TY - JOUR
T1 - A rapid microtechnique for the estimation of muscarinic and nicotinic receptor binding parameters using 96-well filtration plates
AU - Gattu, Mahanandeeshwar
AU - Terry, Alvin V.
AU - Buccafusco, Jerry J.
N1 - Funding Information:
The authors appreciate the assistance of Mrs. Patricia Ryan for her excellent secretarial support in preparation of this manuscript and Le’Sean Davis for his technical assistance. This study was supported by the Department of Veterans Affairs Medical Research Service.
PY - 1995/12
Y1 - 1995/12
N2 - 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 presents 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.
AB - 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 presents 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.
KW - Filtration plate, 96-well
KW - Ligand binding
KW - Microtechnique
KW - Muscarinic receptor
KW - Nicotinic receptor
KW - [H]Cytisine
KW - [H]N-methyl scopolamine
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U2 - 10.1016/0165-0270(95)00100-X
DO - 10.1016/0165-0270(95)00100-X
M3 - Article
C2 - 8788056
AN - SCOPUS:0029623042
SN - 0165-0270
VL - 63
SP - 121
EP - 125
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 1-2
ER -