Influence of phospholipase C on muscarinic acetylcholine receptor binding in rat brain

Treatment of neural membranes from rat cerebral cortex with phospholipase C (phosphatidylcholine cholinephosphohydrolase) inhibited the binding of radiolabelled antagonists to muscarinic acetylcholine receptors. This inhibition was incomplete, was not competitive, and did not appear to be related to the production of inhibitory products. The affinity of carbamylcholine for cortex muscarinic receptors was increased by phospholipase C action. The distribution of receptors between states of high and low affinity was not affected by phospholipase C; rather, the affinity for carbamylcholine of the lowest affinity receptors was selectively increased. This suggests that membrane lipids influence the interaction of the receptor binding subunit with other structures in the synaptic membrane.     

Measurement of acetylcholine turnover rate in discrete areas of rat brain

The turnover rate of ACh was estimated in brain stem, two cortical areas and striatum of rat brain. The turnover rate was highest in the striatum (1.3 μmoles/g/hr); lowest in brain stem (0.092 μmoles/g/hr); and intermediate values were observed in limbic and occipital cortex. The highest ACh concentrations were measured in striatum, those in brain stem were intermediate but in the two cortical areas the ACh concentrations were the lowest. The results of the turnover estimations with the finite difference method yielded values similar to those obtained with the procedure described in this paper. Moreover, once the baseline was established, this method could be reliably used to estimate turnover rate using a single infusion time. The latter simplication would be very useful to compare ACh turnover rate in drug studies.     

Effects of acute and chronic morphine on regional rat brain acetylcholine turnover rate

A simplified method to study the acetylcholine (ACh) turnover rate (TR_ACh) in brain parts of drug treated rats has been presented. In striatum and occipital cortex of rats receiving a large dose of morphine (140 μ moles/kg i.p.) or implanted chronically with morphine pellets, the TR_ACh is influenced in a different manner. The single injection of morphine reduced the synthesis of ACh in cortex but not in striatum. Morphine pellets decreased striatal TR_ACh but failed to alter the TR_ACh in occipital cortex. Naloxone reversed both changes of TR_ACh elicited by morphine although it was devoid of any effect of the synthesis of ACh in rat brain parts. We suggest that morphine may prevent the ACh release from neurons as proposed by others, however, this effect in striatum of rats receiving a single dose of morphine is masked by the simultaneous action of morphine on the dopaminergic nigrostriatal pathway which regulates the turnover rate of striatal ACh.     

Solubilization of muscarinic acetylcholine receptor by zwitterionic detergent from rat brain cortex

The muscarinic acetylcholine receptor was solubilized from rat brain cortex by zwitterionic detergent 3-[(3-chloramidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). About 15% of the binding activity was solubilized and 40% of the activity was destroyed by the detergent. Binding of the muscarinic antagonist [³H]-N-methyl-4-piperidyl benzilate (4NMPB) was saturable. Scatchard analysis revealed a single population of binding sites with K_D value of 0.7 nM and a B_max value of 340 fmoles/mg protein. The homogenate and the CHAPS treated pellet and soluble receptors showed similar affinity for the agonists oxotremorine and carbamylcholine and for the antagonists QNB and atropine. The dissociation of 4NMPB from the soluble receptors appears slightly slower than from the membrane bound receptors.     

Purification of the muscarinic acetylcholine receptor from porcine brain

The muscarinic acetylcholine receptor of porcine cerebrum has been purified to apparent homogeneity by affinity chromatography, with conjugated 3-(2'-aminobenzhydryloxy)tropane (ABT) as described previously (Haga, K., and Haga, T. (1983) J. Biol. Chem. 258, 13575-13579). In a single step purification using 900 ml of digitonin/cholate-solubilized preparations and 300 ml of the ABT-agarose gel, we obtained, in a yield of 10-15%, more than 250 pmol of muscarinic receptors which bind [3H]N-methylscopolamine with a specific activity of 1,000-5,000 pmol/mg of protein (1,000-5,000-fold purification). The muscarinic receptors eluted from the ABT-agarose gel with 0.1 mM atropine were adsorbed to hydroxylapatite and then recovered as a concentrated solution. Muscarinic receptors were further purified by rechromatography with the same gel or by gel permeation high pressure liquid chromatography. The amino acid composition of the purified receptor was determined, and the specific activity of the purified preparation was estimated to be 13,100 pmol/mg of protein on the basis of amino acid composition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptors with or without radioiodination revealed a single, major band with an apparent Mr of 70,000 either by silver staining or radioautogram. The major band corresponded to the band which specifically bound [3H]propylbenzylcholine mustard (irreversible muscarinic ligand). The purified receptor showed essentially the same specificity for muscarinic ligands as unpurified receptors.     

Effects of N-ethylmaleimide on muscarinic acetylcholine receptor subtype autoradiography and inositide response in rat brain

Chemical modification of brain muscarinic acetylcholine receptors (mAChr) with N-ethylmaleimide (NEM) has been employed to investigate mAChr-subtype distribution and mediation of the inositide response. 3H-Pirenzepine and 3H-oxotremorine-M were used to autoradiographically localize the M1- and M2-AChr subtypes, respectively, in brain slices. M1- and M2-AChr distribution were observed to be distinct from each other. The presence of 1 mM NEM selectively reduced the labeling of M2-, but not of M1-AChr. These data support the notion that NEM converts the high-affinity M2-AChr to a lower affinity state, without affecting the affinity of the M1-AChr. Also, regional analysis indicated that the M1- and M2-AChr subtypes were not interconvertible by NEM. NEM at 30 microM enhanced the carbamylcholine stimulated labeling of phosphatidic acid from 32Pi in nerve endings from rat forebrain, suggesting that the low affinity M2-AChr may mediate at least a part of the inositide response to cholinergic stimulation.     

The development of the muscarinic acetylcholine receptor in normal and hyperphenylalaninemic rat cerebrum

The effect of hyperphenylalaninemia on the development of the muscarinic acetylcholine receptor in rat cerebrum has been studied. Rats were subjected to the hyperphenylalaninemic regimen as of 5 days of age. A gradual and steady decrease in the number of binding sites forl-[³H]quinuclidinylbenzilate was observed, with the white matter more affected than the gray matter. A return to normal blood phenylalanine levels after the age of 21 days does not lead to an increase in this number of binding sites.     

Polyamine turnover in different regions of rat brain

The dynamics of the formation and disappearance of polyamines in rat brain have been examined after intraventricular administration of a tracer dose of [³H]putrescine. After 2 days [³H]putrescine was no longer detectable in any brain region examined. [³H] Spermidine and [³H] spermine were formed in all brain areas. In the midbrain, hypothalamus and cerebellum (regions which manifested the greatest initial accumulation of tritium) the specific radioactivity of spermidine declined with a half-life of 16-19 days. However, in areas with a low initial accumulation of tritium (the medulla-pons, internal capsule, cerebral cortex and corpus striatum) the specific radioactivity of spermidine changed very little between 2 and 19 days after the putrescine administration. Levels of [³H]spermine increased continuously in all brain areas for a 14-day period after the putrescine injection.     

The character of the muscarinic receptors in different regions of the rat brain

The binding characteristics of muscarinic receptors have been critically examined in six regions of the rat brain. The binding curves of antagonists are similar for all six areas but the binding curves of agonists show large differences. It is shown that in all regions there are three classes of receptors with similar binding characteristics but that these are present in different proportions. The binding constants to the three receptor types of a range of agonists were examined and evidence was produced in support of the theory that the subclasses of brain receptors are due to a single receptor subunit subject to different conformational constraints.     

Effect of antinociceptive doses of oxotremorine on mouse brain acetylcholine turnover rate

The effect of antinociceptive doses of oxotremorine on the steady-state level and turnover rate of acetylcholine (ACh) was investigated in male Swiss-Webster mice. Oxotremorine produced dose-related increases in ACh levels which attained statistical significance (p ≥ 95; Dunnett's T) with the ED₈₄ antinociceptive dose in two sacrifice methods. The increased steady-state level of ACh was temporally correlated with the peak antinociceptive effect of oxotremorine. ACh turnover rate decreased with increasing doses of oxotremorine. The ACh turnover rate decreased from 11.06 ± 1.62 nmol/g/min to 5.38 ± 0.71 nmol/g/min by decapitation method and 30.20 ± 1.8 nmol/g/min to 19.99 ± 1.6 nmol/g/min by the microwave irradiation method (Ed₈₄ oxotremorine doses). The decrease in turnover rate of ACh produced by antinociceptive doses of oxotremorine is of a lesser magnitude than that produced by tremorogenic doses (1,2).     

Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland.

A family of five subtypes of muscarinic acetylcholine receptors (mAChR) has been identified based on their molecular structures and second signal transduction pathways. In the present study, we examined the antagonist binding profiles of 9 muscarinic antagonists (atropine, 4-DAMP, pirenzepine, oxybutynin, tiquizium, timepidium, propiverine, darifenacin and zamifenacin) for human muscarinic acetylcholine receptor subtypes (m1, m2, m3, m4 and m5) produced by using a baculovirus infection system in Sf9 insect cells, and rat tissue membrane preparations (heart and submandibular gland). In a scopolamine methyl chloride [N-methyl-3H]- ([3H]NMS) binding assay, pirenzepine and timepidium displayed the highest affinities for the m1 and m2 subtypes, respectively, and both zamifenacin and darifenacin had the highest affinities for the m3 subtype, although the selectivities among the five subtypes were less than 10-fold. Propiverine showed a slightly higher affinity for the m5 subtype, whereas none of the drugs used in this study was uniquely selective for the m4 subtype. The binding affinities of muscarinic antagonists for rat heart and submandibular gland strong correlated with those for human cloned m2 and m3 subtypes, respectively. These data suggest that [3H]NMS binding studies using rat heart and submandibular gland might be useful methods which predict the affinities of test drugs for human muscarinic M2 and M3 receptor subtypes.     

Neurochemical effects of the endocannabinoid uptake inhibitor UCM707 in various rat brain regions

To date, UCM707, (5Z,8Z,11Z,14Z)-N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide, has the highest potency and selectivity in vitro and in vivo as inhibitor of the endocannabinoid uptake. Its biochemical, pharmacological and therapeutic properties have been intensely studied recently, but the information on its capability to modify neurotransmitter activity, which obviously underlies the above properties, is still limited. In the present study, we conducted a time-course experiment in rats aimed at examining the neurochemical effects of UCM707 in several brain regions following a subchronic administration (5 injections during 2.5 days) of this inhibitor in a dose of 5 mg/kg weight. In the hypothalamus, the administration of UCM707 did not modify GABA contents but reduced norepinephrine levels at 5 h after administration, followed by an increase at 12 h. Similar trends were observed for dopamine, whereas serotonin content remained elevated at 1 and, in particular, 5 and 12 h after administration. In the case of the basal ganglia, UCM707 reduced GABA content in the substantia nigra but only at longer (5 or 12 h) times after administration. There were no changes in serotonin content, but a marked reduction in its metabolite 5HIAA was recorded in the substantia nigra. The same pattern was found for dopamine, contents of which were not altered by UCM707 in the caudate-putamen, but its major metabolite DOPAC exhibited a marked decrease at 5 h. In the cerebellum, UCM707 reduced GABA, serotonin and norepinephrine content, but only the reduction found for norepinephrine at 5 h reached statistical significance. The administration of UCM707 did not modify the contents of these neurotransmitters in the hippocampus and the frontal cortex. Lastly, in the case of limbic structures, the administration of UCM707 markedly reduced dopamine content in the nucleus accumbens at 5 h, whereas GABA content remained unchanged in this structure and also in the ventral-tegmental area and the amygdala. By contrast, norepinephrine and serotonin content increased at 5 h in the nucleus accumbens, but not in the other two limbic structures. In summary, UCM707 administered subchronically modified the contents of serotonin, GABA, dopamine and/or norepinephrine with a pattern strongly different in each brain region. So, changes in GABA transmission (decrease) were restricted to the substantia nigra, but did not appear in other regions, whereas dopamine transmission was also altered in the caudate-putamen and the nucleus accumbens. By contrast, norepinephrine and serotonin were altered by UCM707 in the hypothalamus, cerebellum (only norepinephrine), and nucleus accumbens, exhibiting biphasic effects in some cases.     

Beneficial metabolic effects of M3 muscarinic acetylcholine receptor deficiency

Most animal models of obesity and hyperinsulinemia are associated with increased vagal cholinergic activity. The M3 muscarinic acetylcholine receptor subtype is widely expressed in the brain and peripheral tissues and plays a key role in mediating the physiological effects of vagal activation. Here, we tested the hypothesis that the absence of M3 receptors in mice might protect against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. In all cases, the lack of M3 receptors greatly ameliorated impairments in glucose homeostasis and insulin sensitivity but had less robust effects on overall adiposity. Under all experimental conditions tested, M3 receptor-deficient mice showed a significant elevation in basal and total energy expenditure, most likely due to enhanced central sympathetic outflow and increased rate of fatty-acid oxidation. These findings suggest that the M3 receptor may represent a potential pharmacologic target for the treatment of obesity and associated metabolic disorders.     

Asymmetric distribution and down-regulation of the muscarinic acetylcholine receptor in rat cerebral cortex

The distribution and down-regulation of the muscarinic acetylcholine receptor (mAChR) were studied in dissociated cells from right (RCC) and left (LCC) cerebral cortex. For this purpose [³H]quinuclidinyl benzilate (QNB) and [³H]pirenzepine (Pz), two muscarinic antagonists, were used. The mAChR binding sites detected with [³H]QNB were asymmetrically distributed between the two hemispheres, the majority being found in the RCC. Asymmetry was also evident in the distribution of the mAChR subtypes (M₁ and M₂) detected with [³H]Pz. Under basal conditions the RCC had roughly 50% more M₁ subtype than the LCC. The pharmacological and kinetic parameters were similar for both antagonists in RCC and LCC, indicating that the observed lateralization was due to a different density of the receptor rather than to different kinetics of binding of the two radioligands. After sustained stimulation with the agonist carbamoylcholine, the receptor sites detected with [³H]Pz, i.e. the M₁ subtype of mAChR, decreased at a higher rate in the RCC (44%) than in the LCC (25% of controls), demonstrating that the down-regulation process is more active in the right than in the left cortex, and thus implying that there is better coupling between the stimulated mAChR and its effector system in the former.     

Effect of immobilization stress on serotonin content and turnover in regions of the rat brain.

Sprague-Dawley rats were stressed by immobilization from 30 to 300 minutes and the effects on serotonin (5-HT) and 5-hydroxy-indoleacetic acid (5-HIAA) content were determined in the cerebral cortex, diencephalon, striatum, hippocampus and the brain stem. In a subsequent study 5-HT turnover rate in these brain areas was estimated by measuring 5-HIAA accumulation 0, 30, 60 and 90 minutes after probenecid. The content of 5-HIAA and the turnover rate of 5-HT were significantly increased in the cerebral cortex shortly after the onset of immobilization. The content of 5-HIAA in the brainstem was increased by immobilization although 5-HT turnover rate was not increased. Short term increases in 5-HIAA content were observed in the striatum and hippocampus. However, no significant changes in 5-HT turnover rate were observed in either of these 2 brain areas. Immobilization did not affect 5-HIAA content or 5-HT turnover in the diencephalon. The sensitivity of the serotonergic system in the cerebral cortex to immobilization stress suggests that this brain region could be used in future studies of the interrelationships between stress and the brain serotonergic system.     

Affinity chromatography of the muscarinic acetylcholine receptor

A novel compound, 3-(2'-aminobenzhydryloxy)-tropane (ABT), and an ABT-agarose gel were synthesized and used for the purification of solubilized muscarinic receptors. ABT had a high affinity with an apparent dissociation constant (Kd) of 7 nM for the muscarinic receptors solubilized from the porcine brain by digitonin. An ABT-agarose gel was prepared by coupling ABT with epoxy-activated Sepharose 6B, and the degree of substitution to the gel was determined to be 4-5 mumol/ml of the gel by UV absorption spectrum. During affinity chromatography using 10 ml of the ABT-agarose gel and 100 ml of the digitonin-solubilized preparation, 70% of muscarinic receptors were adsorbed to the gel, in marked contrast with the adsorption of only 2% of proteins. Approximately 25% of muscarinic receptors applied to the gel were eluted biospecifically with 1 mM muscarinic ligands. The purified fraction showed a high affinity for [3H]quinuclidinyl benzylate with a Kd of 0.4 nM and similar specificity for muscarinic ligands to that of unpurified soluble receptors. The protein concentration of the purified fraction was too low to be determined accurately, but very approximately a purification of 10(3)-fold was indicated.