Characterization of prejunctional and postjunctional muscarinic receptors of the ascending reflex contraction in rat ileum

The ascending reflex contraction of the small intestine involves predominan tly cholinergic neurotransmission. The orally projecting neural excitatory pathway of the myenteric reflex was studied in an in vitro model of rat ile al segments. The contractile response elicited by aboral field stimulation was significantly inhibited by a range of muscarinic receptor antagonists. Methoctramine and tripitramine (both M-2 selective, pIC(50) = 9.3 and 8.8, respectively), darifenacin and hexahydrosiladifenidol (both M-3 selective, pIC(50) = 7.3 and 7.7, respectively), and pirenzepine (M-2 selective, pIC(5 0) = 7.0). In radioligand binding experiments on synaptosomal and smooth mu scle plasma membrane fractions, we examined whether prejunctional or postju nctional muscarinic receptors exist that could potentially contribute to th e reflex contraction. In the synaptosomal fraction, the muscarinic ligand [ H-3]N-methylscopolamine labeled a homogeneous population of receptors (Hill coefficient = 1) with a K-d value of 0.31 +/- 0.09 nM and a B-max value of 185 +/- 16.6 fmol/mg protein. The ratio of potency of subtype-selective mu scarinic receptor antagonists in competition studies was tripitramine (pK(i ) = 8.7 +/- 0.3) > 1/6 x methoctramine (pK(i) = 7.9 +/- 0.02) > 1/25 x dari fenacin (pK(i) = 7.3 +/- 0.2) > 1/316 x hexahydrosiladifenidol (pK(i) = 6.2 +/- 0.1) > 1/2511 x pirenzepine (pK(i) = 5.3 +/- 0.1). In the smooth muscl e plasma membrane fraction, the K-d value was 0.29 +/- 0.05 nM and the B-ma x value was 770 +/- 29 fmol/mg. The competition studies revealed a similar ratio of potency of the respective antagonists. These data suggest that mus carinic M-2 receptors, located at prejunctional and postjunctional sites, a re predominantly involved in the ascending reflex contraction.