The role of M2 muscarinic receptor subtypes in mediating contraction of the pig bladder base after cyclic adenosine monophosphate elevation and/or selective M3 inactivation.

PURPOSE

In the bladder body M2 muscarinic receptors predominate but a smaller population of M3 receptors mediates direct detrusor contraction. M2 receptors have an indirect role by inhibiting cyclic adenosine monophosphate mediated relaxation in the bladder body. We investigated whether a similar mechanism also exists in the bladder base.

METHODS

Experiments were performed on pig detrusor muscle. In receptor binding studies using l-quinuclidinyl [phenyl-4-(3)H] benzilate ([(3)H]QNB) (NEN Life Science Products, Inc., Boston, Massachusetts) displacement experiments were performed with subtype selective antagonists to determine the M2-to-M3 receptor ratio. In functional studies the affinity of these antagonists against carbachol induced contractions was calculated in normal tissues and in tissues after cyclic adenosine monophosphate elevation by pre-contraction with KCl and relaxation with isoprenaline, and/or selective M3 inactivation by incubation with 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) mustard (Sigma Chemical Co., St. Louis, Missouri) in the presence of methoctramine (Sigma Chemical Co.) to protect M2 receptors.

RESULTS

In saturation binding studies receptor density was 130.5 of fmol./mg. protein and the dissociation constant for [(3)H]QNB was 0.27 nM. Displacement of [(3)H]QNB by the M3 selective antagonist 4-DAMP and the M2 antagonist methoctramine indicated an M2-to-M3 ratio of about 3:1. In functional studies 4-DAMP and methoctramine caused competitive antagonism of responses with affinity values of 9.5 and 6.3 in normal tissues, and 9.3 and 6.1, respectively, in cyclic adenosine monophosphate elevated tissues, suggesting the involvement of M3 receptors only. In tissues in which M3 receptors were inactivated and cyclic adenosine monophosphate levels were elevated the affinity of 4-DAMP was significantly reduced to 8.5 but that of methoctramine was significantly increased to 6.5, suggesting the involvement of M2 receptors.

CONCLUSIONS

The M3 subtype appears to mediate contraction of the normal and cyclic adenosine monophosphate elevated tissues of the bladder base. Involvement of M2 receptors was only noted after selective M3 inactivation and cyclic adenosine monophosphate elevation.