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.