Characterization of muscarinic receptor subtypes in pig airways: radioligand binding and northern blotting studies

This study was undertaken to characterize the muscarinic receptor subtypes present in adult pig peripheral lung and airway smooth muscle. The binding of the nonselective muscarinic antagonist [N-methyl-3H]scopolamine ([3H]NMS) to pig airways showed a single class of binding sites with a maximum density of 172 and 450 fmol/mg protein in peripheral lung and airway smooth muscle, respectively. Unlike [3H]NMS, the M1-selective antagonist, [3H]telenzepine, recognized two populations of binding sites in peripheral lung. Approximately 14% of total [3H]telenzepine binding sites displayed high affinity [dissociation constant (Kd) = 0.95 nM], whereas the remaining sites showed low affinity (Kd = 14.2 nM). The high- and the low-affinity [3H]telenzepine binding sites displayed the pharmacological profile of M1 and M2 receptors, respectively. Heterogeneity of pig airways muscarinic receptor was also revealed by competitive binding experiments against [3H]NMS with the M2-selective antagonist methoctramine. This compound recognized 70 and 90% of total receptors with high affinity in airway smooth muscle (Ki = 4.44 nM) and peripheral lung (Ki = 9.82 nM), respectively. This result suggests that the dominant muscarinic receptor in pig airways is of the M2 subtype. Northern blot analysis demonstrated the presence of m1 and m2 mRNAs transcripts in peripheral lung and m2 and m3 mRNAs in airway smooth muscle with no evidence for m4 mRNA.