Evidence is presented for linkage of opioid receptors directly to the stimulatory G protein (guanine nucleotide-binding protein), Gs, in addition to the generally accepted linkage to the inhibitory and "other" G proteins, G; and G., in F-11 (neuroblastoma-dorsal root ganglion neuron) hybrid cells. Treatment of intact F-11 cells with cholera toxin decreased specific binding of the opioid agonist [D-Ala2, D-Leu5jenkephalin to F-11 cell membranes by 35%, with the remaining binding retaining high affinity for agonist. Under these conditions cholera toxin influenced the a subunit of G. (Gsa) but had no effect on the a subunit of Gj/. (Gi/oa), based on ADP-ribosylation studies. Pertussis toxin treatment decreased high-affinity opioid agonist binding by about 50%; remaining binding was also of high affinity, even though pertussis toxin had inactivated Gi/oa selectively and essentially completely. Simultaneous treatment with both toxins had an additive effect, reducing specific binding by about 80%. While opioid agonists inhibited forskolin-stimulated adenylate cyclase activity of F-11 cells as expected, opioids also stimulated basal adenylate cyclase activity, indicative of interaction with G, as well as G;. Cholera toxin treatment attenuated opioidstimulation of basal adenylate cyclase, whereas pertussis toxin treatment enhanced stimulation. In contrast, inhibition by opioid of forskolin-stimulated activity was attenuated by pertussis toxin but not by cholera toxin. It is concluded that a subset of opioid receptors may be linked directly to G. and thereby mediate stimulation of adenylate cyclase. This G,-adenylate cyclase interaction is postulated to be responsible for the novel excitatory electrophysiologic responses to opioids found in our previous studies of sensory neurons and F-11 cells.Opioid receptors are generally known to be linked to pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G proteins) that mediate inhibition (Gi proteins) of neuronal activity and (via Gi adenylate cyclase (AC) (1-6). However, opioid agonists, at concentrations lower than that required to inhibit neuronal activity, elicit excitatory electrophysiologic effects on dorsal root ganglion (DRG) neurons in culture (3,7,8). Considerable circumstantial evidence has implicated GS, a cholera toxin (CTX)-sensitive stimulatory G protein, and cAMP in these excitatory effects (3, 9). We have also found that for DRG neurons and DRG-spinal cord explants, in addition to the predicted inhibition of forskolin-stimulated AC activity by opioids, basal activity is stimulated by opioids (10) via a receptor-mediated process (11).In the present study we have utilized F-11 cells to obtain direct biochemical support for mediation of the dual excitatory/inhibitory effects of opioids by coupling of opioid receptors to both CTX-and PTX-sensitive G proteins. The F-11 cell line, a mouse N18TG2 neuroblastoma-rat primary DRG neuron hybrid, retains a number of properties of sensory (DRG) neurons (12, 13). Also, F-11 cells contain an opioid-i...

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.

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The subthalamic nucleus, a clinically important component of the extrapyramidal motor system, and a lateral area extending into the peduncle contain catecholamine terminals and dopamine receptors coupled to adenylate cyclase. In addition, dopamine agonists administered in vivo enhance glucose utilization in the region. Thus, neuronal function in this region is directly affected by dopamine and dopaminergic drugs.

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