QNB Binding Research Articles

Characterization of muscarinic cholinergic receptors in membrane preparations from rat prostatic adenocarcinoma

The binding characteristics of 3H-quinuclidinyl benzilate (QNB) to muscarinic sites in isolated plasma membrane fractions from R-3327 Dunning tumors (H and AT-1 sublines); ventral, dorsolateral prostate; and urinary bladder of the rat were studied. QNB binding to all preparations, except from AT-1 tumors, was specific, saturable, and of high affinity. The AT-1 tumors completely lacked specific QNB binding. The muscarinic receptor density in H tumors was twofold and twentyfold higher than that in the ventral prostate and dorsolateral prostate respectively. The receptor density in the urinary bladder was approximately twofold higher than that in H tumors. The Kd values in H tumors and ventral prostate were very similar and significantly higher than that in dorsolateral prostate or the urinary bladder. QNB binding in H tumors was strongly inhibited by classical muscarinic receptor antagonists atropine and scopolamine, but poorly by the agonists carbacholine and pilocarpine. In contrast to scopolamine or atropine, inhibition by pirenzepine and AF-DX116 was relatively low. These data indicate that the muscarinic receptor in Dunning H tumors is of M3 type.

Binding Characteristics of the Muscarinic Receptor Subtype in Rabbit Pancreas

The muscarinic receptor in the rabbit pancreas was characterized with the use of the labeled ligand (3H)-(-)-quinuclidinyl-benzylate ((3H)-(-)-QNB). Specific binding of (3H)-(-)-QNB to pancreatic acini was found to be reversible and of high affinity, with an equilibrium dissociation constant (KD) of 68 pmol/l and a receptor density (RT) of 170 fmol/mg protein. Agonist binding behaviour was investigated by displacement of (3H)-(-)-QNB binding by eight agonists like arecoline, arecaïdine-propargylester (APE) and carbachol, yielding only low affinity binding sites. The inhibition of (3H)-(-)-QNB binding by the selective antagonists pirenzepine, hexahydrosiladifenidol (HHSiD) and (11-[2-[diethyl-amino)-methyl)-1-piperidinyl]acetyl)-5,11-dihydro-6H-pyr ido (2,3-b) (1,4) benzodiazepin-6-one) (AF-DX 116) confirmed the M3 nature of the rabbit pancreatic receptor.

Alpha adrenergic drugs inhibit [3H]-QNB binding to muscarinic receptors of rat heart, brain and parotid gland membranes

Alpha adrenergic agonists and antagonists as clonidine, guanfacine, yohimbine, phenylephrine and prazosin inhibited the [3H]-QNB binding to rat brain cortex muscarinic acetylcholine receptor (mAChR, M-1 subtype), heart (M-2 subtype) and parotid gland homogenate (M-3 subtype) in a dose-dependent competitive fashion. Ki values were between 10(-6) and 10(-3) M. Hill coefficients were about 1. No correlation was found between mAChR inhibiting capacity of these drugs and their activity on alpha adrenergic receptors. In contrast, other transmitters, as dopamine, GABA, glutamic acid, histamine, serotonin, isoproterenol and platelet activating factor (PAF) did not affect the QNB binding.

Effects of the Ca 2+-antagonists nifedipine, verapamil, flunarizine and of the calmodulin antagonist trifluoperazine on muscarinic cholinergic receptors in rat cerebral cortex

1. 1. Studies were made of [ 3H]QNB binding to muscarinic receptors in a membrane fraction from the cerebral cortex of rats treated orally for 13 days with the Ca 2+-antagonists nifedipine (20 mg/kg), verapamil (50 mg/kg), flunarizine (10 mg/kg) and with the calmodulin antagonist trifluoperazine (3 mg/kg). 2. 2. The [ 3H]QNB binding capacity ( B max) was decreased by the three Ca 2+-antagonists: nifedipine, verapamil and flunarizine, the decrease was most pronounced with nifedipine. 3. 3. The decrease in the number of muscarinic receptors after nifedipine and flunarizine was accompanied by an increase in their affinity; verapamil decreased both the number and the affinity of muscarinic receptors. 4. 4. The calmodulin antagonist trifluoperazine changed neither the number nor the affinity of muscarinic receptors. 5. 5. It is suggested that continuous treatment with different Ca 2+ or calmodulin antagonists leads to difference in character and degree of alterations in the basic characteristics of muscarinic receptors in rat cerebral cortex.

The interaction of acetylcholine receptors in porcine atrial membranes with three kinds of G proteins.

We developed a simple procedure to detect the interaction of muscarinic receptors in atrial membranes with exogenous GTP-binding proteins (G proteins). The procedure consists of mixing atrial membranes with G proteins in the presence of sodium cholate, diluting the mixture with a salt buffer and then measuring the ligand binding activity. The displacement by carbachol of [3H] QNB binding to muscarinic receptors in the atrial membranes was not affected by guanine nucleotides when the membranes had been treated at 60 degrees C for 30 min or with N-ethylmeleimide (NEM) and became affected by them after mixing the heat- or NEM-treated membranes with G proteins. The displacement curves in the presence of GTP were essentially the same irrespective of the presence or absence of G proteins. Those in the absence of GTP shifted to a lower concentration of carbachol with addition of a higher concentration of G proteins, indicating an increase in GTP-sensitive high affinity agonist binding sites. The highest affinity for carbachol was detected with membranes treated with NEM and then mixed with G proteins. The GTP-sensitive high affinity agonist binding could be detected with any one of three kinds of G proteins (Gi, Go, Gn) which were purified from porcine cerebrum, indicating that the muscarinic receptor m2 subtype may interact with and possibly activate these three kinds of G proteins.

Alteration of acetylcholine metabolism in the brain of zucker fatty rat

The cerebral metabolism of acetylcholine (ACh) and [(3)H]quinuclidinyl benzilate([(3)H]QNB) binding to particulate fraction from the brain were examined using Zucker fatty rat. In all regions of the brain of fatty rat, ACh content showed a lower level than that of lean rat. Choline acetyltransferase activities in the striatum and medulla oblongata of fatty rat were significantly higher than those in lean rat, whereas the activities of acetylcholinesterase in the same brain regions of fatty rat were found to be significantly lower than those in lean rat. The [(3)H]QNB binding to particulate fraction obtained from the striatum of fatty rat was also found to be lower than that in lean rat. These results strongly suggest that the brain of Zucker fatty rat has impairments in the functions of cerebral cholinergic system.

Comparative studies of muscarinic and dopamine receptors in three strains of rat

Cholinesterase activities and characteristics of muscarinic and dopamine form 9 week old male Sprague-Dawley (SD), Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were studied. Plasma cholinesterase activity in WKY was significantly lower (50%) than activity in the other strians. In studies of muscarinic receptors, the number of [ 3H]QNB binding sites in striata from SD rats was lower (18%) than those from WKY and SHR. However, muscarinic receptor properties (K d and B max) were the same in hypothalami. Studies of dopamine receptors revealed that the densities of both D-1 and D-2 receptors in both striata and hypothalami were significantly higher in SHR than in other strains. However, there were no differences in the affinity constant (K d). The higher densities in hypothalami from SHR were mainly due to the high populaion of D-a and D-2 receptors in the posterior hypothalamus. In the anterior hypothalamus, there was no difference in the population of D-2 receptors. These results provide a substantive basis, i.e. demonstration of alterations in drug metabolizing enzymes and receptor populations, on which to build an understanding of the genetic predisposition to the actions of xenobiotic agents.

Effects of Castration and Diabetes Mellitus on Cholinergic Responsiveness and Muscarinic Receptors in the Rat Vas Deferens

Studies were carried out to compare the effects of diabetes mellitus and castration on the muscarinic responsiveness of rat vasa deferentia. One and two months after castration or after streptozotocin-induced diabetes, there were significant decreases in serum testosterone levels, accompanied by significant decreases in vas deferens weights, protein contents, and protein concentrations. However, contractile responses of vasa deferentia from streptozotocin-diabetic or castrated rats to carbachol were significantly increased compared to controls. These changes were accompanied by increases in Kd and decreases in Bmax (pm/tissue) for 3H-quinuclidinyl benzilate (QNB) binding. Contractile responses and QNB binding were also studied in the spontaneously diabetic BB/Wor rat, a less severely diabetic model than the streptozotocin-diabetic rat. Ninety days after the onset of diabetes in the BB rat, there was an increase in the contractile response to carbachol. There were qualitatively similar but smaller changes in vas deferens weights, protein contents, protein concentrations, and QNB binding than in the streptozotocin-diabetic rats. The data show that diabetes and castration increased the contractile responses of vasa deferentia despite decreases in protein content and in the number of muscarinic receptors. The results suggest that muscarinic receptor-effector coupling is more efficient in vasa deferentia from castrated or diabetic rats, resulting in a greater contractile response to cholinergic agonists.

Amiloride inhibits 22Na uptake and [ 3H]QNB binding in rat submandibular cells

Dispersed acini isolated by collagenase digestion of the rat submandibular gland were used to compare the effects of amiloride and furosemide on the uptake of the isotopic tracer 22Na and on the binding of [ 3H]quinuclidinyl benzylate ([ 3H]QNB). In mM concentrations, both inhibitors reduced 22Na uptake in resting cells 34 and 25–29%, respectively. Acetylcholine (1 μM) enhanced uptake 23% and this effect was reduced 45% by amiloride and 26% by furosemide. Amiloride inhibited the binding of [ 3H]QNB to crude membranes prepared from fresh submanidubular glands in a dose-dependent fashion (IC 50=8×10 −6 M). Furosemide (3×10 −8 to 10 −3 M) did not inhibit radioligand binding. Na influx into resting salivary acini thus appears to occur by both amiloride-sensitive and furosemide-sensitive transport systems. The similar inhibition by furosemide of unstimulated and stimulated uptake of 22Na suggests that acetylcholine does not significantly activate the cotransport system within the time frame (i.e., 2 min) of the experiments. Acetylcholine appears to activate an amiloride-sensitive Na/H antiport, but amiloride blocks cholinergic receptors and may thus affect Na transport by receptor blockade. Other actions of amiloride, such as its ability to penetrate into cells and to act as a weak base which alters intracellular pH, may also contribute to the inhibition of Na entry into salivary cells.

Characterization of ion movements and their relationship to muscarinic receptor binding and excitation-contraction coupling in guinea pig ileal longitudinal smooth muscle.

The relationship between ion movements (sodium uptake and potassium release) and agonist-induced contractile responses or muscarinic receptor binding was investigated in the guinea pig ileal longitudinal muscle (GPLM). Sodium uptake and potassium release were agonist-dependent, concentration-dependent, and stereoselective, with the following rank order of maximum ion movement: muscarinic agonists greater than histamine greater than substance P = serotonin. Potassium depolarization did not initiate sodium uptake or potassium release. Sodium uptake was rapid and monophasic, preceding potassium release which was biphasic in nature. Full muscarinic agonists produced equal maximal increases in sodium uptake, while maximal potassium release varied for all muscarinic agonists and in addition differed from sodium uptake in the following ways: time course, stereoselectivity, sensitivity to calcium antagonists, modulation by the guanylyl nucleotide derivative, 5'-guanylylimidodiphosphate (Gpp(NH)p), and inhibition by muscarinic receptor blockade with benzilylcholine mustard. The calcium ionophores A23187 and ionomycin (SQ23377) did not produce any sodium uptake; A23187 but not ionomycin produced potassium release comparable to that evoked by muscarinic agonists. Ion movement in response to combinations of agonists were not additive. Muscarinic agonist binding as measured by competition for [3H]quinuclidinyl benzilate ([3H]QNB) binding, was best described by multiple sites and was regulated by Gpp(NH)p. Excellent correlations were observed between the dissociation constants for binding and sodium uptake, potassium release, and contraction. The best correlations were those between the pharmacologic responses and the high affinity binding site in the absence, and the low affinity site in the presence, of Gpp(NH)p, respectively. Furthermore, the potencies of muscarinic agonists to evoke ion movements and to inhibit [3H]QNB binding were similar, and from one to two orders of magnitude less than those for contraction. It is suggested that contraction and potassium release were mediated by the high affinity, and sodium uptake by the low and average affinity muscarinic agonist binding sites, respectively. These findings suggest an agonist-activated receptor-effector coupling model in GPLM that leads to the activation of sodium uptake, potassium release, and subsequently, contraction.

Effects of nBM lesions on muscarinic-stimulation of phosphoinositide hydrolysis

The nucleus basalis magnocellularis (nBM) is believed to be the major path of cholinergic innervation to the frontal cortex. The cerebral cortex is known to contain muscarinic receptors that are coupled to the hydrolysis of phosphoinositides (PI) (9,14). Adult male Sprague-Dawley rats were unilaterally and bilaterally lesioned at the nBM with the excitotoxin ibotenic acid and killed at 7 or 21 to 23 days postsurgery. In rats unilaterally lesioned 7 days previously, the carbachol dose-response curves in lesioned fronto-parietal cortex were identical to control fronto-parietal cortices. In rats studied 21 to 23 days postsurgery, carbachol dose-response curves were again identical in control vs. lesioned fronto-parietal cortices. Similar results are obtained when bilaterally lesioned rats are compared to sham-operated controls. For each group, the hydrolysis is linear with respect to time until 15 minutes with a maximum reached at approximately 40 minutes. Receptor density, as measured by [ 3H]-QNB binding or agonist competition for [ 3H]-QNB binding, was not changed by any of the lesions studied. These results suggest that the loss of cholinergic innervation from the nBM does not result in compensatory denervation supersensitivity in cerebral fronto-parietal cortical muscarinic receptors.

Mechanism of Agonist‐Induced Down‐Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line

The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.

Muscarinic cholinergic components in the carp brain

The activity of the muscarinic cholinergic system (acetylcholine, ACh; acetylcholinesterase, AChE; choline acetyltransferase, ChAT; muscarinic acetylcholine receptors) was studied in the carp brain. The ACh content (13.9 +/- 1.1 nmol/g wet tissue) was estimated by gas chromatography after microwave irradiation focused to the head. The AChE and ChAT activities were 153 +/- 13 nmol/min/mg protein and 817 +/- 50 pmol/min/mg protein, respectively. The characteristics of [(3)H](?)quinuclidinyl benzilate ([(3)H](?)QNB) and [(3)H]pirenzepine ([(3)H]PZ) binding were also studied in brain membranes. Their specific binding was linearly dependent on the protein content and they appeared to bind with high affinity to a single, saturable binding site. A dissociation constant (K(d)) of 47 +/- 6.3 pM and a maximum number of binding sites (B(max)) of 627 +/- 65 fmol/mg protein were obtained for [(3)H](?)QNB, with a K(d) value of 3.85 +/- 0.67 nM and a B(max) value of 95.3 +/- 6.25 fmol/mg protein for [(3)H]PZ binding. The [(3)H]PZ binding amounted to only 15% of the [(3)H](?)QNB-labeled sites, as estimated from the ratio of the B(max) values of [(3)H](?)QNB and [(3)H]PZ, suggesting a low density of M(1) subtype. Atropine sulfate, atropine methylnitrate and PZ inhibited the binding of both radioligands with Hill slopes (n(H)) close to unity. The n(H) value of AF-DX 116 was close to 1 against [(3)H](?)QNB binding, while it was 0.75 against [(3)H]PZ binding. The displacement curves of oxotremorine and carbachol were shallow for the binding of both radioligands. The rank order of potency of muscarinic ligands against [(3)H](?)QNB binding (K(i) nM) was atropine sulfate (0.55) > atropine methylnitrate (1.61) > PZ (61.19) > oxotremorine (156.3) > AF-DX 116 (307) > carbachol (1301), while in the case of [(3)H]PZ binding it was atropine sulfate (0.24) > atropine methylnitrate (0.34) > PZ (10.38) > AF-DX 116 (55.87) > oxotremorine (62.79) > carbachol (1696). The results indicate the presence of a well-developed muscarinic cholinergic system with predominantly M(2) receptors in the carp brain.

In vitro effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on muscarinic cholinergic receptors in mouse striatum

1. MPTP significantly lowered K d of the binding of [ 3H]QNB to muscarine receptor without affecting B max values compared with those of control. Hill coefficients (nH) of control and MPTP (250 μM) added group were 1.15 ± 0.127 and 0.56 ±0.202, respectively. 2. Prior addition of pargyline to MPTP did not prevent the decrease of [ 3H]QNB binding. The patterns of displacement of [ 3H]QNB by MPTP and MPP + were similar to those by some muscarinic agonists, such as acetylcholine, carbamyl choline and methacholine. 3. These results suggest that MPTP might be muscarinic agonist and might play a role to produce Parkinsonism through directly affecting the muscarinic cholinergic receptors in vivo.

P-332 - Differences in QNB binding between freshly isolated and cultured single cells prepared from longitudinal smooth muscle of guinea pig ileum

P-332 - Differences in QNB binding between freshly isolated and cultured single cells prepared from longitudinal smooth muscle of guinea pig ileum

Muscarinic receptor subtype specificity of 5′-(isobutylthio)-adenosine (SIBA) and its analogs

1. 1. 5′-(Isobutylthio)-adenosine (SIBA) and its analogs, at 100 μM, inhibited [ 3 H]N- methylscopolamine binding to homogenates of whole brain and cortex (mainly M 1 subtype receptors) by 11–30% and to cerebellum (mainly M 2 subtype receptors) by 20–39%. 2. 2. At 0.01–1.0 μM, stimulation of [ 3H]QNB and NMS-inaccessible [ 3H]QNB binding was observed, with the most induced by 1 μM 3-deaza-SIBA. 3. 3. In contrast, [ 3H]pirenzepine ([ 3H]PZ) binding to whole brain and cortex was inhibited in a dose-dependent manner with K i values in the μM range. 4. 4. As antagonists of acetylcholine-induced contraction of guinea pig ileum (mainly M 2 subtype receptors), the analogs were slightly more potent than pirenzepine, but several orders of magnitude less than atropine; the order of potency was opposite that determined for the binding of [ 3H]PZ to cortex. 5. 5. Thus, SIBA and its analogs may have differential effects on muscarinic receptor subtypes and show some specificity for the M 1 receptor subtype.

Muscarinic receptor binding in central airway musculature in chronic airflow obstruction

The hypothesis of an increased muscarinic receptor sensitivity in airway musculature of patients with chronic airflow obstruction (CAO) has been investigated by in vitro radioligand binding studies. The receptor binding profiles were determined in membrane homogenates of tracheal and main bronchial smooth muscle, as well as in segmental bronchial tissue preparations. The number of receptors was measured by binding of the radioactive muscarinic antagonist [3H]-(-)-quinuclidinyl benzilate ([3H]-(-)QNB). The affinity for agonists was investigated by studying the inhibition of [3H]-(-)-QNB binding by the full muscarinic agonist methylfurtrethonium. Binding characteristics were determined (1) in tracheal smooth muscle from 7 patients with chronic airflow obstruction (2) in main bronchial smooth muscle of 5 patients with CAO and (3) in segmental bronchial tissue of 10 patients with CAO. The receptor binding properties were compared with values obtained on airway tissue from 31 subjects without CAO. Muscarinic receptors in smooth muscle preparations of trachea and main bronchus were present in normal density, and showed normal [ 3H]-(-)-QNB binding affinity and methylfurtrethonium binding properties. In segmental bronchial tissue preparations which contain smooth muscle and glandular tissue, also normal receptor numbers and [ 3H]-(-)-QNB affinity values were found. Morphometric examination of these preparations revealed normal amounts of smooth muscle and submucosal glands. This suggests that the excessive mucus production of CAO is not accompanied by an increased muscarinic receptor density in submucosal glands. These observations suggest unaltered muscarinic receptor characteristics in central airway smooth muscle of patients with chronic airflow obstruction.

In vitro and ex vivo evaluation of cyclic aminoalkyl benzilates as potential emission tomography ligands for the muscarinic receptor

A series of muscarinic antagonists were screened as potential receptor imaging agents. (+)2α-tropanyl benzilate (TRB), N-methyl-4-piperidyl benzilate (NMPB) and several analogs amenable to labeling with positron emitting isotopes were evaluated for muscarinic binding to mouse brain tissue in vitro and ex vivo using [ 3H]quinuclidinyl benzilate as the probe. The in vitro assay directly compared the innate binding affinities of the compounds. The rank order of binding (IC 50) was TRB (0.7 nm), QNB (0.8 nm), scopolamine (1.3 nm) and NMPB (1.6 nm). The ex vivo assay was used to gain information regarding the pharmacokinetics and brain penetration of the compounds in live animals. Ex vivo results demonstrated that TRB was rapidly taken up into the brain and was equipotent with QNB in occupying muscarinic binding sites at early time points, but TRB binding decreased twice as fast over time as QNB binding. The results suggest TRB would be a good candidate for radiolabeling and further study.

A role for potassium channels in the regulation of cortical muscarinic acetylcholine receptors in an in vitro slice preparation

The rules underlying muscarinic acetylcholine receptor (mAChR) regulation in an in vitro cortical slice preparation of adult rats were examined following various alterations of bioelectric activity and following agonist stimulation. Muscarinic ACh antagonists [ 3 H]N- methyl scopolamine ([ 3H]NMS) or [ 3H]quinuclidinyl benzylate ([ 3H]QNB) were used to label cell surface vs total (i.e. surface and internal) receptors, respectively. Depolarization of neural membranes for 4 h at 22–37 °C using veratridine or high external potassium (K +) led to a temperature-dependent down-regulation of surface mAChR of 26.2% and 11.3%. Total mAChRs decreased by 37.6% and 8.1%. Addition of picrotoxin and glutamic acid also led to decreases in mAChRs. Increases in inward chloride ion current induced by γ-aminobutyric acid (GABA) or gold chloride had no significant effect on mAChRs. Blockade of calcium channels and synaptic transmission by magnesium or cobalt and postsynaptic calcium channels with nifedipine showed a significant effect on mAChRs only in the latter case. In contrast, agonist stimulation using carbachol led to a large down-regulation for both [ 3H]NMS and [ 3H]QNB (26.1%, 35.9%). ACh decreased [ 3H]QNB binding by 33.9%, but had little effect on [ 3H]NMS binding (6.3%). For [ 3H]QNB binding sites the effects of carbachol appeared to summate with those of veratridine. Down-regulation of [ 3H]NMS labelled mAChRs by carbachol and veratridine had an estimated half-time of 30 min and 2 h, respectively. Neither the effects of veratridine nor carbachol could be antagonized by tetrodotoxin (TTX), showing that the effects were not due to an increase in sodium ion currents. However, a common thread linking the various agents which induce mAChR down-regulation appears to involve changes in potassium (K +) current. Potassium channel blockers tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP) and apamin had little independent effect on mAChR number, but prevented veratridine-induced down-regulation, presumably through a blockade of K +- and Ca 2+-dependent K +-channels. Only TEA and 4-AP diminished carbachol-induced down-regulation suggesting that this effect involves only the non Ca 2+-dependent K +-channels. It thus appears that mAChR regulation in the rat cerebral cortex is linked to changes in active K +-channel currents: activation of the K +-channel by depolarization-induced changes in K + current or by agonist stimulation leading to changes in the selective K + currents stimulate mAChR down-regulation; blockage of the K +-channels prevents this down-regulation.

Identification of drug receptors in porcine dental pulp by the radioligand binding assay

1. 1. The acetylcholine (ACh), histamine and serotonin (5-HT) receptors in porcine dental pulp were characterized by the radioligand binding assay. 2. 2. For [ 3H]nicotine binding site, K d was 8.06 ± 1.65 nM and B max was 270.83 ± 32.68 fmol/mg protein. 3. 3. For [ 3H]QNB binding site, K d was 1.04 ± 0.14 nM and B max was 24.83 ± 3.09 fmol/mg protein. 4. 4. For [ 3H]histamine binding site, K d was 1.22 ± 0.1 nM and B max was 283.15 ± 33.1 fmol/mg protein. 5. 5. For [ 3H]5-HT binding site, K d was 1.41 ± 0.1 nM and B max was 53.1 ± 3.4 fmol/mg protein. 6. 6. These findings indicate that the specific receptors for ACh, histamine and 5-HT are present in the porcine dental pulp, and that the ACh receptor is predominantly nicotinic.