piperidines and enpiperate

The application of PET with NMPB has provided useful information toward understanding the pathogenesis of mesial TLE, and can be used as a tool in the presurgical evaluation. Many other patient populations have yet to be studied with cholinergic neuroreceptor imaging. The use of NAChR radioligands in the study of ADNFLE may be especially rewarding.

Topics: Benzilates; Brain; Calcium Channel Blockers; Epilepsy, Temporal Lobe; Flumazenil; GABA Modulators; Humans; Piperidines; Receptors, Cholinergic; Tomography, Emission-Computed

11C-NMPB is a useful ligand which is used to measure brain muscarinic cholinergic receptors (mAChRs) for its high affinity and high brain uptake. PET images of 11C-NMPB uptake had the highest accumulations in the cerebral cortex and striatum and the lowest in the cerebellum, both in patients with Parkinson's disease (PD) and normal subjects. 11C-NMPB uptake was homogeneous throughout the cerebral cortex in the normal subjects but greater in the frontal cortex of about one half of the PD patients as compared with other cerebral cortical areas. Quantitative analysis showed that the PD patients had high K3 values, an index of mAChR binding, in the frontal cortex. In PD, increased mAChR binding in the frontal cortex may be related to frontal lobe dysfunction. This technique should prove useful for detecting subclinical impairment of the central cholinergic system in PD.

Topics: Animals; Benzilates; Brain; Carbon Radioisotopes; Humans; Parkinson Disease; Piperidines; Receptors, Muscarinic; Tomography, Emission-Computed

An acute effect of triazolam, a potent benzodiazepine agonist, on cholinergic receptor binding in the human brain was measured by PET (positron emission tomography) using [11C]N-methyl-4-piperidylbenzilate ([11C]NMPB), a potent muscarinic cholinergic receptor antagonist. Two PET scans were performed in each subject: (1) control scan; (2) after oral administration of 0.5 mg triazolam or placebo. The previously discussed amnestic effect of triazolam was measured by immediate and delayed recall of meaningful and meaningless syllables. A compartment model employing the radioactivity in the cerebellum as an input function was used for the quantification of receptor binding. The binding parameter, k3, was decreased after triazolam administration in all measured regions, whereas no change was observed after placebo treatment. The reduction compared to the control study varied from 8.6 +/- 3.7% in the temporal cortex to 16.3 +/- 6.3% in the thalamus. Triazolam administration impaired both immediate and delayed recall of syllables, whereas placebo administration had no effects. Benzodiazepine agonists are reported to decrease the cortical acetylcholine release. The decrease of acetylcholine release in the synaptic cleft might be the explanation for the decreased binding of [11C]NMPB.

Topics: Adolescent; Adult; Benzilates; Brain; Humans; Male; Mental Recall; Parasympatholytics; Piperidines; Receptors, Muscarinic; Tomography, Emission-Computed; Triazolam

The binding kinetics of 11C-N-methyl piperidyl benzilate (11C-NMPB) in rhesus monkey brain were studied using animal positron emission tomography (PET) (SHR2000). This study is intended to assess the validity of the method using the cerebellum as a reference region, and to evaluate the effects of anesthesia on 11C-NMPB binding. Two monkeys, anesthetized with ketamine, received intravenous 11C-NMPB alone (370-760 MBq, < 1 microg/kg) or mixed with varying doses of nonradioactive NMPB (3 microg/kg, 10 microg/kg, 30 microg/kg) and were subjected to PET scans for 60 minutes. Regions of interest (ROI) were drawn on reconstructed PET images and a time-activity curve was obtained for each region. 11C-NMPB accumulated densely in the striatum and cerebral cortex with time. In contrast, the tracer accumulation significantly decreased with increased doses of nonradioactive NMPB. In the cerebellum, on the other hand, the accumulation of 11C-NMPB remained low and the tracer was slowly eliminated from the brain following the injection. 11C-NMPB binding in the cerebellum was barely affected by the increased dose of nonradioactive NMPB. We thus concluded that the specific 11C-NMPB binding was negligible in the cerebellum, and performed simplified evaluation of 11C-NMPB binding in each brain region by a graphical method using the cerebellum as a reference region. PET was conducted 26 times, in total both in ketamine-anesthetized and awake monkeys (n = 3 each). Measurements of 11C-NMPB binding showed good run-to-run reproducibility within individual animals. When 11C-NMPB binding was compared between ketamine-treated and awake animals, a significant increase in 11C-NMPB binding was observed in the striatum but not in other brain regions of ketamine-treated animals.

Topics: Animals; Benzilates; Binding Sites; Brain; Brain Mapping; Cerebellum; Macaca mulatta; Male; Metabolic Clearance Rate; Piperidines; Positron-Emission Tomography; Protein Binding; Radiopharmaceuticals; Receptors, Muscarinic; Reference Values; Tissue Distribution

We investigated postischemic alterations in benzodiazepine receptor, D1 dopamine receptor, and muscarinic acetylcholine receptor binding after transient middle cerebral artery (MCA) occlusion in rats using [3H]-flumazenil, [3H]-SCH23390, and [3H]-N-methyl-4-piperidyl benzilate ([3H]-NMPB), respectively, as radioligand. These ligand bindings were determined at 3 and 24 h and at 3 and 7 days after ischemia/reperfusion of MCA by using autoradiographic methods. Ischemic cell injury was clearly detected from 3 h after ischemia/reperfusion and progressively increased from 3-24 h after ischemia/reperfusion of MCA. The area of cell injury reached maximum at 24 h after ischemia/reperfusion of MCA. [3H]-SCH23390 binding was reduced to 47% of the contralateral side at 3 days after ischemia/reperfusion of MCA. After 7 days, [3H]-SCH23390 binding was further reduced by 20% in the striatum. [3H]-NMPB binding was slightly decreased in both the striatum and cerebral cortex at 3 days after ischemia/reperfusion of MCA, and [3H]-NMPB binding in the striatum and cerebral cortex were reduced to 42 and 62% of the contralateral side at 7 days after ischemia/reperfusion of MCA. [3H]-NMPB was also decreased at 24 h. In contrast, [3H]-flumazenil binding was not decreased in the striatum and cerebral cortex within 7 days after ischemia/reperfusion of MCA. These results suggest that [3H]-SCH23390 and [3H]-NMPB binding do not correlate with cell injury by ischemia/reperfusion, although vulnerability to ischemia/reperfusion was observed with these receptors. In addition, central benzodiazepine receptor imaging might be essentially stable to neuronal cell injury induced by transient focal cerebral ischemia in rats, in contrast to the results of PET studies.

Topics: Animals; Autoradiography; Benzazepines; Benzilates; Binding Sites; Binding, Competitive; Cerebral Cortex; Cerebral Infarction; Corpus Striatum; Disease Models, Animal; Down-Regulation; Flumazenil; Infarction, Middle Cerebral Artery; Male; Nerve Degeneration; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Reaction Time; Receptors, Dopamine D1; Receptors, GABA-A; Receptors, Muscarinic; Reperfusion Injury; Sensitivity and Specificity; Tritium

Cerebral cholinergic deficits have been described in Alzheimer disease (AD) and as a result of normal aging. At the present time, there are very limited options for the quantification of cholinergic receptors with in vivo imaging techniques such as PET. In the present study, we examined the feasibility of utilizing [11C]N-methyl-4-piperidyl benzilate (NMPB), a nonselective muscarinic receptor ligand, in the study of aging and neurodegenerative processes associated with cholinergic dysfunction. Based on prior data describing the accuracy of various kinetic methods, we examined the concentration of muscarinic receptors with [11C]NMPB and PET using two- and three-compartment kinetic models. Eighteen healthy subjects and six patients diagnosed with probable AD were studied. Pixel-by-pixel two-compartment model fits showed acceptable precision in the study of normal aging, with comparable results to those obtained with a more complex and less precise three-compartment model. Normal aging was associated with a reduction in muscarinic receptor binding in neocortical regions and thalamus. In AD patients, the three-compartment model appeared capable of dissociating changes in tracer transport from changes in receptor binding, but suffered from statistical uncertainty, requiring normalization to a reference region, and therefore limiting its potential use in the study of neurodegenerative processes. After normalization, no regional changes in muscarinic receptor concentrations were observed in AD.

Topics: Adult; Age Factors; Aged; Aging; Alzheimer Disease; Benzilates; Biological Transport; Brain; Brain Chemistry; Carbon Radioisotopes; Computer Simulation; Female; Humans; Kinetics; Male; Middle Aged; Piperidines; Receptors, Muscarinic; Tomography, Emission-Computed

The novel muscarinic cholinergic ligands (+)N-[11C]methyl-3-piperidyl benzilate ([11C](+)3-MPB) and its stereoisomer [11C](-)3-MPB were evaluated in comparison with [11C]4-MPB in the brains of conscious monkeys (Macaca mulatta) using high-resolution positron emission tomography (PET). The regional distribution patterns of [11C](+)3-MPB and [11C]4-MPB at 60-91 min postinjection were almost identical: highest in the striatum and occipital cortex; intermediate in the temporal and frontal cortices, cingulate gyrus, hippocampus, and thalamus; lower in the pons; and lowest in the cerebellum. The uptake of [11C](+)3-MPB in all regions was higher and the dynamic range of regional uptake differences of [11C](+)3-MPB was better than those of [11C]4-MPB. The levels of [11C](-)3-MPB were much lower in all regions of the brain than [11C](+)3-MPB and [11C]4-MPB. Administration of scopolamine, a muscarinic cholinergic antagonist, at a dose of 50 microg/kg reduced the radioactivity of [11C](+)3-MPB and [11C]4-MPB in all regions except the cerebellum. Time-activity curves of [11C](+)3-MPB peaked in all regions, while those of [11C]4-MPB showed gradual increases with time in all regions except the thalamus, pons, and cerebellum. Two graphical analyses (Logan plot and Patlak plot) with plasma radioactivity as an input function into the brain were applied to evaluate receptor binding in vivo. [11C](+)3-MPB showed linear regression curves on Logan plot analysis and nonlinear curves on Patlak plot in all regions, suggesting that [11C](+)3-MPB bound reversibly to the muscarinic receptors. The in vivo binding parameters as well as uptake at 60-91 min postinjection of [11C](+)3-MPB were consistent with muscarinic receptor density in the brain as reported in vitro.

Topics: Animals; Benzilates; Brain; Carbon Radioisotopes; Consciousness; Hallucinogens; Macaca mulatta; Male; Piperidines; Receptors, Muscarinic; Stereoisomerism; Tomography, Emission-Computed

The effects of changes in regional cerebral blood flow (rCBF) with aging on muscarinic cholinergic receptor binding were evaluated with [15O]H(2)O and N-[11C]methyl-4-piperidyl benzilate (4-MPB) in the living brains of young (5.9+/-1.8 years old) and aged (19.0+/-3.3 years old) monkeys (Macaca mulatta) in the conscious state using high-resolution positron emission tomography (PET). For quantitative analysis of receptor binding in vivo with [11C]4-MPB, metabolite-corrected arterial plasma radioactivity curves were obtained as an input function into the brain, and graphical Patlak plot analysis was applied. In addition, two-compartment model analysis using the radioactivity curve in the cerebellum as an input function (reference analysis) was also applied to determine the distribution volume (DV=K(1)/k(2)') for [11C]4-MPB. With metabolite-corrected arterial input, Patlak plot analysis of [11C]4-MPB indicated a regionally specific decrease in muscarinic cholinergic receptor binding in vivo in the frontal and temporal cortices as well as the striatum in aged compared with young animals, showing no correlation with the degree of reduced rCBF. In contrast, on the reference analysis with cerebellar input of [11C]4-MPB, all regions assayed except the pons showed a significant age-related decrease of DV, and the degree of reduction of DV was correlated with that of rCBF. These results demonstrated the usefulness of kinetic analysis of [11C]4-MPB with metabolite-corrected arterial input, not with reference region's input, as an indicator of the aging process of cortical muscarinic cholinergic receptors in vivo measured by PET with less blood flow dependency.

Topics: Acetylcholine; Aging; Animals; Benzilates; Binding, Competitive; Brain; Brain Mapping; Cerebrovascular Circulation; Consciousness; Macaca mulatta; Magnetic Resonance Imaging; Male; Oxygen Radioisotopes; Parasympatholytics; Pharmacokinetics; Piperidines; Radioligand Assay; Receptors, Muscarinic; Tomography, Emission-Computed

We evaluated the sex-related differences in the decline of the cerebral muscarinic acetylcholinergic receptor (mACh-R) due to aging by using 11C-N-methyl-4-piperidyl benzilate (11C-NMPB) and positron emission tomography (PET). The subjects consisted of 37 (20 males and 17 females) healthy volunteers. The 11C-NMPB uptake was evaluated by the ratio method (regional 11C-NMPB uptake/Cerebellar 11C-NMPB uptake; rNMPB ratio). The correlation between sex, aging, and the rNMPB ratio in normal aging was evaluated by a multiple regression analysis. The rNMPB ratio was higher in females than in males throughout the entire cerebral region (p < 0.01-p < 0.0001) and the rNMPB ratio might thus possibly decline with age more rapidly in females. Our study therefore revealed the existence of sex-related differences in the cerebral mACh-R.

Topics: Adult; Aged; Aging; Benzilates; Brain; Carbon Radioisotopes; Female; Humans; Ligands; Male; Middle Aged; Piperidines; Receptors, Muscarinic; Sex Characteristics; Tomography, Emission-Computed

The muscarinic cholinergic receptor ligands N-[(11)C]ethyl-4-piperidyl benzilate (4-EPB) and N-[(11)C]propyl-4-piperidyl benzilate (4-PPB) were developed and evaluated in comparison with N-[(11)C]methyl-4-piperidyl benzilate (4-MPB) in the conscious monkey brain using positron emission tomography (PET). Time-activity curves of [(11)C]4-EPB, unlike [(11)C]4-MPB, showed peaks within 91 min in regions rich in muscarinic receptors. [(11)C]4-PPB showed no specific binding even in the regions rich in these receptors. These observation demonstrated that increases in [(11)C]alkyl chain length could alter the kinetic properties of receptor ligands for PET.

Topics: Animals; Benzilates; Brain; Isotope Labeling; Ligands; Macaca mulatta; Magnetic Resonance Imaging; Male; Piperidines; Radiopharmaceuticals; Receptors, Muscarinic; Tomography, Emission-Computed

Anticholinergic drug is often used to treat extrapyramidal symptoms. We measured muscarinic cholinergic receptor (mAchR) occupancy by the oral administration of biperiden in eight healthy subjects using positron emission tomography (PET) and [11C]N-methyl-4-piperidylbenzilate (NMPB). After the baseline scan each subject underwent one or two post-dose PET scans. mAchR occupancy was 10-45% in the frontal cortex three hours after the oral administration of 4 mg of biperiden. The occupancy correlated with the plasma concentration of biperiden in a curvilinear manner.

Topics: Adult; Animals; Benzilates; Binding Sites; Biperiden; Brain; Humans; Macaca mulatta; Male; Muscarinic Antagonists; Piperidines; Receptors, Muscarinic; Time Factors; Tomography, Emission-Computed

The present work demonstrates quantitative autoradiography by using positron emission tomography tracers and storage phosphorimaging plates. The uptake and association of [(11)C]N-methyl-4-piperidylbenzilate was measured in rat brain tissue cryosections of various thicknesses. The signal increased with increasing section thickness, but only in 10-micrometer-thick sections did the binding reach the steady state during a 50-min observation time. This violation of the equilibrium condition, potentially combined with perfusion limitations, leads to erroneous increased binding-site density and decreased affinity in the 25- and 50-micrometer-thick sections. For better imaging of receptor distribution it is reasonable to use thicker sections. For quantitative analysis of receptor-binding parameters, the specific properties of ligands at different thicknesses of cryosections need to be considered. Evidence is provided that the nonselective muscarinic antagonist N-methyl-4-piperidylbenzilate binds preferentially to the M(4) subtype of muscarinic acetylcholine receptors.

Topics: Animals; Autoradiography; Benzilates; Cerebral Cortex; Half-Life; Image Processing, Computer-Assisted; Male; Neostriatum; Piperidines; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Spinal Cord; Tomography, Emission-Computed

Apparent muscarinic acetylcholine (mAch) receptor occupancy in mouse cerebral cortex, hippocampus, and striatum by scopolamine, an antagonist, and biperiden, a relatively selective M1 antagonist, was estimated with competitive binding studies using two different radioligands: 3H-N-methyl piperidyl benzilate (3H-NMPB) and 3H-quinuclidinyl benzilate (3H-QNB). Both radioligands labeled mAch receptors in these brain regions, and the relative regional distributions of the specific binding of 3H-NMPB in vivo paralleled the distribution of mAch receptors. 3H-NMPB binding in vivo was much more sensitive to direct competitive inhibition by scopolamine than was 3H-QNB. A similar discrepancy in sensitivity to competitors between 3H-NMPB and 3H-QNB was also observed when biperiden was used as a competitor, indicating that binding to different subtypes of the mAch receptor could not account for the observed differences in sensitivity to competition. An in vivo saturation study suggested that the apparent association rate constant (k on) of 3H-QNB binding might be changed by ligand concentration. The heterogeneity of the free ligand concentration in intact brain was assessed in relation to the ligand concentration dependency of the apparent association rate constant (k on) of 3H-QNB binding. This finding, together with the more favorable accumulation of 3H-NMPB in cerebral cortex, hippocampus, and striatum, leads us to conclude that 3H-NMPB, or its positron emitting counterpart, should be the more favorable radiotracer for the estimation of mAch receptor occupancy by cholinergic drugs in the brain.. mAch receptor, QNB, NMPB, in vivo, mouse.

Topics: Animals; Benzilates; Biperiden; Brain; Male; Mice; Muscarinic Antagonists; Parasympatholytics; Piperidines; Quinuclidinyl Benzilate; Receptors, Muscarinic

Changes in both regional cerebral blood flow (rCBF) and postsynaptic muscarinic cholinergic activity in the rat brain were investigated after ligation of the common carotid arteries (CCAs) bilaterally with (15)O-labeled water (H2(15)O) and [11C]N-methyl-4-piperidylbenzilate, a potent muscarinic receptor antagonist.. PET was performed in the same Wistar rat, 7 days and 1 mo after the CCA ligation. Regional cerebral blood flow and the transfer coefficient k3, the rate of binding of 11C-NMPB, were measured, based on the autoradiographic method and the graphical plotting analysis, respectively.. The levels of rCBF in the frontal cortex of the ligated group were significantly lower than those in the cerebellum and those in sham group, after 7 days and 1 mo postoperation. Although the level of k3 in the frontal cortex 7 days after operation was not altered, it decreased significantly after 1 mo in the ligated group. Neither cortical infarct nor cortical neuronal loss was observed histologically.. Common carotid artery ligation in Wistar rats caused a prolonged cerebral hypoperfusion without degeneration of the cortical neurons and a later decline of postsynaptic cholinergic receptor activity. These findings suggest that the decline in the postsynaptic cholinergic activity that is associated with the prolonged reduction in the cerebral blood supply may reflect pathophysiology that is equivalent to the deterioration of cognitive function in patients with chronic cerebrovascular insufficiency.

Topics: Animals; Benzilates; Brain; Brain Ischemia; Carbon Radioisotopes; Carotid Artery, Common; Cerebrovascular Circulation; Ligation; Male; Muscarinic Antagonists; Oxygen Radioisotopes; Piperidines; Radiopharmaceuticals; Rats; Rats, Wistar; Receptors, Muscarinic; Time Factors; Tomography, Emission-Computed; Water

We studied the cerebral muscarinic acetylcholinergic receptor (mACh-R) by means of 11C-N-methyl-4-piperidyl benzilate (11C-NMPB) and positron emission tomography (PET) in Alzheimer's disease (AD) cases, and the findings were compared with the cerebral blood flow (CBF) and the glucose metabolism (CMRGlc) to evaluate the relationship between the mACh-R and the CBF or the CMRGlc. The subjects consisted of 18 patients with AD and 18 age and sex matched normal volunteers. The patients were clinically diagnosed according to the criteria of the NINDS-ADRDA as having "probable AD" and were thus classified into two groups (mild and moderate AD) according to the severity of dementia determined by DSM-III-R. The CBF was measured by 99mTc-HMPAO SPECT, and the CMRGlc was measured by 18FDG PET. The 11C-NMPB uptake was evaluated by the graphical method and the ratio method (ROIs/Cerebellum). A significant mACh-R decrease and more severe CMRGlc decrease in the cortical region was seen in mild and moderate AD. The decrease in the CBF was not as obvious as that in the mACh-R and the CMRGlc. Our study thus suggested that the mACh-R decreased in patients with AD, and that the 18FDG PET was the most sensitive method for detecting the degenerative regions in patients with AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Benzilates; Brain; Carbon Radioisotopes; Case-Control Studies; Cerebrovascular Circulation; Female; Glucose; Humans; Male; Middle Aged; Piperidines; Receptors, Muscarinic; Tissue Distribution; Tomography, Emission-Computed

Quantification of human brain muscarinic cholinergic receptors was investigated with the use of [11C]N-methyl-4-piperidyl benzylate (NMPB) and positron emission tomography (PET). Whole-brain uptake of NMPB at 90 to 110 minutes after intravenous injection was approximately 10% of the administered dose. The initial cerebral distribution of NMPB corresponded to the pattern of cerebral perfusion; however, at progressively longer postinjection intervals, regional distinctions consistent with muscarinic receptor binding were evident: activity at 90 to 110 minutes postinjection was highest in the striatum and cerebral cortex, intermediate in the thalamus and pons, and lowest in the cerebellum. After the development of a chromatographic system for isolation of authentic [11C]NMPB in plasma, tracer kinetic modeling was used to estimate receptor binding from the cerebral and arterial plasma tracer time-courses. Ligand transport rate and receptor-binding estimates were obtained with the use of compartmental models and analytical methods of varying complexity, including a two-parameter pixel-by-pixel-weighted integral approach and regional least-squares curve-fitting analyses employing both two- and three-compartment model configurations. In test-retest experiments, precision of the methods and their abilities to distinguish altered ligand delivery from binding in occipital cortex during an audiovisual presentation were evaluated. Visual stimulation increased the occipital blood-to-brain NMPB transport rate by 25% to 46% in estimates arising from the various approaches. Weighted integral analyses resulted in lowest apparent transport changes and in a concomitant trend toward apparent binding increases during visual activation. The regional least-squares procedures were superior to the pixel-by-pixel method in isolating the effects of altered tracer delivery from receptor-binding estimates, indicating larger transport effects and unaltered binding. Precision was best (less than 10% test-retest differences) for the weighted integral analyses and was somewhat lower in the least-squares analyses (10-25% differences). The authors conclude that pixel-by-pixel-weighted integral analyses of NMPB distribution introduce transport biases into receptor-binding estimates. Similar confounding effects also are predicted in noncompartmental analyses of delayed radiotracer distribution. The use of regional nonlinear least-squares fitting to two- and three-compartment models, although

Topics: Adult; Benzilates; Biological Transport; Brain; Carbon Radioisotopes; Female; Humans; Male; Occipital Lobe; Parasympathomimetics; Piperidines; Receptors, Muscarinic; Sensitivity and Specificity; Tissue Distribution; Tomography, Emission-Computed

To assess muscarinic acetylcholine receptors (mAChRs) in the brains of patients with progressive supranuclear palsy and Parkinson's disease, and to correlate the cholinergic system with cognitive function in progressive supranuclear palsy and Parkinson's disease.. Positron emission tomography (PET) and [11C]N-methyl-4-piperidyl benzilate ([11C]NMPB) was used to measure mAChRs in the brain of seven patients with progressive supranuclear palsy, 12 patients with Parkinson's disease, and eight healthy controls. All of the patients with progressive supranuclear palsy were demented. The Parkinson's disease group consisted of 11 non-demented patients and one demented patient. The mini mental state examination (MMSE) was used to assess the severity of cognitive dysfunction in all of the subjects. The modified Wisconsin card sorting test (WCST) was used to evaluate frontal cognitive function in the non-demented patients with Parkinson's disease and controls.. The mean K3 value, an index of mAChR binding, was significantly higher for the frontal cortex in the patients with Parkinson's disease than in the controls (p<0.01). By contrast, the patients with progressive supranuclear palsy had no significant changes in the K3 values of any cerebral cortical regions. The mean score of the MMSE in the progressive supranuclear palsy group was significantly lower than that in the control group. Although there was no difference between the Parkinson's disease and control groups in the MMSE, the non-demented patients with Parkinson's disease showed significant frontal lobe dysfunction in the WCST.. The increased mAChR binding in the frontal cortex of the patients with Parkinson's disease may reflect denervation hypersensitivity caused by loss of the ascending cholinergic input to that region from the basal forebrain and may be related to frontal lobe dysfunction in Parkinson's disease. The cerebral cortical cholinergic system may not have a major role in cognitive dysfunction in progressive supranuclear palsy.

Topics: Aged; Benzilates; Brain; Brain Mapping; Carbon Radioisotopes; Cholinergic Fibers; Female; Humans; Male; Middle Aged; Neuropsychological Tests; Parasympatholytics; Parkinson Disease; Piperidines; Receptors, Cholinergic; Receptors, Muscarinic; Supranuclear Palsy, Progressive; Tomography, Emission-Computed

4-N-Methylpiperidyl benzilate (NMPB), a high affinity antagonist for the muscarinic cholinergic receptor, has been synthesized in carbon-11-labeled form through the N-[11C]methylation of 4-piperidylbenzilate. The product was isolated by HPLC, and obtained in yields (> 100 mCi) and specific activities (500-3000 Ci/mmol) sufficient for in vivo evaluation in small animals. Time-dependent regional brain distributions in rats and mice showed high radiotracer uptake and retention in striatum and cortex, and low in cerebellum, consistent with muscarinic cholinergic receptor distributions. Radiotracer retention in tissues could be significantly reduced by pretreatment of animals with a large dose of a competing antagonist, quiniclidinyl benzilate. Whole body biodistribution in rats was used to calculate the expected human internal radiation dosimetry for this new radiopharmaceutical. These animal experiments formed the basis for subsequent introduction of [11C]NMPB into human use with positron emission tomography.

Topics: Animals; Benzilates; Carbon Radioisotopes; Female; Male; Mice; Muscarinic Antagonists; Piperidines; Radiation Dosage; Rats; Rats, Sprague-Dawley; Tissue Distribution

The reaction of propiverinhydrochloride (1) and 1-methyl-4-piperidyl benzilate (2) with the biomimetic system manganese(III)-5,10,15,20-tetrakis(pentafluorophenyl)porphyrin chloride (MnTPFPPCl)/pyridine/hydrogen peroxide affords unchanged 1 and 2 and 15 potential metabolites, which were isolated and identified. These compounds are products of cleavage of the ester bond, of O-dealkylation, aromatic oxidation, respectively of decarboxylation, demethylation, and N-oxidation, Products were identified by TLC, UV, and MS in comparison with authentic samples. Thereby we found a nearly conformity with rat metabolism.

Topics: Animals; Benzilates; Chromatography, Thin Layer; Mass Spectrometry; Oxidation-Reduction; Piperidines; Rats; Spectrophotometry, Ultraviolet

It was previously found that alcuronium increases the binding of [3H]methyl-N-scopolamine to cardiac muscarinic receptors by a positive allosteric action while its effect on the binding of [3H]quinuclidinyl benzilate is negative. The, features of the antagonist's molecule which decide whether its allosteric interaction with alcuronium is positive or negative are not known. In the present work, it was found that alcuronium has a positive allosteric effect also on the binding of [3H]atropine and [3H]methyl-N-piperidinyl benzilate to muscarinic receptors in rat heart atria and that its effect on the binding of [3H]methyl-N-quinuclidinyl benzilate is negative. A comparison of the five radiolabelled antagonists that have been investigated so far indicates that the type of allosteric interaction (positive or negative) is not determined by the presence or absence of the quaternary nitrogen or of the benzilyl moiety in the molecule of the antagonist. Apparently, features of the N-bearing moiety of muscarinic antagonists other than the presence of a charge on nitrogen play a key role in the determination of the type of interaction.

Topics: Alcuronium; Allosteric Regulation; Animals; Atropine; Benzilates; In Vitro Techniques; Male; Muscarinic Antagonists; Myocardium; Piperidines; Quinuclidinyl Benzilate; Rats; Rats, Wistar; Receptors, Muscarinic

Topics: Animals; Benzilates; Biopterins; Brain; Carbon Radioisotopes; Macaca mulatta; Male; Molecular Structure; Organ Specificity; Piperidines; Receptors, Muscarinic; Tomography, Emission-Computed

The effects of age on the binding parameters of [11C]N- methyl-4-piperidylbenzilate ([11C]NMPB), a specific muscarinic cholinergic receptor ligand, were studied. Eighteen healthy male volunteers (18-75 years old) participated. Regional radioactivity in the brain was followed for 60 min by positron emission tomography (PET). Uptake of [11C]NMPB continuously increased in all brain areas with the exception of the cerebellum. For the quantification of receptor binding, a compartment model, in which radioactivity in the cerebellum was used as an input function, was used. The binding parameter, K3, of muscarinic acetylcholine receptors in eight brain regions (pons, hippocampus, frontal cortex, striatum, temporal cortex, thalamus, occipital cortex, parietal cortex) showed an age-related decrease of about 45% over the age range.

Topics: Adolescent; Adult; Aged; Aging; Benzilates; Brain Chemistry; Humans; Ligands; Magnetic Resonance Imaging; Male; Middle Aged; Parasympatholytics; Piperidines; Receptors, Cholinergic; Tomography, Emission-Computed

Plasma concentrations of the anticholinergic diphenylmethane derivative N-methyl-4-piperidinylbenzilate (Po;1) in rats after i.v. and p.o. administration of the drug and in 6 rabbits after i.v. administration of the drug were estimated by a gc capillary column technique in connection with detection by thermoionisation. Furthermore, after i.v. administration of the drug its concentrations in different tissues (heart, lung, liver, kidneys and brain) were estimated in rats. The results show a fast absorption of the drug after p.o. administration and also a fast decrease of 1 plasma concentrations in all plasma concentration curves obtained indicating a fast distribution of 1 in deeper tissue compartments. A high affinity especially to the lung and the heart can be derivated from the high 1 tissue concentrations in the rat. A common feature of all 1 plasma concentration curves obtained is the repeated appearance of concentration peaks which is particularly marked in the individual 1 plasma curves at the rabbits. We observed great interindividual differences in the pharmacokinetic behaviour. Furthermore, some plasma concentrations are below the detection limit of the analytical method used. These are the reasons why curve fitting to usual pharmacokinetic compartment models and the determination of parameters i.e. AUC and MRT is not suitable. However, the results agree with the assumption of a multi-compartment model at which the drug is periodical released in the general circulation. Interactions with biological membranes resulting out of the amphiphily of the drug are some possible reasons for the outstanding pharmacokinetic behaviour.

Topics: Administration, Oral; Animals; Benzilates; Female; Injections, Intravenous; Male; Piperidines; Rabbits; Rats; Rats, Inbred Strains; Species Specificity

The presence and properties of muscarinic receptors on intact rat thymocytes have been studied by the use of the muscarinic antagonists [3H]-3-quinuclidinyl benzilate (3H-QNB), [3H]-4-N-methylpiperidinyl benzilate ([3H]-NMBP) and [3H]-N-methylscopolamine (3H-NMS). The course of binding of 3H-QNB reveals a maximum at 5 min, and shows a subsequent decrease of bound radioactivity, suggesting internalization of the receptor 3H-3-QNB complex. This phenomenon has also been studied by the use of another muscarinic antagonist, 3H-NMPB, which has a faster on and off rate than 3H-QNB, and which may be rapidly displaced by an excess of unlabeled muscarinic antagonist, atropine. Bound 3H-NMPB is highly susceptible to atropine displacement only within the first two minutes of incubation with thymocytes at 37 degrees C. The kinetics of binding of the less lipophilic muscarinic antagonist 3H-NMS to thymocytes, show no maximum as a function of incubation time and 3H-NMS is susceptible to displacement by atropine up to 30 min incubation time with the thymocytes. These data suggest that binding of lipophilic benzilate type antagonists (3H-QNB and 3H-NMPB) may be followed by internalization and/or isomerization of the receptor-benzilate antagnist complex while the 3H-NMS-receptor complex is not subject to these processes.

Topics: Animals; Atropine; Benzilates; Kinetics; Male; Muscarine; N-Methylscopolamine; Piperidines; Quinuclidinyl Benzilate; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives; Thymus Gland

Enpiperate, 4-(3, 5)-exocydopropyl-N-methylpiperidyl diphenyl hydroxy acetate, was synthesized in China. It has been shown to have no effect on the normal tension of rabbit aorta strips or contractions induced by NE. It inhibited the contraction of high K+-depolarized rabbit aorta strips at concentrations of 3.8 and 10.9 mumol/L. The same effect was seen on isolated guinea pig ileum, at a concentration of 10.9 mumol/L, which was depolarized by Ca2+ free high K+ Locke-Ringer's solution and the contraction was extragenous Ca2+ dependent. The inhibition was overcome by increasing the extracellular Ca2+ concentration. In the presence of EDTA (0.1 mmol/L), the NE induced intracellular Ca2+ dependent contraction was inhibited by enpiperate (3.8, 10.9 mumol/L), although no significant difference was found between enpiperate and papaverine. It is suggested that enpiperate is a calcium antagonist and that the antagonism is carried out by blocking the potential dependent calcium channel (PDC) in the membrane of vascular smooth muscles. A intracellular calcium antagonistic action may also be involved.

Topics: Animals; Aorta; Benzilates; Calcium Channel Blockers; Guinea Pigs; Ileum; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Muscle, Smooth, Vascular; Piperidines; Rabbits

Chemical modification of muscarinic M1 receptors in a synaptoneurosomal preparation of rat cerebral cortex by a hydrophilic histidyl-group-specific reagent, diethylpyrocarbonate (DEP), reduces the number of [3H]-4NMPB binding sites in a dose-dependent way. The effect can be reversed by hydroxylamine treatment. No such effect is observed when carbethoxylation with 2.5 mM DEP is carried out in the presence of atropine, 4NMPB, pirenzepine or carbachol. These findings indicate that DEP specifically modifies histidyl residue(s) positioned at the binding site in members of the M1 receptor family. However, treatment with 2.5 mM DEP in the presence of various muscarinic ligands significantly disturbs the binding state of agonists. The results suggest that M1 receptors may have more than one histidyl residue of importance in ligand binding.

Topics: Animals; Atropine; Benzilates; Binding Sites; Carbachol; Cerebral Cortex; Diethyl Pyrocarbonate; Histidine; Hydrogen-Ion Concentration; Oxotremorine; Piperidines; Rats; Receptors, Muscarinic; Synaptosomes

The effects of the voltage-sensitive sodium channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors were studied in homogenates of rat atria. Also studied were the effects of muscarinic ligands on the binding of tritium-labeled batrachotoxin ([3H]BTX) to the same preparation. BTX (1 microM), which induces an open state in sodium channels, enhanced the affinity of binding of several agonists to the muscarinic receptors. Analysis of the data indicated that the effect of BTX was to increase the affinity of the agonists toward the high-affinity sites. Binding of antagonists was not affected by BTX. At higher concentrations of toxin, the density of the high affinity muscarinic sites was also affected. The binding of agonists (but not of antagonists) to muscarinic receptors in turn enhanced the specific binding of [3H]BTX to sodium channels. These effects on the muscarinic receptors and on the sodium channels were inhibited in the presence of Gpp(NH)p at concentrations lower than those bringing about conversion of binding sites from the high affinity to the low affinity conformation. On the basis of these findings we suggest that the opening of sodium channels and the binding of agonists to muscarinic receptors in rat atrial membranes are coupled events which are mediated by guanine nucleotide-binding protein(s). Such a hypothesis is consistent with previously proposed models for signal transduction in the membrane.

Topics: Acetylcholine; Animals; Batrachotoxins; Benzilates; Carbachol; Dibucaine; Dose-Response Relationship, Drug; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Heart Atria; Ion Channels; Kinetics; Male; Mathematics; Myocardium; Oxotremorine; Piperidines; Rats; Receptors, Muscarinic; Sodium; Veratridine

Topics: Alkaloids; Anesthesia, General; Anesthesia, Intravenous; Animals; Antipsychotic Agents; Benzilates; Berberine Alkaloids; Diazepam; Dogs; Drug Interactions; Etorphine; Female; Male; Mice; Morphinans; Piperidines; Rabbits

The expression of muscarinic acetylcholine binding sites and of cholinesterases was studied in extracts prepared from discrete regions of the human fetal brain, between the gestational ages of 14 and 24 weeks. The specific binding of [3H]N-methyl-4-piperidyl benzilate [( 4H]-4NMPB) to muscarinic binding sites ranged between 0.05 and 1.30 pmol/mg protein in the different brain regions, with Kd values of 1.2 +/- 0.2 nM. Binding of the cholinergic agonist oxotremorine fitted, in most of the brain regions examined, with a two-site model for the muscarinic binding sites. The density of muscarinic binding sites increased with development in most regions, with different rates and onset times. It was higher by about sixfold in some areas destined to become cholinergic, such as the cortex and midbrain, than in noncholinergic areas such as the cerebellum. In other areas destined to become cholinergic, such as the hippocampus and the caudate putamen, the receptor density remained low. Average density values increased from 0.1 +/- 0.1 at 14 weeks up to 0.7 +/- 0.4 pmol/mg protein at 24 weeks. The variability in the specific activities of cholinesterase was relatively low, and extracts from different brain regions hydrolyzed from 5 to 30 nmol of [3H]acetylcholine/min/mg protein. These were mostly "true" acetylcholinesterase (EC 3.1.1.7) activities, inhibited by 10(-5) M BW284C51, with minor pseudocholinesterase (EC 3.1.1.8) activities, inhibited by 10(-5) M iso-OMPA. The enzyme from different brain regions and developmental stages displayed similar Km values toward [3H]acetylcholine (ca. 4 X 10(-4) M-1). The ontogenetic changes in cholinesterase specific activities had no unifying pattern and/or relationship to the cholinergic nature of the various brain areas. In most of the brain regions, the arbitrary ratio between the specific activity of cholinesterase and the density of muscarinic binding sites decreased with development, with average values and variability ranges of 83 +/- 50 and 19 +/- 19 at 14 and 24 weeks, respectively. Our findings suggest divergent regulation for cholinergic binding sites and cholinesterase in the fetal human brain and imply that the expression of muscarinic receptors is related to the development of cholinergic transmission, while acetylcholinesterase is also involved in other functions in the fetal human brain.

Topics: Acetylcholine; Acetylcholinesterase; Benzilates; Brain; Embryonic and Fetal Development; Gestational Age; Humans; Kinetics; Neurons; Oxotremorine; Piperidines; Receptors, Muscarinic

The effects of the culture's age and of liposome treatments on the properties of muscarinic receptors in cultured rat heart myocytes prepared from the hearts of newborn (1-3 days old) rats were investigated. In these studies we investigated the binding characteristics of antagonists and agonists to the myocyte muscarinic receptors in young (5 days after plating) vs. older (14 days after plating) cultures. Our findings demonstrate that the aging of the cells in culture is accompanied by a reduction in the muscarinic binding capacity and by alterations in the proportion of high- and low-affinity states toward muscarinic agonists, as well as by striking changes in the mode of coupling of the receptors with guanine nucleotide binding protein(s) [G protein(s)]. The above effects of the culture's age occur concomitantly with alterations in the lipid composition of the cultured myocytes (in 14-day old cultures, the phosphatidylcholine/sphingomyelin ratio is reduced, and the cholesterol level is elevated). In order to explore whether the lipid composition is involved in the mechanism that alters the properties and coupling of the muscarinic receptors, we treated aging cultures with liposomes containing egg phosphatidylcholine. This treatment resulted in 14 day old cultures with a lipid composition similar to that of young cultures, and the treated myocytes demonstrated muscarinic receptor properties similar to those of young myocyte cultures. The implications for the role of membrane lipid composition and organization in determining the properties of the muscarinic receptors and their coupling with G proteins are discussed.

Topics: Acetylcholine; Animals; Animals, Newborn; Benzilates; Carbachol; Cells, Cultured; Guanylyl Imidodiphosphate; Kinetics; Liposomes; Myocardium; Oxotremorine; Piperidines; Rats; Receptors, Muscarinic

The effects of Na+-channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors in homogenates of rat brain and heart were studied. BTX enhanced the affinity for the binding of the agonists carbamoylcholine and acetylcholine to the muscarinic receptors in brainstem and ventricle, but not in the cerebral cortex. Analysis of the data according to a two-site model for agonist binding indicated that the effect of BTX was to increase the affinity of the agonists to the high-affinity site. Guanyl nucleotides, known to induce interconversion of high-affinity agonist binding sites to the low-affinity state, canceled the effect of BTX on carbamoylcholine and acetylcholine binding. BTX had no effect on the binding of the agonist oxotremorine or on the binding of the antagonist [3H]-N-methyl-4-piperidyl benzilate. The local anesthetics dibucaine and tetracaine antagonized the effect of BTX on the binding of muscarinic agonists at concentrations known to inhibit the activation of Na+ channels by BTX. On the basis of these findings, we propose that in specific tissues the muscarinic receptors may interact with the BTX binding site (Na+ channels).

Topics: Aconitine; Animals; Batrachotoxins; Benzilates; Binding, Competitive; Brain; Carbachol; Guanylyl Imidodiphosphate; Ion Channels; Kinetics; Macromolecular Substances; Male; Myocardium; Piperidines; Rats; Receptors, Muscarinic; Sodium; Veratridine

The affinity of selected antipsychotic and antidepressant drugs for the muscarinic receptor was studied in membranes from both human and rat striatum and cerebral cortex. While there are regional differences in the anticholinergic potency of the drugs, there is good agreement between the obtained inhibition constants from the corresponding human and rat striatum (r: 0.98) and from human and rat cerebral cortex (r: 0.96). There is also good agreement between the obtained Ki values within one species: human cerebral cortex versus human striatum (r: 0.99) and for rat cerebral cortex and rat striatum (r: 0.87). Thus, the previously published quantitative estimates of the antimuscarinic activity of psychoactive drugs which were derived from studies on membranes from rat brain give an accurate estimate of the antimuscarinic activity in human brain. The drugs tested in this study include chlorpromazine acetophenazine, haloperidol, sulpiride, remoxipride (FLA-731 (-), a substituted benzamide), amitriptyline and two serotonin uptake blockers: norzimelidine and alaproclate.

Topics: Aged; Animals; Antidepressive Agents; Antipsychotic Agents; Benzilates; Cerebral Cortex; Corpus Striatum; Humans; In Vitro Techniques; Male; Parasympatholytics; Piperidines; Psychotropic Drugs; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Species Specificity

Topics: Animals; Avoidance Learning; Benzilates; Conditioning, Psychological; Discrimination Learning; Female; Glycolates; Male; Parasympatholytics; Piperidines; Quinuclidines; Quinuclidinyl Benzilate; Rats

The mode of interaction of bisquaternary pyridinium oximes with rat brain muscarinic receptors in cerebral cortex and brain stem preparations was studied by the use of the tritium-labeled antagonist N-methyl-4-piperidyl benzilate ( [3H] 4NMPB). Binding of the labeled muscarinic antagonist was inhibited by these drugs, the most potent inhibitors being 1-(2-hydroxyiminoethylpyridinium)-1-(3-cyclohexylcarboxypyridin ium)dimethyl-ether (HGG-42) and its 3-phenylcarboxypyridinium analog (HGG-12) (apparent KI = 1.3-1.7 and 1.8-2.2 microM, respectively). Analysis of the binding properties suggested that binding of the muscarinic antagonist and the bisquaternary pyridinium oximes was nonexclusive. Kinetic binding data provide evidence that the drugs inhibit binding of muscarinic antagonists in an allosteric manner, with a resulting decrease in the rates of both association of [3H]4NMPB to the receptor and its dissociation from it. These effects were observed both in brain stem and in cortical preparations even after pretreatment and washing out of the inhibitors. The selective natures of HGG-12 and HGG-42 were apparent from their irreversible effects on the number of muscarinic binding sites. In brain stem, the presence of these drugs resulted in a loss of about 30% of binding sites, which accounts in part for the apparent decrease in maximal binding capacity observed in the equilibrium binding of [3H]4NMPB. In the cortex, however, only approximately 10% of the muscarinic receptors were lost upon exposure to these drugs. The decrease in the muscarinic receptor population of the brain stem was dependent on both concentration and time and occurred both in vitro and in vivo following injection of HGG-12 into rats. Unlike the in vitro loss of receptor sites, which was irreversible, the in vivo effect was restored 2 hr after the injection. Taken together, the results suggest that the bisquaternary oximes are allosteric inhibitors of the muscarinic acetylcholine receptor and may be capable of distinguishing between receptor states and inducing specific irreversible effects. Because of these properties, the drugs may prove extremely useful as sensitive probes in studies on the nature of the agonist-receptor-effector relationship.

Topics: Animals; Benzilates; Brain; Dose-Response Relationship, Drug; Kinetics; Mathematics; Obidoxime Chloride; Oximes; Piperidines; Pyridinium Compounds; Rats; Receptors, Muscarinic

The role of the functional substituents on the pyridinium ring of bisquaternary pyridinium compounds, mostly oximes, in exerting reversible and irreversible inhibition of binding of [3H]-N-methyl-4-piperidyl benzilate [( 3H]-4NMPB) to rat brain stem muscarinic receptors was studied. The drugs tested, i.e. HGG-42, HGG-12, HGG-52, HI-6, obidoxim, SAD-128 and TMB-4, could reversibly inhibit binding of [3H]-4NMPB, with the highest potency (KI = 1.7 - 6 microM) exhibited by analogs possessing hydrophobic substituents at position 3 or 4 of the pyridinium ring. Bisquaternary drugs possessing an oxime moiety at position 2, but not at position 4 of the pyridinium ring, could also induce about 30% reduction of maximal binding capacity (Bmax) (loss of muscarinic receptors) in addition to their reversible effect. Thus the structural correlates of the reversible and the irreversible effects of these drugs are different.

Topics: Animals; Benzilates; Binding, Competitive; Brain Stem; Cell-Free System; Obidoxime Chloride; Oximes; Parasympatholytics; Piperidines; Pyridinium Compounds; Rats; Receptors, Muscarinic; Structure-Activity Relationship; Trimedoxime

We propose a method for obtaining kinetic parameters of unlabeled ligands which is based on analyzing the effect of their competition on the binding kinetics of a labeled ligand. In this method (competition kinetics), the binding kinetics of a labeled ligand are measured in the absence and in the presence of given concentrations of a competing unlabeled ligand. The rate equations appropriate to the particular kinetic model may be solved employing linear homogeneous differential equations. The rate constants appearing in the final integrated expressions can be evaluated by nonlinear regression. We have demonstrated the validity and applicability of the method to study receptor systems, using the muscarinic receptors in rat brain cortex as an experimental system. We have determined the various kinetic parameters of two muscarinic antagonists (N-methyl-4-[3H]piperidyl benzilate and (-)-N-[3H]methylscopolamine) by following directly their binding kinetics. These parameters were compared with those obtained for the same unlabeled ligands in homogeneous and heterogeneous competition studies. In all cases, the parameters obtained by the competition kinetics method were close to those extracted from direct binding kinetics, and in cases where both types of studies were performed on the same preparations, the parameters obtained by the two methods were essentially identical.

Topics: Animals; Benzilates; Binding, Competitive; Cerebral Cortex; In Vitro Techniques; Kinetics; Male; N-Methylscopolamine; Parasympatholytics; Piperidines; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine Derivatives

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-[3H]piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. In contrast, the major differences between the kinetic binding parameters of agonists and antagonists to the low affinity agonist binding sites are in the association rate constants, which were 2-5 orders of magnitude lower for agonists. This demonstrates that there are basic differences in the interactions of agonists with the low and high affinity sites. Our findings also suggest that isomerization of the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.

Topics: Animals; Benzilates; Binding Sites; Binding, Competitive; In Vitro Techniques; Kinetics; Male; Medulla Oblongata; Parasympathomimetics; Piperidines; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Tritium

The relationship between the steroid hormone 17 beta-estradiol and the muscarinic cholinergic receptors present in the preoptic area (POA), median hypothalamus and posterior hypothalamus of female rats was examined in vitro at various stages of the estrous cycle. Muscarinic receptors varied in a cyclic manner, specifically in the POA, as shown by an increase in the proportion of high-affinity agonist binding sites (RH) to 60% during the proestrus, as compared to RH proportion observed during diestrus-2 and during the afternoon of proestrus (35%). Exposure of POA homogenates to 17 beta-estradiol resulted in conversion of RH to low-affinity agonist binding sites (RL). This effect of the hormone was also restricted to the POA taken from rats during the morning of proestrus. It was blocked by the antiestrogenic drug, clomiphene, and could be prevented by preoccupation of the muscarinic receptors by their own ligands prior to the addition of hormone. It follows that significant changes in POA muscarinic receptors in situ exactly coincided with the known critical time period characterized by high estrogen levels and high levels of estrogen receptors in the POA. These changes in muscarinic receptors might thus conceivably reflect variations in cholinergic activity in the POA during the estrous cycle.

Topics: Animals; Benzilates; Binding, Competitive; Clomiphene; Estradiol; Estrus; Female; Hypothalamus; Oxotremorine; Piperidines; Pregnancy; Preoptic Area; Proestrus; Rats; Receptors, Muscarinic; Time Factors

The effect of Ca2+ on the biochemical characteristics of muscarinic receptors in the adenohypophysis of male and female rats at the various stages of the estrous cycle was investigated in binding experiments using the specific muscarinic antagonist N-methyl-4-piperidyl benzylate ( [3H]-4NMPB) and the muscarinic agonist oxotremorine. By using Ca2+ chelators such as EGTA, and Ca2+ channel blockers such as D-600, we showed that Ca2+ profoundly alters the binding characteristics of both antagonists and agonists to the muscarinic receptors. In female rats the effect of Ca2+ on antagonist binding is mainly on the maximal binding capacity of the receptors, while changes in the dissociation constants are much more moderate. The effect is expressed in the ability of Ca2+ to expose or to eliminate binding sites as a function of the estrous cycle. In agonist binding, the presence of Ca2+ has a pronounced effect on the proportion of high-affinity binding sites, which parallels the changes induced in antagonist binding throughout the estrous cycle. Interestingly, the natural progression of the cycle from diestrus 2 to the estrous stage undergoes a change identical to that occurring in vitro upon Ca2+ removal. D-600 can completely block the effect of Ca2+ on the binding of both [3H]-4NMPB and oxotremorine. The concentration of D-600 required in order to induce such blocking is also dependent on the estrous cycle. It appears that the progression of the estrous cycle is accompanied by changes in the muscarinic receptors which may in turn be coupled to Ca2+ channels.

Topics: Animals; Benzilates; Binding Sites; Binding, Competitive; Calcium; Calcium Channel Blockers; Diestrus; Estrus; Female; Gallopamil; Male; Oxotremorine; Piperidines; Pituitary Gland, Anterior; Pregnancy; Proestrus; Rats; Receptors, Muscarinic

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.

Topics: Animals; Benzilates; Carbachol; Cell Membrane; Cerebral Cortex; Cobalt; Fatty Acids; Guanosine Triphosphate; Medulla Oblongata; Piperidines; Rats; Receptors, Muscarinic; Structure-Activity Relationship

The interaction of the antifibrillatory antiarrhythmic drug, bretylium tosylate, with the muscarinic receptor in tissue homogenates from regions of rat brain and heart was investigated. Competition-binding experiments were carried out with the highly specific tritiated antagonist N-methyl-4-piperidyl benzilate. Bretylium tosylate competitively displaced the labeled antagonist from the muscarinic receptor. The binding of the drug to the two brain preparations was found to be best fitted by a one-site model in each case. On the other hand, in the case of both heart preparations, a two-site model yielded a significantly better fit for the binding data than that given by a single-site model. The low affinity-binding constants in the atrium and the ventricle were similar (approximately 10 microM) to those in the brain regions examined, namely, the cortex and the medullapons. Sites with relatively higher affinity for the drug were detected in the heart only, with equilibrium-binding constants of 0.24 +/- 0.12 microM and 0.97 +/- 0.27 microM for the atrium and the ventricle, respectively. The drug also exerted anti-acetylcholine activity (K1 = 14 +/- 2 microM) measured physiologically in the guinea pig atrium, which correlated well with the concentration of the drug observed to be efficacious clinically (approximately 10 microM).

Topics: Animals; Anti-Arrhythmia Agents; Benzilates; Binding, Competitive; Brain; Bretylium Compounds; Bretylium Tosylate; Cerebral Cortex; Drug Interactions; In Vitro Techniques; Male; Medulla Oblongata; Myocardium; Piperidines; Rats; Receptors, Muscarinic

We have employed a method based on ligand competition experiments, which is capable of detecting interactions among ligand-occupied binding sites, to study the interactions between rat adenohypophysis muscarinic receptors occupied by several muscarinic antagonists. In this method, one examines the binding of a labeled ligand (the primary ligand) in the absence and presence of a competing ligand. The inhibition of binding of the primary ligand by the competing ligand shows significant deviations from that expected assuming a population of noninteracting, heterogeneous binding sites. The deviations seen in the case of competition between N-methyl-4-piperidyl benzilate (4NMPB) and (-)-N-methyl scopolamine (a benzilate and tropate) are more pronounced than in the case of 4NMPB and (-)-3-quinuclidinyl benzilate (two benzilate derivatives). The occurrence of such deviations suggests the existence of site-site interactions among rat adenohypophysis muscarinic receptors. On the other hand, no deviations were observed in competition experiments in homogenates of rat cortex and medulla-pons. This finding correlates with the linear Scatchard plots (with no indications for site-site interactions or heterogeneity) obtained for the binding of muscarinic antagonists in these brain regions. A mathematical analysis demonstrates that the deviations from the expectations of the site-heterogeneity model observed in the rat adenohypophysis system (which shows similar binding patterns for all ligands employed) can occur only if the primary and competing ligands induce different conformational transitions upon binding to the receptor. It is concluded that different muscarinic antagonists can lead to different isomerization states of the receptor in the system.

Topics: Allosteric Regulation; Animals; Benzilates; Binding, Competitive; In Vitro Techniques; Male; Models, Chemical; N-Methylscopolamine; Parasympatholytics; Piperidines; Pituitary Gland, Anterior; Protein Conformation; Quinuclidinyl Benzilate; Rats; Receptors, Muscarinic; Scopolamine Derivatives

Competition between 3H-3-quinuclidinyl benzilate (2 and 18 nM) and varying concentrations of antagonists (4-N-methyl piperidinyl benzilate (4-NMPB), atropine, scopolamine and pirenzepine (0-1000 nM) and agonists (acetylcholine, carbamylcholine, oxotremorine and pilocarpine (0.10(-10) to 10(-2) M)) was studied. Inhibition of specific 3H-3-quinuclidinyl benzilate binding was evaluated by Dixon plots which for both competing agonists and antagonists showed deviations from linearity. The best nonlinear least square fit to the Dixon plots was offered by a hyperbola, parabola or a straight line, depending on the ligand used. The data support a model of equilibrium binding which assumes that 3H-3-quinuclidinyl benzilate and one competing ligand molecule may simultaneously bind to the receptor at high concentrations of the competing ligand.

Topics: Animals; Atropine; Benzilates; Benzodiazepinones; Binding Sites; Binding, Competitive; In Vitro Techniques; Kinetics; Ligands; Male; Models, Biological; Piperidines; Pirenzepine; Quinuclidinyl Benzilate; Rats; Receptors, Cholinergic; Receptors, Muscarinic; Scopolamine

Highly potent photoaffinity probes for muscarinic binding sites were prepared by the incorporation of an azido group into the benzilic acid moiety in two compound, 3-quinuclidinyl benzilate (3QNB) and N-methyl-4-piperidyl benzilate (4NMPB). Inactivation of muscarinic sites in rat cortex depends on the formation of a reversible complex with the azides prior to their photolytic conversion to the highly reactive nitrenes. During photolysis, radiolabeled azido-4NMPB interacted specifically and with high affinity (Kd = 1.06 nM) with the muscarinic receptors, and the ligand could be covalently incorporated into a macromolecule of about 86,000 Mr, presumably the muscarinic receptor. The incorporation was almost stoichiometric when compared to determination of receptor density by reversible ligands. Atropine (10 microM) afforded specific protection (greater than 83%) of the receptor against inactivation by azido-[3H]4NMPB. This compound and the other ligands described here (i.e., amino-4NMPB, amino-3QNB, and azido-3QNB) represent powerful potential probes for the biochemical isolation and characterization of muscarinic receptors.

Topics: Affinity Labels; Animals; Azides; Benzilates; Binding, Competitive; Brain Chemistry; Parasympatholytics; Photochemistry; Piperidines; Quinuclidines; Quinuclidinyl Benzilate; Rats; Receptors, Cholinergic; Receptors, Muscarinic

Topics: Animals; Benzilates; Binding, Competitive; Carbachol; Cats; Gastrointestinal Hormones; Kinetics; Parasympatholytics; Piperidines; Receptors, Cholinergic; Receptors, Muscarinic; Submandibular Gland; Vasoactive Intestinal Peptide

Topics: Autoantibodies; Benzilates; Humans; Immunoglobulin G; Kinetics; Myasthenia Gravis; Parasympatholytics; Piperidines; Quinuclidinyl Benzilate; Receptors, Cholinergic; Receptors, Muscarinic

Topics: Animals; Benzilates; Male; Mathematics; Parasympatholytics; Piperidines; Pituitary Gland, Anterior; Quinuclidinyl Benzilate; Rats; Receptors, Cholinergic; Receptors, Muscarinic

Topics: Aging; Animals; Benzilates; Cerebellar Cortex; Heterozygote; Homozygote; Hyperbilirubinemia, Hereditary; Kinetics; Mice; Mice, Inbred Strains; Parasympatholytics; Piperidines; Rats; Rats, Gunn; Rats, Inbred Strains; Receptors, Cholinergic; Receptors, Muscarinic; Species Specificity

The highly specific tritiated muscarinic antagonist N-methyl-4-piperidyl benzilate [(3H)-4NMPB] was used in direct binding and in competition experiments with the muscarinic agonist oxotremorine to investigate the influence of in vivo endocrine manipulations involving estrogen on muscarinic receptors in the adenohypophysis and hypothalamus. In the adenohypophysis the characteristics of antagonist binding in ovariectomized female rats, like those in androgenized females, resembled those in normal male rats; this trend was reversed in ovariectomized females after implantation of 17 beta-estradiol capsules, with the characteristics of antagonist binding now resembling those in normal female rats at estrus. Agonist binding characteristics also showed some distinct differences between treated and normal female rats: the proportion of high affinity binding sites decreased both in ovariectomized and androgenized rats, while the affinity of these sites became greater as compared to control animals. The results clearly show that changes in the levels of sex steroids, as a result of the above-mentioned endocrine manipulations, influence the behavior of the muscarinic receptors in both areas, and suggest muscarinic participation in the regulation of gonadotropin release in the hypothalamus-pituitary-ovary axis.

Topics: Animals; Benzilates; Biomechanical Phenomena; Female; Gonadal Steroid Hormones; Hypothalamus; Male; Muscarine; Oxotremorine; Piperidines; Pituitary Gland, Anterior; Preoptic Area; Rats; Rats, Inbred Strains

Topics: Animals; Benzilates; Cerebral Cortex; Cholic Acids; Detergents; Kinetics; Piperidines; Rats; Receptors, Cholinergic; Receptors, Muscarinic; Solubility

Topics: Affinity Labels; Animals; Benzilates; Calcium; Female; Kinetics; Piperidines; Pituitary Gland, Anterior; Rats; Rats, Inbred Strains; Receptors, Cholinergic; Receptors, Muscarinic

The ligand binding properties of muscarinic receptors present in rat adenohypophysis were studied using the tritiated antagonists N-methyl-4-piperidyl benzilate, 3-quinuclidinyl benzilate, and methylscopolamine. Equilibrium analysis of antagonists binding as well as competition experiments with several agonists were used to probe the nature of binding sites. The nature of antagonist binding in the adenohypophysis points to heterogeneity of binding sites, in contrast to other brain regions, in which homogeneous populations of muscarinic receptors were detected. Some of the binding properties, e.g. affinity constants, population of high affinity agonist binding sites, etc., clearly indicated differences between male and female rats as well as differences in female rats at various stages of estrous cycle. The nature of these receptors and their possible physiological role are discussed.

Topics: Animals; Benzilates; Binding, Competitive; Estrus; Female; Kinetics; Male; Piperidines; Pituitary Gland, Anterior; Pregnancy; Rats; Receptors, Cholinergic; Receptors, Muscarinic; Sex Differentiation; Sex Factors

Topics: Animals; Benzilates; Estrus; Female; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Male; Oxotremorine; Parasympatholytics; Piperidines; Pituitary Gland, Anterior; Pregnancy; Rats; Receptors, Cholinergic; Receptors, Muscarinic; Sex Factors

The effects of L-T4 and betamethasone treatment of newborn mice on the development of the cholinergic muscarinic receptor in certain brain regions was studied using the potent labeled muscarinic antagonist [3H]4-N-methyl-piperidyl benzilate. Treatment with both L-T4 and betamethasone caused an accelerated accumulation of muscarinic receptors in the cortex 16 days post partum, with a subsequent reduction in level at 30 days. In the cerebellum and caudate putamen, only betamethasone caused a similar early accumulation of muscarinic receptors, while the later effect, namely a reduction in the level at 30 days, was seen with both hormones in these two regions as well as in the hippocampus. The results can explain some behavioral effects observed in other studies after treatment with these hormones.

Topics: Aging; Animals; Animals, Newborn; Benzilates; Betamethasone; Brain; Cerebellum; Cerebral Cortex; Kinetics; Mice; Parasympatholytics; Piperidines; Receptors, Cholinergic; Receptors, Muscarinic; Thyroxine

The ligand binding properties of muscarinic receptors present in whole hypothalamus as well as in its three regions (preoptic area, median and posterior regions) were studied in male rats and mice, as well as in female rats during various stages of the estrous cycle, using the tritiated antagonist N-methyl-4-piperidyl benzilate. Kinetic and equilibrium analysis of antagonist binding as well as competition experiments with agonists, were used to probe the nature of the binding sites and possible differences between specific muscarinic sites in the various preparations. We could detect differences in agonist binding parameters between male and female rats in the preoptic area. In female rats binding of agonist to high affinity state is characterized by a lower affinity (27 nM) than in males (3.7 nM). Secondly, the population of agonist high affinity sites at the proestrous stage is much higher than that at other stages of cycle (66 vs. 38%). In addition, the binding properties of muscarinic receptors following intracisternal 6-hydroxydopamine lesion were investigated. This treatment resulted in a decrease of about one sixth of the total muscarinic receptors in the hypothalamus. The data are best interpreted as a degeneration of existing presynaptic muscarinic receptors located on catecholamine terminals in the hypothalamus. The pre- and postsynaptic nature of the muscarinic receptors, their localization in view of their binding properties, and their possible physiological role are discussed.

Topics: Animals; Benzilates; Female; Hydroxydopamines; Hypothalamus; Kinetics; Male; Oxotremorine; Piperidines; Rats; Receptors, Cholinergic; Receptors, Muscarinic; Sex Factors; Species Specificity

Nine days after medial septal lesion a 20% increase in the number of muscarinic antagonist binding sites in rat hippocampus was observed without any change in the affinity for agonist or antagonist. Chronic atropine treatment (s.c. 5 mg/kg, twice a day for 14 days, 20 mg/kg once a day for 14 days or 100 mg/kg for 4 days and 20 mg/kg for 10 days, once daily) led to an increase in the number of muscarinic antagonist binding sites in rat hippocampus with 35, 80 and 80% respectively and also lowered the affinity for 3H-antagonists in a dose dependent manner. Agonist binding studies also indicated an increase in receptor number and a decrease in affinity. The latter change can possibly be explained by the presence of residual atropine 24 h after the last injection. If this is taken into account we may conclude that muscarinic supersensitivity evoked either by severing the input or by chronic pharmacologic blockade both produced "new receptors' with ligand binding properties similar to the original receptors.

Topics: Animals; Atropine; Benzilates; Cerebral Cortex; Corpus Striatum; Hippocampus; Hypothalamus; Male; Piperidines; Quinuclidinyl Benzilate; Rats; Rats, Inbred Strains; Receptors, Cholinergic; Receptors, Muscarinic; Septum Pellucidum

Topics: Animals; Benzilates; Brain; Female; Hypothalamus; Kinetics; Male; Mice; Oxotremorine; Parasympatholytics; Piperidines; Preoptic Area; Receptors, Cholinergic; Receptors, Muscarinic; Sex Factors; Thermodynamics; Tissue Distribution

Topics: Adrenal Cortex; Adrenal Medulla; Aging; Animals; Benzilates; Female; Hypothalamus; Organ Specificity; Piperidines; Pituitary Gland, Anterior; Preoptic Area; Rats; Rats, Inbred Strains; Receptors, Cholinergic; Receptors, Muscarinic

Characteristics of the binding mechanism to specific muscarinic sites were determined by means of high affinity binding of tritium labeled N-methyl-4-piperidylbenzilate (4-NMPB) to homogenate from various regions of mouse brain: cortex, caudate putamen, thalamus, hippocampus, medulla pons and cerebellum. In vivo experiments confirm results obtained when investigating regional distribution of the muscarinic receptors in vitro. Kinetics as well as equilibrium analysis of the binding in the different brain regions to probe the nature of the binding is described. The combined data strongly support the conclusion that mouse brain exhibits functional heterogeneity of muscarinic receptors.

Topics: Animals; Atropine; Benzilates; Binding, Competitive; Brain; Kinetics; Mice; Parasympatholytics; Piperidines; Receptors, Cholinergic; Receptors, Muscarinic; Scopolamine; Tissue Distribution

The kinetics of the association and dissociation of the radiolabeled high affinity muscarinic antagonist N-[3H]methyl-4-piperidinyl benzilate was studied with the receptor from smooth muscle. Neither the agonists carbamylcholine and oxotremorine nor the antagonist atropine did influence the dissociation rate (k-2) of the receptor . antagonist complex. Binding of the antagonist (A) takes place in two consecutive steps: (formula: see text). Agonists did not seem to influence the dissociation constant (k-1/k+1) of the first binding step, while atropine caused a large increase in this constant. These results indicate that while antagonist-antagonist competition can be explained in terms of competition for the free receptor (R), antagonist-agonist competition cannot be fitted with such a simple model.

Topics: Animals; Atropine; Benzilates; Binding, Competitive; Carbachol; Intestine, Small; Kinetics; Male; Muscle, Smooth; Oxotremorine; Parasympatholytics; Piperidines; Rats; Receptors, Cholinergic; Receptors, Muscarinic

Topics: Animals; Benzilates; Brain; Guanine Nucleotides; Guanosine Triphosphate; Magnesium; Male; Medulla Oblongata; Mice; Oxotremorine; Piperidines; Pons; Receptors, Cholinergic; Receptors, Muscarinic; Structure-Activity Relationship

Topics: Animals; Benzilates; Brain; Cerebral Cortex; Hydrogen-Ion Concentration; Medulla Oblongata; Mice; Oxotremorine; Parasympathomimetics; Piperidines; Pons; Protein Conformation; Receptors, Cholinergic; Receptors, Muscarinic; Structure-Activity Relationship; Temperature

The mechanism of binding of two antagonists, 3-quinuclidinyl benzilate and N-methyl-4-piperidinyl benzilate, to the muscarinic receptor was studied. The pseudo-first order rate constant of association showed a hyperbolic dependence on the concentration of the antagonist(s) indicating that the interaction involves two equilibria. The first binding equilibrium is reached rapidly and is characterized by dissociation constants 2.7 +/- 0.4 nM and 6.7 +/- 2.5 nM in phosphate buffer (0.05 M, pH = 7.4) for 3-quinuclidinyl benzilate and N-methyl-4-piperidinyl benzilate, respectively. The first binding equilibrium is followed by a slower isomerization step of the receptor . antagonist complex. The equilibrium constants for the isomerization step of the complex for both ligands were about 0.15. The overall constant of binding obtained as the product of the above constants shows good agreement with the results of equilibrium binding studies.

Topics: Animals; Benzilates; Cerebral Cortex; Intestine, Small; Kinetics; Male; Mathematics; Muscle, Smooth; Piperidines; Protein Binding; Quinuclidines; Rats; Receptors, Cholinergic; Receptors, Muscarinic