The selectivity of coupling of m1, m3, and m5 muscarinic receptors to activation of the neuronal type of nitric oxide synthase was investigated. Stimulation with the agonist carbachol of all three receptor subtypes expressed in Chinese hamster ovary cells resulted in a rapid and transient activation of the enzyme, as measured by stimulation of guanylate cyclase in reporter neuroblastoma cells. Carbachol was more potent and efficacious at m5 receptors than at the other two receptor subtypes. Stimulation of all three muscarinic receptors resulted in an increased concentration of intracellular calcium, with a time course that preceded activation of nitric oxide synthase. At each receptor subtype, there was a close relationship between the magnitude of the maximal calcium response and that of enzyme activation.

We investigated the coupling of the M2 muscarinic acetylcholine receptors expressed in Chinese hamster ovary cells to activation of neuronal nitric oxide (NO) synthase. Stimulation of guanylate cyclase activity in detector neuroblastoma cells was used as an indirect measure of the generation of NO in Chinese hamster ovary cells. The muscarinic agonist carbachol induced marked time- and concentration-dependent enhancement of the activity of NO synthase. Activation of neuronal NO synthase by M2 muscarinic receptors was associated with a small increase in the concentration of intracellular Ca²⁺. These data suggest the presence of alternate mechanisms of activation of neuronal NO synthase which might be operative in the absence of large changes in the concentration of cellular Ca²⁺. These findings help to understand the mechanisms of activation of NO synthase.

The first putative extracellular domains of both ml and ml muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y), and threonine (T). This triplet is repeated as LYTLYT in m2 receptors. However, it is repeated in a transposed fashion (LYTTYL) in the sequence of ml receptors. In this work we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity in determining the distinct differential drug-receptor interaction at the two receptor subtypes. Mutation of the LYTTYL sequence of ml receptors to the corresponding m2 receptor LYTLYT sequence, however, did not significantly change the binding affinity of the agonist carbachol or the affinity of the majority of a series of receptor antagonists which are able to discriminate between wild-type ml and m2 receptors. The reverse mutation at the m2 receptor also did not modify agonist affinity, but altered affinity of several receptor subtype-selective antagonists. The magnitude of affinity changes, however, was small, and the direction of these changes was opposite to what would be expected if the m2 receptor LYTLYT sequence were important for determining the binding profile of m2-receptor-selective antagonists. Our data suggest that the LYTTYL-LYTLYT sequence differences between ml and m2 muscarinic receptors are not important for determining receptor pharmacology.