Rapamycin was incubated with human liver microsomes and an NADPH regenerating system, the metabolites were purified by semipreparative HPLC, and their structures were elucidated by direct chemical ...ionization and FAB-MS. At least six fractions were isolated containing rapamycin metabolites, indicating that rapamycin is metabolized by the human liver cytochrome P-450 system. One of these metabolites was identified as 41-O-demethyl-rapamycin. A second metabolite was hydroxylated in a yet unknown position. These two metabolites retained immunosuppressive activity in a phytohemagglutinin-stimulated human lymphocyte assay with IC50S of 1 and 1.5 nmol/liter, respectively. Rapamycin was metabolized by rat small intestinal microsomes to at least two metabolites, indicating extra-hepatic metabolism of rapamycin.
Protein kinase C (PKC) has been implicated in the control of epithelial proliferative activity and in the process of malignant transformation. Helicobacter pylori (H.p.) infection is associated with ...increased gastric epithelial cell proliferation and has been linked with gastric carcinoma. In the present study, we report that the H.p. fatty acid cis-9,10-methyleneoctadecanoic acid (MOA) directly activates PKC (Ka 3.3 microM). The effect of MOA upon PKC activation was Ca2+ dependent but did not require phosphatidylserine as phospholipid cofactor. MOA increased the stimulatory effect of phosphatidylserine at low Ca2+ (1 microM) concentrations. These findings indicate that MOA interacts at the phospholipid- and the diacylglycerol-binding domain to elicit PKC activation. Treatment of gastric mucous cells HM02 caused translocation of PKC from the cytosol to the nuclear, mitochondrial and membrane fraction. Furthermore, MOA stimulated 3Hthymidine incorporation into the DNA of HM02 cells. Our results show that the H.p. fatty acid MOA activates PKC and increases DNA synthesis in gastric epithelial cells.
The affinities of seven natural and synthetic prostaglandins PGE2; 16,16-dimethyl PGE2; iloprost (stable prostacyclin analogue); PGF2 alpha; PGD2; BW245c (stable PGD2 analogue); and U46619 (stable ...thromboxane analogue) to the PGE2 binding site of rabbit gastric mucosa were determined by measuring 3HPGE2 displacement from its high-affinity plasma membrane binding sites. In parallel, the potency of each prostaglandin in inhibiting acid generation in vitro was determined by measuring the inhibition of histamine-stimulated 14Caminopyrine accumulation in rabbit parietal cells prepared by enzymatic dispersion and enriched by counterflow elutriation. All seven prostaglandins displaced 3HPGE2 and inhibited histamine-stimulated 14Caminopyrine accumulation in a concentration-dependent manner. For all tested prostaglandins, the IC50 values were in excellent agreement for both variables measured. It is concluded that (a) a PGE2 receptor is localized on the parietal cell and mediates inhibition of acid formation by all prostaglandins and (b) the different in vitro antisecretory potencies of prostaglandins can be attributed to their different affinities to this PGE2 receptor.
Established in vitro models for studies of hepatic drug biotransformation include the use of primary hepatocytes. In normal liver the space of Disse provides the possibility of bilateral attachment ...to extracellular matrix for each hepatocyte. This configuration is disrupted by the cell isolation procedure of normal liver tissue, which delivers suspensions of round shaped cells. In standard culture configurations this unphysiologic cell shape terminates in a morphological dedifferentiation and inability to biotransform drugs. This study analyses the relevance of extracellular matrix geometry in hepatocyte monolayer configurations for expression and activity of cytochrome P450 3A. This enzyme is involved in the biotransformation of a large number of pharmaceuticals including the immunosuppressants tacrolimus and sirolimus. Morphological analysis of primary rat hepatocytes cultured with and without overlay of collagen type I was performed by transmission and scanning electron microscopy. Expression and activity of cytochrome P450 3A was studied by Western blot and the use of two model drugs specific for this enzyme. To this purpose the immunosuppressive drugs tacrolimus and sirolimus were used. Metabolites were analyzed by HPLC and HPLC/MS. Two sided attachment to extracellular matrix induces profound changes of the hepatocellular morphology in vitro resulting in the reconstitution of a polyhedric cell shape. This phenomenon is paralleled by an enhanced expression of cytochrome P450 3A and corresponding metabolic activity. As shown for tacrolimus biotransformation, the model may be useful to study complex metabolic patterns. In addition this model may facilitate studies of the kinetics of hepatocellular drug biotransformation in a setting with prolonged stability.
In this study, a modified, specific assay for measurement of tacrolimus and its metabolites in blood and urine from transplant patients using high-performance liquid chromatography (HPLC) linked to ...mass spectrometry (MS) is described. Samples were prepared for HPLC-MS by modified solid-liquid extraction. The original two-step washing procedure was replaced by a single washing step, and samples were eluted with acetonitrile/water instead of dichloromethane, thus avoiding an evaporation step. Samples were injected automatically every 3 min into the HPLC-MS system. Time-consuming gradient elution was replaced by isocratic elution. This procedure resulted in a lower limit of quantitation of 0.2 microgram/L. The interassay variability was 14.5% for 5 micrograms/L and 15.8% for 25 micrograms/L. The intrassay variability was 11.2% for 5 micrograms/L and 4% for 25 micrograms/L. The recovery for tacrolimus in blood was 90.4% for 1 microgram/L, 78.9% for 10 micrograms/L, and 81.3% for 25 micrograms/L. Measurement of tacrolimus and its metabolites in samples from various transplant patients showed that the main metabolites found in blood and urine are demethyl-tacrolimus, di-demethyl-tacrolimus and demethyl-hydroxy-tacrolimus. Cross validation of the modified HPLC-MS assay with a microparticle enzyme immunoassay showed a significant correlation between the two assays, with r = 0.915.
Tacrolimus (FK 506) is a new, potent immunosuppressive drug for primary and rescue therapy in liver and kidney transplantation. Therapeutic drug monitoring is essential for this drug because of its ...narrow therapeutic window. Blood levels are monitored routinely by enzyme linked immunoassay (ELISA) or by microparticle enzyme immunoassay (MEIA). In a 13-year-old recipient of a liver transplant who had poor hepatic function during the first postoperative week, the authors observed unusually high tacrolimus blood concentrations using either the ELISA (26.6 to 49.0 microg/l) or MEIA (58.5 to 64.5 microg/l). Parent drug levels measured in the same blood samples by high-performance liquid chromatography/mass spectrometry (HPLC/MS) were up to 10-fold lower (5.1 to 9.0 microg/l). The discrepancies between the immunoassay and HPLC/MS results could not be attributed to any of the known metabolites of tacrolimus.
Phosphatidylcholine (PC) is the major phospholipid of the hydrophobic gastric mucosal barrier and is chiefly released from mucous cells into the gastric mucus. Whereas the mucosa contains highly ...unsaturated PC, gastric mucus predominantly contains palmitoyl-oleoyl-PC and palmitoyl-linoleoyl-PC, indicating a selective release of these PC species into the gastric lumen. In order to understand gastric PC metabolism, we investigated synthesis and release of PC in cultivated porcine gastric mucous cells, using dual labelling with methyl-3H-choline and 1-14C-palmitate, in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), indomethacin and prostaglandin E2 (PGE2). Linear incorporation of methyl-3H-choline and 1-14C-palmitate into PC was achieved for at least 8h. In contrast to type II pneumocytes TPA increased PC synthesis in gastric mucous cells but not its release. Indomethacin did not influence PC synthesis, but it decreased the release of newly synthesized PC. PGE2 antagonized the effect of indomethacin on PC release. We conclude that PC release by isolated porcine gastric mucous cells is regulated in a manner different from type II pneumocytes. PC release is impaired by indomethacin and this impairment is restored by PGE2.
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.
To define the mechanisms by which Helicobacter pylori stimulates pepsinogen secretion, the in vitro release of pepsinogen was studied using a preparation of pig chief cell monolayers. Helicobacter ...pylori induced a time- and concentration-dependent release of pepsinogen into the medium, with about a three-fold increase in pepsinogen secretion over controls found after 45 min of incubation. 3x10(7) H. pylori produced 50% of the maximal response found at a H. pylori count of 2x10(8). The action of H. pylori did not depend on the presence of the vacuolating toxin (vacA) and the cytotoxin-associated protein (cagA). Dibutyryl-cAMP and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also markedly stimulated pepsinogen secretion and enhanced the stimulatory effect of H. pylori. Helicobacter pylori-stimulated pepsinogen release was inhibited by lanthanum and the calmodulin antagonist W-7, but not by the L-type Ca2+ channel blocker nifedipine, TMB-8, an agent that blocks the release of Ca2+ from intracellular stores, the protein kinase C inhibitor staurosporine and the protein kinase A inhibitor H-8. It is suggested that H. pylori directly stimulates pepsinogen release from gastric chief cells and that this effect is mediated via the calcium/calmodulin messenger branch.
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