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).

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.

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.

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.

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.

D2 autoreceptors are not involved in the down-regulation of the striatal dopamine transporter caused by alpha-methyl-p-tyrosine.

The mechanisms by which the brain dopamine neuronal transporter is regulated by chronic alteration of dopamine transmission are not well understood. It has been shown previously that chronic inhibition of dopamine synthesis decreases dopamine transporter (DAT) density and function. The purpose of the present study was to determine whether these effects involve dopamine D2 receptors. Chronic treatment with alpha-methyl-p-tyrosine decreased binding of [3H]mazindol and dopamine release by d-amphetamine. The down-regulation of the DAT by alpha-methyl-p-tyrosine was not altered by co-treatment with a D2 receptor agonist or antagonist. However, chronic treatment with a D2 agonist, quinpirole, also decreased mazindol binding and amphetamine-induced release of dopamine. The results indicate that chronic inhibition of dopamine synthesis and stimulation of D2 receptors have similar, but independent, effects on DAT binding and function.

The effect of smoking on energy expenditure and plasma catecholamine and nicotine levels during light physical activity.

A number of studies have found that cigarette smoking causes an acute increase in resting energy expenditure, but the effect on energy expenditure during light physical activity is less clear. Since both smoking and activity have been shown to increase plasma catecholamines, these could produce additive effects on energy expenditure when smoking during light physical activity. In this study, the impact of cigarette smoking on energy expenditure, cardiovascular function, plasma nicotine and plasma catecholamine levels was determined in adult male subjects at rest and while engaged in light physical activity. Smoking at rest resulted in a 3.6% increase in energy expenditure above the resting baseline; whereas the increase in energy expenditure caused by smoking during light physical activity (compared with the light physical activity baseline) was 6.3%. This increase during light physical activity was significantly greater than the increase observed at rest (p < 0.025). As expected, plasma nicotine increased with smoking during both rest and light physical activity. An increase in plasma nicotine was associated with smoking during light physical activity. When this increase was adjusted as a covariate, the difference in smoking-related energy expenditure between light physical activity and rest disappeared, suggesting nicotine accounts for the effect. Plasma epinephrine and norepinephrine levels increased with smoking and showed a significantly greater increase during light physical activity compared to rest. Cigarette smoking caused a significantly greater increase in heart rate during light physical activity than it did while at rest, but there was no significant effect of smoking on mean blood pressure. It was concluded that there is enhanced energy expenditure associated with cigarette smoking during light physical activity when compared with smoking at rest which could be due in part to smoking-induced increases in circulating plasma catecholamines and perhaps nicotine.

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.

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.

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.

Characterization of nicotine-induced desensitization of evoked dopamine release from rat striatal synaptosomes.

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 (t 1/2 = 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.

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.

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.

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.

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.

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.

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.

Stimulation of [3H]dopamine release by nicotine in rat nucleus accumbens.

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 microM 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 monoamines in specific nuclei of the mesolimbic system may be an important determinant of the desire to smoke cigarettes.

Brain and serum levels of choline and lecithin resulting from long-term administration.

Choline and phosphatidylcholine tissue concentrations were examined in mice treated with long-term (18-22 month) dietary choline enrichment, choline deficiency, or phosphatidylcholine enrichment. There were no significant differences found in choline levels among the dietary groups in any of the tissues examined: plasma, erythrocytes, cortex, hippocampus, and striatum. In contrast, the concentration of phosphatidylcholine in both the choline enriched and phosphatidylcholine enriched groups were significantly increased in the cortex, compared to the choline deficient group, and in the striatum, compared to control. No differences in phosphatidylcholine concentration were found in the hippocampus or plasma between any of the dietary groups. These results are in contrast to the reported effects of acute or short-term choline and phosphatidylcholine treatment and indicate that there may be differences between the effects of short-term and long-term administration on the blood and brain levels of choline and phosphatidylcholine.

The effect of cigarette smoking during pregnancy on the cholinergic system in isolated term human placental tissue.

Mothers who smoke cigarettes during pregnancy give birth to babies with lower birth weights than do nonsmoking mothers. One hypothesis to explain this finding is that nicotine depresses the activity of the placental cholinergic system, which has been linked to the placental transport of amino acids and other substances. The levels and activities of several components of the term placental cholinergic system were determined in smokers and nonsmokers to investigate whether this system is involved in the effect of smoking. There were no statistically significant differences in the levels, synthesis or release of acetylcholine in the tissues from smoking and nonsmoking mothers, nor in the activities of the choline uptake system or the enzymes choline acetyltransferase, cholinesterase or sodium/potassium adenosine triphosphatase. The results do not support the hypothesis that the lower birth weights of babies born to smoking mothers is mediated by an effect of nicotine or other tobacco components on the placental cholinergic system.