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1.
Sci Rep ; 7(1): 8836, 2017 08 18.
Article in English | MEDLINE | ID: mdl-28821731

ABSTRACT

Inflammatory bowel disease (IBD) is associated with altered microbiota composition and metabolism, but it is unclear whether these changes precede inflammation or are the result of it since current studies have mainly focused on changes after the onset of disease. We previously showed differences in mucus gut microbiota composition preceded colitis-induced inflammation and stool microbial differences only became apparent at colitis onset. In the present study, we aimed to investigate whether microbial dysbiosis was associated with differences in both predicted microbial gene content and endogenous metabolite profiles. We examined the functional potential of mucus and stool microbial communities in the mdr1a -/- mouse model of colitis and littermate controls using PICRUSt on 16S rRNA sequencing data. Our findings indicate that despite changes in microbial composition, microbial functional pathways were stable before and during the development of mucosal inflammation. LC-MS-based metabolic phenotyping (metabotyping) in urine samples confirmed that metabolite profiles in mdr1a -/- mice were remarkably unaffected by development of intestinal inflammation and there were no differences in previously published metabolic markers of IBD. Metabolic profiles did, however, discriminate the colitis-prone mdr1a -/- genotype from controls. Our results indicate resilience of the metabolic network irrespective of inflammation. Importantly as metabolites differentiated genotype, genotype-differentiating metabolites could potentially predict IBD risk.


Subject(s)
Colitis/etiology , Colitis/metabolism , Gastrointestinal Microbiome , Metabolome , Metabolomics , Phenotype , Animals , Chromatography, High Pressure Liquid , Disease Models, Animal , Disease Susceptibility , Genotype , Inflammatory Bowel Diseases/etiology , Inflammatory Bowel Diseases/metabolism , Male , Mass Spectrometry , Metabolomics/methods , Metagenome , Metagenomics , Mice , Mice, Knockout , RNA, Ribosomal, 16S/genetics
2.
J Pharm Biomed Anal ; 110: 27-33, 2015 Jun 10.
Article in English | MEDLINE | ID: mdl-25796981

ABSTRACT

Transporter proteins expressed in the gastrointestinal tract play a major role in the oral absorption of some drugs, and their involvement may lead to drug-drug interaction (DDI) susceptibility when given in combination with drugs known to inhibit gut wall transporters. Anticipating such liabilities and predicting the magnitude of the impact of transporter proteins on oral drug absorption and DDIs requires quantification of their expression in human intestine, and linking these to data obtained through in vitro experiments. A quantitative targeted absolute proteomic method employing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) together with a quantitative concatenation (QconCAT) strategy to provide proteotypic peptide standards has been applied to quantify ATP1A1 (sodium/potassium-ATPase; Na/K-ATPase), CDH17 (human peptide transporter 1; HPT1), ABCB1 (P-glycoprotein; P-gp), ABCG2 (breast cancer resistance protein; BCRP), ABCC2 (multidrug resistance-associated protein 2; MRP2) and SLC51A (Organic Solute Transporter subunit alpha; OST-α), in human distal jejunum (n=3) and distal ileum (n=1) enterocyte membranes. Previously developed selected reaction monitoring (SRM) schedules were optimised to enable quantification of the proteotypic peptides for each transporter. After harvesting enterocytes by calcium chelation elution and generating a total membrane fraction, the proteins were subjected to proteolytic digestion. To account for losses of peptides during the digestion procedure, a gravimetric method is also presented. The linearity of quantifying the QconCAT from an internal standard (correlation coefficient, R(2)=0.998) and quantification of all target peptides in a pooled intestinal quality control sample (R(2)≥ 0.980) was established. The assay was also assessed for within and between-day precision, demonstrating a <15% coefficient of variation for all peptides across 3 separate analytical runs, over 2 days. The methods were applied to obtain the absolute abundances for all targeted proteins. In all samples, Na/K-ATPase, HPT1, P-gp and BCRP were detected above the lower limit of quantitation (i.e., >0.2 fmol/µg membrane protein). MRP2 abundance could be quantified in distal jejunum but not in the distal ileum sample. OST-α was not detected in 2 out of 3 jejunum samples. This study highlights the utility of a QconCAT strategy to quantify absolute transporter abundances in human intestinal tissues.


Subject(s)
Chromatography, Liquid , Ileum/chemistry , Jejunum/chemistry , Membrane Transport Proteins/analysis , Proteomics/methods , Tandem Mass Spectrometry , Calibration , Cell Membrane/chemistry , Chromatography, Liquid/standards , Enterocytes/chemistry , Humans , Ileum/cytology , Jejunum/cytology , Linear Models , Multidrug Resistance-Associated Protein 2 , Proteomics/standards , Reference Standards , Reproducibility of Results , Tandem Mass Spectrometry/standards
3.
Biopharm Drug Dispos ; 34(1): 2-28, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22927116

ABSTRACT

The use of whole body physiological-based pharmacokinetic (PBPK) models linked with in vitro-in vivo extrapolation (IVIVE) of kinetic parameters from laboratory experiments, has become embedded within many of the pharmaceutical industry and is used even as part of regulatory submissions. These include the influence of transporter proteins on drug disposition, a subject for which we have witnessed an increasing awareness. A combination of the development of high-powered analytical techniques and antibody-based technology, together with a realization that an understanding of absolute transporter protein abundances together with activity can potentially enhance the modelling of transporter kinetics by PBPK-IVIVE link models. This review summarizes the mechanistic approaches to integrate suitable non-biased in vitro transporter kinetic data relevant to the intestine (i.e. 'intrinsic' K(i) , 'intrinsic' K(m) ), by in vitro system modelling for these kinetic inputs with the advantages of, and challenges for, generating these data for input into PBPK models. This step is considered as a prerequisite for mechanistic modelling of the oral absorption for drugs that are substrates for transporters. Various approaches are provided to integrate intestinal transporter expression into PBPK models with a perspective on the incorporation of the absolute abundance/activity of transporters to enhance the predictive power of the models. We define the key intestinal tissue and functional expression-based scaling factors required. The objective is to use these for facilitating the extrapolation from in vitro intestinal transporter assays to the in vivo system, using absolute quantification methodologies. The models could be used to elucidate the complex relationship and relative importance of metabolizing enzymes and transporters in drug disposition and toxicity.


Subject(s)
Membrane Transport Proteins/metabolism , Models, Biological , Pharmaceutical Preparations/metabolism , Animals , Biological Transport , Drug Design , Drug Industry , Drug-Related Side Effects and Adverse Reactions , Humans , Intestinal Absorption
4.
Drug Metab Dispos ; 36(1): 87-94, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17932223

ABSTRACT

Modified release (MR) formulations are used to enhance the safety and compliance of existing drugs by improving their pharmacokinetics. Predicting the likely success of MR formulations is often difficult before clinical studies. A systematic in vitro approach using mouse and human tissues was adopted to rationalize the in vivo pharmacokinetics of 9- and 15-h MR formulations of an alpha-adrenoreceptor antagonist, 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4 tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline (UK-338,003). Immediate release UK-338,003 was well absorbed in humans consistent with moderate Caco-2 cell monolayer permeability. In contrast, 9- and 15-h modified release formulations showed marked reductions in C(max) (47.1 and 68.9%) and AUC(0-72) (32.6 and 54.0%). Colonic intubation resulted in 81.3 and 73.8% reductions in C(max) and AUC(0-72). Mechanistic studies in isolated mouse tissues showed that colonic UK-338,003 permeability (P(app) < 0.5 x 10(-6) cm/s) was at least 40 times lower than that for ileum with marked asymmetry. UK-338,003 was found to be a substrate for P-glycoprotein (PGP) with a weaker interaction for multidrug resistance-associated protein-type transporters in mouse intestine. PGP inhibition dramatically increased colonic UK-338,003 permeability to the levels observed in ileum. Low UK-338,003 apical to basolateral permeability was also observed in ex vivo human distal intestine, but both the asymmetry and increase in permeability after PGP inhibition were significantly lower. In conclusion, the poor absorption of MR UK-338,003 in humans can be explained by a combination of PGP-dependent efflux and low intrinsic permeability in the lower bowel. Regional permeability studies in ex vivo tissues used during drug development can highlight absorption problems in the distal bowel and assess the feasibility of developing successful MR formulations.


Subject(s)
Adrenergic alpha-Antagonists/pharmacokinetics , Intestinal Mucosa/metabolism , Isoquinolines/pharmacokinetics , Mouth Mucosa/metabolism , Receptors, Adrenergic, alpha/metabolism , Sulfonamides/pharmacokinetics , Absorption , Administration, Oral , Administration, Rectal , Adrenergic alpha-Antagonists/administration & dosage , Adrenergic alpha-Antagonists/blood , Adult , Animals , Caco-2 Cells , Chromatography, Liquid , Colon/metabolism , Delayed-Action Preparations , Humans , Ileum/metabolism , Intestinal Absorption , Isoquinolines/administration & dosage , Isoquinolines/blood , Male , Mice , Mice, Knockout , Predictive Value of Tests , Sulfonamides/administration & dosage , Sulfonamides/blood , Tandem Mass Spectrometry
5.
Bangladesh Med Res Counc Bull ; 32(3): 72-7, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17867271

ABSTRACT

Recent studies primarily in man have shown that cod fish oil rich in polyunsaturated fatty acids (PUFA) are beneficial to certain inflammatory diseases such as ulcerative colitis. This study was undertaken to observe any change in intestinal secretion where the tissues have been treated with cod fish and sunflower oils. Male Sprague Dawley rats weighing 200 gms were fed omega-3 fatty acids for 50 days. Changes in colonic secretion (fed with PUFA) were studied in-vitro in an Ussing chamber. Rat colon which were not fed with PUFA served as controls. Basal intestinal short circuit in PUFA group were comparable with control group in stripped rat colon. The results showed significant high short circuit current in cod fish oil and sunflower oil treated tissues. When stripped colonic tissues (fed with PUFA) were stimulated by EC50 of carbachol, bradykinin and prostaglandin; there was no significant changes in Short circuit current. PGE2 and LTB4 levels were measured in rat colon fed with PUFA by using radioimmunoassay. Biochemical changes in PGE2 and LTB4 levels showed LTB4 were significantly raised in both cod fish oil group and sunflower oil group. This study reveals that intestinal permeability increases in the rat colon (fed with PUFA) as indicated by high short circuit current. The high levels of leukotriene in colonic tissues also explains the high basal short circuit current in the present study.


Subject(s)
Colitis, Ulcerative/drug therapy , Colon/drug effects , Fatty Acids, Unsaturated/therapeutic use , Fish Oils/therapeutic use , Animals , Fatty Acids, Omega-3/therapeutic use , In Vitro Techniques , Inflammation , Male , Phospholipids , Rats
6.
Gut ; 52(2): 224-30, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12524404

ABSTRACT

BACKGROUND AND AIMS: Factors that induce luminal bacteria to cross the intestinal epithelium following injury remain poorly defined. The aim of this study was to investigate the interaction between glutamine metabolism, energy supply, and inflammatory mediators in determining the translocation of non-pathogenic bacteria across cultured enterocytes. METHODS: The effect of tumour necrosis factor alpha (TNF-alpha) on translocation of Escherichia coli C25 across Caco-2 epithelial monolayers was studied in the presence of products and inhibitors of glutamine metabolism. Simultaneous measurements of transepithelial electrical resistance (TEER) and flux of lucifer yellow were used to assess effects on the paracellular pathway. Lactate dehydrogenase release was used to monitor enterocyte integrity. Imaging of monolayers in these experimental conditions was undertaken with transmission electron microscopy. RESULTS: Exposure to basolateral TNF-alpha (20 ng/ml) for six hours induced translocation of E coli across Caco-2 but only if accompanied by simultaneous glutamine depletion (p<0.01). Translocation was inhibited by addition of glutamine for two hours (p<0.01) but not by an isonitrogenous mixture of non-glutamine containing amino acids. Inhibition of glutamine conversion to alpha-ketoglutarate, but not blockade of glutathione or polyamine synthesis, also induced translocation in the presence of TNF-alpha. Manipulations that induced bacterial translocation were associated with a marked reduction in enterocyte ATP levels. No effect of these treatments on paracellular permeability or lactate dehydrogenase release was observed. Conditions in which translocation occurred were associated with the presence of bacteria within enterocyte vacuoles but not the paracellular space. CONCLUSIONS: In inflammatory conditions, the availability of glutamine as an enterocyte fuel substrate is essential for the preservation of a functional barrier to microorganisms. In conditions of acute glutamine depletion, cytokine mediated bacterial translocation appears to be primarily a transcellular process.


Subject(s)
Bacterial Translocation/drug effects , Enterocytes/metabolism , Escherichia coli/physiology , Glutamine/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Adenosine Triphosphate/analysis , Aminooxyacetic Acid/pharmacology , Buthionine Sulfoximine/pharmacology , Caco-2 Cells/physiology , Caco-2 Cells/ultrastructure , Cell Membrane Permeability/physiology , Culture Media , Eflornithine/pharmacology , Electric Impedance , Energy Metabolism/physiology , Enzyme Inhibitors/pharmacology , Humans , L-Lactate Dehydrogenase/metabolism , Microscopy, Electron
7.
J Pharmacol Exp Ther ; 303(3): 1095-101, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12438532

ABSTRACT

Information on the extent to which xenobiotics interact with P-glycoprotein (PGP) during transit through the intestine is crucial in determining the influence of PGP on oral drug absorption. We have recently described a novel use of isolated ileum from PGP-deficient mdr1a(-/-) mice to resolve PGP- and non-PGP-dependent drug efflux and provide a definitive measure of intrinsic drug permeability without recourse to inhibitors. The present study uses this approach to investigate the impact of PGP on intestinal permeability of paclitaxel and digoxin in different regions of the mouse intestine (jejunum, ileum, and proximal and distal colon). Absorption of paclitaxel and digoxin in tissues from wild-type mice was low and showed little regional variation. In contrast, absorption of both drugs was markedly higher in mdr1a(-/-) intestine, although the increase was highly region-dependent, with the ileum and distal colon showing the greatest effect and much smaller changes in the jejunum and proximal colon. These effects were accompanied by the abolition of paclitaxel and digoxin secretion in mdr1a(-/-) mice, suggesting that regional variations in intestinal permeability are masked by differential PGP expression, confirmed by immunoblotting studies. Propranolol permeability, which is not influenced by PGP, showed similar regional variation in both wild-type and mdr1a(-/-) tissues, suggesting that differences are at the level of transcellular permeability. These data suggest that the ileum and the distal colon are regions of relatively high transcellular permeability for xenobiotics that are compensated by enhanced expression of PGP.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/biosynthesis , ATP Binding Cassette Transporter, Subfamily B/deficiency , ATP-Binding Cassette Transporters/biosynthesis , Intestinal Absorption/physiology , Intestinal Mucosa/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP-Binding Cassette Transporters/genetics , Animals , Colon/drug effects , Colon/metabolism , Digoxin/pharmacokinetics , Ileum/drug effects , Ileum/metabolism , In Vitro Techniques , Intestinal Absorption/drug effects , Intestinal Mucosa/drug effects , Intestinal Mucosa/physiology , Jejunum/drug effects , Jejunum/metabolism , Male , Membrane Transport Proteins/biosynthesis , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/genetics , Mice , Mice, Knockout , Paclitaxel/pharmacokinetics , Permeability/drug effects , Propranolol/pharmacokinetics
8.
Br J Pharmacol ; 135(8): 2038-46, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11959808

ABSTRACT

1. Intestinal xenobiotic transporters are a significant barrier to the absorption of many orally administered drugs. P-glycoprotein (PGP) is the best known, but several others, including members of the multidrug resistance-associated protein (MRP) family, are also expressed. Definitive information on their precise effect on intestinal drug permeability is scarce due to a lack of specific inhibitors and the difficulty of studying non-PGP activity in the presence of high PGP expression. 2. We have investigated the in vitro use of intestinal tissues from PGP knockout (mdr1a (-/-)) mice as a tool for dissecting the mechanisms of intestinal drug efflux. The permeability characteristics of digoxin (DIG), paclitaxel (TAX) and etoposide (ETOP) were measured in ileum from mdr1a (-/-) and wild-type (FVB) mice mounted in Ussing chambers. 3. DIG and TAX exhibited marked efflux across FVB tissues (B-A : A-B apparent permeability (P(app)) ratio 10 and 17 respectively) which was absent in mdr1a (-/-) tissues, confirming that PGP is the sole route of intestinal efflux for these compounds. The A-B P(app) of both compounds was 3 - 5 fold higher in mdr1a (-/-) than in FVB. 4. Polarized transport of ETOP in FVB tissues was reduced but not abolished in mdr1a (-/-) tissues. Residual ETOP efflux in mdr1a (-/-) tissues was abolished by the MRP inhibitor MK571, indicating involvement of both PGP and MRP. 5. MK571 abolished calcein efflux in mdr1a (-/-) tissues, while quinidine had no parallel effect in FVB tissues, suggesting involvement of MRP but not PGP. 6. Tissues from mdr1a (-/-) mice provide a novel approach for investigating the influence of PGP ablation on intestinal permeability and for resolving PGP and non-PGP mechanisms that modulate drug permeability.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , ATP Binding Cassette Transporter, Subfamily B/deficiency , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP-Binding Cassette Transporters/genetics , Intestinal Absorption/physiology , Intestinal Mucosa/metabolism , Pharmaceutical Preparations/metabolism , ATP Binding Cassette Transporter, Subfamily B/biosynthesis , ATP Binding Cassette Transporter, Subfamily B/physiology , ATP Binding Cassette Transporter, Subfamily B, Member 1/deficiency , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP-Binding Cassette Transporters/biosynthesis , ATP-Binding Cassette Transporters/physiology , Animals , Drug Resistance, Multiple/genetics , Etoposide/metabolism , Ileum/metabolism , In Vitro Techniques , Male , Mannitol/metabolism , Mice , Mice, Knockout , Permeability , Propranolol/metabolism , Reverse Transcriptase Polymerase Chain Reaction
9.
Br J Pharmacol ; 135(6): 1443-8, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11906957

ABSTRACT

1. The diverse physiological actions of somatostatin are mediated by a family of G-protein coupled receptors (SSTRs). Several peptide analogues of somatostatin such as octreotide have been developed for therapeutic use, including treatment of gastrointestinal disorders such as secretory diarrhoea. However, their development as anti-diarrhoeal agents has been limited by poor oral bioavailability, necessitating parenteral administration. This in vitro study investigated the anti-secretory potential of a group of novel, non-peptide, somatostatin-receptor agonists that selectively activate specific SSTR subtypes to assess their potential for oral administration. 2. The ability of the agonists to inhibit forskolin-stimulated chloride secretion was measured using a sensitive bioassay system in isolated rat colonic mucosa. 3. The SSTR-2 selective agonist, L-779,976 was 10-times more potent than octreotide as an inhibitor of secretion when added to the basolateral surface of rat colon. Non-peptide agonists selective for SSTR1 (L-797,591), SSTR3 (L-796,778), SSTR4 (L-803,087) or SSTR5 (L-817,818) showed little or no anti-secretory activity in this preparation. 4. L-779,976 was able to inhibit secretion when applied to the luminal surface at sub-micromolar concentrations suggesting that it can cross the colonic epithelium. The anti-secretory potency of luminal L-779,976 was increased 3 fold in the presence of GF120918, a known inhibitor of P-glycoprotein. 5. Non-peptide somatostatin receptor agonists may provide a basis for the development of new, orally available anti-diarrhoeal therapies.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Amides/pharmacology , Colon/drug effects , Indoles/pharmacology , Receptors, Somatostatin/agonists , Tetrahydroisoquinolines , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Acridines/pharmacology , Amides/chemistry , Amides/metabolism , Animals , Colon/metabolism , Dose-Response Relationship, Drug , Drug Synergism , In Vitro Techniques , Indoles/chemistry , Indoles/metabolism , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Isoquinolines/pharmacology , Male , Rats , Rats, Sprague-Dawley , Receptors, Somatostatin/metabolism
10.
Dig Dis Sci ; 46(11): 2499-503, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11713960

ABSTRACT

Somatostatin inhibits colonic ion secretion in animal models and cultured intestinal cell lines via somatostatin receptor subtype 2 and subtype 1, respectively. In a recent in vitro ion transport study of the human colon, somatostatin was shown to stimulate short-circuit current, a measure of electrogenic ion transport. In this study we have used the reverse-transcription polymerase chain reaction (RT-PCR) and measurements of changes in short-circuit current (Isc) in response to receptor subtype-specific analogs of somatostatin, to define the somatostatin receptor subtype responsible for the stimulation of short-circuit current in human colon. Somatostatin receptor subtypes 1, 2, and 5, but not 3 and 4, were detected in the human colonic epithelium. Measurements of short-circuit current showed somatostatin and octreotide (1 micromol/liter) increased the prostaglandin stimulated short-circuit current by 12.3+/-1 and 11.0+/-1 microA/cm2, respectively. Similarly, analogs selective for somatostatin receptor subtypes 2 and 5 (1 micromol/liter) produced an increase of short-circuit current of 11.7+/-1 and 13.2+/-1 microA/cm2, respectively. However, at a concentration (10 nmol/liter) near the EC50, the somatostatin receptor subtype 2 analog increased short-circuit current by 9+/-1 microA/cm2, whereas the receptor subtype 5 analog had no effect. There was no difference in receptor expression or effect of the peptides related to the anatomical site of tissue collection. In conclusion, human colonic mucosa expresses multiple somatostatin receptor subtypes, of which subtype 2 mediates the stimulatory effect of somatostatin on ion transport.


Subject(s)
Colon/metabolism , Receptors, Somatostatin/metabolism , Somatostatin/pharmacology , Cyclic AMP/metabolism , Humans , Ion Transport , Receptors, Somatostatin/physiology , Reverse Transcriptase Polymerase Chain Reaction
11.
Am J Physiol Cell Physiol ; 281(2): C388-97, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11443038

ABSTRACT

Despite significant advances in the characterization of tight junction (TJ) proteins, little is known about how molecular changes relate to function due primarily to the limitations of conventional paracellular probes. To address this, the paracellular pathway in Caco-2 and T84 cell lines was profiled by measuring the permeabilities of 24 polyethylene glycols (PEG) of increasing molecular radius (3.5--7.4 A) analyzed by mass spectrometry. When combined with a paracellular sieving model, these data provided quantitative descriptors of the pathway under control conditions and after exposure to TJ modulators. PEG profiles in both cell lines conformed to a biphasic process involving a restrictive pore (radius 4.3--4.5 A) and a nonrestrictive component responsible for permeability of larger molecules. PEG profiling revealed significant differences between the effects of EGTA and sodium caprate (C10). The restrictive component of EGTA-treated cells lost all size discrimination due to an increase in pore radius. Sodium caprate had no effect on pore radius but increased permeability via a different mechanism possibly involving increased numbers of functional pores. PEG profiling provides a useful tool for probing the functional regulation of the paracellular route.


Subject(s)
Intestinal Mucosa/metabolism , Models, Biological , Tight Junctions/metabolism , Biomarkers , Caco-2 Cells , Cell Line , Colon/cytology , Colon/metabolism , Decanoic Acids/pharmacology , Egtazic Acid/pharmacology , Humans , Intestines/cytology , Permeability/drug effects , Polyethylene Glycols/pharmacokinetics
12.
J Pharmacol Exp Ther ; 296(2): 584-91, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11160647

ABSTRACT

Intestinal drug efflux mediated by P-glycoprotein and other ABC transporters is widely accepted as a reason for low or variable oral absorption. However, little is known about species and regional differences in P-glycoprotein so the functional and predictive relevance of observations made in cell models such as Caco-2 is uncertain. The aim of this study was to define the kinetics of drug efflux in rat and human intestinal tissues in vitro using the "reference" substrates digoxin and vinblastine. The expression and functional role of other ABC transporters in the transport of these compounds was also investigated. Saturable, verapamil-sensitive efflux of digoxin was observed in all intestinal regions. Apparent affinity of the efflux process varied within a relatively narrow range (50-92 microM), increasing in rat from small to large intestine. In contrast, maximal transporter activity varied over a 4- to 5-fold range with ileum > jejunum > colon. Similar regional differences in efflux were also observed with vinblastine. Maximal efflux levels were similar in Caco-2 and ileum for both substrates, suggesting that Caco-2 may quantitatively predict small intestinal drug efflux. Digoxin efflux kinetics was virtually identical in rat and human colon. Inhibitor studies showed that digoxin and vinblastine efflux in intestinal tissues was mediated by P-glycoprotein, although a minor component could be attributed to multidrug resistance-related protein (MRP)-like transporters in Caco-2. This study has analyzed the differential functional expression of drug efflux along the gastrointestinal tract. Such data will be critical in developing predictive models of P-glycoprotein-mediated efflux using information gathered from in vitro systems.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Intestinal Mucosa/metabolism , Pharmaceutical Preparations/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Animals , Antineoplastic Agents, Phytogenic/metabolism , Caco-2 Cells , Calcium Channel Blockers/pharmacology , Cardiotonic Agents/metabolism , Colonic Neoplasms/metabolism , DNA Primers , Digoxin/metabolism , Epithelium/drug effects , Epithelium/metabolism , Glycoproteins/metabolism , Humans , Intestinal Mucosa/drug effects , Kinetics , Male , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Verapamil/pharmacology , Vinblastine/metabolism
13.
J Physiol ; 528 Pt 1: 165-76, 2000 Oct 01.
Article in English | MEDLINE | ID: mdl-11018115

ABSTRACT

1. Fatty acid-induced cholecystokinin (CCK) secretion in humans and from the enteroendocrine cell line STC-1 depends critically on acyl chain length. 2. Therefore we have characterized the relationship between acyl chain length and the potency of the fatty acid to induce CCK secretion and changes in intracellular Ca2+ concentration ([Ca2+]i) in two enteroendocrine cell lines (STC-1 and GLUTag). We found that the potency of the fatty acid was directly proportional to its chain length and therefore inversely proportional to its solubility. 3. In both cell types, the fatty acid-induced rise in [Ca2+]i in response to decanoic acid (C10), dodecanoic acid (C12) and tetradecanoic acid (C14) was significantly reduced in Ca2+-free medium and largely blocked by nicardipine. Intracellular stores also contributed to the overall shape of the [Ca2+]i peak. Thus all the fatty acids tested caused the release of Ca2+ from stores and influx of extracellular Ca2+, presumably through L-type calcium channels. 4. To probe the site of fatty acid action, we studied the distribution of 14C-labelled dodecanoic acid. This label was rapidly and irreversibly accumulated by both cell types, where it became concentrated about 20-fold. Confocal microscopy of a fluorescent analogue of dodecanoic acid clearly demonstrated that it entered the cytosol and was not merely partitioning in the cell membrane. These data indicate that an intracellular action for fatty acid-induced CCK secretion cannot be eliminated. 5. Dodecanoic acid itself, and not a metabolite, is the agent responsible for triggering Ca2+ entry since a non-metabolizable form of dodecanoic acid (2-bromododecanoic acid) was also capable of inducing a rise in [Ca2+]i in both cell types. 6. In conclusion, the rise in [Ca2+]i in STC-1 and GLUTag cells evoked by medium- to long-chain fatty acids results from the triggering of a specific signalling pathway. Whether triggering occurs through activation of a membrane-bound receptor or at an intracellular site remains to be clarified.


Subject(s)
Calcium/metabolism , Cholecystokinin/metabolism , Enteroendocrine Cells/metabolism , Fatty Acids/metabolism , Intracellular Fluid/metabolism , Calcium Channel Blockers/pharmacology , Cell Line , Cell Membrane Permeability/drug effects , Colon/cytology , Colon/drug effects , Colon/metabolism , Decanoic Acids/metabolism , Decanoic Acids/pharmacology , Dose-Response Relationship, Drug , Enteroendocrine Cells/cytology , Enteroendocrine Cells/drug effects , Fatty Acids/pharmacology , Humans , Lauric Acids/metabolism , Lauric Acids/pharmacology , Molecular Structure , Myristic Acid/metabolism , Myristic Acid/pharmacology , Nicardipine/pharmacology , Serum Albumin, Bovine/pharmacology
14.
J Health Popul Nutr ; 18(1): 49-53, 2000 Jun.
Article in English | MEDLINE | ID: mdl-11014771

ABSTRACT

Recent studies show that enteric nerves are involved in the action of cholera toxin, both in vivo and in vitro. The aim of this study was to investigate in vitro the influence of carbachol, a cholinergic agonist, on the action of cholera toxin. Cultured HT29-19A cell lines and rat ileal mucosa were used in an Ussing chamber for the measurement of short-circuit current induced by cholera toxin. Cyclic AMP was measured from HT29-19A cell lines by standard radio-immunoassay. Pre-treatment of the HT29-19A cell lines with carbachol potentiated cholera toxin-induced secretory response, and enhanced accumulation of cAMP. Carbachol also potentiated the cholera toxin-secretory response in the rat ileal mucosa, but only following pretreatment with the prostaglandin synthesis inhibitor, indomethacin. There was synergistic interaction between cholera toxin and cholinergic neurotransmitter carbachol on the intestinal epithelium. Cholinergic agonists may play a role in regulating the secretory response to the toxin. Such interaction is masked in the intact tissues in vitro due to the release of prostaglandins during isolation.


Subject(s)
Carbachol/pharmacology , Cholera Toxin/toxicity , Cholinergic Agonists/pharmacology , HT29 Cells/metabolism , Ileum/metabolism , Intestinal Mucosa/metabolism , Animals , Cyclic AMP/metabolism , Cyclooxygenase Inhibitors/pharmacology , Drug Synergism , HT29 Cells/drug effects , Humans , Ileum/drug effects , Ileum/innervation , In Vitro Techniques , Indomethacin/pharmacology , Intestinal Mucosa/drug effects , Male , Rats , Rats, Sprague-Dawley
15.
Dig Dis Sci ; 45(11): 2100-7, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11215722

ABSTRACT

In vitro somatostatin is a potent inhibitor of intestinal ion secretion in animal models and cultured human cell lines, providing a rationale for its use in secretory diarrheas. However, the effects of somatostatin on ion transport in native human colonic epithelium have not been reported. In this study the effects of somatostatin and octreotide on the basal short-circuit current and the cAMP- and Ca2+-stimulated short-circuit current were studied in isolated human colonic mucosa mounted in Ussing chambers. Under basal conditions somatostatin and octreotide (1 micromol/liter) stimulated a small, bumetanide-sensitive increase in short-circuit current. Following stimulation of secretion with prostaglandin E2, somatostatin and octreotide further increased the short-circuit current in a dose dependent fashion (ED50 approximately 10 nmol/liter for both). This stimulation of short-circuit current was not affected by pretreatment of the tissue with basolateral tetrodotoxin (1 micromol/liter) or mucosal amiloride (10 micromol/liter). In contrast, somatostatin and octreotide had no effect when secretion was stimulated with 8-bromo-cAMP, and pretreatment of the tissue with somatostatin and octreotide (0.1 micromol/liter) did not alter the secretory response to carbachol. The absence of any inhibitory effect of somatostatin and octreotide on electrogenic secretion in the human colon may explain the variable results obtained when somatostatin or octreotide are used for the treatment of secretory diarrheas.


Subject(s)
Colon/drug effects , Intestinal Mucosa/drug effects , Ion Channels/drug effects , Octreotide/pharmacology , Somatostatin/pharmacology , Animals , Culture Techniques , Dose-Response Relationship, Drug , Epithelium/drug effects , Humans , Intestinal Secretions/drug effects , Membrane Potentials/drug effects , Rats , Rats, Inbred WKY , Secretory Rate/drug effects , Water-Electrolyte Balance/drug effects
16.
Gastroenterology ; 117(6): 1363-9, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10579977

ABSTRACT

BACKGROUND & AIMS: This study has investigated stimulus-secretion coupling of enterochromaffin cells by studying the cellular location and function of voltage-gated Ca(2+) channels within small intestinal crypts. METHODS: Digital fluorescence imaging and electrochemical detection were used to measure intracellular Ca(2+) responses and serotonin (5-hydroxytryptamine [5-HT]) secretion in intact crypts isolated from guinea pig and human duodenum. RESULTS: In fluo-3-loaded crypts, electrical depolarization with high K(+) solution increased cytosolic free [Ca(2+)] only in single cells subsequently identified by immunocytochemistry as enterochromaffin cells. In guinea pig enterochromaffin cells, the L-type Ca(2+) channel agonist FPL 64176 (3 micromol/L) did not change resting intracellular [Ca(2+)] but potentiated the depolarization-evoked increase in [Ca(2+)] (298 +/- 72 nmol/L) by 19 +/- 3-fold. In the majority of human enterochromaffin cells, FPL 64176 alone increased resting [Ca(2+)] by 423 +/- 171 nmol/L. Secretion studies in guinea pig crypts showed that high K(+) and FPL 64176 caused a 12-fold increase in 5-HT release. Noradrenaline caused increases in both enterochromaffin cell [Ca(2+)] and 5-HT release. CONCLUSIONS: Using this approach, we have found that in duodenal crypts, enterochromaffin cells, but not other epithelial cells, contain L-type voltage-gated Ca(2+) channels involved in regulating 5-HT secretion. These data have implications for the pharmacological control of intestinal disorders involving enterochromaffin cell dysfunction.


Subject(s)
Calcium Channels, L-Type/metabolism , Duodenum/metabolism , Enterochromaffin Cells/metabolism , Aniline Compounds/metabolism , Animals , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/analysis , Chromatography, High Pressure Liquid , Duodenum/cytology , Duodenum/drug effects , Enterochromaffin Cells/drug effects , Guinea Pigs , Immunohistochemistry , In Vitro Techniques , Male , Serotonin/metabolism , Xanthenes/metabolism
17.
Am J Physiol ; 277(5): G967-75, 1999 11.
Article in English | MEDLINE | ID: mdl-10564102

ABSTRACT

Somatostatin is a powerful inhibitor of intestinal Cl(-) secretion. We used patch-clamp recording techniques to investigate the effects of somatostatin on low-conductance (23-pS) K(+) channels in the basolateral membrane of human colonic crypts, which are an important component of the Cl(-) secretory process. Somatostatin (2 microM) elicited a >80% decrease in "spontaneous" K(+) channel activity in cell-attached patches in nonstimulated crypts (50% inhibition = approximately 8 min), which was voltage-independent and was prevented by pretreating crypts for 18 h with pertussis toxin (200 ng/ml), implicating a G protein-dependent mechanism. In crypts stimulated with 100-200 microM dibutyryl cAMP, 2 microM somatostatin and its synthetic analog octreotide (2 microM) both produced similar degrees of K(+) channel inhibition to that seen in nonstimulated crypts, which was also present under low-Cl(-) (5 mM) conditions. In addition, 2 microM somatostatin abolished the increase in K(+) channel activity stimulated by 2 microM thapsigargin but had no effect on the thapsigargin-stimulated rise in intracellular Ca(2+). These results indicate that somatostatin peptides inhibit 23-pS basolateral K(+) channels in human colonic crypt cells via a G protein-dependent mechanism, which may result in loss of the channel's inherent Ca(2+) sensitivity.


Subject(s)
Colon/chemistry , Colon/drug effects , Potassium Channel Blockers , Somatostatin/pharmacology , Bucladesine/pharmacology , Calcium/analysis , Calcium/metabolism , Chlorides/metabolism , Colon/metabolism , Cyclic AMP/metabolism , Enzyme Inhibitors/pharmacology , Epithelial Cells/chemistry , Epithelial Cells/metabolism , Epithelial Cells/physiology , GTP-Binding Proteins/metabolism , Humans , Intestinal Mucosa/cytology , Membrane Potentials/drug effects , Patch-Clamp Techniques , Pertussis Toxin , Potassium Channels/metabolism , Second Messenger Systems/drug effects , Second Messenger Systems/physiology , Thapsigargin/pharmacology , Virulence Factors, Bordetella/pharmacology
18.
Pflugers Arch ; 437(4): 596-602, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10089573

ABSTRACT

The most abundant basolateral K+ channels in human colonic crypt cells have a low conductance (23 pS), respond to increases in intracellular Ca2+ and cAMP, and have been implicated in intestinal electrogenic Cl- secretion. The effect of nicardipine on the activity of these K+ channels was examined by patch-clamp recording in the cell-attached and excised inside-out configurations from the basolateral membrane of single crypts isolated from biopsied samples of human distal colon. During cell-attached recordings, addition of 2 micromol/l nicardipine to crypts pretreated with 200 micromol/l dibutyryl cAMP decreased single-channel open probability by 87%, but in parallel studies nicardipine had no effect on the intracellular Ca2+ concentration. Using inside-out patches from crypts pretreated with dibutyryl cAMP (bathed in 1.2 mmol/l Ca2+), the addition of increasing concentrations of nicardipine (200 nmol/l, 2 micromol/l and 20 micromol/l) decreased single-channel open probability in a concentration-dependent manner (IC50 0.47 micromol/l). In additional experiments using stripped rat distal colonic mucosa mounted in conventional Ussing chambers, serosal addition of nicardipine at increasing concentrations (ranging from 200 nmol/l to 20 micromol/l) produced a concentration-dependent inhibition of dibutyryl-cAMP-stimulated electrogenic Cl- secretion (IC50 2 micromol/l). Taken together, these results indicate that nicardipine has a direct inhibitory action on 23-pS basolateral K+ channels in human intestinal crypt cells, which is likely to decrease cAMP-stimulated electrogenic Cl- secretion. These basolateral K+ channels may provide a focal point for the development of new strategies in the treatment of secretory diarrhoeal diseases.


Subject(s)
Colon/physiology , Nicardipine/pharmacology , Potassium Channel Blockers , Animals , Bucladesine/pharmacology , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Cell Membrane/drug effects , Cell Membrane/physiology , Chlorides/metabolism , Colon/drug effects , Colon/ultrastructure , Colonic Diseases, Functional , Humans , Ion Channel Gating/drug effects , Patch-Clamp Techniques , Potassium Channels/physiology , Rats
19.
J Pharmacol Exp Ther ; 288(1): 171-8, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9862768

ABSTRACT

The influence of secretory transporters on intestinal permeability characteristics of the H2 receptor antagonists ranitidine and cimetidine was studied in Caco-2 monolayers and rat intestinal mucosa mounted in Ussing chambers. Both drugs exhibited vectorial transport across rat ileum with significantly greater (2-4-fold) permeability in the serosal-to-mucosal than the mucosal-to-serosal direction, indicative of net mucosal secretion. Mucosal ranitidine secretion was also observed in rat distal colon, although to a lesser degree. Ileal ranitidine secretion was concentration dependent and significantly reduced by the P-glycoprotein (P-gp) substrates verapamil and cyclosporin. In contrast, probenicid, an inhibitor of the multidrug-related protein, had no effect on ranitidine permeability. The paracellular marker mannitol showed no evidence of asymmetric permeability or sensitivity to P-gp inhibitors. Significant expression of P-gp protein in rat intestinal epithelial cells was confirmed by immunoblotting. Caco-2 monolayers, which overexpress P-gp, also showed asymmetric permeability of ranitidine and cimetidine. In this model, ranitidine permeability in the mucosal-to-serosal direction decreased by approximately 95% as monolayer resistance increased from 150 to 500 Omega/cm2, indicating a primarily paracellular route of transport. However, serosal-to-mucosal permeability was insensitive to resistance changes, consistent with a primarily transcellular route in this direction. These data indicate that ranitidine and cimetidine can act as substrates for intestinal P-gp and suggest that the balance between absorptive and secretory mechanisms as a factor in determining intestinal absorption needs to be a routine consideration even for compounds expected to have a predominantly paracellular route of absorption.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cimetidine/pharmacokinetics , Histamine H2 Antagonists/pharmacokinetics , Intestinal Mucosa/metabolism , Ranitidine/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Animals , Biological Transport , Caco-2 Cells , Cell Membrane Permeability/drug effects , Colon/drug effects , Colon/metabolism , Genes, MDR/physiology , Humans , In Vitro Techniques , Intestinal Absorption , Intestinal Mucosa/drug effects , Intestines/drug effects , Male , Rats , Rats, Sprague-Dawley
20.
J Physiol ; 513 ( Pt 1): 11-8, 1998 Nov 15.
Article in English | MEDLINE | ID: mdl-9782155

ABSTRACT

1. The present study has investigated whether fatty acids directly influence peptide release from enteroendocrine cells using STC-1, a mouse intestinal endocrine tumour cell line, previously shown to release cholecystokinin (CCK) in response to other physiological stimuli. 2. Fatty acids elicited a chain length- and dose-dependent stimulation of CCK secretion. Dodecanoic acid (C12) was most effective, producing up to a 5-fold increase in CCK secretion. Fatty acids with less than ten carbon atoms did not increase secretion. The chain length dependence of these effects mimics closely fatty acid-induced CCK secretion previously observed in humans in vivo. 3. Esterification of C12 abolished CCK secretion, indicating a critical role for a free carboxyl group in eliciting secretion. In contrast, modification of the methyl terminus had no effect on C12-induced secretion. The non-metabolizable C12 analogue 2-bromododecanoic acid was equally effective. 4. C12 elicited a marked increase in intracellular calcium levels (200-300 nM) in STC-1 cells which was abolished by the L-type Ca2+ channel antagonist nicardipine. In contrast, C8 produced a smaller and more transient Ca2+ response. C12-induced CCK secretion was also blocked by nicardipine. 5. These data suggest that fatty acids can interact directly with enteroendocrine cells to stimulate CCK secretion via increases in intracellular calcium mediated primarily by L-type Ca2+ channels.


Subject(s)
Calcium/physiology , Cholecystokinin/metabolism , Endocrine System/physiology , Fatty Acids/pharmacology , Animals , Calcium Channel Blockers/pharmacology , Cell Survival/drug effects , Culture Media , Endocrine System/cytology , Endocrine System/drug effects , Mice , Nicardipine/pharmacology , Radioimmunoassay , Stimulation, Chemical , Structure-Activity Relationship , Tachyphylaxis/physiology , Tumor Cells, Cultured
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