REGULATION OF PANCREATIC MUSCARINIC RECEPTORS IN VITRO

提出的研究将探讨的分子结构

The proposed research will probe the molecular structure of muscarinic acetylcholine receptors in the pancreas and lacrimal and parotid glands and define the mechanisms involved in modulation of receptor densities and affinity for agonists. These studies will enlarge understanding of the cellular processes responsible for control of neurotransmitter receptors in epithelia a goal of particular importance as dysfunction in these mechanisms is implicated in the pathology of pancreatitis and cystic fibrosis. To investigate structural characteristics of muscinic receptors, acini prepared from these organs will be labeled with (3H) propylbenzilycholine mustard (PrBCM), an irreversible cholinergic antagonist. After labelling, plasma membranes will be prepared and treated with neuraminidase, endoglycosidase F, and proteases and then solubilized and electrophoresed. Shifts in the electrophoretic mobility of labelled receptor protein induced by these enzymes will reveal the contribution of sialic acid and total glycosyl residues to the apparent molecular size of the muscarinic receptor as well as the size of the receptor domains on the extracellular and cytoplasmic sides of the membrane bilayer. Regulation of muscarinic receptor population size and affinity for agonists also will be examined by investigation of the effects of chronic exposure of cultured acini to acetyl-choline analogues. The subcellular pathways and organelles involved in receptor biosynthesis and degradation will be probed using the reversible muscarinic antagonists, (3H)-N- methylscopolamine and (3H)-quinuclidinyl benzilate and specific inhibitors such as methylamine, monensine, swainsonine, cycloheximide, and tunicamycin. The possible involvement of protein phosphorylation in muscarinic receptor desensitization and down regulation also will be examined through the use of compounds such as phorbol esters, which directly activate protein kinase in the acinar cell. Effects of phosphorylating conditions on the electrophoretic mobility and isoelectric point of solubilized muscarinic receptors also will be examined, utilizing (3H) PrBCM as a tag for the receptor molecule. Additionally, the ability to manipulate the size of the acinar muscarinic receptor population in culture will allow the direct determination of the stoichiometric relationship between the numbers of functional receptors and secretory responsiveness. The proposed studies will address fundamental questions regarding the mechanisms by which exocrine gland secretory cells adjust to varying intensity and duration of neurohumoral stimulation.