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Title: Regulation of muscarinic acetylcholine receptors in the 1321N1 human astrocytoma cell line

The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of 1321N1 human astrocytoma cells was studied. Binding was studied in both intact cells and cell lysates. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand ({sup 3}H)QNB or the hydrophilic radioligand ({sup 3}H)NMS. In contrast, full agonists exhibited markedly lower apparent affinities in intact cells with ({sup 3}H)QNB than with ({sup 3}H)NMS. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptor subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs ({sup 3}H)QNB were markedly higher. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. In other studies, the effects of the protein kinase C activator phorbol 12-myristate, 13-acetate (PMA) on muscarinic receptor downregulation and internalization in 1321N1 cells were determined. PMA alone did not induce muscarinic receptor downregulation but instead decreased both the rate and final extent of downregulation induced by the agonist carbachol. The specificity of other protein kinase C activators for inhibiting carbachol-induced downregulation indicated involvement of protein kinase C. Furthermore,more » the protein kinase C inhibitor staurosporine prevented the inhibitory effect of PMA on downregulation. However, staurosporine did not inhibit agonist-induced downregulation.« less
Authors:
Publication Date:
OSTI Identifier:
5545274
Resource Type:
Miscellaneous
Resource Relation:
Other Information: Thesis (Ph. D.)
Publisher:
Columbia, MO (US); Univ. of Missouri
Research Org:
Missouri Univ., Columbia, MO (United States)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; ACETYLCHOLINE; RECEPTORS; PHORBOL ESTERS; BIOLOGICAL EFFECTS; AFFINITY; BIOCHEMICAL REACTION KINETICS; INHIBITION; LIGANDS; MAN; PARASYMPATHOLYTICS; PHOSPHOTRANSFERASES; TRACER TECHNIQUES; TRITIUM COMPOUNDS; TUMOR CELLS; AMINES; AMMONIUM COMPOUNDS; ANIMAL CELLS; ANIMALS; AUTONOMIC NERVOUS SYSTEM AGENTS; CARCINOGENS; DRUGS; ENZYMES; ESTERS; HYDROGEN COMPOUNDS; ISOTOPE APPLICATIONS; KINETICS; MAMMALS; MEMBRANE PROTEINS; NEUROREGULATORS; ORGANIC COMPOUNDS; PARASYMPATHOMIMETICS; PHOSPHORUS-GROUP TRANSFERASES; PRIMATES; PROTEINS; QUATERNARY COMPOUNDS; REACTION KINETICS; TRANSFERASES; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques; 560300 -- Chemicals Metabolism & Toxicology