ANTIBODIES TO A SYNTHETIC PEPTIDE CAN BE USED TO DISTINGUISH BETWEEN MUSCARINIC ACETYLCHOLINE-RECEPTOR BINDING-SITES IN BRAIN AND HEART

  • 1 September 1988
    • journal article
    • research article
    • Vol. 34  (3) , 327-333
Abstract
Since the reports elucidating the sequence of four subtypes of muscarinic cholinergic receptors appeared, it has been clear that pharmacological approaches to the study of subtypes of these receptors are inadequate to selectively detect one subtype in the presence of the others. One methodology that can provide more selective reagents with which to study these subtypes is immunology. Thus, using the information on the primary sequence of these receptors available in the literature, rabbits were injected with an oligopeptide, CRKIPKRPGSVHRTPSRQ, conjugated to keyhole limpet hemocyanin. This oligopeptide (m1 C-terminal peptide) corresponds to the 17-amino acid sequence of the carboxyl terminus of a rat m1 muscarinic receptor. This portion of the amino acid sequence of the muscarinic receptor protein has been shown to be unique to the m1 receptor and has not been found in the other subtypes of the receptor thus far sequenced. The antisera (anti-m1 antisera) had high titer against the m1 C-terminal peptide in a solid phase radioimmunoassay. The anti-m1 antisera were shown to immunoprecipitate [3H] quinuclidinyl benzilate ([3H]QNB) binding activity solubilized from rat forebrain. [3H]Pirenzepine ([3H]PZ) has been shown to interact with a subset of [3H]QNB binding sites in forebrain and heart. The anti-m1 antisera were shown to immunoprecipitate [3]PZ binding sites in cerebral cortex, hippocampus, and corpus striatum, areas believed to be rich in the m1 subtype of the muscarinic receptor. Although [3H]PZ binding activity was present in receptor preparations solubilized from heart, neither [3H]PZ- nor [3H] QNB-binding activities could be immunoprecipitated from this tissue using the anti-m1 antisera. A monoclonal antibody raised against the porcine atrial muscarinic receptor was shown to immunoprecipitate both [3H]PZ- and [3H]QNB-binding activities solubilized from rat heart, but only [3H]QNB-binding activity could be immunoprecipitated from forebrain using this antibody. Immunoprecipitation of [3H]PZ- and [3H]QNB-binding activity by anti-m1 antisera could be inhibited by the m1 C-terminal peptide. Peptides corresponding to the C-terminal portions of the rat m3 and m4 muscarinic receptor were not inhibitory in the immunoprecipitation assay. This study provides further evidence for subtypes of muscarinic receptors in rat tissues and supports the hypothesis that receptor subtypes defined using PZ can be further subclassified on the basis of differences in primary structure. The data indicate that [3H]PZ binds not only to the m1 muscarinic receptor but also to other muscarinic receptor subtypes in solubilized preparations from rat brain and heart. Therefore, [3H]PZ binding cannot be assumed to measure m1 receptors exclusively in rat tissues. Additionally, the results obtained in this study indicate that the approach of developing antibodies specifically directed toward a single subtype of muscarinic receptor, by using peptides that are uniquely represented in a given subtype, will be fruitful.