Forskolin-stimulated platelet adenylyl cyclase activity is lower in persons with major depression

Forskolin-Stimulated Platelet Adenylyl Cyclase Activity Is Lower in Persons with Major Depression John A. Menninger and Boris Tabakoff

We investigated platelet adenylyl cyclase activity in 17 subjects with a history of major depression ("depressed subjects") and 20 controls. Forskolin was used to directly activate adenylyl cyclase, while guanine nucleotides (Gpp(NH)p) and fluoride ions were used to measure adenylyl cyclase activity modulated through the G proteins. Forskolin-stimulated adenylyl cyclase was significantly lower in the depressed subjects (p < 0.0005). There was a statistically significant difference in basal adenylyl cyclase activity between male depressed subjects and male controls. The basal adenylyl cyclase activity was also lower in female depressed subjects, but this difference did not reach statistical significance (p < 0.2). The adenylyl cyclase activity measured after stimulation with a guanine nucleotide or cesium fluoride did not differ between control and depressed male or female subjects. Severity of current depression and the current use of antidepressant medication were not related to the lower forskolin-stimulated enzyme activity in the depressed subjects. The difference in forskolin-stimulated adenylyl cyclase activity appears to reflect a qualitative difference in the adenylyl cyclase enzyme activity in persons with a histo~ of major depression. © 1997 Society of Biological Psychiatry Key Words: Adenylyl cyclase, forskolin, G proteins, platelets, major depression BIOL PSYCHIATRY 1997;42;30--38

Introduction Depression is a chronic, recurrent illness that represents a major health burden. Regier calculated from the 1988 Epidemiological Catchment Area (ECA) study that 5.8% of the U.S. population will at some time in their life suffer from major depression (Regier et al 1988). Others have placed the lifetime risk for major depressive disorders from 2%-4% of persons in the community (Katon and

From the Department of Pharmacology and Psychiatry, University of Colorado Health Sciences Center, Denver, Colorado. Address reprint requests to Boris TabakofL PhD, 4200 East 9th Avenue, Campus Box C-236, Denver, CO 80262. Received December 21, 1995; revised April 22, 1996.

© 1997 Society of Biological Psychiatry

Schulberg 1992) to approximately 8% (Keller and Hanks 1994). In 1990, depressive disorders afflicted at least 11 million Americans and cost the U.S. economy an estimated $44 billion (Hall and Wise 1995). Since the development of the biogenic amine hypothesis of depression, researchers have been investigating the biochemical determinants of major depression in an attempt to improve diagnosis and treatment of this disorder. The biogenic amine theory of depression as first enunciated in the 1960s (Schildkraut 1965) postulated that depression involves a deficit of noradrenergic activity in certain brain areas. The therapeutic effect of antidepressant drugs was initially theorized to be secondary to an elevation of synaptic levels of the monoamines, thus 0006-3223197/$17.00 PII S0006-3223(96)00245-4

Forskolin-Stimulated Platelet Adenylyl Cyclase Activity

restoring normal adrenergic neuronal transmission. Numerous studies evaluating the levels of various biogenic amines--norepinephrine, serotonin, and dopamine--and their respective metabolites in the brain, blood, urine, and cerebrospinal fluid of depressed patients have failed to provide convincing evidence in support of this theory. There has been a gradual transition in research from studies focused on alterations of the various biogenic amines to research investigating dysfunction of their targets, the beta- and alpha-adrenergic and serotonergic receptors and the molecules (effectors) whose activity these receptors modulate. Adenylyl cyclase is the enzyme which generates the ubiquitous second messenger, cyclic adenosine-3',5'monophosphate (cyclic AMP). The activity of adenylyl cyclase is modulated through a variety of receptors including the adrenergic and serotonergic receptors. Adenylyl cyclase activity has been shown to be altered by chronic antidepressant medication (including tricyclic drugs, atypical antidepressants, and monoamine oxidase inhibitors) and electroconvulsive treatment (Chen and Rasenick 1995; Menkes et al 1983; Newman and Lerer 1989; Ozawa and Rasenick 1991; Yamaoka et al 1988). It has been speculated that the mechanism by which antidepressants produce their beneficial effects involves changes in postreceptor components or processes that include changes in the coupling between the guanine nucleotide binding proteins (G proteins) and the catalytic unit of adenylyl cyclase (Chen and Rasenick 1995; Ozawa and Rasenick 1989). In a postmortem study of brain adenylyl cyclase activity, Cowburn et al recently demonstrated reduced basal, forskolin- and guanosine 5'-O-(3-thiotriphosphate) (GTP~S)-stimulated adenylyl cyclase activity in suicide victims vs matched controls (Cowbum et al 1994). These differences were most apparent in suicides involving death from violent means or suicide victims having a history of depression. Although the studies with antidepressant medications and the study of postmortem brain tissue indicate that further examination of adenylyl cyclase activity would be fruitful, the relative inaccessibility of human brain in live subjects for studies of the relationship between adenylyl cyclase and depression has led researchers to consider human blood cells as a model system which may reflect brain biochemistry. Platelets have been used as models for studying a number of biochemical parameters related to depression including the function of beta- and alpha2adrenoceptors (Stahl 1985), serotonergic dysfunction (Lingjaerde 1990), and adenylyl cyclase activity (Collins and Sandier 1971). While a significant number of studies utilizing platelets of depressed subjects have generated data on the activity of platelet adenylyl cyclase in these

BIOLPSYCHIATRY 1997;42:30-38

31

individuals, a majority of these studies were focused on examining the modulation of adenylyl cyclase activity through activation of particular receptor systems rather than specifically measuring the activity of the catalytic unit of the enzyme. The assay conditions used in such studies were optimized to examine receptor-mediated stimulation (e.g., PGEi,) or receptor-mediated inhibition (e.g., alpha2-adrenergic) of adenylyl cyclase activity. A variety of components, e.g., GTP (Birnbaumer et al 1990) and various other guanine nucleotides (Hudson and Fain 1983; Nelson and Seamon 1986; Wadman et al 1991), and high concentrations of either sodium ions (Motulsky and Insel 1983; Steer and Wood 1981) or calcium ions (Caldwell et al 1992), used in such assays have significant effects on enzyme activity. These assay constituents compromised the ability of the researchers to generate insights into the integrity of the catalytic component of the adenylyl cyclase signal transduction system in depressed subjects. Seamon and Daly have demonstrated that the diterpene, forskolin, activates adenylyl cyclase in the absence of G proteins (Seamon and Daly 1981). Forskolin interacts with a binding site for such alkaloids on the enzyme protein and directly stimulates the catalytic activity of adenylyl cyclase. The recent demonstration of reduced forskolin-stimulated adenylyl cyclase activity in brain of depressed subjects (Cowburn et al 1994) prompted us to reexamine forskolin-stimulated adenylyl cyclase activity in platelets of depressed subjects. We performed our assay of forskolin-stimulated adenylyl cyclase activity under conditions described by Seamon and Daly (1981) and we compared this activity to activity measured when platelet adenylyl cyclase was stimulated via activation of G proteins.

Methods

Subjects Depressed subjects were recruited from the Colorado Psychiatric Hospital and the community. A brief screening tool, derived from the High Mood section of the NIAAA Alcohol Use Disorders and Disabilities Schedule (AUDADIS) (Grant et al 1995) and the psychosis section of the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) (Bucholz et al 1994), was used to exclude subjects with bipolar disorder or a major psychotic disorder. Subjects were included who were either currently or previously depressed for at least 2 weeks. Subjects were requested to remain alcohol free for at least 7 days prior to the blood draw and the full interview. After written consent was obtained, all subjects completed a Beck Depression Inventory (BDI). The BDI is a widely used self-rated depression scale consisting of 21 groups of

32

BIOL PSYCHIATRY 1997;42:30-38

items (Beck et al 1961). Standard venapuncture technique was then used to collect a blood sample into Becton Dickinson vacutainers containing EDTA. Subjects were then assessed by a structured interview conducted by one of the authors (JM). The interview included the WHO/ ISBRA (Word Health Organization/International Society for Biomedical Research on Alcoholism) Interview Schedule, and the Low Mood and Behavior portions of the AUDADIS. The WHO/ISBRA Interview Schedule was adapted from the AUDADIS and is being used in a six-center multi-national project on State and Trait Markers in Alcoholism (Grant et al 1995). The interview generates data on demography, medical and psychiatric conditions, use of prescribed and over-the-counter medications, recent alcohol and drug consumption, prior alcohol and drug-related experiences, and family history of alcohol and drug use related disorders and psychiatric disorders. The family history section of the WHO/ISBRA Interview was adapted from the Family History Section developed by Dr. Marc Schuckit and his colleagues for the Collaborative Study on the Genetics of Alcoholism (COGA) project (Schuckit et al 1994). The Low Mood and Behavior portions of the AUDADIS instrument allow DSM-III-R, DSM-IV, and ICD-10 diagnoses of major depressive disorder and dysthymia. The WHO/ISBRA instrument allows DSM-III-R, DSM-IV and ICD-10 diagnoses of alcohol and drug dependence and abuse which were used as exclusion criteria to limit the subject population to persons with major depression (unipolar) without comorbid alcohol dependence. The AUDADIS has been shown to be highly reliable for the diagnoses of alcohol use disorders and major depression, achieving reliability (~:) coefficients ranging between 0.73-0.76 and 0.600.65, respectively (Grant and Hartford 1995; Grant et al 1995). Control subjects were recruited from university personnel, hospital staff, and the community. They were determined to have no current or prior history of major depression, bipolar disorder, or major psychotic disorder. They were also determined to have no history of alcohol dependence. Our research protocol was approved by the Colorado Multiple Institutional Review Board.

Preparation of Platelet Membranes Blood was collected in vacutainer tubes containing 1.5 mg potassium EDTA per mL. The samples were collected from all subjects between the hours of 10 AM and 3 PM. The platelets were prepared within 4 hours of the time of the blood draw. Blood platelet rich plasma layer was transferred to a fresh centrifuge tube and again centrifuged for 10 minutes at 700xg at room temperature. The upper platelet rich layer was again transferred to a fresh centri-

J.A. Menninger et al

fuge tube and centrifuged at 2800xg for 15 minutes at room temperature. The supernatant fluid was discarded and the platelet pellet was then stored at minus 70°C. Prior to the assay of platelet adenylyl cyclase activity, the platelet pellet was thawed and washed at 4°C. The platelets were suspended, by the use of a hand-held homogenizer, in 1.5 mL of 50 mM Tris-HC1 (pH 7.5) containing 20 mM EDTA, and the suspension was centrifuged at 17,000xg for 10 minutes. This procedure was repeated and the platelet pellet was then suspended in 1.5 mL of 5 mM Tris-HCl (pH 7.5) containing 5 mM EDTA and then centrifuged at 17,000xg for 10 minutes. The platelet pellet was then suspended in 1.5 mL of 5 mM Tris-HC1 (pH 7.5) containing 1 mM EDTA, diluted as necessary with 5 mM Tris-HC1 (pH 7.5) containing 1 mM EDTA, and used immediately for the assays of platelet adenylyl cyclase activity. Protein determination was performed using the Pierce Bicinchoninic Acid (BCA) protein microtiter method (Davis and Radke 1987; Walker 1994).

Adenylyl Cyclase Assay Approximately 10-50 p.g of prepared platelet membrane protein (50 ~zL) was added to 200 IxL of assay buffer consisting of 25 mM Tris-maleate (pH 7.5), 10 mM theophylline, 5 mM MgC12, 0.25 mM ATP, and [a-32p]ATP (1.2-2.0 × 10 6 cprn/assay). Adenylyl cyclase activity was measured either under these basal conditions or with the addition of one of the following: 10 ~xM Gpp(NH)p (5'-guanylyl-imidodiphosphate), l0 mM CsF (cesium fluoride), or l0 ixM forskolin. Following equilibration of the assay mixture at 30°C for 5 minutes, the reaction was initiated by adding 50 IxL of platelet membranes. The reaction mixture was further incubated at 30°C for l0 minutes, and the reaction was terminated by the addition of 750 IxL of ice cold solution containing 4 mM ATP, 1.4 mM cyclic AMP, and 10,000 cpm [3H]cyclic AMP (25-40 Ci/mmol, New England Nuclear DuPont) to each assay tube. [3H]cyclic AMP together with [32p] cyclic AMP generated by adenylyl cyclase were isolated by sequential chromatography on Dowex and alumina columns as described by Salomon and colleagues (Salomon et al 1974) and quantitated by liquid scintillation counting. All reported values were corrected for recovery of [3H]cyclic AMP, and adenylyl cyclase activity was expressed as pmol cyclic AMP generated/mg protein/minute. An aliquot of human erythroleukemia (HEL) cell membranes with known levels of adenylyl cyclase activity was assayed with each group of samples. The HEL membrane preparation was used as a reference standard to control between-assay variability (see Results). The value of the

Forskolin-Stimulated Platelet Adenylyl Cyclase Activity

BIOL PSYCHIATRY 1997;42:30-38

33

Table 1. Characteristics o f the Study Subjects* Depressed subjects

Controls

17 7 men, 10 women mean 39 - 2; range 24-60

20 10 men, 10 women mean 35 - 2; range 22-55 75% (15) 10% (2) 15% (3) mean 17.0 +-- 0.5; median 18; range 12-19 mean 270 +-- 14; range 149347 55% (11) 15% (3) mean 109 ___ 150; median 60; range 0-600 mean 39 -+ 14; median 12; range 7-271 0 0

Currently depressed Beck Depression Score

100% (17) 0 0 mean 15.9 - 0.5; median 16; range 12-19 mean 264 ___ 14; range 167356 59% (10) 12% (2) mean 57 _+ 84; median 12; range 0-276 mean 111 --- 50; median 26; range 9-818 0 29% (5) mean 23 - 3.2; median 17; range 10-58 mean 17 - 2.4; range 1-35 mean 16 - 6.7; median 5; range 1-100 mean 0.9 --- 0.18; median 0.8; range 0.06-3 mean 5.3 4- 1.2; median 5; range 0-12 35% (6) mean 13.5 - 2.2; range 1-38

Ever antidepressant treatment Current antidepressant treatment

82% (14) 65% (1l)

Number of subjects Gender Age Race Caucasian African-American Asian Years of education Platelets (× 109/[,) Ever smokers Current smokers Grams of alcohol consumed in the past 30 days Days since last drank Ever alcohol dependent Ever drug dependent Age at onset of depression Years since onset of depression No. episodes of depression Longest episode of depression (years) Number of months depressed in past year

0 mean 1.3 - 0.4; median 1; range 0 - 8 0 0

* Plus-minus values are standard errors; medians are included for data with nonnormal distributions. Numbers in parentheses indicated the number of subjects in a particular category.

adenylyl cyclase activity obtained with HEL membranes within each day's assay was divided by the HEL membrane activity average over the entire project period. The resulting factor was used to standardize all adenylyl cyclase activity values obtained on a particular day. All assays of adenylyl cyclase activity were performed without knowledge of the diagnostic status of the subjects.

Statistical Analysis Analyses of variance were performed with SigmaStat software. The dependent variables in this analysis were basal, guanylyl-imidodiphosphate-, cesium fluoride-, and forskolin-stimulated adenylyl cyclase activities. Independent variables included group status (i.e., depressed or control) and gender. Separate analyses were done for various depression indices, including Beck Depression Inventory score, years since onset of depression, number of episodes, longest episode, number of months depressed

in the past year, and current antidepressant use. Pearson product moment correlations between dependent variables were also determined with SigmaStat software, p values below 0.05 were considered to be statistically significant.

Results Table 1 summarizes the demographic characteristics of the depressed and control subjects who participated in this study. The two study groups were similar regarding gender, years of education, current smoking and prior smoking history. Both groups had similar and normal platelet counts as well. The depressed subjects were all Caucasian compared to 10% African-American and 15% Asian in the control group. The depressed group were slightly older, and drank less alcohol in the past 30 days than the control group. All subjects in this study avoided alcohol for at least 1 week prior to the assessment. None

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BIOLPSYCHIATRY

J.A. Menninger et al

1997;42:30-38

Table 2. Adenylyl Cyclase Activity in Platelets of Depressed and Control Subjects: Effects of Group and Gender Adenylyl Cyclase activities, basal and stimulated levels* pmol of cyclic AMP/mg of protein/ min--mean ± SEM Gender (n)

Depressed Controls

All (17) Males (7) Females (10) All (20) Males (10) Females (10)

Gpp(NH)p (10 izM)

Basal 10.1 11.3 9.3 14.4 15.3 13.5

+- 0 . 8 t ± 0.6t -+ 1.3 _+ 1.6 ± 1.6 -+ 2.9

37.7 43.5 33.6 43.3 44.7 41.8

CsF (10 mM)

+- 2.2 ± 3.1 -+ 2.5 _+ 2.9 ± 3.7 ± 4.6

97.1 102.5 93.3 106.9 105.2 108.6

± ± ± -± ±

Forskolin (10 ixM)

3.4 6.2 3.5 4.4 5.7 6.9

231.8 271.1 204.4 333.7 347.8 319.7

± 13.0~ ± 19.2J" _+ 11.6~: _+ 20.5 _+ 23.6 +- 34.2

* Gpp(NH)p denotes guanylyl-imidodiphosphate,and CsF, cesium fluoride. There were no group by gender interactions as determined by a two-way analysis of variance. There were significant group effects for basal and forskolin-stimulated activities, using ANOVA followed by the Student-Newman-Keuls procedure: t p < 0.05, ~ p < 0.0005, comparison of depressed subjects to corresponding controls.

of the control or depressed subjects had a diagnosis of alcohol dependence or abuse as ascertained by our interview. A history of drug dependence was noted in 29% of the depressed group. Subjects with depression, on average, had a 17-year history of major depression, 16 total episodes of depression (though the median number of episodes was five), with the longest episode lasting almost 11 months. The depressed group had been depressed for an average of 5.3 months out of the last year. Over one-third (35%) were currently depressed subjects with a BDI score -->15, a standard threshold indicating severe depression (Craven et al 1988). Nearly two-thirds of the depressed group (65%) were currently taking antidepressant medication and more than four-fifths (82%) had taken antidepressant medication at some time in their life. Intraassay variability was determined by assaying basal, forskolin- and CsF-stimulated activity in triplicate; the average coefficient of variation in 17 such experiments was 14.0% for basal activity, 5.0% for forskolin-stimulated activity and 4.3% for CsF-stimulated activity. Interassay variability was also determined using basal and CsF-stimulated adenylyl cyclase activities. Activity in four aliquots from the same preparation of platelets was determined in assays performed on 4 separate days within a 2-week period. The coefficient of variation was 23.4%, 13.5%, and 9.2% for basal, forskolin-stimulated and CsFstimulated activities, respectively. In order to compare our assay conditions with those used in prior studies, in certain cases we included 1 mM CaC12 in our assays and noted that this concentration of CaC12 produced the following reductions in adenylyl cyclase activity: basal, 45%; Gpp(NH)p, 49%; CsF, 37%; and forskolin, 29%. Assays in which 100 mM NaC1 was included resulted in reductions in basal and Gpp(NH)pstimulation of 79% and 78%, respectively, and marked enhancement of CsF- and forskolin-stimulated activity by 140% and 154%, respectively. Under our standard conditions of assay, forskolin-

stimulated adenylyl cyclase activity, but not guanylyl imidodiphosphate-, or cesium fluoride-stimulated activity, was lower in the platelets of depressed subjects as compared to controls (p < 0.0005) (Table 2). The fact that differences in the specific activity of adenylyl cyclase were noted under conditions of stimulation by one agent (forskolin), but not others (guanylyl imidodiphosphate or cesium fluoride) precludes the possibility that such differences were simply due to differences in platelet protein between depressed and control subjects. Forskolin-stimulated adenylyl cyclase specific activity for each of the control and depressed subjects is depicted in Figure 1. Basal activity was also lower in the group of depressed male subjects (p < 0.05), but the differences in basal activity between female depressed and control subjects did

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Male Control Subjects

Male Depressed Subjects

Female Female Control Depressed Subjects Subjects

Forskolin-Stimulated Platelet Adenylyl Cyclase Activity

Table 3. Adenylyl Cyclase Activity in Platelets of Depressed Subjects: Effect of Current Beck Depression Inventory Score Adenylyl Cyclase activities, basal and stimulated levels* pmol of cyclic AMP/mg of proteirdmin-mean _+ SEM Depressed subjects (n) BDI--- 15 (6) BDI < 15 (11)

Basal

Gpp(NH)p (10 ~M)

9 . 1 - 1.4 3 6 . 7 - 3 . 3 10.7 --- 1.0 38.2 -+ 3.1

CsF (10 mM)

Forskolin (10 p,M)

99.1-+3.8 229.4-+25.4 96.0 ± 4.9 233.1 - 15.5

* Gpp(NH)p denotes guanylyl-imidodiphosphate, CsF, cesium fluoride, and BDI, Beck Depression Inventory.

not reach statistical significance (p < 0.2). The difference in forskolin-stimulated activity remained significant irrespective of gender, though the difference was more robust in the women. We also found no significant correlation of forskolin-stimulated activity with age, education level, age at onset of depression, duration of depression problems, number of episodes of depression, longest period of depression, consumption of alcohol in the past month, or prior heaviest alcohol consumption. Focusing on the depressed group, we found no significant correlation (r = -0.01) of forskolin-stimulated adenylyl cyclase (nor any of the other assays, data not shown) with the severity of current depression as self-rated on the Beck Depression Inventory. Dividing the depressed subjects into two groups using the threshold of ->15 as an indication of severe depression also yielded no significant differences between adenylyl cyclase activities (Table 3). The current use of antidepressant medication also did not generate any significant differences in adenylyl cyclase activity (Table 4).

Discussion Differences in adenylyl cyclase activity in the platelets of depressed patients compared to controls have not been a universal finding in previous studies. Researchers have generally not found any difference in basal adenylyl cyclase activity when examining depressed subjects (Abou-Saleh et al 1994; Garcia-Sevilla et al 1990; Kafka et al 1986; Karege et al 1992; Mitrius et al 1983; Mooney et al 1985; Newman et al 1992; Roy and Kafka 1989;

BIOLPSYCHIATRY 1997;42:30-38

35

Wang et al 1974). It is of interest that statistically significant differences in basal adenylyl cyclase activity were noted only in male subjects in our study. This may reflect a true differential effect of gender, or may reflect the difficulty of precisely measuring basal adenylyl cyclase activity in platelet membranes of an individual. We have estimated that the interassay variability for basal activity is approximately 23.4% and therefore it would be difficult to demonstrate the statistical significance of differences if such differences do not substantially exceed the assay variability. Some investigators have noted a significantly diminished prostaglandin El-stimulated platelet adenylyl cyclase activity in depressed patients (Abou-Saleh et al 1994; Kafka et al 1986; Kafka et al 1985; Kanof et al 1988; Murphy et al 1974; Newman et al 1992; Siever et al 1983; Wang et al 1974). The studies that assessed fluoride- and guanine nucleotide (Gpp(NH)p)stimulated platelet adenylyl cyclase activity showed no differences in depressed persons compared to controls (Abou-Saleh et al 1994; Mitrius et al 1983; Newman et al 1992), and although several researchers have also examined forskolin-stimulated adenylyl cyclase activity in the platelets of depressed persons, none have found significant differences from controls (Abou-Saleh et al 1994; GarciaSevilla et al 1990; Karege et al 1992; Newman et al 1992). There are a number of reasons why it is difficult to compare our results with those of the previous studies. First, many of the early studies had relatively small and heterogeneous groups of affective disorder subjects, which included variably both euthymic subjects and patients with a bipolar mood disorder (Kafka et al 1986; Lenox et al 1983; Murphy et al 1974; Siever et al 1983; Siever et al 1984; Wang et al 1974). Second, assay conditions that do not favor measurements of the activity of the catalytic unit of adenylyl cyclase were used. Researchers have generally been interested in receptor mediated activation of adenylyl cyclase activity and/or receptor mediated inhibition of stimulated adenylyl cyclase activity (Abou-Saleh et al 1994; Garcia-Sevilla et al 1990; Kafka et al 1986; Kafka et al 1985; Karege et al 1992; Mooney et al 1985; Newman et al 1992; Roy and Kafka 1989). Assay conditions were therefore employed that favored the receptor mediated stimulation or inhibition of adenylyl cyclase. Such condi-

Table 4. Adenylyl Cyclase Activity in Platelets of Depressed Subjects: Effect of Current Antidepressant Medication Adenylyl Cyclase activities, basal and stimulated levels* pmol of cyclic AMP/mg of protein/min--mean 4- SEM

Depressed subjects (n)

Basal

Gpp(NH)p (10 p,M)

CsF (10 mM)

Forskolin (10 p,M)

On antidepressants (11) Antidepressant-free (6)

11.2 _+ 1.1 8.2 --- 1.0

39.0 +-- 3.0 35.1 _+ 3.2

92.4 _ 3.5 105.6 ± 6.0

246.0 ___ 18.3 205.8 _+ 9.6

* Gpp(NH)p denotes guanylyl-imidodiphosphate, and CsF, cesium fluoride.

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BIOLPSYCHIATRY 1997;42:30-38

tions confound direct measures of catalytic unit activity in the absence of G protein modulation. Adenylyl cyclase catalytic activity is modulated by G proteins, including Gsot and Giot (Bimbaumer et al 1990) and the 13~/subunits of the G proteins (Tang et al 1992). The G proteins are activated by guanine nucleotides, such as Gpp(NH)p, which promote the dissociation of the a and ~3~/subunits of the G proteins (Birnbaumer et al 1990), or by fluoride ions, which interact with the o~ subunit to stabilize an active transition state of this modulatory protein (Sondek et al 1994). The activated tx or the 13"y subunits of the G proteins interact with specific sites on the adenylyl cyclase catalytic protein (Chen et al 1995) to modulate the rate of catalysis. Forskolin is an alkaloid which binds directly to the catalytic unit of adenylyl cyclase at a site distinct from the site of interaction of the G proteins (Seamon et al 1981). Therefore, prior studies which used agents that activate or inhibit catalytic activity of adenylyl cyclase via the G proteins would not necessarily be reflective of the forskolin-stimulated adenylyl cyclase activity. In fact, in agreement with another study in which guanine nucleotides were included in the assays (Abou-Saleh et al 1994), we also found no difference in Gpp(NH)p or fluoride-stimulated adenylyl cyclase activity between depressed and control subjects. Rasenick and others have shown that chronic treatment of rats with antidepressants enhances G protein stimulated adenylyl cyclase activity in the cerebral cortex (Ozawa and Rasenick 1989; 1991); however, when we subdivided the depressed subjects into those taking antidepressant medications and those not on medication, we noted no statistically significant differences in platelet adenylyl cyclase activity measured either in the presence of Gpp(NH)p or cesium fluoride (i.e., agents which stimulate adenylyl cyclase activity via interactions with G protein). Our demonstration of lower forskolin-stimulated adenylyl cyclase activity in subjects with a history of depression mimics best the recent study of Cowburn and colleagues (Cowburn et al 1994) who demonstrated, under assay conditions similar to ours, lower forskolin-stimulated adenylyl cyclase activity in the brains of suicide victims; however, Cowburn et al also found decreases in guanine nucleotide-stimulated adenylyl cyclase activity. Since they found no differences in the quantity of Gs or Gi/Go, they postulated that their results were indicative of an overall quantitative decrease in catalytic unit activity. On the other

J.A. Menninger et al

hand, Ozawa et al reported a significant reduction in G proteins in post-mortem brain of depressed subjects (Ozawa et al 1993). Thus, decreased brain adenylyl cyclase activity measured in the presence of guanine nucleotides may reflect changes in G proteins in addition to or independent of changes in the properties of the catalytic unit of the adenylyl cyclase enzyme. Our studies in platelets may reflect at least a portion of the events taking place in the brains of depressed subjects. We have recently identified, cloned, and characterized an adenylyl cyclase (AC VII) from a human erythroleukemia (HEL) cell line and found that this adenylyl cyclase is present in human brain as well as in peripheral tissues (Hellevuo et al 1993, 1995; Yoshimura et al 1996). The HEL cells are of megakaryocyte lineage and have been used extensively as models of platelet function (Brass et al 1991). It was, therefore, of some interest to note that certain characteristics of the AC VII measured in transiently transfected human embryonic kidney (HEK) cells (Hellevuo et al 1995) are also evident in measurements of platelet adenylyl cyclase activity, i.e., response to phorbol esters (Simmoteit et al 1991). One can currently hypothesize that at least one (AC VII) or more of the nine isoforms of adenylyl cyclase that are present in human brain (Cali et al 1994; Hellevuo et al 1995; Iyengar 1993; Paterson et al 1995) are also present in platelets. Given the results we obtained when adenylyl cyclase activity was stimulated by forskolin compared to the fact that no changes were witnessed when adenylyl cyclase activity was stimulated via G protein, one would have to conclude that the adenylyl cyclase activity differences we witnessed are qualitative changes in the catalytic protein and related to mechanisms concerned with either binding of forskolin or the process of activation of adenylyl cyclase by agents acting at the forskolin binding site. Since individuals with a history of depression but not currently depressed, as well as currently depressed subjects, demonstrated a low forskolin activated adenylyl cyclase activity, one also has to seriously consider whether the witnessed trait is a result of depression or may be an inherent characteristic of individuals predisposed to major depressive illness.

This work was supportedfunds from NIAAA and the BanburyFoundation. We thank Josephine I for her superb assistance in performingthe enzyme assays.

References Abou-Saleh MT, Collins J, George A, Rommelspacher H (1994): Adrenoceptor activity and adenylate cyclase inhibition in depression [letter]. Br J Psychiatry 165:276-277.

Beck AT, Ward CH, Mendelson M, Mock J, Erbaugh J (1961): An inventory for measuring depression. Arch Gen Psychiatry 4:561-571.

Forskolin-Stimulated Platelet Adenylyl Cyclase Activity

Birnbaumer L, Abramowitz J, Brown AM (1990): Receptoreffector coupling by G proteins. Biochim Biophys Acta 1031:163-224. Brass LF, Manning DR, Shattil SJ (1991): GTP-binding proteins and platelet activation. Prog Hemost Thromb 10:127-174. Bucholz KK, Cadoret R, Cloninger CR, et al (1994): A new, semi-structured psychiatric interview for use in genetic linkage studies: a report on the reliability of the SSAGA. J Stud Alcohol 55:149-158. Caldwell KK, Boyajian CL, Cooper DM (1992): The effects of Ca z÷ and calmodulin on adenylyl cyclase activity in plasma membranes derived from neural and non-neural cells. Cell Calcium 13:107-121. Cali JJ, Zwaagstra JC, Mons N, Cooper DM, Krupinski J (1994): Type VIII adenylyl cyclase. A Ca 2+/calmodulin-stimulated enzyme expressed in discrete regions of rat brain. J Biol Chem 269:12190-12195. Chen J, DeVivo M, Dingus J, et al (1995): A region of adenylyl cyclase 2 critical for regulation by G protein beta gamma subunits. Science 268:1166-1169. Chen J, Rasenick MM (1995): Chronic antidepressant treatment facilitates G protein activation of adenylyl cyclase without altering G protein content. J Pharmacol Exp Ther 275:509517. Collins GG, Sandier M (1971): Human blood platelet monoamine oxidase. Biochem Pharmacol 20:289-296. Cowburn RF, Marcusson JO, Eriksson A, Wiehager B, O'Neill C (1994): Adenylyl cyclase activity and G-protein subunit levels in postmortem frontal cortex of suicide victims. Brain Res 633:297-304. Craven JL, Rodin GM, Littlefield C (1988): The Beck Depression Inventory as a screening device for major depression in renal dialysis patients. Int J Psychiatry Med 18:365-374. Davis LC, Radke GA (1987): Measurement of protein using flow injection analysis with bicinchoninic acid. Anal Biochem 161:152-156. Garcia-Sevilla JA, Padro D, Giralt MT, Guimon J, Areso P (1990): Alpha 2-adrenoceptor-mediated inhibition of platelet adenylyl cyclase and induction of aggregation in major depression. Effect of long-term cyclic antidepressant drug treatment. Arch Gen Psychiatry 47:125-132. Grant BF, Hartford TC (1995): Comorbidity between DSM-IV alcohol use disorders and major depression: Results of a national survey. Drug Alcohol Depend 39:197-206. Grant BF, Hartford TC, Dawson DA, Chou PS, Picketing RP (1995): The alcohol use disorder and associated disabilities interview schedule (AUDADIS): Reliability of alcohol and drug modules in a general population sample. Drug Alcohol Depend 39:37-44. Hall RC, Wise MG (1995): The clinical and financial burden of mood disorders. Cost and outcome. Psychosomatics 36:S 1118. Hellevuo K, Yoshimura M, Kao M, Hoffman PL, Cooper DM, Tabakoff B (1993): A novel adenylyl cyclase sequence cloned from the human erythroleukemia cell line. Biochem Biophys Res Commun 192:311-318. Hellevuo K, Yoshimura M, Mons N, Hoffman PL, Cooper DM, Tabakoff B (1995): The characterization of a novel human

BIOLPSYCHIATRY 1997;42:30-38

37

adenylyl cyclase which is present in brain and other tissues. J Biol Chem 270:11581-11589. Hudson TH, Fain JN (1983): Forskolin-activated adenylyl cyclase. Inhibition by guanyl-5'-yl imidodiphosphate. J Biol Chem 258:9755-9761. Iyengar R (1993): Molecular and functional diversity of mammalian Gs-stimulated adenylyl cyclases. FASEB J 7:768775. Kafka MS, Nurnberger JI, Siever L, Targum S, Uhde TW, Gershon ES (1986): Alpha 2-adrenergic receptor function in patients with unipolar and bipolar affective disorders. JAffect Disord 10:163-169. Kafka MS, Siever LJ, Nurnberger JI, et al (1985): Platelet alpha-adrenergic receptor function in affective disorders and schizophrenia. Psychopharmacol Bull 21:599-602. Kanof PD, Johns CA, Davidson M, Siever L J, Coccaro EF, Davis KL (1988): Platelet alpha 2-adrenergic receptor function in psychiatric disorders. Psychiatry Res 23:11-22. Karege F, Bovier P, Widmer J, Gaillard JM, Tissot R (1992): Decrease in epinephrine-induced attenuation of platelet adenylate cyclase activity in depressed patients: relation with plasma electrolytes. Neuropsychobiology 26:129-135. Katon W, Schulberg H (1992): Epidemiology of depression in primary care. Gen Hosp Psychiatry 14:237-247. Keller MB, Hanks DL (1994): The natural history and heterogeneity of depressive disorders: implications for rational antidepressant therapy. J Clin Psychiatry 55:25-31; discussion 32-33, 98-100. Lenox RH, Ellis JE, Van Riper DV, et al (1983): Platelet alpha2-adrenergic receptor activity in clinical studies of depression. In Usdin E, Goldstein M, Friedhoff AJ, Georgotas A (eds), Frontiers in Neuropsychiatric Research. London: MacMillan Press, pp 331-356. Lingjaerde O (1990): Blood platelets as a model system for studying serotonergic dysfunction and effects of antidepressants. Pharmacol Toxicol 66(suppl 3):61-68. Menkes DB, Rasenick MM, Wheeler MA, B itensky MW (1983): Guanosine triphosphate activation of brain adenylate cyclase: enhancement by long-term antidepressant treatment. Science 219:65-67. Mitrius JC, Micuni M, Arora RC, Meltzer HY, U'Prichard DC (1983): Responsiveness of alpha 2-adrenergic receptors is decreased in platelets from depressed patients. Soc Neuroscience Abstracts 9:990. Mooney JJ, Schatzberg AF, Cole JO, Kizuka PP, Schildkraut JJ (1985): Enhanced signal transduction by adenylate cyclase in platelet membranes of patients showing antidepressant responses to alprazolam: preliminary data. J Psychiatr Res 19:65-75. Motulsky HJ, Insel PA (1983): Influence of sodium on the alpha 2-adrenergic receptor system of human platelets. Role for intraplatelet sodium in receptor binding. J Biol Chem 258: 3913-3919. Murphy DL, Donnelly C, Moskowitz J (1974): Catecholamine receptor function in depressed patients. Am J Psychiatry 131:1389-1391. Nelson CA, Seamon KB (1986): Binding of [3H]forskolin to human platelet membranes. Regulation by guanyl-5'-yl imi-

38

BIOL PSYCHIATRY 1997;42:30-38

dodiphosphate, NaF, and prostaglandins E1 and D2. J Biol Chem 261:13469-13473. Newman ME, Lerer B (1989): Modulation of second messenger function in rat brain by in vivo alteration of receptor sensitivity: relevance to the mechanism of action of electroconvulsive therapy and antidepressants. Prog Neuro-Psychopharmacol Biol Psychiatry 13:1-30. Newman ME, Lerer B, Lichtenberg P, Shapira B (1992): Platelet adenylate cyclase activity in depression and after clomipramine and lithium treatment: relation to serotonergic function. Psychopharrnacology 109:231-234. Ozawa H, Gsell W, Frolich L, et al (1993): Imbalance of the Gs and Gi/o function in post-mortem human brain of depressed patients. J Neural Transm Gen Sect 94:63-69. Ozawa H, Rasenick MM (1989): Coupling of the stimulatory GTP-binding protein Gs to rat synaptic membrane adenylate cyclase is enhanced subsequent to chronic antidepressant treatment. Mol Pharmacol 36:803- 808. Ozawa H, Rasenick MM (1991): Chronic electroconvulsive treatment augments coupling of the GTP-binding protein Gs to the catalytic moiety of adenylyl cyclase in a manner similar to that seen with chronic antidepressant drugs. J Neurochem 56:330-338. Paterson JM, Smith SM, Harmar AJ, Antoni FA (1995): Control of a novel adenylyl cyclase by calcineurin. Biochem Biophys R es Commun 214:1000-1008. Regier DA, Boyd JH, Burke JD, Jr., et al (1988): One-month prevalence of mental disorders in the United States. Based on five Epidemiologic Catchment Area sites. Arch Gen Psychiatry 45:977-986. Roy A, Kafka MS (1989): Platelet adrenoceptors and prostaglandin responses in depressed patients. Psychiatry Res 30:181189. Salomon Y, Londos C, Rodbell M (1974): A highly sensitive adenylate cyclase assay. Anal Biochem 58:541-588. Schildkraut JJ (1965): The catecholamine hypothesis of affective disorders: A review of supporting evidence. Am J Psychiatry 122:509-522. Schuckit MA, Hesselbrock V, Tipp J, Anthenelli R, Bucholz K, Radziminski S (1994): A comparison of DSM-III-R, DSM-IV and ICD-10 substance use disorders diagnoses in 1922 men and women subjects in the COGA study. Collaborative Study on the Genetics of Alcoholism. Addiction 89:1629-1638. Seamon K, Daly JW (1981): Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. J Biol Chem 256:9799-9801.

J.A. Menninger et al

Seamon KB, Padgett W, Daly JW (1981): Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc Natl Acad Sci U S A 78:3363-3367. Siever LJ, Kafka MS, Insel TR, Lake CR, Murphy DL (1983): Effect of long-term clorgyline administration on human platelet alpha-adrenergic receptor binding and platelet cyclic AMP responses. Psychiatry Res 9:37-44. Siever LJ, Kafka MS, Targum S, Lake CR (1984): Platelet alpha-adrenergic binding and biochemical responsiveness in depressed patients and controls. Psychiatry Res 11:287-302. Simmoteit R, Schulzki HD, Palm D, Mollner S, Pfeuffer T (1991): Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters. FEBS Lett 285:99-103. Sondek J, Lambright DG, Noel JP, Hamm HE, Sigler PB (1994): GTPase mechanism of G proteins from the 1.7-A crystal structure of transducin alpha-GDP-A1F-4. Nature 372:276279. Stahl SM (1985): Platelets as pharmacologic models for the receptors and biochemistry of monaminergic neurons. In Longenecker GL (ed), The Platelets: Physiology and Pharmacology. Orlando, FL: Academic Press, Inc., pp 307-340. Steer ML, Wood A (1981): Inhibitory effects of sodium and other monovalent cations on human platelet adenylate cyclase. J Biol Chem 256:9990-9993. Tang WJ, Iniguez-Lluhi JA, Mumby S, Gilman AG (1992): Regulation of mammalian adenylyl cyclases by G-protein alpha and beta gamma subunits. Cold Spring Harb Symp Quant Biol 57:135-144. Wadman IA, Famdale RW, Martin BR (1991): Evidence for regulation of human platelet adenylate cyclase by phosphorylation. Inhibition by ATP and guanosine 5'-[beta-thio]diphosphate occur by distinct mechanisms. Biochem J 276: 621-630. Walker JM (1994): The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol 32:5-8. Wang YC, Pandey GN, Mendels J, Frazer A (1974): Platelet adenylate cyclase responses in depression: implications for a receptor defect. Psychopharmacologia 36:291-300. Yamaoka K, Nanba T, Nomura S (1988): Direct influence of antidepressants on GTP binding protein of adenylate cyclase in cell membranes of the cerebral cortex of rats. J Neural Transm 71:165-175. Yoshimura M, Ikeda H, Tabakoff B (1996): Mu opioid receptors inhibit dopamine-stimulated activity of type V adenylyl cyclase but enhance the dopamine-stimulated activity of type VII adenylyl cyclase. Mol Pharmacol 50:43-51.