QNB Binding Research Articles

Muscarinic cholinergic receptor binding in rat mast cells.

Specific [3H]-QNB binding was present in isolated, purified, intact rat mast cells and in crude membrane preparations. The binding is saturable, time- and temperature-dependent. Cholinergic agents inhibit selectively the binding: atropine is the most effective of the antagonists while oxotremorine is the most potent of the muscarinic agonists. It is concluded that rat mast cells are provided with muscarinic cholinergic receptors.

Quantitation of tolerance development after chronic oxotremorine treatment

A new procedure was developed to quantitate the tolerance which develops as mice are chronically infused with the muscarinic agonist, oxotremorine. Cumulative dose-response curves were constructed for the effects of oxotremorine on body temperature and rotarod performance by administering sequential injections to individual animals. These dose-response curves compare favorably to those constructed by injecting individual animals with one of several doses. The sequential injection technique was used to assess the magnitude of tolerance development to oxotremorine. A linear relationship between oxotremorine infusion rate (dose) and magnitude of change of the ED 50 value for impairment of rotarod performance was observed, with animals receiving an infusion rate of 1.0 mg/kg/hr showing a 24-fold increase in ED 50. Dose-response curves for tolerant animals were parallel to those constructed for naive animals. The oxotremorine dose required to decrease body temperature to 35°C (ED 35°) was 80-fold greater than control in the group treated with 1.0 mg/kg/hr. The dose-response curves for tolerant animals were not parallel to those seen in naive animals. Time courses of recovery from a challenge dose of oxtoremorine suggest little change in metabolism occurred during chronic infusion. Chronic oxotremorine infusion resulted in a decrease in the total number of QNB binding sites. Both high- and low-affinity sites were reduced in number. Since no change in K 1 for the muscarinic agonist, carbamylcholine, was observed, it seems unlikely that a change occurs in the affinity of the muscarinic receptor for agonists. Significant change in receptor number was detected only in animals that received higher doses of oxotremorine. Chronic oxotremorine treatment had no effect on choline uptake by synaptosomes prepared from any of five brain regions.

Regional adaptation of muscarinic receptors and choline uptake in brain following repeated administration of diisopropylfluorophosphate and atropine

The specific [ 3H]QNB binding and high-affinity uptake of [ 14C]choline in 8 brain regions (cerebral cortex, hippocampus, hypothalamus, thalamus, striatum, midbrain, cerebellum and brainstem) after repeated administration of DFP and atropine to guinea-pigs were simultaneously measured. Following repeated DFP administration, AChE was markedly depressed in each brain region. In these animals, there was a significant decrease in specific [ 3H]QNB binding in the cerebral cortex, hippocampus and striatum, whereas the [ 3H]QNB binding in the rest of brain regions was unchanged. Scatchard analysis revealed a 36% decrease in the B max value for the striatal [ 3H]QNB binding without a change in the K d value, suggesting a change in the receptor density. In contrast, repeated atropine administration produced a significant enhancement in the [ 3H]QNB binding only in the hippocampus and striatum. The B max value in the striatum increased by 21% without a change in the K d value. In addition to the receptor alteration, high affinity uptake of [ 14C]choline in the hippocampus and striatum was significantly decreased by DFP treatment, while that in the striatum increased by atropine treatment. Thus the present study has demonstrated that a prolonged activation and blockade of central muscarinic receptors resulted in specific adaptation in both the receptor density and ACh availability at the synapses in the cerebral cortex, hippocampus and striatum.

Changes in muscarinic binding in distant brain regions showing neuronal losses after folic acid injections into the substantia innominata.

Injection of folic acid (FA) into the nucleus substantia innominata (NSI) was found to decrease [3H]quinuclidinyl benzilate ([3H]QNB) binding in the frontal cortex, pyriform cortex, amygdala, and the NSI itself without changing the KD. Binding in the thalamus, caudate nucleus, hippocampus, and substantia nigra was not affected. [3H]Flunitrazepam binding was unchanged in all eight regions studied. Previous work indicates FA injections into the NSI produce epileptiform activity and cause loss of GABAergic and possibly other neurons in the frontal and pyriform cortices, the amygdala, and thalamus. The reductions of [3H]QNB binding in the first three of these regions are interpreted as indicating that many of the neurons lost are cholinoceptive, a finding that supports the previous hypothesis that activation of cholinergic projections from the NSI is an important part of the mechanism of cell loss in these regions.

Effect of electroconvulsive shock on muscarinic cholinergic receptors in rat cerebral cortex and hippocampus.

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

GTP increases airway muscarinic antagonist binding sites: An effect regulated by Mg 2+

Using [ 3H]QNB, we demonstrated that GTP and Gpp(NH)p increased muscarinic receptor high affinity sites in bovine tracheal muscle preparations; however, neither the dissociation constant of [ 3H]QNB binding sites nor pulmonary β-adrenergic receptor sites was altered. The GTP effect on increasing the receptor sites was relatively small (16%), sensitive (ED 50 0.48 μM) and specific (Gpp(NH)p > GTP = GDP > GMP ⪢ ATP). Mg 2+ potentiated this increase by up to 94.8%, whereas Na +, K + and Ca 2+ had no such effect.

The preclinical antipsychotic evaluation of HRP 913, a novel benzisoxazole derivative

AbstractHRP 913 {1‐[3‐(6‐fluoro‐1,2‐benzisoxazole‐3‐yl)propyl]‐4‐(2‐oxo‐1‐benzimidazolinyl)} piperidine demonstrated preclinical antipsychotic activity with features that may provide a clinical advantage over current therapy. It was effective in blocking amphetamine stereotypy in rats (ED50 = 0.4 mg/kg, i.p.), amphetamine circling in SN‐lesioned rats (ED50 = 0.3 mg/kg, i.p.), apomorphine stereotypy in rats (ED50 = 0.8 mg/kg, i.p.), but not apomorphine circling in SN‐lesioned rats (up to 10 mg/kg, i.p.). It also blocked Sidman avoidance in rats (ED50 = 0.17 mg/kg, i.p.) and monkeys (ED50 = 0.2 mg/kg, p.o.) and blocked intracranial self‐stimulation in rats (0.09 mg/kg, i.p.). A unique biphasic effect on catalepsy was found. Monkey EPS studies demonstrate a potential for EPS that is lower than some existing clinical standards. HRP 913 displaced 3H‐spiroperidol from rat striatal sites (IC50 = 6.0 × 10−9 M) and inhibited WB‐4101 binding (IC50 = 2.6 × 10−8 M) with only slight effect on QNB binding. HRP 913 does not appear to have marked α‐blocking properties in vivo. HRP 913 is a potent dopamine antagonist and is predicted to have less side effects than current therapy.

Evidence against the presence of spare muscarinic receptors in the rabbit urinary bladder

AbstractIn a recent study by Anderson and Marks [1982], indirect evidence was presented for the existence of “150‐fold muscarinic receptor excess” in the rabbit urinary bladder. This conclusion was based on the quantitative comparison of the ability of carbamylcholine to both directly contract bladder strips and to inhibit (quinuclidinyl benzilate) binding. In order to investigate the presence of “spare receptors” in the bladder directly, we have determined the effect of benzilylcholine mustard (a noncompetitive cholinergic inhibitor) on both bethanechol stimulation of muscle‐strip contraction and on [3H]QNB binding (muscarinic receptor density). The results of these studies indicate that there is no significant “muscarinic receptor excess” in the rabbit urinary bladder.

The muscarinic cholinergic receptors in the posterior hypothalamus of hypertensive and normotensive rats

The density of [ 3H]quinuclidin-3-yl benzilate ([ 3H]QNB) binding sites in the posterior hypothalamus was determined in spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats at the ages of 1, 3, 11 and 50 weeks. In SHR, even at the early age of 1 week which is prehypertensive, the values obtained were 1.5 times greater than those of age-matched WKY rats. The values of the equilibrium dissociation constant (K D) did not differ between SHR and WKY rats of the same age. In the pons medulla, however, the density of [ 3H]QNB binding sites was not different between the two strains of rats of matching age. Isolation-induced hypertension in adult Wistar rats and an increase in the density of [ 3H]QNB binding sites in the posterior hypothalamus were observed to arise concomitantly. A hypothesis is offered whereby a relative increase in ACh receptor sites in the posterior hypothalamus is a primary cause of hypertension in the models considered.

Differential alterations of cholinergic muscarinic receptors during chronic and acute tolerance to organophosphorus insecticides

Male mice treated for 2 weeks with the anticholinesterase insecticide disulfoton ( O, O-diethyl S-[2-(ethylthio)-ethyl] phosphorodithioate; 10 mg per kg per day) became tolerant to the hypothermic and antinociceptive effects of disulfoton itself and of oxotremorine, a muscarinic cholinergic agonist. Homogenates of brain and ileum from tolerant animals exhibited reduced binding of the specific muscarinic antagonist [ 3H]quinuclidinyl benzilate ([ 3H]QNB). In forebrains of tolerant animals, the number of receptors ( B max) was decreased 40% with no change in the affinity constant. Acetylcholinesterase (AChE) activity was 15% of control. Forty-eight hours after a single injection of disulfoton (10 mg/kg) mice were more resistant than their controls to the hypothermic and antinociceptive effects of a second administration of the same insecticide and of oxotremorine. Tolerance was not present 96 hr after a single administration of disulfoton. A single injection of disulfoton produced 74, 65 and 27% inhibition of AChE activity after 4, 48 and 96 hr respectively. Four hours after a second injection at 48 or 96 hr, 73 or 72% inhibition was found. [ 3H]QNB binding of animals treated with a single injection of disulfoton and of controls did not differ at either time point. An increase in the K i for inhibition of [ 3H]QNB binding by unlabeled oxotremorine was observed in forebrain from mice killed 48 hr after a single injection of disulfoton, indicating a decreased affinity of the muscarinic receptor for agonists. Binding of [ 3H]oxotremorine-M was decreased significantly 48 hr after a single injection of disulfoton and after chronic treatment. It is suggested that a differential down-regulation of muscarinic receptors occurs in acute and chronic tolerance, involving agonist and antagonist binding sites and depending on duration of exposure.

3H]QNB binding to muscarinic receptors in intact avian salt gland cells.

3H]QNB binding to muscarinic receptors in intact avian salt gland cells.

The effect of chronic lithium pretreatment on rat brain muscarinic receptor regulation

The effect of two weeks of lithium (Li) pretreatment on up and down regulation of muscarinic receptors in rat brain was measured in two groups of rats. After two weeks of feeding 0.2% LiCl in ground pellets, one group of rats was injected with atropine (3 mg/kg daily for 5 days) and another group with diisopropyl fluorophosphate, a cholinesterase inhibitor (1.2 mg/kg). Li pretreatment completely abolishes the significant 23% rise in QNB binding induced by atropine but does not prevent the significant 16% decline in QNB binding induced by DFP. These results suggest that chronic Li pretreatment prevents supersensitivity but does not prevent subsensitivity of rat brain muscarinic receptors.

Antinociceptive and hypothermic effects of trimethyltin

Trimethyltin (TMT) induced a dose-dependent antinociceptive and hypothermic effect in mice. Antinociception was not attenuated by naloxone but was reversed by atropine. TMT, however, was ineffective in displacing ( 3H)-QNB binding in vitro and did not affect ( 3H)-QNB binding or acetylcholinesterase activity after in vivo administration. The ethyl ester of nipecotic acid, a specific inhibitor of synaptosomal GABA uptake, exerted a similar antinociceptive effect that could be blocked by atropine. The GABA receptor antagonist bicuculline attenuated antinociception induced by TMT and nipecotic acid ethyl ester but not by morphine or oxotremorine. γ-Vinyl GABA, an irreversible inhibitor of GABA metabolism, prolonged TMT but not morphine-induced antinociception. In contrast, neither the dose-response nor the time course of TMT-induced hypothermia were affected by any of the drugs tested. The findings suggest that the GABAergic system may be involved in TMT induced antinociception; however, the mechanism responsible for the hypothermic effect of TMT is not apparent.

The biphasic response of muscarinic cholinergic receptors in cultured heart cells to agonists. Effects on receptor number and affinity in intact cells and homogenates.

A biphasic time course of the agonist-mediated loss of muscarinic cholinergic receptors has been demonstrated in cultured chick embryo heart cells by radioligand binding studies using the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB). This agonist-mediated receptor loss was associated with decreased affinity of the receptor for agonist as judged by competitive binding of the agonist carbamylcholine with [3H]QNB to cell homogenates (Galper, J. B., and Smith, T. W. (1980) J. Biol. Chem. 255, 9571-9579). In the current studies the concentration dependence of agonist-mediated receptor loss was also found to be biphasic. The apparent shift of affinity following brief (15 min) agonist exposure coincided with the agonist-mediated loss of a subclass of high affinity receptors with an IC50 for carbamylcholine inhibition of [3H]QNB binding of 3.9 x 10(-7) M. Those receptors remaining constituted a subclass of low affinity receptors with IC50 = 8.2 x 10(-5) M. The data further suggest that an apparent decrease in agonist affinity after guanine nucleotide exposure represents conversion of high affinity receptors to a similar low affinity state, IC50 = 8.6 x 10(-5) M. The rapid loss of [3H]QNB binding sites in the presence of agonist did not require interaction of agonist with intact cells, but also occurred if cells were homogenized and then subjected to a brief (15 min) exposure to agonist. The slow loss over 3 h of [3H]QNB binding sites could only be demonstrated in intact cells incubated with agonist prior to homogenization. To probe further the later phase of agonist-mediated receptor loss, we developed new assay methods for determining muscarinic antagonist binding to intact cells. In control cells, binding of the hydrophobic antagonist [3H]QNB was quite similar in extent to binding of the more hydrophilic antagonist [3H]methylscopolamine ([3H]MS), with Kd values of 0.11 and 0.47 nM, respectively. Kinetic analysis of the binding of these two ligands was performed to determine whether they might distinguish between two states of the receptor. Both ligands bound to the receptor by a two step mechanism consistent with the formation of a low affinity complex followed by conversion to a high affinity complex. However, the ratio of reverse to forward rate constants of the second step of [3H]MS binding was roughly 100-fold greater than that for the more hydrophobic ligand [3H]QNB. Comparison of the time course of agonist-induced receptor loss as measured by binding of [3H]MS or [3H]QNB was consistent with muscarinic agonist mediation of a stepwise alteration in receptor configuration from a form that bound both [3H]MS and [3H]QNB to a form that bound only [3H]QNB and thence to a form that bound neither [3H]MS nor [3H]QNB. The relationship of such a sequential mechanism to agonist-induced changes in the relationship of the receptor to the cell membrane and agonist-induced endocytosis of the receptor is discussed.

The subcellular fractionation of the bovine caudate nucleus.

Two synaptosomal fractions could be obtained from bovine caudate nucleus on sucrose density gradients one of which had a much greater capacity for 'high affinity' choline uptake than the other but comparable amounts of CAT and choline kinase activity. Specific binding of QNB was widely distributed among all the subcellular fractions except the mitochondrial fraction and in quantitative terms by far the greatest amount was in the microsomal fraction. Only the microsomal fraction contained measurable amounts of glycerophosphocholine phosphodiesterase.

Interactions between chemical and electrical kindling of the rat amygdala

Holtzman rats were implanted with a chemitrode into the left basolateral amygdala, which could then be stimulated electrically (400 μA, 1 s, AC) or chemically by injection of carbachol (1 μl, 2.7 nmoles, sterile, isotonic). Group A received a daily injection of carbachol and developed kindled seizures. Group B received carbachol mixed with equimolar atropine, which blocked seizures and kindling. After 20 injections, both groups were stimulated electrically once a day and kindled at similar rates. Two additional groups received electrical or sham stimulation, followed by carbachol kindlind. No transfer effects were observed. Four additional groups received 27 nmoles of atropine through the chemitrode, followed 15 min later by electrical stimulation, sham stimulation, carbachol injection or saline injection, respectively. Atropine completely blocked carbachol kindling but did not alter the rate of electrical kindling. No difference in the number of QNB binding sites was observed in the amygdala of rats sacrificed two weeks after full electrical kindling. The lack of interaction between electrical and carbachol kindling and the failure of atropine to block electrical kindling of the amygdala suggest that the activation of local muscarinic synapses, while essential for carbachol kindling, is not required for electrical kindling of the rat amygdala.

Memory and postsynaptic cholinergic receptors in aging mice

Significant retention deficits were observed on passive avoidance tasks (step-down and step-through) in 15-, 20-, and 25-month-old male C57BL/6 mice compared with 4- and 8-month-old mice. In contrast, cholinergic muscarinic receptor binding ([ 3H]quinuclidinyl benzilate) in cerebral cortex, striatum, hippocampus, and cerebellum in these same animals revealed no difference in this 4- to 25-month age range. In a separate comparison of 4- and 29-month-old female mice, [ 3H]QNB binding was significantly decreased in the older group in cerebral cortex, hippocampus, and striatum. Environmental enrichment, compared with an impoverished environment, significantly improved retention in mice on 24-hr step-down performance but affected QNB binding only minimally (6–7% decrease of QNB binding in cerebral cortex and hippocampus). Benzodiazepine ([ 3H]flunitrazepam) receptor binding was significantly (12–15%) decreased in 29-month-old mice compared with 4-month-old mice in the cerebral cortex, hippocampus, cerebellum, and brain stem.

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.

Molecular mechanism of the effects of guanine nucleotide and sulfhydryl reagent on muscarinic receptors in smooth muscles studied by radiation inactivation

The molecular sizes of the units concerned in 3-quinuclidinyl benzilate (QNB) binding and in the effects of guanine nucleotide and sulfhydryl reagent on the inhibition of QNB binding by carbachol in smooth muscle of guinea pig ileum were determined to be 76,000, 179,000 and 107,000, respectively by the radiation inactivation method. One or more subunits (GTP subunit) other than the receptor subunit in a muscarinic receptor appeared to be involved in the effect of guanine nucleotide. When guanine nucleotide was present, the receptor subunit seemed to be dissociated from the GTP subunit.

Regulation by isoproterenol of muscarinic acetylcholine receptor numbers and sensitivity in rat submandibular, but not lacrimal, glands

Chronic administration of DL-isoproterenol, a β-adrenergic agonist, to male Sprague-Dawley rats increased submandibular gland weights by 3 to 4-fold. This increase resulted from a combination of hyperplasia and hypertrophy of secretory cells. Possible effects of this drug regimen on submandibular gland muscarinic acetylcholine receptors were examined by analysis of the binding of the cholinergic antagonist, L-quinuclidinyl [ 3H]benzilate, to receptors in gland homogenates. Parallel investigations of receptors in exorbital lacrimal glands, an organ that is not grossly affected by chronic isoproterenol treatment, were also carried out. [ 3H]QNB bound to submandibular receptors with a K d of 37.8±6.3 pM in control rats and 41.0±4.0 pM in isoproterenol-treated animals, a non-significant difference ( P > 0.05). In contrast, the maximal binding level ( B max) is isoproterenol-treated rats, 1.52±0.10 fmol/ μg DNA, was depressed by approx. 30% ( P<0.05) from that of 2.22±0.16 fmol/ μg DNA in control animals. In lacrimal glands, both K d (61.3±5.3 vs. 53.2±4.0 pM) and B max (1.74±0.24 vs. 1.78±0.17 fmol/ μg DNA) were unchanged by isoproterenol treatment. The affinity of glandular muscarinic receptors for cholinergic agonists was also examined by competition experiments using carbachol. This agonist inhibited [ 3H]QNB binding to receptors in homogenates from both glands in a dose-dependent fashion. Inhibition constant ( K i) for this interaction were similar in control and isoproterenol-treated lacrimal glands; 53.6±5.4 μM and 66.6±7.9 μM, respectively ( P>0.05). In submandibular glands, isoproterenol treatment elicited a highly significant ( P < 0.01) shift in K i from 17.3±1.4 μM to 68.3±5.2 μM. These results demonstrate that chronic administration of isoproterenol to rats results in a reduction in receptor numbers and a decrease in their sensitivity to cholinergic agonists in submandibular, but not lacrimal, glands.