MISSISSIPPI COBRE: CORE C: IN VITRO PHARMACOLOGY CORE

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The In Vitro Pharmacology Core screens compounds for their ability to interact with specific targets of interest. The primary targets are neuronal receptors and transporters for neurotransmitters and neuromodulators that mediate neuropsychiatric effects. The core also screens compounds for their ability to interact with ligand-independent ion channels. The core is also designed to assess functionality of compounds at the sites where they interact, e.g., to assess whether they have agonistic or antagonistic properties. Such assays include assessment of GTPgammaS binding, ion transport, and assays for cAMP and inositol trisphosphate formation. The primary methodology employed by the core thus far to test samples submitted to the core is by competition binding assays using radioligands specific for targets of interest e.g., 3H-DAMGO for mu opioid receptors. In the future the core will also make use of non-radioactive methodologies. When possible, the core uses membrane preparations of human receptors transfected into cell lines. However, when such receptors are not available suitable membrane preparations obtained from animal sources are used. Evaluation of samples for interactions with targets involves 1 to 3 steps. The initial step is a primary screen in which a single concentration (e.g. 10 ¿M for compounds and 10 mg/ml of extract or isolated fraction) is tested for its ability to compete for radioligand binding to the target site. If the primary screen reveals more than 50% inhibition of specific binding of the radioligand, a secondary screen is carried out. The secondary screen uses 4 to 6 concentrations of the sample ranging from 10 ¿M down to 100 pM at log unit intervals. The competition curve is evaluated to determine an IC50 value by non-linear regression analysis using a one-site competition equation (Prism, Graphpad Software). The IC50 value is converted to a KI value based on simultaneous evaluation of the saturation binding kinetics of the radioligand. For more detailed analyses, e.g., for radioligands that do not appear to have Hilll slopes = 1 or which may show different affinities, 12 point competition binding assays are run as noted above and either one-site with Hill coefficient not set at one, or multiple binding site analyses are used to fit the data using Prism. To date, the Core has assays for mu, delta and kappa opioid receptors, and CB1 cannabinoid receptors. In development are assays for CB2, and OFQ receptors and Ca2+ signaling. Additional receptor, ion channel, and transporter binding assays are in the planning stage. These core functions involve the use of state of the art technologies as well as time-tested laboratory methodologies. Using multi-label reader technologies and radioactive binding techniques, the core is set up to rapidly assess specific activities of compounds and extracts held in the repositories (Core A) at the University of Mississippi National Center for Natural Products Research and of compounds synthesized by the Chemistry Core (Core B). These studies can be performed on tissues harvested from animals or from cultured cells. Mechanistic studies into the actions of extracts and their compounds can be assessed using biochemical kinetic analyses as well as cellular electrophysiology and imaging techniques, which will be added in the 2nd year. This exploratory ability is accomplished through the cores four functional components; cell culture facility, functional testing facility, biochemistry, receptor pharmacology and histology facility, and the soon to be added electrophysiology and imaging facility. The In vitro Pharmacology Core has made significant equipment purchases this year including a PerkinElmer Topcount with 12 PMT readers and a PerkinElmer EnVision multilabel reader with a dispensing unit. The core has also purchased cell culture incubators and laminar flow hoods for the cell culture facility and electronic multichannel pipettors to facilitate microplate assays.