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Biomedical subjects

K F Sewing

Publications and source records attributed to K F Sewing.

At least 73 records · Page 4Linked to original sources
PubMed1995

A novel bioreactor design for in vitro reconstruction of in vivo liver characteristics.

We have constructed a bioreactor aimed at imitating the three-dimensional micro- and macroenvironment of the liver. In vivo hepatocytes are arranged in plates of cell monolayers and are specifically attached with both sinusoidal surfaces to the space of Disse which contains extracellular matrix. Nonparenchymal cells are located on the other side of the space of Disse toward the sinusoid. For supporting monolayer hepatocytes with bipolar attachment to the extracellular matrix, we used a double gel culture technique that sandwiches hepatocytes between two layers of collagen. In double gel cultures, albumin production increases during an adaptive period to the in vitro environment. In contrast to conventional single gel hepatocytes, double gel hepatocytes maintain expression of sinusoidal microvilli and a polyhedric cell shape in culture as seen by transmission electron microscopy. Albumin production in the bioreactor was stable. The organotypical bioreactor concept is an example of organ mimicry and may provide the basis for the organ-otypical development of a full-sized hybrid artificial liver.

Albumins↗
PubMed1995

Reconstruction of liver tissue in vitro: geometry of characteristic flat bed, hollow fiber, and spouted bed bioreactors with reference to the in vivo liver.

Bioreactors currently being developed for hybrid artificial livers vary greatly with respect to their microenvironment. The specific architecture modifies the relationship parenchymal and nonparenchymal cells have with the exchange surfaces of the bioreactor. Most designs are either based on hollow fiber, spouted bed, or flat bed devices. This diversity is contrasted by the uniform and unique organization of the in vivo liver. The liver cells are arranged as plates and both sinusoidal surfaces of the hepatocytes are enclosed within the matrix of the space of Disse. In this study we intended to define the in vivo liver tissue characteristics in a manner useful for an organotypical approach to hepatic tissue engineering. Transmission electron microscopy of an in vivo liver was utilized to describe these ratios. The ratios defined in this study are based on the constant hepatocellular expression of two sinusoidal surfaces. A relationship is established between the expression of the sinusoidal surfaces and their use as attachment and exchange surfaces inside a bioreactor. The presence of biliary surfaces and nonparenchymal cell surfaces is compared. The functional relevance of an in vivo like extracellular matrix geometry for oxidative biotransformation of primary hepatocytes in vitro was studied using the two model drugs cyclosporin and rapamycin. The generation of the hydroxylated cyclosporin metabolites AM 9 and AM 1 and four rapamycin metabolites was analyzed by high performance liquid chromatography (HPLC). It is shown that the cell-specific biotransformation rates at 1 week in culture in matrix overlayed hepatocytes was 5-10 times that of hepatocytes without matrix overlay. Bilaminar membrane (BLM) bioreactors were used to reconstruct extracellular matrix geometry, three-dimensional cell plates, and sinusoidal analogs in between cell plates.

Animals↗
PubMed1995

Bismuth subcitrate and omeprazole inhibit Helicobacter pyloriF1-ATPase.

The effects of various types of antiulcer agents against Helicobacter pylori F1-ATPase were studied. ATPase was released into the aqueous phase (i.e., solubilized) by sonication. The enzyme activity depended on Mg2+, but not Ca2+. The maximum activity occurred at an ATP/Mg2+ ratio of 1/5 and at pH 7.5. Mg(2+)-dependent ATPase activity was inhibited by sodium azide and the monovalent cations K+ and Na+, but not by oligomycin, dicyclohexylcarbodiimide, ouabain, or SCH 28080. The antiulcer agents ranitidine, pirenzepine, aluminum hydroxide, and sucralfate failed to influence H. pylori F1-ATPase. In contrast, bismuth subcitrate and the H+/K(+)-ATPase inhibitor omeprazole inhibited the enzyme. Inhibition was prevented and reversed by the mercaptan glutathione, indicating that both drugs interfere with sulfhydryl groups of the enzyme. The data suggest that bismuth subcitrate and omeprazole owe their antibacterial activity against H. pylori, at least in part, to inhibition of F1-ATPase, an enzyme involved in bacterial energy metabolism.

Anti-Ulcer Agents↗
PubMed1995

Effects of flavonoids on parietal cell acid secretion, gastric mucosal prostaglandin production and Helicobacter pylori growth.

The effect of the flavonoids flavone, flavanone and quercetin on parietal cell acid production, H+/K(+)-ATPase activity, gastric mucosal prostaglandin E2 biosynthesis and Helicobacter pylori growth was studied. All flavonoids inhibited acid production in isolated parietal cells in response to histamine and dibutyryl-cAMP stimulation (IC50 values between 26 and 139 mumol/l) and inhibited H+/K(+)-ATPase activity. Inhibition of H+/K(+)-ATPase activity was dependent on the ATP concentration. Fluorescence measurements showed that flavanone reacts with ATP. These findings indicate that the inhibitory action of flavonoids on H+/K(+)-ATPase activity is related to their ability to complex ATP. Flavone and flavanone (10 and 100 mumol/l) stimulated prostaglandin E2 production in isolated gastric mucosal cells. Furthermore, the compounds inhibited Helicobacter pylori growth in a concentration-dependent manner. From these finding it appears that flavonoids are a group of compounds which could have a therapeutic potential for treatment of gastrointestinal diseases associated with Helicobacter pylori infection.

Adenosine Triphosphate↗
PubMed1995

Metabolism of the immunosuppressant tacrolimus in the small intestine: cytochrome P450, drug interactions, and interindividual variability.

The small intestinal metabolism of tacrolimus, which is used as an immunosuppressant in transplantation medicine, was investigated in this study. Tacrolimus was metabolized in vitro by isolated human, pig, and rat small intestinal microsomes. The metabolites generated were identified by HPLC/MS. Tacrolimus and its metabolites were quantified using HPLC or HPLC/MS. The cytochrome P450 (CYP) enzymes responsible for tacrolimus metabolism in small intestine were identified using specific CYP antibodies and inhibitors. For characterization of the interindividual variability, microsomes were isolated from small intestinal samples of patients who had undergone resection for various reasons. In an in vitro model using pig small intestinal microsomes, 32 drugs were analyzed for their interactions with tacrolimus metabolism. After incubation with human, rat, and pig small intestinal microsomes, the metabolites 13-O-demethyl and 13,15-O-demethyl tacrolimus were identified. The metabolism of tacrolimus by human small intestine was inhibited by anti-CYP3A, troleandomycin, and erythromycin, indicating that, as in the liver, CYP3A enzymes are the major enzymes for tacrolimus metabolism in the human small intestine. Metabolism of tacrolimus by small intestinal microsomes isolated from 14 different patients varied between 24 and 110 pmol/13-O-demethyl tacrolimus/min/mg microsomal protein, with a mean +/- SD of 54.2 +/- 29.2 pmol/min/mg. Of 32 drugs tested, 15 were found to inhibit small intestinal tacrolimus metabolism: bromocryptine, corticosterone, cyclosporine, dexamethasone, ergotamine, erythromycin, ethinyl estradiol, josamycin, ketoconazole, nifedipine, omeprazole, progesterone, rapamycin, troleandomycin, and verapamil. All of these drugs inhibited tacrolimus metabolism by human liver microsomes as well. It is concluded that tacrolimus is metabolized by cytochrome CYP3A enzymes in the small intestine. The rate of the CYP3A enzymatic activities varies about 5 times from patient to patient, and drugs that interfere with the in vitro metabolism of tacrolimus in the liver also inhibit its small intestinal metabolism.

Animals↗
PubMed1995

Characterization of CCK receptor-mediated effects on intracellular calcium of porcine chief cells.

Effects of cholecystokinin (CCK) receptor agonists and antagonists on intracellular calcium ¿[Ca2+]i¿ of isolated porcine chief cells were determined by FURA2 fluorometry. CCK-8 increased [Ca2+]i with an EC50 of 6 nmol/l. The CCKB receptor preferring agonists gastrin-17-I, desulfated CCK-8 and CCK-4 had only small stimulatory effects (< 12% of maximal CCK-8 effect, EC50's in the low nanomolar range) and did not inhibit the CCK-8 response, suggesting that they were acting at CCKB but not, as partial agonists, at CCKA receptors. A71378 had its main stimulatory effect in low and a slight additional effect in high concentrations (EC50 80 pmol/l and > 1 mumol/l), respectively, while A72962 had its main stimulatory effect in high concentrations (EC50 > 1 mumol/l). The CCK receptor antagonists L364.718, L365.260, CBZ-CCK27-32 and dibutyryl cGMP inhibited CCK-8 (100 nmol/l) response concentration-dependently with IC50's of 540 pmol/l, 2mumol/l, 3 mumol/l and 250 mumol/l, respectively. These results suggest that CCK effects on [Ca2+]i of porcine chief cells are mainly (> 80%) mediated via CCKA receptors, which differ from guinea-pig and rabbit chief cell receptors by a higher distinction capacity between selective CCKA and CCKB, receptor agonists (A71378 versus A72962) and antagonists (L364.718 versus L365.260) and by the apparent lack of activation by desulfated CCK-8 and gastrin-17-I. Isolated porcine chief cells therefore appear to be a favourable "in vitro" system to characterize CCKA receptor specific compounds.

Animals↗
PubMed1994

High-performance liquid chromatographic analysis of phospholipids from different sources with combined fluorescence and ultraviolet detection.

An isocratic high-performance liquid chromatographic (HPLC) system was developed for the separation of major phospholipid classes, i.e., phosphatidylcholine, sphingomyelin, lysophosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. Phospholipids were detected with ultraviolet absorption at 205 nm and subsequent fluorescence detection. Fluorescence of the phospholipids (excitation, 340 nm; emission, 460 nm) was achieved by postcolumn formation of mixed micelles with 1,6-diphenyl-1,3,5-hexatriene. For ultraviolet absorption there were great differences depending on the saturation of phospholipid fatty acids but for fluorescence the sensitivity was almost identical for all phospholipids except phosphatidylinositol and lysophosphatidylcholine. Dipalmitoylphosphatidylcholine showed nearly no ultraviolet but good fluorescence response. Ultraviolet to fluorescence ratio was characteristic for different phospholipids and for identical phospholipids from different sources. Quantification of phosphatidylcholine and phosphatidylethanolamine with HPLC using N-monomethylphosphatidylethanolamine (dioleoyl) as an internal standard gave the same results as phospholipid phosphorus quantification after thin-layer chromatography.

Chromatography, High Pressure Liquid↗
PubMed1994

Role of carbonic anhydrase in basal and stimulated bicarbonate secretion by the guinea pig duodenum.

The role of carbonic anhydrase in the process of proximal duodenal mucosal bicarbonate secretion was investigated in the guinea pig. In a series of experiments in vivo, the duodenum was perfused with 24 mmol/liter NaHCO3 solution (+ NaCl for isotonicity) to ensure that active duodenal HCO3- secretion against a concentration gradient was measured. Acetazolamide (80 mg/kg) was infused intravenously to examine the role of carbonic anhydrase on basal and agonist-stimulated HCO3- secretion. Acetazolamide abolished basal HCO3- secretion and significantly decreased HCO3- secretion after stimulation with dibutyryl 5'-cyclic adenosine monophosphate (dBcAMP, 10(-5) mol/kg), dibutyryl 5'-cyclic guanosine monophosphate (dBcGMP, 10(-5) mol/kg), prostaglandin E2 (PGE2, 10(-6) mol/kg), PGF2 alpha (10(-6) mol/kg), tetradecanoyl-phorbol-acetate (TPA, 10(-7) mol/kg), glucagon (10(-7) mol/kg), vasoactive intestinal polypeptide (VIP, 10(-8) mol/kg), and carbachol (10(-8) mol/kg). Utilizing a fluorescence technique, we could detect the enzyme carbonic anhydrase in equal amounts in villous and crypt cells of the proximal duodenal epithelium; no activity was demonstrated in tissues pretreated with acetazolamide. In conclusion, carbonic anhydrase is required for both basal and stimulated duodenal HCO3- secretion.

Acetazolamide↗
PubMed1994

Measurement of blood concentrations of FK506 (tacrolimus) and its metabolites in seven liver graft patients after the first dose by h.p.l.c.-MS and microparticle enzyme immunoassay (MEIA).

1. Blood and urine concentrations of the macrolide immunosuppressant FK506 and its metabolites were measured in seven orthotopic liver transplant patients after the first oral dose of FK506 (0.04 +/- 0.02 mg kg-1) used as primary immunosuppressant. A specific h.p.l.c.-MS assay was used, allowing the measurement of parent drug and eight metabolites. Results were compared with those obtained using a microparticle enzyme immunoassay (MEIA). 2. Blood drug concentrations were described by an open two compartment model with first-order absorption giving the following mean data: tmax: 1.9 (h), Cmax: 17.4 (microgram l-1), AUC: 328.1 (microgram l-1 h), t1/2,1: 0.74 (h). The terminal elimination half-life was estimated at about 26 h using the h.p.l.c.-MS assay. 3. The metabolites found in blood were demethyl-FK506 and demethyl-hydroxy-FK506, while in urine FK506 and eight of its metabolites were detected.

Adult↗
PubMed1994

Interaction of Helicobacter pylori and its fatty acids with parietal cells and gastric H+/K(+)-ATPase.

Helicobacter pylori and the fatty acids produced by this organism were compared for their acid inhibitory activity in isolated parietal cells and their interaction with gastric H+/K(+)-ATPase. H pylori (intact organisms, sonicates, methanolic extracts, and extracts from culture medium) and the fatty acids cis 9,10-methyleneoctadecanoic acid and tetradecanoic acid inhibited at fairly high concentrations histamine- and dibutyryl cyclic adenosine monophosphate stimulated acid production in isolated parietal cells, dissipated (with a slow onset) the H+/K(+)-ATPase created H+ gradient in gastric membrane vesicles, and inhibited H+/K(+)-ATPase activity in a concentration dependent manner. The inhibitory potency of H pylori and the fatty acids in relation to H+/K(+)-ATPase depended on the amount of membrane protein. Bovine serum albumin prevented enzyme inhibition and proton dissipation from gastric vesicles. The data indicate that H pylori establishes its antisecretory action in parietal cells by blocking H+/K(+)-ATPase activity and also by a detergent action at the apical parietal cell membrane. The fatty acids cis 9,10-methyleneoctadecanoic acid and tetradecanoic acid are probably the acid inhibitory factors secreted by H pylori.

Animals↗
PubMed1994

Phospholipid synthesis in isolated porcine gastric mucous cells.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the major phospholipids of the gastric mucosal surface barrier and chiefly originate from mucous cells. Among these phospholipids PC with palmitic acid as its hydrophobic moieties is believed to protect the gastric mucosa by its hydrophobic properties. We investigated the phospholipid synthesis of isolated porcine gastric mucous cells in vitro and incubated them in the presence of radiolabelled precursors. Incorporation of 3H-choline into PC and of 14C-ethanolamine into PE was linear at 1, 10, and 100 mumol/l substrate concentration for at least 6 h. Half-maximal rate of precursor incorporation was achieved at 21 and 15 mumol/l of choline and ethanolamine, respectively. Ethanolamine inhibited PC synthesis and choline inhibited PE synthesis. A small amount of radioactivity originating from 14C-ethanolamine and from the methyl groups of 3H-methionine were incorporated into PC. Palmitic acid was incorporated into PC more than PE. Indomethacin did not influence the de novo synthesis of PC and PE via the Kennedy pathway, but inhibited the incorporation of 3H-methionine into PC. These results indicate that in gastric mucous cells PC and PE synthesis de novo depends on the concentrations of choline and ethanolamine. The palmitic acid content of PC depends on the availability of palmitic acid as a substrate: indomethacin-induced mucosal damage is not explained by modulation of phospholipid synthesis de novo.

Animals↗
PubMed1994

Comparative activities of agonists of active duodenal bicarbonate secretion in the guinea pig.

The comparative activity of agonists of duodenal bicarbonate secretion was studied in the anesthetized guinea pig, where the duodenal lumen was perfused with 24 mmol/l NaHCO3 to ensure active secretion of bicarbonate. Agonists were infused alone and in combination. Dibutyryl 3',5'-cyclic adenosine monophosphate, vasoactive intestinal polypeptide (VIP) and prostaglandin E2 (PGE2) were strong stimulants of bicarbonate secretion. Theophylline, dibutyryl 3',5'-cyclic guanosine monophosphate, glucagon and prostaglandin F2 alpha (PGF2 alpha) were weaker agonists, and secretin had no effect. Combinations of any two of VIP, PGE2 and glucagon depressed bicarbonate secretion, whereas combinations of PGE2 and PGF2 alpha, VIP and PGE2, and glucagon and PGF2 alpha increased bicarbonate secretion. The data indicate that cAMP and other secondary messengers may mediate duodenal bicarbonate secretion.

Adenosine Monophosphate↗
PubMed1994

Cyclic adenosine monophosphate is the second messenger of prostaglandin E2- and vasoactive intestinal polypeptide-stimulated active bicarbonate secretion by guinea-pig duodenum.

In a guinea-pig model we determined the intracellular events mediating the response of duodenal epithelial cells to vasoactive intestinal polypeptide (VIP) and prostaglandin (PG) E2. Intravenous administration of VIP (10(-9) to 10(-7) mol/kg) and PGE2 (10(-9) to 10(-6) mol/kg) dose-dependently increased duodenal epithelial bicarbonate secretion against an HCO3- concentration gradient, measured by a luminal perfusion technique, in anaesthetized guinea-pigs up to 4.5-fold. This secretion could be mimicked by intraduodenal dibutyryl cyclic adenosine monophosphate (dBcAMP; 10(-9) to 10(-7) mol/kg). Secretin (10(-9) mol/kg) and PGF 2 alpha (10(-9) to 10(-7) mol/kg), both given intravenously, were without effect or considerably less efficient. For VIP and PGE2, specific receptors coupled to adenylate cyclase could be demonstrated in homogenates of isolated duodenal epithelial cells. VIP and PGE2 stimulated adenylate cyclase activity up to sixfold, whereas PGF2 alpha and secretin were considerably less potent and efficient. VIP and PGE2 increased intracellular cyclic AMP levels up to fivefold and ninefold, respectively. This was followed by an increase in cytosolic protein kinase A activity. Bicarbonate secretion was maximal at 30 min. Examination of the subcellular distribution of protein kinase A showed a predominant cytosolic location. These data support the notion the PGE2 and VIP cause bicarbonate secretion by the serial activation of adenylate cyclase and protein kinase A in duodenal epithelial cells.

Adenylyl Cyclases↗
PubMed1994

Use of organotypical cultures of primary hepatocytes to analyse drug biotransformation in man and animals.

1. In conventional single-gel culture systems for primary hepatocytes, rapid loss of drug metabolizing capacities is a common feature and parallels general loss of function. An organotypical (double gel) culture technique for primary hepatocytes is established by enclosing the cells within two layers of extra cellular matrix. This serves to imitate the in vivo microenvironment within the space of Dissé. Using rat hepatocytes, this technique has been shown previously to maintain protein synthetic functions in vitro and to allow more efficient P450A-dependent biotransformation of drugs than a standard single-gel culture system. 2. The aim was to test the capacity of this organotypical culture model for primary rat and human hepatocytes to generate drug metabolites in a typical species-dependent pattern. 3. Urapidil, an antihypertensive drug, was used as a test compound, since it is metabolized in vivo in a species-dependent manner in rat and man. 4. Primary rat and human hepatocytes were cultured within two layers of collagen and exposed to 2.25 micrograms/ml urapidil for periods of 1-24 h at 3 days in culture. Urapidil metabolites were measured using hplc. 5. Metabolite M1 (hydroxylated product) was produced preferentially in human hepatocyte cultures, and metabolites M2/M3 (O-demethylated, N-demethylated product) were preferentially generated in rat cultures. This corresponded to the in vivo pattern found in man and rat, respectively. 6. Since in vitro urapidil metabolism by human and rat hepatocytes cultured in a double-gel system reflects that in vivo, it is suggested that information from such a system may be useful to predict the metabolic pathway of novel xenobiotics and to direct further toxicological evaluation.

Adult↗
PubMed1994

Effects of prostaglandins on [Ca2+]i and adenylate cyclase activity in isolated porcine gastric mucous cells.

In porcine gastric mucous cells, isolated enzymatically from the fundic mucosa and enriched by counterflow centrifugation, PGE2 (1 microM) increased adenylate cyclase activity to 225% and, distinct from that documented for other species, also [Ca2+]i, measured fluorimetrically with Fura2/AM, in Ca(2+)-containing and Ca(2+)-free incubation medium to 182% and 165% of control values, respectively. PGF2 alpha, PGD2, the stable prostacyclin analogue iloprost and the thromboxane-mimetic U46619 had no significant effects on adenylate cyclase activity and [Ca2+]i. Histamine (10 microM) stimulated adenylate cyclase activity to 236% of control value, an effect which could be blocked by the H2-receptor antagonist ranitidine. However, histamine and the activators of the cAMP system forskolin and dibutyryl cAMP had no significant effect on [Ca2+]i, indicating that an activation of the adenylate cyclase/cAMP system per se does not result in an increase in [Ca2+]i. These data suggest that prostanoids stimulate adenylate cyclase activity and [Ca2+]i in gastric mucous cells via activation of EP-receptors linked to both second messenger systems.

Adenylyl Cyclases↗
PubMed1993

Isolated brush cells of the rat stomach retain their structural polarity.

The brush cells (BC) are highly polarized elements occurring in epithelia of endodermal origin. They have a preferential topographical distribution in the organs in which they reside. In the stomach of the rat, BC prevail near the transitional zone separating the forestomach from the glandular stomach. Thus, a method was developed to isolate and recover BC from this organ with the aim of investigating the changes they may undergo after dissociation. Strips of the rat stomach were severed from the very proximal border of the glandular region and incubated in Hanks' balanced salt solution containing pronase. After sedimentation of the dissociated cells (crude sediment containing all stomach epithelial cell types) two successive cell fractions were prepared on performed Percoll gradient in an attempt to enrich BC in a defined layer. BC were recovered in a fraction at a density close to 1.03 g/ml where they represented about 2% of all cells. The isolated BC changed their form from columnar to pear-shaped; however, they maintained their structural polarity over 2 h as demonstrated by light microscopy, transmission-and scanning-electron microscopy. The fine structure of BC was always satisfactorily preserved. Maintenance of the structural polarity of isolated BC is contrary to the general rule according to which all conventional epithelial cells examined to date lose their polarity after isolation. This result is discussed in relation to morphological findings in isolated sensory cells (hair cells, photoreceptor cells) leading to the suggestion that BC are more similar to these than to conventional epithelial cells.

Animals↗