title: M3 muscarinic receptors on murine HSDM1C1 cells: further functional, regulatory, and receptor binding studies.
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 microM), (+)muscarine (EC50 = 4.5 microM) and cis-dioxolane (EC50 = 0.72 microM) 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 microM muscarine during the last 24h of [3H]myo-inositol-labeling resulted in a concentration-dependent reduction in the cis-dioxolane 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 microM) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Institute of Pharmacology, Syntex Discovery Research, Palo Alto, California 94304, USA.
authors.forename: N A
authors.initials: NA
authors.lastname: Sharif
authors.forename: Z P
authors.initials: ZP
authors.lastname: To
authors.forename: K H
authors.initials: KH
authors.lastname: Wong
authors.forename: R E
authors.initials: RE
authors.lastname: Delmendo
authors.forename: R L
authors.initials: RL
authors.lastname: Whiting
authors.forename: R M
authors.initials: RM
authors.lastname: Eglen
mesh_terms: D000818:Animals; D016014:Linear Models; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism*; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000378:metabolism / Q000502:physiology*; D014316:Tritium; D014407:Tumor Cells, Cultured
chemical_list: D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D014316:Tritium