A rapid microtechnique for the estimation of muscarinic and nicotinic receptor binding parameters using 96-well filtration plates.
A microtechnique for the assay of muscarinic and nicotinic receptor binding parameters employing a 96-well filtration multiscreen system has been developed. Utilizing rat cerebral cortex as the receptor source, saturation analysis of [3H]NMS binding revealed a mean Bmax and Kd value of 1.95 pmol/mg protein and 0.71 nM, respectively. In competition studies with atropine a Ki value of 1.67 nM was determined. Nicotinic receptor binding studies were also performed with this technique with [3H]Cytisine. The binding constants for both ligands correlated well with established literature values; however, in the nicotinic assays concentrations of ligand at 20 times the Kd were required to develop adequate number of counts. Compared to presently available receptor binding techniques, this newly developed system for muscarinic and nicotinic binding is very simple and convenient to use and offers the advantages of speed, accuracy and reproducibility. In addition, a large number of samples can be assayed. This report also present the first use of the 96-well filtration multiscreen system for the estimation of muscarinic and nicotinic receptor binding constants. This methodology should prove to be useful for determining the binding characteristics of potential cholinergic ligands.
Gattu
M
M
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta 30912, USA.
Terry
A V
AV
Buccafusco
J J
JJ
D000470:Alkaloids / Q000378:metabolism; D000818:Animals; D001392:Azocines; D001667:Binding, Competitive; D002540:Cerebral Cortex / Q000737:chemistry*; D005374:Filtration; D019832:N-Methylscopolamine; D010276:Parasympatholytics / Q000378:metabolism; D011807:Quinolizines; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000378:metabolism; D014316:Tritium / Q000378:metabolism; D000093843:Quinolizidine Alkaloids
D000470:Alkaloids; D001392:Azocines; D019832:N-Methylscopolamine; D010276:Parasympatholytics; D011807:Quinolizines; D011976:Receptors, Muscarinic; D011978:Receptors, Nicotinic; D012602:Scopolamine Derivatives; D014316:Tritium; C004712:cytisine; D000093843:Quinolizidine Alkaloids