Alpha - Regan Ure.epub (request)
Ronald
α 1-adrenoceptors are widely distributed and are activated either by norepinephrine released from sympathetic nerve terminals or by epinephrine released from the adrenal medulla. Receptor activation mediates a variety of functions, including contraction of smooth muscle, cardiac stimulation, cellular proliferation and activation of hepatic gluconeogenesis and glycolysis. In the CNS, the activation of α 1-adrenoceptors results in depolarization and increased neuronal firing rate.
The K i-value of the test drug is the concentration, at which 50 % of the receptors are occupied by the test drug. The affinity constant K i [mol/l] is recorded and serves as a parameter to assess the efficacy of the test drug.
Binding of 3H-WB 4101 to α1-adrenergic receptors in brain is used to test hypotensive activity as a possible side effect of neuroleptic drugs. The test E.5.1.6 is described in the chapter on neuroleptic activity.
The purpose of this assay is to assess the interaction of hypotensive agents with central α 2-receptors and determine possible clonidine-like mechanisms of action. Clonidine binding may also be relevant to the activity of other classes of drugs such as antidepressants that interact with α 2-receptors.
Tissue homogenates are incubated for 20 min at 25 C with 3 nM 3H-clonidine and varying drug concentrations, and immediately filtered under reduced pressure on Whatman GF-B filters. The filters are washed with 3 five ml volumes of 0.05 M Tris buffer pH7.7, and transferred to scintillation vials. Specific clonidine binding is defined as the difference between total bound radioactivity and that bound in the presence of 1 μM clonidine.
The existence of presynaptic receptors which regulate the evoked release of neurotransmitters has been functionally demonstrated in both peripheral and central nervous system (Langer 1981; Starke 1981; Raiteri et al. 1984; Miller 1998). Presynaptic adrenergic α 2-receptors regulate the evoked release of norepinephrine, comprising a short negative feedback loop. Alpha-2 agonists, such as clonidine and guanabenz, inhibit evoked release and alpha-2 antagonists, such as yohimbine and idazoxan, enhance evoked release.
The assay is used as a biochemical screen for agents which enhance or inhibit release of [3H]norepinephrine (3H-NE) and is particularly useful for testing receptor function of α 2-adrenergic agonists and antagonists.
The procedures used emphasize delicate care of slices. By treating slices with great care, one is able to incubate at low tracer concentrations of 3H-NE (25 nM), thus minimizing nonspecific labeling of releasable pools other than those in noradrenergic nerve terminals. It also permits the use of low (and more physiological) stimulation parameters,which allow the neurons to recover easily between stimulations and do not flood the synaptic cleft with released NE, which would compete with any applied drug thus decreasing sensitivity.
For most assays, a 1mMstock solution of the test compound is made up in a suitable solvent and diluted such that the final concentration in the assay is 1 μM. Higher or lower concentrations may be used depending on the potency of the drug.
To establish a stable baseline, control buffer is pumped through the chamber for 1 h at a flow rate of 0.7 ml/min before the first stimulation. One hour is allowed to pass before the second stimulation. When drugs are used, each concentration is prepared in a separate flask in control buffer and allowed to equilibrate with the tissue slice 20 min before the second stimulation. The experiment is stopped 40 min after the second stimulation. Stimulation parameters are set at 5 Hz (2 ms duration) for 60 s, with 1 ms delay and voltage setting of 440 SIU (25f0Ω).
The effects of drugs can be reported as S2/S1 ratios. To normalize the data, drug effects can be estimated by first calculating S2/S1 values for control and drug-treated slices and then expressing the S2/S1 value for the drug-treated slices as a percentage of the S2/S1 value for the control slices for each experiment.Each condition should be tested in slices from each animal.
Imidazoline receptors constitute a family of nonadrenergic high-affinity binding sites for clonidine, idazoxan, and allied drugs. Drugs selectively binding to imidazoline receptors are expected to have less side effects than clonidine (Ernsberger et al. 1992, 1997; Molderings et al. 1992; Limon et al. 1992). One major subclass, the I1 receptors, being mainly distributed in the brain and brain stem, partly mediates the central hypotensive action of clonidine-like drugs. The I2 receptors, an other subclass, are mitochondrial, not G protein coupled, and have diversified functions. They may be involved in neuroprotection for cerebral ischemia. Two binding sites of [3H]p-aminoclonidine, α 2 adrenoceptors and imidazoline binding sites, could be separated (Ernsberger et al. 1987;Bricca et al. 1988; Kamisaki et al. 1990). At least 3 subtypes of imidazoline/ guanidinium-receptive sites have been found by photoaffinity labeling (Lanier et al. 1993).
Whole bovine brains and adrenal glands are obtained from a local slaughterhouse. The lateral medulla oblongata is isolated by a sagittal section through the lateral margin of the pyramids and then bisected. The ventral half is defined as the ventrolateral medulla.
Data are obtained as disintegrations per min and transferred to the Equilibrium Binding Data Analysis program (McPherson 1985). Then, several experiments are analyzed simultaneously with the LIGAND program for non-linear curve fitting (Munson and Rodbard 1980). IC 50 values are estimated from inhibition curves by non-linear curve fitting (Mutolsky and Ransnas 1987). Protein assay data are also analyzed by non-linear curve fitting (McPherson 1985).
Molderings and Gthert (1995) determined electrically or K+-evoked tritium overflow from superfused rabbit aortic strips pre-incubated with [3H]noradrenaline in order to characterize presynaptic imidazoline receptors which mediate noradrenaline release and compared them with I1- and I2-imidazoline radioligand binding sites.
β-adrenoceptors are widely distributed, found at both central and peripheral sites, and are activated either via norepinephrine released from sympathetic nerve terminals or via epinephrine released from the adrenal medulla. Important physiological consequences of β- adrenoceptor activation include stimulation of cardiac rate and force, relaxation of vascular, urogenital and bronchial smooth muscle, stimulation of renin secretion from the juxtaglomerular apparatus, stimulation of insulin and glucagon secretion from the endocrine pancreas, stimulation of glycogenolysis in liver and skeletal muscle and stimulation of lipolysis in the adipocyte.
While it was initially thought that cardiac stimulation involved primarily the β 1-adrenoceptor, it now appears that all of the receptor subtypes may be involved. Bronchodilation appears to be mediated by the β 2-adrenoceptor. The β 3-adrenoceptor is responsible for lipolysis in white adipose tissue and thermogenesis in the brown adipose tissue found in rodents. Renin release appears to be mediated by the β 1-adrenoceptor (Waitling 2006).
The binding reaction is started by adding 100 μl membrane suspension per incubation sample (approx. 2 mg protein/ml). The samples are incubated for 60 min in a shaking water bath at 25 C. The reaction is stopped by rapid vacuum filtration of the total incubation volume over glass fiber filters. Thereby the membranebound radioactivity is separated from the free activity. Filters are washed immediatelywith approx. 20 ml icecold rinse buffer per sample. The retained membranebound radioactivity on the filter is measured after addition of 2 ml liquid scintillation cocktail per sample in a Packard liquid scintillation counter.
A 1 mM propranolol stock solution is made up in distilled water and further diluted 1:20 in distilled water to give 50 μMpropranolol solution. Twenty μl of dilute stock solution is added to 3 tubes to determine nonspecific binding (yields a final concentration of 1 μM in a 1 ml assay).
The tissue homogenates are incubated for 15 min at 25 C with 1 nM 3H-DHA and varying drug concentrations. With each binding assay, triplicate samples are incubated with 1 μM ()-propranolol under identical conditions to determine nonspecific binding. The assay is stopped by vacuum filtration through Whatman GF/B filters which are washed 3 times with 5 ml of icecold 0.05Tris buffer, pH8.0. The filters are counted in 10 ml of Liquiscint scintillation cocktail.
A compound with β 2-selectivity would be less likely to produce cardiac effects but more likely to produce bronchiolar constriction. The test is used to determine the affinity of compounds for the β 2-adrenergic receptor subtype. A measure of receptor subtype selectivity can be determined when data are compared with those obtained in the β 1-adrenergic assay in rat cerebral cortex.
A 1 mM propranolol stock solution is made up in distilled water and further diluted 1:20 in distilled water to give 50 μMpropranolol solution. Twenty μl of dilute stock solution are added to 3 tubes to determine nonspecific binding (yielding a final concentration of 1 μM in a 1 ml assay).
The tissue homogenates are incubated for 15 min at 25 C with 1 nM 3H-DHA and varying drug concentrations. In each binding assay, triplicate samples are incubated with 1 μM ()-propranolol under identical conditions to determine nonspecific binding. The assay is stopped by vacuum filtration through Whatman GF/B filters which are washed 3 times with 5 ml of icecold 0.05 Tris buffer, pH8.0. The filters are counted in 10 ml of Liquiscint scintillation cocktail.
Chinese hamster ovary cells (CHO-K1 cells; CCL61, American Type Culture Collection, Rockville, Md., USA) were transfected with plasmid DNA for stable expression using the calcium phosphate precipitation method (Chen and Okayama 1987) as described for the rat A1 adenosine receptor (Freund et al. 1994). Positive clones were selected with 600 μg/ml of the neomycin analog G-418, and single clonal lines were isolated by limiting dilution. Expression of the receptor was verified by radioligand binding.
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