Glutathione S-transferase in mucus of rat small intestine.

Glutathione S-transferases in the small intestine function in detoxification of electrophilic compounds ingested in foods, dietary supplements, and orally administered drug preparations. Although the required substrate glutathione (GSH) is synthesized in the intestinal enterocytes, the rate of synthesis is slow compared to both the maximal GST activity and the rate of uptake of luminal GSH. GSH is supplied to the intestinal lumen in the bile, and normal luminal concentrations in the rat are about 250 microM. The present study was designed to test the hypothesis that exogenous GSH is used for intestinal conjugation by glutathione S-transferase. The results show that 250 microM of extracellular GSH stimulated conjugation of 1-chloro-2,4-dinitrobenzene by approximately 300% in rat intestinal enterocyte preparations. However, an unexpected finding was that most of this stimulated activity did not depend upon uptake of GSH by the enterocytes but was due to glutathione S-transferase associated with mucus. Immunohistochemistry of glutathione S-transferase in the intact small intestine confirmed that a portion of the GST is present in the mucus layer. The presence of this detoxication enzyme in the extracellular mucus layer provides a novel mechanism for preventing direct contact of potentially toxic dietary electrophiles with the intestinal enterocytes.     

Rational selection of antisense oligonucleotide sequences.

The purpose of this review is to identify rational selection procedures for the identification of optimal antisense oligonucleotide sequences. The review is firstly focused on how to find optimal hybridization sites, and secondly on how to select sequences that bind to structured RNA. The methods reviewed range from the more empirical testing of large numbers of mRNA complementary sequences to the more systematic techniques, i.e. RNase H mapping, use of combinatorial arrays and prediction of secondary structure of mRNA by computational methods. Structures that bind to structured RNA, i.e. aptastrucs and tethered oligonucleotide probes, and foldback triplex-forming oligonucleotides are also discussed. Relating to selection of antisense sequences by aid of computational analysis, valuable www addresses are given along with examples of folded structures of mRNA.     

Highly loaded nanoparticulate carrier using an hydrophobic antisense oligonucleotide complex.

Antisense oligonucleotides, and particularly those with phosphorothioate backbones, have emerged as potential gene specific therapeutic agents and are currently undergoing evaluation in clinical trials for a variety of diseases. In the area of HIV-1 therapeutics, targeting of oligonucleotides to infected cells, such as macrophages, would be highly desirable. The present study was designed to prepare and characterize oligonucleotide-loaded nanoparticles for this purpose. Due to their hydrophilic characteristics, oligonucleotides are difficult to entrap in polymeric particles. Here, the oligonucleotides were first complexed with cetyltrimethylammonium bromide. The oligonucleotide-loaded nanoparticles were prepared by the emulsification-diffusion method and subsequently purified. In comparison with previous studies, a high oligonucleotide-loading was achieved; 2.5, 5 and 10% oligonucleotide loading were assessed. If the initial oligonucleotide content was 4%, this method produced a final oligonucleotide loading of 1.9% with an entrapment efficiency of 47%. The integrity of the oligonucleotide and of the polymer, in the final freeze-dried product, was retained.     

Cimetidine absorption and elimination in rat small intestine.

The purpose of this study was to determine the characteristics of intestinal absorption and metabolism of cimetidine. The initial finding of the appearance of cimetidine sulfoxide in rat and human jejunum from cimetidine perfusions had prompted an isolation of mucosal membrane transport and enterocyte metabolism contributions in earlier membrane vesicle and microsomal studies, respectively. In this report, perfusion studies in rat small intestine detail regional differences in intestinal elimination. Cimetidine S-oxide appears to a significantly greater extent in the jejunum compared with the ileum. Jejunal metabolite appearance is shown to be a function of the pH-dependent intracellular uptake of cimetidine. Cimetidine permeability decreases with increasing perfusion concentration in both jejunum and ileum. Similar permeability magnitudes and concentration dependence are observed in both regions. Perfusion studies with inhibitors of cimetidine mucosal transport and inhibitors of microsomal S-oxidation provide an inhibition profile suggesting that jejunal cimetidine permeability decreases with increasing intracellular cimetidine concentration. The data support a reduction in paracellular cimetidine absorption as controlled by intracellular cimetidine. This inference is drawn on the basis of mass balance. Because significant appearance of cimetidine S-oxide was previously found in human jejunal perfusions, this region-dependent intestinal elimination process detailed in rats may be relevant to drug plasma-level double peaks observed in clinical studies. Saturation of jejunal metabolism at typical oral doses may limit paracellular absorption of cimetidine in the jejunum and contribute to the double peak phenomenon and to absorption variability.     

Down-regulation of a gene-expression by an antisense BNA oligonucleotide

We recently developed a novel nucleic acid analogue, Bridged Nucleic Acid (BNA), one of the most promising artificial nucleic acids for antisense and/or antigene methodology. The antisense effects of BNA modified oligonucleotides targeting the Internal Ribosomal Entry Site (IRES) in Hepatitis C Virus (HCV) RNA were evaluated. As a result, it was found that the antisense BNA oligonucleotides efficiently suppressed the targeted gene-expression in a sequence specific manner. Although the stem region in the mRNA is generally thought to be out of target for the antisense strategy, BNA oligonucleotide targeting the stem region in the HCV-IRES gave a positive antisense effect, also. It is quite noteworthy.     

Gab2 antisense oligonucleotide blocks rat basophilic leukemic cell functions

Adapter molecule Grb2-associated binder-like protein 2 (Gab2) plays a critical role in FcepsilonRI-induced mast cell degranulation and activation. The present study aimed to investigate the pharmacological effects of an antisense oligonucleotide (ASO) targeted at Gab2 on the immune responses of rat basophilic leukemic (RBL)-2H3 cells. Gab2 ASOs were rationally designed and transfected into RBL-2H3 cells. Gab2 mRNA and protein knockdown was confirmed by real-time RT-PCR and immunoblotting, respectively. Effects of Gab2 ASO on FcepsilonRI-induced release of histamine and beta-hexosaminidase was measured by EIA and an enzymatic assay, respectively; signaling events by immunoblotting; and cytokine mRNA expression by RT-PCR. Effects of Gab2 ASO on cell adhesion and migration were performed on fibronectin-coated 96-well plate and transwells cell culture chambers, respectively. We have characterized a phosphorothioate-modified ASO targeted at Gab2 mRNA that was able to knockdown Gab2 mRNA and protein in RBL-2H3 cells. Gab2 ASO significantly blocked IgE-mediated mast cell release of beta-hexosaminidase and histamine; phosphorylation of Akt, p38 mitogen-activated protein kinase and PKCdelta; and up-regulation of cytokine mRNA levels (e.g. IL-4, -6, -9 and -13, and TNF-alpha). In addition, Gab2 ASO markedly prevented mast cell adhesion to fibronectin-coated plates and restrained random migration of RBL-2H3 cells in cell culture chambers. Our findings show that Gab2 knockdown in RBL-2H3 cells by ASO strategy can suppress many aspects of the mast cell functions and, therefore, a selective Gab2 ASO may have therapeutic potential for mast cell-dependent allergic disorders.     

Characterization of ethiofos absorption in the rat small intestine.

The absorption characteristics of ethiofos were studied using the rat in situ intestine circulating perfusion technique. Slow absorption kinetics were observed for ethiofos with varying rates of absorption and metabolism/degradation in situ as a function of buffer and absorption enhancers. In most cases less than 10 per cent of the radiolabeled compound is lost from the circulating perfusate in 90 min. In addition, over the same time period greater than 40 per cent of the intact parent compound was lost by degradation. Much of the difference can be accounted for in the formation of the free thiol metabolite. WR-1065, suggesting ester hydrolysis or metabolic activity. Good stability was observed in all perfusate systems ex vivo indicating that the degradation occurs in situ. The disodium salt of ethylenediaminetetraacetic acid (EDTA) was shown to be an effective absorption enhancer of ethiofos. The enhancement of intestinal absorption by EDTA was dose-dependent resulting in a 20-fold increase in blood levels of ethiofos in the portal blood. Follow-up studies in the rhesus monkey confirm this observation. Salicylate and dimethylsulfoxide (DMSO) also resulted in absorption enhancement although to a lesser degree than that seen after EDTA treatment. Addition of several alkaline phosphatase inhibitors did not significantly improve absorption of ethiofos in the rat small intestine. Proposed mechanism(s) for intestinal absorption and absorption enhancement of ethiofos are discussed.     

Inhibition of post-trabeculectomy fibrosis via topically instilled antisense oligonucleotide complexes co-loaded with fluorouracil

Trabeculectomy is the mainstay of surgical glaucoma treatment, while the success rate was unsatisfying due to postoperative scarring of the filtering blebs. Clinical countermeasures for scar prevention are intraoperative intervention or repeated subconjunctival injections. Herein, we designed a co-delivery system capable of transporting fluorouracil and anti-TGF-β2 oligonucleotide to synergistically inhibit fibroblast proliferation via topical instillation. This co-delivery system was built based on a cationic dendrimer core (PAMAM), which encapsulated fluorouracil within hydrophobic cavity and condensed oligonucleotide with surface amino groups, and was further modified with hyaluronic acid and cell-penetrating peptide penetratin. The co-delivery system was self-assembled into nanoscale complexes with increased cellular uptake and enabled efficient inhibition on proliferation of fibroblast cells. In vivo studies on rabbit trabeculectomy models further confirmed the anti-fibrosis efficiency of the complexes, which prolonged survival time of filtering blebs and maintained their height and extent during wound healing process, exhibiting an equivalent effect on scar prevention compared to intraoperative infiltration with fluorouracil. Qualitative observation by immunohistochemistry staining and quantitative analysis by Western blotting both suggested that TGF-β2 expression was inhibited by the co-delivery complexes. Our study provided a potential approach promising to guarantee success rate of trabeculectomy and prolong survival time of filtering blebs.     

A method for stabilization of monoamine oxidases in homogenates of rat intestine epithelium

In a homogenate of epithelium isolated from the small intestine of male Wistar rats, the amine oxidase activity with 10(-3)M tyramine was 9200 +/- 200 nmol (g tissue)-1 h-1 of which 91% was due to the A form of monoamine oxidase (MAO) and 9% to the B form. Semicarbazide-sensitive amine oxidase activity was not detected with either 10(-3)M tyramine or 10(-4)M benzylamine as substrate. However, it was detectable in the homogenate of the gut residue where the activity with 10(-4)M benzylamine was 3600 +/- 200 nmol (g tissue)-1 h-1. The MAO activity, in homogenates of epithelium prepared with 0.1 M sodium phosphate pH 7.4, was stable at 4 degrees C for at least 6 h whilst at minus 20 degrees C it decreased by 70% within 24 h. Incorporation of 10% (v/v) glycerol into the homogenization medium stabilized the enzymes. The total activity and proportions due to MAO-A and MAO-B and kinetic constants for tyramine and 5-hydroxytryptamine, did not alter during 5 weeks storage at -20 degrees C. The ability to store tissue homogenates should facilitate studies of intestinal amine oxidases.     

Absorption of iron from iron succinyl-protein complexes by mouse small intestine.

The absorption of iron from iron succinyl-protein complexes was investigated in mice. 59Fe-labelled succinyl-casein and -albumin complexes, [59Fe]ferritin and 59FeSO4, at doses of 20 or 200 micrograms of iron, were administered orally to normal mice or mice with absorption enhanced by chronic hypoxia. 59Fe from iron succinyl-protein was well absorbed in normal mice (greater than 10% of dose) and showed enhanced absorption in hypoxic mice (greater than 40% of dose). Intestinal uptake was predominantly by the duodenum for all compounds studied. In-vivo absorption of 59Fe from an iron succinyl-protein complex was studied using tied-off segments of mouse duodenum, jejunum or ileum of normal or hypoxic mice. Incubation for up to 15 min in duodenum or 60 min in ileum showed very little absorption of 59Fe. No enhancement of absorption was seen in hypoxic mice. It was concluded that absorption of the intact iron succinyl-protein complex cannot explain absorption seen after oral dosing.     

Hydrogen peroxide reduces beta-adrenoceptor function in the rat small intestine.

Incubation of isolated rat intestinal segments with hydrogen peroxide (H2O2) led to a decreased beta-adrenoceptor response. The maximal relaxation induced by isoprenaline was lowered while the EC50 remained unaffected. The effect of H2O2 in the small intestine increased slightly from duodenum to ileum. In the ileum, 10(-4) M H2O2 led to a 10% decrease of the maximal relaxation due to isoprenaline and 1 mM decreased the maximal response to about 50%. We further investigated the level at which the isoprenaline response was impaired. The relaxation caused by the stable cAMP analog, dibutyryl-cAMP, or by the adenylate cyclase activator, forskolin, was not affected or affected less than by isoprenaline. When the response to isoprenaline was expressed relative to the maximal response to dibutyryl-cAMP or forskolin, pretreatment with H2O2 led to a decreased isoprenaline response relative to the response to dibutyryl-cAMP or forskolin. This might indicate that exposure to H2O2 leads to a disturbance in receptor-mediated cAMP production. The adenylate cyclase unit is probably not affected since the response to forskolin is relatively resistant to H2O2. Our conclusion is that pretreatment of isolated intestinal segments with H2O2 leads to disturbed beta-adrenoceptor coupling, probably due to altered membrane integrity.     

Drug absorption from the irradiated rat small intestine in situ.

The absorption of acidic drugs phenobarbitone and sulphafurazole, basic drugs mecamylamine and quinidine, and a neutral drug isoniazid was studied in situ. Rats were irradiated 750 rad whole-body with 60Co and the absorption experiment was done three and six days thereafter using the cannulated small intestine of urethane-anaesthetized rats. Drug disappearance from the intestinal lumen and drug levels in the whole blood and intestinal wall were measured. In control rats phenobarbitone showed the most rapid absorption and mecamylamine the slowest. Irradiation retarded the disappearance of all drugs from the intestinal lumen on the third postirradiation day. Fluid absorption was also diminished. On the sixth postirradiation day the absorption of phenobarbitone, sulphafurazole and mecamylamine had returned to the control level, but the absorption of quinidine and isoniazid was still retarded. After i.v. administration of drugs they were not significantly excreted into the intestinal contents and irradiation did not modify excretion. The distribution of drugs between the intestinal fluid and the intestinal wall was complete in the first 10 min of experiment. Mecamylamine and quinidine were lowered in the whole blood by irradiation. Blood levels of drugs did not correlate well to the rate of disappearance of drugs from the intestinal lumen. The reversible changes in absorption induced by irradiation are probably secondary effects of irradiation on intestinal morphology, permeability and transport capacity, composition, and possibly blood flow.     

The absorption of warfarin from the rat small intestine in situ.

The in situ absorption from the rat small intestine of the weakly acidic drug, warfarin (pKa 5-05), at 200 mug ml-1 in the instilled fluid with initial pH levels of 3, 5, 7 or 8 has been examined. These initial pH's in the buffer changed rapidly towards neutrality. The buffers at pH's 3 and 5 probably caused different amounts of warfarin precipitation, which resulted in different rates of warfarin disappearance from the instilled fluid which paralleled the initial rates of accumulation of warfarin in (or on) the intestinal wall. Where greater drug precipitation had probably occurred the initial rates of absorption into the plasma were slower. At the initial pH of 3 and by solubilization of warfarin with propylene glycol, the rate of absorption was similar to that from a fluid of pH 7. Propylene glycol in 15% solution did not affect the system significantly. The relatively high transfer of warfarin into octanol from buffer solution at pH 7 might indicate that the small fraction of unionized drug (1 : 100) at pH 7 is enough for remarkable transfer of this highly lipid-soluble drug.     

First-pass hydrolysis of a propranolol ester derivative in rat small intestine.

To evaluate the first-pass hydrolysis of O-isovaleryl-propranolol (isovaleryl-PL), which was used as a model ester-compound, rat intestinal jejunum and blood vessels were perfused simultaneously. The membrane permeability of isovaleryl-PL was greater than that of PL because it was more lipophilic. Isovaleryl-PL was almost completely hydrolyzed to PL and isovaleric acid (IVA) in epithelial cells at a rate limited by its uptake. Based on pH partitioning, PL and IVA were transported into both vascular (pH 7.4) and luminal sides (pH 6.5). Therefore, when isovaleryl-PL was perfused into the jejunal lumen, more than 90% permeated into the blood vessel as PL. In addition, PL appeared in the lumen at a rate 6-fold greater than that in blood vessels. When isovaleryl-PL was perfused, its disappearance (50.5+/-1.95 nmol/min) was the sum of the absorption and secretion rates of PL. In contrast, IVA was transported into blood vessels rather than the jejunal lumen. In addition, the calculated degradation clearance from in vitro hydrolysis (Km 13.7+/-1.71 microM, Vmax 29.1+/-3.81 nmol/min/mg protein) was 3.42 ml/min/10 cm jejunum, which was 24-fold greater than the observed degradation clearance (CLdeg) (0.14+/-0.02 ml/min/10 cm jejunum). These findings indicate that in addition to the liver, the intestine markedly contributes to first-pass hydrolysis.     

Autoradiographic localisation of opiate receptors in rat small intestine

[3H]Naloxone and [3H]dihydromorphine are selective ligands for opiate receptors. Using an in vitro autoradiographic technique, binding of these ligands has been demonstrated to the villi and crypts in rat small intestine. These results indicate the presence of opiate receptor sites in the small intestine which suggests further a role for endogenous opiates in the transport functions of intestinal mucosa.     

Limited interaction between tacrolimus and P-glycoprotein in the rat small intestine.

The significance of intestinal P-glycoprotein (P-gp) in determining the oral bioavailability of tacrolimus has been still controversial. In this study, we reevaluated the interaction of tacrolimus with P-gp in the rat small intestine, by evaluating its absorption from the rat small intestine and its modulating effect on the absorption of known P-gp substrates (digoxin, methylprednisolone, and vinblastine). Intestinal absorption of tacrolimus itself was as extensive as other P-gp modulators such as cyclosporine and verapamil. While cyclosporine and verapamil significantly increased the absorption of methylprednisolone and vinblastine through potent inhibition of intestinal P-gp, tacrolimus failed to achieve this. When cyclosporine and tacrolimus were intravenously administered to rats, digoxin absorption was significantly increased by cyclosporine but not by tacrolimus. When tacrolimus was coadministered with clotrimazole, a specific CYP3A inhibitor, into the rat small intestine, the area under the curve of tacrolimus blood concentrations increased more than seven-fold compared with that of tacrolimus alone. Our present results strongly suggest that the interaction between tacrolimus and P-gp is limited in the rat small intestine and that extensive metabolism by CYP3A enzymes is more responsible for the low oral bioavailability of tacrolimus. It was considered that the extensive absorption of cyclosporine and verapamil was closely associated with their potent ability to inhibit intestinal P-gp.     

Drug absorption from in situ rat small intestine during metoclopramide administration.

The effect of metoclopramide on the absorption of drugs in solution in the small intestinal lumen of rats in situ was studied. Metoclopramide in doses up to 50 mg/kg sc did not significantly modify the disappearance of isoniazid and quinidine from the small intestinal lumen. At the end of the absorption experiments, quinidine in the whole blood of the experimental animals was increased after metoclopramide. The blood level did not correlate to the drug disappearance from the intestinal lumen. The results probably differ from those obtained when drugs are given orally to subjects treated with metoclopramide.     

KATP通道激活介导大鼠小肠的缺血预处理

AIM: To study whether the protective effects of ischemic preconditioning against rat small intestine ischemia/reperfusion injury could be mediated by KATP channel opener. METHODS: Preconditioning (Pc) was induced by 3 cycles of 8-min superior mesenteric artery (SMA) occlusion and 10-min reperfusion before prolonged ischemia. Cromakalim (Cro 75 micrograms.kg-1) and glibenclamide (Gli 8 mg.kg-1) were injected i.v. 10 min before prolonged ischemia and Pc, respectively. RESULTS: Compared with ischemic reperfusion (IR) group, Pc before prolonged ischemia (Pc + IR) decreased LDH release [(380 +/- 55) vs (559 +/- 49) U.L-1, P < 0.05], attenuated intestinal edema [wet weight/dry weight (WW/DW), 5.6 +/- 0.6 vs 6.34 +/- 0.29, P < 0.05], ameliorated intestinal histological damage (grading scale, 3.4 vs 5.7, P < 0.01), and improved reperfusion-induced hypotension. These effects of Pc were mimicked by Cro [LDH, (298 +/- 40) vs (559 +/- 49) U.L-1, P < 0.05; WW/DW, 5.6 +/- 0.4 vs 6.34 +/- 0.29, P < 0.05; grading scale, 3.6 vs 5.7, P < 0.01] and abolished in the presence of Gli [LDH, (624 +/- 44) vs (559 +/- 49) U.L-1; WW/DW, 6.6 +/- 0.6 vs 6.34 +/- 0.29; grading scale, 5.7 vs 5.7; P > 0.05] compared with IR group, respectively. CONCLUSION: Ischemic preconditioning on the rat small intestine is mediated by activation of KATP channels.     

Curcumin reduces indomethacin-induced damage in the rat small intestine

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in clinical medicine. Their utility is, however, often limited by the adverse effects they produce in the gastrointestinal tract. Oxidative stress has been shown to occur in the small intestine in response to the oral administration of indomethacin, an NSAID commonly used in toxicity studies. In view of this, the effect of curcumin, an agent with anti-oxidant properties, was evaluated on indomethacin-induced small intestinal damage in a rat model. Rats were pretreated with various doses of curcumin (20 mg kg(-1), 40 mg kg(-1) and 80 mg kg(-1)) before administering indomethacin at 20 mg kg(-1). Various parameters of oxidative stress and the extent of small intestinal damage produced by indomethacin, with and without pretreatment with curcumin, were measured. Macroscopic ulceration was found to occur in the small intestine in response to indomethacin. The viability of enterocytes from indomethacin-treated animals was significantly lower than those from control animals. Drug-induced oxidative stress was also evident as seen by increases in the levels of malondialdehyde and protein carbonyl and in activities of pro-oxidant enzymes such as myeloperoxidase and xanthine oxidase in indomethacin-treated rats. Concomitant decreases were seen in the activities of the antioxidant enzymes catalase and glutathione peroxidase in these animals. Pretreatment with curcumin was found to ameliorate these drug-induced changes. Thus, curcumin appears to hold promise as an agent that can potentially reduce NSAID-induced small intestinal damage.