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In an attempt to assess the structural requirements of hexahydro-sila-difenidol for potency and selectivity, a series of analogues modified in the amino group and the phenyl ring were investigated for their affinity to muscarinic M1- (rabbit vas deferens), Mr (guinea-pig atria) and Mr (guinea-pig ileum) receptors. All compounds were competitive antagonists in the three tissues. Their affinities to the three muscarinic receptor subtypes differed by more than two orders of magnitude and the observed receptor selectivities were not associated with high affinity. The pyrrolidino and hexamethyleneimino analogues, compounds substituted in the phenylring with a methoxy group or a chlorine atom as weil as p-fluoro-hexahydro-difenidol displayed the same affinity profile as the parent compound, hexahydro-sila-difenidol: M1 = M3 > M2 • A different selectivity patternwas observed for p-fluoro-hexahydro-sila-difenidol: M3 > M1 > M2 • This compound exhibited its highest affinity for M3-receptors in guinea-pig ileum (pA 2 = 7.84), intermediate affinity for M1-receptors in rabbit vas deferens (pA 2 = 6.68) and lowest affinity for the Mrreceptors in guinea-pig atria (pA 2 = 6.01). This receptor selectivity profile of p-fluoro-hexahydro-sila-difenidol was confirmed in ganglia (M1), atria (M2 ) and ileum (M 3 ) of the rat. Furthermore, dose ratios obtained with either pirenzepine (Mt) or hexahydrosila- difenidol (M2 and M3) and the p-fluoro analogue used in combination suggested that the antagonism was additive, implying mutual competition with a single population of muscarinic receptor subtypes. These results indicate that p-fluoro-hexahydro-sila-difenidol represents a valuable tool for characterization of muscarinic receptor subtypes.
Five different musearlnie receptor subtypes ean be distinguished by the differenees in their amino aeid sequence, the eoupled signal transduetion system, pharmaeologieal binding properties and aetivation of ionie fluxes. The present study served to eharaeterize the binding profile of musearlnie receptors in human eolon eareinoma eells (HT-29) using seleetive musearlnie antagonists. The affinities of the compounds were eompared with their poteney to inhibit cholinergieally-aetivated phosphoinositide metabolism. Pirenzepine displaced [\(^3\)H]N-methyl-scopolamine binding and inhibited inositolphosphate (IP) release with potencies typieal of those of non-M\(_1\) receptors. The M\(_3\) subtype-selective antagonists sila-hexocyelium and hexahydro-sila-difenidol bad high affinity to the musearlnie reeeptors in HT-29 cells (K0 = 3.1 nM and 27 nM, respectively) and inhibited IP release at nanomolar concentrations. The M\(_2\) receptor antagonists, AF-DX 116 and methoctramine, had low antimusearinic poteneies. Our results demonstrate that HT-29 human colon earcinoma cells contain an apparently pure population of M\(_3\) receptors. These cells could serve as a model system for further investigations coneerning regulatory and signal transduction mechanisms associated with glandular muscarinic M\(_3\) receptors.