Subtype-selective up-regulation by chronic nicotine of high-affinity nicotinic receptors in rat brain demonstrated by receptor autoradiography

Subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) are differentially sensitive to up-regulation by chronic nicotine exposure in vitro. To determine whether this occurs in animals, rats were implanted with minipumps containing saline +/- nicotine (6.0 mg/kg/rat/day) for 14 days. Autoradiography with [125I]epibatidine using 3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride (A-85380) or cytisine as selective competitors allowed quantitative measurement in 33 regions of 3 families of nAChR binding, with properties of alpha4beta2, alpha3beta4, and alpha3/alpha6beta2. Chronic nicotine exposure caused increases of 20 to 100% for alpha4beta2-like binding in most regions surveyed. However, binding to this subtype was not increased in some regions, including habenulopeduncular structures, certain thalamic nuclei, and several brainstem regions. In 9 of 33 regions, including catecholaminergic areas and visual structures, alpha3/alpha6beta2-like binding represented >10% of total binding. Binding to this subtype was up-regulated by nicotine in only two of these nine regions: the nucleus accumbens and superior colliculus. alpha3beta4-Like binding represented >10% of total in 15 of the 33 regions surveyed. Binding to this subtype was increased by nicotine in only 1 of these 15 regions, and actually decreased in subiculum and cerebellum. These studies yielded two principal findings. First, chronic nicotine exposure selectively up-regulates alpha4beta2-like binding, with relatively little effect on alpha3/alpha6beta2-like and alpha3beta4-like binding in vivo. Second, up-regulation by chronic nicotine exposure shows considerable regional variation. Differential subtype sensitivity to chronic nicotine exposure may contribute to altered pharmacological response in individuals who smoke or use nicotine replacement therapy.     

Heterogeneity of high-affinity nicotinic [3H]acetylcholine binding sites

Studies were conducted on high-affinity, nicotinic binding of 3H-labeled acetylcholine ([3H]ACh) to membrane preparations derived from the TE671 human clonal line and the PC12 rat pheochromocytoma. For comparative purposes and to extend results obtained by others in previous studies, [3H]ACh binding to membrane preparations derived from rat brain and from the electric tissue of Torpedo californica also was characterized. In each case, specific [3H]ACh binding (KD values of about 10 nM) could be fit by linear Scatchard and logit-log curves (slope of the latter of about 1.0) indicating that binding occurred to a single class of noninteracting sites, except that a better fit to PC12 cell membrane binding data was obtained using a two-site model. Quantitation of high-affinity binding sites for [3H]ACh and radiolabeled alpha-bungarotoxin and studies of unlabeled toxin competition for [3H]ACh binding indicated that toxin and agonist sites on TE671 cell or Torpedo membranes were closely related, but that toxin and agonist sites had limited or no physical overlap on PC12 cell or rat brain membranes. Generally, drugs of the bisonium and bis-choline series were more effective inhibitors of [3H]ACh binding to TE671 cell or Torpedo membranes, but nicotine and cytisine interacted with PC12 cell or rat brain sites with highest affinity. These results suggest that nicotinic agonists bind with high affinity to specific, membrane-bound sites on presumptive nicotinic ACh receptors expressed on each of these preparations. Moreover, the data are consistent with structural heterogeneity of these nicotinic receptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The comparative pharmacology and up-regulation of rat neuronal nicotinic receptor subtype binding sites stably expressed in transfected mammalian cells

We stably transfected human embryonic kidney cells (HEK 293 cells) with genes encoding rat neuronal nicotinic receptor alpha2, alpha3, or alpha4 subunits in combination with the beta2 or beta4 subunit to generate six cell lines that express defined subunit combinations that represent potential subtypes of rat neuronal nicotinic acetylcholine receptors (nAChRs). These cell lines were designated KXalpha2beta2, KXalpha2beta4, KXalpha3beta2, KXalpha3beta4, KXalpha4beta2, and KXalpha4beta4. The Kd values of [3H](+/-)epibatidine ([3H]EB) binding to membranes from these six cell lines ranged from approximately 0.02 to 0.3 nM. The pharmacological profiles of the agonist binding sites of these putative nAChR subtypes were examined in competition studies in which unlabeled nicotinic ligands, including 10 agonists and two antagonists, competed against [3H]EB. Most nicotinic ligands examined had higher affinity for the receptor subtypes containing the beta2 subunit compared with those containing the beta4 subunit. An excellent correlation (r > 0.99) of the binding affinities of the 10 agonists was observed between receptors from KXalpha4beta2 cells and from rat forebrain tissue, in which [3H]EB binding represents predominantly alpha4beta2 nAChRs. More important, the affinities (Ki values) for the two tissues were nearly identical. The densities of the binding sites of all six cell lines were increased after a 5-day exposure to (-)-nicotine or the quaternary amine agonist carbachol. These data indicate that these cell lines expressing nAChR subunit combinations should be useful models for investigating pharmacological properties and regulation of the binding sites of potential nAChR subtypes, as well as for studying the properties of nicotinic compounds.     

Determinants of agonist binding affinity on neuronal nicotinic receptor beta subunits

The alpha and beta subunits of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are thought to contribute "principal" and "complementary" components to the agonist binding site, respectively. At least six loops of amino acid sequence (A, B, and C from alpha; D, E, and F from beta) are involved. We demonstrated previously that receptors containing the beta2 subunit had consistently higher affinities for a variety of agonists than beta4-containing receptors. For example, the affinity of the alpha2beta2 receptor for epibatidine, ACh, nicotine, and dimethylphenylpiperazinium (DMPP) exceeds that of alpha2beta4 by 9-, 61-, 87-, and 120-fold, respectively. Using saturation and competition analysis of receptors formed by chimeric beta subunits coexpressed with alpha2 in Xenopus laevis oocytes, we have now identified sequence segment 54-63 (corresponding to loop D) as a major determinant of affinity for epibatidine, ACh, nicotine, and DMPP. We then analyzed a series of mutant beta2 subunits in which each residue that differs between beta2 and beta4 in this region was changed from what occurs in beta2 to what occurs in beta4. The N55S, V56I, and E63T mutations each resulted in a loss of affinity for ACh and nicotine of 3- to 4-fold, whereas the T59K mutation resulted in a 7-fold loss of ACh and nicotine affinity. These mutations had little or no effect on epibatidine and DMPP affinity. The positive charge introduced by the T59K mutation does not appear to underlie loss of agonist affinity, because a similar loss of affinity was observed when a negative charge (T59D) was introduced at this position.     

Dependence of nicotinic acetylcholine receptor recovery from desensitization on the duration of agonist exposure

When subjected to prolonged exposure to nicotinic agonists, nicotinic acetylcholine receptors undergo desensitization, resulting in an inactive receptor that does not allow for the passage of ions. The induction of desensitization of diverse nicotinic acetylcholine receptor subtypes in muscle, ganglia, or brain is likely to play important modulatory roles in synaptic transmission. Furthermore, nicotinic receptor desensitization may contribute to behavioral changes in humans or animals subjected to prolonged nicotine exposure pharmacologically or through the use of tobacco products. We investigated the recovery from desensitization of muscle-type nicotinic acetylcholine receptors in TE671/RD cells induced by exposure to acetylcholine or nicotine. Rates of recovery from desensitization are dependent on the length of agonist exposure and on the agonist used to induce desensitization. Increasing the time of exposure results in an increase in the time constant of recovery for both agonists. The recovery from nicotine-induced desensitization is consistently faster than the recovery from acetylcholine-induced desensitization regardless of whether nicotine or acetylcholine is used to assess levels of desensitization. These findings suggest the existence of more than one state of receptor desensitization and that nicotinic agonists vary in their efficiency of inducing receptors to states of differing depths of desensitization.     

Genetic analysis of nicotine-induced seizures and hippocampal nicotinic receptors in the mouse

A classical F2 and backcross genetic design was used to examine the relationship between sensitivity to nicotine-induced seizures and hippocampal nicotinic receptors. Nicotine was administered by i.v. infusion through cannulas implanted in the right jugular veins of mice from the C3H and DBA mouse strains and their derived F1, F2 and backcross (F1 X C3H and F1 X DBA) generations. Nicotine-induced seizure sensitivity was assessed by infusing nicotine at a 2.0 mg/kg/min concentration until the onset of a clonic seizure. After seizure testing, mice were sacrificed and the binding of alpha-bungarotoxin (BTX) was determined in three brain regions: cortex, midbrain and hippocampus. The pattern of results for the six generations for seizure sensitivity was similar to that observed for hippocampal BTX binding. Genetic analyses indicate that receptor concentrations in the hippocampus and seizure sensitivity are both influenced by a relatively simple genetic system which involves one or more genes acting in an additive fashion. In addition, the genetic correlation which is indicative of the extent to which two characters are influenced by the same genes, supports the relationship between nicotine-induced seizure sensitivity and hippocampal BTX binding. The results suggest that those animals which are sensitive to nicotine-induced seizures have a greater concentration of nicotinic receptors as determined by the binding of BTX than animals which are less sensitive to the convulsant effects of nicotine.     

Naltrexone modulation of nicotinic acetylcholine receptor activity

The narcotic antagonist naltrexone (NTX) has both depressant and excitatory effects on nicotinic acetylcholine receptor activity, and the present study was primarily concerned with mechanisms involved in the latter modulatory action. Single channel data were recorded in cell-attached mode from cultured embryonic chick skeletal muscle myotubes at 10 degrees C under control conditions (0.15 or 0.3 microM acetylcholine, ACh) or in the presence of NTX (0.15 microM ACh plus 0.1-5 microM NTX). Distributions of open and closed times indicated that there were at least two open and four closed channel states in all cases, and evidence for at least two gateways between the open and closed classes was provided by autocorrelation data. In the absence of NTX the proportion of long (greater than 2 msec) openings increased with increasing ACh concentration, suggesting that the channel in both the mono- and biliganded forms of the receptor could open. Three possible six-state models of channel kinetics which were compatible with these findings could not be distinguished on the basis of conventional Markov modeling of open and closed times; however when account was taken of burst distributions, one model was clearly superior. Estimated transition rates in this model showed that enhanced receptor activity in the presence of NTX was due mainly to increased cooperativity of ACh binding. The results suggest that in the chick nicotinic ACh receptor, low micromolar concentrations of NTX may cause enhancement and blockade of activity by binding to a site which is allosterically coupled to several activation and gating processes.     

Postischemic alteration of muscarinic acetylcholine and adenosine A1 binding sites in gerbil brain

We studied the alterations in the binding of muscarinic cholinergic and adenosine A₁ receptors following transient cerebral ischemia in Mongolian gerbils and examined the effects of the novel vinca alkaloid derivative vinconate and pentobarbital against the alterations in the binding of these receptors. Animals were allowed to survive for 5 h and 7 days after 10 min of cerebral ischemia induced by bilateral occlusion of common carotid arteries. [³H]Quinuclidinyl benzilate (QNB) and [³H]cyclohexyladenosine (CHA) were used to label muscarinic cholinergic and adenosine A₁ receptors, respectively. The [³H]QNB and [³H]CHA bindings showed no significant alteration in the gerbil brain 5 h after ischemia. However, these bindings in the striatium, the hippocampal CA1 sector, and the hippocampal CA3 sector revealed a significant reduction 7 days after ischemia. The [³H]CHA binding also showed a significant decline in the dentate molecular layer 7 days after ischemia. Intraperitoneal application of vinconate (100 and 300 mg/kg) 10 min and pentobarbital (40 mg/kg) 30 min before ischemia showed a mild reduction in the [³H]CHA binding in the brain 5 h after ischemia. Especially, the reduction was found in the hippocampal CA1 sector and the dentate molecular layer. However, the [³H]QNB binding revealed no significant alteration in the brain 5 h after ischemia. Seven days after ischemia, both drugs prevented a marked reduction in the [³H]CHA binding in the striatium, but not in the hippocampal CA1 sector, the hippocampal CA3 sector, and the dentate molecular layer. By contrast, vinconate and pentobarbital failed to prevent the reduction in the [³H]QNB binding in the striatum. Morphological study indicated that vinconate and pentobarbital ameliorated the neuronal damage to the striatum, but not the hippocampal damage 7 days after ischemia. This histological finding was relatively consistent with the alteration in the [³H]CHA binding. these receptor autoradiographic and histological data suggest that vinconate and pentobarbital can protect the brain from both cellular and functional consequences of ischemia. These findings are of interest in relation to the mechanisms of ischemic brain damage.     

雌激素对穹隆-海马伞切断与卵巢切除大鼠脑内烟碱型乙酰胆碱受体的干预作用

目的:观察雌激素对阿尔茨海默病大鼠脑内不同部位烟碱型乙酰胆碱受体表达的干预作用。方法:实验于2003-11在青岛大学医学院脑病研究所进行。取健康雌性Wistar大鼠10只,随机分为穹隆-海马伞切断+卵巢切除组和雌激素组,每组5只大鼠。所有大鼠在脑立体定位仪上切断穹隆-海马伞,并切除双侧卵巢,建立模拟伴有体内雌激素水平下降的阿尔茨海默病动物模型。雌激素组大鼠术后第3天皮下注射雌二醇1mg/kg,以后每7d给药1次,30d后应用免疫组织化学技术观察两组阿尔茨海默病大鼠海马CA1区、皮质区、杏仁复合体区和基底前脑Meynert核等部位的烟碱型乙酰胆碱受体阳性细胞的表达。结果:经补充后10只大鼠进入结果分析。雌激组大鼠脑海马CA1区、脑皮质、杏仁复合体区、Meynert核区的烟碱型乙酰胆碱受体阳性细胞数均高于穹隆-海马伞切断+双侧卵巢切除组(30.20±8.65),(64.40±3.52)个/视野;(41.95±9.63),(96.81±9.13)个/视野;(38.18±9.19),(84.50±5.48)个/视野;(18.18±1.65),(89.33±10.24)个/视野;P<0.01。结论:补充外源性雌激素治疗后阿尔茨海默病大鼠各脑区内的烟碱型乙酰胆碱受体表达显著增加,从而提高了胆碱能神经元的功能。     

Nicotinic acetylcholine receptor agonist SIB-1508Y improves cognitive functioning in chronic low-dose MPTP-treated monkeys.

Monkeys that receive chronic low-dose 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) administration have difficulty performing numerous cognitive tasks. This study further examines the extent to which chronic low-dose MPTP exposure affects performance of a visual memory task [variable delayed response (VDR)] with both attentional and short-term memory components and assesses the effects of the novel neuronal nicotinic acetylcholine receptor agonist SIB-1508Y and levodopa on cognitive task performance. Before MPTP treatment, these monkeys displayed a delay-dependent decrement in performance on the VDR task and performed well on delayed matching-to-sample and visual pattern discrimination tasks. Chronic low-dose MPTP treatment caused a shift to a delay-independent pattern of responding on the VDR task, such that short-delay trials were performed as poorly as long-delay trials. There were also deficits in performing the delayed matching-to-sample task, whereas visual discrimination performance remained intact. SIB-1508Y normalized the pattern of response on the VDR task by significantly improving performance on short-delay trials and on the delayed matching-to-sample task. These effects lasted up to 24 to 48 h after SIB-1508Y administration. Neither levodopa nor nicotine significantly improved task performance. These results suggest that chronic low-dose MPTP exposure results in a cognitive disturbance that can be corrected by the nicotinic acetylcholine receptor agonist SIB-1508Y but not by levodopa. Thus, SIB-1508Y may be useful in the treatment of the cognitive deficits in Parkinson's disease.     

Loperamide binding to opiate receptor sites of brain and myenteric plexus.

Loperamide, a new antidiarrheal agent, was tested to determine whether its biological activity involves binding to opiate receptor sites. Loperamide and morphine competitively inhibited 3H-naloxone binding to homogenates a guinea-pig brain and myenteric plexus. The Kp values obtain in the presence of Na+ were: morphine, 9.60-10(-9) M (brain), 1.66-10(-7) M (myenteric plexus); loperamide, 7.20-10(-9) M (brain), 1.33-10(-7) M (myenteric plexus); naloxone, 4.78-10(-10) M (brain), 1.27-10(-9) M (myenteric plexus. In the absence of Na+, binding a loperamide and morphine to brain homogenate was enhanced while the binding of naloxone was reduced. Morphine (IC50 = 7.5-10(-8) M) and loperamide (IC50 = 6.9-10(-9) M) inhibited the electrically induced contractions of longitudinal muscle from guinea-pig ileum, and naloxone competitively antagonized these effects. The Kd value calculated for the interaction of naloxone with binding sites associated with the contracting muscle was between 0.98-10(-9) M and 1.85-10(-9) M. In the mouse hot plate test, subcutaneous administration of morphine (minimal effective dose = 6.6 mugmol/kg) and loperamide (minimal effective dose = 78 mugmol/kg) delayed the response to heat stimuli and this effect was completely blocked by prior administration of naloxone. In the anesthetixed dog, intravenous administration of morphine (100 mug/kg) and loperamide (100 mug/kg) enhanced the contractile activity of circular muscle in proximal and distal duodenum, distal ileum and proximal colon but duodenal longitudinal muscle was relaxed; these effects were completely reversed by subsequent administration of naloxone. It is concluded that loperamide binds to opiate receptor sites and possesses opiate agonist activity both in vivo and in vitro.     

D-2 dopamine receptors in aging mouse striatum: determination of high- and low-affinity agonist binding sites

Striatal D-2 dopamine receptors in homogenates from aged male C57BL/6J mice were examined for high and low-affinity agonist binding. High-affinity dopamine binding requires the ternary complex of the D-2 receptor and a guanine nucleotide binding regulatory protein (N). Thus, changes in the interaction of D-2 and N could contribute to previously reported changes in agonist binding in aged rodents and humans. Qualitative experiments indicated no age-change in the ability of guanine nucleotides to reduce the apparent potency of dopamine at D-2 receptors. Also, no age differences were observed in the ability of guanine nucleotides to decrease N-[3H]propylnorapomorphine binding, suggesting that the ability of guanine nucleotides to dissociate D-2 and N was similar with age. Quantitative determination of the high- (RH) and low-affinity (RL) agonist binding components of striatal D-2 dopamine receptors in the absence of guanine nucleotides indicated differences in the density of RH, and the percentage of D-2 receptors measured as RH, between the ages of 3 and 12 months. No changes in RH or percentage of RH occurred after midlife. In contrast, the total D-2 receptor population, [3H]spiperone maximum binding, declined progressively from 3 to 24 months. Age-changes were restricted to D-2 receptor density; the dissociation constants for agonist and antagonist binding were similar across age. The data suggest that age-changes in striatal D-2 dopamine receptors can occur in the density of the D-2 receptor and in the mechanism that confers the property of high-affinity agonist binding upon the D-2 receptor.     

Antagonist-induced opioid receptor up-regulation. II. Regionally specific modulation of mu, delta and kappa binding sites in rat brain revealed by quantitative autoradiography

The neurochemical basis for the supersensitivity to mu or kappa opiate agonists observed after chronic naloxone infusion (reported in the preceding paper) has been investigated using the technique of quantitative in vitro autoradiography. The binding of [3H]-D-Ala2MePhe4Glyol enkephalin (3H-DAGO) to mu opioid sites was increased in many brain regions after chronic administration (7 days) of a low dose of naloxone (0.5 mg/kg/hr). The greatest increases were seen in the hippocampal strata moleculare and lucidum. The binding of [3H]-D-Ala2-D-Leu5 enkephalin (3H-DADL) to delta sites and of [3H]-bremazocine to kappa sites were not significantly affected in any region. In contrast, after administration of a higher dose of naloxone (3.0 mg/kg/hr), the binding to mu, delta and kappa sites was increased in many brain areas, suggesting that up-regulation is observed only when a sufficient proportion of the receptors in vivo are occupied by the antagonist. However, a different regional pattern of up-regulation was seen for each class of opiate binding site. The greatest increases in kappa binding in the brain were seen in the periaqueductal gray and the hippocampal molecular and pyramidal cell layers. The substantia nigra showed up-regulation of kappa but not of mu sites. Differences in the pattern of mu and kappa binding site up-regulation were also noted in the spinal cord. This regionally specific modulation of opiate binding sites suggests that, for each receptor type, the level of tonic activation varies between individual brain areas. In addition, the data demonstrate that all three types of opiate receptor can undergo up-regulation "in vivo."     

Potential role of the alpha4 and alpha6 nicotinic receptor subunits in regulating nicotine-induced seizures

Several studies have shown that genetic factors influence sensitivity to nicotine-induced seizures in the mouse. We used recombinant inbred (RI) strains derived from the Long-Sleep (LS) and Short-Sleep (SS) mouse lines to assess the possibility that polymorphisms associated with one or more of the nicotinic receptors cosegregate with differential sensitivity to nicotine-induced seizures. Restriction fragment length polymorphisms (RFLPs) associated with the alpha2, alpha3, alpha4, alpha5, and alpha6 nicotinic receptors were identified in the LS and SS mouse lines, but the RI strains were polymorphic for only the alpha4 and alpha6 RFLPs. The RI strains were tested for sensitivity to nicotine-induced seizures. Strain and gender effects on seizure sensitivity were obtained as assessed by ED(50) values and latency to seizure. Those RI strains with the LS-like alpha4 RFLP were, on average, more sensitive to nicotine-induced seizures than were those RI strains with SS-like alpha4 RFLP. The alpha6 nicotine receptor may also play a role in modulating nicotine-induced seizures, but this effect is markedly influenced by gender. Females of the RI strains with the LS-like alpha6 RFLP were more sensitive to nicotine than were females of the strains with the SS-like alpha6 RFLP. Similar trends were seen in the males, but these trends were not significant. Thus, these strain differences may be due to polymorphisms associated with both the alpha4 and alpha6 nicotinic receptors, but gender also plays an important role in regulating sensitivity to nicotine-induced seizure.     

Dietary choline intake modulates benzodiazepine receptor binding and gamma-aminobutyric acidA receptor function in mouse brain

Several lines of evidence suggest that dietary choline intake influences the metabolism of membrane phospholipids with possible effects on GABAergic neurotransmission. Based on these findings, the present experiments determined whether chronic choline supplementation or deficiency alters GABAergic function at the level of the gamma-aminobutyric acid (GABA)/benzodiazepine-chloride channel complex. To accomplish this, mice were fed diets containing 0% (deficient), 0.2% (basal) or 2.0% (supplemented) choline chloride for 28 days, and behavior, ligand binding at several sites in the complex and chloride uptake were determined in various brain regions. For both rotarod ataxia and open-field activity, mice receiving choline supplementation had a decreased response to clonazepam compared to those receiving basal and deficient diets. Choline supplementation significantly increased the in vivo binding of [3H]Ro15-1788 to cortex and cerebellum by 19% and 24%, respectively, and in vitro studies in cortical membranes indicated a significant 36% increase in the maximal number of [3H]flunitrazepam binding sites without a change in affinity, as compared to basal controls. In contrast, [3H]Ro15-1788 binding in vivo in all brain regions from mice fed the deficient diet decreased significantly to 20 to 58% of control values. Dietary choline intake did not alter GABA levels in brain, the binding of [35S]t-butylbicyclophosphorothionate to the chloride channel or the coupling between GABA and either the t-butylbicyclophosphorothionate site or the benzodiazepine site. However, the function of the GABAA receptor, determined by muscimol-stimulated chloride uptake into cortical synaptoneurosomes, was increased significantly in tissue from the supplemented group as compared to both control and deficient groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of d-tubocurarine with the nicotinic acetylcholine receptor/channel molecule

The interactions of d-tubocurarine (d-TC) with the ionic channel of the nicotinic acetylcholine receptor were studied by biochemical methods in Torpedo electric organ membranes and by electrophysiological methods on frog sciatic nerve-sartorius muscle preparation. Torpedo membranes were treated with alpha-bungarotoxin to inhibit the acetylcholine receptor sites, then binding of [3H]perhydrohistrionicotoxin to the ionic channel sites was studied and found to be inhibited by d-TC. At 37 degrees C, the Ki of d-TC was 10 microM, and at 22 degrees C it was 100 microM. The affinity of d-TC for the ionic channel sites relative to that of perhydrohistrionicotoxin was constant at temperatures from 2-20 degrees C, but increased at higher temperatures up to 37 degrees C. The peak endplate current amplitude was depressed with 1 to 2 microM d-TC in a voltage-dependent manner, with considerable departure from linearity at 10 and 30 degrees C. The effect of d-TC on spontaneous miniature endplate currents was similar and slightly more potent. The time constant of endplate current decay was decreased by d-TC (1 and 2 microM) at temperatures of 10, 15 and 30 degrees C. The channel lifetime was reduced by d-TC, but channel conductance was unaffected. It is suggested that d-TC interacts with both the acetylcholine receptor sites as well as its ionic channel sites in closed and open conformations.     

Molecular effects of dimethylanatoxin on the peripheral nicotinic acetylcholine receptor.

N,N-dimethylanatoxin (DMAnTX), the quaternary derivative of the potent nicotinic agonist (+)-anatoxin-a (AnTX), has been evaluated for potency and efficacy at nicotinic acetylcholine receptors of frog motor endplates and Torpedo electric organs. DMAnTX was only weakly effective in eliciting contracture of the frog rectus abdominis and was orders of magnitude less potent than AnTX. Biochemical assay showed that DMAnTX was a weak inhibitor of [125I]alpha-bungarotoxin binding to the receptors in frog muscle and Torpedo electroplaque membranes: the IC50 values were 60 and 14 microM, respectively. A low frequency of single channel currents recorded from isolated interosseal fibers at concentrations from 20 to 100 microM of DMAnTX and the stimulation of [3H]perhydrohistrionicotoxin [( 3H]H12-HTX) binding (half-maximal at 0.3 microM) confirmed the weak activation of the receptor. DMAnTX also exhibited antagonist effects. In muscle twitch assays, 100 microM of DMAnTX effectively decreased the tension induced by nerve stimulation, although DMAnTX did not affect muscle membrane action potentials. The binding of [3H] perhydrohistrionicotoxin was also inhibited at high micromolar concentrations of DMAnTX. Combination of DMAnTX with acetylcholine in single channel current experiments demonstrated that DMAnTX possesses ion channel blocking properties, which become apparent at low micromolar concentrations, and DMAnTX enhances the desensitization induced by acetylcholine above 10 microM AnTX. The difference in agonist potency between AnTX and DMAnTX may be attributed to a change in conformation of the molecular skeleton induced by the N-methyl groups.     

Irreversible and quaternary muscarinic antagonists discriminate multiple muscarinic receptor binding sites in rat brain

The maximal number of binding sites (Bmax) of [3H]quinuclidinyl benzilate (QNB) binding was greater than the Bmax of N-[3H]methylscopolamine (NMS) binding to homogenates of rat brain. The competition of NMS for [3H]QNB demonstrated that NMS discriminates multiple muscarinic binding sites. Similarly, pirenzepine competition of [3H]QNB binding also revealed multiple muscarinic binding sites. Pirenzepine competition for [3H]NMS also was shallow and demonstrated the presence of binding sites with similar affinities to those labeled by [3H]QNB. These data were consistent with the presence of at least three populations of muscarinic binding sites with similar affinities for [3H]QNB: the M1 and M2 binding sites having high and low affinity for pirenzepine, respectively, but which cannot be discriminated by [3H]NMS, and a third site with high affinity for [3H]QNB which has low affinity for NMS. The classical muscarinic antagonists, atropine and scopolamine, also appear to have slightly different affinities for the putative M1 and M2 binding sites. The use of the irreversible antagonists, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and propylbenzilylcholine mustard (PBCM), were used to elucidate the distinct properties of these multiple muscarinic binding sites. Both PBCM and EEDQ irreversibly decreased the Bmax of [3H]QNB and [3H]NMS binding in cortex. PBCM did not appear to discriminate putative M1 and M2 binding sites but selectively alkylated the high affinity NMS and QNB binding sites. In contrast, EEDQ modified the low affinity NMS binding sites such that they still bound [3H]QNB but their affinity for other muscarinic antagonists was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)