Effect of long-term nicotine treatment on [3H]nicotine binding sites in the rats brain

Male rats were receiving nicotine (base), 50 mg/kg in their drinking fluid for 9 and 41 weeks, respectively. A markedly reduced intake of fluid was observed during both regimes of nicotine treatment. At withdrawal of nicotine after 9 weeks of treatment the intake of water was immediately increased up to control level while in rats with 41 weeks of exposure the intake of drinking fluid increased to 65% over the control level still 14 days after withdrawal of nicotine. Tolerance to nicotine was measured after 7 days of abstinence in rats treated for 9 weeks. No significant change in cholineacetyltransferase (ChAT) activity or number of muscarinic binding sites in brain was observed after 9 or 41 weeks of nicotine treatment. Twenty-four hours after withdrawal from the 41 weeks nicotine treatment there was a significant increase of 46% in the number of [3H] nicotine binding sites in the hippocampus while the number of binding sites was decreased by 44% in the cortex and unchanged in the midbrain. On day 14th of abstinence these changes had disappeared. Following 9 weeks of nicotine treatment no change in [3H] nicotine binding was observed after 24 h or 7 days of abstinence.     

Mecamylamine prevents tolerance but enhances whole brain [3H]epibatidine binding in response to repeated nicotine administration in rats

RATIONALE: Chronic administration of nicotine in rats results in upregulation of neuronal nicotinic receptors. Upregulation has been proposed to reflect receptor desensitization, which may underlie functional tolerance to nicotine's effects. However, evidence indicates that tolerance and upregulation do not always parallel each other, suggesting that either upregulation does not always reflect desensitization, or mechanisms other than receptor desensitization account for tolerance to nicotine. OBJECTIVES: The present studies examined tolerance to nicotine-induced antinociception and changes in receptor binding after two regimens of intermittent nicotine injections in rats. The role of receptor activation in upregulation and tolerance was also examined by co-administering nicotine with the non-competitive antagonist, mecamylamine. METHODS: Sprague-Dawley rats were administered a short (once-daily, s.c. for 6 days (0.35 mg/kg)) or long (twice-daily for 11 days (0.66 mg/kg)) series of injections and tolerance to nicotine-induced antinociception and [3H]epibatidine binding in whole brain were measured. RESULTS: The short series of injections resulted in tolerance to nicotine-induced antinociception, but failed to increase [3H]epibatidine binding. In contrast, the long series of injections resulted in both tolerance and increased receptor binding. Once-daily pairings of mecamylamine (1 mg/kg, s.c.) with nicotine (0.35 mg/kg) for 6 days blocked the development of tolerance, indicating receptor activation is necessary for tolerance to occur. Pairing mecamylamine with nicotine (0.66 mg/kg) twice daily for 11 days blocked tolerance but produced a greater increase in [3H]epibatidine binding than nicotine alone. CONCLUSIONS: A dissociation of tolerance from receptor upregulation was observed in the present study. The finding that receptor activation may be necessary for tolerance but not upregulation is discussed within the context of possible mechanisms controlling tolerance to nicotine.     

3H]acetylcholine and [3H](-)nicotine label the same recognition site in rat brain

High affinity binding sites for [3H]acetylcholine and [3H](-)nicotine in rat brain were compared with respect to key characteristics, any one of which should distinguish them if they are different. The density of binding sites for each ligand varied approximately 4-fold in five areas of rat forebrain, but in each of these areas and in human cerebral cortex as well, the densities of [3H]acetylcholine- and [3H](-)nicotine-binding sites were indistinguishable. The affinity of [3H](-)nicotine was higher than that of [3H]acetylcholine, but nicotinic cholinergic drugs competed for the sites labeled by the two ligands with similar affinities; and in each case, the site labeled displayed marked stereoselectivity for the enantiomers of nicotine. The binding of [3H]acetylcholine and [3H](-)nicotine was decreased to the same extent by preincubation of tissues with dithiothreitol, and the binding was restored by subsequent treatment with 5,5'-dithiobis-2-nitrobenzoic acid, indicating that a disulfide bond is required at or near the binding site for each ligand. Treatment of rats with nicotine for 10 days increased the density of binding sites for both ligands, and treatment with the cholinesterase inhibitor soman for 9 days decreased the density of binding sites for both ligands. Taken together, these results indicate that [3H]acetylcholine and [3H](-)nicotine bind to the same nicotinic cholinergic recognition site in rat brain.     

Subchronic treatment of rats with nicotine: interconversion of nicotinic receptor subtypes in brain

A significant increase in the number of cortical high-affinity (-)-[3H]nicotine binding sites was measured in rats treated with nicotine (0.45 mg/kg) twice daily for 18 days. Competition experiments with (-)-[3H]nicotine and various concentrations of unlabelled (-)-nicotine revealed that the proportion of high-affinity nicotine binding sites was significantly increased in the nicotine-treated group while the proportion of low-affinity nicotinic binding sites was similarly significantly reduced compared to the controls. In addition there was a significant decrease in the affinity of both subtypes of nicotinic binding sites.     

Exposure to nicotine enhances the behavioral stimulant effect of nicotine and increases binding of [3H]acetylcholine to nicotinic receptors

Rats were given daily injections of nicotine sulfate in doses ranging from 0.1 to 0.4 mg/kg. The behavioral effect of these injections was measured as locomotor activity in photocell cages. Repeated administration of the same dose to each rat resulted in an enhancement of the stimulant effect of nicotine. This enhanced behavioral effect was quite pronounced within 5 days of repeated injection. Tissue from the cerebral cortex of these rats, exposed to nicotine for 5 days, was assayed for binding of [3H]acetylcholine to nicotinic receptors. These relatively small doses of nicotine resulted in 18-26% increases in cortical nicotinic receptors, compared to saline-treated rats. Rats exposed to 0.2 mg/kg of nicotine for 5 days and then given saline for 7 days still showed an enhanced behavioral response to nicotine on the eighth day after exposure, and nicotinic binding in the cortex was still elevated. However, 21 days after exposure to nicotine both the behavioral response to nicotine and the binding values had returned to the same values as those of saline-treated rats. These data imply that increased binding of [3H]acetylcholine to nicotinic sites and the enhanced behavioral effect of nicotine are functionally linked.     

Nicotinic acetylcholine receptors in the rat stomach: I. (-)-[3H]nicotine binding

The nicotinic acetylcholine receptors in the rat stomach were characterized by means of a radioligand binding assay with (-)-[3H]nicotine as ligand. Saturation binding studies on the gastric fundus membranes revealed the presence of two binding sites with dissociation constant (KD) values of 3.1 and 289 nM, and maximum binding capacity (Bmax) values of 3.6 and 76 fmol/mg protein, respectively. The Bmax of the high affinity binding site was greatest in the cardia, followed by fundal mucosa, fundal muscle, and, finally antrum. The IC50 values of cholinergic drugs to inhibit (-)-[3H]nicotine binding in fundus membranes were as follows: (-)nicotine, 0.12 nM; cytosine, 9.3 nM; acetylcholine, 17.7 nM; carbachol, 700 nM; hexamethonium, 2270 nM. The IC50 values of alpha-bungarotoxin, d-tubocurarine and atropine were greater than 100 microM. The muscarinic acetylcholine receptors were also characterized with [3H]quinuclidinyl benzilate and the choline acetyltransferase activity was measured. These results suggest that nicotinic acetylcholine receptors as well as muscarinic acetylcholine receptors are present in the rat stomach and that the regional distribution of these receptors is uneven.     

Some effects of oestrogens on [3H]-mepyramine binding in rat brain

Oestrogen depress [3H]-mepyramine binding in the amygdala, hypothalamus and cerebral cortex of rats. These effects of oestrogens are prevented by concurrent administration of inhibitors of cyclic 3'5' AMP phosphodiesterase, suggesting that the hormones act by reducing the activity of adenylate cyclase.     

The effect of acute dyflos (DFP) treatment on [3H]nicotine binding to mouse brain homogenate

One possible adaptive mechanism that might arise due to inhibition of cholinesterase in the brain is a down regulation of central cholinergic receptors. Therefore studies were performed to determine the effect of acute dyflos exposure on [3H]nicotine binding. Specific [3H] nicotine binding was demonstrated to be saturable, reversible, stereospecific, and inhibited by a number of nicotinic compounds. Scatchard analysis of specific [3H] nicotine binding produced a curvilinear plot that was resolved into high- and low-affinity sites with Kd values of 6.1 +/- 2.5 and 114 +/- 13 nM, and Bmax values of 11.8 +/- 3.5 and 182 +/- 24 fmol (mg protein)-1, respectively. The nicotinic binding sites in brain homogenate from dyflos-treated mice that were killed 20 min or 10 h after exposure did not exhibit significant alterations in binding parameters from control mouse brain homogenate. However, brain homogenate from treated mice that were killed 24 h after exposure resulted in statistically significant differences in the low-affinity K(D) and Bmax values from controls. Since no alterations were found in the high-affinity binding parameters and dyflos had only a minimal effect on the low-affinity site at 24 h, it was concluded that nicotinic receptor down regulation does not appear to be the mechanism through which the mouse functionally adapts to cholinesterase inhibition caused by acute dyflos treatment.     

Studies on the binding of [3H]flumazenil and [3H]sarmazenil in post-mortem human brain

Two benzodiazepine analogues, [³H]flumazenil and [³H]sarmazenil, were used to study the GABA_A/benzodiazepine receptor complex in human post-mortem brain using in vitro receptor assays on homogenates and whole hemisphere autoradiography. Both radioligands bound in a saturable manner to single binding sites in the tissue preparations from any brain region. The highest levels of binding were found in the cortical regions and in cortex cerebelli. Both [³H]flumazenil and [³H]sarmazenil were excellent radioligands for autoradiography with high binding in cerebral and cerebellar cortex with no or very low binding in areas with white matter. The addition of a high concentration of flumazenil or clonazepam did not inhibit the binding of [³H]sarmazenil to granule cells in the cerebellum while the binding of [³H]flumazenil was abolished completely in all regions. The results show that with the two different radioligands, one an antagonist and one a partial inverse agonist, the binding pattern to GABA_A/benzodiazepine receptor complex is approximately similar in most brain regions. The additional binding seen in the cerebellum with [³H]sarmazenil is suggested to be due to binding to an α₆-containing complex.     

Effects of perinatal nicotine exposure on development of [3H]hemicholinium-3 binding sites in rat neonate brain

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

Chronic benzodiazepine treatment increases [3H]muscimol binding in mouse brain

High affinity [3H]muscimol receptors were analyzed in the forebrain and cerebellum of mice that had been treated on a chronic regimen with chlordiazepoxide and clonazepam. In the forebrain, only clonazepam induced an increase in the number of muscimol binding sites 2 hr after drug treatment. In the cerebellum, both the chlordiazepoxide and clonazepam-treated animals showed an increased number of muscimol binding sites 2 hr after drug treatment. At 26 hr after drug treatment, only the clonazepam-treated animals still revealed an increased number of cerebellar muscimol binding sites. Chronic benzodiazepine administration, therefore, induced an increase in the apparent number of high affinity muscimol binding sites in both the forebrain and cerebellum.     

Differential effects of triethyllead on synaptosomal [3H]dopamine vs. [3H]acetylcholine and [3H]gamma-aminobutyric acid release.

In vitro exposure to tetraethyllead (Et4Pb, 10 microM) did not alter the release of [3H] dopamine (DA), [3H]acetylcholine (ACh), or [3H]gamma-aminobutyric acid (GABA) from superfused synaptosomes isolated from rat brain striatum, hippocampus, and cortex, respectively. On the other hand, a concentration-dependent increase in the spontaneous release of these transmitters was observed following exposure to triethyllead (Et3Pb, 0.1-10 microM). The magnitude of 1 microM Et3Pb-induced [3H]DA release was 5-fold greater than that observed for [3H]ACh or [3H]GABA release. Removal of [Ca2+]e did not alter the Et3Pb-induced increase in the release of these three transmitter substances, nor did Et3Pb alter synaptosomal 45Ca efflux. EtePb-induced [3H]ACh and [3H]GABA release, but not [3H]DA release, was blocked by lowering [Na+]e from 140 to 50 mM. Similarly, the release of [3H]ACh and [3H]GABA, but not [3H]DA, induced by either Na,K-ATPase inhibition or veratridine (a Na(+)-ionophore), was attenuated by lowering [Na+]e from 140 to 50 mM. However, Et3Pb did not inhibit isolated synaptic membrane Na,K-ATPase, nor did the magnitude or temporal patterns of Et3Pb-induced transmitter release resemble transmitter release induced by Na,K-ATPase inhibition. Et3Pb and veratridine, but not Na,K-ATPase inhibition, produced an increase in synaptosomal [3H] deoxyglucose phosphate (dGluP) efflux, suggesting that both compounds increase membrane permeability. A Et3Pb-induced increase in membrane permeability is further supported by electrophysiological studies using the frog neuromuscular junction in which Et3Pb was found to reduce both the input resistance and membrane potential of muscle cells. As with [3H]ACh and [3H]GABA release, the Et3Pb-induced increase in synaptosomal [3H]dGluP efflux was attenuated by lowering [Na+]e.(ABSTRACT TRUNCATED AT 250 WORDS)     

Buffer effects on high affinity [3H]-prazosin binding in brain and spinal cord

[3H]-Prazosin binding was characterized in cortical and spinal membranes from Fischer 344N and Sprague-Dawley rats. Estimates of Bmax and Kd values were comparable with earlier studies of these regions in the central nervous system (CNS). However, the Kd obtained using Tris buffer system was greater than when HEPES or phosphate buffer was used. These data indicate that high affinity [3H]-prazosin binding in the homogenates of tissue from the CNS is affected critically by buffer selection.     

Dopamine D-2 receptors in canine brain: ionic effects on [3H]neuroleptic binding

In order to determine the effects of monovalent cations on the binding of 3H-neuroleptics to canine striatal dopamine D-2 receptors, a study of the effects of ions on the binding of two 3H-neuroleptics from different drug classes was undertaken. Dopamine D-2 receptors are selectively labeled in a sodium-sensitive manner by the benzamide ligand [3H]YM-09151-2. In the presence of 120 mM NaCl, [3H]YM-09151-2 had a dissociation constant of 55 pM and a maximal receptor density of 34 pmol/g of tissue. In the absence of NaCl, the dissociation constant was 440 pM with no change in the receptor density. The binding of [3H]YM-09151-2 (52 pM) was increased by 700% with 150 mM Na+, 440% with 500 mM Li+ and 290% with 500 mM K+. The ion concentrations producing half-maximal increases in binding were 4 mM Na+, 8.5 mM Li+ and 115 mM K+. The ionic strength control, N-methyl-D-glucamine, did not increase [3H]YM-09151-2 binding. [3H]Spiperone binding was much less affected by monovalent cations. Analysis of saturable binding showed that these changes were due to changes in binding affinity, independent of changes in receptor density. The sulfhydryl alkylating agent, N-ethylmaleimide, inactivated [3H]YM-09151-2 binding. Sodium ions at 120 mM protected approximately 40% of the susceptible sites while lithium and potassium ions (120 mM) did not. The anion exchange blocker, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS), has been shown to be capable of blocking the effect of Na+ on the binding of agonists to alpha 2-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin enhancement of [3H]-gamma-aminobutyric acid and [3H]muscimol binding in rat brain

The pineal hormone, melatonin, enhanced the sodium-independent binding of [3H]-gamma-aminobutyric acid ([3H]GABA) and [3H]muscimol in the rat cerebral cortex in vitro. This effect was augmented by preincubation of synaptic membranes with melatonin but was abolished by preincubation with Triton X-100. Saturation binding studies using [3H]GABA (2.5 to 1000 nM) indicated that the melatonin-induced enhancement of binding is due to an increase in low-affinity GABAA binding sites. These findings suggest that the central effects of melatonin involve modulation of GABAergic function.     

Up-regulation of brain [3H]diazepam binding sites in chronic caffeine treated rats

Brain [3H]diazepam and [3H]L-phenylisopropyladenosine binding sites in caffeine treated (75 mg/kg/day, i.p. 12 days) and caffeine withdrawn (30 days) rats were examined. Treatment with caffeine (75 mg/kg/day) for 12 days increases the Bmax (maximum binding capacity) for [3H]diazepam binding by 30.9% whereas the same treatment increases the Bmax for [3H]L-PIA binding by 165%. The Bmax for [3H]diazepam binding sites returns to slightly below control levels but [3H]L-PIA binding sites remain elevated after 30 days of caffeine withdrawal. These findings suggest that the up-regulation of [3H]diazepam binding sites seen in caffeine treated rats may not be adequately explained by a direct antagonism of caffeine on benzodiazepine receptors. Other modes of interaction therefore must be considered.