Nitric oxide control of the dorsal aorta and the intestinal vein of the Australian short-finned eel Anguilla australis

This study investigated the mechanisms by which nitric oxide (NO) regulates the dorsal aorta and the intestinal vein of the Australian short-finned eel Anguilla australis. NADPH diaphorase histochemistry and immunohistochemistry using a mammalian endothelial nitric oxide synthase (NOS) antibody could not demonstrate NOS in the endothelium of either blood vessel; however, NOS could be readily demonstrated in the endothelium of the rat aorta that was used as a control. Both blood vessels contained NADPH diaphorase positive nerve fibres and nerve bundles, and immunohistochemistry using a neural NOS antibody showed a similar distribution of neural NOS immunoreactivity in the perivascular nerves. In vitro organ bath physiology showed that a NO/soluble guanylyl cyclase (GC) system is present in the dorsal aorta and the intestinal vein, since the soluble GC inhibitor oxadiazole quinoxalin^-1 (ODQ; 10^–5 mol l^–1) completely abolished the vasodilatory effect of the NO donor, sodium nitroprusside (SNP; 10^–4 mol l^–1). In addition, nicotine (3x10^–4 mol l^–1) mediated a vasodilation that was not affected by removal of the endothelium. The nicotine-mediated dilation was blocked by the NOS inhibitor, Nω-nitro-arginine (L-NNA; 10^–4 mol l^–1), and ODQ (10^–5 mol l^–1). More specifically, the neural NOS inhibitor, Nω-propyl-L-arginine (10^–5 mol l^–1), significantly decreased the dilation induced by nicotine (3x10^–4 mol l^–1). Furthermore, indomethacin (10^–5 mol l^–1) did not affect the nicotine-mediated dilation, suggesting that prostaglandins are not involved in the response. Finally, the calcium ionophore A23187 (3x10^–6 mol l^–1) caused an endothelium-dependent dilation that was abolished in the presence of indomethacin. We propose the absence of an endothelial NO system in eel vasculature and suggest that neurally derived NO contributes to the maintenance of vascular tone in this species. In addition, we suggest that prostaglandins may act as endothelially derived relaxing factors in A. australis.

Copper exposure induces trafficking of the Menkes protein in intestinal epithelium of ATP7A transgenic mice

The final steps in the absorption and excretion of copper at the molecular level are accomplished by 2 closely related proteins that catalyze the ATP-dependent transport of copper across the plasma membrane. These proteins, ATP7A and ATP7B, are encoded by the genes affected in human genetic copper-transport disorders, namely, Menkes and Wilson diseases. We studied the effect of copper perfusion of an isolated segment of the jejunum of ATP7A transgenic mice on the intracellular distribution of ATP7A by immunofluorescence of frozen sections. Our results indicate that ATP7A is retained in the trans-Golgi network under copper-limiting conditions, but relocalized to a vesicular compartment adjacent to the basolateral membrane in intestines perfused with copper. The findings support the hypothesis that the basolateral transport of copper from the enterocyte into the portal blood may involve ATP7A pumping copper into a vesicular compartment followed by exocytosis to release the copper, rather than direct pumping of copper across the basolateral membrane.

Role of P-glycoprotein in the intestinal absorption of tanshinone IIA, a major active ingredient in the root of Salvia miltiorrhiza Bunge

The extracts from the roots of Salvia miltiorrhiza Bunge (Danshen) are widely and traditionally used in the treatment of angina pectoris, acute myocardial infarct, hyperlipidemia and stroke in China and other Asian countries. In this study, we have investigated the role of P-glycoprotein (P-gp) in the intestinal absorption of tanshinone IIA (TSA), a major active constituent of Danshen, using several in vitro and in vivo models. The oral bioavailability of TSA was about 2.9

Role of the ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB, one of the major active diterpenoids from the root of Salvia miltiorrhiza

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (C_max) and area under the plasma concentration-time curve (AUC) with increasing dosage. The time to C_max (T_max) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (P_app) based on TSB disappearance from the lumen (P_lumen) were 6.2- to 7.2-fold higher (p < 0.01) than those based on drug appearance in mesenteric venous blood (P_blood). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The P_app values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC_0-24hr in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.

Lupin (Lupinus albus) protein isolate (L-ISO) has adequate nutritional value and reduces large intestinal weight in rats after restricted and ad libitum feeding.

Background: A protein isolate from white lupin (Lupinus albus; L-ISO) has potential as a novel human food ingredient, but its nutritional effects are unknown.

Methods: We evaluated protein quality and effects on body composition in rats of isoenergic diets of L-ISO, lactalbumin, or casein with both restricted (10-day) and ad libitum (28-day)intake. The diets were equivalent in protein per se, but supplementation was used to balance essential amino acid levels.

Results: In both studies, the rats consumed similar amounts of each diet, and no effect of diet on the gain:feed ratio was observed--though gain:N ratio and net protein utilization were slightly lower for the L-ISO diet. Lower large intestinal weights after the L-ISO than after the lactalbumin diet were observed in both studies. The L-ISO diet resulted in lowered body fat percentage in the 10-day study but in an elevated level in the 28-day study. Liver composition (DNA, RNA, glycogen, and fat) and plasma levels of some amino acids (His, Thr, Ala, Pro, Tyr, Val and Met) were affected by diet, but no effects on plasma lipid, glucose, or uric acid were observed.

Conclusion: The L-ISO diet did not affect feed intake and has adequate nutritional quality in rats whilst modifying large intestinal weight in a potentially beneficial manner--suggesting potential for this protein in human nutrition.

St. Johns wort attenuates irinotecan-induced diarhea via down-regulation of intestinal pro-inflammatory cytokines and inhibition of intestinal epithelial apoptosis

Diarrhea is a common dose-limiting toxicity associated with cancer chemotherapy, in particular for drugs such as irinotecan (CPT-11), 5-fluouracil, oxaliplatin, capecitabine and raltitrexed. St. John's wort (Hypericum perforatum, SJW) has anti-inflammatory activity, and our preliminary study in the rat and a pilot study in cancer patients found that treatment of SJW alleviated irinotecan-induced diarrhea. In the present study, we investigated whether SJW modulated various pro-inflammatory cytokines including interleukins (IL-1β, IL-2, IL-6), interferon (IFN-γ) and tumor necrosis factor-α (TNF-α) and intestinal epithelium apoptosis in rats. The rats were treated with irinotecan at 60 mg/kg for 4 days in combination with oral SJW or SJW-free control vehicle at 400 mg/kg for 8 days. Diarrhea, tissue damage, body weight loss, various cytokines including IL-1β, IL-2, IL-6, IFN-γ and TNF-α and intestinal epithelial apoptosis were monitored over 11 days. Our studies demonstrated that combined SJW markedly reduced CPT-11-induced diarrhea and intestinal lesions. The production of pro-inflammatory cytokines such as IL-1β, IFN-γ and TNF-α was significantly up-regulated in intestine. In the mean time, combined SJW significantly suppressed the intestinal epithelial apoptosis induced by CPT-11 over days 5–11. In particular, combination of SJW significantly inhibited the expression of TNF-α mRNA in the intestine over days 5–11. In conclusion, inhibition of pro-inflammatory cytokines and intestinal epithelium apoptosis partly explained the protective effect of SJW against the intestinal toxicities induced by irinotecan. Further studies are warranted to explore the potential for STW as an agent in combination with chemotherapeutic drugs to lower their dose-limiting toxicities.

A mechanistic study of the intestinal absorption of crypotanshinone, the major active constituent of Saliva miltiorrhiza

The nature of intestinal absorption of most herbal medicine is unknown. Cryptotanshinone (CTS) is the principal active constituent of the widely used cardiovascular herb Salvia miltiorrhiza (Danshen). We investigated the oral bioavailability of CTS in rats and the mechanism for its intestinal absorption using several in vitro and in vivo models:1) Caco-2 cell monolayers; 2) monolayers of MDCKII cells overexpressing P-glycoprotein
(PgP); and 3) single-pass rat intestinal perfusion with mesenteric vein cannulation. The systemic bioavailabilities of CTS after oral and intraperitoneal administration at 100 mg/kg were 2.05 and 10.60%, respectively. In the perfused rat intestinal model, permeability coefficients based on CTS disappearance from the luminal perfusate (P_lumen) were 6.7- to 10.3-fold higher than permeability coefficients based on drug appearance in venous blood (P_blood). P_blood significantly increased in the presence of the P-gP inhibitor, verapamil. CTS transport across Caco-2 monolayers was pH-, temperature- and ATP-dependent. The transport from the apical (AP) to the basolateral (BL) side was 3- to 9-fold lower than that from the BL to the AP side. Inclusion of verapamil (50 µM) in both AP and BL sides abolished the polarized CTS transport across Caco-2 cells. Moreover, CTS was significantly more permeable in the BL to AP than in the AP to BL direction in MDCKII and MDR1-MDCKII cells. The permeability coefficients in the BL to AP direction were significantly higher in MDCKII cells overexpressing PgP. These findings indicate that CTS is a substrate for PgP that can pump CTS into the luminal side.

Effect of dietary fatty acids on the intestinal permeability of marker drug compounds in excised rat jejunum

The aim of this study was to explore the effects of diets containing saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and ω-3 and ω-6 polyunsaturated fatty acids (ω-3 and ω-6 PUFA, respectively) on the passive and active transport properties of rat jejunum using marker compounds. Rats were fed diets supplemented with 18.4% (w/w) lipid (4 groups) or standard rat chow (1 group) for a period of 30 days. At the end of the dietary period, mucosal scrapings were taken for the determination of membrane phospholipids, and the apparent jejunal permeability of radiolabelled marker compounds was determined using modified Ussing chambers. Changes in the phospholipid content of the brush border membrane reflected the different lipid content of the diets. The passive paracellular permeability of mannitol was not significantly affected by the fatty acid composition of the diet, although there was a trend toward decreased mannitol permeability in the rats fed both the ω-3 and ω-6 PUFA diets. In comparison, the transcellular diffusion of diazepam was reduced by 20% (P < 0.05) in rats fed diets supplemented with ω-3 and ω-6 PUFA. In the lipid-fed rats, the serosal to mucosal flux of digoxin, an intestinal P-glycoprotein substrate, was reduced by 20% (P < 0.05) relative to the chow-fed group, however there were no significant differences between the different lipid groups. The active absorption of D-glucose via the Na+-dependent transport pathway was highest in the SFA, MUFA and PUFA ω-3 dietary groups, intermediate in the low-fat chow group and lowest in the PUFA ω-6 group, and was positively correlated with short-circuit current. These studies indicate that dietary fatty acid changes can result in moderate changes to the active and passive transport properties of excised rat jejunum.

Influencing health through intestinal microbiota modulation and probiotics

Science has progressed fast in providing answers to probiotic health benefits to the consumers. This symposium also proves that progress is fast. However, several challenges still need to be solved and more effective strains and strain combinations discovered. This will pave the way from good probiotics to specific products for clearly identified target populations. The intestinal microbiota and its interaction(s) with probiotics challenges researchers to turn to the next new page to discover new approaches and treatment modalities that utilize probiotics as means of providing good nutrition with clear health benefits to all consumers.

Thermodynamics of lipophilic drug binding to intestinal fatty acid binding protein and permeation across membranes

Intestinal fatty acid binding protein (I-FABP) is present at high levels in the absorptive cells of the intestine (enterocytes), where it plays a role in the intracellular solubilization of fatty acids (FA). However, I-FABP has also been shown to bind to a range of non-FA ligands, including some lipophilic drug molecules. Thus, in addition to its central role in FA trafficking, I-FABP potentially serves as an important intracellular carrier of lipophilic drugs. In this study we provide a detailed thermodynamic analysis of the binding and stability properties of I-FABP in complex with a series of fibrate and fenamate drugs to provide an insight into the forces driving drug binding to I-FABP. Drug binding and selectivity for I-FABP are driven by the interplay of protein−ligand interactions and solvent processes. The Gibbs free energies (ΔG°) determined from dissociation constants at 25 °C ranged from −6.2 to −10 kcal/mol. The reaction energetics indicate that drug binding to I-FABP is an enthalpy−entropy driven process. The relationship between I-FABP stability and drug binding affinity was examined by pulse proteolysis. There is a strong coupling between drug binding and I-FABP stability. The effect of an I-FABP protein sink on the kinetics and thermodynamics of tolfenamic acid permeation across an artificial phospholipid membrane were investigated. I-FABP significantly decreased the energy barrier for desorption of tolfenamic acid from the membrane into the acceptor compartment. Taken together, these data suggest that the formation of stable drug−I-FABP complexes is thermodynamically viable under conditions simulating the reactant concentrations likely observed in vivo and maybe a significant biochemical process that serves as a driving force for passive intestinal absorption of lipophilic drugs.

Characterization of lipophilic drug binding to rat intestinal fatty acid binding protein

Intestinal fatty acid binding protein (I-FABP) is present at high levels in the absorptive cells of the intestine (enterocytes) where it plays a role in the intracellular solubilization of fatty acids (FA). However, I-FABP has also been shown to bind to a range of non-FA ligands, including some lipophilic drug molecules, albeit with generally lower affinity than FA. The significance of these lower affinity interactions with exogenous compounds is not known. In this manuscript, we describe further characterization of drug-rat I-FABP binding interactions using a thermal-shift assay. A structural explanation of the observed affinity of rat I-FABP for different drugs based on spectroscopic data and modeling experiments is presented. In addition, immunocytochemistry has been used to probe the expression of I-FABP in a cell culture model reflective of the absorptive cells of the small intestine. Taken together, these data suggest a possible role for I-FABP in the disposition of some lipophilic drugs within the enterocyte.

Examination of the role of intestinal Fatty acid-binding protein in drug absorption using a parallel artificial membrane permeability assay

Transcellular diffusion across the absorptive epithelial cells (enterocytes) of the small intestine is the main route of absorption for most orally administered drugs. The process by which lipophilic compounds transverse the aqueous environment of the cytoplasm, however, remains poorly defined. In the present study, we have identified a structurally diverse group of lipophilic drugs that display low micromolar binding affinities for a cytosolic lipid-binding protein—intestinal fatty acid-binding protein (I-FABP). Binding to I-FABP significantly enhanced the transport of lipophilic drug molecules across a model membrane, and the degree of transport enhancement was related to both drug lipophilicity and I-FABP binding affinity. These data suggest that intracellular lipid-binding proteins such as I-FABP may enhance the membrane transport of lipophilic xenobiotics and facilitate drug access to the enterocyte cytoplasm and cytoplasmic organelles.

The interaction of lipophilic drugs with intestinal fatty acid-binding protein

Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen {approx} bezafibrate > S-ibuprofen >> nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.

Relationship between alcohol abuse upper gastro-intestinal haemorrhage and nutritional status during acute hospital admission

This thesis is concerned with the effect of alcohol consumption on the pathogenesis of bleeding from the upper gastrointestinal tract via nutritional pathways. Altered nutritional status is a frequently recognised clinical accompaniement of heavy alcohol consumption in hospitalized patients. Similarly, upper gastrointestinal bleeding is frequently accompanied by the presence of heavy alcohol consumption. Nevertheless, the clinical quantification of alcohol intake is often descriptive, so that a link between alcohol use and upper gastrointestinal haemorrhage via nutritional mechanisms has been only generally defined. In the literature review, the methods of defining alcohol use and abuse, using interview, biochemical and haematological techniques are noted. The relationship between alcohol abuse and nutrient imbalances is reviewed, especially in relation to possible effects on the gastrointestinal tract, appetite and eating habits. A further section reviews the relationship between alcohol use and anatomical lesions of the upper gastrointestinal tract likely to lead to bleeding. Following the chapter in which the methods used in this thesis are described. Chapter 4 seeks to describe the study population and its subgroups in this thesis in relation to interview, biochemical and haematological methods. Alcohol use is defined in relation to (1) a clinical classification of heavy or light drinking, based on a questionnaire administered in Casualty, (2) a quantified method of determining alcohol consumption during a subsequent ward dietetic assessment, (3) in relation to a biochemical definition (recent drinking and non-drinking), and a classification of (1) and (2) called, for the purposes of this thesis, 'alcohol abusers' and 'nonabusers'. Heavy, regular and recent drinkers and alcohol abusers tend to be male and younger than light, infrequent and nonrecent drinkers and nonabusers. Chapter 5 relates the nutritional status of those patients admitted acutely to hospital in relation to the groups defined in Chapter 4, Nutritional status is defined in terms of food intake, anthropometry, biochemical and haematological parameters. Different methods of defining alcohol use give rise to different patterns of nutritional impairment. Chapter 6 relates the nutritional status of those patients admitted acutely to hospital in relation to the presence or absence of an endoscopically defined site of upper gastrointestinal bleeding. A difference is seen between those bleeding from a Mailory-weiss tear and other sites of bleeding, similarly, biochemical differences in nutritional status emerge between those patients who presented in shock, and those who did not. Chapter 7 explores the relationships between biochemical markers of nutritional status and haemostatic variables in the groups of abusers/non-abusers, the various sites of primary bleeding/controls, and shock/non-shock. Serum copper appears to be related to altered haemostasis in a manner not apparently described elsewhere.