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1.
In isolated rat hearts L-alphacetylmethadol (LAAM) produced a concentration-dependent decrease in the spontaneous beating rate. This effect was completely prevented by 1.0 microM atropine. Chronic treatment of rats with LAAM increased the number of striatal dopamine receptors measured by [3H]spiroperidol binding. The affinity of these binding sites for [3H]spiroperidol was unchanged by LAAM treatment. There were no significant changes in the number or affinity of binding sites for the labeled muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) with chronic LAAM treatment. The ability of LAAM, nor-LAAM, or dinor-LAAM to antagonize the binding of [3H]spiroperidol (40 pM) or [3H]QNB (125 pM) to striatal membrane fragments was tested. The measured affinity constants for LAAM and metabolites were 100-3000 times higher than the affinity constants of unlabeled spiroperidol at [3H]spiroperidol binding sites. The affinity constants of LAAM and metabolites at muscarinic binding sites were 10-20 times higher than pilocarpine and 5000-8000 times higher than atropine. These results suggest that LAAM can produce some of its effects by acting as a weak agonist at muscarinic receptor sites. 相似文献
2.
Rats were administered one electroconvulsive shock daily for 7 days (ECS X 7) and were killed 24 hours after the last treatment. Muscarinic cholinergic receptor number, as determined by [3H] quinuclidinyl benzilate [( 3H]QNB) binding, was significantly reduced in the cerebral cortex. A parallel group of rats was trained on a passive avoidance task 24 hours following the last ECS and tested for retention of the original avoidance response 24 hours later; these animals exhibited a profound amnesia. Animals tested 1 hour following training were not amnestic, indicating that learning was unimpaired. Animals trained 7 days following ECS X 7 were not amnestic and [3H] QNB binding changes were not demonstrable at this time. A single ECS which does not significantly affect cortical [3H] QNB binding, did not induce amnesia in rats trained 24 hours after the treatment and tested 24 hours later. The parallel, cumulative nature of ECS-induced muscarinic receptor down-regulation and ECS-induced anterograde amnesia suggests a possible causative relationship. 相似文献
3.
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) 相似文献
4.
The Bmax for [3H]QNB binding in the bladders of alcohol preferring (AA) rats was only approximately 60% of that in the alcohol non-preferring (ANA) rats. No significant change in Bmax for [3H]QNB binding in bladder was observed between alcohol insensitive (AT) and alcohol sensitive (ANT) rats. No significant change in Kd for [3H]QNB binding in bladder was observed between the four different rat lines studied. Therefore, alcohol preference but not sensitivity is associated with a decrease in muscarinic receptor density in the rat bladder. Because all of the rats used in this study were ethanol-naive, the decrease in muscarinic receptor density in the bladders of alcohol preferring rats is associated with genetic factors inherent to this rat line. Further studies are needed to determine if these observations are tissue specific or specific to the m2 subtype, which predominates in the rat bladder. 相似文献
5.
The effectiveness of several detergents and salts in solubilizing the muscarinic acetylcholine receptor (identified by its atropine-sensitive [ 3H]3-quinuclidinyl benzilate (QNB) binding) from bovine striatal membranes is reported. The highest density of receptor is obtained by extraction with 1% digitonin-0.1 mM EDTA. Although the total solubilized muscarinic receptors (sites/ml) are increased and the nonspecific binding is decreased when 1 M NaCl is included in this extraction medium, the receptor density (sites/mg protein) is lower. The solubilized receptors have the same specific QNB binding affinity, and sensitivity to a variety of drugs, as the membrane-bound muscarinic receptors. 相似文献
6.
Fractional [ 3H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [ 3H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [ 3H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 M) significantly decreased stimulation-induced fractional [ 3H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [ 3H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [ 3H]ACh release from striatum of both age groups is modulated mainly by M 3 muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo- 3H]inositol decreased with aging, carbachol-stimulated [ 3H]myo inositol-1-fosfat (IP 1) accumulation was found similar in striatal, cortical and hippocampal slices. 相似文献
7.
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. 相似文献
8.
The β-adrenergic and muscarinic cholinergic receptors in the splenic homogenates of control and 6-hydroxydopamine (6-OHDA) treated rats were characterized. The specific binding of [ 3H]dihydroalprenolol (DHA) and [ 3H]quinuclidinyl benzilate (QNB) in the rat spleen were saturable and of high affinity and showed pharmacological specificity of splenic β-adrenergic and muscarinic cholinergic receptors. Following 6-OHDA treatment, the Bmax value for specific [ 3H](-)DHA binding to the rat spleen was significantly increased by 26 percent and 22 percent compared to control at 2 and 3 weeks without a change in the Kd. In contrast, there was a 38 percent decrease in the Bmax for [ 3H](-)QNB in the 6-OHDA treated rat spleen at 2 and 3 weeks respectively without a change in the Kd. The Bmax value at 5 weeks was significantly greater than that at 2 or 3 weeks. The splenic norepinephrine (NE) concentration was markedly reduced by the 6-OHDA treatment at 1 to 3 weeks, while there was a significant recovery in the splenic NE concentration at 5 weeks. Thus, our results strongly suggest that we are biochemically localizing muscarinic cholinergic receptors on the sympathetic nerves of the rat spleen and that the β-adrenergic receptors of the spleen are localized postsynaptically. 相似文献
9.
Hypoglycemic brain injury is a common and serious complication of insulin therapy associated with diabetes. This study evaluated
the effect of insulin-induced hypoglycemia and STZ-induced diabetes on striatal cholinergic receptors and enzyme expression
and on motor function. Cholinergic enzymes: AChE and ChAT gene expression, radioreceptor binding assay and immunohistochemistry
of muscarinic M1, M3 receptors and α7nAChR were carried out. Motor performance on grid walk test was analysed. AChE and ChAT
expression significantly downregulated in hypoglycemic and diabetic rats. Total muscarinic and Muscarinic M3 receptor binding
decreased in hypoglycemic rats compared to diabetic rats whereas muscarinic M1 receptor binding increased in hypoglycemic
rats compared to diabetic rats. Real-time PCR analysis and confocal imaging of muscarinic M1, M3 receptors confirmed the changes
in muscarinic receptor binding in hypoglycemic and diabetic rats. In hypoglycemic rats, α7nAChR expression significantly up
regulated compared to diabetic rats. Grid walk test demonstrated the impairment in motor function and coordination in hypoglycemic
and hyperglycemic rats. Neurochemical changes along with the behavioral data implicate a role for impaired striatal cholinergic
receptor function inducing motor function deficit induced by hypo and hyperglycemia. Hypoglycemia exacerbated the neurobehavioral
deficit in diabetes which has clinical significance in the treatment of diabetes. 相似文献
10.
[3H]Pirenzepine [( 3H]PZ) and [3H] (-)Quinuclidinylbenzilate [( 3H] (-)QNB) specific binding to soluble rat brain muscarinic cholinergic receptors was assessed as a function of time subsequent to receptor solubilization. The soluble brain muscarinic receptor is stable at 4 degrees C when assayed by [3H] (-)QNB binding (t 1/2 = 80 hrs). In contrast the pirenzepine state of the receptor decays rapidly (t 1/2 = 3.0 hrs). Prior occupation of the receptor with [3H] (-)QNB or [3H]PZ increases the receptor stability by two to five fold (t 1/2 QNB greater than 1,000 hrs; t 1/2 PZ = 6.5 hrs). These data indicate that pirenzepine binds to an allosteric state of the muscarinic receptor and that caution should be employed in the assignment of receptor subtypes based solely upon the binding of ligands which recognize unique conformational states. 相似文献
11.
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. 相似文献
12.
To further analyze functionally important cholinergic receptors on lymphocytes, we studied the binding of the muscarinic antagonist Quinuclidinyl benzilate (QNB) to murine splenic lymphocytes. Studies of displacement of [ 3H]QNB by unlabelled QNB on lymphocytes revealed at least two binding sites. Scatchard analysis of equilibrium binding isotherms also distinguished two sites with apparent Kds of 480 nM and 16 μM. There was greater specific QNB binding to B cell-enriched lymphocyte fractions than to T cell fractions. Lymphocyte binding demonstrated temperature-dependent dissociability, and specific binding occurred on isolated lymphocyte membranes as well. Both muscarinic and nicotinic ligands competed for QNB binding to lymphocytes with low and nearly equal affinity. Therefore, QNB binding sites on lymphocytes appear to be of low affinity and of mixed muscarinic and nicotinic character. 相似文献
13.
The presence of muscarinic receptors in sheep and rat pineals was detected by binding of [ 3H]quinuclidinyl benzilate ([ 3H]QNB), a potent and specific muscarinic antagonist. [ 3H]QNB binding to sheep pineal membrane resuspensions was saturable and reversible, with a rate constant for association at 37°C of 6×10 8M ?1min ?1 and a rate constant for dissociation of 1×10 ?2min ?1. Kinetic and saturation experiments yielded an equilibrium dissociation constant of 13–18 pM and a concentration of binding sites equivalent to 1.1 pmol/g of original wet weight. This is only about 5% of the level of β-adrenergic receptors. Competition by a variety of cholinergic drugs confirmed the muscarinic nature of the binding sites. Experiments in rats failed to detect a significant decrease in pineal [ 3H]QNB binding following bilateral superior cervical ganglionectomy, suggesting that the binding sites are not localized exclusively on sympathetic terminals. 相似文献
14.
The effects of apomorphine on the binding properties of striatal muscarinic receptors were investigated using the specific muscarinic antagonist, [ 3H](?)3-quinuclidinyl benzilate ([ 3H](?)QNB). When binding measurements were made in 50 mM sodium/HEPES buffer, pH 7.4, containing Mg +2, the binding of [ 3H](?)QNB was consistent with the presence of two binding sites; 57% of the sites had a high affinity dissociation constant of 0.030 nM whereas the remaining sites had a low affinity dissociation constant of 0.64 nM. Apomorphine (1.0 μM) enhanced the binding of [ 3H](?)QNB by an apparent conversion of low to high affinity sites. A variety of other agents were screened for their ability to enhance [ 3H](?)QNB binding, and a pattern generally consistent with a dopaminergic effect was observed although some evidence for a β-adrenergic effect was demonstrable. The potent neuroleptics haloperidol, spiperone and sulpiride failed to antagonize the apomorphine enhancement of [ 3H](?)QNB binding as well as some adrenergic antagonists. However, the potent inhibitors of the dopamine-sensitive adenylate cyclase, α-flupenthixol and fluphenazine, specifically blocked the apomorphine enhancement of [ 3H](?)QNB binding with K i values of approximately 0.1 μM. 相似文献
15.
Quantitative measurements were made of choline acetyltransferase (CAT) activity, acetylcholinesterase (AChE) acitivity and cholinergic muscarinic receptor binding ([ 3H]QNB) in eight areas of a cross-section of the rat medulla oblongata. A fourth cholinergic parameter, high-affinity choline uptake, was measured in three groups of these areas. CAT, AChE and [ 3H]QNB binding were found to be highest in the hypoglossal nucleus and the dorsal motor nucleus of the vagus; the lowest value was in the area which contains the inferior olive and the corticospinal tract. The distribution of AChE and CAT acitivities varied approximately 7- to 10-fold among the eight regions examined, whereas that of the muscarinic receptor varied only about 4-fold. The Na +-dependent high-affinity choline uptake varied approximately 20-fold from the region with the lowest activity (inferior olivary nucleus and corticospinal tract) to that with the highest activity (tissue areas containing the dorsal motor nucleus, hypoglossal nucleus, nucleus of the solitary tract and nucleus cuneatus). The four cholinergic parameters are statistically correlated throughout all the areas of the medulla which were studied. 相似文献
16.
Abstract: House fly heads contain a binding site for 3-quinuclidinyl benzilate (QNB) that is quite similar in pharmacology to the muscarinic acetylcholine receptor of vertebrate tissues. The house fly site binds [ 3H]QNB reversibly with a K d of 260 PM and B max of 1 pmol/g of heads from direct binding measurements. The K d calculated from the ratio of the dissociation rate constant (2 × 10 −4 sec −1) to the association rate constant (2.5 × 10 6 M −1 Sec −1) was 80 pM. The house fly site binds (-)quinuclidinyl benzilate preferentially, as do classic muscarinic receptors. The binding is also sensitive to other muscarinic antagonists and agonists. Nicotinic and other drugs are no more effective on the house fly site than they are on the rat brain muscarinic receptor itself. These binding studies suggest that the house fly QNB binding site is a muscarinic receptor. 相似文献
17.
Summary In the chick embryo a cholinesterase activity appears in various organ anlagen which has been correlated with morphogenetic movements (Drews 1975). The cholinesterase activity is present in the mesenchyme of the limb bud during aggregation of the central chondrogenic core. In the present study binding of tritium labelled quinuclidinyl benzilate (( 3H)QNB), a muscarinic antagonist, to homogenates of chick limb buds was investigated by a filtration assay. In the homogenate of limb buds at Stage 24 specific binding of ( 3H)QNB was demonstrated. Determination of binding constants and inhibition of binding by agonists and antagonists was studied at Stage 25/26. Specific binding was defined by the difference in binding in the absence and presence of atropine (1 M). Specific binding of ( 3H)QNB reflected a muscarinic receptor. The K d in two experiments was 0.11 nM and 0.16 nM, the binding capacity was 15.7 fmol ( 3H)QNB/mg protein and 12.0 fmol ( 3H)QNB/mg protein, respectively. Data on displacement of specific bound ( 3H)QNB by various nicotinic and muscarinic ligands confirmed the muscarinic nature of the receptor. Muscarinic ligands inhibited the ( 3H) QNB binding, whereas nicotinic ligands caused no inhibition at pharmacological concentrations. I conclude that a specific muscarinic acetylcholine receptor is part of the cholinergic system whose presence is indicated by cholinesterase activity in the chondrogenic core of the limb bud during morphogenesis. 相似文献
18.
It is known that quinuclidinyl benzilate (QNB) binds specifically and with high affinity to the cholinergic muscarinic receptor and that behaves as a potent antagonist of this receptor. We have analysed
-[3H]QNB binding to rat CNS membranes after the administration of the convulsant 3-mercaptopropionic acid (MP) (150 mg·kg−1, i.p.). The studies were done in rats killed at two stages: during and after seizures. No changes in [3H]QNB binding to hippocampus and cerebral cortex membranes were found. [3H]QNB binding increased about 40 and 80% in striatum and cerebellum membranes, respectively. The changes were observed both in seizure and postseizures states. The study was extended to the assay of [3H]QNB binding kinetic constants in the anatomical areas modified by the convulsant. The analysis of the saturation curves indicated an increase in the binding affinity but no change in the number of binding sites. Hill number values were near the unit suggesting a non-cooperative interaction between the ligand and the receptor, and the labelling of a homogeneous population of receptor sites. The results suggest the participation of some cholinergic pathways in the development and maintenance of MP-induced seizures. 相似文献
19.
Human erythrocyte ghosts contain a small population of muscarinic cholinergic receptors, as evidenced by their high affinity binding of radiolabeled quinuclinidinyl benzilate ([ 3H]QNB). The apparent K D is 1.3 × 10 ?9 M and the receptor sites are saturated at a QNB concentration of 5 nM. The number of sites is 23 fmoles/mg membrane protein. The pharmacological profile of the specific binding is similar to that of neural membranes. The binding is not stereoselective for the d and 1 isomers of QNB, a situation which prevails in the muscarinic receptors of another peripheral cholinergic system, the rat iris, but not in the central nervous system. 相似文献
20.
Although prior studies have supported the validity of measuring total muscarinic receptor binding in postmortem brain, there has not been a study of postmortem effects on muscarinic receptor subtypes, M1 and M2, defined by high and low affinity for pirenzepine, respectively. We have examined in rat brain the effect of postmortem delay at room temperature, storage at 4 degrees C and -20 degrees C, and multiple freeze/thaw cycles on total muscarinic binding, measured with [3H]quinuclidinylbenzilate ([3H]QNB) and on M1 muscarinic binding, measured with [3H]pirenzepine ([3H]Pir). We found that delay at room temperature up to 4 h, or storage at 4 degrees C for 24 h or at -20 degrees C for 4 weeks, or 3 freeze/thaw cycles had no effect on [3H]QNB or [3H]Pir binding. Exposure of brain to room temperature for 15 h, however, led to an increase in [3H]QNB binding, without change in [3H]Pir. Scatchard analysis showed an increase in binding sites without a change in affinity. We conclude that [3H]QNB and [3H]Pir are valid measures of total and M1 muscarinic binding, respectively, under these circumstances, but that caution must be used in the interpretation of indirect measures of M2 binding. 相似文献
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