[125/123I] 5-Iodo-3-pyridyl ethers. syntheses and binding to neuronal nicotinic acetylcholine receptors.

Three 3-pyridyl ether nicotinic ligands-(S)-5-Iodo-3-[(2-pyrrolidinyl)-methoxy]pyridine (5-iodo-A-85865), (S)-5-Iodo-3-[1-(methyl)-2-pyrrolidinyl-methoxy]pyridine (5-Iodo-A-84543), and (S)-5-iodo-3-[1-methyl-(2-azetidinyl)-methoxy]pyridine (5-iodo-N-Me-A-85380) were labeled with I-125/I-123, and their ability to label high-affinity brain nicotinic acetylcholine receptors (nAChRs) was evaluated. The most promising ligand, [123/125I] 5-iodo-A-85865, showed approximately 65% inhibition of radioactivity uptake in thalamus in mice pretreated with cytisine. Preliminary SPECT imaging studies with [123I] 5-iodo-A-85865 revealed a distribution profile consistent with nAChRs (thalamus > frontal cortex > cerebellum) and a more rapid pharmacokinetic profile relative to azetidinyl 3-pyridyl ether based ligands.

Axotomy and nerve growth factor regulate levels of neuronal nicotinic acetylcholine receptor alpha3 subunit protein in the rat superior cervical ganglion.

Neuronal nicotinic acetylcholine receptors (nAChRs) play a significant role in sympathetic transmission in the superior cervical ganglia (SCG), with most of the signal carried by a nAChR containing an alpha3 subunit. Work has shown that transection of the postganglionic nerves (axotomy) of the SCG results in a decrease in mRNA transcripts for alpha3, alpha5, alpha7 and beta4 and in protein expression of alpha7 and beta4. To evaluate effects of axotomy on alpha3 protein in the SCG, quantitative immunoblotting was used to demonstrate a dramatic decrease (> 80%) in the levels of this subunit 4 days after axotomy. Similarly, immunocytochemistry showed a marked decline in the number and the intensity of stained neurons for the alpha3 subunit as well as tyrosine hydroxylase. Ganglia explanted into culture for 4 days also showed a substantial decrease in alpha3 subunit protein. This decrease was partially prevented by the addition of nerve growth factor (NGF) to the culture medium at the time of explantation. Additionally, this decrease was reversed by the addition of NGF to the culture medium following 4 days in culture in the absence of NGF. These findings suggest that the loss of alpha3 subunit contributes to the reported decrease in ganglionic synaptic transmission that follows axotomy, and that NGF plays an important role in regulating the expression of alpha3-containing nAChRs in the SCG.

Blockade of rat alpha3beta4 nicotinic receptor function by methadone, its metabolites, and structural analogs.

The opioid agonist properties of (+/-)-methadone are ascribed almost entirely to the (-)-methadone enantiomer. To extend our knowledge of the pharmacological actions of methadone at ligand-gated ion channels, we investigated the effects of the two enantiomers of methadone and its metabolites R-(+)-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium perchlorate (EDDP) and R-(+)-2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline hydrochloride (EMDP), as well as structural analogs of methadone, including (-)-alpha-acetylmethadol hydrochloride (LAAM) and (+)-alpha-propoxyphene, on rat alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs) stably expressed in a human embryonic kidney 293 cell line, designated KXalpha3beta4R2. (+/-)-methadone inhibited nicotine-stimulated 86Rb+ efflux from the cells in a concentration-dependent manner with an IC50 value of 1.9 +/- 0.2 microM, indicating that it is a potent nAChR antagonist. The (-)- and (+)-enantiomers of methadone have similar inhibitory potencies on nicotine-stimulated 86Rb+ efflux, with IC50 values of approximately 2 microM. EDDP, the major metabolite of methadone, is even more potent, with an IC50 value of approximately 0.5 microM, making it one of the most potent nicotinic receptor blockers reported. In the presence of (+/-)-methadone, EDDP, or LAAM, the maximum nicotine-stimulated 86Rb+ efflux was markedly decreased, but the EC50 value for nicotine stimulation was altered only slightly, if at all, indicating that these compounds block alpha3beta4 nicotinic receptor function by a noncompetitive mechanism. Consistent with a noncompetitive mechanism, (+/-)-methadone, its metabolites, and structural analogs have very low affinity for nicotinic receptor agonist binding sites in membrane homogenates from KXalpha3beta4R2 cells. We conclude that both enantiomers of methadone and its metabolites as well as LAAM and (+)-alpha-propoxyphene are potent noncompetitive antagonists of alpha3beta4 nAChRs.

Agonist regulation of rat alpha 3 beta 4 nicotinic acetylcholine receptors stably expressed in human embryonic kidney 293 cells.

Effects of agonists on rat alpha 3 beta 4 nicotinic acetylcholine receptors expressed in KX alpha 3 beta 4R2 cells [human embryonic kidney 293-derived cells] were studied. The potencies of seven agonists varied over a 7000-fold range, with a rank order of epibatidine >> A85380 > cytisine approximately 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) approximately nicotine > acetylcholine > carbachol. The efficacies of all of the agonists studied here were similar except for DMPP, which seemed to be a partial agonist compared with nicotine and acetylcholine. Nicotine and carbachol desensitized the receptors in a time- and concentration-dependent manner. The EC(50) values for nicotine and carbachol to desensitize the receptors during a 60-min exposure were 3 and 51 microM, respectively, indicating that these agonists are more potent at desensitizing the receptors than at activating them. The function of the receptors recovered from agonist-induced desensitization rapidly and almost completely. The half-time for recovery of function from desensitization after a 60-min treatment with nicotine increased with the concentration of nicotine used to desensitize the receptors. In contrast, no such concentration dependence for time to recovery of function was found when carbachol was used to desensitize the receptors. We propose that this difference may be due to the cell permeability of nicotine, allowing it to enter and be sequestered inside of cells and then slowly diffuse out to maintain receptor desensitization. After a 5-day exposure to 100 microM nicotine, the receptors were completely desensitized, but receptor function recovered to 83% of control values with a half-time of about 10.5 min. Although the number of nicotinic receptor binding sites measured with (+/-)-[(3)H]epibatidine was increased during the chronic treatment with nicotine, no increase in function was detected.

Neuronal nicotinic acetylcholine receptor alpha3 subunit protein in rat brain and sympathetic ganglion measured using a subunit-specific antibody: regional and ontogenic expression.

A synthetic peptide corresponding to the C-terminus of the alpha 3 subunit of the rat neuronal nicotinic acetylcholine receptor (nAChR) was used to generate a rabbit polyclonal alpha 3 antibody. The specificity of this antibody was characterized by immunoblotting, immunohistochemical and immunoprecipitation techniques. Using this antibody, the relative densities of the alpha 3 subunit were quantitatively determined in different brain regions and in superior cervical ganglion (SCG). Among these regions, SCG, interpeduncular nucleus (IPN) and pineal gland showed the highest levels of alpha 3 protein expression. Habenula and superior colliculi had intermediate levels of expression. Low levels were found in cerebral cortex, hippocampus and cerebellum. The ontogenic profile of the alpha 3 subunit in the SCG was also determined. The alpha 3 protein level is low at postnatal day (P 1), but increases rapidly during the first seven postnatal days. This level then plateaus and remains stable through postnatal day 35. These findings suggest that neuronal nAChRs containing the alpha 3 subunit participate in important roles in specific regions of the rat brain and the SCG.

Evidence of a functional alpha7-neuronal nicotinic receptor subtype located on motoneurons of the dorsal motor nucleus of the vagus.

In vitro autoradiography using 125I-alpha-bungarotoxin (alpha-BGTx) and anti-alpha7 immunohistochemistry were performed on the dorsal motor nucleus of the vagus (DMV) of sham and chronically vagotomized rats to determine whether the alpha7-nicotinic acetylcholine receptor (nAChR) is located postsynaptically on DMV neurons whose axons contribute to the vagus nerve. Intense bilateral 125I-alpha-BGTx binding and anti-alpha7 immunostaining were observed in coronal brain sections containing the DMV of sham-vagotomized animals. Unilateral cervical vagotomy resulted in ipsilateral losses of 125I-alpha-BGTx binding and anti-alpha7 immunostaining from the DMV. Simultaneous staining of rat brainstem sections with anti-alpha7 and anti-choline acetyltransferase (ChAT) antibodies (to identify cholinergic DMV neurons that project into the vagus nerve) revealed that every DMV neuron that was stained for ChAT showed alpha7-staining as well. In vagotomized animals, no ChAT-positive neurons expressing alpha7-nAChRs remained in the ipsilateral DMV. We conclude that the alpha7-nAChR subtype is located postsynaptically on DMV neurons. To test whether the alpha7-nAChR is similar to the alpha7-homomeric nAChR, experiments were performed in anesthetized rats, and compounds were microinjected into the DMV while monitoring intragastric pressure (IGP). alpha-BGTx and strychnine antagonized nicotine-induced increases in IGP; no antagonism was observed with methyllycaconitine, a compound known to block the homomeric alpha7-nAChR subtype. Recovery from alpha-BGTx-induced antagonism of the nicotine response was observed. We conclude that there is a nAChR containing the alpha7-subunit in the DMV that is different from the homomeric alpha7-nAChR subtype.

Brainstem nicotinic receptor subtypes that influence intragastric and arterial blood pressures.

The purpose of this study was to investigate the effect of microinjection of nicotine and nicotinic receptor antagonists into the dorsal motor nucleus of the vagus (DMV) or medial subnucleus of the tractus solitarius (mNTS) on intragastric (IGP) and arterial blood pressures (BP) in anesthetized rats. Nicotine microinjected into the DMV (10-300 pmol) produced dose-related increases in IGP (ED(50) = 89 pmol); no significant changes were noted for BP. Ipsilateral vagotomy abolished nicotine-induced increases in IGP. Nicotine microinjected into the mNTS in a dose range of 0.1 to 300 pmol produced dose-related decreases in IGP (ED(50) = 0.6 pmol) and BP (ED(50) = 5.4 pmol). Bilateral vagotomy abolished nicotine-induced decreases in IGP while having no effect on BP. In rats treated with daily s.c. injections of nicotine (0.8 mg/kg of base) for 10 days, microinjections of nicotine into the DMV produced similar increases in IGP. BP responses from the mNTS were not affected by chronic treatment. However, nicotine microinjections into the mNTS no longer produced a decrease in IGP in these chronically treated animals. alpha-Bungarotoxin (100 pmol) significantly blocked nicotine-evoked increases in IGP from the DMV while having no effect on nicotine-induced responses elicited from the mNTS. Hexamethonium (10 and 100 pmol) microinjected into the mNTS dose-dependently blocked nicotine-induced effects but did not interfere with the action of nicotine at the DMV. Our data indicate that nicotine-induced changes in IGP result from nicotine acting at two sites, the DMV and mNTS; and that at least three different nicotinic receptors in the dorsal medulla oblongata can influence gastrointestinal and cardiovascular function.

Dextromethorphan and its metabolite dextrorphan block alpha3beta4 neuronal nicotinic receptors.

Dextromethorphan (DM), a structural analog of morphine and codeine, has been widely used as a cough suppressant for more than 40 years. DM is not itself a potent analgesic, but it has been reported to enhance analgesia produced by morphine and nonsteroidal anti-inflammatory drugs. Although DM is considered to be nonaddictive, it has been reported to reduce morphine tolerance in rats and to be useful in helping addicted subjects to withdraw from heroin. Here we studied the effects of DM on neuronal nicotinic receptors stably expressed in human embryonic kidney cells. Studies were carried out to examine the effects of DM on nicotine-stimulated whole cell currents and nicotine-stimulated (86)Rb(+) efflux. We found that both DM and its metabolite dextrorphan block nicotinic receptor function in a noncompetitive but reversible manner, suggesting that both drugs block the receptor channel. Consistent with blockade of the receptor channel, neither drug competed for the nicotinic agonist binding sites labeled by [(3)H]epibatidine. Although DM is approximately 9-fold less potent than the widely used noncompetitive nicotinic antagonist mecamylamine in blocking nicotinic receptor function, the block by DM appears to reverse more slowly than that by mecamylamine. These data indicate that DM is a useful antagonist for studying nicotinic receptor function and suggest that it might prove to be a clinically useful neuronal nicotinic receptor antagonist, possibly helpful as an aid for helping people addicted to nicotine to refrain from smoking, as well as in other conditions where blockade of neuronal nicotinic receptors would be helpful.

Activation and Ca2+ permeation of stably transfected alpha3/beta4 neuronal nicotinic acetylcholine receptor.

The alpha3/beta4 rat neuronal nicotinic acetylcholine receptor, stably transfected in human embryonic kidney cells, was examined using the whole-cell-clamp technique and 2-dimensional confocal imaging. Application of agonists (nicotine, cytisine, epibatidine) activated a large (100-200 pA/pF) inwardly rectifying monovalent current, with little current at voltages between 0 and +40 mV. Rapid application of nicotine and cytisine indicated EC50 values of congruent with22 and congruent with64 microM, respectively, and suggested second order binding kinetics (Hill coefficient approximately 2). The time constant of desensitization (decay) of nicotine-activated current was concentration-dependent (typically approximately 10 s at 30 microM versus approximately 1.0 s at 100-1000 microM), but not voltage-dependent and was significantly smaller than the approximately 200 s reported for the alpha3/beta4 receptor expressed in Xenopus oocytes. Nicotine-activated current was rapidly and reversibly blocked by coapplication of mecamylamine and d-tubocurarine. At -80 mV holding potentials, the current was also suppressed by approximately 25% either upon complete removal or elevation of Ca2+ to 10 mM. Total replacement of Na+ by Ca2+ also completely blocked the current. On the other hand, evidence for permeation of Ca2+ was indicated by increased inward current at -40 mV upon elevation of Ca2+ from 2 to 10 mM, as well as a rise in the cytosolic Ca2+ proportional to the current carried by the receptor. These findings are consistent with the idea that Ca2+, in addition to its channel-permeating properties, may also regulate the receptor from an extracellular site. Our results suggest that the alpha3/beta4 neuronal nicotinic acetylcholine receptor, when stably expressed in human embryonic kidney 293 cells, has desensitization kinetics and Ca2+ regulatory mechanisms somewhat different from those described for the receptor expressed in Xenopus oocytes.

Increased nicotinic receptors in brains from smokers: membrane binding and autoradiography studies.

Chronic administration of nicotine increases the density of neuronal cholinergic nicotinic receptors in cells and in rodent brain, and similar increases have been reported in brains from human smokers. To further examine this phenomenon, we measured nicotinic receptor binding sites in brain regions from matched populations of smokers and nonsmokers. We first measured binding of [3H](+/-)epibatidine ([3H]EB) and [3H]cytisine in homogenate preparations from samples of prefrontal and temporal cerebral cortex. Binding of each radioligand was significantly higher (250-300%) in both cortical regions from brains of smokers. Frozen sections from each of the cerebral cortical regions and the hippocampus were used for autoradiographic analysis of [3H]EB binding. In cerebral cortex, binding was most dense in layer VI in the prefrontal cortex and layers IV and VI in the temporal cortex. Densitometric analysis of [3H]EB binding sites revealed marked increases of 300 to 400% of control in all cortical regions examined from smokers' brains. Binding in the hippocampal formation was heterogeneously distributed, with dense areas of binding sites seen in the parasubiculum, subiculum, and molecular layer of the dentate gyrus, and the lacunosum-moleculare layer of the CA1/2. Binding of [3H]EB was significantly higher in all six regions of the hippocampus examined from brains of smokers compared with nonsmokers. These increases ranged from 160% of control in parasubiculum to 290% in the molecular layer of the dentate gyrus. The increase in nicotinic receptors in the cerebral cortex and hippocampus of smokers may modify the central nervous system effects of nicotine and contribute to an altered response of smokers to nicotine.

Liquid chromatographic studies with immobilized neuronal nicotinic acetylcholine receptor stationary phases: effects of receptor subtypes, pH and ionic strength on drug-receptor interactions.

Nicotinic acetylcholine receptor (nAChR) alpha3-subunits, beta4-subunits, alpha3/beta4-subunit combination and alpha4/beta2-subunit combination were immobilized on chromatographic stationary phases and the binding affinities of the different nAChR subtypes were chromatographically evaluated. The observed relative binding affinities of epibatidine were alpha4/beta2>alpha3/beta4 and epibatidine did not bind at alpha3-subunits and beta4-subunits. No significant difference in binding affinities was observed on the alpha4/beta2 nAChRs immobilized in immobilized artificial membrane (IAM) particles and those sterically immobilized on Superdex 200 beads. The effects of mobile phase pH and ionic strength on the binding affinities of the alpha3/beta4 nAChRs support were also investigated. The results are consistent with the proposed ligand-nAChR binding model in which a cationic center exists at the binding site.

Nicotinic receptor binding sites in rat primary neuronal cells in culture: characterization and their regulation by chronic nicotine.

We have characterized high affinity neuronal nicotinic acetylcholine receptors labeled by [3H]cytisine in primary neuronal cell cultures from fetal rat brains. After 15 days in culture, the highest density of [3H]cytisine binding sites (Bmax approximately 57 fmol/mg protein) was found in cells from the brainstem, which includes the following subcortical brain areas: the septum, thalamus, hypothalamus, midbrain, pons and medulla. A lower density of sites was found in cells from the cerebral cortex, hippocampus, and caudate nucleus. [3H]Cytisine binds to receptors in primary cells from the brainstem and cerebral cortex with a Kd of approximately 0. 5 nM, and the binding is inhibited by the agonists nicotine, acetylcholine, and epibatidine with IC50 values of 1 to 20 nM, and by carbachol and the antagonist dihydro-beta-erythroidine with IC50 values of 0.5 to 1.5 microM. Chronic treatment of neuronal cultures with nicotine for 7 days differentially affected the number of nicotinic receptors in cells from different brain areas; it significantly increased the number of nicotinic binding sites in cells from the cerebral cortex, hippocampus, and caudate, but not in cells from the brainstem. The nicotine-induced increase of receptors in cerebral cortical cultures was not blocked by either mecamylamine or dihydro-beta-erythroidine. These results indicate that primary cultures of rat neuronal cells provide a good model system in which to study and compare the properties and regulation of native neuronal nicotinic acetylcholine receptors.

Pharmacology of neuronal nicotinic acetylcholine recceptors: effects of acute and chronic nicotine.

Neuronal nicotinic acetylcholine receptors mediate nicotine's diverse effects on the brain, spinal cord and autonomic nervous system. These receptors are composed of alpha and beta subunits. Eight different alpha and three different beta subunits have been identified in vertebrate nervous systems, giving rise to the possibility of multiple subtypes of nicotinic receptors, defined by their constituent subunits. The pharmacological and channel conductance properties of the recombinant receptor subtypes studied in cellular expression systems differ from one another. In addition, the regulation of the receptor density and function during and after acute and chronic exposure to nicotine appears to differ among the subtypes. The predominant receptor subtypes in specific brain regions and peripheral neurons are beginning to be identified and their characteristics studied using new ligands and methods. As more is learned of the differences among the receptor subtypes, it should be possible to identify which specific subtype mediates a specific function within the nervous system and which subtypes are associated with the reinforcing and addictive actions of nicotine.

Immobilized nicotinic receptor stationary phase for on-line liquid chromatographic determination of drug-receptor affinities.

Nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels which mediate nicotinic cholinergic transmission in the nervous system. A nAChR subtype composed of alpha3 and beta4 subunits (alpha3/beta4 subtype), prepared from a stably transfected KXalpha3beta4R2 cell line, has been immobilized in the phospholipid monolayer of an immobilized artificial membrane (IAM) liquid chromatography (LC) stationary phase. Approximately 60 mg of protein was immobilized per gram IAM particles. The resulting phase was used for the rapid on-line chromatographic determination of drug binding affinities to nAChRs. Relative binding affinities were determined by frontal chromatography for (+/-)-epibatidine (Kd: 0.27 +/- 0.05 nM) > A85380 (Kd: 17.2 +/- 0.5 nM) > (-)-nicotine (Kd: 88 +/- 33 nM) > carbachol (Kd: 1280 +/- 30 nM) > atropine (Kd: 14,570 +/- 2600 nM). These results are consistent with the affinity rank order obtained from binding assays using membrane homogenates. The immobilized receptor LC stationary phase was stable and reproducible. This approach opens up the possibility of the production of a variety of immobilized receptor LC stationary phases which may be used for direct determination of drug-receptor binding interactions and for the rapid on-line screening of combinatorial pools for drug candidates.

Rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a transfected cell line: pharmacology of ligand binding and function.

We stably transfected human kidney embryonic 293 cells with the rat neuronal nicotinic acetylcholine receptor (nAChR) alpha3 and beta4 subunit genes. This new cell line, KXalpha3 beta4R2, expresses a high level of the alpha3/beta4 receptor subtype, which binds (+/-)- [3H]epibatidine with a Kd value of 304+/-16 pM and a Bmax value of 8942 +/- 115 fmol/mg protein. Comparison of nicotinic drugs in competing for alpha3/beta4 receptor binding sites in this cell line and the binding sites in rat forebrain (predominantly alpha4/beta2 receptors) revealed marked differences in their Ki values, but similar rank orders of potency for agonists were observed, with the exception of anatoxin-A. The affinity of the competitive antagonist dihydro-beta-erythroidine is >7000 times higher at alpha4/beta2 receptors in rat forebrain than at the alpha3/beta4 receptors in these cells. The alpha3/beta4 nAChRs expressed in this cell line are functional, and in response to nicotinic agonists, 86Rb+ efflux was increased to levels 8-10 times the basal levels. Acetylcholine, (-)-nicotine, cytisine, carbachol, and (+/-)-epibatidine all stimulated 86Rb+ efflux, which was blocked by mecamylamine. The EC50 values for acetylcholine and (-)-nicotine to stimulate 86Rb+ effluxes were 114 +/- 24 and 28 +/- 4 microM, respectively. The rank order of potency of nicotinic antagonists in blocking the function of this alpha3/beta4 receptor was mecamylamine > d-tubocurarine > dihydro-beta-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompetitive mechanism, whereas dihydro-beta-erythroidine blocked the function competitively. The KXalpha3 beta4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.

Nicotinic receptor mediates spontaneous GABA release in the rat dorsal motor nucleus of the vagus.

Spontaneous postsynaptic currents were investigated in neurons of the caudal portion of the dorsal motor nucleus of the vagus using the patch-clamp technique to study the effect of neuronal nicotinic acetylcholine receptor activation on synaptic transmission. In voltage-clamped neurons, bath application of nicotine (1-30 microM) elicited a concentration-dependent increase in the frequency of the spontaneous synaptic currents. The effect was also observed with application of the nicotinic receptor agonists epibatidine (10 nM) and cytisine (10 microM). Mecamylamine (20 microM) and curare (50 microM), two nicotinic receptor antagonists, both decreased the effect of 3 microM nicotine on the frequency of the spontaneous postsynaptic currents. This effect of 3 microM nicotine was also blocked by 20 microM bicuculline, a competitive antagonist of the GABA(A) receptor; in contrast, it was not affected by 1 mM kynurenic acid, an antagonist of the ionotropic glutamate receptor. In the presence of 1 microM tetrodotoxin, 3 microM nicotine was unable to affect the synaptic activity. Our findings suggest the existence of nicotinic receptors on GABAergic axons projecting to the vagal motoneurons. Because the effect is completely abolished by 1 microM tetrodotoxin, the nicotinic receptors are not localized on the presynaptic nerve terminal and their action on the GABA release requires the propagation of an action potential from their location to the synaptic terminal. This effect of nicotinic receptor activation on spontaneous GABA release in the dorsal motor nucleus of the vagus may have an important role in the regulation of gastrointestinal motility.

[125I]IPH, an epibatidine analog, binds with high affinity to neuronal nicotinic cholinergic receptors.

An analog of epibatidine (EB) was synthesized with an iodine atom in the 2 position of the pyridyl ring. This analog, (+/-)-exo-2-(2-iodo-5-pyridyl)-7-azabicyclo[2.2.1]heptane (IPH), as well as its two stereoisomers, displayed high affinity for neuronal nicotinic receptors; therefore, radioiodinated IPH, [125I]IPH, was synthesized with specific radioactivities consistently > 1000 Ci/mmol, and its properties as a radioligand for neuronal nicotinic receptors were evaluated. The characteristics of [125I]IPH binding in tissue homogenates appeared to be virtually identical to those reported for [3H]epibatidine binding; but the high specific radioactivity of [125I]IPH greatly facilitated measurements of nicotinic receptors in tissues with relatively low receptor densities and/or where tissues are in limited supply. Autoradiography with [125I]IPH provided clear localization of nicotinic receptors in brain and adrenal gland after film exposure times of < or = 2 days. We conclude that [125I]IPH will be a very useful radioligand for the study of neuronal nicotinic receptors in brain and in peripheral ganglia.

Differential regulation of neuronal nicotinic receptor binding sites following chronic nicotine administration.

Chronic nicotine administration to rats produces an increase in neuronal nicotinic receptors in the CNS. Moreover, the up-regulated sites labeled by [3H]cytisine in cerebral cortex appear to be composed exclusively of alpha4 and beta2 subunits. It is unknown whether receptor subtypes that do not bind [3H]cytisine with high affinity are also affected. In the present studies, we tested the hypothesis that nicotine treatment differentially alters the density of neuronal nicotinic receptor subtypes in rat nervous tissues. Thus, we compared the binding of [3H]cytisine with that of [3H]epibatidine to nicotinic receptors in brain, spinal cord, and adrenal gland from rats that had been injected twice daily with nicotine or saline vehicle for 10 days. Chronic nicotine treatment led to an increase in nicotinic receptor binding sites in the cerebral cortex and in the dorsal lumbar spinal cord, but not in the thalamus. It is important that virtually all of the observed increases could be accounted for by a selective effect on the fraction of receptors exhibiting high affinity for both [3H]cytisine and [3H]epibatidine. In contrast, no change in [3H]epibatidine binding was seen in the adrenal gland, a tissue that does not exhibit high-affinity [3H]cytisine binding. These data indicate that, under the conditions used here, nicotine up-regulates the alpha4beta2 nicotinic receptor subtype, which can be labeled by [3H]cystisine and [3H]epibatidine, but not non-alpha4beta2 subtypes, which can be labeled by [3H]epibatidine.

[3H]epibatidine labels nicotinic receptors in rat brain: an autoradiographic study.

Epibatidine, a frog skin alkaloid, is a nicotinic receptor agonist with potent analgesic activity. We used in vitro receptor autoradiographic techniques to map binding sites for [3H]epibatidine in rat brain, and those for another nicotinic agonist, [3H]cytisine. Both [3H]epibatidine and [3H]cytisine are excellent radioligands for autoradiography. [3H]Epibatidine in particular binds with very high affinity and extremely low nonspecific binding. In the present study, the distribution of binding was almost identical for both radioligands and closely resembled that of previously published autoradiographic studies with two other nicotinic ligands, [3H]acetylcholine and [3H]nicotine. Brain regions demonstrating the highest levels of [3H]epibatidine binding included the interpeduncular nucleus, medial habenular nucleus, fasciculus retroflexus, superficial gray layer of the superior colliculus and numerous thalamic nuclei, including the anteroventral, dorsal lateral geniculate and gelatinosus nuclei. Quantitative analysis revealed that [3H]epibatidine binding was greater than [3H]cytisine binding in several regions. In particular, [3H]epibatidine clearly labeled the optic nerve, optic chiasm and optic tract, whereas [3H]cytisine binding to these structures did not exceed background levels. Other regions in which [3H]epibatidine binding was greater than that of [3H]cytisine included the medial habenula, fasciculus retroflexus, olivary pretectal nucleus and superficial gray layer of the superior colliculus. The differences in labeling between these two nicotinic ligands may arise from receptor heterogeneity in brain nicotinic receptors and the ability of [3H]epibatidine to label more than one subtype of nicotine receptor.