Characterization of Muscarinic Receptors in the Human Bladder Mucosa: Direct Quantification of Subtypes Using 4-DAMP Mustard

Muscarinic Receptor Binding of the Novel Radioligand, [3H]Imidafenacin in the Human Bladder and Parotid Gland

“…Furthermore, our previous study with M 1 -M 5 -receptor knockout mice revealed that expression of the M 3 subtype was higher in the salivary gland than in the bladder (19 (20). Consistent with these findings, the predominance of the M 3 receptor in the human parotid gland was demonstrated directly by a radioligand binding assay using N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), an irreversible inactivating agent of the M 3 subtype (14). Since imidafenacin exhibited greater selectivity for the M 3 than M 2 subtype in CHO-K1 cells expressing human muscarinic receptors (4), the high affinity of [ 3 H]imidafenacin for muscarinic receptors in the human parotid gland reflects M 3 -subtype selectivity.…”

“…We previously demonstrated that imidafenacin bound muscarinic receptors in the human 4), indicating a close correlation between the binding affinity of imidafenacin to bladder muscarinic receptors and its pharmacological effects in the bladder. Previous studies (2,12,14) showed that muscarinic receptors were present on not only detrusor but also urothelium, a epithelial lining of the urinary tract. The current study measured specific [ 3 H]imidafenacin binding in the human detrusor including the urothelium.…”

“…They were immediately transported to the laboratory, and the adventitia and connecting fatty tissues were removed. All specimens of the human normal parotid gland were taken from areas macroscopically free of tumors (14). They were then stored at −80°C until used.…”

Fesoterodine, Its Active Metabolite, and Tolterodine Bind Selectively to Muscarinic Receptors in Human Bladder Mucosa and Detrusor Muscle

“…Furthermore, the predominance of the M 3 receptor in the human parotid gland was demonstrated directly by a radioligand binding assay using N-(2-chloroethyl)-4-piperidinyldiphenylacetate (4-DAMP mustard), an irreversible inactivating agent of the M 3 subtype. 11 The significantly greater affinity of fesoterodine and 5-HMT in the bladder compared with the parotid gland could be attributable to the greater density of M 2 than M 3 receptors in the bladder tissue. Also, it must be remembered that such in vitro receptor binding of fesoterodine could be significantly influenced by pharmacokinetics such as intestinal absorption, distribution, metabolism, and elimination after systemic administration, such as has been reported for imidafenacin.…”

“…11,12 The tissues were carefully minced with a pair of scissors and homogenized using the Kinematica Polytron homogenizer in 19 volumes of ice-cold 30 mM Na þ /HEPES buffer (pH 7.5). The homogenates were then centrifuged at 40,000g for 20 minutes.…”