Long-lasting inactivation of nicotinic receptor function in vitro by treatment with high concentrations of nicotine.

Exposure of nicotinic acetylcholine receptors (nAChRs) to brief pulses of nicotine results in the stimulation of dopamine release, whereas prolonged treatment with low concentrations of nicotine (approximately 10 nM) produces a reversible blockade of a subsequent nicotine challenge as a result of nAChR desensitization. We and others have observed that, following prolonged treatment with stimulating (microM) concentrations of nicotine, there is incomplete recovery from desensitization. In this study we investigated this nonrecoverable component by characterizing the ability of nicotine to stimulate [3H]dopamine release from rat striatal synaptosomes following recovery from nicotine-induced desensitization. Brief (12 s) exposure to 30 microM nicotine, or longer exposure (> or = 5 min) to 0.3 microM nicotine produced a long-lasting decrease in nAChR function with an apparent IC50 of 0.7 microM. The maximal inactivation achieved was approximately 50%. Recovery of nAChR function did not return even after 5 h, whereas recovery from desensitization occurred within 20 min. Determinations of the concentration of nicotine in the superfusate indicated that residual nicotine could not account for the observed decrease in response as a consequence of desensitization. These results indicate that high concentrations of nicotine can produce a long-lasting nAChR inactivation which can be distinguished from reversible nAChR desensitization.

Attenuation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity by tobacco smoke.

Several epidemiological studies have indicated that there may be an inverse relationship between smoking and Parkinson's disease. The purpose of this study was to determine whether chronic exposure to cigarette smoke alters the parkinsonian-like neurochemical changes caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice. Following 4 weeks of brief, intermittent exposure to smoke, mice were treated with MPTP, 10 mg/kg. Smoke exposure was found to reduce the decrease in striatal dopamine and metabolite levels caused by MPTP. Although smoke exposure inhibited cerebral MAO-B activity, tissues from smoke-treated mice were able to metabolize MPTP in a normal fashion. This suggests that inhibition of cerebral MAO may not be a major mechanism for the apparent protective effect of cigarette smoke.

Chemistry and biological activities of N,N-dimethylaminoethyl acrylate, a choline acetyltransferase inhibitor.

N,N-Dimethylaminoethyl acrylate (acryl-DMA) was synthesized as a tertiary nitrogen choline acetyltransferase (ChAc) inhibitor which would be able to penetrate biological membranes to inhibit ChAc in the nerve terminal. The synthesis from dimethylaminoethanol and acrylyl chloride was described and the hydration with times in an aqueous medium measured by NMR spectroscopy was presented. The autohydrolysis in water was found to be 1.75 x 10(-8) mol/min at pH 7.4 and 5.0 mM concentration. The enzymatic hydrolysis was unaffected by cholinesterases. Acryl-DMA was capable of inhibiting ChAc extracted from rat brain with I50 of 5.02 x 10(-4) M. The inhibition was reversible and displayed uncompetitive kinetics with respect to both substrates, choline and acetyl-CoA. Neither the hydrolysis nor the hydration products of acryl-DMA could inhibit ChAc. Although acryl-DMA was hydrated rapidly and completely within 1 hr at high pH (9.0), the time course of inhibition ability of acryl-DMA in aqueous medium at physiological pH was found to decrease rather slowly and by 36% in 1 hr, indicating that acryl-DMA can survive from hydration at physiological pH. Acryl-DMA was also tested for its ability to block electrically induced muscle contractions in both isolated skeletal and smooth nerve-muscle preparations. The ED50's obtained were less than 5 x 10(-4) M in both cases.

Investigations of the cholinergic modulation of GABA release in rat thalamus slices.

The thalamus receives a dense cholinergic projection from the pedunculopontine tegmentum. A number of physiological studies have demonstrated that this projection causes a dramatic change in thalamic activity during the transition from sleep to wakefulness. Previous anatomical investigations have found that muscarinic type 2 receptors are densely distributed on the dendritic terminals of GABAergic interneurons, as well as the somata and proximal dendrites of GABAergic cells in the thalamic reticular nucleus. Since these structures are the synaptic targets of cholinergic terminals in the thalamus, it appears likely that thalamic pedunculopontine tegmentum terminals can activate muscarinic type 2 receptors on GABAergic cells. To test whether activation of muscarinic type 2 receptors affects the release of GABA in the thalamus, we have begun pharmacological studies using slices prepared from the rat thalamus. We have found that the application of the nonspecific muscarinic agonist, methacholine, and the muscarinic type 2-selective agonist, oxotremorine.sesquifumarate, diminished both the baseline, and K(+) triggered release of [(3)H]GABA from thalamic slices. This effect was calcium dependent, and blocked by the nonselective muscarinic antagonist atropine, the muscarinic type 2-selective antagonist, methoctramine, but not the muscarinic type 1 antagonist, pirenzepine. Thus, it appears that one function of the pedunculopontine tegmentum projection is to decrease the release of GABA through activation of muscarinic type 2 receptors. This decrease in inhibition may play an important role in regulating thalamic activity during changes in states of arousal.

Investigation of the rate-limiting step in the synthesis of acetylcholine by the human placenta.

The uptake of [3H]choline and its conversion to [3H]acetylcholine were investigated in term human placental tissue in vitro. Although the net uptake of [3H]choline increased throughout a 45 min incubation period, intracellular [3H]choline levels reach a plateau after 2 min. There was a constant increase in [3H]acetylcholine levels throughout the incubation period. After 45 min, 36.5 per cent of the total intracellular tritium was recovered as acetylcholine by high-voltage electrophoresis. The effects of the choline acetyltransferase inhibitors, 2-benzoylethyltrimethyl-ammonium chloride (BETA) and 4-naphthylvinyl pyridine (NVP), and an inhibitor of choline uptake, hemicholinium-3 (HC-3), were also investigated for their influence on the uptake and metabolism of [3H]choline. A significant depression in both [3H]choline uptake and [3H]acetylcholine synthesis could be demonstrated with all three compounds, although with somewhat different time courses and activities. An analysis of the accumulation of [3H]acetylcholine in relation to the uptake and intracellular levels of [3H]choline as well as the patterns of inhibition produced by the inhibitors indicates that, unlike nervous tissue, the rate-limiting step in the synthesis of acetylcholine in human placental tissue is the transacetylation reaction catalysed by choline acetyltransferase.

Nicotine self-administration in rats: estrous cycle effects, sex differences and nicotinic receptor binding.

RATIONALE: Research on smoking behavior and responsiveness to nicotine suggests that nicotine's effects may depend on the sex of the organism. OBJECTIVE: The present study addressed four questions: 1) Will female rats self-administer nicotine? 2) Does self-administration by females vary as a function of estrous cycle? 3) Does self-administration by females differ from that of males? 4) Does self-administration of nicotine result in up-regulation of nicotinic receptor binding and are these changes similar in males and females? METHODS: Male and female Sprague-Dawley rats were allowed to self-administer nicotine at one of four doses (0.02-0.09 mg/kg, free base) on both fixed and progressive ratio schedules of reinforcement. RESULTS: Females acquired nicotine self-administration across the entire range of doses. Acquisition of self-administration at the lowest dose was faster in females than males. However, few sex differences were found in the number of active responses, number of infusions, or total intake of nicotine during stable fixed ratio self-administration. In contrast, females reached higher break points on a progressive ratio. For both schedules, females had shorter latencies to earn their first infusion of each session and demonstrated higher rates of both inactive and timeout responding. There was no effect of estrous cycle on self-administration during either fixed or progressive ratio sessions. Self-administered nicotine resulted in average arterial plasma nicotine levels between 53 and 193 ng/ml and left hemi-brain levels between 174 and 655 ng/g, depending on dose. Nicotine self-administration produced similar up-regulation of nicotinic receptor binding sites in males and females, as reflected by increased right hemi-brain binding of [3H]-epibatidine, when compared to the brains of untreated control rats. CONCLUSIONS: These results suggest that while males and females may regulate their intake of nicotine similarly under limited access conditions, the motivation to obtain nicotine is higher in females.

A sensitive and specific radioenzymatic assay for the simultaneous determination of choline and phosphatidylcholine.

A radioenzymatic procedure for the simultaneous measurement of picomol quantities of phosphatidylcholine and choline is described. Phospholipase-D from Streptomyces chromofuscus hydrolyzes phosphatidylcholine to choline and phosphatidic acid. Choline kinase in the presence of [32P]ATP phosphorylates the choline to form [32P]phosphorylcholine. The phosphorylcholine, isolated by ion exchange chromatography, is measured by scintillation spectroscopy. Using a chloroform: methanol separation, phosphatidylcholine and choline can be measured from the same sample. Different sources of phospholipase-D were compared for their ability to hydrolyze phosphatidylcholine to choline and phosphatidic acid. Phospholipase-D from Streptomyces chromofuscus was found to result in almost complete hydrolysis. As a measure of specificity, lysophosphatidylcholine and sphingomyelin were assayed using this method and were found to result in, at most, only 2% of the amount of phosphorylcholine produced compared to phosphatidylcholine. This procedure allows for the simultaneous measurement of choline and phosphatidylcholine in brain and serum samples with a very high degree of sensitivity and specificity.

In vivo effects of some tertiary alkylaminoethyl esters with choline acetyltransferase inhibitory properties.

The tertiary nitrogen derivatives of two choline esters, chloroacetylcholine and acrylcholine, known to inhibit choline acetyltransferase (ChAc) in vitro, were tested for their effects in the whole animal, including peripheral and central cholinergic systems. These esters are N,N-dimethylaminoethyl chloroacetate (Cl-DMA) and N,N-dimethylaminoethyl acrylate (acryl-DMA). The peripheral preparations studied included a neuromuscular junction, a sympathetic ganglion and a postganglionic parasympathetic exocrine preparation. Both Cl-DMA and acryl-DMA blocked responses in these preparations when injected intravenously. The LD50 values for Cl-DMA and acryl-DMA were 640 mg/kg and 183 mg/kg, respectively. Cl-DMA and acryl-DMA were also able to inhibit brain ChAc when injected intravenously by 32% and 18.5%, respectively. The brain levels of acetylcholine (ACh) were significantly reduced by about 25% with Cl-DMA but not significantly with acryl-DMA when the animals were forced to exercise after injection. It is obvious that ChAc inhibition is not effective in decreasing ACh levels significantly under normal conditions.

Inhibition of uptake of 1-methyl-4-phenylpyridinium ion and dopamine in striatal synaptosomes by tobacco smoke components.

To determine whether the attenuation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity by tobacco smoke exposure is caused by inhibition of the neuronal uptake of 4-phenylpyridinium ion (MPP+), various tobacco components and a smoke extract were tested for inhibitory activity in striatal synaptosomes. A dimethylsulfoxide extract of tobacco smoke filtrate was found to inhibit the uptake of MPP+ and dopamine. These results suggest that inhibition of the neuronal dopamine uptake mechanism may account for the protective effects of smoke exposure on MPTP-induced neurotoxicity.

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.

Two week nicotine treatment selectively increases bone vascular constriction in response to norepinephrine.

This study was designed to determine if nicotine treatment alters the constrictor and/or dilator function of the vessels which regulate blood flow to intact bone. Nicotine (1.7 mg/kg/day) or nicotine-free, phosphate-buffered saline was administered subcutaneously to mature male rats for 2 weeks via osmotic mini-pumps. On the 14th day, the rats were anesthetized and in vivo experiments were performed to quantitate the changes in arterial blood pressure and perfusion of the intact tibia (measured by laser Doppler flowmetry) in response to two constrictor agonists (norepinephrine, NE and arginine vasopressin, AVP) and two vasodilator agents (acetylcholine, ACh and sodium nitroprusside, SNP). Dose-response curves were generated by plotting the change in the bone vascular resistance index (mmHg/bone perfusion units) evoked by each dose of agonist. In addition, bone arteriolar expression of endothelial nitric oxide synthase protein was quantitated by Western blot analysis. Nicotine treatment significantly enhanced the constriction of the bone vasculature in response to NE, but not to AVP. Vascular dilation in response to ACh and SNP was not changed by nicotine. These results indicate that nicotine selectively accentuates the constrictor response of the bone vasculature to exogenous NE. This enhanced constriction to NE is not due to impaired endothelial cell release of nitric oxide or diminished smooth muscle response to nitric oxide. Since NE and AVP activate similar cell signaling mechanisms to induce constriction, the selective enhancement of NE-induced constriction suggests that nicotine alters a mechanism unique to NE signaling; possibly the number or binding affinity of alpha adrenergic receptors. Since endogenous NE regulates basal blood flow to bone, the effect of nicotine to augment NE-induced constriction could lead to a chronic reduction in blood flow to bone.

Effects of long-term dietary choline and phosphatidylcholine administration on muscarinic receptors in aged mouse brain.

It has been suggested that choline and/or phosphatidylcholine may be beneficial in improving the memory deficits associated with aging and Alzheimer's disease. The effects of long-term choline or phosphatidylcholine treatment on cholinergic receptors in the brain have been investigated. Mice were maintained on one of four diets from 50 days of age until sacrificed at 20-24 months. [3H]-QNB binding in the cortex, hippocampus and striatum was specific, saturable and of high affinity. Animals treated with a phosphatidylcholine-enriched diet displayed a down-regulation of muscarinic receptors in the cortex and hippocampus, reflected by a decrease in Bmax. There were no significant differences in the binding affinities among the treatment groups. Choline levels were unaffected by the various diets, however phosphatidylcholine treatment resulted in an increase in phosphatidylcholine in the cortex and somewhat in the hippocampus. This study indicates that choline and phosphatidylcholine have different effects after long-term dietary administration. Phosphatidylcholine treatment results in a down-regulation of muscarinic receptors in certain brain areas which appears to be related to an increase in phosphatidylcholine concentration. Any potentially beneficial effects derived from chronic phosphatidylcholine treatment must overcome the apparent down-regulation of muscarinic receptors which may occur.

Relationships between chemical structure and inhibition of human placental choline acetyltransferase by keto analogs of acetylcholine.

Seven keto analogs of acetylcholine were synthesized and evaluated as inhibitors of human placental choline placental choline acetyltransferase. Their potencies for inhibition of horse serum cholinesterase and stimulation of cholinergic receptors in the longitudinal ileal muscle of the guinea pig were investigated. The most potent and selective inhibitor of choline acetyltransferase was (2-benzoylethyl)trimethylammonium chloride with an I50 of 3 X 10(-6) M. It exhibited considerably low activities at muscarinic and nicotinic receptors and cholinesterases. Its high potency for inhibiting choline acetyltransferase was atrributed to: (a) its cationic terminal, a site for an electron acceptor interaction; (b) an aryl moiety for hydrophobic and electron donor contributions; and (c) a positive charge on the carbon atom adjacent to the benzene ring due to the presence of the carbonyl group, which interacts with the nucleophilic residue on the enzyme.

Ergocryptine and other ergot alkaloids stimulate the release of [3H]dopamine from rat striatal synaptosomes.

Ergocryptine is an ergot alkaloid that affects dopaminergic activity principally by interacting with D2-type receptors. In this study the ability of ergocryptine and several other ergot alkaloids to release [3H]dopamine from isolated nerve endings was demonstrated using in vitro superfusion of rat striatal synaptosomes. Ergocryptine, ergocristine, and bromocryptine produced an elevation in baseline dopamine release of approximately 400% with effective concentrations (EC50) of approximately 30 microM. Ergotamine, ergonovine, ergovaline, and ergocornine were devoid of activity. The time-course of the ergocryptine-stimulated release was relatively slow compared with amphetamine, nicotine, or K+-stimulated [3H]dopamine release; the maximal increase in release required a 5-min treatment. A number of receptor antagonists were examined for their ability to block ergocryptine-stimulated release. Of the dopaminergic, adrenergic, serotonergic, GABA-ergic, and cholinergic antagonists examined, only phentolamine produced a moderate attenuation in evoked release. Omission of Ca++ from the medium did not affect ergocryptine-evoked release. Following ergocryptine treatment, the synaptosomes were fully responsive to other stimulant. The results indicate that, in addition to interacting with dopamine receptors, several ergot alkaloids may produce dopaminergic effects by increasing the release of dopamine from central nerve endings. Several mechanisms to account for the evoked neurotransmitter release are discussed.

Nicotine does not reduce blood flow to healthy bone in rats.

Given the increased incidence of orthopedic complications among smokers, we tested the null hypothesis that nicotine, the most vasoactive substance in cigarettes, does not reduce blood flow to long bones. Nicotine was administered to adult rats at a rate of 2.4 or 3.6 mg/kg/d for 2 weeks to determine if nicotine has a dose-dependent effect on bone blood flow. Control rats received nicotine-free solution. After 2 weeks, the rats were anesthetized. The microsphere technique was used to measure flow to femurs and tibias. Blood was collected to measure plasma nicotine. The lower dose established a plasma level of 14 ng/mL (SEM, 4 ng/mL); the higher dose elevated nicotine to 43 ng/mL (SEM, 11 ng/mL). Neither dose altered blood flow to tibias or femurs. A higher dose or longer treatment may be required to reduce bone blood flow. Alternatively, nicotine may not reduce blood flow to healthy bone at any dose but may delay bone healing by other mechanisms (ie, inhibiting angiogenesis and/or osteogenesis).

Effects of chronic choline and lecithin on mouse hippocampal dendritic spine density.

Dendritic spines, which project from the dendrites of central neurons, are thought to contribute to the amount of contact area available for synaptic connections. The density of these spines has been found to correlate with learning and memory function, and there is a progressive decrease in dendritic spine density with aging. In addition, experimental animals given a choline-enriched diet have an increase in neocortical spine density compared to controls. In this study, the dendritic spine density of hippocampal pyramidal cells was examined in aged mice which had received life-long choline enriched, choline deficient or lecithin enriched diets. These treatments had no effect on hippocampal dendritic spine density compared to control. The results indicate that dietary supplementation may have different effects in different brain areas and that the relative increase in learning and memory function in aged animals given a choline or lecithin enriched diet is not due to an increase in hippocampal dendritic spine density.

Dose-response relationship for nicotine-induced up-regulation of rat brain nicotinic receptors.

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 2x, 4x, or 8x/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.

Evidence for functional activity of up-regulated nicotine binding sites in rat striatal synaptosomes.

A number of studies have found that the chronic administration of nicotine causes an increase in the density of nicotinic binding sites in the brain, but it is not known whether these additional binding sites are functionally active receptors. In this study, the effects of 1-week administration of the potent nicotinic agonist, (+)-anatoxin-a (96 nmol/day via osmotic minipumps), was assessed on [3H]nicotine binding and [3H]dopamine uptake and release in rat striatal synaptosomes. Chronic (+)-anatoxin-a treatment resulted in a 32% increase in the Bmax of [3H]nicotine binding in anatoxin-treated animals compared to control. There was a 43% increase in the activity of 3 microM nicotine to release [3H]dopamine from synaptosomes of anatoxin-treated animals, but the release induced by 20 mM K+ depolarization was unaffected. There was no effect of chronic (+)-anatoxin-a treatment on the uptake of [3H]dopamine. A strong positive correlation (r = 0.64) was found between the density of [3H]nicotine binding sites and the nicotine-induced stimulation of [3H]dopamine release in individual animals. These results indicate that (+)-anatoxin-a, like nicotine, produces an up-regulation of nicotine binding sites following chronic administration, and that these additional sites are functional receptors capable of mediating the release of dopamine from striatal synaptosomes.

Pharmacological studies of N,N-dimethylaminoethyl chloroacetate and N,N-dimethylaminoethyl acrylate as inhibitors of choline acetyltransferase in isolated skeletal and smooth muscle preparations.

Two tertiary amine esters, N,N-dimethylaminoethyl chloroacetate (Cl-DMA) and N,N-dimethylaminoethyl acrylate (acryl-DMA), which have recently been shown to be inhibitors of choline acetyltransferase (ChAc) were investigated to determine their actions in isolated skeletal and smooth muscle preparations. Both compounds caused neuromuscular blockade in indirectly stimulated nerve-muscle preparations (ED50 values of Cl-DMA were 6.9 -42.0 X 10(-4) M and those of acryl-DMA were 1.2-5.8 X 10(-4) M). The blockade was completely or partially reversible after drug washout. A comparison of the ED50 values for neuromuscular blockade with the ID50 values for ChAc inhibition suggested that the acryl-DMA compound might not cause neuromuscular blockade via ChAc inhibition because the potency ratios (ED50/ID50) of Cl-DMA were higher than 1, whereas those of acryl-DMA were equal to or lower than 1. This was borne out by further experiments on isolated neuromuscular preparations which showed that the site of action for acryl-DMA was post-junctional, whereas that for Cl-DMA was prejunctional. In addition, the weak stimulating properties of Cl-DMA and acryl-DMA were investigated in isolated skeletal and smooth muscle. Cl-DMA was shown to be a partial cholinergic agonist, whereas acryl-DMA was a nonspecific stimulant not involving cholinergic receptors. Although both Cl-DMA and acryl-DMA are inhibitors of ChAc, only Cl-DMA appears to have sufficient specificity for use as a possible ChAc inhibitor in vivo.