Cholinergic enzyme activities and muscarinic binding in the cerebral cortex of rats of different age and sex

1. The choline acetyltransferase and acetylcholinesterase activities in the cerebral cortex and hippocampus and muscarinic binding in the cerebral cortex did not differ significantly between male and female Wistar rats. 2. Choline acetyltransferase activities in the cerebral cortex and hippocampus of rats were not altered during ageing. 3. Acetylcholinesterase activities in these same brain areas were markedly decreased during ageing, possibly reflecting a loss of postsynaptic enzyme activity. 4. When measured using 3H-pirenzepine, binding to the postsynaptic muscarinic receptors was slightly higher in 26-month-old rats than in 12-month-old rats; total muscarinic binding measured using 3H-quinuclidinyl benzilate did not alter during ageing. 5. The present study does not support the hypothesis that in the rat brain the number of postsynaptic muscarinic binding sites decreases during ageing.     

Weak binding of 10-hydroxymetabolites of nortriptyline to rat brain muscarinic acetylcholine receptors

The relative affinities of nortriptyline (NT) and its 10-hydroxymetabolites to rat brain muscarinic acetylcholine receptors have been determined by competition with 3H-quinuclidinyl benzilate binding. It is shown that the major NT metabolite, E-10-OH-NT, has only 1/18 the affinity of NT for the muscarinic receptor. Since this metabolite is equipotent to NT in inhibiting neuronal noradrenaline uptake, it is suggested that it might be of clinical value as an antidepressant by virtue of having less anticholinergic side-effects than NT itself.     

A muscarinic receptor type in human lymphocytes: a comparison of 3H-QNB binding to intact lymphocytes and lysed lymphocyte membranes

Human blood lymphocytes from normal blood donors exhibited specific binding of the muscarinic antagonist 3H-quinuclidinyl benzilate (3H-QNB). The 3H-QNB binding to intact viable lymphocytes as well as to lysed lymphocyte membranes "P2" was saturable and displaceable by both muscarinic agonists and antagonists. For the lysed lymphocyte membranes "P2" a single binding site with a Bmax of 109 pmol/g protein and a Kd of 15 nM was obtained. Intact viable lymphocytes also showed one binding site with a Kd of 24 nM and a Bmax of 1556 pmol/g protein. The higher Bmax value might be explained in terms of uptake of the ligand when using intact cells or through loss of binding sites when using lysed lymphocyte membranes "P2". IC50 values were lower by a factor of 10(2) for atropine and scopolamine and by 10(4) for pirenzepine when lysed lymphocyte membranes "P2" were used instead of intact viable lymphocytes.     

Changes in brain muscarinic acetylcholine receptors and behavioral responses to atropine and apomorphine in chronic atropine-treated rats

Chronic blockade of cholinergic transmission with atropine resulted in a decrease in atropine-induced activity in the rats, whereas apomorphine - induced locomotion was enhanced. Maximal binding of ³H-quinuclidinyl benzilate (QNB), a muscarinic antagonist, to homogenate of cerebral cortex, striatum and hippocampus was significantly higher in chronic atropine-treated rats than in control animals. No difference was observed in K_D value of the specific ³H-QNB binding or in ID₅₀ value of oxotremorine in inhibiting ³H-QNB binding. No change in the specific binding of ³H-spiroperidol, a dopaminergic antagonist, was observed in those three regions of brains of chronic atropine-treated rats when it was compared with that of control animals. The role of brain muscarinic acetylcholine receptors in behavioral responses is discussed relating an effect of dopaminergic neurons on cholinergic activities.     

Fatty acid incorporation increases the affinity of muscarinic cholinergic receptors for agonists

Incorporation of unsaturated fatty acids into membrane fragments from rat brain cortex and medulla pons selectively increased the affinity of the muscarinic agonist, carbamylcholine. The affinity and number of binding sites for the labeled antagonist, N-[3H]methyl-4-piperidyl benzilate was unchanged. The effect on agonist binding was most prominent in the cortex, in which carbamylcholine IC50 values were decreased up to 5-fold. Selectivity of the effect was observed with fatty acids of chain length 18-20 carbons, unsaturation in position 11-12, and a cis conformation of the double bond being most effective. The effects of fatty acids on agonist binding were due primarily to alterations in the affinity constants for the binding reaction, with minor increases in the proportion of high-affinity sites. Transition metals selectively increased the percentage of high-affinity sites in the cortex, but in cis-vaccenic-acid-treated membranes more than additive effects of the metal were observed; both were reversed by GTP. GTP also reversed binding parameters in cis-vaccenic-acid-treated medulla membranes to control level. We conclude that the primary effect of the active fatty acids is to alter the thermodynamic properties of muscarinic agonist binding without markedly inducing interconversion.     

Muscarinic cholinergic and histamine H1 receptor binding of phenothiazine drug metabolites

In vitro binding affinities of chlorpromazine, fluphenazine, levomepromazine, perphenazine and some of their metabolites for dopamine D2 receptors, alpha 1- and alpha 2 adrenoceptors in rat brain were previously reported from our laboratories. The present study reports the in vitro binding affinities of the same compounds for muscarinic cholinergic receptors and for histamine H1 receptors in rat brain, using 3H-quinuclidinyl benzilate and 3H-mepyramine as radioligands. Chlorpromazine, levomepromazine, and their metabolites had 5-30 times higher binding affinities for muscarinic cholinergic receptors than fluphenazine, perphenazine and their metabolites. Levomepromazine was the most potent and fluphenazine the least potent of the four drugs in histamine H1 receptor binding. 7-Hydroxy levomepromazine, 3-hydroxy levomepromazine and 7-hydroxy fluphenazine had only 10% of the potency of the parent drug in histamine H1 receptor binding, while the 7-hydroxy-metabolites of chlorpromazine and perphenazine had about 75% of the potency of the parent drug in this binding system. Their histamine H1 receptor binding affinities indicate that metabolites may contribute to the sedative effects of chlorpromazine and levomepromazine.     

Effects of Pro-Leu-Gly-NH2 and cyclo (Leu-Gly) on the binding of 3H-quinuclidinyl benzilate to striatal cholinergic muscarinic receptors

The effects of Pro-Leu-Gly-NH2 (melanotropin release inhibiting factor, MIF) and its analog, cyclo (Leu-Gly) on the mouse and rat striatal cholinergic muscarinic receptors labeled with 3H-quinuclidinyl benzilate (QNB) were investigated. 3H-QNB bound to the rat striatal muscarinic receptors at a single high affinity site with receptor density (Bmax value) of 1200 fmol per mg protein and an apparent dissociation constant (Kd value) of 53.5 pM. At 140 pM concentration of 3H-QNB, the specific binding to the receptors was 724 fmol per mg protein. MIF in a concentration range of 10(-9) to 10(-4) M did not alter the binding of 3H-QNB but at 10(-3) M decreased the binding by 25%. Cyclo (Leu-Gly), on the other hand, in the concentration range of 10(-9) to 10(-3) M had no effect on the binding of 3H-QNB. A single injection of MIF (3 or 10 mg/kg IP) to rats did not alter the Bmax or the Kd value of 3H-QNB to bind to the striatal membranes. 3H-QNB bound to the mouse striatal muscarinic receptors at a single high affinity site with a Bmax value of 991 fmol/per mg protein and a Kd value of 21 pM. Neither acute administration of MIF (3 or 10 mg/kg IP) nor chronic treatment of the peptide (2, 8 or 32 mg/kg IP, daily for 5 days) to mice could influence the binding of 3H-QNB to the striatal muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of muscarinic acetylcholine receptors in the avian salt gland

Electrolyte and fluid secretion by the avian salt gland is regulated by activation of muscarinic acetylcholine receptors (R). In this study, these receptors were characterized and quantitated in homogenates of salt gland from domestic ducks adapted to conditions of low (freshwater, FW) and high (saltwater, SW) salt stress using the cholinergic antagonist [3H]-quinuclidinyl benzilate (QNB). Specific binding of the antagonist to receptors in both FW- and SW-adapted glands reveals a single population of high affinity binding sites (KdFW = 40.1 +/- 3.0 pM; KdSW = 35.1 +/- 2.1 pM). Binding is saturable; RLmaxFW = 1.73 +/- 0.10 fmol/micrograms DNA; RLmaxSW = 4.16 +/- 0.31 fmol/micrograms DNA (where L is [3H]QNB and RL the high affinity complex). Calculated average cellular receptor populations of 5,800 sites/cell in FW-adapted glands and 14,100 sites/cell in SW-adapted glands demonstrate that upward regulation of acetylcholine receptors in the secretory epithelium follows chronic salt stress. The receptor exhibits typical pharmacological specificities for muscarinic cholinergic antagonists (QNB, atropine, scopolamine) and agonists (oxotremorine, methacholine, carbachol). In addition, the loop diuretic furosemide, which interferes with ion transport processes in the salt gland, competitively inhibits [3H]QNB binding. Preliminary studies of furosemide effects on [3H]QNB binding to rat exorbital lacrimal gland membranes showed a similar inhibition, although the diuretic had no effect on antagonist binding to rat brain or atrial receptors.     

Effects of phospholipase A2 on numbers of histamine H1 receptors in guinea pig lungs

Effects of phospholipase A2 on numbers of histamine H1 receptors and muscarinic acetylcholine receptors were estimated in guinea pig lungs. Histamine H1 and muscarinic acetylcholine receptors in lung membranes were studied by the direct binding technique using 3H-pyrilamine and 3H-quinuclidinyl benzilate, respectively. The control group had two orders (high-affinity and low-affinity) of binding sites of histamine H1 receptors. Pretreatment of lung membranes with phospholipase A2 destroyed high-affinity binding sites, and these sites could not be detected after treatment. Low-affinity sites were not affected by the treatment. In contrast, the numbers of muscarinic acetylcholine receptors did not change significantly in spite of the addition of phospholipase A2. These results indicate that resistance of receptors against phospholipase A2 treatment varies among receptors and even between the binding sites in the same receptor. These variations might modulate pathological conditions associated with inflammation in which phospholipase is activated. Easy establishment of histamine tachyphylaxis might be explained by this mechanism.     

Identification of Muscarinic Receptors in Rat Cerebral Cortical Microvessels

Microvessels isolated from rat cerebral cortex consist mainly of capillaries (greater than 85%). Fresh, intact microvessel preparations have been analyzed by radioligand binding techniques for muscarinic receptors. Scatchard analysis of specific quinuclidinyl benzilate (QNB) binding indicates that microvessels possess a large number of muscarinic sites (914 fmol/mg protein) of high affinity (KD = 0.034 nM). The association and dissociation rate constants (0.37 min-1 nM-1 and 0.0067 min-1, respectively) yield an equilibrium KD of 0.018 nM. Displacement of [3H]QNB by muscarinic ligands and control substances is typical of muscarinic receptors. The results indicate that cerebral microvessels possess a large population of muscarinic receptors.     

Regulation of the Muscarinic Acetylcholine Receptor: Effects of Phosphorylating Conditions on Agonist and Antagonist Binding

Incubation of rat brain synaptic membranes under phosphorylating conditions (i.e., in the presence of Mg2+, ATP, and cyclic AMP) leads to a loss in muscarinic acetylcholine receptors, detectable as specific binding of the muscarinic antagonist L-[3H]quinuclidinyl benzilate. A role for protein phosphorylation in this receptor loss is indicated by the finding that 5'-adenylyl imidodiphosphate, a nonhydrolysable analogue of ATP, does not support receptor loss. Furthermore, receptor loss is inhibited by adenosine and 2-deoxyadenosine, both of which inhibit protein kinase activity. The loss of muscarinic receptors is calmodulin dependent, and it has been demonstrated here that this requirement is probably at the level of calmodulin-dependent phosphorylation. An investigation of the effects of phosphorylation on the binding of the agonist carbachol to synaptic membranes from the cortex and cerebellum demonstrated that phosphorylation altered the relative proportions of the super-high-, high-, and low-affinity binding sites. The results were consistent with an apparent conversion of high- into super-high-affinity sites. In the presence of 5'-guanylyl imidodiphosphate, agonist binding demonstrated the properties expected of a population of largely low-affinity sites. This conversion of super-high- and high-affinity sites into low-affinity sites by 5'-guanylyl imidodiphosphate was partially inhibited by phosphorylation.     

Brain Soluble Fractions Which Modulate Na+, K+-ATPase Activity Likewise Modify Muscarinic Receptor

Two brain soluble fractions, named peaks I and II, which respectively stimulate and inhibit neuronal Na⁺, K⁺-ATPase activity, have been isolated by gel filtration in Sephadex G-50. Since cholinergic transmission seems related to such enzyme activity, in this study we evaluated the effect of brain peak I, peak II, a more purified fraction II-E and commercial ouabain, on specific binding of the muscarinic antagonist [³H]quinuclidinyl benzilate to membranes from rat cerebellum, hippocampus and cerebral cortex. We found that binding was increased by peak I and decreased by peak II, II-E and ouabain, all effects proving concentration-dependent. Since the changes exerted on the muscarinic receptor followed a pattern similar to the one already described for synaptosomal membrane Na⁺, K⁺-ATPase activity, both systems seem to interact at a functional level.     

Aging and rat brain muscarinic receptors as measured by quinuclidinyl benzilate binding

Measurement of cholinergic muscarinic receptor binding in various rat brain areas using the ligand [³H]quinuclidinyl benzilate indicates that receptor binding is decreased in striatum and cerebellum of aged female rats (22 months old) as compared to younger rats (4 months old). Decreases were not observed in cortex, hippocampus, hypothalamus, or amygdala areas. Further examination of [³H]quinuclidinyl benzilate binding in subcellular fractions of aged and young rat cerebellum and striatum indicated a decrease in binding in the crude nuclear and crude synaptosomal fractions. Binding data indicate the observed decrease in specific ligand binding is due to a decrease in number of binding sites while receptor affinity does not appear to change.Supported by the Research Service of the Veterans Administration and by Research Grant NS 13227 from NINCDS.     

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

Heterogeneity of solubilized muscarinic cholinergic receptors: binding and hydrodynamic properties

Previous studies have described the conversion, after detergent solubilization, of the multiple populations of membrane-bound muscarinic agonist binding sites to a population of uniform affinity. This paper describes the solubilization of at least two receptor species, distinct in their agonist binding characteristics, which are capable of interconversion by transition metal ions. This finding enabled a more detailed examination of the molecular properties and regional differences of brain muscarinic receptors than was previously possible. Muscarinic receptors (mAChR) obtained from the rat cerebral cortex or medulla pons were solubilized using digitonin or the zwitterion detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). The equilibrium binding of the antagonist [3H]-4-N-methylpiperidyl benzilate ([3H]4NMPB) to detergent-solubilized receptors resembled binding to neural membranes and exhibited subnanomolar affinity, saturability, and simple mass action kinetics. Agonist binding to soluble preparations was measured by competition of [3H]4NMPB binding sites. Saturation isotherms for agonist binding to digitonin- and Chaps-solubilized mAChR obtained from various brain regions appear flattened and have Hill coefficients in the range 0.52-0.78. Computerized modelling techniques indicate that the best fit to the experimental data is provided by a model specifying two soluble muscarinic agonist binding sites with differing dissociation constants, KH and KL, respectively. Solubilization of cerebral cortex membranes with Chaps or digitonin resulted in a population with a composition of high- and low-affinity sites similar to that found in the membrane-bound state. In contrast, solubilization of the medulla pons resulted in an approximately 40% loss of high-affinity sites. Solubilized receptors retained the sensitivity to transition metals ions, but were insensitive to guanine nucleotides. Density gradient centrifugation indicated that Chaps-solubilized mAChR are composed of two molecular forms with S20,W equal to 9.9 S and 14.9 S. The 14.9 S species comprises approximately 30% of the total binding activity in the cortex and approximately 40% in the medulla. We identify the 14.9 S species as being associated with a guanylnucleotide binding protein because treatment of medulla membranes with guanylylimidodiphosphate prior to solubilization results in disappearance of 14.9 S with 9.9 S unchanged. Sedimentation of cortical mAChR in the presence of Cu+2 leads to an increase in 14.9 S to almost 50% of the total binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased muscarinic cholinergic receptors in prefrontal cortices of medicated schizophrenics

Alterations in 3H-quinuclidinyl benzilate binding sites associated with muscarinic cholinergic receptors were investigated in orbito-frontal and medial frontal cortices from 12 schizophrenics, 6 on-drug and 6 off-drug cases, and from 10 controls. Significantly lower affinities of the sites were found in both areas of schizophrenics than controls. An increase in receptor number was shown only in the orbito-frontal cortex from schizophrenics. On-drug group of schizophrenics did, however, show a significant increase in receptor number and a significant decrease in affinity in both areas, while there were no significant differences in any binding parameters of off-drug schizophrenics from controls. Also in the caudate the similar results were obtained. It is, thus, concluded that alterations in muscarinic cholinergic receptors of schizophrenic patients result from long-term medication with antimuscarinic actions.     

The effect of chronic ethanol consumption on muscarinic receptors in rat brain

Ethanol (15% v/v) was administered in the drinking water to male Wistar rats over period of 3 months. Binding properties of muscarinic receptors were studied in synaptosomes from selected brain areas using [³H]quinuclidinyl benzilate and its displacement by the selective antagonist, pirenzepine and the agonist, carbachol. Dissociation constants (K_d) of all three ligands in the cerebral cortex, hippocampus and striatum of ethanol-treated groups did not differ from those in controls. Density of [³H]quinuclidinyl benzilate binding sites in the cortex of ethanol-treated animals was approx. 50% higher than in controls (2.06 ± 0.2 and 1.32 ± 0.2 pmol/mg of protein respectively, mean ± SD, n = 6, P < 0.001). This was largely attributable to an increase in M₁ binding sites as shown by pirenzepine displacement studies. In the hippocampus and striatum binding capacity of muscarinic receptors was not affected by ethanol treatment. Synthesis of acetylcholine in cerebral cortex prisms from ethanol-treated animals was not inhibited under resting conditions, but stimulation of synthesis by high K⁺ concentration was significantly altenuated by comparison with controls. These results suggest that chronic ethanol consumption induces changes in cholinergic neurotransmission in selected brain areas.     

Interaction of the neuromuscular blocking drug atracurium with muscarinic acetylcholine receptors.

On isolated rat heart atria, atracurium competitively antagonized the negative chronotropic effect of methylfurmethide, shifting the concentration-response curve to the right without diminishing the agonist's maximal effect; Kd calculated from dose ratios was 3.0 mumol/l. On the longitudinal muscle of rat ileum, atracurium antagonized the effect of methylfurmethide in a non-competitive manner; at 50 mumol/l atracurium, the maximum response to methylfurmethide was diminished by about 50%. Atracurium antagonized the binding of (3H)quinuclidinyl benzilate [3H)QNB) to muscarinic binding sites in the atria, ileal longitudinal muscle and cerebellum with IC50 values of 5-8 mumol/l, and in brain cortex of 25 mumol/l. Atracurium was little efficient, however, in antagonizing the binding of N-(3H-methyl) scopolamine [3H)NMS) to muscarinic binding sites. Complete blockade was not achieved at concentrations up to 1 mmol/l. Concentrations required to diminish the binding by 50% were 10 - 1000 times higher for (3H)NMS than for (3H)QNB. Atracurium brought about the dissociation of (3H)QNB-receptor complexes, but its effect was considerably stronger at a concentration of 30 mumol/l than at 1 mmol/l. Atracurium slowed down the dissociation of (3H)QNB-receptor complexes observed after the addition of atropine. The effects of atracurium on the dissociation of (3H)NMS-receptor complexes were similar to those on (3H)QNB-receptor complexes, but a high concentration of atracurium (1 mmol/l) produced a transient increase in (3H)NMS binding preceding its subsequent dissociation. Although the observations of the antagonism by atracurium of the effect of methylfurmethide on the heart atria, and of the inhibition of the specific binding of (3H)QNB to the atria, ileal smooth muscle, cerebellum and brain cortex are compatible with the assumption of a competitive interaction, the discrepancy between the effects of atracurium on the binding of (3H)QNB and (3H)NMS indicates that atracurium does not bind to the same binding site as (3H)QNB and (3H)NMS. It appears that most effects of atracurium on muscarinic receptors are allosteric and that both negative and positive cooperatives play a role in interactions between atracurium and muscarinic ligands.     

Effect of Electroconvulsive Shock on Muscarinic Cholinergic Receptors in Rat Cerebral Cortex and Hippocampus

Abstract: Single electroconvulsive shock (ECS) induced no change in [³H]quinuclidinyl benzilate ([³H]QNB) binding to muscarinic cholinergic receptors in rat cortex and hippocampus. ECS administered once daily for 7 days induced a significant reduction in [³H]QNB binding in both brain areas. Concurrent ECS reversed the significant increase in cortical [³H]QNB binding induced by chronic atropine administration. These findings may have relevance to the antidepressant or amnestic effects of electroconvulsive therapy.     

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.