Platelet Adenylyl Cyclase Activity: A Biological Marker for Major Depression and Recent Drug Use

Platelet Adenylyl Cyclase Activity: A Biological Marker for Major Depression and Recent Drug Use Lisa M. Hines and Boris Tabakoff, on behalf of the WHO/ISBRA Study on State and Trait Markers of Alcohol Use and Dependence Investigators Background: Adenylyl cyclase (AC) is an enzyme that can regulate the physiologic effects of numerous drugs and hormones through the production of cyclic adenosine-3=,5=-monophosphate (cAMP). Some studies suggest that certain measures of AC activity are lower among depressed subjects. We examined the relationship between various measures of AC activity and major depression, taking into account potential confounders, such as drug use and gender. Methods: We assessed the relationship between platelet levels of AC activity and lifetime diagnosis of major depression among 1481 participants (226 subjects with a history of major depression and 1255 control subjects) in an international, cross-sectional study initiated by the World Health Organization and the International Society on Biomedical Research on Alcoholism. Results: After accounting for recent drug use, subjects with a history of major depression had markedly lower mean levels for all measures of platelet AC activity compared with control subjects. The adjusted odds ratios for major depression comparing the bottom to the top quartile of AC activity were 2.69 for basal (95% confidence interval [CI] 1.30 –5.56), 3.72 for cesium fluoride-stimulated (95% CI 1.54 – 8.98), 6.20 for forskolin-stimulated (95% CI 2.04 –18.80), and 2.20 for Gpp(NH)p-stimulated (95% CI 1.03– 4.70). Conclusions: Subjects with major depression have lower platelet AC activity levels, and this relationship is dramatically attenuated by various types of drug use. Key Words: Adenylyl cyclase, depression, biomarker, platelets, drug use, epidemiology

I

t has been estimated that up to 20% of the United States population suffers from mild depression, with 2%–5% suffering from severe forms of the illness (Nestler et al 2002). Furthermore, depression is the cause of substantial medical, personal, social, and economic burden, particularly among the medically ill and the elderly (McDaniel 2000). The relatively slow progress in elucidating the intricate neurobiology of depression has been attributed to both the complex and heterogeneous nature of this disorder. The DSM-IV (American Psychiatric Association 1994) criteria for diagnosing depression are based on a wide range of symptoms that differ according to the individual, reflecting the heterogeneous nature of this disorder. Subtypes of depression classified by symptoms have been proposed; however, there is currently no biological evidence to support that these subtypes are etiologically different (Akiskal 2000; Blazer 2000; Nestler et al 2002). The identification of state or trait markers for major depression would facilitate more effective prevention and treatment methods, as well as provide valuable clues to the underlying etiologies. There is some evidence to suggest that adenylyl cyclase (AC) activity might serve as a biological marker for major depression (Cowburn et al 1994; Hoffman et al 2002; Menninger and Tabakoff 1997; Menninger et al 2000). Adenylyl cyclase is an enzyme that converts adenosine 5=-triphosphate (ATP) to the ubiquitous intracellular second messenger, cyclic adenosine3=,5=-monophosphate (cAMP). Other major components involved in cAMP production are various extracellular signal receptors and heterotrimeric guanine nucleotide-binding regula-

From the Department of Pharmacology (LH, BT), University of Colorado Health Sciences Center, Denver, Colorado. Address reprint requests to Lisa M. Hines, Sc.D., University of Colorado Health Sciences Center at Fitzsimons, School of Medicine, Department of Pharmacology, Mail Stop F-8303, P.O. Box 6511, Aurora, CO 800450511; E-mail: [email protected]. Received January 14, 2005; revised March 29, 2005; accepted May 27, 2005.

0006-3223/05/$30.00 doi:10.1016/j.biopsych.2005.05.040

tory proteins (G proteins) that couple the signals generated at receptors to the catalysis of cAMP formation. The production of cAMP stimulates the activities of cAMP-dependent protein kinases (e.g., protein kinase A), which then modulate metabolism and gene expression in various cells and tissues (Montminy 1997; Sunahara et al 1996; Tang and Hurley 1998). Adenylyl cyclase activity is regulated by different receptors, including adrenergic and serotonergic receptors. Many drugs, hormones, and neurotransmitters produce their physiologic effects by stimulating or inhibiting the catalytic activity of AC and thus affecting the concentration of cAMP within the cell (Rang et al 1995). It has been proposed that the beneficial effect of antidepressant medications could be attributed to changes in the postreceptor components involved in cAMP production, such as alterations in the coupling between G proteins and the catalytic unit of AC (Donati and Rasenick 2003). Thus, AC activity level might represent a good candidate for a state and/or trait marker of major depression. Clinical and epidemiologic research has also provided suggestive evidence regarding the association between AC activity level and major depression, although it has been somewhat inconsistent. Initial human studies with postmortem brain demonstrated reduced basal, forskolin-, and guanosine 5=-O-(3thiotriphosphate)-stimulated AC activity in suicide victims, particularly those having a history of depression, compared with control subjects (Cowburn et al 1994). Because of the inaccessibility of human brain, researchers have used human blood cells (e.g., platelets) as a model for brain biochemistry (Best et al 1977; Collins and Sandler 1971; Lingjaerde 1990; Stahl 1985; Wadman et al 1991). Adenylyl cyclase activity can be measured in human platelets obtained from venipuncture. With the use of reagents that induce changes in the properties of the catalytic unit of AC (e.g., forskolin) and/or by changes in G proteins [e.g., cesium fluoride (CsF) and Gpp(NH)p], modulation of AC activity can also be assessed. Forskolin can stimulate AC activity in the absence of G proteins by binding directly to the catalytic unit (Seamon and Daly 1981; Seamon et al 1981). A few studies have found significantly lower levels of forskolin-stimulated AC activity in platelets among subjects with major depression compared with those without (Hoffman et al 2002; Menninger and Tabakoff BIOL PSYCHIATRY 2005;58:955–962 © 2005 Society of Biological Psychiatry

956 BIOL PSYCHIATRY 2005;58:955–962 1997; Menninger et al 2000); however, one small study did not find any difference in forskolin-stimulated AC activity among those afflicted with posttraumatic stress disorder (Weizmann et al 1994). The data have not been consistent for other measurements for AC activity. In a secondary analysis, Hoffman et al (2002) observed minor differences for unadjusted forskolin- and CsFstimulated AC activities, but not basal or Gpp(NH)p-stimulated AC activities, when comparing subjects with a history of major depression with those without. The inconsistencies observed for the different measures of AC activity among the previous studies could be attributed to confounding factors that were not accounted for. It is known that many drugs and hormones influence AC activity, as well as the downstream effects of AC activity. Given that AC activity is stimulated or inhibited by a number of drugs and that depressed subjects are more likely to use either prescribed medications and/or other drugs (Agosti et al 2002; Nunes and Levin 2004), various types of medications could have a dramatic impact on the relationship between history of major depression and AC activity level. The goal of this study was to evaluate the relationship between platelet AC activity and major depression, taking into account potential confounding variables, such as various types of drug use and gender.

Methods and Materials Participants In 1988, the World Health Organization (WHO) and the International Society on Biomedical Research on Alcoholism (ISBRA) initiated a multi-center international study with a goal of identifying state or trait markers of alcohol and drug abuse. The participating clinical centers included the University Central Hospital of Helsinki, Helsinki, Finland; the Royal Prince Alfred Hospital, University of Sydney, Sydney, Australia; the Escola Paulista de Medicina, University of São Paulo, São Paulo, Brazil; the School of Health Sciences, Sapporo Medical University, Sapporo, Japan; and the Douglas Hospital Research Center, Montreal, Quebec, Canada. In this cross-sectional study, each of the centers was responsible for recruiting subjects through advertisements and personal contacts. The original goal was to collect men and women across three age groups: 18 –29, 30 – 44, and 45– 60 years. These age groups were selected to evaluate differences between earlyonset and late-onset problem drinking and alcoholism, as well as the utility of various biological markers in relationship to age. Within each age group, efforts were made to achieve representation in four categories of alcohol consumption: nondrinkers, light drinkers (⬍140 g/week for women and ⬍210 g/week for men), heavy drinkers (ⱖ140 g/week for women and ⱖ210 g/week for men), and alcoholics (according to DSM-IV and ICD-10 criteria) being admitted for the first time to an in-patient treatment program. Recruitment occurred in locales already established by the clinical centers: for instance, the Helsinki center recruited nondrinkers and drinking subjects from occupational health care units, whereas alcoholics were recruited from detoxification units. A preliminary study of approximately 20 subjects recruited by each center (101 subjects total) was conducted to ascertain the logistical soundness of the study (Tabakoff 1996). For the actual study, the recruitment period occurred over an approximately 5-year interval, for which the target number was 600 subjects per study site. By the end of the study period, there were a total of 1863 participants who completed the WHO/ISBRA Interview Schedule and provided both blood and urine samples at one of www.sobp.org/journal

L.M. Hines and B. Tabakoff the participating centers. Because the policy of the centers in Helsinki and Sydney had evolved to focus on treatment of dependent subjects (who were predominantly male), these centers recruited only male participants in the other specified categories of alcohol consumption by the end of the study. The descriptive characteristics of the study populations and details of the WHO/ISBRA Collaborative Project have been previously described in detail (Glanz et al 2002; Menninger et al 2000; Tabakoff 1996). The WHO/ISBRA Interview Schedule is a comprehensive questionnaire regarding alcohol and drug use, psychiatric disorders associated with substance abuse, and family history of alcohol abuse and mental disorders (Glanz et al 2002). The instrument was translated into different languages with the assistance of the staff at the National Institutes of Health and the staff of the participating clinical centers. Several meetings and training sessions were held for personnel of all centers to familiarize them with the use of the interview instrument and collection of biological samples. Developed in concert with the Alcohol Use Disorders and Associated Disabilities Interview Schedule by the National Institute on Alcohol Abuse and Alcoholism (NIAAA), all diagnoses of alcohol abuse or psychiatric disorders were obtained from the WHO/ISBRA questionnaire by use of computerized algorithms based on DSM-IV criteria (Grant et al 1995). Subjects provided written consent, and all centers participating in this study received institutional review board (or similar body) approval for the protocol in accordance with the National Institutes of Health Guidelines for Protection from Risk of Human Subjects. All centers reviewed the completed interview instruments and then forwarded them to the data repository at the Division of Epidemiology at the NIAAA (Bethesda, Maryland). Before finalizing data entry, all information was checked for consistency and accuracy by consultation between clinical center staff and the data repository, as necessary. Measurements for platelet AC activity were successfully conducted on 79% of the WHO/ISBRA study population (1481 subjects). All analyses in the present study were conducted with de-identified data collected from these subjects. AC Activity Assays At the time of interview, blood samples were collected by standard venipuncture technique into vacutainers containing ethylenediaminetetraacetic acid (EDTA) for preparation of plasma and platelets. Platelet AC activity measurements were conducted by researchers who were blinded to case– control status. Samples were assayed in batches, which included standards and both subjects with a history of major depression (cases) and control subjects. Within 2 hours of collection, blood samples were centrifuged at 700 g for 10 min at room temperature. The platelet-rich plasma layer was transferred to a fresh centrifuge tube, and this procedure was repeated. The upper platelet-rich layer was transferred again to a fresh tube for 15-min centrifugation (2800 g) at room temperature. The platelet pellet was recovered and stored at ⫺70°C until being shipped on dry ice to the coordinating center in Helsinki, Finland and then to Denver, Colorado for analysis. The platelet pellet was thawed and washed twice at 4°C by suspension in 1.5 mL of 50 mmol/L Tris-Hcl (pH 7.5) containing 20 mmol/L EDTA, followed by centrifugation at 17,000 g for 10 min. The final pellet was resuspended in 1.5 mL of 5 mmol/L Tris-HCl (pH 7.5) containing 5 mmol/L EDTA, with a hand-held Teflon homogenizer. The homogenate was diluted with 5 mmol/L

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L.M. Hines and B. Tabakoff Tris-HCl (pH 7.5) containing 1 mmol/L EDTA to attain protein concentration within 200 –1000 ␮g/uL and immediately assayed for platelet AC activity. Protein determinations were performed with the Pierce Bicinchoninic Acid protein microtiter method (Pierce Biotechnology, Rockford, Illinois). Platelet AC activity measurements were conducted by researchers who were blinded to case– control status. Samples were assayed in batches, which included both cases and control subjects. Fifty microliters of the prepared platelet membrane protein (approximately 10 –50 ␮g) was added to 200 ␮L of assay buffer (25 mmol/L Tris-maleate at pH 7.5, 10 mmol/L theophylline, 4 mmol/L MgCl2, .25 mmol/L ATP, and [␣-32P] ATP at 1.2–2.0 ⫻ 106 cpm/assay). Adenylyl cyclase activity was measured in duplicate under these conditions (i.e., basal activity) and with addition of either 10 ␮mol/L Gpp(NH)p, 10 mmol/L CsF, or 10 ␮mol/L forskolin. After equilibrating the assay mixture at 30°C for 5 min, the reaction was initiated by adding the platelet membranes. The reaction mixture was incubated at 30°C for 10 min, and the reaction was terminated by the addition of 750 ␮L of an ice-cold solution containing 4 mmol/L ATP, 1.4 mmol/L cAMP, and 10,000 cpm [3H]cAMP (25– 40 Ci/mmol). Adenylyl cyclase– generated [3H]cAMP and [32P]cAMP were isolated by sequential chromatography on Dowex and alumina columns, as described by Salomon et al (1974) and quantitated by liquid scintillation counting. All reported values for AC activity, corrected for the recovery of [3H]cAMP, are expressed as picomoles of cAMP generated per milligram of protein per minute. An aliquot of human erythroleukema (HEL) cell membranes, with known and stable levels of AC activity, was assayed with each group of samples and used as a reference standard. To correct for interassay variability across the different groups of samples, all values of AC activity obtained with HEL membranes within each day’s assay were divided by the HEL cell membrane activity averaged over the entire project period. The resulting factor was used to standardize all AC activity values obtained on a particular day. Without HEL standardization, we previously reported interassay coefficients of variation as 23.4% for basal, 9.2% for CsF-stimulated, and 13.5% for forskolin-stimulated AC activity. With HEL standardization, interassay coefficients of variation were 15.4%, 10.8%, and 11.9%, respectively (Menninger et al 2000).

(yes/no). These variables were selected on the basis of previous work with this study population (Hoffman et al 2002). To investigate the influence of drug use on the relationship between AC activity and major depression, we systematically removed subjects who reported recent use (in the 30 days before blood draw) of addictive drugs, analgesics, and/or antidepressants. Addictive drugs included any of the following: sedatives, tranquilizers, painkillers, stimulants, marijuana, cocaine, heroin, methamphetamines, inhalants, hallucinogens, or steroids. Recent analgesic/antipyretic or antidepressant use was also based on the 30 days before the interview. All data collected from study subjects was self-reported. Linear regression models were stratified by gender to examine the gender-specific relationships between major depression and AC activity levels. To estimate the independent effect of gender on AC activity levels, we restricted multivariate models to nondepressed subjects who were not recently taking any of the previously described drugs. To evaluate the effect of endogenous estrogen levels on AC activity, AC activity levels were regressed on the previously described factors among the female control subjects who were not recently taking any of the previously described drugs. Because we did not have information on menopausal status at the time of blood draw, we used age (⬍40 years vs. ⬎50 years) as a surrogate for menopausal status. Among all subjects who did not report recent use of addictive drugs, analgesics, or antidepressants, we used logistic regression models to estimate the odds ratios and 95% confidence intervals (CIs) for levels of biomarkers predicting lifetime diagnosis of major depression. The distributions of AC activity levels were divided into quartiles according to the distribution in control subjects who were not currently taking any of the previously described drugs. We estimated the risk of depression in each quartile relative to the highest quartile and the significance of trend in relative risk across quartiles. Both crude odds ratios and odds ratios adjusting for the previously described covariates were computed. Multivariate logistic regression models adjusting for the previously described factors were also used to evaluate the relationship between the ratios of stimulated to basal AC activity levels and major depression. Ratios were computed by dividing stimulated AC activity levels [CsF, forskolin, and Gpp(NH)p] by basal AC activity levels.

Statistical Analysis All statistical analyses were performed with a statistical software package (SAS 8.02; SAS Institute, Cary, North Carolina). Among the 1481 subjects included in this study, measurements were missing for basal (n ⫽ 3), Gpp(NH)p-stimulated (n ⫽ 40), and forskolin-stimulated (n ⫽ 35) AC activity levels. Outliers (⬎3 interquartile ranges) and normality of AC activity measurements were assessed. Because of the small number of outlying values, no exclusions were made. The outcome variables (basal, CsFstimulated, forskolin-stimulated, and Gpp(NH)p-stimulated AC activity levels) resembled a normal distribution. To minimize any effect of influential outlying values, median values were used when computing odds ratios. Chi-square tests and t tests were used to compute p values when comparing the characteristics of cases and control subjects. Univariate and multivariate linear regression models were used to examine the relationship between history of major depression and platelet AC activity levels. All multivariate models were adjusted for recruitment site (Helsinki, Montreal, São Paolo, Sapporo, Sydney), gender, age (years), family history of alcohol dependence or abuse (yes/no), and lifetime alcohol dependence

Results As illustrated in Table 1, characteristics often associated with depression were more prevalent among subjects with a lifetime diagnosis of major depression compared with control subjects. This was also observed for various types of recent drug use, specifically antidepressants and known addictive drugs (i.e., sedatives, tranquilizers, painkillers, stimulants, marijuana, cocaine, heroin, methamphetamines, inhalants, hallucinogens, or steroids). The unadjusted mean levels were significantly different between the two groups for forskolin- and CsF-stimulated AC activities. Although the correlations between the different measures of AC activity are relatively strong, ranging from .46 to .75, the particular factors associated with platelet AC activity level were not identical for the different measurements of AC activity (Hoffman et al 2002). The variables included in these analyses represent the strongest and most consistent predictors, or potential confounders, of the relationship between AC activity and major depression. Among this study population of 1481 subjects, we also observed significantly lower unadjusted mean levels of forskolinwww.sobp.org/journal

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Table 1. Descriptive Characteristics of the Study Population

Characteristics

Lifetime Diagnosis of Major Depression (DSM-IV) (n ⫽ 226)

No Lifetime Diagnosis of Major Depression (DSM-IV) (n ⫽ 1255)

128 (56.6) 98 (43.4) 37.4 (11.2)

901 (71.8) 354 (28.2) 37.2 (12.2)

8 (3.5) 137 (60.6) 59 (26.1) 0 (0) 22 (9.7) 78 (34.5) 72 (31.9) 37 (16.4) 167 (73.9) 162 (71.7)

226 (18.0) 386 (30.8) 346 (27.6) 70 (5.6) 227 (18.1) 195 (15.5) 366 (29.2) 21 (1.7) 507 (40.4) 671 (53.5)

Gender, n (%) Male Female Age, y (SD) Collaborating Center, n (%) Helsinki, Finland Montreal, Canada São Paolo, Brazil Sapporo, Japan Sydney, Australia Addictive Drug Use in Past 30 Days, n (%) Analgesic Use in Past 30 Days, n (%) Antidepressant Use in Past 30 Days, n (%) Lifetime Alcohol Dependence, n (%) Family History of Alcohol Dependence or Abuse, n (%) Platelet Adenylyl Cyclase Activities, pmol/mg/minb Basal Gpp(NH)p-stimulated Cesium fluoride-stimulated Forskolin-stimulated

Pa ⬍.001

15.9 (6.6) 79.9 (28.0) 115.5 (29.5) 310.8 (83.2)

15.9 (6.8) 77.5 (29.9) 121.8 (31.2) 331.5 (102.6)

.81 ⬍.001

⬍.001 .41 ⬍.001 ⬍.001 ⬍.001 .99 .26 .005 .001

All variables are expressed as means (SD), unless otherwise indicated. Addictive drugs include sedatives, tranquilizers, painkillers, stimulants, marijuana, cocaine, heroin, methamphetamine, inhalants, hallucinogens, and steroids. a 2 ␹ or t test. b For basal adenylyl cyclase activity levels, n ⫽ 1252 control subjects; for Gpp(NH)p, n ⫽ 218 cases and 1223 control subjects; for forskolin, n ⫽ 219 cases and 1227 control subjects.

and CsF-stimulated AC activity when comparing subjects with versus without a history of major depression. Without taking into account recent drug use, this association was only statistically significant for CsF-stimulated activity after adjusting for age, recruitment site, gender, family history of alcohol dependence or abuse, and lifetime history of alcohol dependence. Given that a number of drugs can either stimulate or inhibit AC activity and that depressed subjects are more likely to be using either prescribed medications or other drugs, we used the WHO/ISBRA data set to assess the potential impact of drug use on the relationship between major depression and AC activity. Because of the complexity of simultaneously adjusting for the numerous types of drugs, various dosages, and the concomitant effects of multiple drug use, we examined the effect of recent drug use by excluding recent licit and/or illicit drug users. Interestingly, the relationship between major depression and AC activity level was markedly confounded by addictive drug use (i.e., sedatives, tranquilizers, painkillers, stimulants, marijuana, cocaine, heroin, methamphetamines, inhalants, hallucinogens, steroids), analgesics/antipyretic use, and antidepressant use within the 30 days before blood draw (Table 2). As reflected from the clear decreasing trend in both the unadjusted and adjusted AC activity levels, the strength of the relationship grew increasingly stronger for all AC measurements with the subsequent exclusion of each type of drug user. When adjusting for recruitment site, gender, age, family history of alcohol dependence or abuse, and lifetime alcohol dependence, mean levels of platelet AC activity (pmol cAMP/mg protein/min) were lower in cases compared with control subjects for all measures of AC activity (difference in adjusted means of cases relative to control subjects: basal, ⫺2.08 [SE .83]; CsF-stimulated, ⫺12.27 [SE 3.58]; forskolinstimulated, ⫺36.80 [SE 11.70]; Gpp(NH)p-stimulated, ⫺6.26 [SE www.sobp.org/journal

3.54]). Although the exclusion of all recent drug users resulted in a decrease in sample size of approximately 60% for depressed subjects and 40% for control subjects, the sample size was more than adequate to achieve highly significant associations with major depression for basal (p ⫽ .01), CsF-stimulated (p ⫽ ⬍0.001), and forskolin-stimulated (p ⫽ .002) AC measurements and borderline significance for Gpp(NH)p-stimulated AC measurements (p ⫽ .08). There is evidence to suggest that the relationship between major depression and AC activity levels might be gender specific (Menninger and Tabakoff 1997). Among nondepressed subjects who were not recently using any of the previously described drugs, the adjusted mean levels for all measures of stimulated AC activity were lower for women compared with men (Table 3). The observed relationship between major depression and AC activity level was apparent for both men and women, as was the confounding effect of recent drug use. The relationship between major depression and forskolin-stimulated AC activity levels seemed to be stronger in men than in women (difference in adjusted means of cases relative to control subjects ⫽ ⫺43.63 [SE 15.80] for men and ⫺26.24 [SE 16.09] for women); however, the removal of all recent drug users from the gender-stratified analyses compromised the power to accurately estimate the relationship among female subjects. We also attempted to evaluate the potential influence of endogenous estrogen levels on AC activity by regressing AC activity levels on the previously described factors among the female control subjects only. Because we did not have information on menopausal status at the time of blood draw, we used age (⬍40 years vs. ⬎50 years) as a surrogate for menopausal status. In addition to excluding all recent drug users, we excluded all reported hormone users. We did not observe any

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Table 2. Difference in Unadjusted and Adjusted Mean Platelet Adenylyl Cyclase Activity Levels in Depressed Versus Nondepressed Subjects

Adenylyl Cyclase Activity Basal n (depressed/nondepressed) Diff. unadjusted mean (SE) Diff. adjusted mean (SE) Cesium Fluoride n (depressed/nondepressed) Diff. unadjusted mean (SE) Diff. adjusted mean (SE) Forskolin n (depressed/nondepressed) Diff. unadjusted mean (SE) Diff. adjusted mean (SE) Gpp(NH)p n (depressed/nondepressed) Diff. unadjusted mean (SE) Diff. adjusted mean (SE)

Excluding Recent Users of Addictive Drugs, Analgesics, or Antidepressants

p

Excluding Recent Users of Addictive Drugs

p

Excluding Recent Users of Addictive Drugs or Analgesics

226/1252 ⫺.007 (.49) .06 (.51)

.99 .90

148/1059 ⫺1.18 (.60) ⫺.90 (.62)

.05 .15

106/757 ⫺2.36 (.74) ⫺2.02 (.76)

226/1255 ⫺6.34 (2.24) ⫺4.81 (2.31)

.005 .04

148/1060 ⫺8.66 (2.74) ⫺6.51 (2.80)

.002 .02

107/757 ⫺13.46 (3.30) ⫺9.98 (3.35)

⬍.001 .003

89/743 ⫺16.25 (3.54) ⫺12.27 (3.58)

⬍.001 ⬍.001

219/1227 ⫺20.74 (7.33) ⫺8.52 (7.23)

.005 .24

142/1035 ⫺33.10 (9.05) ⫺17.43 (8.82)

⬍.001 .05

102/737 ⫺49.92 (10.99) ⫺30.63 (10.74)

⬍.001 .005

84/724 ⫺56.24 (12.07) ⫺36.80 (11.70)

⬍.001 .002

.26 .84

142/1032 ⫺.63 (2.64) ⫺2.90 (2.65)

.81 .27

101/734 ⫺4.39 (3.25) ⫺5.09 (3.26)

.18 .12

83/721 ⫺6.26 (3.54) ⫺6.26 (3.54)

.08 .08

All Subjects

218/1223 2.46 (2.18) ⫺.44 (2.21)

p

.001 .008

88/743 ⫺2.49 (.81) ⫺2.08 (.83)

p

.002 .01

Difference (Diff.) represents the unadjusted or adjusted mean platelet adenylyl cyclase activity levels (picomoles cyclic adenosine monophosphate per milligram protein per min) for subjects with a lifetime diagnosis of major depression minus those without. All differences are expressed as means (SE). Multivariate models were adjusted for recruitment site, gender, age, family history of alcohol dependence or abuse, and lifetime alcohol dependence. Recent users are defined as individuals who reported any use of any addictive drugs, analgesics, or antidepressants during the 30 days before blood draw. Addictive drugs represent sedatives, tranquilizers, painkillers, stimulants, marijuana, cocaine, heroin, methamphetamine, inhalants, hallucinogens, and steroids.

significant differences for any of the AC activity levels when comparing nondepressed older women with nondepressed younger women; however, the sample size for this analysis was relatively small (n ⫽ 115–118). When estimating the odds ratio for major depression according to quartile of AC activity among all non– drug users, subjects in the lowest quartile for all measurements of AC activity had a significantly increased risk of major depression (Table 4). After adjusting for the previously described covariates, the odds ratios for major depression, comparing the bottom and top quartiles of AC activity, were 2.69 for basal (95% CI 1.30 –5.56), 3.72 for CsF-stimulated (95% CI 1.54 – 8.98), 6.20 for forskolin-stimulated (95% CI 2.04 –18.80), and 2.20 for Gpp(NH)p-stimulated (95% CI 1.03– 4.70). There were highly significant trends in relative risk across quartiles for all AC activity measurements (p ⬍ .008) except Gpp(NH)p-stimulated (p ⫽ .10). When simultaneously adjusting for all AC measurements, the correlation between AC Table 3. Effect of Gender on Adjusted Mean Platelet Adenylyl Cyclase Levels Among Nondepressed Subjects Who Are Not Recent Drug Users Adenylyl Cyclase Activity

Difference (SE)

p

Basal Cesium Fluoride-Stimulated Forskolin-Stimulated Gpp(NH)p-Stimulated

⫺.51 (.70) ⫺5.78 (3.10) ⫺21.41 (9.93) ⫺7.90 (2.98)

.46 .06 .03 .008

Difference represents the adjusted mean platelet adenylyl cyclase activity levels (picomoles cyclic adenosine monophosphate per milligram protein per min) for women minus men. All differences are expressed as means (SE). Multivariate models were adjusted for recruitment site, age, family history of alcohol dependence or abuse, and lifetime alcohol dependence. Recent users are defined as individuals who reported any use of any addictive drugs, analgesics, or antidepressants during the 30 days before blood draw. Addictive drugs represent sedatives, tranquilizers, painkillers, stimulants, marijuana, cocaine, heroin, methamphetamine, inhalants, hallucinogens, and steroids.

activity measurements (R2 ⫽ .46 –.75) attenuated the effect of each AC activity measurement when considered alone. A trend in relative risk was still observed across quartiles for basal, forskolin-, and CsF-stimulated AC activity measurements; however, the sample size was not sufficient to accurately estimate the independent effects of the various measurements of AC activity. Overall, the data suggested that forskolin- and CsF-stimulated AC activity levels were the strongest predictors of major depression. Given that basal and stimulated AC levels are correlated, it is possible that subjects with below- or above-average differentials between basal and stimulated AC activity levels might possess inherent differences that affect AC function, such as genetic variation in the AC protein. Thus, we considered using the ratio of stimulated to basal AC activity levels as a potential state or trait marker of major depression. On the basis of the adjusted relatives risks for depression as predicted by quartiles for the ratio of stimulated to basal AC activity levels, there was no clear evidence that subjects with a below- or above-average differential were at higher risk of major depression. Furthermore, the ratio of stimulated to basal AC activity levels was not a better predictor of major depression than the absolute values of platelet AC activity.

Discussion In this study, all measures of platelet AC activity (basal, CsF-, Gpp(NH)p-, or forskolin-stimulated) were associated with an increased risk for a lifetime diagnosis of major depression. Importantly, this relationship is markedly attenuated by recent drug use. When taking into account the various types of drug use, subjects in the lowest quartile of either basal, CsF-, Gpp(NH)p-, or forskolin-stimulated AC activity had a two- to sixfold increased risk of major depression compared with subjects in the highest quartile. www.sobp.org/journal

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Table 4. Odds Ratio of Lifetime Diagnosis of Major Depression According to Platelet Adenylyl Cyclase Activity Levels, Excluding All Recent Users of Addictive Drugs, Analgesics, or Antidepressants

Basal Median (IQR) pmol cAMP/mg protein/min n Unadjusted OR (95% CI) Adjusted OR (95% CI) Cesium Fluoride Median (IQR) pmol cAMP/mg protein/min n Unadjusted OR (95% CI) Adjusted OR (95% CI) Forskolin Median (IQR) pmol cAMP/mg protein/min n Unadjusted OR (95% CI) Adjusted OR (95% CI) Gpp(NH)p Median (IQR) pmol cAMP/mg protein/min n Unadjusted OR (95% CI) Adjusted OR (95% CI)

AC Activity Quartile 1

AC Activity Quartile 2

AC Activity Quartile 3

AC Activity Quartile 4

8 (6–10) 193 2.48 (1.27–4.84) 2.69 (1.30–5.56)

13 (12–14) 196 2.14 (1.08–4.23) 1.96 (.95–4.04)

17 (15–18) 232 1.25 (.61–2.56) 1.54 (.72–3.29)

23 (21–26) 210 1.00 1.00

90 (81–96) 217 5.10 (2.20–11.78) 3.72 (1.54–8.98)

108 (103–112) 209 4.07 (1.73–9.57) 3.46 (1.42–8.41)

127 (122–134) 207 3.10 (1.29–7.46) 3.04 (1.23–7.55)

160 (149–174) 199 1.00 1.00

⬍.001 .007

224 (198.5–243) 216 8.80 (3.06–25.25) 6.20 (2.04–18.80)

291 (275–309) 206 6.28 (2.14–18.41) 4.91 (1.59–15.14)

357 (338.5–381) 200 5.05 (1.69–15.08) 3.96 (1.28–12.25)

458 (428–505) 186 1.00 1.00

⬍.001 .001

45 (35–51) 201 1.82 (.93–3.56) 2.20 (1.03–4.70)

65 (61–68.5) 200 1.21 (.59–2.48) 1.34 (.63–2.85)

82 (77–88) 204 1.64 (.83–3.22) 1.76 (.86–3.61)

112 (101–127) 199 1.00 1.00

.17 .10

p for Trend

.002 .006

Median value and interquartile range represent adenylyl cyclase (AC) activity in picomoles cyclic adenosine monophosphate (cAMP) per milligram protein per minute. Multivariate models were adjusted for recruitment site, gender, age, family history of alcohol dependence or abuse, and lifetime alcohol dependence. Recent users are defined as individuals who reported any use of any addictive drugs, analgesics, or antidepressants during the 30 days before blood draw. Addictive drugs represent sedatives, tranquilizers, painkillers, stimulants, marijuana, cocaine, heroin, methamphetamine, inhalants, hallucinogens, and steroids. CI, confidence interval; IQR, interquartile range; OR, odds ratio.

These findings confirm previous clinical and epidemiologic research with suggestive evidence to support a relationship between major depression and certain measures of platelet AC activity (Cowburn et al 1994; Hoffman et al 2002; Menninger and Tabakoff 1997; Menninger et al 2000). With a relatively small sample size (n ⫽ 37), the first study to explore this relationship found associations with forskolin-stimulated AC activity and basal AC activity (male subjects only). No association was observed between antidepressant treatment and AC activity. The association with forskolin-stimulated platelet AC activity was also observed in a secondary analysis with the Australian subset of the WHO/ISBRA population; however, this study was also limited by the small number of subjects with a history of major depression (Menninger et al 2000). Using the WHO/ISBRA study population, Hoffman et al (2002) identified the best predictors of basal, CsF-, forskolin-, and Gpp(NH)p-stimulated platelet AC activity through multivariate prediction models generated with a purposeful selection algorithm. With this approach, history of major depression was a modest predictor for forskolin- and CsF-stimulated AC activity levels only. The inconsistencies previously observed regarding the relationship between major depression for all AC measurements and antidepressant use is likely attributable to both small sample size and the need to account for various types of drug use. It is known that marijuana and other cannabinoids can affect AC activity through receptors that are coupled to the inhibitory guanine nucleotide-binding protein (Gi) (Howlett 1995; Prather et al 2000). Nine isoforms of the mammalian AC enzyme, with differing regulatory properties, are known to exist. In contrast to many of the AC isoforms, the type VII isoform of AC (ADCY7) is insensitive to inhibition by Gi␣, but is stimulated by the ␤␥ subunits of Gi. Thus, acute cannabinoid exposure has been www.sobp.org/journal

shown to increase activity of ADCY7, which is suspected to be the predominant isoform in human platelets and is also found in the brain (Federman et al 1992; Yoshimura et al 1996). It is possible that the observed relationship between platelet AC activity and major depression could be predominantly attributed to the ADCY7 isoform. The relationship between AC activity and major depression is further strengthened with our recent data from ADCY7 transgenic mice. Compared with wild-type male mice, we observed fewer depression-like symptoms with male ADCY7 transgenic mice and more with heterozygous knockouts (Hu 2004). Interestingly, the converse was observed for females, suggesting that the downstream effects of AC activity could potentially be modified by inherent gender differences, such as endogenous estrogen levels. Our data suggest that there are gender differences in AC activity levels; however, these differences with respect to the relationship with major depression need to be further elucidated. The relationship between major depression and AC activity levels is observed for both men and women, as is the confounding effect of recent drug use. The relationship between forskolinstimulated AC activity and major depression might be more profound in men than in women. There was no indication that endogenous estrogen levels affect AC activity levels when age (⬍40 vs. ⬎50 years) was used as a surrogate for menopausal status; however, the exclusion of all recent drug users from the gender-specific analyses substantially reduced the power among women to adequately assess these relationships. Limitations of this study should be considered. Although measuring AC activity in platelets is more practical, it is not clear whether it is a true representation of brain biochemistry. There might also be some misclassification in diagnosing depression due to cultural differences in the perception of the diagnostic

L.M. Hines and B. Tabakoff symptoms among this diverse population. In addition, the overrepresentation of alcohol-dependent subjects among the WHO/ ISBRA study population might compromise the generalizability of these results to other populations; however, the fact that major depression and alcohol dependence are common comorbidities exemplifies the relevance of these findings to a large subset, or potential subtype, of depressed subjects. Finally, the crosssectional study design prohibits any conclusions regarding whether this marker can serve as a prognostic or diagnostic biomarker. Longitudinal studies are needed to assess this relationship. To date, WHO/ISBRA represents the largest study population with platelet AC measurements and detailed information regarding a broad range of factors. This study is the first to address the relationship between major depression and platelet AC activity levels, accounting for various types of drug use. In summary, low levels of platelet AC activity are associated with an increased risk of major depression. Importantly, the relationship between platelet AC activity and major depression is markedly attenuated by recent licit and/or illicit drug use. Gender differences with respect to this relationship need to be further elucidated. Our findings suggest that AC activity level is a good candidate for a state or trait marker of major depression. Prospective studies are needed to determine whether this marker can be used as a prognostic or diagnostic tool for major depression. The pursuit to identify genetic determinants of AC activity might provide insight into genetic predisposition for major depression. This work was supported, in part, by the National Institute of Alcohol Abuse and Alcoholism (NIAAA), The Banbury Fund, Lohocla Research Corporation, and the World Health Organization (WHO). We thank Josephine Tsao (University of Colorado Health Sciences Center, Aurora, Colorado) for carrying out all adenylyl cyclase assays and the WHO/International Society on Biomedical Research on Alcoholism (ISBRA) Investigators for their contributions to this study. The WHO/ISBRA Investigators are: Katherine Conigrave, F.A.F.P.H.M., Ph.D. (Royal Prince Alfred Hospital, Camperdown, NSW, Australia); Maurice Dongier, M.D. (Douglas Hospital Research Centre, Quebec, Canada); Howard Edenberg, Ph.D. (Indiana University School of Medicine, Indianapolis, Indiana); C.J. Peter Eriksson, Ph.D. (National Public Health Institute, Helsinki, Finland); Maria Lucia Formigoni, Ph.D. (Federal University of São Paulo, São Paulo, Brazil); Bridget Grant, R.N., Ph.D. (NIAAA, National Institutes of Health [NIH], Bethesda, Maryland); Anders Helander, Ph.D. (Karolinska Institutet and University Hospital, Stockholm, Sweden); Paula Hoffman, Ph.D. (University of Colorado Health Sciences Center, Aurora, Colorado); Kalervo Kiianmaa, Ph.D. (National Public Health Institute, Helsinki, Finland); Tsukasa Koyama, M.D. (Hokkaido University, Sapporo, Japan); Lucie Legault, B.Sc. (Douglas Hospital Research Centre, Quebec, Canada); Ting-Kai Li, M.D. (NIH, U.S. Department of Health and Human Services, Bethesda, Maryland); Taina Methuen, M.D. (University of Helsinki, Helsinki, Finland); Maristela Monteiro, M.D., Ph.D. (Management of Substance Dependence, WHO, Geneva, Switzerland); Toshikazu Saito, M.D., Ph.D. (Sapporo Medical University, Sapporo, Japan); Mikko Salaspuro, M.D. (University Center Hospital of Helsinki, Helsinki, Finland); John Saunders, M.D. (Princess Alexandra Hospital, Brisbane, Australia); Boris Tabakoff, Ph.D. (University of Colorado Health Sciences Center, Aurora, Colorado); Sergio Tufik, M.D. (Department of Psychobiology, Universidade Federal de São Paulo, Brazil); John Whitfield, Ph.D.

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