Presence of m3 subtype muscarinic acetylcholine receptors and receptor- mediated increases in the cytoplasmic concentration of Ca2+ in Jurkat, a human leukemic helper T lymphocyte line

T Kaneda, Y Kitamura and Y Nomura

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.

Recent studies have demonstrated the presence and the regulatory function of several neurotransmitters in the immune system. In the present study, we examined the presence of acetylcholine receptors, using pharmacological and molecular biological assays, and their transmembrane control and functions, using a biochemical assay, in a cloned human leukemic helper T lymphoma cell line, Jurkat. Several muscarinic agonists, such as acetylcholine, carbachol, muscarine, and oxotremorine-M (Oxo-M), at 100 microM caused a transient elevation of the free cytosolic Ca2+ concentration ([Ca2+]i), in contrast to the tonic elevation of [Ca2+]i induced by 10 micrograms/ml phytohemagglutinin (PHA). It appeared that the elevation induced by Oxo- M, the most potent [Ca2+]i elevator, was more effectively inhibited by p-fluorohexahydrosiladifenidol hydrochloride (p-F-HHSiD) and 4- diphenylacetoxy-N-methylpiperidine methiodine than by pirenzepine and 11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H- pyrido[2,3-b] [1,4]benzodiazepine-6-one (AF-DX 116), suggesting that a pharmacological M3 subtype of muscarinic receptors is involved in the elevation of [Ca2+]i. Northern blot analysis showed that the m3 type of receptors are expressed in Jurkat cells. Scatchard analysis of [3H]quinuclidinyl benzilate binding to intact cells indicated a Kd of 14.1 nM and a Bmax of 45,370 binding sites/cell. [3H]Quinuclidinyl benzilate binding to cell membranes was also inhibited by p-F-HHSiD rather than by pirenzepine and AF-DX 116. Oxo-M induced formation of inositol trisphosphate, and 5'-O-(2-thio)diphosphate inhibited the formation. Cholera toxin treatment inhibited the PHA-induced [Ca2+]i rise but did not affect the Oxo-M-induced rise. Neither pertussis nor butulinus (type C) toxin affected the rise induced by Oxo-M or PHA. Thus, bacterial toxin-insensitive GTP-binding proteins seem to be involved in the Oxo-M-induced increase in [Ca2+]i. Treatment with 12-O- tetradecanoylphorbol 13-acetate abolished the Oxo-M-induced [Ca2+]i rise but did not affect that induced by PHA. m3 Muscarinic receptors thus appear to cause Ca2+ mobilization from intracellular stores via bacteria toxin-insensitive GTP-binding proteins, phospholipase C activation, and inositol trisphosphate formation in Jurkat cells. Protein kinase C seems to negatively modulate the m3 receptor system.

Volume 43, Issue 3, pp. 356-364, 03/01/1993
Copyright © 1993 by American Society for Pharmacology and Experimental Therapeutics

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