QNB Binding Sites Research Articles

Characterization of a new muscarinic toxin from the venom of the Brazilian coral snake Micrurus lemniscatus in rat hippocampus

AimsWe have isolated a new muscarinic protein (MT-Mlα) from the venom of the Brazilian coral snake Micrurus lemniscatus. Main methodsThis small protein, which had a molecular mass of 7,048Da, shared high sequence homology with three-finger proteins that act on cholinergic receptors. The first 12 amino acid residues of the N-terminal sequence were determined to be: Leu-Ile-Cys-Phe-Ile-Cys-Phe-Ser-Pro-Thr-Ala-His. Key findingsThe MT-Mlα was able to displace the [3H]QNB binding in the hippocampus of rats. The binding curve in competition experiments with MT-Mlα was indicative of two types of [3H]QNB-binding site with pKi values of 9.08±0.67 and 6.17±0.19, n=4, suggesting that various muscarinic acetylcholine receptor (mAChR) subtypes may be the target proteins of MT-Mlα. The MT-Mlα and the M1 antagonist pirenzepine caused a dose-dependent block on total [3H]inositol phosphate accumulation induced by carbachol. The IC50 values for MT-Mlα and pirenzepine were, respectively, 33.1 and 2.26 nM. Taken together, these studies indicate that the MT-Mlα has antagonist effect on mAChRs in rat hippocampus. SignificanceThe results of the present study show, for the first time, that mAChRs function is drastically affected by MT-Mlα since it not only has affinity for mAChRs but also has the ability to inhibit mAChRs.

Binding studies on two functional cardioselective antimuscarinic compounds.

Vecuronium and himbacine are antimuscarinic compounds which in functional studies exhibited a ca 6- and 10-fold higher potency at cardiac muscarinic receptors than at ileal muscarinic receptors. However in binding studies both compounds failed to differentiate between [3H](-)-QNB binding sites in guinea-pig atrial and ileal muscle homogenates. In the latter experiments, the dissociation constants of vecuronium in atria and ileum and that of himbacine in ileum were lower than the values determined functionally. The basis for the lack of cardioselectivity in binding studies is not known. These compounds add to the list of functional cardioselective muscarinic receptor antagonists that failed to display selectivity in binding studies with [3H](-)-QNB.

Assessment of Muscarinic Receptor Subtypes in Human and Rat Lower Urinary Tract by Tissue Segment Binding Assay

Muscarinic receptors in the human and rat lower urinary tract (urinary bladder detrusor muscle and mucosa, and prostate) were identified by intact tissue segment binding assays with two radioligands, and the effects of prolonged receptor activation in vitro on muscarinic receptors were examined. Hydrophilic [3H]-NMS and hydrophobic [3H]-QNB bound to the detrusor muscle segments with the same density, suggesting that the muscarinic receptors were localized at the plasma membrane. While the density of muscarinic receptor was higher in detrusor muscle than in the bladder mucosa and prostate, there was no species-specific difference either in density or in subtype distribution (M1, M2, and M3subtypes in detrusor; M2and M3subtypes in bladder mucosa; and M1and M2subtypes in prostate). Incubation of detrusor strips with carbachol decreased [3H]-NMS binding sites within 20 min, followed by a reduction of [3H]-QNB binding sites after a 60-min lag phase. The loss of the binding sites over 3 h after carbachol treatment was the same (approximately 40%) for both radioligands. The present intact tissue segment binding assay reveals tissue-specific and plasma membrane distribution of distinct muscarinic receptor subtypes and their dynamic changes (internalization and down-regulation) in lower urinary tract of humans and rats.

Muscarinic receptor expression and receptor-mediated detrusor contraction: comparison of juvenile and adult porcine tissue

Urinary bladder function is known to mature during fetal and postnatal development, including changes in neurotransmitter regulation of detrusor contraction. However, only few experimental data are available about muscarinic receptor antagonist function in the urinary bladder from young animals. In the present study, we compare the muscarinic receptor-mediated contractions in juvenile and adult porcine detrusor and the effects of antimuscarinic compounds. Urinary bladders from young (8-12 weeks; 12- to 35-kg body weight) and mature pigs (>40 weeks; >100 kg) were compared. Muscarinic receptor expression was assessed by real time polymerase chain reaction and radioligand binding. Muscle contraction was measured with a force transducer; L-type Ca2+ currents (ICa,L) of isolated detrusor myocytes were recorded with standard voltage clamp technique. Juvenile and adult detrusor expressed similar quantities of the messenger RNA of M2 and M3 receptors. The number of [3H]QNB-binding sites and their affinity for the radioligand were also similar between juvenile and adult detrusor. In contrast, maximum contractile responses to the muscarinic receptor agonist carbachol were slightly larger in juvenile than adult bladders. On the other hand, carbachol was slightly less potent in juvenile than in adult tissue. The M3 antagonist DAU 5884 and the spasmolytic drug propiverine inhibited contractile responses with comparable efficacies and potencies in juvenile and adult tissue. ICa,L was somewhat smaller in juvenile than in adult cells. Taken together, these data suggest that expression and function of M2 and M3 receptors are similar in the detrusor of juvenile and mature pigs. Therefore, similar responses to antimuscarinic compounds could be expected in young and adult patients.

The inhibitory effects of alphaxalone on M1 and M3 muscarinic receptors expressed in Xenopus oocytes.

Alphaxalone is a neurosteroid anesthetic, but its mechanisms of action are not completely understood. Muscarinic receptors are involved in a variety of neuronal functions in the brain and autonomic nervous system, and much attention has been paid to them as targets of anesthetics. In this study, we investigated the effects of alphaxalone on M(1) and M(3) muscarinic receptors using the Xenopus oocyte expression system. Alphaxalone inhibited acetylcholine-induced currents in oocytes expressing M(1) receptors at clinically relevant concentrations. Alphaxalone also suppressed acetylcholine-induced currents in oocytes expressing M(3) receptors. The half-maximal inhibitory concentration values for the inhibition of M(1)- and M(3)-mediated currents were 1.8 +/- 0.6 micro M and 5.3 +/- 1.0 micro M, respectively. GF109203X, a selective protein kinase C inhibitor, had little effect on the inhibition of acetylcholine-induced currents by alphaxalone in oocytes expressing these receptors. Alphaxalone inhibited the specific binding of [(3)H]quinuclidinyl benzilate to oocytes expressing M(1) or M(3) receptors. These findings suggest that alphaxalone at clinically relevant concentrations inhibits the function of M(1) and M(3) receptors through a protein kinase C-independent mechanism by interfering with the [(3)H]quinuclidinyl benzilate binding sites on the receptors. Alphaxalone, a neurosteroid anesthetic, inhibited the function of muscarinic M(1) and M(3) receptors and the specific binding of [(3)H]quinuclidinyl benzilate ([(3)H]QNB) to oocytes expressing these receptors. These findings suggest that alphaxalone inhibits these receptors by interfering with the QNB binding sites.

Effects of Perinatal Nicotine Exposure on Development of [3H]Hemicholinium-3 Binding Sites in Rat Neonate Brain

In this study, [3H]hemicholinium-3 ([3H]HC-3) binding, which labels the presynaptic high affinity-choline transport sites, was examined in two brain regions, cerebral cortex and midbrain, of nicotine-treated and -untreated rat neonates. In nicotine-untreated neonates, [3H]HC-3 binding sites of cerebral cortex increased from 64 fmol/mg protein at postnatal day 7 to 142 fmol/mg protein at postnatal day 35. In nicotine-treated neonates, the development of [3H]HC-3 binding sites in cerebral cortex was significantly retarded, compared with control neonates on the 7th, 14th and 21st postnatal days. In parallel with this, the development of muscarinic receptor in cerebral cortex, which was detected by [3H]quinuclidinyl benzylate ([3H]QNB) binding, was also retarded by nicotine treatment. However, in midbrain, neither [3H]HC-3 nor [3H]QNB binding sites at postnatal day 14 was affected by nicotine treatment. These results strongly suggest that perinatal treatment with nicotine inhibits presynaptic and postsynaptic development of the cholinergic pathway in cerebral cortex but not in midbrain of rat neonate.

Antagonism of muscarinic receptors in the rabbit iris-ciliary body by 8-OH-DPAT and other 5-HT1A receptor agonists.

Physiological studies have shown that serotonin and 5-HT1A agonists can influence muscarinic function in the rabbit iris-ciliary body (ICB). The purpose of this study was to examine whether a direct interaction exists between muscarinic and 5-HT1A receptors in the ICB. At high concentrations, the 5-HT1A agonist 8-OH-DPAT attenuated the carbachol-induced stimulation of inositol phosphates (InsPs) production, but this was not blocked by the presence of 5-HT1A antagonists. In contrast, serotonin failed to influence carbachol-induced InsPs formation. Moreover, 8-OH-DPAT but not serotonin displayed affinity for [3H]QNB binding sites in the ICB. The combined data suggest that activation of 5-HT1A receptors in the ICB does not cause a modulation of muscarinic receptor-stimulated phosphoinositide turnover. The data instead suggest that, at high concentrations, 8-OH-DPAT acts as an antagonist at muscarinic receptors and in this way influences muscarinic receptor function. The mechanism of 5-HT-induced modulation of muscarinic function in the ICB therefore remains to be elucidated.

Muscarinic Acetylcholine Receptors on Rat Thymocytes: Their Possible Involvement in DNA Fragmentation.

Several studies have shown that the nervous (and hormonal) system controls immune functions. In the present study, we examined the presence of muscarinic acetylcholine receptors and the effect of carbachol on DNA fragmentation in adult rat thymocytes. Rat thymocytes possessed high affinity binding sites for the muscarinic antagonist [3H]3-quinuclidinyl benzilate (QNB). The average number of binding sites per cells was 3000, and the equilibrium dissociation constant of [3H]QNB on intact cell was approximately 80 nM. The binding was inhibited by an M1- and M3-selective antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodine (4-DAMP). Hydrocortisone (100 mg/kg, s.c.) treatment of rats for 2 days prior to sacrifice increased the average number of [3H]QNB binding sites on thymocytes by 82 +/- 33%. The gel electrophoresis of DNA extracted from carbachol-treated thymocytes revealed a ladder pattern typical of intranucleosomal fragmentation. The addition of oxotremorine-M also induced DNA fragmentation and the effects of muscarinic agonists were inhibited by the addition of atropine or 4-DAMP. The results suggest the existence of muscarinic receptors and the possible involvement in apoptosis in thymocytes.

Muscarinic Acetylcholine Receptors on Rat Thymocytes: Their Possible Involvement in DNA Fragmentation

Several studies have shown that the nervous (and hormonal) system controls immune functions. In the present study, we examined the presence of muscarinic acetylcholine receptors and the effect of carbachol on DNA fragmentation in adult rat thymocytes. Rat thymocytes possessed high affinity binding sites for the muscarinic antagonist [3H]3-quinuclidinyl benzilate (QNB). The average number of binding sites per cells was 3000, and the equilibrium dissociation constant of [3H]QNB on intact cell was approximately 80 nM. The binding was inhibited by an Ml- and M3-selective antagonist, 4-diphenylacetoxy-7V-methylpiperidine methiodine (4-DAMP). Hydrocortisone (100 mg/kg, s.c.) treatment of rats for 2 days prior to sacrifice increased the average number of [3H]QNB binding sites on thymocytes by 82 + 33%. The gel electrophoresis of DNA extracted from carbachol-treated thymocytes revealed a ladder pattern typical of intranucleosomal fragmentation. The addition of oxotremorine-M also induced DNA fragmentation and the effects of muscarinic agonists were inhibited by the addition of atropine or 4-DAMP. The results suggest the existence of muscarinic receptors and the possible involvement in apoptosis in thymocytes.

Long‐term carbachol treatment‐induced down‐regulation of muscarinic M2‐receptors but not m2 receptor mRNA in a human lung cell line

1. The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. We have investigated the effect of homologous stimulation on the regulation of M2 muscarinic receptor protein and gene in human embryonic lung fibroblasts (HEL 299 cells). 2. Saturation studies performed with the non-selective hydrophilic ([3H]-N-methyl-scopolamine, [3H]-NMS) and lipophilic (3H]-quinuclidinyl benzilate, [3H]-QNB) muscarinic antagonists revealed a single class of high affinity binding sites. 3. Carbachol (1 mM) induced a rapid down-regulation of [3H]-NMS binding sites. Within 12 h, the process had approached steady state with 40 to 60% loss of receptors at 12 and 24 h. 4. The loss of [3h]-QNB binding sites (40% reduction at 24 h) occurred at a slower rate than did loss of [3H]-NMS binding sites as a result of receptor sequestration. 5. Carbachol treatment was accompanied by a functional desensitization of the receptor after 24 h of agonist treatment. In untreated cells, forskolin induced a large increase in cyclic AMP accumulation which was inhibited significantly by carbachol. The inhibitory effect of carbachol on forskolin-induced cyclic AMP accumulation was lost following 24 h carbachol stimulation. 6. The steady state level of muscarinic m2 mRNA measured by Northern blot analysis was not affected by carbachol had no effect on the stability of m2 mRNA. 7. The rate of transcription of m2 muscarinic receptor gene as measured by nuclear RNA run-on assay was unaltered by carbachol stimulation. 8. These results suggest that homologous sequestration, desensitization, and down-regulation of M2 modifications of m2 muscarinic receptor mRNAs.

Nerve growth factor treatment restores [3H]QNB binding site density in adult rat subjected to cortical infarction.

The effects of unilateral cortical devascularization and nerve growth factor treatment on muscarinic cholinoceptors labelled with [3H]QNB were investigated in rat cerebral cortex. The lesion induced a significant ipsilateral decrease in the density (Bmax) but not in the affinity (kD) of [3H]QNB binding sites. Intraventricular infusion of 2.5 S mouse nerve growth factor (NGF) increased the density of binding sites such that Bmax values of lesioned animals treated with NGF were not different from control subjects.

O-275 - Pharmacological studies on the respiratory tract (Rept. 185): Repeated antigenic challenge induces airway hyperresponsiveness to ACh without changing [3H]QNB binding sites.

O-275 - Pharmacological studies on the respiratory tract (Rept. 185): Repeated antigenic challenge induces airway hyperresponsiveness to ACh without changing [3H]QNB binding sites.

Upregulation of nicotinic receptors following continuous infusion of nicotine is brain-region-specific

Rats receiving 4 mg nicotine/kg/day via implanted minipumps sustained plasma nicotine concentrations of 40 ng/ml throughout two weeks of nicotineinfusion. Numbers of brain [ 3H]nicotine binding sites were increased by about 50% in cortex and hippocampus whereas numbers of [ 3H]nicotine binding sites in striatum were unaffected by nicotine treatment at either of the timepoints examined (7, 14 days). Cortical [ 125I]α-bungarotoxin and [ 3H]QNB binding sites were also unchanged. The regional selectivity of nicotinic receptor modulation may reflect the low dose of nicotine used and the mode of administration. The changes observed may be pertinent to the continuous administration of nicotine in man, via transdermal nicotine patches.

Effects of propentofylline, a NGF synthesis stimulator, on alterations in muscarinic cholinergic receptors induced by basal forebrain lesion in rats

Basal forebrain (BF) lesions induced by ibotenic acid produced increases in the B max and K d values of [ 3H]QNB binding sites in the frontal cortex, parietal cortex, and hippocampus. Twenty-eight-day successive administration of propentofylline (10 and 25 mg/kg, p.o.) significantly reduced the K d values of [ 3H]QNB binding sites, to the levels of those in a sham group, in a dose-dependent manner. Moreover, propentofylline (25 mg/kg, p.o.) significantly reduced the B max value of [ 3H]QNB binding sites compared with that in a vehicle-treated BF-lesioned group. These results suggest that successive administration of propentofylline ameliorates changes in muscarinic cholinergic receptors through improving presynaptic cholinergic dysfunction.

Muscarinic receptor subtypes in human nasal mucosa: characterization, autoradiographic localization, and function in vitro.

Muscarinic receptors play important roles in the regulation of glandular secretion and vasomotor tone in human nasal mucosa. M1, M2, and M3 muscarinic receptor subtypes were pharmacologically characterized in human inferior turbinates by receptor-binding assays using [3H](-)quinuclidinyl benzilate (QNB, identifies total muscarinic receptors) and [3H]-pirenzepine (PZ). Receptors were localized by autoradiography, and their function examined in vitro by assaying mucus secretion from cultured nasal mucosal explants. In competition assays, PZ was employed as a selective muscarinic antagonist for M1 receptors, gallamine and AF-DX 116 for M2 receptors, and 4-DAMP for M3 receptors. These ligands are selective at low nanomolar concentrations, but can interact with other muscarinic receptors at higher concentrations. It is not known if they can interact with putative M4 and M5 muscarinic receptor subtypes. Using [3H](-)QNB, total muscarinic receptor binding was 688.4 +/- 49.6 fmol/mg protein (Bmax), with a Kd of 1.47 +/- 0.13 nM. [3H]-PZ bound to 45% of the total QNB binding sites. In competition experiments, 4-DAMP displaced [3H](-)QNB with the lowest IC50, followed by PZ and AF-DX 116. Autoradiograms demonstrated that [3H](-)QNB binding was completely displaced by 4-DAMP, partially displaced by PZ, but not displaced by gallamine or AF-DX 116, and suggested that M1 and M3 subtypes coexist in submucosal glands. The localization of M1 receptors on submucosal glands was confirmed by direct labeling with [3H]-PZ. [3H]-PZ also labeled vessels, but with a low silver grain density. Autoradiographic [3H]-QNB binding was displaced by 4-DAMP and atropine, but not by PZ, gallamine, or AF-DX 116. In studies of mucus secretion in vitro, 4-DAMP significantly inhibited methacholine-induced secretion. Although less effective, PZ also had significant inhibitory effects. Neither gallamine nor AF-DX 116 had any inhibitory effect. M1 receptors (PZ binding sites) may regulate glandular secretion while M3 receptors (4-DAMP binding sites) may regulate glandular secretion and vasomotor tone in human nasal mucosa.

Characterization of muscarinic receptors in rat kidney

Muscarinic receptors in mammalian kidney seem to be involved in diuresis. In this study we give a detailed characterization of receptors in rat kidney. Specific binding of [ 3H](−)-quinuclidinylbenzilate ([ 3H]QNB) to membranes of rat kidney cortex was saturable and of high affinity. A dissociation constant of 0.063 ± 0.003 nM and a receptor density of 1.46 ± 0.07 pmol/g wet weight were obtained. The dissociation kinetics could be best described by assuming a mono-exponential function (k −1 = (0.52 ± 0.1) × 10 −4 s −1). The binding of [ 3H]QNB reached a maximum in 60 min at 0.6 nM at 37°C. Competition experiments with the enantiomers of benzetimide confirmed the muscarinic nature of the [ 3H]QNB binding sites. The inhibition constants of pirenzepine (0.23 ± 0.02 μM), (±)-hexahydrosiladifenidol (0.040 ± 0.002 μM, AF-DX 116 (1.45 ± 0.07 μM), methoctramine (1.67 ± 0.02 μM) and gallamine (78 ± 3 μM) classified this receptor as an M 3 receptor. Inhibition of[ 3H]QNB binding by the agonists methylfurtrethonium, arecoline, isoarecoline methiodide, arecaidine propargyl ester and McN-A-343 displayed monophasic inhibition curves. With (±)-cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide in two out of four experiments a small (11%) population of high affinity agonist sites could be detected. The potassium sparing diuretic amiloride inhibited [ 3H]QNB binding (36 ± 3 μM). Although in a way related to the amiloride binding site, the muscarinic receptors in rat kidney are unlikely to be the primary target of diuretic action of this drug.

Muscarinic receptor localization and function in rabbit carotid body

Acetylcholine and muscarinic agonists inhibit chemosensory activity in the rabbit carotid sinus nerve (CSN). Because the mechanism of this inhibition is poorly understood, we have investigated the kinetics and distribution of muscarinic receptors in the rabbit carotid body with the specific muscarinic antagonist [ 3H]quinuclidinylbenzilate ([ 3H]QNB). Equilibrium binding experiments identified displaceable binding sites (1 μM atropine) with a K d = 71.46pM and a B max = 9.23pmol/g tissue. These binding parameters and the pharmacology o the displaceable [ 3H]QNB binding sites are similar to specific muscarinic receptors identified in numerous other nervous, muscular and glandular tissues. Comparisons of specific binding in normal and chronic CSN-denervated carotid bodies suggest that muscarinic receptors are absent on afferent terminals in the carotid body; however, nearly 50% of the specific [ 3H]QNB binding is lost following chronic sympathectomy, suggesting the presence of presynaptic muscarinic receptors on the sympathetic innervation supplying the carotid body vasculature. Autoradiographic studies have localized the remainder of [ 3H]QNB binding sites to lobules of type I and type II parenchymal cells. In separate experiments, the muscarinic agonists, oxotremorine (100 μM) and bethanechol (100 μM) were shown to inhibit both the release of catecholamines and the increased CSN activity evoked by nicotine (50 μM) stimulation of the in vitro carotid body. Our data suggest that muscarinic inhibition in the rabbit carotid body is mediated by receptors located on type I cells which are able to modulate the excitatory actions of acetylcholine at nicotinic sites.

Regulation of Brain Nicotinic Receptors by Chronic Agonist Infusion

Several studies have demonstrated that chronic treatment with nicotine elicits an increase in the number of brain nicotinic receptors. To determine whether this effect is elicited by other nicotinic agonists found in tobacco, the effects of chronic infusion with nicotine on brain nicotinic receptors were compared with those after anabasine and lobeline. C57BL/6 mice were infused with saline or equimolar doses (18.5 mumol/kg/h) of nicotine, anabasine, or lobeline for 8 days. Nicotinic receptors, quantified by the binding of [3H]nicotine and [125I]iodo-alpha-bungarotoxin (alpha-[125I]BTX), and muscarinic receptors, quantified by the binding of [3H]quinuclidinyl benzilate ([3H]QNB), were then assayed in eight brain regions. An increase in [3H]nicotine binding was observed in all regions except cerebellum following chronic infusion with nicotine and anabasine, whereas lobeline did not alter the number or affinity of these binding sites. This increase was due to changes in Bmax and not in the affinity of the receptor for the ligand (KD). A slight increase in alpha-[125I]BTX binding was observed in cortex following chronic anabasine infusion. [3H]QNB binding sites were largely unaltered following chronic infusion with any of the nicotinic analogs. The levels of the agonists in the brain were also determined after chronic treatment, and the amounts of lobeline and anabasine were found to be higher than that of nicotine. Thus, the failure of lobeline to elicit changes in nicotine binding is not due to reduced brain concentrations.

Autoradiographic localization of muscarinic acetylcholine receptors in the rat pulmonary vascular tree

The pharmacological characteristics and the anatomical localization of muscarinic receptors in the pulmonary vascular tree were investigated in lung sections of Wister-Kyoto (WKY) and spontaneously hypertensive rats (SHR). [ 3H]Quinuclidinyl benzylate ([ 3H]QNB) was bound by sections of rat lung in a manner consistent with the labeling of muscarinic acetylcholine receptors, with a dissociation constant value (K d) of 0.41 ± 0.3 nM in WKY rats and of 0.37 ± 0.2 nM in SHR. The density of muscarinic acetylcholine receptors was higher in sections of lung of WKY rats than of SHR. In the pulmonary vasculature these sites were associated with the smooth muscle of the medial layer of different size branches of the pulmonary artery and vein. No [ 3H]QNB binding sites were found within the endothelium in the blood vessels of either WKY rats or SHR. The density of [ 3H]QNB binding sites was significantly lower in the smooth muscle of pulmonary vein and its branches in SHR. There were no significant hypertension-dependent changes in the density and pattern of muscarinic receptors of pulmonary artery smooth muscle.

Developmental changes of [ 3H]QNB-binding sites in growth cone membranes isolated from the fetal and neonatal rat brain

Developmental changes of [ 3H]QNB-binding sites in growth cone membranes isolated from the fetal and neonatal rat brain