SUMMARYIt is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M 1 -M 4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [ 3 H]Nmethylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (Ϫ)-eburnamonine on the M 2 and M 4 receptors and by brucine on the M 1 and M 3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M 1 , M 3 , and M 4 receptors, for pentylthio-TZTP on the M 2 receptors, and for arecoline on the M 3 receptors. (Ϫ)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M 2 receptors and for pilocarpine on the M 4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (Ϫ)-eburnamonine and pilocarpine and (Ϫ)-eburnamonine and oxotremorine-M on the M 2 receptors (25-and 7-fold increases in affinity, respectively) and between brucine and pentylthio-TZTP on the M 2 and brucine and carbachol on the M 1 receptors (8-fold increases in affinity). The discovery that it is possible to increase the affinity of muscarinic receptors for their agonists by allosteric modulators offers a new way to subtype-specific pharmacological enhancement of transmission at cholinergic (muscarinic) synapses.It has long been known that the affinity of muscarinic receptors for their agonists and antagonists can be diminished by compounds acting allosterically (for reviews, see Refs. 1-3). It was discovered, however, that the affinity of cardiac muscarinic receptors for the muscarinic antagonist NMS can be increased by the neuromuscular blocker alcuronium (4, 5). Subsequently, it was shown that the positive allosteric action of alcuronium on the binding of NMS is specific for the M 2 and M 4 muscarinic receptor subtypes (Ref.6, but see Refs. 7 and 8 for conflicting data concerning the M 3 and M 4 subtypes) and that it also applies to the binding ...

Studies on the metabolic pathway of the acetyl group for acetylcholine synthesis. Biochem. Phormucol. 29, 167-174. Gibson G. E.. Jope R., and Blass J. P. (1975) Decreased synthesis of acetylcholine accompanying impaired oxidation of pyruvic acid in rat brain minces. Biochem. J . 148, 17-23. Glover V. A. S. and Potter L. T. (1971) Purification and properties of choline acetyltransferase from ox brain striate nuclei. J . Nerrrochrm. 18, 571-580. Goldberg A. M. and Hanin I., eds (1976) Biology ofcholinergic Function. Raven Press, New York. Grewaal D. A. and Quastel J. H. (1973) Control of synthesis and release of radioactive acetylcholine in brain slices from the rat.

1. The synthesis of acetylcholine (ACh) has been measured in homogenates of the sciatic nerve, normal and denervated extensor digitorum longus (e.d.l.) muscles, and central (innervated) and peripheral (non-innervated) parts of the diaphragm of the rat. The synthesis proceeded under conditions accepted as optimal for the activity of choline acetyltransferase (ChAT). In view of the finding that cardiac carnitine acetyltransferase (CarAT) is able to acetylate choline (White & Wu, 1973), the possible contribution of CarAT to the synthesis of ACh in the muscles was investigated by using bromoacetylcholine (BrACh) as an inhibitor of ChAT and bromoacetylcarnitine (BrACar) as an inhibitor of CarAT.2. BrACh at a concentration of 2 mum inhibited the synthesis of ACh in nerve homogenates by 98%, in the homogenates of normal e.d.l. muscles by 53%, in denervated e.d.l. muscles by less than 5%; in the central part of the diaphragm BrACh inhibited ACh synthesis by 65%, and in the peripheral part by 13%. Comparative inefficiency of BrACh in inhibiting the synthesis of ACh in muscle homogenates was not due to its inactivation; the inhibitory effect of BrACh on the neural synthesis of ACh was preserved in the presence of muscle homogenates.3. BrACar at a concentration of 20 mum inhibited the synthesis of ACh in homogenates of the nerve by 18%, in those of normal e.d.l. muscles by 67%, in denervated e.d.l. muscles by 90%; in the central part of the diaphragm it inhibited the synthesis by 29%, and in the peripheral part by 76%.4. The inhibitory effects of BrACh and BrACar on the synthesis of ACh in muscle homogenates were roughly additive.5. Within 2 days of transection of the sciatic nerve, the BrACh-sensitive synthesis of ACh in the e.d.l. muscle diminished by 28%, whereas the BrACh-insensitive synthesis of ACh did not change. At 4 days after denervation, the rate of BrACh-sensitive synthesis decreased to 3% of control values.6. The results indicate that at least two enzymes are responsible for the synthesis of ACh in muscle homogenates is probably catalysed by CarAT. associated with intramuscular nerves, and probably corresponds to ChAT. The other enzyme is comparatively insensitive to BrACh, is sensitive to BrACar, and is probably localized in the muscle fibres. The BrACh-insensitive and BrACar-sensitive synthesis of ACh in muscle homogenates is probably catalysed by CarAT.7. Under the conditions used in the present experiments, CarAT was responsible for approximately one half of the synthesis of ACh in the homogenates of innervated e.d.l. muscles and for all ACh synthesized after denervation.8. The results provide an explanation for earlier findings of residual ACh synthesis in homogenates of denervated muscles, without resort to the idea that ChAT is localized in muscle fibres. It is proposed that CarAT catalyses some synthesis of ACh also in intact muscles, and that it is responsible for the synthesis of ACh observed during incubation of whole denervated muscles. It is not clear what is the physiological function of the synth...

No abstract