Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

Abstract

High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands 3H-N-methylscopolamine (3H-NMS), 3H-quinuclidinyl benzilate (3H-QNB), and 3H-pirenzepine. 3H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, 3H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by 3H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for 3H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure. In conclusion, both M1 and M2 muscarinic receptor subtypes and an adenylate cyclase system linked to at least some of these receptors are present as intrinsic components of myelin. The possibility that some of these muscarinic receptors may be involved in regulation of phosphinositide metabolism and the protein kinase activities of myelin is considered.

Calcium signaling in cultured rat oligodendrocytes

“…Receptor-mediated Cai responses were observed in both cultured and acutely dissociated P21 OLGs, although interpretation of the results from the latter experiments may be limited by the presence of 443% contaminating cells. Nonetheless, these results provide further evidence for the existence of neurotransmitter signal transduction systems in OLGs and myelin, as first suggested by the work of Ritchie et al (1987) and Larocca et al (1987b) based on receptor binding and phosphoinositide hydrolysis assays. For example, M, and M, muscarinic receptors linked to phosphoinositide hydrolysis and CAMP formation have been found in rat brain myelin (Larocca et al, 198713) and cultured OLGs, respectively (Cohen and Almazan, 1994).…”

“…For example, M, and M, muscarinic receptors linked to phosphoinositide hydrolysis and CAMP formation have been found in rat brain myelin (Larocca et al, 198713) and cultured OLGs, respectively (Cohen and Almazan, 1994). In addition, GTP-binding proteins have been reported to be present in myelin (Larocca et al, 1987a).…”

Identification of GTP‐ binding proteins in myelin and oligodendrocyte membranes

“…The indicated position of rus in Figure 7B was verified by subjecting this GTPlabeled electroblot to anti-ras antibody, which clearly visualized the major ras bands, although at a diminished tured oligodendrocytes. Not surprisingly, G, and G,, regulatory components of CAMP-dependent signal-transducing networks, are readily apparent in oligodendrocytes and, given the presence of muscarinic receptors and adenylate cyclase in the CNS myelin sheath (Larocca et al, 1987), the demonstration of these two G proteins in association with highly purified myelin membranes Although the function of G,, protein may overlap that of other G proteins (Spiegel, 1987), there are indications that it may regulate ion channels (Freissmuth et al, 1989), in addition to other functions it might have. Since not all cell types possess this G protein (Gilman, 1987), we think it is significant that oligodendrocytes do and that the association of this protein with myelin membranes persists despite numerous procedures designed to rid myelin of adventitiously associated cellular elements.…”

Acetylcholine agonists stimulate mitogen-activated protein kinase in oligodendrocyte progenitors by muscarinic receptors

“…Earlier studies demonstrated the presence of m1 and m2 muscarinic acetylcholine receptors (mAChRs) in purified myelin isolated from adult rat brain (Larocca et al, 1987b). These receptors were found to couple to phosphoinositide hydrolysis and inhibition of adenylyl cyclase (Larocca et al, 1987a;Khan and Morell, 1988).…”