Muscarinic M3 receptors mediate total inositol phosphates accumulation in murine HSDM1C1 fibrosarcoma cells

Muscarinic receptors in murine fibrosarcoma HSDM1C1 cells were characterized using both radioligand binding and total inositol phosphates accumulation (IPs). Muscarinic agonists elicited a concentration-dependent enhancement of IPs accumulation with a maximum of 14-fold stimulation above basal level. The following potencies ( - log EC₅₀) were observed for the full agonists: ( + )-cis-dioxolane 5.4, oxotremorine-M 5.3, ( + )-muscarine 5.2 and carbachol 5.0. Bethanechol (4.1) and arecoline (5.0) were partial agonists, evoling 43 and 55%, respectively of the maximum level of stimulation to ( + )-cis-dioxolane, whereas pilocarpine and McN-A-343 were inactive as agonists (1 μmol/1-1 mmol/1) The apparent affinities for muscarinic antagonists ( - log K_B) estimated by Schild regression were: 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) 9.2, dicyclomine 7.0, pirenzepine 6.9, (±)-p-F-HHSiD (para-fluoro-hexahydro-siladifenidol) 7.0, AF-DX 116 6.2, methoctramine 5.7. In saturation binding studies using ³H]N-methylscopolamine a homogeneous population of sites was identified, with a density of 145 pmol/mg protein. In competition radioligand binding studies, the following apparent affinities ( - log K_i) were observed: 4-DAMP 9.7, dicyclomine 8.3, (±)-p-F-HHSiD 7.6, AF-DX 116 6.8, methoctramine 6.6 and gallamine 6.8. In binding studies all antagonists studied recognized a single population of sites, as judged by the Hill coefficients from the displacement isotherms. These data are consistent with HSDM1C1 cells expressing an apparent homogeneous muscarinic M₃ population that mediates a large level of total IPs accumulation. This clonal line may provide a useful model to further elucidate relationship between endogenous muscarinic M₃ receptor stimulation and IPs accumulation. Additionally, these studies illustrate the importance of characterizing muscarinic receptor subtypes by null methods when using biochemical responses in isolated cells.