PSYCHOTROPIC DRUGS & MUSCARINIC RECEPTOR TYPES

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In our initial studies, we found that the human neuroblastoma (SH-SY5Y) cell line consistently expresses a high density of mAChRs (about 220 fmol/mg protein or 25,000 receptors/cell). Most of the mAChRs in the SH-SY5Y cells showed high affinity for [3H]PZ, suggesting an M1 nature of these muscarinic receptors. To our knowledge, the SH-SY5Y cell line is the only cell line with high affinity [3H]PZ binding to_the mAChRs on the intact cells. The SH-SY5Y cells have a functional PI system and an adenylate cyclase system. The existence of these effector systems offers a unique opportunity to study the effector coupling mechanisms of the M1 receptors in a homogeneous neuronal cell line. Our initial data suggest that these M1 receptors are coupled to the PI system. In this proposal, we will further examine the pharmacological properties of the mAChRs in SY-SY5Y cells using several new selective muscarinic ligands and compare these data with a transfected B82 system which is thought to contain only one type of mAChr. i.e., M1. Furthermore, we will also examine the second messenger system coupled to the receptors in order to obtain a better understanding of the molecular basis for the function of the mAChRs in both cell lines. Although our initial pharmacological studies indicate that the predominant mAChRs on the SH-SY5Y cells are of the M1 type, since there could be more than one mAChR showing high affinity for PZ (40), an unambiguous definition of this receptor requires a knowledge of its primary structure by gene cloning and sequencing. However, this will only be done if the mAChRs in the SH-SY5Y cell line is unique. This study will yield clone(s) which express a constant population of M1 receptors with pharmacological and biological properties of the native M1 receptors. This study will provide in depth information on the functional mechanism of neural mAChRs and their genetic regulation. This knowledge will have importance in the drug treatment of CNS disorders with cholinergic deficits such as senile dementia of the Alzheimer's type (SDAT), Huntington's disease (HD) and Parkinson's disease(PD). Furthermore, these cell lines will be used as models for the development of selective drugs which act at a single type of mAChR thereby improving therapeutic effects while reducing side effects.