Astrocytic Alkalinization By Therapeutically Relevant Lithium Concentrations: Implications For Myo-Inositol Depletion

IntroductionThe most widely accepted theory for the mechanism of the therapeutic effect of the lithium ion(Li⁺) in bipolar disorder is the inositol depletion hypothesis.This hypothesis was proposed on the basis of an uncompetitive inhibition of the regeneration of myo-inositol,needed for continued phospholipase C(PLC) signaling,from inositol monophosphate by low concentrations of Li⁺.Later an additional mechanism for Li⁺-mediated myo-inositol depletion was reported,i.e.,an inhibition of the cellular uptake of myo-inositol observed in astrocytes treated chronically with Li⁺.Cellular uptake is important,because the only cells within the brain that synthesize myo-inositol (from glucose) are endothelial cells.These findings do not explain why Li+ treatment is effective against both manic and depressive phases,but additional studies of myo-inositol uptake showed that uptake inhibition in Li⁺-treated astrocytoma cells only occurred at high concentrations of rnyo-inositol,whereas the uptake at a low concentration of myo-inositol was enhanced following Li⁺ treatment.As a possible explanation for this result it was pointed out that myo-inositol uptake in astrocytes occurs by the aid of two inositol uptake carriers,the high-affinity Na⁺-dependent inositol transporter(SMIT) and the lower affinity H⁺-dependent inositol transporter(HMIT),where SMIT shows an increased activity at higher pH,whereas HMIT shows a reduced activity at higher pH.Accordingly myo-inositol uptake by the low-affinity HMIT would be reduced and that by the high-affiny SMIT increased,if Li⁺ treatment caused an alkalinization of the astrocytes. That this scenario may be relevant for the mechanism of action by Li⁺ treatment and/or prophylaxis of both mania and depression in bipolar patients is supported by magnetic resonance spectroscopy(MRS) studies,which have shown a decrease of pH in the brain of bipolar patients,including drug-free patients and led to the conclusion that cerebral pH may be decreased in bipolar disorder.In the present study we have therefore used primary cultures of mouse astrocytes, which functionally react to chronic treatment with Li⁺,to investigateâ…°) whether therapeutically relevant concentrations of Li⁺(0.5,1.0 and 2.0 mM) have acute and/or chronic effect on steady-state intracellular pH(pH_i);â…±) whether Li⁺ has acute and/or chronic effects on the Na⁺/H⁺ exchanger(NHE),the most important bicarbonate/CO₂-independent acid-extruding transporter in mammalian cells;â…²) whether Li⁺ regulates mRNA and/or protein expression of NHE1,a major NHE isoform in cultured astrocytes;andâ…³) the magnitude of the intracellular Li⁺ concentrations during continued Li⁺ treatment.Experimental MethodsThe Primary astrocytes were dissociated from the neophallia of the cerebral hemispheres.After treated with dBcAMP for 1 week,astrocytes were acute or chronic treated with lithium carbonate or lithium chloride,which both provide Li⁺ concentration of 2.0,1.0,or 0.5 mM.After treatment,the astroyctes were loaded with 5μM fluorescent pH-sensitive indicator,BCECF-AM.Then the steady state pHi was measured.The activity of NHE1 was determined by acute NH₄Cl-prepulse technique. After chronic treatment,the cells were collected for RT-PCR and Western-Blot to detect mRNA and protein expression of NHE1.The results are analysised with one-way ANOVA by Fisher's LSD multiple comparison test for unequal replications.The level of significance was set at P＜0.05.Results1.Intracellular concentration of Li⁺.The intracellular concentration of Li⁺ increased rapidly during Li⁺ treatment with little,if any difference between cells treated for 0.5 hr,1 hr,or 3 days and a maximum ratio between intracellular and extracellular Li⁺ of 0.40(after treatment with 0.5 mM Li⁺ for 3 days).After treatment for 3 weeks there was a doubling of the intracellular Li⁺ concentration at 1 and 2.0 mM extracellular Li⁺,although the intracellular concentration of Li⁺ remained well below its extracellular concentration,creating an extracellular/intracellular Li⁺ gradient of at most 0.50.2.pH_i. In astrocytes that had not been treated with Li⁺ the average pH_i was between 7.0 and 7.20 in 3-4-week-old cultures and around 7.30 in 6-7-week-old cultures,with values between 6.8 and 7.4 in individual cultures.Acute treatment with 1.0 mM lithium carbonate(2.0 mM Li⁺) for between 0.5 and 2 h caused no statistically significant change in pH_i,but chronic treatment with 2.0 mM Li⁺ for 3 days or 1,2,3 and 4 weeks increased it by～0.20 at all time points(P＜0.05);similar chronic treatment with a Li⁺ concentration of 1.0 mM caused an increase of about 0.10,that was statistically significant at 2 and 3 weeks(P⁰.05).A Li⁺ concentration of 0.5 mM did not significantly change pHi.Similar results were obtained after treatment with lithium chloride for 2 weeks,indicating that the effect was evoked by the lithium ion not the accompanying anion.3.NHE Activity.In untreated cells 2.0 mM Li⁺ exerted an acute effect on NHE activity,measured asâ–³pH_i/â–³t during recovery after NH₄Cl challenge.The activity after 30 min of continued exposure to 0.5,1 or 2.0 mM Li⁺ was significantly(P＞0.05) increased by almost 50,slightly above 50 and almost 100%.Again,similar effects were observed with lithium chloride.The effect onâ–³pH_i/â–³t of chronic treatment with 0.5 mM Li⁺ during 2 weeks was not statistically significant and appeared smaller than that in cells that had not been chronically treated,even when Li⁺ was present during the measurement of NHE activity.However,after treatment with 1 or 2.0 mM Li⁺ there was a statistically significant(P＜0.05) increase of almost the same magnitude as during acute treatment. When Li⁺ was withdrawn immediately before the measurement of NHE activity,there was a tendency towards a decrease of NHE activity in cells treated with Li⁺ for two weeks.After treatment with a Li⁺ concentration of 0.5 mM there was little,if any effect but after treatment with 1 or 2.0 mM Li⁺,the activity of NHE was reduced by～10 and～20%,respectively,a statistically significant effect(P＜0.05).4.mRNA expression of NHE.mRNA Expression of NHE1 was examined after 1 or 3 days,or 1,2,3 or 4 weeks of treatment with 0.5,1 or 2.0 mM Li+.It was unchanged after 1 day,regardless of the concentration of Li⁺ and at all times studied after exposure to 0.5 mM Li⁺.However it was significantly decreased to～30%(P＜0.05) of control after treatment with 1 or 2.0 mM Li⁺ for between 3 days and 4 weeks.5.Protein expression of NHE1Regardless whether the cultures were treated with LiCl or lithium carbonate, treatment with 0.5,1,or 2.0 mM Li⁺ for 3 weeks had no significant effect on protein expression of NHE1.DiscussionBy studying the range 0.5-2.0 mM lithium,the pharmacologically relevant plasma concentration range has been included.The observation that the intracellular Li⁺ concentration is lower than its extracellular concentration is consistent with the observation of Li⁺ brain concentrations in the human brain in vivo between 0.23 and 0.55 mM at a plasma Li⁺ concentration of 0.7 mM.Accumulation of Li⁺ is likely to occur both via the Na⁺/H⁺ exchanger⁺,in agreement with the observation that Li⁺ inhibits Na⁺ uptake by this transporter by 30%at 1.0 mM Li⁺,and the Na⁺,K⁺-ATPase, consistent with an inhibition of K⁺ uptake.The uptake must be balanced by extrusion of Li⁺,also mediated by the Na⁺,K⁺-ATPase,which is in agreementt with the observation that Li⁺ has affinity for both the extracellular K⁺-activated site and the intracellular Na⁺-activated site of the Na⁺,K⁺-ATPase.The observed values for pHi in control cultures are similar to literature data for mammalian astrocytes in the absence of bicarbonate/CO₂.The intracellular alkalinization by Li⁺ is consistent with the conclusion in a review by Aronson that inward gradients of Li⁺ stimulate H⁺ efflux by an exchange of external Li⁺ for internal H⁺ by a Li⁺-mediated stimulation at the extracellular site of NHE.The inward gradients of Li⁺ also improve the activity of NHE in astrocytes with acute or chronic treatment with Li⁺.The down-regulation of NHE 1 mRNA in response to continuous stimulation by Li⁺ is in agreement with NHE1 being the predominant NHE isoform in astrocytes.The tendency towards a smaller Li⁺-mediated stimulation of NHE activity in cells during chronic treatment with Li⁺ than in acutely treated cells,and the reduction in NHE activity immediately after withdrawal of Li⁺ after chronic treatment are consistent with a down-regulation of NHE in response to continued stimulation.The mechanism of this homeostatic regulation is unknown but a similar response occurs in the case of K⁺ uptake,the rate of which becomes normalized(i.e.,upregulated) after chronic treatment with Li⁺.There is precedence from other systems,e.g.,experimentally induced heart failure that down-regulation of mRNA does not necessarily imply that expression of the corresponding protein is also down-regulated,even after a considerable length of time.Although NHE responded acutely to extracellular Li⁺,the change in intracellular pH required chronic treatment,especially at the lower Li⁺ concentrations.Moreover,in the clinical setting,there will be another lag phase until changes in uptake of myo-inositol lead to changes in myo-inositol content,all of which may contribute to the considerable length of time required for Li⁺ treatment to become effective.That the intracellular alkalinization in response to Li⁺ treatment may be relevant for its efficacy in treatment of bipolar disorder is suggested by a preliminary finding that chronic treatment with therapeutically relevant concentrations of carbamazepine or valproate also leads to an increased pHi in cultured astrocytes,albeit by a different mechanism (stimulation of Na/HCO₃ cotransporter).Besides increasing myo-inositol uptake by SMIT and decreasing myo-inositol uptake by HMIT an increased pHi has additional effects on astrocytes.It may be of special interest that cPLA₂ is stimulated by elevated pHi,since chronic treatment with Li⁺,carbamazepine or valproic acid has been found to increase this enzyme in cultured astrocytes and with the exception of valproic acid also in the brain in vivo.Another reaction that is stimulated by increased phi is the astrocyte-specific racemation of L-serine,generating D-serine,an important agonist at the glycine-binding site of the NMDA receptor.An ecto-nucleotidase,degrading ATP to AMP and pyrophosphate is also stimulated by increased pH,at least in C6 glioma cells,but since this is an extracellular enzyme its activity is decreased by enhanced NHE activity.An increased extracellular acidity also decreases NMDA receptor activity.To what extent these other effects of increased pHi in astrocytes might affect astrocytic activity or whole-brain function is unknown,as is their possible correlation with bipolar disorder.The influx of Na⁺ by stimulation of NHE1 might affect intracellular ion content and cell swelling.Several studies show that stimulation of NHE1 might lead to uptake of water,which could explain the slight increase in brain water in Li⁺-treated rats and the occurrence of pseudotumor cerebri as a side effect of Li⁺ treatment in humans.Thus Li⁺-mediated stimulation of astrocytic NHE1 exchanger might be associated with,not only therapeutic effects of Li⁺,but also some of its side effects. ConclusionChronic treatment with a pharmacologically relevant Li⁺ concentration increases pHi in astrocytes,the mechanism is that inward gradients of Li⁺ sitmulate H⁺ efflux by an exchange of external Li⁺ for internal H⁺ by a Li⁺-mediated stimulation at the extracellular site of NHE.The astrocytic alkalinization may create conditions for decreased uptake of high myo-inositol concentrations and increased uptake of low concentrations.The pharmacological relevance of this effect is supported by literature data suggesting brain acidosis that increased that carbamazepine and valproate also increase pH_i in astrocytes.Stimulation of NHE1-stimulated sodium ion uptake might also trigger uptake of chloride ions and osmotically obliged water.