Peter P. Rowell

Nicotinic Stimulation of [3H]Acetylcholine Release from Mouse Cerebral Cortical Synaptosomes

The effects of nicotine and 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) on the release of newly synthesized [3H]acetylcholine in mouse cerebral cortical synaptosomes were examined. Nicotine and DMPP produced increases in [3H]acetylcholine release, over the level of spontaneous release, of 24% and 30%, respectively, of a maximum depolarization‐induced release produced by 50 mM potassium. The maximum effect was achieved at a concentration of 1 × 10‐4M for both agents. The time course of release indicated a slow onset of action, reaching a maximum effect at 15 min of incubation. Both nicotine and DMPP also produced a slightly greater release of total tritium, measured in the absence of cholinesterase inhibition, than of [3H]acetylcholine. The release induced by nicotine was completely antagonized by hexamethonium and was largely (58%) calciumdependent. Nicotine also produced an increase in [3H]choline accumulation into synaptosomes. These results indicate that the nicotinic agonists nicotine and DMPP can produce a moderate enhancement of acetylcholine release by a receptor‐mediated action on cholinergic nerve terminals in the central nervous system.

Stimulation of [3H]Dopamine Release by Nicotine in Rat Nucleus Accumbens

Abstract: The mesolimbic system of the brain has been shown to be involved in the reward properties of a number of agents. It is possible that release of monoamines by nicotine in this brain area could be related to the pleasurable aspects related to cigarette smoking. In this investigation, the effect of nicotine on the release of [3H]dopamine in the nucleus accumbens of the rat was studied. It was shown that nicotine produced a concentration‐dependent increase in [3H]dopamine release at concentrations of 0.1 μM and above. The increase in release was found to be almost completely calcium dependent. The nicotine‐induced release was only partially blocked by the nicotinic antagonists hexamethonium and d‐tubocurarine. A number of cholinergic agonists, as well as other compounds, were tested for their capacity to mimic the effect of nicotine. At equimolar concentrations there was, at most, only 50% of the activity of nicotine. The results of this study demonstrate that nicotine stimulates the release of dopamine in the nucleus accumbens at concentrations similar to those in the blood of cigarette smokers. This suggests that the release of mono‐amines in specific nuclei of the mesolimbic system may be an important determinant of the desire to smoke cigarettes.

Dose‐Response Relationship for Nicotine‐Induced Up‐Regulation of Rat Brain Nicotinic Receptors

Abstract: The chronic administration of nicotine to animals has been shown to result in an increase in brain nicotinic acetylcholine receptor (nAChR) density. It has been suggested that this agonist‐induced receptor up‐regulation is a consequence of long‐term nAChR desensitization in vivo. In this study, the effects of different nicotine doses and administration schedules as well as the resulting blood and brain nicotine levels were determined to assess the effect of in vivo nicotine concentration on nAChR density in the brain. Rats with indwelling subcutaneous cannulas were infused for 10 days with 0.6–4.8 mg/kg/day nicotine either 2×, 4×, or 8×/day or by constant infusion. The nAChR density in cortical, striatal, and hippocampal tissue measured by [3H]cytisine binding as well as the corresponding plasma and brain nicotine levels measured by GC analysis were determined. The results showed a dose‐dependent increase in nAChR density with significant increases achieved at 2.4 mg/kg/day in all three brain areas. It is surprising that at this dose there was little difference between the constant infusion of nicotine and twice‐daily administration, whereas more frequent periodic injections were actually less effective at up‐regulating nAChRs. An analysis of the blood and brain levels of nicotine compared with the concentrations that produce nAChR desensitization suggests that in vivo desensitization alone is not sufficient for nAChR up‐regulation to occur.

Subsets of acetylcholine-stimulated 86Rb+ efflux and [125I]-epibatidine binding sites in C57BL/6 mouse brain are differentially affected by chronic nicotine treatment

Nicotinic cholinergic receptor (nAChR) sites that bind nicotine with high affinity (likely alpha4beta2-nAChR) increase following chronic nicotine treatment. Effects of chronic treatment on other nAChR binding sites and functional responses of nAChRs are less well studied. Therefore, C57BL/6 mice were intravenously infused for 10 days with saline or nicotine (five doses, 0.25-4.0 mg/kg/h) and nAChR function and three different nicotinic binding sites in 12 brain regions were assessed. Plasma nicotine and cotinine increased linearly with dose. 86Rb+ efflux with higher sensitivity to acetylcholine tended to decrease with increasing dose, whereas efflux with lower sensitivity to acetylcholine tended to increase. As anticipated, likely alpha4beta2-nAChR [125I]-epibatidine binding sites increased with treatment (estimated dosage for one-half maximal increase was 0.44 mg/kg/h, plasma nicotine approximately 20 ng/ml). 86Rb+ efflux with higher sensitivity to acetylcholine and cytisine-sensitive [125I]-epibatidine binding are predominantly alpha4beta2-nAChR. A high correlation between these parameters was observed across brain regions and slopes of these regression lines decreased with treatment dose, suggesting a decrease in function per unit receptor. Likely alpha3beta4-nAChR binding sites were unaffected even at the highest dose (4.0 mg/kg/h, approximately 210 ng/ml). A third set of diverse nAChR binding sites increased in some brain regions, but only after high-dose treatment.Nicotinic cholinergic receptor (nAChR) sites that bind nicotine with high affinity (likely α4β2-nAChR) increase following chronic nicotine treatment. Effects of chronic treatment on other nAChR binding sites and functional responses of nAChRs are less well studied. Therefore, C57BL/6 mice were intravenously infused for 10 days with saline or nicotine (five doses, 0.25-4.0 mg/kg/h) and nAChR function and three different nicotinic binding sites in 12 brain regions were assessed. Plasma nicotine and cotinine increased linearly with dose. 86Rb+ efflux with higher sensitivity to acetylcholine tended to decrease with increasing dose, whereas efflux with lower sensitivity to acetylcholine tended to increase. As anticipated, likely α4β2-nAChR [125I]-epibatidine binding sites increased with treatment (estimated dosage for one-half maximal increase was 0.44 mg/kg/h, plasma nicotine ≈20 ng/ml). 86Rb+ efflux with higher sensitivity to acetylcholine and cytisine-sensitive [125I]-epibatidine binding are predominantly α4β2-nAChR. A high correlation between these parameters was observed across brain regions and slopes of these regression lines decreased with treatment dose, suggesting a decrease in function per unit receptor. Likely α3β4-nAChR binding sites were unaffected even at the highest dose (4.0 mg/kg/h, ≈210 ng/ml). A third set of diverse nAChR binding sites increased in some brain regions, but only after high-dose treatment.

Impaired spatial working memory and altered choline acetyltransferase (CHAT) immunoreactivity and nicotinic receptor binding in rats exposed to intermittent hypoxia during sleep

Exposure to intermittent hypoxia (IH), such as occurs in sleep-disordered breathing (SDB), is associated with cognitive impairment, neurodegeneration, oxidative stress, and inflammatory responses within rodent brain regions such as the basal forebrain. In this region, damage to cholinergic neurons correlates with working memory deficits in a number of neurodegenerative disorders, suggesting that degeneration of cholinergic systems may also contribute to the working memory impairments observed after IH exposures. We therefore examined basal forebrain choline acetyltransferase (CHAT) immunohistochemistry, nicotinic receptor binding in the prefrontal cortex (PFC), and working memory, in male rats tested on a delayed matching to place (DMP) task in the water maze following exposure to either room air (RA) or intermittent hypoxia (IH; alternating 90s epochs of 21% and 10% O(2) during sleep). IH-treated animals displayed impaired working memory with respect to controls, along with significant reductions in CHAT-stained neurons in the medial septal nucleus, in both the vertical and horizontal limbs of the diagonal band, and the substantia inominata after 14 days of IH exposure. In addition, increases in nicotinic binding and receptor affinity in the PFC were observed after 14 days of IH exposure. Thus, a loss of cholinergic neuronal phenotype in the basal forebrain may contribute to the cognitive impairments associated with CIH exposure. However, compensatory mechanisms may also be activated in other brain regions, and may provide potential therapeutic targets for the cognitive impairments associated with SDB.

Characterization of Nicotine-Induced Desensitization of Evoked Dopamine Release from Rat Striatal Synaptosomes

Abstract: Nicotine has been shown to stimulate neurotransmitter release from brain tissue by acting on presynaptic receptors. In this study, the ability of nicotine pretreatment to produce functional desensitization was investigated in rat striatal synaptosomes in which the release of [3H]dopamine was measured with an in vitro superfusion system. Pretreatment of synaptosomes with low concentrations of l‐nicotine resulted in a decrease in the ability of a subsequent nicotine challenge to evoke [3H]dopamine release. The IC50 for nicotine‐induced desensitization was found to be 12 nM with a maximum inhibition of >90% at 300 nM. Nicotine pretreatment did not affect the release evoked by amphetamine, veratridine, or 15 mM K+. The onset of nicotine‐induced desensitization occurred with a t1/2 of 43 s at 30 nM nicotine. The temperature dependence of onset yielded a Q10 of 1.2.Recovery from desensitization was slower (t1/2 = 4.33 min), and both the onset and recovery appeared to follow a single first‐order process. Several intermittent schedules of nicotine treatment were found to be effective at inducing and maintaining desensitization. The results of this study show that nonstimulating concentrations of nicotine can produce a complete functional desensitization of subsequent nicotine‐induced neurotransmitter release.

Nanomolar concentrations of nicotine increase the release of [3H]dopamine from rat striatal synaptosomes

Nicotine stimulates the release of several neurotransmitters from brain tissue by acting on presynaptic nicotinic acetylcholine receptors (nAChR). In this study, an in vitro superfusion system was used to measure the nicotine-evoked release of [3H]dopamine (DA) from rat striatal synaptosomes. A 2-min exposure to micromolar nicotine produces a rapid increase in [3H]DA release. With continued exposure the response declines, apparently due to conversion of the nAChRs to a high-affinity desensitized conformation. In contrast, prolonged exposure to nanomolar concentrations of nicotine, while not producing an immediate response, leads to a gradual cumulative enhancement in [3H]DA release. This effect is calcium-dependent and blocked by the nicotinic antagonist, dihydro-beta-erythroidine. It is suggested that the gradual DA release in response to low concentrations of nicotine occurs as a result of either open channel properties of the desensitized receptor or an equilibrium between the high-affinity desensitized and active states of the nAChRs.

Structure-activity relationships of enkephalins containing serially replaced thiomethylene amide bond surrogates

An isomeric series of four leucine-enkephalin analogs containing the thiomethylene ether unit as an amide bond replacement in all positions have been prepared by solid phase methods. The resulting pseudopeptides divulged widely differing retentive behaviors on reversed phase high performance liquid chromatography (HPLC). An analog containing the Phe psi[CH2S]Leu dipeptide replacement at the 4-5 position exhibited binding close to the parent, leucine enkephalin; its guinea pig ileum (GPI) activity was the highest of the analogs tested. Another compound, Tyr psi[CH2S]Gly1-2]-Leu-enkephalin, also displaced 3H-etorphine well in the binding assay, but caused increased contractions in the GPI assay at low concentrations. The Phe psi[CH2S]Leu results are not compatible with the necessity of a beta-turn structure for agonist activity in the GPI assay.

Oral administration of nicotine: Its uptake and distribution after chronic administration to mice

An investigation was made of the suitability of administering nicotine to experimental animals by inclusion in the drinking water. It was found that, after an initial accommodation period of several weeks, nicotine could be administered up to a concentration of 100 micrograms/ml with no decrease in fluid intake or weight gain compared to control. An analysis of the steady-state plasma levels and distribution of nicotine was made in mice which had received nicotine in the drinking water at a concentration of 60 micrograms/ml. The average daily dose of nicotine received by these animals was 17.2 mg/kg. The steady-state plasma level of nicotine was 34.4 ng/ml, representing 6% of the total compound present at steady-state as determined by thin-layer chromatography. The distribution of nicotine or metabolite in mice which had received [methyl-14C]-nicotine orally was determined. Whole-body autoradiography, as well as direct tissue counting, demonstrated that nicotine accumulates in a number of areas, particularly the salivary gland, nasal epithelium, uterus, and liver. There was relatively little material in the blood or brain. This investigation indicates that ad libitum oral administration is an acceptable method for maintaining experimental animals on nicotine for long periods of time.

Effects of Subchronic Nicotine Administration on Central Dopaminergic Mechanisms in the Rat

Nicotine was administered acutely and subchronically (14 days) to determine whether various synaptic mechanisms are selectively altered in the nigrostriatal and mesolimbic dopaminergic systems in the rat. When added to tissue preparations in vitro, nicotine had no effects on tyrosine hydroxylase, synaptosomal uptake of [3H]dopamine or binding of [3H]spiperone to D2 receptors in either system. However, acute treatment in vivo stimulated tyrosine hydroxylase activity in the nucleus accumbens. This effect was prevented by pretreatment with a nicotinic antagonist, suggesting that it was mediated by nicotinic receptors. Since subchronic exposure to nicotine had no effect on tyrosine hydroxylase, it appears that tolerance develops to this action. In vivo treatment with nicotine did not alter dopamine uptake or receptor binding. The results suggest that, in doses which result in moderate plasma levels, nicotine has selective stimulant actions on nerve terminals of the mesolimbic system.