Effects Of Anti-Unipolar Or Anti-Bipolar Drugs On Astrocytes

IntroductionUnipolar depression and bipolar depression are both common mental disorders, and unipolar depression is usually called depression,and bipolar depression is also called manic-depressive illness.Clinically,anti-unipolar depression drugs are mainly serotonin-specific reuptake inhibitors(SSRIs),fluoxetine as represented,and lithium salts(Li⁺),valproic acid(VPA),and carbamaze-pine(CBZ)are the three classical anti-bipolar drugs.But the accurate pharmacological mechanisms of these drugs have not been clear and need to be further investigated.In brain tissue,phospholipases A₂(PLA₂)mainly includes three subtypes: secretory phospholipase A₂(sPLA₂),cytosolic phospholipase A₂(cPLA₂),and calcium-independent phospholipase A₂(iPLA₂).These PLA₂ could specifically hydrolyze the acyl ester bond at the sn-2 position of glycerol in membrane phospholipids to produce lysophospholipids and free fatty acids(such as arachidonic acid,AA).cPLA₂ activity and arachidonic acid release is linked to dopamine, glutamate,serotonin receptors through different coupling mechanisms.These mechanisms modulate the release of arachidonic acid and levels of second messengers in brain tissue.The majority of cPLA₂ in astrocytes is cPLA_2a.In intact brain, scientists have established that after 6 weeks(Li⁺ and CBZ)or 30 days(VPA)of treatment in rats with doses leading to therapeutically relevant drug levels,each drug decreased the in vivo turnover of arachidonic acid by deacylation from glycerophospholipids followed by re-acylation.Chronic treatment with Li⁺ for 6 weeks decreased the expression of cPLA_2ain rat brain.Serotonin[5-hydroxytryptamine(5-HT)]is an important neurotransmitter in the CNS that has seven subtypes of receptors termed 5-HT₁ through 5-HT₇.5-HT₂ receptor comprises of 5-HT_2A,5-HT_2Band 5-HT_2Creceptors which all express on astrocytes.All three 5-HT₂ receptors are Gq/11 protein-coupled and their stimulation activates phospholipase C(PLC),generating diacylglycerol(DAG)and inositol 1,4,5-trisphosphate(IP3)by hydrolysis of phosphatidyl-inositol 4,5-bisphosphate(PIP2) and leading to an increase of free cytosolic calcium concentration[Ca²⁺]_i.Acute exposure to fluoxetine similarly increases[Ca²⁺]_i in astrocytes by a direct 5-HT₂ receptor action.Recently some reports shows that extracellular-signal regulated kinases(ERK) plays an important role in CNS,for example,in synaptic plasticity and memory formation.Fluoxetine causes ERK phosphorylation,followed by inducing expressions of certain genes.The Fos family of transcription factors includes c-Fos,FosB,Fra-1 and Fra-2 as well as smaller FosB(splice variants of FosB).The activity of all Fos family members is modulated by different kinases,i.e.,MAPK,PKA or PKC,and then Fos protein influences protein stability,DNA-binding activity and the transactivating potential of the transcription factors.In primary cultured astrocytes with dibutyryl cyclic AMP(dBcAMP),there is no serotonin transporter.In the present work,we studied of fluoxetine and anti-bipolar drugs:â‘ the regulation of cPLA₂ by chronic treatment with three anti-bipolar drugs;â‘¡whether fluoxetine-mediated 5-HT_2Breceptor stimulation in astrocytes caused by ERK phosphorylation,followed by effecting the expressions of transcription factors (c-Fos and FosB).MethodsPrimary cultured of mouse astrocytes.â‘ After treating with lithium carbonate, valproate sodium and carbamazepine for 1-4 weeks,measuring the mRNA and protein expressions of three PLA₂ subtypes by RT-PCR and Western blot;â‘¡In astrocytes control or RNA interfered with 5-HT_2Breceptor siRNA,determining the phosphorylation level of ERK_1/2with fluoxetine for 20 min,then checking the mRNA and protein expressions of c-Fos and FosB with fluoxetine for 1 or 4 hours,in the absence or presence of SB204741(5-HT_2Breceptor antagonist)or U0126(an inhibitor of ERK phosphorylation).The results are analysised with one-way ANOVA by SPSS12.0 software.P＜0.05 indicates the statically significant difference.Results1,The chronic effect of three anti-bipolar drugs is enzyme-specificAfter 2 weeks,lithium carbonate(0.25 mM,0.5 mM and 1 mM),carbamazepine (25μM and 50μM),valproate sodium(100μM and 1 mM)could upregulate the mRNA and protein of cPLA_2a,only the time duration was different.But these drugs had non-function to sPLA₂ and iPLA₂,indicating the prolonged treatment with three bipolar drugs having cPLA_2aenzyme-specificity.2,The chronic effect of anti-bipolar drugs to cPLA_2ais drug-specificTopiramate,an anticonvulsant as well as CBZ and VPA,but,which has no anti-bipolar effect.The expression of cPLA_2acould not be regulated by 100μM topiramate for 1-4 weeks.Suggesting the chronic regulation of cPLA_2awith three bipolar drugs being drug-specificity.3,ERK phosphorylation induced by fluoxetine via 5-HT_2BreceptorIn common cultured astrocytes,fluoxetine could increase the phosphorylation level of ERK_1/2in 20 min,but this function was abolished in astrocytes RNA interfered with 5-HT_2Breceptor siRNA.And ERK phosphorylation induced by fluoxetine could be inhibited by GF109203X(PKC inhibition)or BAPTA/AM(a chelator of intracellular Ca²⁺).4,The expression of c-Fos and FosB increased by fluoxetine10μM fluoxetine could respectively increase the mRNA and protein expressions of c-Fos and FosB,which could be inhibited by SB204741 or U0126.Indicating the regulation of c-Fos and FosB by fluoxetine via 5-HT_2Breceptor,and dependenting on ERK phosphorylation mediated by fluoxetine.Discussion 1,The expression of PLA₂ regulated by three anti-bipolar drugsLower concentrations of lithium carbonate led within the period studied only to an up-regulation,but it is possible that longer exposure times might have resulted in a down-regulation.The concept that the length of the treatment period may affect the response is supported by the findings with carbamazepine and valproic acid where the effect of higher concentrations(50μM;1 mM)peaked after 2-3 weeks of treatment and was abolished or greatly reduced after 4 weeks,whereas that to the lower concentration(25μM;100μM)was pronounced after 4 weeks,but also developed more slowly.In contrast,topiramate,which has no anti-bipolar effect had no effect on cPLA_2aexpression,and mRNA expression of iPLA₂ and sPLA₂ was unaltered by Li+, VPA,and CBZ.Thus,the observed effects(up-regulation and down-regulation)show both enzyme specificity and drug specificity,and it was in a systematic fashion dependent upon drug concentration and the length of the treatment period.The concentrations used in the present study are probably pharmacologically relevant.In the present context,it is of interest that cPLA_2ais the only PLA₂ that has specificity for phospholipid substrates containing arachidonic acid(AA).Moreover,the released arachidonic acid can be further oxidized to eicosanoids,converted to endocannabinoids or re-acylated in the membrane,a process that in the past has been quantitatively greatly underestimated.2,The mechanism of c-Fos and FosB expression regulated by fluoxetineRNA interference experiments showed that fluoxetine in cultures of well-differentiated astrocytes exclusively acts on 5-HT_2Breceptors.The potency of fluoxetine in the induction of ERK_1/2transactivation was similar to that previously reported for its effect on[Ca²⁺]i and in agreement with its affinity for the 5-HT_2B receptor.In the present work,we demonstrated that the upregulation of c-Fos and FosB expression by fluoxetine in astrocytes was inhibited by U0126,suggesting that the effects of fluoxetine on gene regulation in astrocytes were induced by 5-HT_2B receptor-mediated ERK_1/2phosphorylation.Whether or not such an effect might be of importance for the therapeutic effect of serotonin-specific uptake inhibitors(SSRIs), depends upon whether other SSRIs also stimulate 5-HT_2Breceptors at realistic extracellular brain concentrations.Fluoxetine and paroxetine have approximately similar therapeutic potency and recent preliminary experiments have shown that the concentration dependence for paroxetine-mediated ERK phosphorylation is virtually identical to that found for fluoxetine.Conclusion1,The expression of cPLA_2amRNA and protein regulated by lithium carbonate, CBZ and VPA had similar trend,and this function was anti-bipolar drug-specific and enzyme-specific.2,Fluoxetine up-regulated the expression of c-Fos and FosB by stimulation of 5-HT_2Breceptor and ERK phosphorylation.