M3 muscarinic receptors on murine HSDM1C1 cells: Further functional,regulatory, and receptor binding studies |
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Authors: | Dr. N. A. Sharif Z. P. To K. H. Wong R. E. Delmendo R. L. Whiting R. M. Eglen |
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Affiliation: | (1) Institute of Pharmacology, Syntex Discovery Research, 94304 Palo Alto, California;(2) Molecular Pharmacology Unit, Alcon Laboratories Inc. (R2-19), 6201 South Freeway, 76123 Forth Worth, Texas |
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Abstract: | In the present studies, the pharmacology and regulation of the functional muscarinic receptors on HSDM1C1 cells were probed using phosphoinositide (PI) turnover assays. In addition, the receptor binding of the putative M3-selective radioligand, [3H]4-DAMP, to cell homogenates was characterized. Carbachol (EC50=9 M), (+)muscarine (EC50=4.5 M) and cis-dioxolane (EC5=0.72 M) were full agonists which stimulated PI turnover by 13.3±1.0 fold above basal values. The potencies of numerous agonists in this assay system were relatively similar to their affinities in receptor binding assays. Exposure of HSDM1C1 cells to 10 nM–10 M muscarine during the last 24h of [3H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cisdioxolane affinity and maximal PI response induced by subsequent treatment with cis-dioxolane. pertussis toxin (5–2000 ng/ml) caused a partial reduction in the cis-dioxolane-induced PI turnover. Likewise, exposure of the HSDM1C1 cells to an active phorbol ester (TPA) resulted in a partial inhibition of the cis-dioxolane-induced (100 M) PI turnover. The half-maximal effect of TPA was produced at 1.8±0.3 nM. [3H]4-DAMP binding to cell homogenates was of high affinity (Kd=0.19±0.04 nM) and moderate capacity (Bmax=201±22 fmol/mg protein). The pharmacological specificity (4-DAMP>p-FHHSiD>dicyclomine>pirenzepine>methoctramine>AFDX-116 >gallamine) resembled that for [3H]NMS binding and correlated well with that observed for inhibition of PI turnover. These studies further support the identification of M3 receptors on HSDM1C1 cells. These receptors have been shown to be influenced by pertussis toxin, an active phorbol ester and to exhibit desensitization. |
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Keywords: | Muscarinic M3 receptors HSDM1C1 cells PI turnover [3H]4-DAMP binding Receptor binding |
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