Richard P. Bazinet

Chronic Carbamazepine Decreases the Incorporation Rate and Turnover of Arachidonic Acid but Not Docosahexaenoic Acid in Brain Phospholipids of the Unanesthetized Rat: Relevance to Bipolar Disorder

BACKGROUND The basis for carbamazepines efficacy in treating bipolar disorder is not agreed on. One hypothesis is that, similar to lithium and valproate (antibipolar drugs), carbamazepine might selectively decrease the kinetics of arachidonic acid (AA) in brain phospholipids. METHODS To assess whether it targets brain AA kinetics, we administered carbamazepine (25 mg/kg/day, IP) to rats for 30 days and then determined its effect compared with that of vehicle on incorporation and turnover rates of AA and docosahexaenoic acid (DHA) in brain phospholipids. In unanesthetized rats that had received carbamazepine or vehicle, [1-14C]AA or [1-14C]DHA was infused intravenously, and arterial blood plasma was sampled until the animal was killed at 5 min and its brain, after being microwaved, was used for acyl-coenzyme A (acyl-CoA) and phospholipid fatty acid analysis. RESULTS Chronic carbamazepine, compared with vehicle, decreased the rate of incorporation of AA-CoA (27%-29%) and turnover of AA (25%-27%) but not of DHA-CoA or DHA in brain phospholipids. CONCLUSIONS The results, which are comparable to published findings after chronic administration of lithium and valproic acid to rats, support the hypothesis that drugs effective against mania in bipolar disorder act by selectively downregulating the incorporation rate of AA-CoA and turnover of AA in brain phospholipids.

Selective remodeling of cardiolipin fatty acids in the aged rat heart

BackgroundThe heart is rich in cardiolipin, a phospholipid acylated in four sites, predominately with linoleic acid. Whether or not aging alters the composition of cardiolipin acyl chains is controversial. We therefore measured the fatty acid concentration of cardiolipin in hearts of 4, 12 and 24 month old rats that consumed one diet, adequate in fatty acids for the duration of their life.ResultsThe concentration (nmol/g) of linoleic acid was decreased in 24 month old rats (3965 ± 617, mean ± SD) vs 4 month old rats (5525 ± 656), while the concentrations of arachidonic and docosahexaenoic acid were increased in 24 month old rats (79 ± 9 vs 178 ± 27 and 104 ± 16 vs 307 ± 68 for arachidonic and docosahexaenoic acids, 4 months vs 24 months, respectively). Similar changes were not observed in ethanolamine glycerophospholipids or plasma unesterified fatty acids, suggesting specificity of these effects to cardiolipin.ConclusionThese results demonstrate that cardiolipin remodeling occurs with aging, specifically an increase in highly unsaturated fatty acids.

Chronic NMDA administration to rats up-regulates frontal cortex cytosolic phospholipase A2 and its transcription factor, activator protein-2

Excessive N‐methyl‐d‐aspartate (NMDA) signaling is thought to contribute to bipolar disorder symptoms. Lithium and carbamazepine, effective against bipolar mania, are reported in rats to reduce brain transcription of an arachidonic acid selective calcium‐dependent cytosolic phospholipase A2 (cPLA2), as well as expression of one of its transcription factors, activator protein (AP)‐2. In this study, we determined if chronic administration of NMDA (25 mg/kg i.p.) to rats would increase brain cPLA2 and AP‐2 expression, as these antimanic drugs are known to down‐regulate excessive NMDA signaling. Administration of a daily subconvulsive dose of NMDA to rats for 21 days decreased frontal cortex NMDA receptor (NR)‐1 and NR‐3A subunits and increased cPLA2 activity, phosphorylation, protein, and mRNA levels. The activity and protein levels of secretory phospholipase A2 or calcium‐independent phospholipase A2 were not changed significantly. Chronic NMDA also increased the DNA‐binding activity of AP‐2 and the protein levels of its α and β subunits. These changes were absent following acute (3 h earlier) NMDA administration. The changes, opposite to those found following chronic lithium or carbamazepine, are consistent with up‐regulated arachidonic acid release due to excessive NR signaling and may be a contributing factor to bipolar mania.

Topiramate does not alter the kinetics of arachidonic or docosahexaenoic acid in brain phospholipids of the unanesthetized rat.

Interest in the potential therapeutic utility of topiramate for treating bipolar disorder was stimulated by published reports of investigator-initiated open label clinical studies. Because chronic lithium, carbamazepine and valproate decrease the turnover of arachidonic acid (AA) but not docosahexaenoic acid (DHA) in brain phospholipids of the awake rat, we tested if topiramate would produce similar results. Rats received either topiramate (20 mg/kg twice per day) or vehicle for 14 days and then while unanesthetized were infused intravenously with either [1-14C] AA or [1-14C] DHA for 5 min while blood was collected from the femoral artery at fixed times. Topiramate did not alter the incorporation rate of AA or DHA from their respective brain acyl-CoA pool into brain phospholipids, nor the turnover of AA and DHA in brain phospholipids. The results of our study indicate that topiramate does not possess a pharmacological property that three drugs with proven efficacy in treating bipolar disorder have in common.

RETRACTED: Chronic Administration of Carbamazepine Down-regulates AP-2 DNA-binding Activity and AP-2α Protein Expression in Rat Frontal Cortex

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of author Stanley Rapoport, with approval from Biological Psychiatry Editor, John H. Krystal, MD. The National Institutes of Health has found that Dr. Jagadeesh S. Rao engaged in research misconduct by falsifying data in Figures 1, 3, and 5 of the aforementioned manuscript. No other authors were implicated in the data falsification

Chronic Treatment with Mood Stabilizers Increases Membrane GRK3 in Rat Frontal Cortex

BACKGROUND G-protein receptor kinases (GRKs) are a family of serine/threonine kinases involved in the homologous desensitization of agonist activated G-protein coupled receptors (GPCRs). G-protein coupled receptor supersensitivity, possibly as a result of decreased GRK, has been suggested in affective disorders. METHODS We used immunobloting to determine if chronic, therapeutically relevant doses of lithium (Li+), carbamazepine (CBZ), and valproate (VPA), would increase GRK2/3 protein levels in rat frontal cortex. RESULTS Chronic Li+ (24%) and CBZ (44%) significantly increased GRK3 in the membrane but not cytosol fractions. Chronic VPA had no effect on GRK3. G-protein receptor kinase 2 protein levels were unchanged by all treatments. The GRK3 membrane to cytosol ratio was increased significantly in Li+ and CBZ treated rats. CONCLUSIONS These results show that chronically administered Li+ and CBZ, but not VPA, increase the translocation of GRK3 from cytosol to membrane, possibly correcting supersensitivity of GPCRs in bipolar disorder.

D-Amphetamine Stimulates D2 Dopamine Receptor-Mediated Brain Signaling Involving Arachidonic Acid in Unanesthetized Rats

In rat brain, dopaminergic D2-like but not D1-like receptors can be coupled to phospholipase A2 (PLA2) activation, to release the second messenger, arachidonic acid (AA, 20:4n-6), from membrane phospholipids. In this study, we hypothesized that D-amphetamine, a dopamine-releasing agent, could initiate such AA signaling. The incorporation coefficient, k∗ (brain radioactivity/integrated plasma radioactivity) for AA, a marker of the signal, was determined in 62 brain regions of unanesthetized rats that were administered i.p. saline, D-amphetamine (2.5 or 0.5 mg/kg i.p.), or the D2-like receptor antagonist raclopride (6 mg/kg, i.v.) before saline or 2.5 mg/kg D-amphetamine. After injecting [1-14C]AA intravenously, k∗ was measured by quantitative autoradiography. Compared to saline-treated controls, D-amphetamine 2.5 mg/kg i.p. increased k∗ significantly in 27 brain areas rich in D2-like receptors. Significant increases were evident in neocortical, extrapyramidal, and limbic regions. Pretreatment with raclopride blocked the increments, but raclopride alone did not alter baseline values of k∗. In independent experiments, D-amphetamine 0.5 mg/kg i.p. increased k∗ significantly in only seven regions, including the nucleus accumbens and layer IV neocortical regions. These results indicate that D-amphetamine can indirectly activate brain PLA2 in the unanesthetized rat, and that activation is initiated entirely at D2-like receptors. D-Amphetamines low-dose effects are consistent with other evidence that the nucleus accumbens, considered a reward center, is particularly sensitive to the drug.

Brain Arachidonic Acid Cascade Enzymes are Upregulated in a Rat Model of Unilateral Parkinson Disease

Arachidonic acid (AA) signaling is upregulated in the caudate-putamen and frontal cortex of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, a model for asymmetrical Parkinson disease. AA signaling can be coupled to D2-like receptor initiated AA hydrolysis from phospholipids by cytosolic phospholipase A2 (cPLA2) and subsequent metabolism by cyclooxygenase (COX)-2. In unilaterally 6-OHDA- and sham-lesioned rats, we measured brain expression of cPLA2, other PLA2 enzymes, and COX-2. Activity and protein levels of cPLA2 were significantly higher as was COX-2-protein in caudate-putamen, frontal cortex and remaining brain on the lesioned compared to intact side of the 6-OHDA lesioned rats, and compared to sham brain. Secretory sPLA2 and Ca2+-independent iPLA2 expression did not differ between sides or groups. Thus, the tonically increased ipsilateral AA signal in the lesioned rat corresponds to upregulated cPLA2 and COX-2 expression within the AA metabolic cascade, which may contribute to symptoms and pathology in Parkinson disease.

In vivo imaging of disturbed pre- and post-synaptic dopaminergic signaling via arachidonic acid in a rat model of Parkinson's disease

BACKGROUND Parkinsons disease involves loss of dopamine (DA)-producing neurons in the substantia nigra, associated with fewer pre-synaptic DA transporters (DATs) but more post-synaptic dopaminergic D2 receptors in terminal areas of these neurons. HYPOTHESIS Arachidonic acid (AA) signaling via post-synaptic D2 receptors coupled to cytosolic phospholipase A2 (cPLA2) will be reduced in terminal areas ipsilateral to a chronic unilateral substantia nigra lesion in rats given D-amphetamine, which reverses the direction of the DAT, but will be increased in rats given quinpirole, a D2-receptor agonist. METHODS D-amphetamine (5.0 mg/kg i.p.), quinpirole (1.0 mg/kg i.v.), or saline was administered to unanesthetized rats having a chronic unilateral lesion of the substantia nigra. AA incorporation coefficients, k* (radioactivity/integrated plasma radioactivity), markers of AA signaling, were measured using quantitative autoradiography in 62 bilateral brain regions following intravenous [1-(14)C]AA. RESULTS In rats given saline (baseline), k* was elevated in 13 regions in the lesioned compared with intact hemisphere. Quinpirole increased k* in frontal cortical and basal ganglia regions bilaterally, more so in the lesioned than intact hemisphere. D-amphetamine increased k* bilaterally but less so in the lesioned hemisphere. CONCLUSIONS Increased baseline elevations of k* and increased responsiveness to quinpirole in the lesioned hemisphere are consistent with their higher D2-receptor and cPLA2 activity levels, whereas reduced responsiveness to D-amphetamine is consistent with dropout of pre-synaptic elements containing the DAT. In vivo imaging of AA signaling using dopaminergic drugs can identify pre- and post-synaptic DA changes in animal models of Parkinsons disease.

Chronic D-amphetamine depresses an imaging marker of arachidonic acid metabolism in rat brain

Acute d-amphetamine (d-Amph) administration to rats leads to the release of arachidonic acid (AA, 20:4n-6) as a second messenger following indirect agonism at dopamine D2-like receptors in the brain. We hypothesized that chronically administered d-Amph in rats also would alter brain AA metabolism and signalling. To test this, adult male rats were injected i.p. daily for 2 wk with saline or 2.5 mg/kg d-Amph. After a 1-d washout, the unanaesthetized rats were injected acutely with i.v. saline, 1 mg/kg quinpirole (a D2-like receptor agonist) or 5.0 mg/kg SKF-38393 (a D1-like receptor agonist), followed by i.v. [1-14C]AA. The AA incorporation coefficient k* (brain radioactivity/integrated plasma radioactivity), a marker of AA signalling and metabolism, was quantified using autoradiography in each of 62 brain regions. Compared with chronic saline, chronic d-Amph widely decreased baseline values of k* in brain regions having D2-like receptors. On the other hand, chronic amphetamine did not alter the k* responses to quinpirole seen in chronic saline-treated rats. SKF-38393 had minimal effects on k* in both chronic saline-treated and amphetamine-treated rats, consistent with D1-like receptors not being coupled to AA signalling. The ability of chronic d-Amph after 1-d washout to down-regulate baseline values of k* probably reflects neuroplastic changes in brain AA signalling, and may correspond to depressive behaviours noted following withdrawal from chronic amphetamine in humans and in rats.