Cigarette Smoking Saturates Brain α4β2 Nicotinic Acetylcholine Receptors FREE

Tobacco dependence (primarily through cigarette smoking) is a major risk factor for death and disability worldwide.^1- 2 This condition is relatively resistant to treatment, as evidenced by the fact that most smokers endorse a desire to quit³ but very few are able to do so on their own⁴ and fewer than half are able to quit long-term even with comprehensive treatment.^3,5 A greater understanding of the mechanisms that underlie tobacco dependence may aid in the development of improved treatments for this condition.

Of the thousands of components of tobacco smoke, nicotine is the one that is most closely linked to tobacco dependence.⁶ Nicotine administration provides positive reinforcement^7- 8 and ameliorates a range of behavioral states that accompany smoking abstinence, including irritability,⁹ anxiety,⁹ and deficits in cognitive performance.^10- 12 Extensive animal research demonstrates that the interaction of nicotine with nicotinic acetylcholine receptors (nAChRs)¹³ (along with associated actions of nicotine) activates dopamine pathways projecting to the nucleus accumbens,^14- 19 leading to positive reinforcement.^8,20

The nAChR containing α₄ and β₂ subunits (α₄β₂* subtype) is the predominant receptor subtype in the mammalian brain, and the α₄²¹ and β₂^22- 25 subunits have been linked to the positive-reinforcing (and cognitive function–enhancing) effects of nicotine. Nicotine has high affinity for α₄β₂* receptors, and therefore, these receptors are considered as primary targets for the actions of nicotine during cigarette smoking. Studies with engineered mutant mice suggest that α₄β₂* nAChRs are necessary and sufficient to exhibit in vivo effects of smoking, such as tolerance and sensitization.²¹

The affinity of nicotine for the α₄β₂* nAChR measured in vitro is in the range of 0.5 to 14 nmol,²⁶ which is equal to a concentration of 0.01 to 2.3 ng/mL. Typical human smokers have venous plasma nicotine concentrations of 10 to 50 ng/mL during the day.²⁷ Based on these reports, we hypothesized that human cigarette smoking results in nearly complete saturation of α₄β₂* nAChRs.

2-[¹⁸F]fluoro-3-(2(S)azetidinylmethoxy) pyridine (2-F-A-85380, abbreviated as 2-FA) is a radiotracer recently developed for the in vivo imaging of α₄β₂* nAChRs with positron emission tomography (PET).^28- 30 Positron emission tomography studies in nonhuman primates demonstrate that receptor binding of 2-FA³¹ (and another PET radiotracer for α₄β₂* nAChRs³²) can be decreased by administration of nicotine or inhalation of tobacco smoke. Recent studies have demonstrated the safety of this radiotracer for use in humans.^30,33- 35 Using 2-FA and PET, we sought to determine the effect of cigarette smoking on brain α₄β₂* nAChR occupancy in tobacco-dependent smokers.

Eleven tobacco-dependent smokers (≥20 cigarettes per day), who were recruited through advertisements in local newspapers and the Internet, participated in the study. Eight subjects were scanned only once in an experimental (smoking) condition, while 3 subjects underwent 2 scanning sessions, 1 in an experimental (smoking) condition and 1 in a control (no smoking) condition, as described later, so that a total of 14 PET scanning sessions were performed for this study. Subjects met DSM-IV criteria for nicotine dependence but were otherwise healthy.

Initial screening consisted of an anonymous telephone interview in which medical, psychiatric, and substance abuse histories were obtained. Qualified subjects were then assessed in person using screening questions from the Structured Clinical Interview for DSM-IV³⁶ 2 days prior to PET scanning. The central inclusion criterion was the DSM-IV diagnosis of nicotine dependence, while any history of an Axis I psychiatric or substance abuse/dependence diagnosis other than nicotine dependence was exclusionary. Other exclusion criteria were pregnancy and current use of medications or any history of a medical condition that might affect the central nervous system at the time of scanning (eg, current treatment with a β-blocker or analgesic medication or history of head trauma with loss of consciousness or epilepsy). Women of childbearing potential had a urine pregnancy test. Subjects who occasionally used alcohol, caffeine, or other drugs, but did not meet criteria for abuse or dependence, were allowed to participate in the study but were instructed to abstain for the 2 days prior to PET scanning. Subjects who drank more than the equivalent of 2 cups of coffee per day (300 mg of caffeine per day) were also excluded, as were subjects who experienced caffeine withdrawal symptoms (such as irritability, flushing, or headache) temporally associated with caffeine ingestion. After complete description of the study to subjects, written informed consent was obtained using forms approved by the local institutional review board.

During the initial visit, additional screening data were obtained, including the smoker's profile form (which includes smoking history data, such as current smoking level, years smoked, brand of cigarette smoked, and quit periods) and scores on the Fagerström Test for Nicotine Dependence,^37- 38 the Beck Depression Inventory,³⁹ the Spielberger State-Trait Anxiety Inventory,⁴⁰ and the Shiffman-Jarvik Withdrawal Scale.⁴¹ An exhaled carbon monoxide (CO) level was obtained using the MicroSmokerlyzer (Bedfont Scientific Ltd, Kent, England) at the initial visit to verify smoking status (subjects were considered to be active smokers if a CO level of ≥8 ppm was obtained).

Participants underwent the following sequence of procedures (described in greater detail later): abstinence from cigarettes and nicotine-containing products for 2 days, a bolus-plus–continuous infusion 2-FA PET scanning session including smoking between 0 and 3.5 cigarettes, blood sampling for plasma nicotine levels and withdrawal symptom monitoring during the PET scanning session, and structural magnetic resonance imaging (MRI) of the brain within 1 week of PET scanning to aid in localization of regions of interest on PET scans.

After the initial screening, participants were instructed to begin smoking/nicotine abstinence at 6 PM 2 nights prior to PET scanning. They reported to our laboratory at 1 PM the day after initiating abstinence. At that time, an exhaled CO level was measured and a brief clinical interview was performed. Participants were deemed to be compliant with study protocol if they reported no smoking since 6 PM the previous night and had an exhaled CO measurement of 8 ppm or less. Subjects were then seen the following day for PET scanning and were required to report continuous abstinence since 2 nights previously and have an exhaled CO level of 3 ppm or less to undergo PET scanning.

At noon on the day of PET scanning, subjects arrived at the Greater Los Angeles Veterans Affairs Healthcare System PET Center, and abstinence was verified as described earlier. Each participant then had an intravenous catheter placed at 12:45 PM in a room adjacent to the PET scanner. At 1 PM, bolus-plus–continuous infusion 2-FA was initiated. In this study, the amount of 2-FA administered as a bolus was equal to the amount infused over 500 minutes (K_bolus = 500 minutes).^42- 43 Consistent with this paradigm, 144 MBq (mean ± SD, 3.88 ± 0.16 mCi) of 2-FA was administered as an intravenous bolus, followed by continuous infusion of 138 MBq (mean ± SD, 3.72 ± 0.15 mCi) in 57.6 mL of saline over the next 480 minutes (7.2 mL per hour) for a total effective dose of radioactivity of 187.5 MBq (mean ± SD, 5.08 ± 0.21 mCi). All PET scans were obtained as series of 10-minute frames.

Three PET scanning sessions were performed without smoking, as control sessions. For 2 of these sessions, subjects were positioned in the scanner before administration of 2-FA, and PET scanning began at the time of bolus injection and continued for 8 hours with 7 scheduled breaks and no smoking to demonstrate the full time-activity curves for the 2-FA method used herein (Figure 1). For the third session, a subject preferred not to be scanned for the full 8 hours and was scanned without smoking, using the shorter (5 hours) scanning protocol as in the experimental sessions described next.

For the 11 experimental sessions with smoking, subjects received the bolus injection of 2-FA in a room adjacent to the PET scanner. They then remained seated in this room for the next 3 hours to allow the radiotracer to reach a relatively steady state. At 4 PM, brain scanning commenced and continued for 60 minutes. At 5 PM (4 hours after the initiation of 2-FA administration), subjects had a 10-minute break in scanning, during which they smoked between 0 and 3 cigarettes of their favorite brand (because we were interested in studying α₄β₂* nAChR occupancy from typical smoking conditions). All subjects smoked regular (not light) cigarettes with similar nicotine yields (range, 1.2-1.4 mg).⁴⁴ The 5 smoking levels for this study were no smoking (n = 3); a single puff, which included only lighting a cigarette and inhaling (measured as roughly one twelfth of a cigarette) (n = 2); 3 puffs of a cigarette (measured as approximately one quarter of a cigarette) (n = 3); a full cigarette (n = 3); and satiety (2½ -3 cigarettes) (n = 3). Subjects were then scanned for 3 hours 50 minutes more with 3 scheduled 10- or 15-minute breaks. Scanning ended at 9 PM.

Blood samples (5 mL) for assay of venous plasma nicotine levels were drawn immediately before and at 10 and 185 minutes following the smoke break from a dual port in the intravenous catheter placed for 2-FA infusion. Two subjects who smoked a full cigarette had a full series of venous plasma nicotine levels drawn prior to the smoke break and at 2, 10, 30, 65, and 185 minutes after the smoke break. Samples were centrifuged, and concentrations of venous plasma nicotine were determined in the laboratory of Peyton Jacob III, PhD, at the University of California, San Francisco, by gas chromatography with nitrogen-phosphorus detection,⁴⁵ using 5-methylnicotine as an internal standard. The lower limit of quantitation was 1 ng/mL.

Cigarette craving was monitored with the Urge to Smoke Scale,^46- 47 an analog scale (range, 0-6) with 10 craving-related questions. This scale was filled out at the beginning of the first break in scanning (prior to smoking), at the end of the first break (after smoking), and during each of the 3 remaining breaks in scanning.

The PET scans were obtained on a General Electric Advance NXi scanner (General Electric Medical Systems, Milwaukee, Wis) with 35 slices in 3-dimensional mode, transaxial resolution full width at half maximum 5.2 to 7.7 mm.⁴⁸ Scans were acquired as series of 10-minute frames. Attenuation correction scanning was performed with the germanium rotating rod source built into the General Electric scanner for 5 minutes at the end of the scanning session, and this attenuation correction was applied to all scans from the session. F 18 was prepared with the CP-45 variable proton energy negative ion cyclotron (The Cyclotron Corporation) and 2-FA was prepared by a published method.⁴⁹ Specific activities ranged from 122 to 241 GBq/μmol (range, 3.3-6.5 Ci/μmol).

An MRI of the brain was obtained within a week of PET scanning to aid in localization of brain regions on the PET scans, with the following specifications: 3-dimensional Fourier-transform spoiled gradient-recalled acquisition with repetition time = 30 milliseconds, echo time = 7 milliseconds, 30° angle, 2 acquisitions, and 256 × 192 view matrix. The acquired volume was reconstructed as roughly 90 contiguous 1.5-mm-thick transaxial sections.

Magnetic resonance imaging to PET coregistration was performed using an automated image registration method.⁵⁰ Regions of interest were drawn on MRI and transferred to coregistered PET scans. Regions of interest included the thalamus, brainstem, cerebellum, prefrontal cortex, and corpus callosum. The thalamus, brainstem, and cerebellum were analyzed as whole structures while representative sections of the prefrontal cortex (middle frontal gyrus parallel to the body of cingulate) and genu of the corpus callosum (on sagittal images) were drawn. These brain regions were chosen based on prior reports indicating a range of nAChR densities in these regions from highest (thalamus) to lowest (corpus callosum).^51- 54 Mean ± SD volumes for the thalamus, brainstem, and cerebellum were 4.97 ± 0.22, 18.37 ± 3.41, and 95.71 ± 13.77 cm³, respectively. The expected ratio of specific to nonspecific binding for the thalamus based on a study of nonhuman primates using 2-FA PET was roughly 2:1.²⁹

The total concentration of tissue radioactivity (C_T) without smoking can be expressed as follows: C_T = C_SB + C_F + C_NB, where C_SB is the concentration of specifically bound radioligand, C_F is the concentration of free radioligand, and C_NB is the concentration of nonspecifically bound radioligand. After smoking, C"_T = C"_SB + C_F + C_NB. In subjects who smoked, C"_SB<C_SB because nicotine, its metabolites, or perhaps other components of tobacco smoke displace some of the specifically bound radiotracer.

At equilibrium conditions and assuming that C_F and C_NB are unchanged as a result of smoking, the fractional displacement (FD) of radiotracer can be expressed as the following:

where

is the fraction of the tissue radioactivity (before smoking) that represents specifically bound radioligand and is therefore the maximum fraction of radiotracer that can be displaced.

At a tracer dose of radioligand (C_SB<B_max), where B_max is the receptor concentration, we can derive the following equation from equation 1 (again assuming equilibrium):

where

[Ni] and [Ach] are the tissue concentrations of nicotine and acetylcholine, respectively, and K_[Ni] and K_[ACh] are the equilibrium dissociation constants for nicotine and acetylcholine, respectively.

The tissue nicotine concentration is assumed to be directly proportional to the amount of cigarette smoked and to the venous plasma nicotine concentration. Therefore, equation 3 can be expressed as:

where ED₅₀ and [D] are in units of cigarettes smoked (or fractions thereof) and EC₅₀ (plasma) (simply called EC₅₀ in the rest of this article) and plasma nicotine concentration [Ni] are in units of nanograms per milliliter or nanomole (nM).

Maximum fractional displacement (R) is also related to the commonly used parameter binding potential (BP*), which is defined as the ratio of VD_SB to VD_NDR,⁵⁵ where VD_SB and VD_NDR are the volumes of distribution for the specific binding and nondisplaceable radioactivity compartments, respectively. Consistent with equation 3, R is defined by the equation:

and BP* can be determined from R, using the equation:

For all drawn regions of interest, displacement of radiotracer was found from before to after smoking (even in the lowest nAChR density region, the corpus callosum). This observation limited using the reference region approach for calculations of specific binding. In addition, this evaluation revealed that the highest levels of specific binding were in the thalamus, brainstem, and cerebellum, so that these regions were used for analysis herein, and each of the 3 regions was analyzed independently. For calculation of the dose-dependent effects of smoking on receptor occupancy, we calculated fractional displacement of total radioactivity for each subject and each smoking condition.

C_T and C"_T values were determined by averaging data from the six 10-minute frames (1 hour) prior to the smoking break (180-240 minutes [mean, 3.5 hours] after bolus injection) and from the last seven 10-minute frames of the PET scanning session (395-480 minutes, including a 15-minute break, [mean, 7.3 hours] after bolus injection) (Figure 2 and Figure 3).

The displacement of total radioactivity (C_T minus C"_T) for each cigarette dose or venous plasma nicotine level was expressed as a percentage of C_T and plotted as a function of amount of cigarette smoked (Figure 4A) or venous plasma nicotine concentration. In these plots, the asymptotic portion of the curve represents the maximum fractional displacement of radioactivity. The plotted data were fitted to equations 5 and 6 mentioned earlier, using nonlinear regression with SigmaStat software (Systat Software, Inc, Richmond, Calif) to determine ED₅₀ (percentage of a cigarette needed to displace 50% of the radiotracer), EC₅₀ (venous plasma nicotine concentration that was associated with 50% radiotracer displacement), and R (maximum fractional displacement). Percentage of receptor occupancy was also calculated by dividing the fractional displacement (as determined in equation 1) by R.

We performed these calculations for the raw C_T value data and for data corrected for the imperfect steady state found in the no-smoking (control) scans (see “Results” section, paragraphs 2 and 4). The BP* values were calculated based on the R value for each region using equation 8.

Subjects were adults (mean ± SD age, 36.6 ± 11.8 years; 8 men, 3 women) who smoked 22.2 ± 2.8 (mean ± SD) regular cigarettes per day and had been smoking for a mean ± SD of 17.5 ± 10.5 years. Six subjects were African American, 4 subjects were white, and 1 subject was Asian American. One participant's longest previous quit period was 2 years, while the others had longest quit periods ranging from a few days to 1 year. Mean ± SD total scores for the Fagerström Test for Nicotine Dependence (5.9 ± 2.2) and the Beck Depression Inventory (3.1 ± 2.5) and mean ± SD per item scores for the Spielberger State-Trait Anxiety Index (1.8 ± 0.5) and the Shiffman-Jarvik Withdrawal Scale (4.2 ± 0.7) indicated that subjects were moderately dependent on tobacco and had low levels of depression and anxiety during the study. At the time of screening, subjects had exhaled CO levels consistent with tobacco dependence (mean ± SD, 18.1 ± 5.7 ppm), while at the time of scanning, subjects had low exhaled CO levels (mean ± SD, 1.9 ± 1.0 ppm), consistent with their compliance with the abstinence protocol of the study. For all subjects, venous plasma nicotine levels were lower than the level of detection of the laboratory for this study (<1 ng/mL) prior to the smoking break in scanning and were the highest in the first sample after smoking, with a half-life of 145 minutes.

Time-activity curves for the control (no-smoking) scans (Figure 1 and Figure 2A-C) demonstrate that the distribution of radioligand reached a near equilibrium state 3.5 hours after initiation of 2-FA administration, with tissue concentrations maintained in a near steady state for the remainder of the scanning sessions. The percentage change per hour in the no-smoking group was calculated by determining the difference in average radioactivity for each region of interest between the period at which 2-FA reached an approximate steady state (180-240 minutes after infusion initiation) and the final 70 minutes of scanning (395-480 minutes after infusion initiation, including a 15-minute break) and dividing by the number of hours (3.8) between the midpoint of these 2 periods. This calculation resulted in average increases in radioactivity for the thalamus, brainstem, and cerebellum of 12%, 12%, and 10%, respectively (3.2%, 3.2%, and 2.6% per hour, respectively). For the last 70 minutes of scanning (last 85 minutes of the scanning session, including a 15-minute break), an almost complete steady state (indicating equilibrium between plasma and brain tissue) was reached, with change in radioactivity in the thalamus, brainstem, and cerebellum of only 0.0 ± 1.0% (mean ± SE), 1.0 ± 1.0% (mean ± SE), and 1.0 ± 1.0% (±mean ± SE), respectively (determined by linear regression).

For the scans with cigarette smoking, decreases in total radioactivity for the 3 studied brain regions with the highest radioactivity accumulation (thalamus, brainstem, and cerebellum) were clearly dose dependent (Figure 2 and Figure 3). A single puff of a cigarette was the only amount of smoking that was followed by recovery of receptor availability within the 3-hour 50-minute time frame after smoking (Figure 2A-C), while the medium to high levels of smoking (one quarter of a cigarette to satiety) resulted in new steady-state conditions at 3 hours after smoking (Figure 2 and Figure 3). Smoking to satiety resulted in a profound decrease of radioactivity in all brain regions studied (Figure 2D). The dose-response curves for displacement of total and specifically bound radioactivities are shown in Figure 4A-C.

The mean ± SE effective dose of a cigarette required to occupy 50% of receptors (ED₅₀) for the 3 primary regions of interest at 3.1 hours after smoking was 0.19 ± 0.05 (Table 1). After correcting for the imperfect steady state found in the control group that did not smoke (by multiplying the prebreak mean total radioactivities for the thalamus, brainstem, and cerebellum by 1.12, 1.12, and 1.10, respectively), ED₅₀ was calculated to be 0.13 ± 0.03 (mean ± SE) of a cigarette (Table 1). The mean ± SE venous plasma nicotine concentration associated with 50% occupancy of receptors (EC₅₀) was determined from the dose response curve (Figure 4D) to be 0.87 ± 0.16 ng/mL (5.3 ± 1.0 nM) (Table 1). Apparent binding potential (BP*) values for the thalamus (Table 1) were similar to those in another published report using 2-FA.²⁹

The mean receptor fractional occupancies at 3.1 hours after smoking 1 puff, 3 puffs, 1 full cigarette, and to satiety were 33%, 75%, 88%, and 95%, respectively (Figure 4) (Table 2). Earlier than 3.1 hours after smoking, we would hypothesize that the fractional occupancy of nAChRs would be even higher. For example, assuming a t_1/2 (half-life) of venous plasma nicotine of 2.5 hours,²⁷ we would expect that smoking 1 cigarette would result in venous plasma nicotine levels at 1 and 2.5 hours after smoking to be roughly 1.8 and 1.2 times higher than the levels at 3.1 hours. These higher nicotine levels would lead to estimated receptor occupancies at 1 and 2.5 hours of 93% and 90%, respectively.

Craving was only alleviated with higher smoking levels (1 full cigarette or satiety). For the 5 smoking levels (none, 1 puff, 3 puffs, 1 cigarette, and satiety), changes in mean ± SD Urge to Smoke Scale scores (0- to 6-point scale) from before to after smoking were −0.8 ± 0.7, 0.2 ± 0.2, 0.1 ± 0.4, −4.6 ± 1.2, and −4.8 ± 1.1, respectively. Craving relief was temporary and elevated craving levels returned later during the scanning session. For example, a mean ± SD change of −2.3 ± 1.6 in Urge to Smoke Scale score was found from before to 2.5 hours after smoking for the group that smoked 1 full cigarette.

The central findings of this study indicate that typical daily smokers have nearly complete saturation of brain α₄β₂* nAChRs throughout the day. The ED₅₀ (0.13 cigarette) is very small compared with smoking levels (1 to 2 cigarettes per hour) found in tobacco-dependent smokers. The EC₅₀ of venous plasma nicotine (0.87 ng/mL or 5.4 nM) found herein is very low compared with venous plasma nicotine levels (which range from 10-37, 10-50, and 19-50 ng/mL for trough, afternoon, and peak levels, respectively)^27,56 found in daily smokers. This EC₅₀ estimate indicates that 96% to 98% of α₄β₂* nAChRs are occupied during the day in tobacco-dependent smokers. Additionally, the EC₅₀ found herein is in the range of concentrations causing desensitization of 50% of α₄β₂* nAChRs. As demonstrated recently,⁵⁷ 100 seconds of coincubation of cells expressing α₄β₂* nAChRs with 10 nM of nicotine (1.6 ng/mL) resulted in 70% receptor desensitization. Thus, results of this study in the context of other α₄β₂* nAChR research indicate a nearly complete shift from other potential nAChR states (such as resting and intermediate desensitized)⁵⁸ to the high-affinity desensitization state during the day in tobacco-dependent smokers. Such prolonged desensitization may be responsible for the up-regulation of these receptors found in smokers.^59- 60

In our study, craving was only reduced with near total occupancy of nAChRs, with up to one quarter of a cigarette doing little to alleviate craving, despite up to 75% receptor occupancy, whereas smoking 1 full cigarette or to satiety resulted in significant craving reductions and at least 88% and 95% receptor occupancy, respectively. Similarly, though roughly 50% of the presmoking craving level returned in the 1 full cigarette group 2.5 hours after smoking, the calculated receptor occupancy by nicotine for that time was roughly 90% (as described earlier).

Continued smoking (in tobacco-dependent smokers) despite near total α₄β₂* nAChR occupancy/desensitization may be explained in several ways. First, smokers may continue to smoke to avoid having free unbound receptors. In this situation, free (nondesensitized) receptors would be responsible for cigarette craving, and nicotine binding and desensitization of these nAChRs may alleviate craving. This action of nicotine may provide positive reinforcement and craving alleviation through frequency-sensitive dopamine release^61- 62 and/or decreased γ-aminobutyric acid tone.¹⁹ Second, it is possible that positive reinforcement from smoking is due to activation of other α₄β₂* nAChR subtypes that are not desensitized by high concentrations of nicotine and that are not labeled by 2-FA because of a smaller number of receptors or low affinity. Therefore, we cannot exclude the possibility that activation of other α₄β₂* nAChRs could be involved in tobacco dependence, and it may be important to identify such receptors in future research. And third, factors other than nicotine binding to nAChRs may be at least partly responsible for craving alleviation and positive reinforcement (which is supported by studies demonstrating that denicotinized cigarettes partially alleviate craving⁶³). In any case, because α₄β₂* nAChRs are the predominant receptor subtype in humans and 2-FA labels almost the entire population of these receptors,⁶⁴ the conclusion from our study still indicates that near complete desensitization of nAChRs has an important role in the pathophysiology of tobacco dependence.

This study should be interpreted in the context of several limitations. First, cigarette smokers vary in their rate and depth of inhalation of cigarettes, and it is recognized that these interindividual differences could have affected the ED₅₀ calculation (though, as noted earlier, all subjects smoked cigarettes with similar nicotine contents and the total amount smoked was measured). Second, test-retest data on the bolus-plus-infusion method used have not yet been published, although there was relatively little variability within study subgroups (based on smoking dosage) compared with the robust effects of cigarette smoking and the ED₅₀ and EC₅₀ determined herein remained relatively stable from region to region. Third, repeated breaks in scanning (which were necessary for subject comfort) may have led to slight shifts in overall radioactivity determinations due to small differences in position from before to after each scanning break, though (as noted earlier) a head holder and laser light positioning were used to attempt to accurately reposition subjects. And fourth, venous rather than arterial nicotine levels were used for study calculations, and arterial levels are reported to be higher than venous at the time of smoking.⁶⁵ However, study calculations were performed on venous nicotine levels 3.1 hours after smoking, a time at which venous and arterial levels would be expected to be similar,⁶⁵ so that our results should provide a reasonable approximation of EC₅₀ for arterial, as well as venous, nicotine levels.

Finally, the association between cigarette smoking and high receptor occupancy found herein has implications for the study of indirect cigarette/nicotine exposures. Studies of fetal exposure to maternal smoking,⁶⁶ neonate exposure to breast milk of mothers who smoke,⁶⁷ and prolonged environmental tobacco smoke exposure^68- 69 all show evidence of venous plasma nicotine levels higher than 1 ng/mL in those who are indirectly exposed, with fetal levels being even higher than maternal levels. Our study indicates that even low levels of exposure result in substantial occupancy of brain α₄β₂* nAChRs, suggesting significant occupancy and desensitization of α₄β₂* nAChRs in subjects indirectly exposed to nicotine and pointing to the need for further research to address this issue.

Correspondence: Arthur L. Brody, MD, 300 UCLA Medical Plaza, Suite 2200, Los Angeles, CA 90095 (abrody@ucla.edu).

Submitted for Publication: July 18, 2005; final revision received November 6, 2005; accepted November 8, 2005.

Funding/Support: This study was supported by National Institute on Drug Abuse (NIDA) grants RO1 DA15059 and DA20872 (Dr Brody) and RO1 DA14093 (Dr London), a Veterans Affairs Type I Merit Review Award (Dr Brody), Tobacco-Related Disease Research Program grants 11RT-0024 (Dr Brody) and 10RT-0091 (Dr London), Office of National Drug Control Policy contract DABT63-00-C-1003 (Dr London), and the NIDA intramural research program (Drs Chefer and Mukhin).

Acknowledgment: We thank the laboratory of Peyton Jacob III, PhD, for determining venous plasma nicotine levels and Josephine Ribe, BS, and Michael Clark, BS, for performing positron emission tomography and magnetic resonance imaging, respectively.