The aims of this proposal are to seek out the pharmacological and neurophysiological functions of the M2 muscarinic receptor in the ileum, trachea and urinary bladder. It is known that the M3 muscarinic receptor elicits contraction in a variety of smooth muscles which respond to muscarinic agonists, yet the most abundant subtype of the muscarinic receptor is the M2, accounting for approximately 80% of the total density of muscarinic receptors in several smooth muscles. Surprisingly, the function of the M2 receptor in smooth muscle is essentially unknown. It is likely that the M2 receptor may cause a 'disinhibition of contraction' by preventing the relaxation elicited by other receptors which stimulate adenylate cyclase, like the beta-adrenergic receptor. Consequently, interactions between subtypes of the muscarinic receptor and other heterologous receptors will be investigated with respect to signaling mechanisms and contractility in smooth muscle. Part of the strategy for dissecting out the functional role of the M2 receptor in smooth muscle will involve the development of novel irreversible antagonists which inactivate M3 muscarinic receptors selectively. These agents should have widespread application as tools in a variety of neuropharmacological studies investigating the functional roles of subtypes of the muscarinic receptor. Moreover, the research described in this proposal could provide the basis for the development of more selective drugs for the treatment of paralytic ileus and glaucoma, and in determining how drugs used in psychiatry interfere with muscarinic cholinergic mechanisms. The specific experimental protocols are: 1) to characterize the muscarinic receptor binding properties of the irreversible muscarinic antagonist 4-DAMP mustard and to identify conditions where this antagonist blocks M3-stimulated phosphoinositide hydrolysis selectively without affecting M2-mediated inhibition of adenylate cyclase activity; 2) to develop new, more selective 4-DAMP mustard analogs; 3) to classify the subtypes of the muscarinic receptor mediating phosphoinositide hydrolysis and inhibition of adenylate cyclase activity in smooth muscle using subtype selective muscarinic antagonists; 4) to identify the subtypes of the muscarinic receptor present in smooth muscle using radioligand binding techniques; 5) to identify receptors which stimulate cyclic AMP accumulation in smooth muscle in a manner that is opposed by activation of the M2 muscarinic receptor; 6) to identify novel mechanisms of muscarinic receptor cross talk in smooth muscle; 7) to determine whether activation of M2 muscarinic receptors in smooth muscle prevents the relaxation mediated by the receptors identified in aim #2; and 8) to determine whether drugs of psychopharmacological interest interfere with muscarinic receptor signaling mechanisms in smooth muscle.
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