Subtypes of muscarinic cholinergic receptors have been described using both pharmacological and molecular biological tools. It appears that there are at least three receptors identifiable with drugs while the genes for five muscarinic receptors have been cloned. Thus, the pharmacological tools in current use appear inadequate to identify definitively a given subtype in a tissue expressing multiple subtypes. Thus, we have developed a set of antibodies selective for each of the molecularly defined subtypes of muscarinic receptor. These antibodies will be used to examine the regulation of muscarinic receptors in rats as well as in clonal cell lines maintained in culture. In addition to these tools to identify selectively a given subtype, we will utilize cells that normally do not express muscarinic receptors but have been transfected stabily with cDNA encoding a given genetically defined subtype of muscarinic receptor.
The specific aims of the project are: 1. To characterize each of the known subtypes of muscarinic receptors utilizing cell lines expressing only a single genetically defined subtype. Several radioligands will be employed to determine if any can be utilized as a """"""""selective"""""""" radioligand under precisely defined conditions. The second. messenger systems associated with each subtype will be examined. 2. To examine the regulation of each of the subtypes of muscarinic receptors In cell lines of """"""""natural"""""""" origin. The phenomena of desensitization and down-regulation will be examined for each receptor subtype. The regulation by protein kinase C will be examined. Potential phosphorylation or palmitoylation of the receptor(s) will be studied. The identity of the G protein(s) to which the receptor(s) couple will be determined. The effects of various hormones on receptor expression will be studied. The possibility that mRNA encoding a given subtype is regulated will be examined. 3. To examine the regulation of subtypes of muscarinic receptors In rats. The effects of chronic administration of an antagonist (e.g. atropine), a cholinesterase inhibitor (e.g. tetrahydroaminoacridine), and a direct acting agonist (e.g. oxotremorine) on the expression of each of the subtypes will be examined using antibodies selective for each subtype. Since muscarinic receptors are involved in many aspects of normal and abnormal physiology and behavior, drugs affecting these receptors could be clinically useful. Up till now, however, side effects have limited the usefulness of such agents; but with a fuller understanding of the subtypes of muscarinic receptors it may be possible to target specific subtypes with drugs to ameliorate the symptoms of, for example, Alzheimers disease, Parkinson's disease, peptic ulcer, and hypertension.
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