piperidines and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

Topics: Amino Acid Sequence; Animals; Brain; Cholic Acids; Chromatography; Mice; Molecular Sequence Data; Piperidines; Receptors, Neurotensin; Receptors, Neurotransmitter; Transduction, Genetic

A key challenge in the development of robust bioanalytical methods, for the determination of drug analyte in human urine samples, is the elimination of potential analyte losses as a result of non-specific adsorption to container surfaces in which the samples are collected, stored or processed. A common approach to address adsorption issues is to treat the urine samples with additives that serve to increase analyte solubility and/or minimise interaction with the container surfaces. A series of adsorption experiments were performed on human urine samples containing an adsorption-prone in-house development compound (AZD9164). A roller-mixing methodology was employed to maximise sample interaction with container surfaces and quantification of analyte was performed by LC-MS/MS following minimal sample preparation. In the absence of any urine additive, adsorptive losses averaged 35% but were highly variable between different lots of urine. In the presence of a range of additives, including the surfactants Tween 80, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) and sodium dodecylbenzenesulphonate (SDBS), analyte adsorption was shown to be eliminated. Of particular academic interest was the finding that adsorptive losses could also be reduced upon the addition of phospholipid. The presence of additive generally had no marked impact on the analyte MS response but the use of an isotopically labelled internal standard satisfactorily compensated for instances in which ion suppression was observed, e.g. in the presence of Tween 80. Since metabolite profiling/identification investigations are often performed on urine samples originating from early clinical pharmacology studies, the elution of selected additives was also monitored by MS. CHAPS, dimethylacetamide (DMA) and HP-β-cyclodextrin eluted as single chromatographic peaks in, or just after, the column void volume whilst polymeric Tween 80, and to a lesser extent SDBS, eluted over a wide retention time window. The potential of the latter surfactants to obscure the detection of unknown metabolites is significant and therefore their use in urine samples, upon which metabolite investigations are to be performed, is not recommended. Upon consideration of other factors such as additive cost and toxicity, CHAPS was selected for use in development of the validated assay.

Topics: Adsorption; Cholic Acids; Chromatography, Liquid; Humans; Piperidines; Quinuclidines; Reproducibility of Results; Surface-Active Agents; Tandem Mass Spectrometry

The expression of the 3 currently known neurotensin receptors was studied in human cancer cells of prostatic, colonic or pancreatic origin by means of RT-PCR analysis and binding experiments. All the cells selected for this work have been shown to exhibit a growth response to neurotensin. We found that the 7 transmembrane domain, levocabastine insensitive receptor (NTR1) is expressed in most but not all of the cells studied whereas the 7 transmembrane domain, levocabastine sensitive receptor (NTR2) is present in none of these cells. The 100 kDa-type I neurotensin receptor (NTR3) is expressed in all the cells assayed. Moreover, we demonstrated that neurotensin can stimulate the growth of CHO cells stably transfected with the NTR3. Taken together, our results strongly suggest that the NTR3 subtype could be involved in the growth response of human cancer cells to neurotensin.

Topics: Animals; Cell Membrane; CHO Cells; Cholic Acids; Colonic Neoplasms; Cricetinae; Drug Resistance, Neoplasm; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Male; Neurotensin; Pancreatic Neoplasms; Piperidines; Prostatic Neoplasms; Protein Binding; Protein Structure, Tertiary; Receptors, Neurotensin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transfection; Tumor Cells, Cultured

Serotonin-S2 receptors from rat frontal cortex were solubilized with a mixture of 6.8 mM CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulphonate) and 1.4 M sodium chloride. This new solubilization procedure solubilized about 40% of the membrane-bound serotonin-S2 receptors in an active form. The solubilized receptors were not sedimented after 1 h of centrifugation at 100 000 X g and they passed freely through 0.20 micron filters. The solubilized preparation showed high affinity binding of [3H]ketanserin and revealed a typical serotonin-S2 receptor profile: binding could be displaced by nanomolar concentrations of different serotonin antagonists and by micromolar concentrations of serotonin agonists. Compounds belonging to other pharmacological classes were poorly, or not active. Upon density gradient sedimentation, Svedberg coefficients of approximately 5 S were found on sucrose gradients made with H2O or D2O as the solvent. This was much lower than the value of 11.5 S previously reported from lysophosphatidylcholine-solubilized serotonin-S2 receptors.

Topics: Animals; Brain Chemistry; Centrifugation, Density Gradient; Cholic Acids; Filtration; In Vitro Techniques; Ketanserin; Microsomes; Nerve Tissue Proteins; Piperidines; Rats; Rats, Inbred Strains; Receptors, Serotonin; Serotonin Antagonists; Sodium Chloride

Topics: Animals; Benzilates; Cerebral Cortex; Cholic Acids; Detergents; Kinetics; Piperidines; Rats; Receptors, Cholinergic; Receptors, Muscarinic; Solubility