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Biomedical subjects

F Lauterbach

Publications and source records attributed to F Lauterbach.

At least 19 recordsLinked to original sources
PubMed1994

Gastric fundic inhibition of sugar transport across the intestinal mucosa of guinea-pig.

A low molecular weight peptide designated gastric fundic factor (GFF), extracted from porcine fundic mucosa and administered to the serosal surface of mucosal sheets from guinea-pig intestine, decreased the transport of luminal glucose across the sheets by up to 70%. The results show that gastric fundic inhibition of glucose absorption observed in different animal models in vivo can be reproduced in vitro, and suggest that the intestinal mucosa itself is the target for peptide hormone(s) released by the gastric fundic mucosa. Simultaneous transport of alpha-aminoisobutyric acid, a nonmetabolisable amino acid, through the jejunal mucosa was unaffected as was paracellular permeation by inulin. However, amino acid transport was also reduced when GFF was administered to sheets of ileal mucosa. The intestinal mucosal sheet in vitro is a sensitive and convenient model with which to follow the purification of GFF to homogeneity.

Aminoisobutyric Acids↗
PubMed1993

Compartmentation of intestinal drug sulphoconjugation. Incorporation of luminal and contraluminal [35S]sulphate into 1-naphthol by the isolated mucosa of guinea pig jejunum and colon.

Compartmentation of 1-naphthol metabolism was inferred from the metabolite pattern and distribution in the isolated mucosa of guinea pig intestine mounted in a flux chamber (Sund and Lauterbach, Arch Pharmacol Toxicol 58: 74-83, 1986). To verify the existence of these compartments the dependence of [35S]sulphate incorporation into 1-naphthol sulphate on the side of administration of 1-naphthol and [35S]sulphate was determined. Isolated mucosae were pre-equilibrated with [35S]-sulphate (5 x 10(6) cpm/mumol, 1 mM) for 30 min and subsequently incubated for 15 min with 50 microM 1-naphthol. The three 1-naphthol sulphate fractions (luminal side, blood side and tissue) were assayed by HPLC and liquid scintillation counting; their specific activity was calculated as percentage of the specific activity of the inorganic sulphate administered. 1-Naphthol glucuronide was also measured. In jejunal experiments: after luminal administration of 1-naphthol, 1-naphthol sulphate appeared almost exclusively in the luminal solution; its specific activity approached 70% and 3%, when [35S]sulphate was added to the luminal and blood side, respectively. After introducing 1-naphthol and [35S]sulphate on the blood side, a high and similar specific activity of 50-60% was observed in all three 1-naphthol sulphate fractions (luminal and blood side, tissue) though adding [35S]sulphate to the lumen side decreased the specific activity to 10-20%. In experiments on colonic mucosa: a specific activity both of luminal and blood side 1-naphthol sulphate of more than 50% was observed with blood side [35S]sulphate irrespective of the side of 1-naphthol administration. When [35S]sulphate was placed on the luminal side the specific activity of blood side 1-naphthol sulphate dropped to only 3%, and that of luminal 1-naphthol sulphate ranged between 6% and 20%. Supplementary experiments in which mucosae were exposed to 1-naphthol and [35S]sulphate for 45 min without preincubation showed a tendency to decrease the lumen: blood distribution ratio of 1-naphthol sulphate. However, the general pattern of 1-naphthol sulphate specific activity remained unchanged. The experiments provide further evidence that the jejunal conjugation of phenolic drugs is being performed in two major compartments, which are accessible from the lumen ("luminal compartment") and blood ("systemic compartment") side. The luminal compartment seems practically inaccessible to blood side sulphate as is the systemic compartment for luminal 1-naphthol. In the colonic mucosa, a major "systemic compartment" receiving its sulphate from the blood side is the site for most of the events, but a minor "luminal compartment" seems to be involved as well.

Animals↗
PubMed1993

2-Naphthol metabolism and metabolite transport in the isolated guinea pig mucosa: further evidence for compartmentation of intestinal drug metabolism.

As an extension of a previous study on the metabolism of 14C-1-naphthol (1-N) by the isolated guinea pig mucosa (Sund & Lauterbach 1986), the isomeric compound 2-naphthol (2-N, 50-130 nmol/ml) has now been examined. 14C-Labelled drug was added to the luminal or contraluminal fluid bathing the two sides of jejunal or colonic mucosal sheets in a symmetrical set up. After aerobic incubation for 45 min. at 37 degrees, the fluid compartments and the tissue were analysed for parent drug and metabolites. Like 1-N 2-N was transformed into its sulphate and glucuronide. In the jejunum, 2-N was more extensively sulphated than 1-N, whereas in the colon the metabolite profiles (sulphate:glucuronide ratio) of the two isomers were similar. Generally, the metabolism rate of 2-N, its metabolite profile and metabolite transport pattern (lumen: blood distribution ratio) as well as the tissue accumulation of parent drug and metabolites, depended on the side of drug administration and on the tissue studied. Thus, changing the drug administration had a pronounced impact on the jejunal metabolism and transport, but caused only minor effects in the colon. In summary, this study emphasizes that drug metabolism and metabolite transport differ in the small and large intestine. The data further support the hypothesis that jejunal drug metabolism takes place in two compartments, of which the most active one is accessible from the lumen side and the other from the blood side. The possibility that colonic drug metabolism may also involve compartmentation should be considered although the present study provided very little evidence for this.

Animals↗
PubMed1991

Cloning and nucleotide sequence of the structural genes encoding the formate dehydrogenase of Wolinella succinogenes.

The formate dehydrogenase of Wolinella succinogenes is a membraneous molybdo-enzyme which is involved in phosphorylative electron transport. The gene (fdhA) encoding the largest subunit was isolated from a gene bank by immunological screening. The fdhA gene was located in an apparent transcriptional unit (fdhA,B,C,D) together with three more structural genes. The N-terminal sequences of three polypeptides present in the isolated enzyme were found to map within the fdhA, B and C structural genes. A polypeptide corresponding to fdhD was not detected in the enzyme preparation. This suggested that the functional formate dehydrogenase was made up of three or four different subunits. The genes fdhA and C encode larger preproteins which differ from the corresponding mature proteins by N-terminal signal peptides. The N-terminal half of the mature FdhA is homologous to the larger subunits of the formate dehydrogenases of E. coli (formate-hydrogenlyase linked) and Methanobacterium formicicum as well as to three bacterial reductases containing molybdenum. It harbours a conserved cysteine cluster and two more domains which may be involved in binding the molybdenum cofactor. FdhB may represent an iron-sulphur protein, twelve cysteine residues of which are arranged in two clusters which are typical of ligands of the iron-sulfur centers in ferredoxins. FdhC is a hydrophobic protein with four predicted transmembrane segments, which appears to be identical with the cytochrome b present in the isolated formate dehydrogenase. It may form the membrane anchor of the enzyme and react with the bacterial menaquinone.

Amino Acid Sequence↗
PubMed1990

The fumarate reductase operon of Wolinella succinogenes. Sequence and expression of the frdA and frdB genes.

The genes of the fumarate reductase of Wolinella succinogenes are organized in an operon. The three structural genes in the order frdC, frdA, frdB, are preceded by a common promoter (Körtner et al. 1990) and followed by a terminator of transcription. The proteins encoded by the genes are identical with the subunits present in the isolated enzyme. FrdA and FrdB are hydrophilic proteins consisting of 656 and 238 amino acids, respectively. The 12 cysteine residues present in FrdB form 3 ferredoxin-like clusters, whereas the 12 cysteines of FrdA are not clustered. Expression of FrdA and FrdB in Escherichia coli from a plasmid containing a DNA fragment with both genes in full length, gave rise to the EPR signals of the bi- and trinuclear iron-sulfur centers of the enzyme. Only the binuclear center was seen on the expression of FrdB together with a C-terminal fragment of FrdA (130 amino acid residues). Neither of the two centers was detected on the expression of FrdA together with a N-terminal fragment of FrdB including cysteine cluster I. Sequence comparison of FrdA and FrdB with the corresponding subunits of the fumarate reductases of E. coli or Proteus vulgaris or to those of the succinate dehydrogenases of E. coli or Bacillus subtilis revealed strong homologies (28-36% identical amino acid residues). Part of the homologous peptide stretches could be assigned to domains that are involved in the binding of the substrate of the FAD prosthetic group of the enzyme.

Amino Acid Sequence↗
PubMed1990

Wolinella succinogenes fumarate reductase contains a dihaem cytochrome b.

The fumarate reductase operon of Wolinella succinogenes is made up of three structural genes (frd-CAB). The frdC gene was located next to the promoter region and identified as the cytochrome b structural gene encoding 256 amino acid residues. The N-terminal amino acid sequences of seven fragments derived from the cytochrome b moiety of the enzyme all mapped within the frdC gene. This suggested that the enzyme contained only one species of cytochrome b. Re-evaluation of earlier measurements of subunit composition, haem B content and molecular weight led to the conclusion that the enzyme contained one molecule of cytochrome b with two haem B groups. The hydropathy plot of the amino acid sequence predicted five membrane-spanning hydrophobic segments, the first four of which contained a single histidine residue each. These residues could form the axial ligands to the two haem B groups. FrdC was found to be homologous with the cytochrome b (SdhC) of the Bacillus subtilis succinate dehydrogenase, but not with the hydrophobic subunits of the fumarate reductase or succinate dehydrogenase of Escherichia coli.

Amino Acid Sequence↗
PubMed1987

Cloning and expression of the genes of two fumarate reductase subunits from Wolinella succinogenes.

The fumarate reductase complex of the anaerobic bacterium Wolinella succinogenes catalyzes the electron transfer from menaquinol to fumarate. Two structural genes coding for subunits of the enzyme have been cloned in Escherichia coli. The genes were isolated from a lambda EMBL3 phage gene bank by immunological screening and subcloned in an expression vector. The genes frdA and frdB, which encode the FAD protein (Frd A, Mr 79,000) and the iron-sulfur protein (Frd B, Mr 31,000) of the fumarate reductase complex, were cloned together with a W. succinogenes promoter. The gene order was promoter-frdA-frdB. The FAD protein and the iron-sulfur protein were expressed in the correct molar mass in E. coli from the clones. The identity of the frdA gene and the suggested polarity were confirmed by comparing the amino-terminal sequence of the Frd A protein with that predicted from the 5'-terminal nucleotide sequence of frdA. The frdA and frdB genes are present only once in the genome. A region downstream of frdB, possibly a gene encoding cytochrome b of the fumarate reductase complex, hybridizes with a second site in the genome.

Amino Acid Sequence↗
PubMed1987

1-Naphthol metabolism and metabolite transport in the small and large intestine. II: Effect of sulphate and phosphate ion omission, and of 2,6-dichloro-4-nitrophenol in the isolated guinea pig mucosa.

A previous study (Sund & Lauterbach 1986) in the isolated guinea pig mucosa showed a complex pattern of 1-naphthol (I) metabolism and metabolite (glucuronide = II and sulphate = III) transport in relation to tissue studied (jejunum and colon) and administration side (lumen versus blood side). In the present paper aspects of I metabolism and II and III transport have been further studied. The experiments involved: Omission of inorganic sulphate in the incubation solution at one particular side or at both sides, to see if and how intestinal sulphoconjugation depended on side of sulphate ion entry, and if II and III efflux might be linked to sulphate ion influx. Similar omission experiments with inorganic phosphate, and Incubation in presence of 2,6-dichloro-4-nitrophenol (IV), a drug claimed to be a selective inhibitor of sulphoconjugation. The experiments showed: In the jejunum, sulphate ion caused a much stronger stimulation of III formation from the lumen than from the blood side, when I was added at the luminal side. In the colon, however, the sulphate ion was more effective on the blood side than on the lumen side, regardless of side at which I was added. More experiments are needed to clarify if conjugate efflux is affected by sulphate ion omission as well. Omission of inorganic phosphate did neither affect I metabolism nor II and III efflux. IV (present at both sides at once) had complex effects, involving inhibition of II and III synthesis as well as their efflux, and, in part, a change in their normal lumen: blood distribution pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Intestinal permeation of nonquaternary amines: a study with telenzepine and pirenzepine in the isolated mucosa of guinea pig jejunum and colon.

Previous studies which demonstrated an intestinal secretion of quaternary ammonium compounds in the jejunum were extended to nonquaternary amines. Telenzepine and pirenzepine, two structurally similar compounds differing in lipid solubility, were administered either to the lumen or blood side of isolated mucosae of guinea pig jejunum and colon mounted in a flux chamber. Transepithelial permeation of both drugs from blood to lumen side was 3.2 to 18 times higher than in the reverse, absorptive direction. The indicated net secretion was highest with telenzepine in the colon. At 7 degrees C transepithelial permeation was restricted to paracellular shunt fluxes. In the colon, the highest cellular concentrations were observed after blood side administration of telenzepine and pirenzepine (500 and 150% of that of the incubation solution). At 7 degrees C these values were reduced to 1 of 5 and 1 of 8 of the values at 37 degrees C. At pH 6.6, 7.4 or 8.2, no differences in the colonic secretion of telenzepine were seen; cellular concentration increased with increasing pH only after luminal administration. Under all conditions, the colon acidified the luminal solution by roughly 0.4 pH units. Phenoxybenzamine, shown previously to inhibit the jejunal secretion of quaternary ammonium compounds, inhibited the secretion of pirenzepine and telenzepine in both gut segments, but did not affect blood-to-lumen permeation of N-methylscopolamine in the colon. In addition, phenoxybenzamine reduced the cellular content in the colon but had little or no effect on pirenzepine or telenzepine content in the jejunum. The results are consistent with a model in which the nonquaternary amines are secreted in the jejunum by two serial transport steps specific for the base cations; an additional diffusion of the free bases is of importance in the basolateral membrane. In the colon, carrier-mediated influx increases with lipophilicity of the base cations, whereas efflux across the brush border is limited to diffusion of the free bases; concomitant luminal acidification and formation of base cations maintains the diffusion gradient.

Animals↗
PubMed1986

Drug metabolism and metabolite transport in the small and large intestine: experiments with 1-naphthol and phenolphthalein by luminal and contraluminal administration in the isolated guinea pig mucosa.

The metabolism and metabolite transport of the monophenol 1-naphthol (I) and the diphenol phenolphthalein (II) have been studied in a symmetrical setup of the isolated jejunal and colonic mucosa from the guinea pig (Lauterbach 1977). In both tissues, the main metabolites of I were its sulphoconjugate and glucuronide, but the rate of metabolism, relative proportion of the metabolites and their distribution pattern varied with tissue and drug administration side in the following manner: By luminal administration (50 nmol/ml) in the jejunum, the metabolism was nearly complete within 45 min., more sulphate (1.5-3x) than glucuronide was formed, and both metabolites were predominantly transferred back to the lumen. By blood-side administration, the metabolism was less complete due to a significant decrease of the sulphated fraction. In consequence, more glucuronide (1.5-3x) than sulphate was formed. Moreover, the efflux pattern of the metabolites changed completely; the greater part of the glucuronide fraction now being conveyed to the blood side, whereas the sulphate tended to distribute in a 1:1 fashion on the lumen and blood side. The colonic mucosa behaved in a dissimilar way, since neither I metabolism nor metabolite efflux pattern in this tissue was influenced significantly by drug administration side. More sulphate (1.5-3x) than glucuronide was formed by both routes, and the metabolite distribution was similar to that observed by blood side administration in the jejunum. The changes described above were associated with changes in tissue accumulation of free I and metabolites; accumulation of I by luminal administration in jejunum was insignificant and that of the metabolites small. The main change caused by a higher concentration of I (130 nmol/ml) was a decrease in the sulphated fraction in the colon. II was metabolized at a slower rate than I, and a significant tissue accumulation of free II was observed in all instances. The monoglucuronide was the main metabolite. Only minor amounts of II monosulphate were formed, making its distribution pattern difficult to ascertain. The distribution of II monoglucuronide on the other hand was generally similar to that described for its I analogue.

Animals↗
PubMed1984

Effect of hyperglycaemia on sugar transport in the isolated mucosa of guinea-pig small intestine.

The effect of hyperglycaemia on sugar transport was studied by comparing transepithelial permeation and tissue content of 3-O-methyl-D-glucose (3-O-MG), beta-methyl-D-glucoside (beta-MDG) and D-glucose in isolated mucosae of guinea-pig jejunum mounted in a flux chamber. Sugars were administered either to the luminal or the blood side of mucosae prepared either from normal animals or those maintained in a hyperglycaemic state by I.V. glucose infusion for 12 h. In control animals, absorptive sugar fluxes increased in the order glucose greater than beta-MDG greater than 3-O-MG. Only beta-MDG was accumulated in the tissue beyond the medium concentration. Permeation of 3-O-MG and beta-MDG in the direction blood-to-lumen was mainly paracellular as indicated by the strict correlation with the simultaneous permeation of polyethylene glycol (mol. wt. 900). Luminal addition of 10(-3) M-phlorhizin increased permeation and decreased tissue content of beta-MDG and D-glucose when administered on the blood side, suggesting that these sugars are recaptured at the brush border even from vigorously mixed solutions. For flux coefficient calculation the preparation was regarded as a three-compartment system. With all three sugars, the influx coefficient was higher at the luminal, but lower at the basolateral membrane than the corresponding efflux coefficient. 3-O-MG displayed the highest basolateral influx coefficient of all three sugars, being even higher than its luminal influx coefficient. The luminal influx coefficient of beta-MDG was 22 times greater, and its basolateral efflux coefficient 2.5 times less than the corresponding values for 3-O-MG, resulting in cellular beta-MDG accumulation. D-Glucose was suited best for transepithelial transport, having a luminal influx coefficient only 1.6 times less, and a basolateral efflux coefficient almost 10 times greater than those for beta-MDG. Prolonged hyperglycaemia increased the lumen-to-blood permeation of all three sugars 1.3-2-fold. No significant differences in tissue content to control values were observed after 45 min (3-O-MG, D-glucose) or 90 min (beta-MDG) incubation. Therefore, flux coefficients increased by the same factors in luminal and basolateral membranes, i.e. 1.7, 1.3 and 1.7 for 3-O-MG, beta-MDG and D-glucose, respectively. These results indicate that changes in both the luminal and basolateral membranes play a role in the increase of sugar transport in hyperglycaemia and that a regulatory mechanism might exist between the transport systems located in both membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

3-O-Methylglucose↗
PubMed1978

Intestinal secretion of sulphanilic acid by the isolated mucosa of guinea pig jejunum.

Tissue uptake and transepithelial permeation of 35S-sulphanilic acid were studied in the isolated guinea pig jejunal mucosa. Methoxy-3H-inulin added simultaneously served as a marker for the extracellular space and permeability of paracellular shunt pathways in the preparation. The uptake of sulphanilic acid from the blood side exceeded that from the luminal side 4--7 fold. The permeation of the acid was strongly correlated to the permeation of inulin. At 5 micrometer and 2.5 mM sulphanilic acid under aerobic conditions, the regression lines for the permeation from lumen to blood pass almost through the origin, while the regression lines for the permeation from blood to lumen intersect the ordinate at a positive Y-value. In anaerobiosis, at 25 mM sulphanilic acid, or with addition of p-toluene sulphonic acid only one regression line is obtained for the permeation in both directions. It is concluded that besides a permeation of sulphanilic acid across inulinpermeable shunt pathways an active transport system exists, which transfers the acid from the blood to the luminal side. This system is saturable, depends on aerobic energy and exhibits mutual inhibition by a structurally related compound. The results are comparable to those previously obtained with cardiac glycosides and quaternary ammonium compounds, in the same preparation. Thus, the intestinal mucosa is able to secrete the same classes of compounds which are secreted by the liver and the kidney.

Aerobiosis↗
PubMed1977

Secretion of monoquaternary ammonium compounds by guinea pig small intestine in vivo.

In anesthetized guinea pigs N-(3H)methylscopolamine (NMScop), N1-(14C)methylnicotinamide (NMN), and (14C)tetraethylammonium (TEA), administered intravenously, were secreted against a concentration gradient into the lumen of the small intestine. The concentration ratio of unmetabolized ammonium base in the intestinal lumen to that in the plasma was 4.3 and 6.5 for NMScop and NMN, respectively, 75 min after the intravenous injection of 1 nmole/g body weight of the individual compounds. The corresponding value for TEA after 180 min was 2.0. The establishment of the concentration gradient between intestinal lumen and plasma was diminished with increasing doses. An excess of NMN inhibited the uphill transport of NMScop. Since the electrical potential difference across the intestinal epithelium and a 'fluid circuit' mechanism cannot solely account for the observed accumulation of the monoquaternary ammonium compounds in the intestinal lumen, the evidence presented supports previous in vitro findings that the small intestine is capable of actively secreting organic cations.

Animals↗