayama, Japan. Of the strains tested, 96% demonstrated in vitro adherence to the rabbit intestinal mucosa, of which 36, 20, and 43% belonged to the strongly, moderately, and weakly adhesive groups, respectively. Of the 27 strains which appeared to be enterotoxigenic in the experiments using rabbit ileal loops, 74% belonged to the strongly adhesive group. All strains of V. mimicus at early log phase showed cell-mediated hemagglutination, and 70% of strongly hemagglutinative strains belonged to the strongly adhesive group, implying a possible correlation between cell-mediated hemagglutination and bacterial adherence. However, no significant correlation could be detected in the production of putative exocellular pathogenic factors and bacterial adherence or enterotoxigenicity.

Effects of concanavalin A and pokeweed lectins on microvillar membrane proteins during the organ culture of rabbit intestinal mucosa

The effects of pokeweed lectin ( PWL) and concanavalin A (Con A) on microvillar membrane ( MVM) proteins during the organ culture of rabbit ileal explants for 24 hours were compared with the known effects of enteropathogenic Escherichia coli ( EPEC). PWL resulted in the accelerated release of brush border enzymes into the culture medium, accompanied by decreased tissue activities and an increase in the total activity present in the tissue and culture medium. Con A had less effect on the release and tissue activities of brush border enzymes and the total activity was not increased. Sucrose density gradient centrifugation of the culture medium showed that MVM enzymes were predominantly particulate, consistent with their release as vesicles. Centrifugation of ileal explants showed that PWL, but not Con A, resulted in a decrease in the modal density of the brush border which was consistent with a lower glycoprotein-to-lipid ratio. These findings suggest that PWL, in common with EPEC, may cause the disruption and vesiculation of microvilli and the compensatory stimulation of MVM protein synthesis.

Interactions of intestinal mediators in the mode of action of cholera toxin.

Cholera toxin (CT) and prostaglandin E2 (PGE2) increased the synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) in rabbit intestinal mucosa, which appeared to be responsible for inducing the release of 5-hydroxytryptamine (5-HT) from enterochromaffin cells into the intestinal lumen. With isolated intestinal cells, CT induced the synthesis of PGE2 more efficiently from epithelial cells than from lamina propria cells; however, the basal amount of this eicosanoid produced by lamina propria cells was approximately six-fold more than that formed by the epithelial cells. The CT-induced stimulation of arachidonate metabolism appeared to be generalised in nature, as PGF2 alpha and leukotrienes were synthesised in addition to PGE2. Injection of dibutyryl cAMP into the intestinal lumen in vivo markedly reduced both basal levels of PGE2, as well as CT-induced levels of PGE2, released into the luminal fluid. Similarly, when biopsy samples of tissue from rabbit intestinal loops, challenged in vivo with dibutyryl cAMP, were washed and incubated in vitro, the amount of PGE2 synthesis remained below basal levels. In contrast, when biopsy samples of normal small intestinal tissue were exposed in vitro to dibutyryl cAMP, PGE2 synthesis increased. Thus, cAMP appeared to down-regulate the levels of intestinal eicosanoids in vivo, despite its innate capacity to evoke PGE2 synthesis from mucosal tissue in vitro. Thus, the data indicate that CT-induced mediators exhibit interactive effects that alter their cellular concentrations, that in turn could affect the biological responses.

Modulation of beta-lactoglobulin transport in rabbit ileum

This study was undertaken to investigate the mechanisms of beta-lactoglobulin transport and its regulation in rabbit intestinal mucosa. beta-Lactoglobulin, the major bovine milk whey protein, was slightly degraded by ileal brush-border membranes, indicating the presence of phosphoramidon-insensitive neutral endoproteases. Modulation of 14C-radiolabeled beta-lactoglobulin transport across rabbit ileum mounted in Grass diffusion cells was studied in Ringer solution in the presence and absence of (in mM) 25 glucose, 10 galactose, and 0.5 colchicine. The transport was also measured in Dulbecco's modified Eagle's medium as a more "balanced" medium. The transport of the degraded products and of [3H]polyethylene glycol-4000, which was used as an extracellular marker, was enhanced by addition of glucose, presumably indicating passage through the tight junctions. The transepithelial passage of intact protein was estimated to be 10-20% by trichloroacetic acid precipitation and high-performance liquid chromatography, thus indicating the effective transport of the intact protein. The passage of the intact protein was inhibited by both colchicine, a microtubule-depolymerizing agent, and galactose by 50 and 30%, respectively. Galactose-like glycoconjugates, known to be present on the membrane, might be involved in the interaction between the protein and the brush border.

Characterization of two forms of peptide YY, PYY(1–36) and PYY(3–36), in the rabbit

Peptide YY (PYY) has been purified as a 36 amino acid peptide from intestinal extracts of several mammalian species including pig, rat, dog, and man. The primary structure of rabbit PYY is still unknown, although rabbit tissues have extensively been used for characterization of PYY receptor subtypes and receptor subtype-mediated actions. We report the purification and primary structure of PYY(1–36) (PYY-I) from rabbit intestinal mucosa, and the existence of a second endogenous molecular form of PYY, PYY(3–36) (PYY-II). The amino acid sequence of PYY-I is YPSKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide. Rabbit PYY differs from porcine PYY, which is identical to rat and canine PYY, by two amino acid substitutions at positions 3 (Ser instead of Ala) and 18 (Asp instead of Ser), whereas rabbit PYY and human PYY differ by only one residue at position 3 (Ser instead of Ile). The existence of two endogenous forms of PYY in the rabbit, with PYY-II lacking the aminoterminal dipeptide Tyr-Pro of PYY-I, is consistent with previously reported findings, demonstrating the existence of PYY-II in man and dog (9,11). We have previously demonstrated that PYY-I is an unselective Y 1/Y 2 agonist, whereas PYY-II is a highly selective Y 2 agonist. Thus, proteolytic processing of PYY-I controls the peptide's receptor selectivity. The existence of PYY-I and PYY-II in the rabbit supports the assumption of a physiological role of Y receptor heterogeneity for PYY.

PH-dependent transepithelial transport of cephalexin in rabbit intestinal mucosa

Previous studies have demonstrated carrier-mediated cephalexin (CPX) transport in rat jejunum under basal conditions. The purpose of this study was to compare fluxes of cephalexin (CPX) and benzylpenicillin (BZP) across rabbit intestine to those reported in rat intestine, to determine whether these fluxes exhibit regioselectivity and to investigate the pH dependence of these fluxes. Fluxes of CPX were examined in rabbit jejunal, ileal and distal colonie mucosa in Ussing chambers. Concurrently, transepithelial (TE) potential difference, TE electrical conductance and mannitol fluxes were determined to evaluate tissue viability and integrity. Mucosal (m)-to-serosal(s) fluxes of CPX were ~ 3-times the s-to-m fluxes (0.66 vs 0.18% h −1 3 cm −2, respectively) in both jejunum and ileum under short-circuit conditions. Distal colonic m-to-s and s-to-m fluxes were ~ 0.18% h −1 3 cm −2. Inhibition of Na +/K +-ATPase activity with ouabain or incubation with the Na +H + exchange inhibitor, amiloride, reduced by 70 and 40%, respectively, the m-to-s fluxes of CPX in the ileum. Effects of changes in luminal pH on TE transport of CPX and mannitol were studied in ileal and colonic mucosa. At a luminal pH of 5.5, ileal m-to-s transport of CPX was increased to 2.85 ± 0.12% h −1 3 cm −2 while mannitol flux and electrical properties were unchanged. In distal colon, pH 5.5 in the luminal bathing solution did not alter CPX or mannitol fluxes. pH dependent carrier-mediated transport was not exhibited by BZP. Thus CPX appears to be absorbed by a pH dependent, carrier-mediated mechanism confined to the small intestine whereas BZP absorption appears to occur by an entirely passive process.