The biological basis of nicotine addiction
Abstracts
Initial Award Abstract |
|
No one has discovered an effective cure for compulsive smoking. Nicotine addiction is almost certainly the reason behind compulsive smoking but treatments that use nicotine skin patches and inhalers do not work very well. There are two reasons why these treatments do not always work. The first reason is that nicotine inhalers and skin patches do not reproduce the rapid nicotine delivery to the brain or the sensory cues, like the taste and smell of burning tobacco, that smokers get from cigarettes. Non-smokers may wonder what makes tobacco smoke so attractive, but smokers have learned to associate it with the euphoria they get from smoking. The second reason why smoking treatments do not always work is that we do not yet understand the biological basis of nicotine addiction. Nicotine addiction, like any other biological disorder, cannot be cured until we know exactly what is going wrong.
Experiments on rats show that nicotine releases chemicals in the reward center of the brain. These chemicals probably trigger the euphoria that smokers experience from smoking. However, like other types of drug addiction, there is more to nicotine addiction than just the rewarding effects of the drug. Smokers continue smoking throughout the day even though the reward diminishes. The ability of nicotine to depress nicotinic receptors in the brain for a long time after it stops activating them probably accounts for compulsive smoking.
Nicotinic receptors normally respond to a chemical messenger called acetylcholine but, once these receptors are inactivated by nicotine, it takes a long time for them to recover enough to be able to respond to acetylcholine. This reduction in receptor activity may prompt smokers to take in more nicotine to attempt to reestablish the normal level of receptor activity. Taking in more nicotine partially compensates for the loss of the inactive receptors but, once the activating effects of nicotine wear off, receptor depression takes over again. This cycle of receptor activation and depression is repeated over and over again throughout the day, resulting in compulsive smoking. The goal of our research is to study the long-lasting depression produced by nicotine at the cellular level and determine the biochemical mechanism that underlies it. We hypothesize that the long-lasting effects of nicotine on brain nicotinic receptors are due to an enzymatic modification of the receptor triggered by nicotine binding. This modification may be the attachment or removal of a phosphate group. Our experiments will test this hypothesis and could point the way to more effective treatments for nicotine addiction. |
Final Report |
|
A recent theory of addiction suggests that the ability of a drug to activate and persistently sensitize the brain reward system is what makes it addictive. This ability creates a positive feedback cycle that enhances the psychologically rewarding effects of the drug and encourages repeated use. However, the effects of nicotine do not appear to be entirely consistent with this theory. Nicotinic receptors in the brain containing a4 and (32 subunits bind nicotine and stimulate the reward system of the brain. Chronic nicotine exposure increases the density of these receptors in the brain but it does not always lead to an increase in the brain nicotinic response. In fact, chronic nicotine exposure appears to irreversibly inactivate a large fraction of rat brain nicotinic receptors expressed in frog oocytes. Thus, chronic nicotine exposure does not always lead to sensitization of the nicotinic response. The specific aims of our research were to (1) characterize nicotine-induced irreversible receptor inactivation and (2) discover the biochemical mechanism responsible for inactivation.
To study inactivation, we expressed rat brain (c4p2) nicotinic receptors in Xenopus oocytes and measured their acetylcholine (ACh) response before and after a prolonged incubation in nicotine. The results showed that the x4(32 ACh response remained depressed for 24 h after a 1 or 12 h incubation in 10 pM nicotine, compared to controls incubated for an equal time in nicotine-free media. However, a 12 h incubation in 101 .LM nicotine did not affect the number of total or surface a4(32 receptors measured by [3H]epibatidine binding. Thus, a 12 h exposure to 10 [LM nicotine appeared to irreversibly inactivate brain nicotinic receptors expressed in oocytes. We initially hypothesized that receptor phosphorylation or de-phosphorylation was responsible for this irreversible inactivation. To test this hypothesis, we used biochemical techniques to demonstrate that rat a4P2 nicotinic receptors were phosphorylated in Xenopus oocytes. Then, we constructed a chimeric ocl/oc4 subunit that removed all the phosphorylation sites in the a4 subunit. Surprisingly, prolonged nicotine incubations still inactivated receptors formed from this chimera and wild-type (32 subunits even though all the phosphorylation sites were removed and the receptor displayed no detectable phosphorylation. These results suggested that changes in receptor phosphorylation were not responsible for nicotine-induced inactivation. We then hypothesized that nicotine uptake and release from the oocytes could cause apparent irreversible inactivation. Nicotine has a pKa of 7.84. Thus, it is only partially ionized at pH 7.4 (74%). Uncharged nicotine could diffuse across the plasma membrane and accumulate inside intracellular acidic vesicles because it is a weak base. Therefore, oocytes could slowly take up and release nicotine into the wash media. Because the equilibrium dissociation constant of a4(32 nAChRs for nicotine is small (4-7 nM), even minute amounts of nicotine released into the wash media could desensitize these receptors and make it appear, as if they were irreversibly inactivated. To test this hypothesis, oocytes expressing (x4(32 receptors were co-incubated with un-injected oocytes that were pre-soaked in 10 g.M nicotine for 12 h. The nicotine-soaked un-injected oocytes released enough nicotine to desensitize the (x4(32 receptors and account for all the apparent irreversible inactivation caused by a 12 h exposure to 10 pM nicotine. These results showed that nicotine release from the oocytes caused the apparent irreversible inactivation of nicotinic receptors following prolonged nicotine incubations. To confirm this hypothesis, we incubated oocytes expressing a4(32 receptors in a saturating concentration of ACh for 12 h or a very low nicotine concentration (5 nM) and measured the effects of the incubation on the ACh response. ACh is fully ionized at pH 7.4 and cannot easily cross the plasma membrane or accumulate inside acidic vesicles. A 12 h incubation in 300 pM ACh or in 5 nM nicotine (which resulted in negligible nicotine release from the oocytes) did not irreversibly inactivate (x402 receptors. We also measured the amount of nicotine released by nicotine-soaked oocytes using radioactive nicotine. Oocytes soaked for 12 h in radioactive nicotine released enough nicotine into the media to desensitize (x4(32 receptors. Finally, we showed that even continuous rapid rinsing with nicotine-fee saline fails to prevent nicotine-soaked oocytes from releasing enough nicotine at the surface of the plasma membrane to desensitize a4P2 receptors. Thus, our results conclusively show that nicotine does not irreversibly inactivate brain nicotinic receptors expressed in frog oocytes but, rather, desensitization caused by nicotine release from the oocytes into the wash media makes it appear as if the receptors are irreversibly inactivated.
The results of our research have several important implications for nicotine addiction. First, they show than nicotine-induced inactivation is not involved in nicotine addiction. Second, they suggest that nicotine is addictive because it persistently sensitizes the reward system of the brain by increasing the density of nicotinic receptors in the brain. Thus, the well documented finding that smoking increases the density of nicotinic receptors in the human brain suggests that chronic tobacco use sensitizes smokers to the effects of nicotine and creates a positive feedback cycle that promotes nicotine addiction. The next step in this research would be to study nicotine uptake and release in mammalian cells expressing brain nicotinic receptors. |
Publications
| The alpha4 subunit of rat alpha4 beta2 nicotinic receptors is phosphorylated in vivo |
| Periodical: Brain Research. Molecular Brain Research |
Index Medicus: |
| Authors: Viseshakul N, Figl A, Lytle C, Cohen B |
ART |
| Yr: 1998 |
Vol: 59 |
Nbr: |
Abs: |
Pg: 100-104 |
| Two mutations linked to nocturnal frontal lobe epilepsy cause use-dependent potentiation of the nicotinic ACh response |
| Periodical: Journal of Physiology |
Index Medicus: |
| Authors: Figl A, Viseshakul N, Shafaee N, Forsayeth J, Cohen BN |
ART |
| Yr: 1998 |
Vol: 513 |
Nbr: 3 |
Abs: |
Pg: 655-670 |
| Use- and concentration-dependent potentiation by two mutations linked to nocturnal epilepsy |
| Periodical: Society for Neuroscience Abstracts |
Index Medicus: |
| Authors: Figl A, Viseshakul N, Forsayeth J, Cohen BN |
ABS |
| Yr: 1998 |
Vol: 24 |
Nbr: |
Abs: |
Pg: 519 |
| The biological basis of nicotine addiction |
| Periodical: Proceedings of the TRDRP Annual Investigator's Meeting |
Index Medicus: |
| Authors: Cohen BN |
ABS |
| Yr: 1998 |
Vol: |
Nbr: |
Abs: |
Pg: 103 |
| Pharmacological similarities between native brain and heterologously expressed receptors |
| Periodical: British Journal of Pharmacology |
Index Medicus: |
| Authors: Shafaee N, Huong M, Truong A, et al |
ART |
| Yr: 1999 |
Vol: 128 |
Nbr: |
Abs: |
Pg: 1291-1299 |
| The beta subunit dominates the relaxation kinetics of heteromeric neuronal nicotinic receptors |
| Periodical: Journal of Physiology |
Index Medicus: |
| Authors: Figl A, Cohen BN |
ART |
| Yr: 2000 |
Vol: 524 |
Nbr: 3 |
Abs: |
Pg: 685-699 |
| Pharmacological differences between native brain and heterologously expressed alpha 4 beta 2 nicotinic receptors |
| Periodical: Brain Research. Molecular Brain Research |
Index Medicus: |
| Authors: Truong A, Xing X, Forsayeth JR, Dwoskin LP, Crooks PA, Cohen BN |
ART |
| Yr: 2001 |
Vol: |
Nbr: |
Abs: |
Pg: |
| A mutation associated with epilepsy enhances desensitization of the alpha4 beta2 neuronal nicotinic receptor |
| Periodical: Biophysical Journal |
Index Medicus: |
| Authors: Figl A, Viseshakul N, Forsayeth J, Cohen BN |
ABS |
| Yr: 1997 |
Vol: 72 |
Nbr: |
Abs: A150 |
Pg: |
| Two ADNFLE mutations cause use-dependent potentiation of the nicotinic ACh response |
| Periodical: Annals of the New York Academy of Sciences |
Index Medicus: |
| Authors: Figl A, Viseshakul N, Shafaee N, Forsayeth J, Cohen BN |
ABS |
| Yr: 1998 |
Vol: |
Nbr: |
Abs: PII-8 |
Pg: |
| ADNFLE mutations reduce Ca2+ -induced potentiation of the ACh response |
| Periodical: Society for Neuroscience Abstracts |
Index Medicus: |
| Authors: Rodriques-Pinguet NO, Jia L, Li M, Figl A, Truong A, Cohen BN |
ABS |
| Yr: 0 |
Vol: |
Nbr: |
Abs: |
Pg: |
| The alpha4 subunit of rat alpha4 beta2 nicotinic receptors is phosphorylated in vivo |
| Periodical: Brain Research. Molecular Brain Research |
Index Medicus: |
| Authors: Viseshakul N, Figl A, Lytle C, Cohen B |
ART |
| Yr: 1998 |
Vol: 59 |
Nbr: |
Abs: |
Pg: 100-104 |
| Two mutations linked to nocturnal frontal lobe epilepsy cause use-dependent potentiation of the nicotinic ACh response |
| Periodical: Journal of Physiology |
Index Medicus: |
| Authors: Figl A, Viseshakul N, Shafaee N, Forsayeth J, Cohen BN |
ART |
| Yr: 1998 |
Vol: 513 |
Nbr: 3 |
Abs: |
Pg: 655-670 |
| Use- and concentration-dependent potentiation by two mutations linked to nocturnal epilepsy |
| Periodical: Society for Neuroscience Abstracts |
Index Medicus: |
| Authors: Figl A, Viseshakul N, Forsayeth J, Cohen BN |
ABS |
| Yr: 1998 |
Vol: 24 |
Nbr: |
Abs: |
Pg: 519 |
| The biological basis of nicotine addiction |
| Periodical: Proceedings of the TRDRP Annual Investigator's Meeting |
Index Medicus: |
| Authors: Cohen BN |
ABS |
| Yr: 1998 |
Vol: |
Nbr: |
Abs: |
Pg: 103 |
| Pharmacological similarities between native brain and heterologously expressed receptors |
| Periodical: British Journal of Pharmacology |
Index Medicus: |
| Authors: Shafaee N, Huong M, Truong A, et al |
ART |
| Yr: 1999 |
Vol: 128 |
Nbr: |
Abs: |
Pg: 1291-1299 |
| The beta subunit dominates the relaxation kinetics of heteromeric neuronal nicotinic receptors |
| Periodical: Journal of Physiology |
Index Medicus: |
| Authors: Figl A, Cohen BN |
ART |
| Yr: 2000 |
Vol: 524 |
Nbr: 3 |
Abs: |
Pg: 685-699 |
| Pharmacological differences between native brain and heterologously expressed alpha 4 beta 2 nicotinic receptors |
| Periodical: Brain Research. Molecular Brain Research |
Index Medicus: |
| Authors: Truong A, Xing X, Forsayeth JR, Dwoskin LP, Crooks PA, Cohen BN |
ART |
| Yr: 2001 |
Vol: |
Nbr: |
Abs: |
Pg: |
| A mutation associated with epilepsy enhances desensitization of the alpha4 beta2 neuronal nicotinic receptor |
| Periodical: Biophysical Journal |
Index Medicus: |
| Authors: Figl A, Viseshakul N, Forsayeth J, Cohen BN |
ABS |
| Yr: 1997 |
Vol: 72 |
Nbr: |
Abs: A150 |
Pg: |
| Two ADNFLE mutations cause use-dependent potentiation of the nicotinic ACh response |
| Periodical: Annals of the New York Academy of Sciences |
Index Medicus: |
| Authors: Figl A, Viseshakul N, Shafaee N, Forsayeth J, Cohen BN |
ABS |
| Yr: 1998 |
Vol: |
Nbr: |
Abs: PII-8 |
Pg: |
| ADNFLE mutations reduce Ca2+ -induced potentiation of the ACh response |
| Periodical: Society for Neuroscience Abstracts |
Index Medicus: |
| Authors: Rodriques-Pinguet NO, Jia L, Li M, Figl A, Truong A, Cohen BN |
ABS |
| Yr: 0 |
Vol: |
Nbr: |
Abs: |
Pg: |
