Muscarinic cholinergic receptors (MR) reduce intracellular cyclic AMP levels through both the inhibition of adenylate cyclase (AC) and the activation of phosphodiesterase (PDE). The mechanisms whereby these MR-mediated events occur will be examined in model cell lines that: (1) only express the MR-AC mechanism (NG108-15 neuroblastoma x glioma cells); (2) only express the MR-PDE mechanism (1321N1 human astrocytoma cells); and (3) express both mechanisms (WI-38 fibroblasts). Islet activating protein (IAP) will be purified from the culture medium of Bordetella pertussis and used to 32P-ADP ribosylate and inactivate the guanine nucleotide regulatory protein (Ni) that is involved in coupling of inhibitory receptors to AC. By examining the effects of IAP on MR responsiveness of these three cell lines and by identifying the peptide substrates for IAP-induced 32P-ADP ribosylation, we will establish the selectivity of IAP action and provide information concerning any role of Ni in the MR-PDE mechanism. The role of Ni in the GTP-sensitive interaction of agonists and antagonists with MR will be examined by identifying solubilized ligand MR Ni complexes on molecular sieves. A reconstitution system will be established in which purified Ni will be incorporated into membranes in which Ni has been inactivated with N-ethylmaleimide. Reconstitution experiments also will be used to investigate the status of Ni in 1321N1 membranes. MR-stimulated 45Ca++ influx will be studied in 1321N1 cells under a variety of conditions with the goal of comparing this phenomenon to MR-mediated activation of PDE. The mechanisms of cholinergic agonist-induced desensitization of the MR-PDE effect and cholinergic agonist-induced enhancement of the responsiveness of AC to stimulatory agonists will be examined. Experiments will be carried out to determine whether the two MR-mediated effects in WI-38 fibroblasts occur through a common receptor population or two receptor subtypes. The completion of these studies should help place in perspective the mechanism of MR-mediated inhibition of AC with the more classical MR-mediated mechanisms that occur through coupling to a Ca++ channel, should help identify the role of putative MR subtypes in these two mechanisms, and should increase our understanding of the molecular basis of cholinergic action. Since cholinergic dysfunction has received increased emphasis in regard to brain disease and since knowledge of MR subtypes and the mechanism of their modification of cellular function in peripheral tissues has important implications in rational drug therapy, studies such as these have significant health relevance.
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