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1.
Neosurugatoxin, a Specific Antagonist of Nicotinic Acetylcholine Receptors   总被引:8,自引:6,他引:2  
Neosurugatoxin (NSTX) (3 nM-30 nM), recently isolated from the Japanese ivory mollusc (Babylonia japonica) exerted a potent antinicotinic action in the isolated guinea pig ileum. Specific [3H]nicotine binding to rat forebrain membranes was saturable, reversible, and of high affinity. Nicotinic cholinergic agonists exhibited a markedly greater affinity for [3H]nicotine binding sites than a muscarinic agonist, oxotremorine. Although alpha-bungarotoxin had no effect on [3H]nicotine binding, low concentrations (1 nM-1 microM) of NSTX inhibited [3H]nicotine binding in the forebrain membranes and its IC50 value was 69 +/- 6 nM. On the other hand, NSTX did not affect muscarinic receptor binding in the brain. These data indicate that NSTX may be of appreciable interest as a neurotoxin with a selective affinity for ganglionic nicotinic receptors.  相似文献   

2.
Benzodiazepine receptors were labeled with [3H] diazepam following intravenous injection in rats. Binding of [3H] diazepam in vivo to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [3H] diazepam invivo was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [3H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [3H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain invivo.  相似文献   

3.
High affinity and saturable binding sites for [3H] imipramine have been demonstrated on human platelet membranes. These binding sites appear to be specific for tricyclic antidepressants and their pharmacologically-active metabolites. In contrast, inactive tricyclic compounds such as the parent iminodibenzyl and iminostilbenes do not inhibit [3H] imipramine binding. The binding of [3H] imipramine to human platelets is of high affinity (Kd ? 1.4nM), saturable (Bmax ? 625 fmols/mg prot), and sensitive to proteolytic degradation. The effects of various drugs and neurotransmitter agonists and antagonists suggests that these binding sites are pharmacologically distinct from the previously reported binding of tricyclic antidepressants to alpha-adrenergic, muscarinic-cholinergic, and histaminergic receptors. The binding characteristics of [3H] imipramine to platelets is similar to that in rat and human brain and may thus serve as a useful model in elucidating the pharmacological and physiological significance of these binding sites.  相似文献   

4.
Preliminary results on the binding of [3H]Boc[Nle28,31]CCK27?33, designated [3H]Boc[diNle]CCK7, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc[diNle]CCK7 displaces [125I]CCK8 from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK8. The order of potencies to displace [3H]Boc[diNle]CCK7 from its binding sites was the same on mouse brain and rat pancreas: [3HBoc[diNle]CCK7>CCK8, Boc-CCK7> non-sulfated CCK8, the pancreas binding sites being more discriminative than the brain binding sites. Thus, [3H]Boc[diNle]CCK7 is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments.  相似文献   

5.
The ability of a series of tetrahydroisoquinoline (THIQ) alkaloids to inhibit the binding of radioligands to catecholamine receptors in the CNS has been examined. (+) THP was the most potent inhibitor of [3H] dihydroalprenolol binding to β-adrenergic receptors and of [3H] haloperidol to dopaminergic receptors and was the least potent inhibitor of [3H] WB-4101 binding to α-adrenergic receptors. Other THIQ alkaloids examined such as salsoline, salsolinol, and reticuline were less potent than (+) THP in inhibiting radioligand binding to β-adrenergic and dopaminergic receptors, and more potent than (+) THP in inhibiting radioligand to α-adrenergic receptors. The marked potency of (+) THP in inhibiting radioligand binding to β-adrenergic receptors (IC50 ~ 10?7 M) was confirmed by the potency of this compound in inhibiting (?) isoproternol elicited accumulations of cyclic AMP in brain slice preparations. These data indicate that, if formed invivo during alcohol consumption, THIQ derivatives such as THP may affect catecholamine neurons in the CNS.  相似文献   

6.
Up to now the only drugs known to be able to inhibit the binding of benzodiazepines to rodent brain receptors are members of this chemical family.Zopiclone (RP 27 267), a new drug with a pharmacological profile similar to that of chlordiazepoxide and nitrazepam but entirely different chemically from benzodiazepines, has been tested for its ability to inhibit benzodiazepine binding. In vitro and in vivo studies have shown that zopiclone is able to inhibit the binding of [3H] diazepam and [3H] flunitrazepam to brain receptors. The potency of zopiclone is quite comparable to that of diazepam and nitrazepam in vitro and to that of chlordiazepoxide in vivo.These results confirm the pharmacological similarities existing between zopiclone and the benzodiazepines.  相似文献   

7.
α-[3H]Bungarotoxin was prepared by catalytic reduction of iodinated α-bungarotoxin with tritium gas. Crude mitochondrial fraction from rat cerebral cortex bound 40 · 10?15 ?60 · 10?15 moles of α-[3H]bungarotoxin per mg of protein. This binding was reduced by 50% in the presence of approx. 10?6 M d-tubocurarine or nicotine, 10?5 M acetylcholine, 10?4 M carbamylcholine or decamethonium or 10?3 M atropine. Hexamethonium and eserine were the least effective of the drugs tested. Crude mitochondrial fraction was separated into myelin, nerve endings, and mitochondria. The highest binding of toxin per mg of protein was found in nerve endings, as well as the greatest nhibition of toxin binding of d-tubocurarine. Binding of α-[3H]bungarotoxin to membranes obtained by osmotic shock of the crude mitochondrial fraction indicates that the receptor for the toxin is membrane bound. 125I-Labeled α-bungarotoxin, prepared with Na 125I and chloramine T, was highly specific for the acetylcholine receptor in diaphragm, however, it was less specific and less reliable than α-[3H]bungarotoxin in brain. We conclude that a nicotinic cholinergic receptor exists in brain, and that α-[3H]bungarotoxin is a suitable probe for this receptor.  相似文献   

8.
[3H]Flunitrazepam was used to characterize benzodiazepine binding sites in human brain. The benzodiazepine binding sites exhibited high affinity, pharmacological specificity and saturability in their binding of [3H]flunitrazepam. The dissociation constant (KD) of [3H]flunitrazepam binding was determined by three different methods and found to be in the range of 2–3 nM. The potency of several benzodiazepine analogs to inhibit specific [3H]-flunitrazepam binding invitro correlated well with their potency in several invivo human and animal tests. The density of [3H]-flunitrazepam binding sites was highest in the cerebrocortical and rhinencephalic areas, intermediate in the cerebellum, and lowest in the brain stem and commissural tracts.  相似文献   

9.
[3H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [3H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (Kd) of 1.5 nM was observed when the [3H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (Kd 20 nM) was observed when [3H] QNB concentration was above 20 nM. Using 10 nM [3H] QNB for binding, the second order association rate constant (k1) of 0.064 nM?1 min?1 and first order dissociation rate constant (k2) of 0.078 min?1(T12 8 min) were observed. k2/k1 = Kd of 1.22 nM is in good agreement with Kd = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [3H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [3H] QNB binding than the muscarinic agonist.  相似文献   

10.
Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [3H]-quinuclidinyl benzilate ([3H]-QNB). Receptor related binding of [3H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, KD, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [3H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC50 values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [3H]-QNB binding. The levo isomer of QNB was about seventy times more effective at inhibiting binding than its dextro isomer while dextro benzetimide was greater than two thousand fold more active than levo benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [3H]-QNB binding sites in a stereoselective manner, the levo isomer being eighty-five times more potent than the dextro isomer.  相似文献   

11.
Kiss  J. P.  Windisch  K.  De Oliveira  K.  Hennings  E. C. P.  Mike  A.  Szász  B. K. 《Neurochemical research》2001,26(8-9):943-950
The aim of this study was to investigate the mechanisms involved in the effect of nicotinic agonists on the [3H]norepinephrine ([3H]NE) release from rat hippocampal slices. The stimulatory effect of nicotine, cytisine, epibatidine and anatoxin-A was completely blocked by the nicotinic antagonist mecamylamine (10 M). In contrast, the effect of dimethylphenylpiperazinium (DMPP) was only partially inhibited by mecamylamine but was completely blocked by the NE uptake inhibitor desipramine (DMI, 10 M). Finally, the effect of lobeline was not affected by mecamylamine and was only partially blocked by DMI. Our data indicate that the majority of nicotinic agonists increase the release of [3H]NE exclusively via stimulation of nicotinic acetylcholine receptors (nAChRs). DMPP, in addition to the stimulation of nAChRs, also evokes a carrier-mediated release. Lobeline has no stimulatory effect on nAChRs, induces a carrier-mediated release and has a further action of unidentified mechanism. Our results suggest that special caution is required for the interpretation of data, when DMPP or lobeline are used as nicotinic agonists.  相似文献   

12.
Characterization of temperature-sensitive [3H]serotonin (5-HT) binding sites (1 and 4 nM Kd sites) revealed complex inhibition by neuroleptics and serotonin antagonists. There was no simple correlation with affinities for S1 and S2 receptors. In vivo pretreatment (48 h before) with mianserin did not alter Bmax or Kd for the 1 nM Kd [3H]5-HT site, although [3H]ketanserin (S2) densities were decreased by 50%. This suggested that possible S2 components of [3H]5-HT binding must be negligeable, even though ketanserin competed with high affinity (IC50 = 3 nM) for a portion of the 1 nM Kd [3H]5-HT site. Low concentrations of mianserin inhibited the 1 nM Kd [3H]5-HT site in a non-competitive manner, as shown by a decrease in Bmax with no change in Kd after in vitro incubation. The complex inhibition data may therefore represent indirect interactions through another site.  相似文献   

13.
High-dose carbachol (10?3 M) has previously been shown to cause NaCl absorption in short-circuited rabbit ileum. The mechanism of this effect may be norepinephrine release induced by carbachol activation of presynaptic nicotinic receptors on adrenergic neurons. Norepinephrine then interacts with postsynaptic α-adrenergic receptors on intestinal mucosal cells to stimulate neutral NaCl absorption and inhibit electrogenic bicarbonate secretion. The present paper examines the in vitro intestinal ion transport effects of DMPP an agent which is more specific than carbachol on nicotinic cholinergic receptors. DMPP (10?5 M) caused a transient increase followed by prolonged depression of the short-circuit current, increased NaCl absorption and increased tissue conductance. This effect was antagonized by hexamethonium and phentolamine. It is concluded that nicotinic cholinergic agents stimulate norepinephrine release from adrenergic nerves and effect intestinal ion transport just as norepinephrine does.  相似文献   

14.
Biochemical and electrophysiological studies were conducted on the electric organ of the electric fish of the Nile, Malapteruruselectricus, in order to determine if transmission was chemically mediated. There was no binding of [3H] acetylcholine, [3H] quinuclidinyl benzilate or [3H]-perhydrohistrionicotoxin; but low acetylcholinesterase activity was observed, as was binding of [125I] α-bungarotoxin. The latter binding was detectable at 0.85 ± 0.07 pmol/g tissue, and was totally inhibited by 1 μM α-bungarotoxin or 100 μM d-tubocurarine. A tetrodotoxin-sensitive action potential was measured which was Na+- dependent. Depolarization (30–40 mV) was caused by carbamylcholine, and this was blocked by d-tubocurarine or α-bungarotoxin. The data suggest that this electric organ which may be a rich source for electrically excitable channels, is innervated by nicotonic cholinergic motoneurons, but the concentrations of acetylcholine receptors and acetylcholinesterase are very low.  相似文献   

15.
Antibodies against peripheral nicotinic acetylcholine receptors (nAChR) were used to determine the proportion of brain α-bungarotoxin binding sites that are immunologically related to the peripheral nAChR. The α-bungarotoxin binding component partially purified from rat brain was labelled with [125I]α-bungarotoxin and reacted with increasing concentrations of rabbit anti(nAChR) antisera. At least 75% of the brain protein could be immunoprecipitated by rabbit anti(rat muscle junctional nAChR) antiserum (M) whereas an antiserum against Torpedo nAChR (J) was without effect and clearly failed to cross-react with the brain component. Both antisera precipitated 100% of [125I]α-bungarotoxin-labelled nAChR from Torpedo marmorata. The lower precipitation of the brain protein was not a consequence of [125I]α-bungarotoxin dissociating during the precipitation. We conclude that the majority of α-bungarotoxin binding sites in brain are clearly recognised by the crossreacting antiserum.Release of [3H]dopamine from striatal synaptosomes could be elicited by nicotine in a dose-dependent manner and the response was prevented by the ganglionic blocker mecamylamine, although antagonism by α-bungarotoxin was less clearcut. Preincubation of the synaptosomes with antiserum M resulted in a statistically significant decrease in the [3H]dopamine response to nicotine at all agonist concentrations tested. Antiserum J, however, had no consistent effect on the response. Thus the actions of the antisera parallel their ability to recognise the brain α-bungarotoxin binding component. We conclude that the cholinergic regulation of dopamine release is in part mediated through a nAChR that is immunologically related to the nAChR of the neuromuscular junction and to the α-bungarotoxin binding component that can be isolated from rat brain.  相似文献   

16.
Various enzymes and proteins reagents inhibited [3H]prostaglandin F2α binding to bovine corpus luteum cell membranes. Studies were undertaken (a) to explore further on the dose response relationships with the above agents, (b) to investigate the mechanism of inhibition of binding with respect to receptor affinities and number and (c) to assess whether decreased binding reflected changes in receptors and/or other membrane components.Preincubation of membranes with phoshpolipase A, trypsin, pronase, lipase, tetranitromethane, dinitrofluorobenzene, acetic anhydride and N-ethylmaleimide resulted in moderate to drastic inhibitions of [3H]prostaglandin F2α binding. The dose-dependent inhibition of binding by enzymes, but not by protein reagents (except for N-ethylmaleimide), exhibited a biphasic pattern: at lower concentrations, the loss of binding was low and relatively plateaued, but at higher concentrations, the losses were dramatic. The drastic reduction in binding by trypsin was due to destruction rather than solubilization of receptors from membranes. Phospholipase A was intrinsically more effective than phospholipases C and Ca2+ was not required for its inhibition of [3H]prostaglandin F2α binding. Protein reagents inhibition of binding was differently influenced by added Ca2+ i.e., loss of binding increased with some (N-ethylmaleimide), decreased with others (tetranitromethane, dinitrofluorobenzene and azobenzene sulfenylbromide). These results are interpreted to indicate that Ca2+ induced conformational changes in membranes which may result in exposure of new groups and burying of already exposed modifiable groups.Treatment of membranes wiht trypsin and N-ethylmaleimide selectively abolished high affinity prostaglandin F2α receptors. The low affinity receptors were present but their numbers as well as their affinity were decreased. Lipase, phospholipase A, acetic anhydride, dinitrofluorobenzen and tetranitromethane appear to decrease binding by totally abolishing all prostaglandin F2α receptors or by severely reducing their affinities.The occupancy of receptors by prostaglandin F2α afforded considerable protection against trypsin, phospholipase A, lipase and dinitrofluorobenzene. These data indicated that the inhibition of binding by the above agents, at least in part, can be attributable to changes in receptor sites alone.  相似文献   

17.
The influence of GABA on the affinity of flunitrazepam (FLU) for benzodiazepine receptor subtypes (type I and II) was studied by measurement of the competitive inhibition of [3H]FLU and [3H]propyl beta-carboline-3-carboxylate ([3H]PCC) binding. When assays were carried out at 0°C using a low concentration (0.040 nM) of [3H]PCC so that the type I receptors were selectively labelled, no significant effect of GABA (10?4 M) on the FLU[3H]PCC competition curve was detected. In contrast, when assays were carried out at 0°C using [3H]FLU or a high concentration of [3H]PCC to achieve [3H]ligand receptor occupancy of both type I and type II receptors, GABA (10?4 M) caused a significant increase in the affinity of FLU as measured by FLU[3H]FLU and FLU[3H]PCC competition experiments. Collectively, these data suggest that the influence of GABA on benzodiazepine receptor binding is mediated, primarily, by the type II receptor. It was also noted that the PCC[3H]FLU competition curve had a Hill coefficient of approximately 1 at 37°C as compared to the results of experiments at 0°C during which a Hill coefficient of approximately 0.7 was calculated.  相似文献   

18.
Norharman competitively inhibits specific binding of [3H]-diazepam in mouse brain homogenates. In vivo this β-carboline produces a striking rigid catatonic-like appearance which is abolished by diazepam. It also causes a rapid tremor but has little anticonvulsant effect. Measurement of in vivo concentrations and receptor occupancy demonstrate that these biological effects occur at doses which occupy a large proportion of benzo-diazepine receptors. It may represent a ligand of the benzo-diazepine receptors whose effects are opposite those of diazepam.  相似文献   

19.
Binding characteristics of benzodiazepine receptors were studied with synaptosomal and microsomal membranes from rabbit brain invitro utilizing [methyl-3H]diazepam. In synaptosomal membranes, both high and low affinity binding sites were identified with the dissociation constants (Kd) of 4.92 nM and 83.8 nM, respectively. However, only the high affinity site was identified with Kd of 3.96 nM with microsomal membranes. Benzodiazepine binding sites appear to include at least two subpopulations of receptors, one with high affinity and another with low affinity binding site.  相似文献   

20.
Properties of [3H] diazepam binding to rat peritoneal mast cells   总被引:6,自引:0,他引:6  
Benzodiazepine binding to rat peritoneal mast cells was investigated using [3H] diazepam as the radioactive probe. The specific binding of [3H] diazepam reaches equilibrium within 10–15 min, is saturable and is linear with cell number. Scatchard analysis of equilibrium binding indicates the existence of only one class of binding sites with a KD = 90 ± 10 nM and Bmax of 261 ± 60 fmoles/106 cells. The binding of [3H] diazepam is temperature dependent, the highest amount is bound at 0°C and shows a pH-optimum between pH 6.8 – 7.4. The binding of [3H] diazepam is reversible with t12 = 1.2 ± 0.2 min. Based on the relative potency of clonazepam and Ro5-4864 in displacing the specific [3H] diazepam binding, the binding sites in the mast cell are similar to those in the peripheral tissues like lung, liver, and kidney and are different from those in the brain. These data indicate that the mast cells have benzodiazepine binding sites which are of the peripheral type.  相似文献   

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