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Mechanism of apparent long-term nicotinic inactivation

Institution: University of California, Riverside
Investigator(s): Li Jia, M.S.
Award Cycle: 2000 (Cycle 9) Grant #: 9DT-0156 Award: $20,565
Subject Area: Nicotine Dependence
Award Type: Dissertation Awards

Initial Award Abstract
Nicotine is an important psychologically rewarding substance in tobacco. The action of nicotine on brain nicotinic receptors drives the habitual use of tobacco. Nicotine exposure for different times and at different doses is reported to produce a range of physiological effects in laboratory animals and humans ranging from elevated locomotor activity, seizures, changes in body temperature, enhancement of cognition and attention, relief of depression, and anxiolysis.

Tolerance and withdrawal symptoms are two important features of nicotine addiction. Tolerance is the reduction in drug response that occurs after chronic exposure. Withdrawal symptoms are the adverse side-effects that occur after chronic drug exposure ends. The development of nicotine tolerance forces smokers to increase their nicotine intake to compensate for the reduced psychological effects of the drug. It also contributes to habitual smoking by reducing the adverse physiological effects of nicotine. Withdrawal symptoms also contribute to nicotine addiction by making smoking cessation more traumatic.

Nicotine addiction, like any other biological disorder, cannot be cured until we know its biological basis. Nicotine receptors are normally activated by a chemical messenger called acetylcholine. After long nicotine exposures, it takes a long-time for these receptors to be able to respond to acetylcholine. Taking in more nicotine partially compensates for this loss of inactive receptors. Thus, the development of nicotine tolerance increases the number of cigarettes, a smoker must smoke to maintain a constant psychological reward from nicotine.

The goal of our research is to study the mechanism responsible for apparent long-term nicotinic receptor inactivation. We hypothesize that the long-lasting effects of nicotine on brain nicotinic receptors are due to the release of nicotine from brain neurons that have taken up nicotine. Our experiments will test this hypothesis and determine if this mechanism is a factor in persistent nicotine tolerance in smokers.

Final Report
The nicotinic receptor mediates nicotine addiction. Chronic nicotine exposure causes apparent persistent inactivation of these receptors. Our preliminary data suggested that the nicotine released by nicotine-soaked oocytes desensitized 42 receptors and made them appear as if nicotine persistently inactivated the 42 receptors expressed in the oocytes. The first aim of the project was to characterize how the chronic nicotine exposure causes the apparent persistent inactivation of 42 receptors expressed in Xenopus oocytes. The second aim of the project was to determine the mechanisms are involved in the nicotine uptake process in oocytes. The third aim was to determine the subcellular localization of nicotine in Xenopus oocytes. The fourth aim was to study the mechanism of apparent long-term nicotinic receptor inactivation in primary cultured neurons.

We previously found that 12 h incubation in 10 M nicotine reduces the ACh response by 76% for a 24 h period. Our latest results show that a 12 h incubation in 10 M nicotine does not affect total and surface 42 receptor expression in the oocytes. Thus, the 12 h nicotine incubation does not reduce the ACh response by decreasing 42 expression. We also found that nicotine release from the oocytes accounts for all the apparent irreversible 42 inactivation produced by a 12 h incubation in 10 M nicotine. ACh cannot not cross the cell membrane easily or accumulate inside intracellular acidic vesicles. Prolonged exposure (1 12 h) to a saturating concentration of ACh does not irreversibly inactivate 42 receptors and also does not cause the apparent irreversible 42 receptor inactivation. These results suggest that nicotine uptake and release from the oocytes causes apparent irreversible inactivation. Surprisingly, rapid continuous superfusion does not prevent nicotine release from the oocytes from desensitizing 42 receptors during the wash. Our results show conclusively that reversible desensitization of the 42 receptors, caused by nicotine release from the oocytes, accounts for all the apparent irreversible receptor inactivation produced by a 12 h incubation in 10 M nicotine.

It has been shown that the exposure of physiological concentration of nicotine causes persistent long-term inactivation of nicotinic receptors, this apparent long-term inactivation of nicotinic receptors drives nicotine intake and results in compulsive smoking. Our results suggest the long-lasting effects of nicotine on brain nicotinic receptors are due to the release of nicotine from brain tissues that have taken up nicotine during smoker smoking. It provides very important information for the clinical treatment of nicotine addiction.