Abstract
We investigated the coupling of muscarinic receptor (M) subtypes to phosphoinositide hydrolysis in ileum and urinary bladder using muscarinic receptor knockout mice. In urinary bladder from wild-type mice, the muscarinic agonist oxotremorine-M, elicited a robust phosphoinositide response characterized by an EC50 value of 0.22 μM and a maximal response (Emax) of 32.8% conversion of [3H]inositol-labeled phosphoinositides into [3H]inositol phosphates. A similar response was observed in urinary bladder from M2 knockout mice, whereas no measurable response was observed in urinary bladder from M3 and M2/M3 knockout mice. In ilea from wild-type and M2 knockout mice, substantial phosphoinositide responses to oxotremorine-M were measured, characterized by EC50 values of 0.37 and 0.52 μM and Emax values of 35.8 and 34.7%, respectively. Oxotremorine-M also elicited phosphoinositide hydrolysis in ilea from M3 and M2/M3 knockout mice, although these responses were less sensitive (EC50 values of 1.6 and 1.4 μM; Emax values of 31.2 and 20.8%, respectively). The response in ileum from the M2/M3 knockout was significantly smaller than that from the M3 knockout. The muscarinic phosphoinositide response in ilea from M2/M3 knockout mice originated in the smooth muscle and exhibited a profile for competitive antagonism consistent with an M1 mechanism. These data suggest a major role for the M3 receptor in eliciting phosphoinositide hydrolysis in the ileum and urinary bladder and minor roles for the M1 and M2 in ileum.
Footnotes
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This work was supported by Pfizer (to F.J.E.), National Institutes of Health Grant NS 30882 (to F.J.E.), a grant from Pharmacia (to M.M.) and the Detrol LA Research Grant Program from Pfizer (to M.M.), the Industrial Technology Research Grant 02A09001a from The New Energy and Industrial Technology Development Organization (NEDO) of Japan (to M.M.), and Grant-in-aid for Scientific Research on Priority Areas 16067101 from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to M.M.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.103093.
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ABBREVIATIONS: M, muscarinic receptor; KO, knockout; KRB, Krebs' Ringer Bicarbonate; AF-DX 116, [[2-[(diethylamino) methyl]-1-piperidinyl]-acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]-benzodiazepine-6-one; [3H]NMS, [3H]N-methylscopolamine; PLC-β, phospholipase-Cβ.
- Received February 16, 2006.
- Accepted May 3, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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