Development of an antiserum against m3 muscarinic receptors: distribution of m3 receptors in rat tissues and clonal cell lines

Abstract

A synthetic oligopeptide (QCDKRKRRKQQYQQRQSV) corresponding to a carboxyl-terminal sequence of the rat m3 receptor (amino acids 561-578) was coupled to carrier proteins and used to generate a polyclonal antiserum. This serum selectively immunoprecipitates at least 90% of the m3 receptors expressed by A9 cells transfected with the cDNA encoding the m3 muscarinic receptor but does not precipitate receptors from cells transfected with cDNA encoding m1, m2, m4, or m5 receptors. Using this m3 antiserum, the density of m3 receptors in various regions of rat brain was quantified. Areas expressing the highest density of m3 receptors are the cortex, hippocampus, striatum, and olfactory tubercle, with 232 fmol/mg, 197 fmol/mg, 140 fmol/mg, and 130 fmol/mg, respectively. Hindbrain regions (i.e., cerebellum, thalamus/hypothalamus, and pons/medulla) expressed fewer m3 receptors, both as a percentage of total muscarinic receptors (5-6%) and in terms of absolute receptor density (12-70 fmol/mg). A panel of subtype-selective antisera (m1, m2, and m3) was used to determine receptor distribution in several peripheral tissues of the rat (lung, ileum, and bladder). The m2 receptor subtype constitutes the majority of total receptors in the bladder (86%), lung (91%), and ileum (69%). The m3 receptor was found at lower densities in these tissues (5-11%), whereas the m1 receptor is present in highest amounts in the ileum (17%). Human clonal cell lines, in which regulation of muscarinic receptors has been commonly studied, were also examined. The SK-N-SH neuroblastoma line, which has been reported to express M3 receptors, on the basis of pharmacology and molecular size, was found to express a mixture of subtypes (m1 = 31%, m2 = 21%, m3 = 43%). Interestingly, SH-SY-5Y and SH-IN cells, both derived from SK-N-SH cells, exhibit predominantly m3 receptors (74% for SH-SY-5Y; 58% for SH-IN), with lower levels of m1 and m2 receptors (5% and 8% for SH-SY-5Y; 4% and 23% for SH-IN, respectively.) Another commonly studied cell line, 132-1-N1 astrocytoma cells, reportedly expressing M3 receptors, based upon mRNA measurements and second messenger linkage, also expresses a predominance of m3 receptors (91% of total). This m3-selective antiserum should prove useful not only for localizing and quantifying m3 muscarinic receptors but also for examining mechanisms involved in the regulation of receptor expression in human tissues or animal models of disease, as well as in cell culture.