Chronic lithium downregulates cyclooxygenase-2 activity and prostaglandin E(2) concentration in rat brain.

Rats treated with lithium chloride for 6 weeks have been reported to demonstrate reduced turnover of arachidonic acid (AA) in brain phospholipids, and decreases in mRNA and protein levels, and enzyme activity, of AA-selective cytosolic phospholipase A(2)(cPLA(2)). We now report that chronic lithium administration to rats significantly reduced the brain protein level and enzyme activity of cyclooxygenase-2 (COX-2), without affecting COX-2 mRNA. Lithium also reduced the brain concentration of prostaglandin E(2) (PGE(2)), a bioactive product of AA formed via the COX reaction. COX-1 and the Ca(2+)-independent iPLA(2) (type VI) were unaffected by lithium. These and prior results indicate that lithium targets a part of the AA cascade that involves cPLA(2) and COX-2. This effect may contribute to lithium's therapeutic action in bipolar disorder.

Energy consumption by phospholipid metabolism in mammalian brain.

Until recently, brain phospholipid metabolism was thought to consume only 2% of the ATP consumed by the mammalian brain as a whole. In this paper, however, we calculate that 1.4% of total brain ATP consumption is consumed for the de novo synthesis of ether phospholipids and that another 5% is allocated to the phosphatidylinositide cycle. When added to previous estimates that fatty acid recycling within brain phospholipids and maintenance of membrane lipid asymmetries of acidic phospholipids consume, respectively, 5% and 8% of net brain ATP consumption, it appears that phospholipid metabolism can consume up to 20% of net brain ATP consumption. This new estimate is consistent with recent evidence that phospholipids actively participate in brain signaling and membrane remodeling, among other processes.

Chronic nutritional deprivation of n-3 alpha-linolenic acid does not affect n-6 arachidonic acid recycling within brain phospholipids of awake rats.

Using an in vivo fatty acid model and operational equations, we reported that esterified and unesterified concentrations of docosahexaenoic acid (DHA, 22 : 6 n-3) were markedly reduced in brains of third-generation (F3) rats nutritionally deprived of alpha-linolenic acid (18 : 3 n-3), and that DHA turnover within phospholipids was reduced as well. The concentration of docosapentaenoic acid (DPA, 22 : 5 n-6), an arachidonic acid (AA, 20 : 4 n-6) elongation/desaturation product, was barely detectable in control rats but was elevated in the deprived rats. In the present study, we used the same in vivo model, involving the intravenous infusion of radiolabeled AA to demonstrate that concentrations of unesterified and esterified AA, and turnover of AA within phospholipids, were not altered in brains of awake F3-generation n-3-deficient rats, compared with control concentrations. Brain DPA-CoA could be measured in the deprived but not control rats, and AA-CoA was elevated in the deprived animals. These results indicated that AA and DHA are recycled within brain phospholipids independently of each other, suggesting that recycling is regulated independently by AA- and DHA-selective enzymes, respectively. Competition among n-3 and n-6 fatty acids within brain probably does not occur at the level of recycling, but at levels of elongation and desaturation (hence greater production of DPA during n-3 deprivation), or conversion to bioactive eicosanoids and other metabolites.

Chronic valproate treatment decreases the in vivo turnover of arachidonic acid in brain phospholipids: a possible common effect of mood stabilizers.

Both (Li(+)) and valproic acid (VPA) are effective in treating bipolar disorder, but the pathway by which either works, and whether it is common to both drugs, is not agreed upon. We recently reported, using an in vivo fatty acid model, that Li(+) reduces the turnover rate of the second messenger arachidonic acid (AA) by 80% in brain phospholipids of the awake rat, without changing turnover rates of docosahexaenoic or palmitic acid. Reduced AA turnover was accompanied by down-regulation of gene expression and protein levels of an AA-specific cytosolic phospholipase A(2) (cPLA(2)). To see if VPA had the same effect on AA turnover, we used our in vivo fatty acid model in rats chronically administered VPA (200 mg/kg, i.p. for 30 days). Like Li(+), VPA treatment significantly decreased AA turnover within brain phospholipids (by 28-33%), although it had no effect on cPLA(2) protein levels. Thus, both mood stabilizers, Li(+) and VPA have a common action in reducing AA turnover in brain phospholipids, albeit by different mechanisms.

Intravenously injected [1-14C]arachidonic acid targets phospholipids, and [1-14C]palmitic acid targets neutral lipids in hearts of awake rats.

The differential uptake and targeting of intravenously infused [1-14C]palmitic ([1-14C]16:0) and [1-14C]arachidonic ([1-14C]20:4n-6) acids into heart lipid pools were determined in awake adult male rats. The fatty acid tracers were infused (170 microCi/kg) through the femoral vein at a constant rate of 0.4 mL/min over 5 min. At 10 min postinfusion, the rats were killed using pentobarbital. The hearts were rapidly removed, washed free of exogenous blood, and frozen in dry ice. Arterial blood was withdrawn over the course of the experiment to determine plasma radiotracer levels. Lipids were extracted from heart tissue using a two-phase system, and total radioactivity was measured in the nonvolatile aqueous and organic fractions. Both fatty acid tracers had similar plasma curves, but were differentially distributed into heart lipid compartments. The extent of [1-14C]20:4n-6 esterification into heart phospholipids, primarily choline glycerophospholipids, was elevated 3.5-fold compared to [1-14C]16:0. The unilateral incorporation coefficient, k*, which represents tissue radioactivity divided by the integrated plasma radioactivity for heart phospholipid, was sevenfold greater for [1-14C]20:4n-6 than for [1-14C]16:0. In contrast, [1-14C]16:0 was esterified mainly into heart neutral lipids, primarily triacylglycerols (TG), and was also found in the nonvolatile aqueous compartment. Thus, in rat heart, [1-14C]20:4n-6 was primarily targeted for esterification into phospholipids, while [1-14C]16:0 was targeted for esterification into TG or metabolized into nonvolatile aqueous components.

85 kDa cytosolic phospholipase A2 is a target for chronic lithium in rat brain.

The mechanism by which chronic lithium exerts its therapeutic effect in brains of bipolar patients is not known. One possibility, suggested by our demonstration in the rat brain, is that chronic lithium inhibits turnover of arachidonic acid (AA) by reducing the activity of an AA-specific phospholipase A2 (PLA2). To test this further, mRNA levels of two AA-specific PLA2s, cytosolic PLA2 (cPLA2) type IV and intracellular PLA2 (iPLA2) type VIII, and protein level of cPLA2 were quantified in the brain of rats given lithium for 6 weeks. Chronic lithium markedly reduced brain mRNA and protein level of cPLA2, but had no effect on mRNA level of iPLA2. These results suggest that the final common path effect of chronic lithium administration is to reduce turnover of AA in brain by down-regulating cPLA2.

Effects of maturation on the phospholipid and phospholipid fatty acid compositions in primary rat cortical astrocyte cell cultures.

Phospholipid and phospholipid fatty acid compositional changes were studied in rat cortical astrocytes during dibutyryl cyclic adenosine monophosphate (dBcAMP, 0.25 mM) treatment starting after 14 days in culture (DIC). After 15 DIC, ethanolamine- and choline glycerophospholipid levels were increased 1.2- and 1.3-fold, respectively in treated compared to control cells. However, after 21 and 28 DIC, these levels were not significantly different between groups. Both groups had an increase in phosphatidylserine levels with increasing time in culture. Similarly, ethanolamine plasmalogen levels were transiently elevated after 21 DIC, but returned to previous levels after 28 DIC. The phospholipid fatty acid compositions for the acid stable and labile ethanolamine- and choline glycerophospholipids indicated that in dBcAMP treated cells, 20:4 n-6 and 22:6 n-3 proportions were elevated with increasing time in culture relative to control cells. As 20:4 n-6 proportions increased, there was a concomitant decrease in 20:3 n-9 proportions, suggesting an up regulation of n-6 series elongation and desaturation. In contrast, in control cells, the 20:4 n-6 proportions decreased with a corresponding increase in the 20:3 n-9 proportions. Thus, in treated cells, the cellular phospholipid fatty acid composition was dramatically different than control cells, suggesting that dBcAMP treatment may act to increase fatty acid elongation and desaturation.

Phospholipase A2 and its role in brain tissue.

Phospholipase A2 (PLA2) is the name for the class of lipolytic enzymes that hydrolyze the acyl group from the sn-2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of the PLA2-catalyzed reaction can potentially act as second messengers themselves, or be further metabolized to eicosanoids, platelet-activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can potentially alter many ongoing cellular processes. The presence of PLA2 in the central nervous system, accompanied by the relatively large quantity of potential substrate, poses an interesting dilemma as to the role PLA2 has during both physiologic and pathologic states. Several different PLA2 enzymes exist in brain, some of which have been partially characterized. They are classified into two subtypes, Ca2+-dependent and Ca2+-independent, based on their catalytic dependence on Ca2+. Under physiologic conditions, PLA2 may be involved in phospholipid turnover, membrane remodeling, exocytosis, detoxification of phospholipid peroxides, and neurotransmitter release. However, under pathological situations, increased PLA2 activity may result in the loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and the accumulation of lipid peroxides. These processes, along with loss of ATP, may be responsible for the loss of membrane phospholipid and subsequent neuronal injury found in ischemia, spinal cord injury, and other neurodegenerative diseases. This review outlines the current knowledge of the PLA2 found in the central nervous system and attempts to define the role of PLA2 during both physiologic and pathologic conditions.

Separation of neutral lipids by high-performance liquid chromatography: quantification by ultraviolet, light scattering and fluorescence detection.

The recent increased use of cell cultures to model physiological events, in particular signal transduction and traumatic injury, has produced a need for a quantitative, high-performance liquid chromatographic separation of neutral lipid classes with a high degree of resolution and reproducibility. We report an isocratic separation using a Phenomenex Selectosil silica column (5 microns). Two solvents were used, 1.2% 2-propanol in n-hexane containing 0.1% acetic acid (90%) and n-hexane (10%) at a flow-rate of 0.6 ml/min. Column temperature was maintained at 55 degrees C and this temperature was critical for baseline resolution of 1,3-diacylglycerol and cholesterol. The use of 10% n-hexane permitted the resolution of low polarity compounds such as butylated hydroxytoluene, triacylglycerols and cholesteryl esters. All of the detectors used produced standard curves with linearity over a wide concentration range.

Altered lipid metabolism in the presence and absence of extracellular Ca2+ during combined oxygen-glucose deprivation in primary astrocyte cultures.

The effect of combined oxygen-glucose deprivation (COGD) on lipid metabolism in primary rat cortical astrocyte cultures was studied in both the presence and absence of extracellular Ca2+. In this study, increases in intracellular Ca2+ from internal Ca2+ stores were not inhibited nor were internal Ca2+ levels buffered. Combined oxygen-glucose deprivation resulted in a quantitative reduction in phospholipid levels and an increase in free fatty acid and lysophospholipid levels. Four hours after the onset of COGD, ethanolamine- and choline glycerophospholipid levels were decreased by 40 and 46% from control levels in the presence of Ca2+, respectively. A similar decrease was found 6 hr after onset of COGD in the absence of Ca2+. These changes were accompanied by elevated levels of the corresponding lysophospholipids. However, the increases in lysophospholipid content did not account for the entire loss of ethanolamine- or choline glycerophospholipid. Phosphatidylserine was reduced in both the presence and absence of extracellular Ca2+ but phosphatidylinositol was only decreased in the absence of Ca2+. Statistically significant increases in total fatty acid (FA) and polyunsaturated fatty acid (PUFA) levels occurred at 30 min and 3 hr after the onset of COGD in the absence and presence of Ca2+, respectively. Arachidonic acid levels were increased in both groups by 1 hr. These increases in FA, PUFA, and specifically arachidonic acid were time-dependent and increased over the 12 hr of COGD. Collectively, these results indicate the activation of an acylhydrolase mechanism in the possible presence of an inhibited reacylation pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatography separation and quantitation of methylprednisolone from rat brain.

A sensitive, reliable method for the extraction, separation, and quantitation of methylprednisolone from rat brain is reported. The method can accurately quantitate methylprednisolone levels between 9.8 and 2500 ng/injection using a two-step HPLC separation and monitoring absorbance at 254 nm. A 90% extraction recovery of methylprednisolone (interday variation of 9.0% and an intraday variation of 0.0 to 7.7%) from rat cortex was obtained with a double extraction method using low toxicity solvents. These solvents are known to quantitatively extract the neutral lipids and phospholipids from brain. Combined with the ability to separate the neutral lipid and methylprednisolone fractions for further separation, and the ability to separate all phospholipid classes in the first run, this method offers great utility combined with the reliable, high extraction recovery and sensitive quantitation of methylprednisolone.