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Agents effective against mania in bipolar disorder are reported to decrease turnover of arachidonic acid (AA) in phospholipids and expression of calcium-dependent AA-selective cytosolic phospholipase A2 (cPLA2) in rat brain. In contrast, fluoxetine, an antidepressant that is reported to switch bipolar depressed patients to mania, increases cPLA2 expression and AA turnover in rat brain. We therefore hypothesized that antidepressants that increase switching to mania generally increase cPLA2 and AA turnover in brain. To test this hypothesis, adult male CDF-344 rats were administered imipramine and bupropion, with reported high and low switching rates, respectively, at daily doses of 10 and 30mgkg−1 i.p., respectively, or i.p. saline (control) for 21 days. Frontal cortex expression of different PLA2 enzymes and AA turnover rates in brain when the rats were unanesthetized were measured. Compared with chronic saline, chronic imipramine but not bupropion significantly increased cortex cPLA2 mRNA activity, protein and phosphorylation, expression of the cPLA2 transcription factor, activator protein-2α(AP-2α) and AA turnover in phospholipids. Protein levels of secretory phospholipase A2, calcium-independent phospholipase A2, cyclooxygenase (COX)-1 and COX-2 were unchanged, and prostaglandin E2 was unaffected. These results, taken with prior data on chronic fluoxetine in rats, suggest that antidepressants that increase the switching tendency of bipolar depressed patients to mania do so by increasing AA recycling and metabolism in brain. Mania in bipolar disorder thus may involve upregulated brain AA metabolism.
Persons with bipolar disorder (BD) experience episodes of depression more frequently and for longer periods than episodes of mania.1 Bipolar depression is a major cause of psychiatric morbidity and mortality and poses a major public health concern. A challenge in treating bipolar depression is the tendency of many antidepressants, particularly tricyclic antidepressants and serotonin selective reuptake inhibitors, to induce episodes of mania or to increase cycle frequency or symptom intensity.2,3 The causes for the tendency of antidepressants to ‘switch’ depressed bipolar patients to mania are not understood.
Antimanic mood stabilizers, lithium, valproic acid and carbamazepine, when given chronically to rats to produce plasma concentrations therapeutically relevant to BD, reduce arachidonic acid (AA; 20:4n−6) turnover in phospholipids and expression of AA cascade enzymes in rat brain.4 AA is a polyunsaturated fatty acid found predominately in the stereo-specifically numbered (sn)-2 position of membrane phospholipids, and can be released from this position by certain phospholipase A2 (PLA2) enzymes.5 These enzymes can be activated through their coupling to certain neuroreceptors by a G-protein or by calcium,6–10 or in conditions of neuroinflammation through their coupling to cytokine receptors.11–13 The unesterified AA that is released by a PLA2 can be converted to eicosanoids or other active metabolites, subjected to β-oxidation, or reesterified into the sn-2 position of membrane phospholipids via a long-chain acyl-CoA synthetase and an acyltransferase.14–19 Released AA and its metabolites can influence a number of brain processes, including signal transduction, transcription, neuronal activity, apoptosis, inflammation and blood flow.20–26
The agents lithium, carbamazepine (5H-dibenz[b, f]azepine-5-carboxamide; 5-carbamoyl-5H-dibenz[b, f]azepine) and valproic acid (2-propylpentanoic acid), which are effective in the manic phase of BD, are reported to decrease brain AA turnover rate in membrane phospholipids in unanesthetized rats without changing the turnover rate of docosahexaenoic acid (DHA; 22:6n−3),27–31 another polyunsaturated fatty acid usually found in the sn-2 position. The effect of chronically administered lithium and carbamazepine on AA turnover was ascribed to a selective decrease in the mRNA, protein and activity levels of AA-selective Ca2+-dependent cytosolic PLA2 (cPLA2),32,33 whereas chronic valproate inhibited the activity of an AA-selective acyl-CoA synthetase that is involved in recycling AA back into membrane phospholipids.27,34
In contrast to the three antimanic agents, another mood stabilizer, lamotrigine (3,5-diamino-6-(2,3- dichlorophenyl)-astriazine), which is preferred for treating bipolar depression and rapid cycling, neither decreased AA turnover in brain phospholipids of unanesthetized rats35 nor changed cPLA2 expression, whereas it decreased transcription and protein of cyclooxygenase (COX)-2.36 Topiramate (2,3:4,5-bis-Oisopropylidene- β-d-fructopyranose sulfamate), which had been thought effective against BD37 and shown to reduce hyperactivity in a quinpirole rat model,38 did not significantly change expression of any of the measured enzymes in the AA cascade, including cPLA2, or alter AA or DHA turnover in rat brain phospholipids.39,40 Supporting this lack of effect, four double-blind placebo-controlled trials later demonstrated that topiramate was ineffective in BD,41 and it was withdrawn as a treatment.
A number of antidepressants when administered to bipolar depressed patients cause them to ‘switch’ to mania more frequently than expected.2,3,42,43 Because antimanic mood stabilizers when given chronically to rats downregulate brain AA turnover and AA enzymes, we thought it possible that the increased switching tendency caused by some of these anti-depressants might be due to their upregulating the brain AA cascade. This hypothesis is consistent with evidence that lamotrigine, which does not upregulate AA turnover but reduces COX-2 expression in rat brain,35,36 does not increase switching, whereas fluoxetine, which upregulates AA turnover and cPLA2 mRNA, protein and activity in rat brain,44,45 is reported to do so.42,46,47
To further test the hypothesis, in the present study, we examined brain AA metabolism and expression of brain PLA2 and COX enzymes in rats treated chronically with two additional antidepressants, imipramine (5-[3-(dimethylamino) propyl]-10,11- dihydro-5H-dibenz [b,f]-azepine monohydrochloride) and bupropion ((±)-1- (3-chlorophenyl)-2-[(1,1 dimethylethyl)amino]-1- propanone hydrochloride). Imipramine, a tricyclic antidepressant, is beneficial for unipolar depression, and is thought to act by inhibiting serotonin and noradrenalin reuptake transporters. At a clinically relevant dose (10mgkg−1 body weight), imipramine decreases depression scores in rodents, as measured by a variety of tests.48–51 Bupropion is thought to act by inhibiting both noradrenalin and dopamine reuptake, but has no significant serotonergic effect.52 Acute or chronic bupropion given to rats had an antidepressant effect by reducing immobility time in the forced swim test,53–55 and improved behavior in another animal model of depression.56 Because imipramine but not bupropion is reported to increase switching in bipolar depressed patients,43,47,57–59 we hypothesized that chronic imipramine but not bupropion would increase markers of AA metabolism, including AA turnover in phospholipids and cPLA2 expression, in rat brain.
Chemicals and reagents, including bupropion and imipramine, were purchased from Sigma Chemicals (St Louis, MO, USA) unless otherwise indicated. The study was conducted following the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (Publication no. 80-23) and was approved by the Animal Care and Use Committee of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Animal Care and Use Committee. Male CDF-344 rats, weighing 180–190 g (Charles River Laboratories; Wilmington, MA, USA), were acclimatized for 1 week in an animal facility with controlled temperature, humidity and light cycle, ad libitum access to food (NIH-31) and water. Rats were assigned randomly to an acute or a chronic treatment (imipramine and bupropion) group or to a control group. Chronic drug-treated rats received 10mgkg−1 day−1 imipramine or 30mgkg−1 day−1 bupropion dissolved in saline (0.9%) once daily for 21 days intraperitoneally (i.p.), whereas controls received the same volume of saline once daily i.p., also for 21 days. For acute studies (to separate chronic from acute drug action), rats were randomized into three treatment groups that received a single i.p. injection of imipramine (10mgkg−1) or bupropion (30mgkg−1) or an equivalent volume of saline 3 h before a study.
The chronic dosing regimens for the two drugs are reported to decrease depression scores in rodents, as measured by a variety of behavioral and biochemical tests.48–51,60 On the last day of a dosing regimen, a rat was injected with its appropriate treatment 3 h before its brain was removed for enzyme, protein, prostaglandin E2 (PGE2) or kinetic analysis. For molecular analysis, 3 h after the last injection, the rat was anesthetized with CO2 and decapitated. The frontal cortex was rapidly dissected and frozen in 2-methylbutane at −50 °C, then stored at −80 °C until use. For PGE2 measurements, the rat was lightly anesthetized with sodium pentobarbital (50mgkg−1; Abbott Laboratories, Chicago, IL, USA) and subjected to head-focused microwave irradiation to inactivate enzymes and stop brain metabolism (5.5kW, 4.8 s; Cober Electronics, Stamford, CT, USA).61,62
For the kinetic radiotracer study, the rat was anesthetized with 1–3% halothane, and polyethylene catheters were inserted into the femoral artery and vein as reported.31 The rat was allowed to recover from surgery for 3 h, with its hindquarters loosely wrapped and taped to a wooden block. During recovery, arterial blood pressure was monitored, and temperature was maintained at 37 °C by means of a rectal probe and a heating element (Indicating Temperature Controller; Yellow Springs Instruments, Yellow Springs, OH, USA). Blood pH, pCO2 and pO2 were monitored using a Rapidlab 248 pH/blood gas analyzer (Bayer, East Walpole, MA, USA), and heart rate and blood pressure were analyzed using a CyberSense monitor (Model BPM01 CyQ 302, Nicholasville, KY, USA).
[1-14C]AA (50 mCimmol−1, >98% pure, Moravek Biochemicals, Brea, CA, USA) was prepared in saline containing 50mgml−1 fatty acid-free bovine serum albumin (Sigma) as described previously.31,61 Three hours after the last injection, unanesthetized rats were infused intravenously (i.v.) for 5 min with 1ml containing 170 μCi kg−1 AA at a rate of 0.223 (1−e−1.92t) ml min−1 with a computer-controlled variable rate infusion pump (No. 22; Harvard Apparatus, South Natick, MA, USA) to achieve a steady-state plasma-specific activity within 1 min.30,31,63 Arterial blood samples were collected at 0, 15, 30, 45, 90, 180, 240 and 300 s during infusion to determine radioactive and cold concentrations of non-esterified AA in plasma. Five minutes after starting infusion, the rat was anesthetized with sodium pentobarbital (50mgkg−1, i.v.) and subjected to head-focused microwave irradiation to stop brain metabolism (5.5kW, 4.8 s; Cober Electronics, Norwalk, CT, USA).64,65 The brain was excised, bisected sagittally and stored at −80 °C for further analysis.
Total lipids were extracted from frozen plasma and from one brain hemisphere by the method of Folch.66 Heptadecanoic acid (17:0) was added as an internal standard to plasma before extraction. The extracts were separated by thin layer chromatography on silica gel plates (Whatman, Clifton, NJ, USA). Unesterified fatty acids were separated using a mixture of heptane:diethylether:glacial acetic acid (60:40:2 by volume),29 and phospholipids (choline glycerol-phospholipid, ChoGpl; phosphatidylserine, PtdSer; phosphatidylinositol, PtdIns; ethanolamine glycerophospholipid, EtnGpl) were separated in chloroform:- methanol:H2O:glacial acetic acid (60:50:4:1 by volume)67 and identified with unlabeled standards in separate lanes. Phospholipid and standard bands were visualized with 6-p-toluidine-2-naphthalenesulfonic acid (Acros, Fairlawn, NJ, USA) under ultraviolet light. Each band was removed and analyzed for radioactivity by liquid scintillation counting. Phospholipid bands were also individually scraped and 200 μl toluene was added with a known amount of di-17:0-PtdCho for quantification before methylation. Fatty acid methyl esters were formed by heating the phospholipid scrapes in 1% H2SO4 in methanol at 70 °C for 3 h.68 The methyl esters were separated on a 30m×25mm inner diameter capillary column (SP-2330; Supelco, Bellefonte, PA, USA), using gas chromatography with a flame ionization detector (Model 6890N; Agilent Technologies, Palo Alto, CA, USA). Runs were initiated at 80 °C, with a temperature gradient to 160 °C (10 °Cmin−1) and 230 °C (3°Cmin−1) in 31 min, and held at 230 °C for 10 min. Peaks were identified by retention times of fatty acid methyl ester standards (Nu-Chek-Prep, Elysian, MN). Fatty acid concentrations (nmol g−1 brain or nmol ml−1 plasma) were calculated by proportional comparison of gas chromatography peak areas to that of the 17:0 internal standard. Tracer identification and separation were performed on fatty acid methyl esters of pooled plasma samples (at the end of the infusion) and pooled brain total lipid extracts as described above. The fatty acid methyl esters were separated as described previously with slight modifications69 using a high-performance liquid chromatography (HPLC) (Beckman, Fullerton, CA, USA) equipped with an in-line UV/vis detector (λ= Gilson, Middleton, WI, USA) and an in-line scintillation counter (β-RAM; IN/US System, Tampa, FL, USA) with a Luna C18 column (Phenomenex, Torrance, CA, USA). Initial conditions were set to a 1 ml min−1 gradient system composed of (A) 100% H2O and (B) 100% acetonitrile. The gradient started with 85% B, for 30 min, and then increased to 100% B over 10 min where it was held for 20 min before returning to 85% B over 5 min.
Acyl-CoA species were isolated from the remaining half brain by the method of Deutsch.70 Weighed brain and a known amount of 17:0-CoA as an internal standard were placed in a 15ml conical vial before sonication of the brain in 25mM potassium phosphate. Isopropanol (2 ml) was added to the vial and the homogenate was sonicated again. Saturated ammonium sulfate (0.25 ml) was added and the sample was lightly shaken by hand. Acetonitrile (4 ml) was added and the sample was vortexed for 10 min before centrifugation. The upper phase was extracted and 10 ml of 25mM potassium phosphate was added. Each sample was run three times through an activated oligonucleotide purification cartridge (Applied Biosystems, Foster City, CA, USA), washed with 10 ml of 25mM potassium phosphate and eluted with 400 μl isopropanol:1mM glacial acetic acid (75:25 by volume). Samples were dried under nitrogen and reconstituted in 100 μl isopropanol:1mM glacial acetic acid (75:25 by volume) for HPLC analysis. Acyl-CoA species were separated using HPLC (Beckman) with a Symmetry C-18, 5 μm column (250×.6mm; Waters-Millipore Corp., Milford, MA, USA). Conditions were set to a 1 ml min−1 gradient system composed of (A) 75mM potassium phosphate and (B) 100% acetonitrile. The gradient started with 44% B, increased to 49% over 25 min and then to 70% over 5 min, remained at 70% for 9 min and returned to 44% over 4 min and was held there for 4 min (end of run). Concentrations of acyl-CoA species and their associated radioactivity were measured using peak area analysis from HPLC chromatograms relative to 17:0-CoA and liquid scintillation counting. These values were used to calculate the specific activities of arachidonoyl-CoA.
The model for determining in vivo kinetics of brain fatty acids in rats is described in detail elsewhere.18,30 Briefly, unidirectional incorporation coefficients, (ml s−1 g−1) of [1-14C]AA, representing incorporation from plasma into brain phospholipid compartments i, were calculated as:
is radioactivity of brain lipid i at time T = 5 min (time of termination of experiment), t is time after beginning infusion and is the plasma concentration of labeled AA during infusion. Integrals of plasma radioactivity were determined by trapezoidal integration. The synthesis of AA from its dietary precursor linoleic acid (18:2n−6) represents less than 0.5% of the plasma AA flux into brain.71 Thus, net rates of incorporation of non-esterified AA from plasma into brain phospholipid i, Jin,i, and from brain arachidonoyl-CoA, JFA,i, were calculated as:
cpl (nmol ml−1) is the concentration of unlabeled non-esterified AA in plasma. A ‘dilution factor’ λ is defined as the steady-state ratio during [1-14C]AA infusion of specific activity of the brain arachidonoyl- CoA pool to plasma unesterified AA specific activity,
Cytoplasmic and nuclear extracts were prepared from rat frontal cortex, as previously described.45 Briefly, the frontal cortex was homogenized in 10mM HEPES, pH 7.9, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 10mM KCl, buffer with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA) using a Teflon-glass homogenizer. After adding 0.5% NP-40, five additional strokes were performed. The suspension was incubated for 30 min on ice, and then centrifuged in a microcentrifuge at 13 000 g for 1 h at 4 °C. To the nuclear pellet, solution B (20mM HEPES, pH 7.9, 1mM EDTA, 1mM EGTA, 1mM DTT, 0.4M NaCl) and a protease inhibitor cocktail (Roche) were added. Tubes were mixed and placed on a small rotatory shaker for 30 min. Finally, the mixture was centrifuged at 13 000 g for 1 h at 4 °C. The supernatant containing the proteins from the nuclear extracts was transferred to a fresh tube. Protein concentrations of cytoplasmic and nuclear extracts were determined using Bio-Rad Protein Reagent (Bio-Rad, Hercules, CA, USA). The composition of extracts from each fraction was confirmed using specific antibodies (data not shown).
Supernatants corresponding to the cytosolic fraction were assayed for cPLA2 activity, using appropriate assay kits (Cayman, Ann Arbor, MI, USA). PGE2 was extracted according to the method of Radin.73 A portion of the extract was dried under nitrogen and assayed for PGE2 using a polyclonal enzyme-linked immunosorbent assay according to the manufacturer’s instructions (Oxford Biomedical Research, Product No. EA 02, Oxford, MI, USA).
Proteins from cytoplasmic (50 μg) and nuclear (50 μg) extracts were separated on 4–20% SDS-polyacrylamide gels (PAGE) (Bio-Rad). Following SDS-PAGE, the proteins were electrophoretically transferred to a nitrocellulose membrane. Protein blots were incubated overnight at 4 °C in TBS buffer, containing 5% nonfat dried milk and 0.1% Tween-20, with specific primary antibodies (1:1000 dilution) for the group IVA cPLA2, group IIA secretory phospholipase A2 (sPLA2), group VIA calcium-independent phospholipase A2 (iPLA2),5 COX-1 (1:1000) (Santa Cruz Biotech, Santa Cruz, CA, USA), activator protein-2α (AP-2α), AP-2β, NFκB p65, p50 and COX-2 (1:1000) (Cell Signaling, Beverly, MA, USA). Protein blots were incubated with appropriate HRP-conjugated secondary antibodies (Cell Signaling) and visualized using a chemiluminescence reaction (Amersham, Piscataway, NJ, USA) on X-ray film (XAR-5, Kodak). Optical densities of immunoblot bands were measured using Alpha Innotech Software (Alpha Innotech, San Leandro, CA, USA) and were normalized to β-actin (Sigma) to correct for unequal loading. All the experiments were carried out three times with 10 independent samples per group. Values are expressed as percent of control.
Total RNA and cDNA were prepared as described27 from frontal cortex and measured by real time quantitative RT-PCR, using the ABI PRISM 7000 sequence detection system (Applied Biosystems). Specific primers and probes for cPLA2, purchased from TaqMan gene expression assays (Applied Biosystems), consisted of a 20× mix of unlabeled PCR primers and Taqman minor groove binder (MGB) probe (FAM dye-labeled). The fold change in gene expression was determined using the ΔΔCT method.74 Data are expressed as the relative level of the target gene (cPLA2) in the chronic imipramine- or bupropion- administered animals normalized to the endogenous control (β-globulin) and relative to the control rats (saline injected) (calibrator), as described previously. 32,45 All the experiments were carried out twice in triplicate with 10 independent samples per group.
A one-way analysis of variance with Tukey’s pairwise post hoc test was used to compare means between rats administered chronic bupropion, chronic imipramine or chronic saline (controls) (SAS 9.1.3, Cary, NC, USA). Statistical significance was taken as P≤0.05. Data are presented as means±standard deviation (s.d.) of independent measurements. Western blot and real time RT-PCR data are presented as means ±s.e.m. of independent measurements.
Rats chronically administered imipramine weighed 14% and 11% less than did controls or rats given bupropion, respectively (261±14, 224±12, 253±14 g for control, imipramine and bupropion, respectively, P < 0.001). There was no significant difference in body temperature, blood pressure, heart rate, arterial blood pH, pCO2 or pO2 between treatment groups and controls.
As in our previous reports,28,35,40,44,62,64,75 HPLC separation of fatty acid methyl ester derivatives from total lipid extracts of pooled plasma confirmed that greater than 98% of radioactivity represented labeled AA after 5 min of infusion of [1-14C]AA across treatment groups. HPLC separation of fatty acid methyl ester derivatives from pooled brain total lipid extracts also showed that greater than 95% of total brain radioactivity was in the form of [1-14C]AA across treatment groups (data not shown). Percent radioactivities from plasma and brain total lipid extracts were comparable between the treated and control rats, and are similar to published data.28,35,40,44,62,64,75
Chronic or acute imipramine and bupropion did not significantly change the plasma concentration of unlabeled unesterified AA (Table 1), or of any other measured plasma unesterified fatty acid (data not shown), compared to control. There was no significant difference in the concentration of brain arachidonoyl- CoA or of any other measured acyl-CoA species between treatment groups and controls (Table 1). There was no significant difference in the unlabeled esterified fatty acid concentration including AA in brain phospholipids between treatment groups and controls (Table 1).
The integral of plasma radioactivity (denominator of Equation (1)) after the 5 min of [1-14C]AA infusion was significantly higher in chronic and acute imipramine-treated rats (217 072±98 701, 156 648±15 886 nCi s−1 ml−1, respectively) compared to chronic and acute bupropion-treated rats (125 064±13 459, 134 865±15 792 nCi s−1 ml−1, respectively) and chronic and acute controls (125 166±14 417, 121 588±12 547 nCi s−1 ml−1, respectively). Chronic imipramine, but not bupropion, increased (Equation (1)) of AA in ChoGpl, PtdIns, EtnGpl and total phospholipids by 24%, 26%, 15% and 21% compared to controls, respectively (Table 2). Chronic imipramine, but not chronic bupropion, increased Jin,i (Equation (2)) in ChoGpl, PtdIns, EtnGpl and total phospholipids by 38%, 39%, 29% and 35% compared to controls, respectively (Table 2). λ (Equation (3)) was not significantly different between imipramine and bupropion-treated rats and controls (Table 3). Chronic imipramine, but not chronic bupropion, increased JFA,i (Equation (4)) in ChoGpl, PtdSer, PtdIns, EtnGpl and total phospholipids by 71%, 41%, 72%, 58% and 66% compared to controls, respectively (Table 3). The turnover rate of AA (FFA,i, Equation (5)) was significantly higher within brain ChoGpl (80%), PtdSer (38%), PtdIns (63%), EtnGpl (47%) and total phospholipids (59%) in chronic imipramine-treated rats compared to controls (Table 3). In contrast, acute imipramine and bupropion did not alter any measured kinetics of AA (Tables 1–3).
Chronic imipramine, but not bupropion, significantly increased brain cPLA2 mRNA, protein and phosphorylated protein by 46%, 78% and 103%, respectively, compared to control levels (Figures 1a–c). Chronic imipramine, but not chronic bupropion, significantly increased cPLA2 activity by 26% compared to control (Figure 1d). sPLA2 and iPLA2 protein levels did not differ significantly between the groups (Figure 2).
Cyclooxygenase-1 and COX-2 protein levels and the concentration of PGE2, one of their major metabolites, were not changed significantly after chronic imipramine or bupropion, compared to control (Figure 3).
Activator protein (AP)-2 and nuclear factor-kappa B (NF-κB) are recognized on the promoter region of cPLA2 and regulate cPLA2 transcription.76,77 Chronic imipramine, but not chronic bupropion, significantly increased the brain level of AP-2α protein by 48% (Figure 4a). AP-2β, NF-κB p65 and NF-κB p50 protein levels did not differ significantly between the groups (Figures 4b–d). The increase in AP-2 may have been responsible in part for the increased cPLA2 mRNA.
Acute imipramine and bupropion did not significantly change brain protein levels of cPLA2, sPLA2, iPLA2, COX-1 or COX-2 compared to the control group (Figure 5).
Chronically administered imipramine, but not bupropion, increased AA incorporation and turnover in rat brain phospholipids and increased brain activity, protein, and mRNA levels of AA-selective cPLA2 78 as well as the level of its transcription factor, AP-2α. Activity and protein levels of sPLA2 and iPLA2 were not significantly affected by either drug. None of these changes was observed at 3 h following a single acute injection of imipramine or bupropion, indicating that they reflected the effect of chronic imipramine administration.
These findings, as well as published evidence that drug-induced changes in AA incorporation and turnover in rat brain phospholipids correlate with altered mRNA, protein and activity levels of cPLA2,4,62,79 and expression of its AP-2 transcription factor, suggest that the elevated AA incorporation and turnover rates following chronic imipramine were caused by increased cPLA2 activity. Similar correlated increases in cPLA2 and AA turnover follow chronic administration to rats of the antidepressant, fluoxetine.44
Phosphorylation of cPLA2 protein by mitogenactivated protein kinase (MAPK) can activate cPLA2,80,81 and imipramine is reported to activate the MAPK pathway in hippocampus-derived neuronal stem cells.82 Thus, MAPK activation may have accounted for the increased cPLA2 phosphorylation by chronic imipramine. The increased cPLA2 protein caused by chronic imipramine can be ascribed to an increase in cPLA2 mRNA and increased levels of the transcription factor AP-2α. Earlier studies also reported that increased AP-2 protein and binding activity were associated with increased cPLA2 mRNA and activity following chronic administration of N-methyl-D-aspartate (NMDA) to rats to produce excitotoxicity.62,79 Chronic fluoxetine in rats increased brain cPLA2 mRNA by increasing the RNA stabilizing protein AUF rather than by increasing AP-2.45
Neither chronic imipramine nor bupropion changed COX protein or PGE2 in the rat frontal cortex. Chronic fluoxetine, which increased rat frontal cortex cPLA2 activity and protein, also did not change brain COX expression or PGE2 concentration.44,45 On the other hand, lithium, carbamazepine and valproic acid, when given chronically to rats, decreased brain COX-2 protein and PGE2, and (for lithium and carbamazepine) cPLA2 expression, and lamotrigine decreased COX-2 transcription.4,83,84
Because chronic imipramine, like chronic fluoxetine, increased rat brain cPLA2 expression and AA turnover, without changing COX-2 or PGE2 levels, AA signaling may be related more directly to mood regulation than COX-2 activity or PGE2 formation. The observations that fluoxetine and imipramine increased AA signaling and cPLA2 expression, and effects of the antimanic agents in reducing these parameters, also may mean that bipolar mania is correlated with increased release of AA and increased AA metabolism through non-COX pathways.
Imipramine and fluoxetine, but not bupropion, increase the tendency of bipolar depressed patients to ‘switch’ to mania.42,43,47,57–59,85 Imipramine, which inhibits synaptic reuptake of norepinephrine, serotonin and dopamine, is reported to cause an exceptionally high rate of manic and hypomanic reactions.43,57,86 Bupropion, which inhibits dopamine and norepinephrine but not serotonin reuptake, has a low switch rate,52,87 whereas fluoxetine, which primarily inhibits serotonin reuptake, produces a high switch rate.
The data in this paper, taken with published data on fluoxetine administration, suggest that antidepressant agents that increase switching of bipolar depressed patients to a manic state do so by increasing cPLA2 activity and AA turnover in brain phospholipids. The increased switching tendency may be related to the ability of the drugs to reduce synaptic serotonin uptake and increase serotonin in the synaptic cleft. cPLA2 activation and AA release from phospholipid can be initiated by activation of postsynaptic serotonergic 5-HT2A/2C receptors.6–9 Studies with additional antidepressants could test this suggestion.
Increased brain AA turnover and metabolism may be associated with bipolar mania. In rats that have been subjected to dietary deprivation of n−3 poly-unsaturated acids, brain cPLA2 activity, protein and mRNA are upregulated and scores on tests of aggression and depression, BD-like behaviors, are elevated compared with animals on an adequate diet.88,89 Further, gene expression of cPLA2, sPLA2 and COX- 2 appears increased in postmortem frontal cortex from BD compared to control subjects.90
This work was entirely supported by the Intramural Research Program of the National Institute on Aging, National Institutes of Health.