![]() Saturation Experimentsĥ0 μl 3H-prazosin (12 concentrations, 5 × 10 −11–5 × 10 –9 M)ĥ0 μl incubation buffer non-specific binding:ĥ0 μl 3H-prazosin (4 concentrations, 5 × 10 −11– × 10 −9 M)ĥ0 μl 3H-prazosin (1 constant concentration, 2–3 × 10 –10 M)ĥ0 μl incubation buffer without or with non-labeled test drug (15 concentrations, 10 −10–10 −3 M) The total volume of each incubation sample is 200 μl (microtiter plates). Experimental Courseįor each concentration samples are prepared in triplicate. After homogenization by Ultra-Turrax, the membrane suspension is stirred under cooling for 20–30 min until the start of the experiment. The pellets are resuspended in a volume of ice-cold rinse buffer, yielding a membrane suspension with a protein content of 1.0–1.5 mg/ml. with bovine serum albumin as a standard.Īt the day of the experiment the required volume of the membrane suspension is slowly thawed and centrifuged at 40,000 g (4 ☌) for 20 min. ![]() Protein content of the membrane suspension is determined according to the method of Lowry et al. The membrane suspension is immediately stored in aliquots of 5–20 ml at −77 ☌. ![]() The final pellets are dissolved (by Ultra-Turrax) in preparation buffer B, corresponding to 1 g ventricle wet weight/4 ml buffer. 300 ml preparation buffer B, homogenized by Ultra-Turrax and centrifuged as before. The resulting pellets are resuspended in approx. The pellets are discarded the supernatant is collected, and centrifuged again at 40,000 g for 20 min. Ventricles are homogenized by Ultra-Turrax (1 g tissue/ 20 ml preparation buffer A), the homogenate is filtered through gauze, and centrifuged at 2000 g (4 ☌) for 10 min. 30 g from 40 rats) are minced with a scalpel into 2–3 mm pieces. After removal of the atria, ventricles (approx. Male Sprague–Dawley rats (200–300 g) are sacrificed by decapitation and the dissected hearts are placed in ice-cold preparation bufferA. The assay is used to evaluate the concentrationbinding characteristics of drugs atria the α 1-adrenoceptor. Thus, the lower the concentration range of the test drug, in which the competition reaction occurs, the more potent is the test drug. If the test drug exhibits any affinity to α-adrenoceptors, it is able to compete with the radioligand for receptor binding äsites. A constant concentration of the radioligand 3H-prazosin (0.2–0.3 nM) is incubated with increasing concentrations of a non-labeled test drug (0.1 nM–1 mM) in the presence of plasma membranes from rat heart ventricles. The α-adrenoreceptor population of plasma membranes from rat heart ventricles consists only of the α1-adrenoreceptor subtype. In the CNS, the activation of α 1-adrenoceptors results in depolarization and increased neuronal firing rate. Receptor activation mediates a variety of functions, including contraction of smooth muscle, cardiac stimulation, cellular proliferation and activation of hepatic gluconeogenesis and glycolysis. 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. A.1.1 In Vitro Methods A.1.1.1 α 1-Adrenoreceptor Binding PURPOSE AND RATIONALE
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