3H-QNB Binding Research Articles

Development of muscarinic cholinergic receptor binding in the visual system of monocularly deprived and dark reared rats.

In 25 day old rats monocularly deprived by unilateral eyelid suture on postnatal day 10 (MD), [3H]quinuclidinyl benzylate (3H-QNB) binding was significantly reduced in the visual cortex (VC) of both sides, but elevated in both superior colliculi (SC). Muscarinic receptor binding in the frontal cortex (FC), a non-visual brain area, in the lateral geniculate nucleus (LGN), and in the retina was not affected. In 25 day old rats raised in complete darkness from birth (DR) similar changes in 3H-QNB binding were found in VC and SC. However, binding levels were also decreased in the FC and significantly increased in the retina. In adult (6 month old) MD and DR rats the differences in 3H-QNB binding as compared to age-matched controls had disappeared completely in all visual brain areas studied. Detailed Scatchard analyses indicate that the alterations in the 3H-QNB binding were due to changes in receptor number only.

Lipid peroxides in brain during aging and vitamin E deficiency: Possible relations to changes in neurotransmitter indices

Lipid peroxide levels, were found to be significantly higher in brains of 18 month old as compared to 4 month old rats, with particularly large increases occuring in the olfactory bulb, globus pallidus, cerebral cortex and caudate-putamen (CP). Eighteen month old rats fed a vitamin E deficient diet for 9 months before sacrifice had lipid peroxide levels significantly higher than age-matched controls in the cerebral cortex, hippocampus and hypothalamus. Age-related decreases were seen in choline acetyltransferase, acetylcholinesterase and 3H-QNB binding in some but not all brain regions, while GABA transaminase and MAO showed age-related increases. No age-related change was seen in tyrosine hydroxylase in the CP or in 3H-dihydroalprenol (DHA) or 3H-spiroperidol binding in the cortex. As compared with controls, vitamin E deficient rats showed decreases of 38% in cortical 3H-DHA binding, of 3 in 3H-QNB binding in the CP and of 23% and 12% in choline acetyltransferase in the CP and cerebellum, respectively. There were no completely consistent regional correlations between significant changes in lipid peroxidase levels and any neurotransmitter indices studied except for MAO which was only measured in the caudate-putamen.

Distribution of ( 3H) QNB binding in dog gastric smooth muscle pre- and post vagotomy

Tritiated quinuclidinyl benzilate [( 3H)QNB] was used to characterize muscarinic cholinergic receptors in membrane fragments prepared from the circular smooth muscle of the dog stomach. In preliminary experiments the effect of protein, incubation time, temperature and pH on QNB binding were evaluated. Muscarinic cholinergic antagonists and agonists inhibited QNB binding in a concentration-dependent manner, but the nicotinic antagonist hexamethonium and adrenergic compounds were not effective in displacing QNB from binding sites. Scatchard plot analysis of binding data showed an asymetric receptor distribution in the stomach. The cardia bound 425fmol of QNB/mg protein with a K d of 0.05nM, the fundus 267fmol/mg protein with a K d of 0.09nM and the antrum 147 fmol/mg protein with a K d of 0.14nM. In a second series of experiments, binding of QNB was measured in dogs which had been vagotomized three weeks earlier. Vagotomy had no effect on the apparent K d but disrupted the asymetric receptor distribution seen in the normal dog such that the Bmax of the cardia fell to a value of 222fmol/mg protein.

Desensitization of the alpha adrenergic receptor system in guinea pig vas deferens

Desensitization induced by alpha adrenergic (α-Ad) stimulation was investigated in organ cultured vas deferens of guinea pig. Brief exposure (1–2 min) of the muscle to noradrenaline (NA) caused short-term desensitization to both NA and acetylcholine (ACh), but not to high K +. After removing the agonist this desensitization completely disappeared within 15 min. Prolonged exposure to NA (i.e., cultured with NA for 3–24 hr) elicited longterm desensitization to NA, ACh and K + (50 mM), but it did not change the maximal contraction by high K + (154 mM). After removing NA from the culture medium the response to the agonist was restored to normal within 24 hr, but not within 15 min. The number and affinity of α-Ad and muscarinic ACh receptors, which were measured by the binding of 3H-WB4101 and 3H-QNB, respectively, were not changed in the muscle during these treatments. Moreover, long-term desensitization, but not short-term desensitization, was depressed by the concomitant presence of cycloheximide. The possible mechanisms of desensitization were discussed in comparison with those of various receptor systems.

Benzilycholine mustard and spare receptors in guinea pig ileum

A comparison was made of muscarinic receptor occupancy by the irreversible antagonist benzilylcholine mustard (BCM) as determined from shifts in the dose-response curve to a muscurinic agonist and from 3H-QNB binding to homogenates of BCM-treated tissue. Major discrepancies were found. A low concentration of BCM (3×10 −8M/15 min.) produced a parallel dose-response curve shift corresponding to 98–99% receptor occupancy by BCN, whereas 3H-QNB binding revealed only 48% receptor occupancy. Possible origins of this discrepancy are discussed. High concentrations of BCM (5×10 −5M, min.) fail to completely alkylate all 3H-QNB binding sites even though response is completely lost. Although significant (64%) recovery of response occurs after prolonged tissue washing (240 min.) this is not accompanied by an increase in 3H-QNB binding. The small fraction (∼5%) of sites inaccessible to BCM and with reduced affinity for 3H-QNB may represent a subpopulation of muscarinic receptors.

Regulation of muscarinic ligand binding sites by nerve growth factor in PC12 phaeochromocytoma cells.

The rat phaeochromocytoma cell line PC12 (ref. 1) possesses many characteristics of adrenal chromaffin cells, including the ability to synthesize, store and release catecholamines in response to acetylcholine and other depolarizing agents2–4. When exposed to 2.5S nerve growth factor (NGF), PC12 cells cease to divide, and exhibit a number of neurone-like characteristics, including the extension of neurites, development of voltage-sensitive ion channels and electrical excitability, and increased capacity to synthesize and release acetylcholine3–8. Biochemical and pharmacological evidence indicates that both adrenal chromaffin cells and sympathetic neurones contain, in addition to ganglionic-type nicotinic receptors, muscarinic cholinergic receptors proposed to modulate catecholamine release9–13. Catecholamine release from PC12 cells is directly stimulated by nicotinic, but not muscarinic, cholinergic agonists3. Although ion flux through acetylcholine-activated channels in PC12 cells is inhibited by high concentrations of the potent muscarinic antagonist quinuclidinyl benzilate (QNB)14, the possibility that these cells may contain functional muscarinic receptors has been largely overlooked. We show here that PC12 cells contain saturable, high-affinity binding sites for 3H-(—)-quinuclidinyl benzilate (3H-QNB), which seem pharmacologically comparable with muscarinic receptors described in rodent brain15,16, smooth muscle17,18 and adrenal medulla12. Moreover, treatment with NGF produces a marked elevation of specific 3H-QNB binding. PC12 cells thus may provide a useful system for investigating the biochemical mechanisms, cellular distribution and regulation of muscarinic receptors by hormones and trophic signals.

Effect of phencyclidine on [ 3H]QNB binding

Intraperitoneal injection of phencyclidine before intravenous injection of [ 3H] Quinuclidinyl benzilate (QNE, 1.6 μg/kg) significantly increased the amount of radioactivity found in the brains of female C57BL/6J mice one hour after the 3H-QNB administration. This effect was found in hypothalamus, cortex, hippocampus and striatum and was decreased by pretreatment of the animal with atropine. The magnitude of the enhancement varied as a function of dose but did not change across the time span studied. These data are in contrast to our findings and those of others of inhibition of the specific binding of 3H-QNB to muscarinic cholinergic receptors by PCP in vitro . When atropine or PCP was administered in vivo and the tissue later analyzed in vitro , no effects of the drugs were observed on 3H-QNB binding. The reasons for the differences remain a matter of speculation.

Studies on 125I-αbungarotoxin binding sites in rat brain

The distribution and properties of 125I-αbungarotoxin ( 125I-αBTX) binding in rat brain using micropunched tissue homogenates were examined with a binding technique. Highest level of 125I-αBTX binding was observed in the hypothalamus, followed by hippocampus, cortex, globus pallidus, nucleus caudatus and nucleus accumbens. Although high levels of 3H-quinuclidinyl benzilate ( 3H-QNB) binding in the striatum were found at dorso-lateral sites and low levels in the central regions, the level of 125I-αBTX binding within the striatum was almost equal. There was no significant correlation between 125I-αBTX binding and choline uptake which could be useful as a marker for functional cholinergic nerve terminals. The inhibition of specific 125I-αBTX binding to the hippocampal homogenates by nicotine, d-tubocurarine and other nicotinic drugs was studied. The results of these studies suggest that αBTX binding sites in the brain are not likely the binding sites of nicotine or the physiological ACh R sites.

BINDING EXPERIMENTS OF MUSCARINIC ACETYLCHOLINE AND DOPAMINE RECEPTORS IN HUMAN BRAINS WITH EMPHASIS ON A CASE OF STRIATONIGRAL DEGENERATION

In the regional distribution of /-3H-quinuclidinyl benzilate (3H-QNB) binding in human brains of neurologically unaffected cases, it was highest in the caudate nucleus which was followed by the putamen, amygdala, cerebral corteces and olfactory bulb and lowest in the substantia nigra. As to the regional distribution of 3H-spiroperidol binding in human control brains, it was highest in the caudate nucleus and was followed by the cerebral corteces, amygdala and lowest in the cerebellar cortex. In a case of striatonigral degeneration (SND), 3H-QNB binding in the putamen and thalamus was lowered and 3H-spiroperidol binding was decreased in the putamen, frontal and parietal corteces, Ammon’s horn, amygdala and substantia nigra as compared to human brains of control cases. These results were noteworthy since no pathological changes were observed in the thalamus, cerebral corteces, amygdala and Ammon’s horn in this case. The 3H-spiroperidol binding was increased by injection of 6-hydroxy-dopamine (6-OHDA) into the substantia nigra of rat brains by 17% as compared to the contralateral intact side. Conversely, 3H-spiroperidol binding was decreased by injection of kainic acid (KA) into the striatum of rat brains by 43% as compared to the contralateral intact side. This meant that dopamine receptors labelled by 3H-spiroperidol were at least partially localized at the postsynaptic site of the nigrostriatal dopamine neuron.

Binding experiments of muscarinic acetylcholine and dopamine receptors in human brains with emphasis on a case of striatonigral degeneration.

In the regional distribution of I-3H-quinuclindinyl benzilate (3H-QNB) binding in human brains of neurologically unaffected cases, it was highest in the caudate nucleus which was followed by the putamen, amygdala, cerebral corteces and olfactory bulb and lowest in the substantia nigra. As to the regional distribution of 3H-spiroperidol binding in human control brains, it was highest in the caudate nucleus and was followed by the cerebral corteces, amygdala and lowest in the cerebellar cortex. In a case of striatonigral degeneration (SND), 3H-QNB binding in the putamen and thalamus was lowered and 3H-spiroperidol binding was decreased in the putamen, frontal and parietal corteces, Ammon's horn, amygdala and substantia nigra as compared to human brains of control cases. These results were noteworthy since no pathological changes were observed in the thalamus, cerebral corteces, amygdala and Ammon's horn in this case. The 3H-spiroperidol binding was increased by injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra of rat brains by 17% as compared to the contralateral intact side. Conversely, 3H-spiroperidol binding was decreased by injection of kainic acid (KA) into the striatum of rat brains by 43% as compared to the contralateral intact side. This meant that dopamine receptors labelled by 3H-spiroperidol were at least partially localized at the postsynaptic site of the nigrostriatal dopamine neuron.

Characterization of autonomic receptors in the rat sublingual gland by biochemical and radioligand assays.

The autonomic receptors of the rat sublingual gland were characterized by radioligand binding and by specific functional responses involving the release of K+ and the generation of cyclic AMP in vitro. Both muscarinic cholinergic and alpha2-adrenergic receptors were present in moderately high density in the sublingual gland, as judged by the binding of the specific radioligands 3H-QNB and 3H-clonidine (Bmax in pmol/g tissue=18.4±2.4 and 9.9±1.3, respectively). By contrast, although alpha1 and beta-adrenergic receptors were detected, they were not present in large numbers. The Bmax (pmol/g tissue) for the binding of 3H-prazosin and 3H-DHA were, respectively, 3.2±0.6 and 3.6±0.4. Stimulation of muscarinic cholinergic receptors with carbamylcholine (2×10−5 M) caused a net release of K+ from sublingual slices incubated in an enriched, oxygenated Krebs Ringer bicarbonate medium of 35±7% after 10 min of incubation. Norepinephrine, phenylephrine, clonidine and isoproterenol did not induced K+ release from the slice preparation, but actually reduced the basal or unstimulated release of K+. As in the submandibular and parotid glands, the release of K+ from sublingual slices had two components, a passive efflux and an active uptake which depended on the activation of an ouabain-sensitive Na+, K+ ATPase. The release of K+ was also dependent on the presence of Ca2+ (2.7 mM) in the incubation medium. Stimulation of beta-adrenergic receptors with isoproterenol (10−5 M) caused a 9-fold increase in the content of glandular cyclic AMP (from 4.0±0.6 to 36.0±2 pmol/mg/10 min). A similar increase was observed with norepinephrine (10−4 M) in the presence of phentolamine (10−4 M). Clonidine at concentrations of 10−5 and 10−4 M reduced the cyclic AMP content to below basal levels. The rat sublingual gland has functional cholinergic receptors and can release K+ in vitro in the presence of cholinergic agents. In contrast to the other major salivary glands, it has a low density of alpha1-adrenergic receptors and fails to release K+ upon stimulation with alpha1-agonists. The functional significance of a low density of beta-adrenergic receptors is still unclear, although there is a definite glandular cyclic AMP response upon stimulation with isoproterenol. Abundant alpha2-adrenergic receptors in the sublingual gland are apparently negatively coupled to adenylate cyclase.

The muscarinic cholinergic binding kinetics of the human urinary bladder

AbstractDetermination of (1) the absolute number of cholinergic receptors, (2) the cholinergic binding kinetics, and (3) the direct antimuscarinic activity of a series of uropharmacologic agents was accomplished by radioligand binding techniques in tissue derived from human urinary bladder. The competition between 3H‐quinuclidinyl benzilate (3H‐QNB) binding and selective antagonists provided the first quantitative assessment of cholinergic receptor characteristics in the human bladder fundus and represents a potential method for assessing the antimuscarinic binding potential of uropharmacologic agents.

Decreased 3H-L-quinuclidinyl benzilate binding and muscarine receptor subsensitivity after chronic gamma-butyrolactone treatment.

A significant decrease in the number of 3H-quinuclidinyl benzilate (3H-QNB) binding sites in striatal membranes was observed in rats exposed to gamma-butyrolactone (GBL) during 3 weeks. This decrease in 3H-QNB binding was not due to a direct effect of GBL on muscarine receptors since no alterations of binding were produced by high concentrations of GBL or of its active metabolite gammahydroxybutyric acid (GHB) when added in vitro. Challenge doses of pilocarpine were less effective in producing catalepsy and tremors in rats chronically treated with GBL. It is proposed that the partial inhibition of the nigro-striatal dopaminergic pathway produced by chronic GBL treatment leads to a persistent activation of the intrastriatal cholinergic interneurons, which are tonically inhibited by dopaminergic nigrostriatal neurons. The activation of striatal cholinergic interneurons results in a decreased number of muscarine receptors and in a lower sensitivity to cholinomimetic agents. Moreover, tolerance to the anesthetic effect of GBL was found to occur after chronic treatment with this drug. The brain levels of GHB at the time of regaining of the righting reflex were 50% higher in GBL pretreated animals then in controls. Indicating the tolerance to GBL-induced "sleep" is due to a decrease sensitivity of the effector to GHB rather than to a faster inactivation of elimination of the drug.

Cholinergic receptors in human hippocampus-- regional distribution and variance with age

The anterio-posterior distribution of cholinergic receptor binding sites in human hippocampus (five parts) as well as the effect of age (age range 3 days - 85 years) on receptor properties has been studied. Muscarinic binding sites was measured using labelled quinuclidinyl benzilate ( 3H-QNB) as ligand and labelled tubocurarine ( 3H-TC) was used for measurement of nicotine-like binding sites. The highest number of 3H-QNB binding sites in human hippocampus was measured at 3 days and 3 weeks of age and the lowest at 82 years of age. The proportion of high and low affinity muscarinic binding sites respectively was about the same at all ages investigated. A decrease in 3H-QNB binding sites with age was found in the anterior parts of the hippocampus (age range 55–84 years). When individual data for number of 3H-TC binding sites were plotted against corresponding number of 3H-QNB binding sites a strong correlation was observed in most of the different regions of the hippocampus.

Decreased muscarinic binding sites in small intestine from mice treated with neostigmine

It has been reported by several authors that animals given repeated sublethal doses of an organophosphate, acetylcholinesterase (AChE) inhibitor, develop tolerance to its toxicity. This phenomenon seems to be due, at least partially, to a decrease of central and peripheral cholinergic receptors. In the present study, we report that a decrease of muscarinic receptors, as measured by [ 3H]-quinuclidinyl benzilate ( 3H-QNB) binding, occurs in the small intestine of mice treated with the carbamate, AChE inhibitor, neostigmine. Male mice were given neostigmine in the drinking water at daily increasing concentrations (from 20 to 1000 ppm). Methylatropine (20mg/kg, i.p.) was administered twice a day for the same period to two groups of control and neostigmine-treated animals. AChE activity was inhibited 60–70% in small intestine and diaphragm and [ 3H]-QNB binding was significantly reduced in the small intestine of neostigmine-treated mice; both the number of receptors and the affinity were lower than control. This decrease was not present in the tissue of mice given methylatropine together with neostigmine. Administration of methylatropine alone caused a significant increase of [ 3H]-QNB binding in the small intestine.

Muscarinic acetylcholine receptor in chick limb bud during morphogeneis.

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 chicken 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 microM). Specific binding of (3H)QNB reflected a muscarinic receptor. The Kd 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.

Trazodone does not competitively inhibit binding at muscarinic cholinoceptors

1. 1. The muscarinic acetylcholine receptor binding of trazodone, a new nontricyclic antidepressant, was investigated in vitro using 3H-3-quinuclidinyl benzilate ( 3H-QNB). 2. 2. Saturation binding experiments in the presence of varying concentrations of trazodone revealed a dose-dependent decrease in the maximum number of 3H-QNB binding sites with no effect on the dissociation constant. 3. 3. This may represent a nonspecific drug-membrane interaction similar to that observed with local anesthetics.

Biochemical characterization of solubilized muscarinic acetylcholine receptors

Several in part new methods (salt extraction, phospholipase treatment, transfer to serumlipoproteins, nonionic detergens mixtures) have been extensively examined in order to obtain a solubilized muscarinic acetylcholine receptor with properties suitable for further purification. An optimal mixture of digitonin and gitonin has been found which forms a tightly bound detergent wall around the receptor protein as indicated by ion exchange and monolayer studies and which allows to solubilize the receptor from enriched synaptosomal membranes with a high yield and stability. The specific 3H-QNB binding of this preparation is nearly unchanged between pH 7.5 and 10. The isoelectric point of the muscarinic acetylcholine receptor has been determined by isoelectric focusing to be between pH 4.5 and 4.6.

Effect of long-term L-dopa administration on the dopaminergic and cholinergic (muscarinic) receptors of striatum in 6-hydroxydopamine lesioned rats

L-Dihydroxyphenylalanine (L-Dopa) (200 mg/kg/day) was administered for 30 days to the rats whose nigrostriatal dopamine pathway was lesioned unilaterally with 6-hydroxydopamine and the receptor binding of 3H-spiperone and 3H-quinuclidinyl benzilate ( 3HQNB) was measured in the dopaminergic and muscarinic cholinergic receptors of the striatum. 3H-spiperone binding increased by 73% and 3HQNB binding decreased by 14% in the lesioned side when compared to the control side of L-Dopa-non-treated rats. 3H-spiperone binding was measured in the lesioned sides of L-Dopa-treated and L-Dopa-non-treated rats and was found to have decreased by 21% in the former. In the control side of the L-Dopa-treated lesioned rats, however, 3H-spiperone binding increased by 27% when compared to the opposite striatum of the same rats. 3HQNB binding in the lesioned side of L-Dopa-treated rats was not significantly different from that of the control side statistically. These results suggest that changes in functional equilibrium between the dopaminergic and cholinergic mechanisms influence the muscarinic cholinergic receptors and that supersensitivity of dopamine receptors after lesion of the nigrostriatal pathway also remains after long-term L-Dopa treatment.

Muscarinic receptor binding and behavioral effects of atropine following chronic catecholamine depletion or acetylcholinesterase inhibition in rats

Rats were subjected to one of two experimental treatments: (1) intraventricular infusion of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA), known to permanently reduce brain dopamine and norepinephrine levels, or (2) chronic administration of the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). Both treatments are believed to produce relative overactivity of cholinergic systems and to suppress forward locomotion. The anticholinergic agent atropine sulfate yielded excessive forward walking in otherwise chronically akinetic 6-OHDA-treated rats, whereas atropine slightly decreased locomotion in controls. The hypothesis that such supersensitivity to atropine may be related to a reduction in the density of muscarinic cholinergic receptors was not supported: First, 3H-quinuclidinyl benzilate (QNB) binding to membrane preparations was not decreased in the 6-OHDA-treated rats; secondly, atropine did not induce excessive forward locomotion in the DFP-treated rats in which 3H-QNB binding was decreased. There were other changes in the DFP-treated rats consistent with muscarinic receptor alteration, including tolerance to the locomotor suppressive effects of DFP, cross tolerance to the cholinergic agonist pilocarpine, and exaggerated atropine-induced increases in core temperature and stereotypy. It is concluded that 6-OHDA and DFP produce different long-term changes in cholinergic brain systems and atropine-sensitive behaviors.