In clinical Urology, muscarinic drug therapy with both agonists (bethanechol) and antagonists (oxybutynin) is used to a considerable extent. However, the muscarinic receptors in the bladder have not been unequivocally determined at the subtype level. This is an important distinction since it appears that the cellular response to neuronally released acetylcholine can be either excitatory or inhibitory depending on the muscarinic receptor subtype stimulated. On the basis of the action of antagonist 3 muscarinic receptor subtypes (designated by upper case """"""""M"""""""", M1 - M3) can be defined although none of the antagonists yet studied is highly selective for one receptor subtype over all of the others. Therefore previous pharmacological characterization of muscarinic subtypes represents the composite properties of a heterogenous mixture of receptor subtypes. Two recent, interrelated advances have made it possible to determine the density, cellular location and physiological regulation of muscarinic receptor subtypes: the cloning of genes for muscarinic receptor subtypes and development of subtype specific antibodies. The molecular biology approach has resulted in the cloning of a family of muscarinic genes which share the dame proposed overall structure and a large degree of protein sequence homology. Five muscarinic receptors (designated by lower case """"""""m"""""""", m1-15) are known and there are indications that more may exist. Two laboratories have generated antibodies that specifically recognize muscarinic receptor subtypes Both of these antibodies are available for the studies proposed and experiments are described for generation of other (m3, m4...) subtype specific antibodies. In addition cDNA clones for muscarinic receptor subtypes m1-m4 are available in this laboratory. A combination of subtype specific immunoprecipitation of solubilized receptors, analysis of receptor G-protein interactions, solution hybridization and in situ hybridization will be used to characterize muscarinic receptor subtypes in normal, adult bladders of rat, rabbit and human. In addition these techniques will be used to study the regulation if bladder muscarinic receptor subtype following chronic drug treatments and physiological intervention. These studies should provide important insights into the mechanism of cholinergic mediated bladder emptying and could form the foundation for development of muscarinic drugs with selective effects on the bladder. In addition these studies will determine the dynamics of muscarinic receptor subtype regulation in animal models and should provide correlates of chronic clinical use of muscarinic agonists and antagonists and bladder hyper reflexia.
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