Transport of quinolinic acid into rabbit and rat brain

The transport metabolism of [³H]quinolinic acid in the central nervous system of rabbits and rats were studied. In vitro [³H]quinolinic acid was not readily accumulated by isolated choroid plexus. After the intraventricular injection of tracer quantities of [³H]quinolinic acid, the [³H]quinolinic acid did not enter the brain as readily as concurrently injected [¹⁴C]mannitol and was not metabolized, The permeability-surface area constant for [³H]quinolinic acid at the rat blood-brain barrier was 1.5±1.3×10^–5 sec^–1 compared to 2.8±0.4×10^–5 sec^–1 for [³H]mannitol. Our results suggest that: 1) [³H]quinolinic acid is transported in the CNS by passive diffusion and 2) is not metabolized.     

Chronic clozapine reduces rat brain arachidonic acid metabolism by reducing plasma arachidonic acid availability

Chronic administration of mood stabilizers to rats down‐regulates the brain arachidonic acid (AA) cascade. This down‐regulation may explain their efficacy against bipolar disorder (BD), in which brain AA cascade markers are elevated. The atypical antipsychotics, olanzapine (OLZ) and clozapine (CLZ), also act against BD. When given to rats, both reduce brain cyclooxygenase activity and prostaglandin E₂ concentration; OLZ also reduces rat plasma unesterified and esterified AA concentrations, and AA incorporation and turnover in brain phospholipid. To test whether CLZ produces similar changes, we used our in vivo fatty acid method in rats given 10 mg/kg/day i.p. CLZ, or vehicle, for 30 days; or 1 day after CLZ washout. [1‐¹⁴C]AA was infused intravenously for 5 min, arterial plasma was collected and high‐energy microwaved brain was analyzed. CLZ increased incorporation coefficients and rates J_in,i of plasma unesterified AA into brain phospholipids i, while decreasing plasma unesterified but not esterified AA. These effects disappeared after washout. Thus, CLZ and OLZ similarly down‐regulated kinetics and cyclooxygenase expression of the brain AA cascade, likely by reducing plasma unesterified AA availability. Atypical antipsychotics and mood stabilizers may be therapeutic in BD by down‐regulating, indirectly or directly respectively, the elevated brain AA cascade of that disease.     

The absence of ABCD2 sensitizes mice to disruptions in lipid metabolism by dietary erucic acid

ABCD2 (D2) is a peroxisomal transporter that is highly abundant in adipose tissue and promotes the oxidation of long-chain MUFA. Erucic acid (EA, 22:1ω9) reduces very long chain saturated fatty acids in patients with X-linked adrenoleukodystrophy but promotes dyslipidemia and dilated cardiomyopathy in rats. To determine the role of D2 in the metabolism of EA, we challenged wild-type and D2 deficient mice (D2 KO) with an enriched EA diet. In D2 KO mice, dietary EA resulted in the rapid expansion of adipose tissue, adipocyte hypertrophy, hepatic steatosis, and the loss of glycemic control. However, D2 had no impact on the development of obesity phenotypes in two models of diet-induced obesity. Although there was a significant increase in EA in liver of D2 KO mice, it constituted less than 2% of all fatty acids. Metabolites of EA (20:1, 18:1, and 16:1) were elevated, particularly 18:1, which accounted for 50% of all fatty acids. These data indicate that the failure to metabolize EA in adipose results in hepatic metabolism of EA, disruption of the fatty acid profile, and the development of obesity and reveal an essential role for D2 in the protection from dietary EA.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Modulation of phosphoinositide metabolism in rat brain slices by excitatory amino acids,arachidonic acid,and GABA

In rat brain slices the synthesis of [³H]phosphoinositides and the production of [³H]inositol monophosphate (IP₁) induced by norepinephrine (NE) were inhibited by glutamate. Calcium concentrations were varied to test if these inhibitory effects of glutamate were mediated by a calcium-dependent process. Although reducing calcium or addition of the calcium antagonist verpamil reduced the inhibitory effects of glutamate, these results were equivocal because reduced calcium directly decreased agonist-induced [³H]phosphoinositide synthesis. The inhibitory effects of glutamate were mimicked by quisqualate in a dose-dependent manner, but none of a variety of excitatory amino acid receptor antagonists modified the inhibition caused by quisqualate. It is suggested that glutamate activates a quisqualate-sensitive receptor (for which an antagonist is not available) and causes inhibition of phosphoinositide hydrolysis mediated in part by a direct or indirect inhibitory effect of calcium on phosphoinositide synthesis. Modulatory effects of arachidonic acid were examined because glutamate and calcium can activate phospholipase A₂. Arachidonic acid caused a rapid and dose-dependent inhibition of [³H]phosphoinositide synthesis and of NE-stimulated [³H]IP₁ production. A similar inhibition of the response to carbachol also occurred. The inhibition caused by arachidonic acid was unchanged by addition of inhibitors of cyclooxygenase or lipoxygenase. Activation of phospholipase A₂ with melittin caused inhibitory effects similar to those of arachidonic acid. Inhibitors of phospholipase A₂ were found to impair phosphoinositide metabolism, likely due to their lack of specificity for phospholipase A₂. Further studies were carried out in slices that were prelabelled with [³H]inositol in an attempt to separate modulatory effects on [³H]phosphoinositide synthesis and agonist-stimulated [³H]IP₁ production. Several excitatory amino acid agonists inhibited NE-stimulated [³H]IP₁ production. This inhibitory inter-action could be due to impaired synthesis of [³H]phosphoinositides because, even though the slices were prelabeled, addition of unlabelled inositol reduced NE-stimulated [³H]IP₁ production, indicating that continuous regeneration of [³H]phosphoinositides is required. In contrast to the inhibitory effects of the excitatory amino acids, gamma-aminobutyric acid (GABA) enhanced the response to NE in cortical and hippocampal slices. GABA also enhanced the response to carbachol in hippocampal and striatal slices and to ibotenic acid in hippocampal slices. Baclofen potentiated the response to NE similarly to the effect of GABA and baclofen partially blocked the inhibitory effect of arachidonic acid but did not alter that of quisqualate.Abbreviations AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid AP₄ dl-2-amino-4-phosphonobutyric acid - BPB bromphenacyl bromide - BSA bovine serum albumin - CNQX 6-cyano-7-nitroquinoxaline-2,3-dione - DFMO -difluoromethylornithine - DIDS diisothiocyanotostilbene-2,2-disulfonic acid - EGTA ethyleneglycol-bis-N - N, N N-tetraacetic acid - GABA -aminobutyric acid - GDEE glutamate diethyl ether - -GG -glutamylglycine - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - NDGA nordihydroguaiaretic acid - NE norepinephrine - NMDA N-methyl-d-aspartate     

Dietary n-6 polyunsaturated fatty acid deprivation increases docosahexaenoic acid metabolism in rat brain

Dietary n-6 polyunsaturated fatty acid (PUFA) deprivation in rodents reduces brain arachidonic acid (20:4n-6) concentration and 20:4n-6-preferring cytosolic phospholipase A(2) (cPLA(2) -IVA) and cyclooxygenase (COX)-2 expression, while increasing brain docosahexaenoic acid (DHA, 22:6n-3) concentration and DHA-selective calcium-independent phospholipase A(2) (iPLA(2) )-VIA expression. We hypothesized that these changes are accompanied by up-regulated brain DHA metabolic rates. Using a fatty acid model, brain DHA concentrations and kinetics were measured in unanesthetized male rats fed, for 15 weeks post-weaning, an n-6 PUFA 'adequate' (31.4 wt% linoleic acid) or 'deficient' (2.7 wt% linoleic acid) diet, each lacking 20:4n-6 and DHA. [1-(14) C]DHA was infused intravenously, arterial blood was sampled, and the brain was microwaved at 5 min and analyzed. Rats fed the n-6 PUFA deficient compared with adequate diet had significantly reduced n-6 PUFA concentrations in brain phospholipids but increased eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid n-3 (DPAn-3, 22:5n-3), and DHA (by 9.4%) concentrations, particularly in ethanolamine glycerophospholipid (EtnGpl). Incorporation rates of unesterified DHA from plasma, which represent DHA metabolic loss from brain, were increased 45% in brain phospholipids, as was DHA turnover. Increased DHA metabolism following dietary n-6 PUFA deprivation may increase brain concentrations of antiinflammatory DHA metabolites, which with a reduced brain n-6 PUFA content, likely promotes neuroprotection and alters neurotransmission.     

Exogenous arachidonic acid mediates permeability of human brain microvessel endothelial cells through prostaglandin E2 activation of EP3 and EP4 receptors

The blood–brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (ARA; 5,8,11,14‐cis‐eicosatetraenoic acid) is a conditionally essential polyunsaturated fatty acid [20:4(n ? 6)] and is a major constituent of brain lipids. The current study examined the transport processes for ARA in confluent monolayers of human brain microvascular endothelial cells (HBMEC). Addition of radioactive ARA to the apical compartment of HBMEC cultured on Transwell^® inserts resulted in rapid incorporation of radioactivity into the basolateral medium. Knock down of fatty acid transport proteins did not alter ARA passage into the basolateral medium as a result of the rapid generation of prostaglandin E₂ (PGE₂), an eicosanoid known to facilitate opening of the blood–brain barrier. Permeability following ARA or PGE₂ exposure was confirmed by an increased movement of fluorescein‐labeled dextran from apical to basolateral medium. ARA‐mediated permeability was attenuated by specific cyclooxygenase‐2 inhibitors. EP₃ and EP₄ receptor antagonists attenuated the ARA‐mediated permeability of HBMEC. The results indicate that ARA increases permeability of HBMEC monolayers likely via increased production of PGE₂ which acts upon EP₃ and EP₄ receptors to mediate permeability. These observations may explain the rapid influx of ARA into the brain previously observed upon plasma infusion with ARA.

     

Arachidonic acid and glucose uptake by freshly isolated human adipocytes

Fatty acid (FA) and glucose transport into insulin-dependent cells are impaired in insulin resistance (IR; type 2 diabetes mellitus). Studies done on the effects of FAs on glucose uptake, and the influence of insulin on FA uptake by adipocytes, have yielded contradictory results. In this study, isolated human adipocytes were exposed to arachidonic acid (AA) and to insulin, and FA uptake as well as glucose uptake was measured. AA uptake into adipocyte membranes and nuclei was also investigated. Glucose uptake was inhibited by 57 +/- 8% after 30 min of exposure to arachidonate. AA was significantly taken up into adipocyte membranes (49.6 +/- 29% and 123 +/- 74%) at 20 and 30 min of exposure, respectively, and into nuclei (147.6 +/- 19.2%) after 30 min. Insulin stimulated AA uptake (24.1 +/- 14.1%) at 30 min by adipocytes from a non-obese subject, while inhibiting it (16.6 +/- 12%) in adipocytes from an obese subject. These results suggest that: (1) AA inhibits glucose uptake by adipocytes exposed over a short period, probably by a membrane-associated mechanism, (2) insulin-dependent AA uptake is dependent on the body mass index (BMI) of the donor and the insulin sensitivity of their adipocytes.     

Dietary manipulation with high marine fish oil intake of fatty acid composition and arachidonic acid metabolism in rat cerebral microvessels

Male weanling Wistar rats were maintained on one of two semisynthetic diets, differing only in the type of oil used: (i) 10% by weight marine fish oil (MFO group) containing 20% eicosapentaenoic acid (EPA) and 17% docosahexaenoic acid (DHA), or (ii) 10% by weight sunflower oil (SFO group). The control group was kept on standard diet for 4 weeks. Blood-free microvessels were isolated from brain cortex by a rapid micromethod, and their fatty acid composition was determined by gas chromatography. It was found that the proportion of n-3 fatty acids (including EPA and DHA) increased significantly in the microvessels of the MFO group, accompanied by a decrease of the n-6 fatty acid series. The changes in fatty acid composition of endothelial cells were not significant in the SFO group in comparison to the control. The amounts of lipoxygenase and cyclooxygenase metabolites were determined. Dietary fish oil decreased the percentage of total products of arachidonate by 50%, while the SFO diet had no effect on it. The amount of lipoxygenase products in the MFO group decreased significantly from 16931±3131 dpm to 6399±357 dpm/300 mg wet weight of brain. Significantly less PGF-1, PGF-2 and 12-hydroxyhepta-decatrienoic acid (HHT) were found in the capillaries of MFO treated animals, in comparison to the SFO group. The ratios of vasoconstrictor and vasodilator metabolites of arachidonate cascade were not modifed by the diets. Our results suggest that fish oil diet reduces the arachidonate cascade in cerebral microvessels. This effect may explain for the efficiency of n-3 fatty acids in vascular diseases.     

Chronic lithium administration attenuates up-regulated brain arachidonic acid metabolism in a rat model of neuroinflammation

Neuroinflammation, caused by a 6-day intracerebroventricular infusion of lipopolysaccharide (LPS) in rats, is associated with the up-regulation of brain arachidonic acid (AA) metabolism markers. Because chronic LiCl down-regulates markers of brain AA metabolism, we hypothesized that it would attenuate increments of these markers in LPS-infused rats. Incorporation coefficients k* of AA from plasma into brain, and other brain AA metabolic markers, were measured in rats that had been fed a LiCl or control diet for 6 weeks, and subjected in the last 6 days on the diet to intracerebroventricular infusion of artificial CSF or of LPS. In rats on the control diet, LPS compared with CSF infusion increased k* significantly in 28 regions, whereas the LiCl diet prevented k* increments in 18 of these regions. LiCl in CSF infused rats increased k* in 14 regions, largely belonging to auditory and visual systems. Brain cytoplasmic phospholipase A(2) activity, and prostaglandin E(2) and thromboxane B(2) concentrations, were increased significantly by LPS infusion in rats fed the control but not the LiCl diet. Chronic LiCl administration attenuates LPS-induced up-regulation of a number of brain AA metabolism markers. To the extent that this up-regulation has neuropathological consequences, lithium might be considered for treating human brain diseases accompanied by neuroinflammation.     

Uptake of a fluorescent-labeled fatty acid by spiroplasma floricola cells

12-(1-pyrene)dodecanoic fatty acid (P12) uptake by Spiroplasma floricola BNR-1 cells was characterized with regard to its kinetics, specificity, metabolism and susceptibility to protein and lipid inhibitors. The uptake process depended on temperature and pH, and exhibited biphasic saturation kinetics with a very low (2.7 M) and a high (37 M) apparent K _m value. Lauric, myristic, palmitic, stearic and oleic fatty acids did not compete with P12 for transport. The fluorescence of P12 was exclusively recovered in the neutral lipid fraction, suggesting that this fatty acid is not further utilized for phospholipid biosynthesis. Valinomycin, carbonylcyanide m-chlorophenyldrazone (CCCP), dicyclohexylcarbodiimide (DCCD), and pronase strongly reduced P12 uptake by cells, but not by membrane vesicles, affecting the high affinity (low K _m) component of the uptake system. Uptake of P12 by cells, as well as by membrane vesicles, was very sensitive to glutaraldehyde, chlorpromazine, phospholipase A₂₁ and ascorbate with FeCl₃, which affected the low affinity (high K _m) component of a transport system. Digitonin stimulated P12 uptake. We suggest that the incorporation of P12 into spiroplasma cell membrane is a two-step process: a high specificity energy-dependent and protease-sensitive binding to the outer surface of membrane, and a low specificity and energy-independent diffusion and partition into the membrane lipid environment.     

Docosahexaenoic acid synthesis from alpha-linolenic acid by rat brain is unaffected by dietary n-3 PUFA deprivation

Rates of conversion of alpha-linolenic acid (alpha-LNA, 18:3n-3) to docosahexaenoic acid (DHA, 22:6n-3) by the mammalian brain and the brain's ability to upregulate these rates during dietary deprivation of n-3 polyunsaturated fatty acids (PUFAs) are unknown. To answer these questions, we measured conversion coefficients and rates in post-weaning rats fed an n-3 PUFA deficient (0.2% alpha-LNA of total fatty acids, no DHA) or adequate (4.6% alpha-LNA, no DHA) diet for 15 weeks. Unanesthetized rats in each group were infused intravenously with [1-(14)C]alpha-LNA, and their arterial plasma and microwaved brains collected at 5 minutes were analyzed. The deficient compared with adequate diet reduced brain DHA by 37% and increased brain arachidonic (20:4n-6) and docosapentaenoic (22:5n-6) acids. Only 1% of plasma [1-(14)C]alpha-LNA entering brain was converted to DHA with the adequate diet, and conversion coefficients of alpha-LNA to DHA were unchanged by the deficient diet. In summary, the brain's ability to synthesize DHA from alpha-LNA is very low and is not altered by n-3 PUFA deprivation. Because the liver's reported ability is much higher, and can be upregulated by the deficient diet, DHA converted by the liver from circulating alphaLNA is the source of the brain's DHA when DHA is not in the diet.     

Abnormal n-6 fatty acid metabolism in cystic fibrosis is caused by activation of AMP-activated protein kinase

Cystic fibrosis (CF) patients and model systems exhibit consistent abnormalities in PUFA metabolism, including increased metabolism of linoleate to arachidonate. Recent studies have connected these abnormalities to increased expression and activity of the Δ6- and Δ5-desaturase enzymes. However, the mechanism connecting these changes to the CF transmembrane conductance regulator (CFTR) mutations responsible for CF is unknown. This study tests the hypothesis that increased activity of AMP-activated protein kinase (AMPK), previously described in CF bronchial epithelial cells, causes these changes in fatty acid metabolism by driving desaturase expression. Using CF bronchial epithelial cell culture models, we confirm elevated activity of AMPK in CF cells and show that it is due to increased phosphorylation of AMPK by Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ). We also show that inhibition of AMPK or CaMKKβ reduces desaturase expression and reverses the metabolic alterations seen in CF cells. These results signify a novel AMPK-dependent mechanism linking the genetic defect in CF to alterations in PUFA metabolism.     

A mechanism accounting for the low cellular level of linoleic acid in cystic fibrosis and its reversal by DHA

Specific fatty acid alterations have been described in the blood and tissues of cystic fibrosis (CF) patients. The principal alterations include decreased levels of linoleic acid (LA) and docosahexaenoic acid (DHA). We investigated the potential mechanisms of these alterations by studying the cellular uptake of LA and DHA, their distribution among lipid classes, and the metabolism of LA in a human bronchial epithelial cell model of CF. CF (antisense) cells demonstrated decreased levels of LA and DHA compared with wild type (WT, sense) cells expressing normal CFTR. Cellular uptake of LA and DHA was higher in CF cells compared with WT cells at 1 h and 4 h. Subsequent incorporation of LA and DHA into most lipid classes and individual phospholipids was also increased in CF cells. The metabolic conversion of LA to n-6 metabolites, including 18:3n-6 and arachidonic acid, was upregulated in CF cells, indicating increased flux through the n-6 pathway. Supplementing CF cells with DHA inhibited the production of LA metabolites and corrected the n-6 fatty acid defect. In conclusion, the evidence suggests that low LA level in cultured CF cells is due to its increased metabolism, and this increased LA metabolism is corrected by DHA supplementation.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Rat brain arachidonic acid metabolism is increased by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide

In a rat model of acute neuroinflammation, produced by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we measured brain activities and protein levels of three phospholipases A2 (PLA2) and of cyclo-oxygenase-1 and -2, and quantified other aspects of brain phospholipid and fatty acid metabolism. The 6-day intracerebral ventricular infusion increased lectin-reactive microglia in the cerebral ventricles, pia mater, and the glial membrane of the cortex and resulted in morphological changes of glial fibrillary acidic protein (GFAP)-positive astrocytes in the cortical mantel and areas surrounding the cerebral ventricles. LPS infusion increased brain cytosolic and secretory PLA2 activities by 71% and 47%, respectively, as well as the brain concentrations of non-esterified linoleic and arachidonic acids, and of prostaglandins E2 and D2. LPS infusion also increased rates of incorporation and turnover of arachidonic acid in phosphatidylethanolamine, plasmenylethanolamine, phosphatidylcholine, and plasmenylcholine by 1.5- to 2.8-fold, without changing these rates in phosphatidylserine or phosphatidylinositol. These observations suggest that selective alterations in brain arachidonic acid metabolism involving cytosolic and secretory PLA2 contribute to early pathology in neuroinflammation.     

Valproyl-CoA and esterified valproic acid are not found in brains of rats treated with valproic acid,but the brain concentrations of CoA and acetyl-CoA are altered

Sodium valproate and lithium are used to treat bipolar disorder. In rats, both reduce the turnover of arachidonic acid in several brain phospholipids, suggesting that arachidonate turnover is a common target of action of these mood stabilizers. However, the mechanisms by which these drugs reduce arachidonate turnover in brain are not the same. Lithium decreases turnover by reducing the activity and expression of the 85-kDa type IVA cytosolic phospholipase A₂ (cPLA₂); valproate does not affect cPLA₂ activity or expression. To test whether valproate alters neural membrane order by direct esterification into phospholipid or by interrupting intermediary CoA metabolism, we measured valproyl-CoA, esterified valproate, and short chain acyl-CoAs in brains from control rats and rats treated chronically with sodium valproate. Valproyl-CoA and esterified forms of valproate were not found in brain with detection limits of 25 and 37.5 pmol/g brain^–1, respectively. Valproate treatment did result in a 1.4-fold decrease and 1.5-fold increase in the brain concentrations of free CoA and acetyl-CoA when compared to control. Therefore the reduction of brain arachidonic acid turnover by chronic valproate in rats is not related to the formation of valproyl-CoA or esterified valproate, but may involve changes in the intermediary metabolism of CoA and short chain acyl-CoA.     

Uptake of arachidonic acid into membrane phospholipids: Effect on chloride transport across cornea

Summary We demonstrate that arachidonic acid (AA) stimulation of chloride transport across frog cornea is mediated via two independent pathways: (1) stimulation of prostaglandins and cAMP synthesis, and (2) a direct physical change in the membrane produced by substitution of different phospholipid acyl chains. AA is well known as a precursor in the synthesis of prostaglandins, which have been shown to stimulate cAMP synthesis and chloride transport in frog cornea. We show that frog cornea can convert exogenous AA to PGE₂, but that in the presence of 10^–5 m indomethacin both the conversion to PGE₂ and stimulation of cAMP are completely blocked. However, with indomethacin the action of AA to stimulate chloride transport (as measured by SCC) remains, but peak height of the response is reduced to 57% of that found when AA alone is given. Similarly, we show that propranolol completely blocks cAMP stimulation, but stimulation of SCC is reduced to 45% of the original response. Therefore, cAMP appears to be responsible for roughly half of the observed stimulation in SCC. By gas chromatographic analysis we show that significant quantities of AA can rapidly substitute into membrane phospholipids of corneal epithelium and L929 cells following the addition of AA to the medium. Modification of membrane phospholipid structure can affect membrane viscosity, membrane-bound enzyme activity, and the distribution and lateral mobility of integral proteins. It seems likely that such alterations in the properties of the membrane may modulate the rate of chloride transport, and this may constitute the second mechanism. Upon addition of AA, both mechanisms appear to stimulate chloride transport simultaneously, and are apparently additive. We show that prolonged exposure to AA results in a large incorporation of AA into phospholipid and consequently, a perturbation in the ratio of unsaturated to saturated fatty acids. We also find evidence of a compensatory cellular mechanism that alters the ratio of endogenously synthesized fatty acids and tends to reduce the membrane-perturbing effect of AA.     

Uptake of Ga-67 into rat cerebral hemisphere and cerebellum. Comparison with Fe-59.

Transferrin and transferrin receptors play an important role in the transport of iron into the brain. To determine whether gallium enters the brain by the same mechanism, uptakes of Ga and 59Fe have been compared under controlled conditions. Rates of gallium penetration into brain (K) were four times slower than those for 59Fe. Kin for Ga when infused with citrate were 0.88 ± 0.24 and 0.94 ± 0.39 x 10 ml gh for cerebral hemisphere and cerebellum, respectively. When infused as the transferrin complex, Ga uptake into the brain was not different from that when infused with citrate. The presence of the anti-transferrin receptor antibody OX-26 significantly reduced uptake of Fe by 60% and 64% into cerebral hemisphere and cerebellum, respectively. By contrast, pretreatment of rats with OX-26 enhanced the uptake of Ga into brain, particularly when infused with citrate; mean increases in uptake of Ga were 120% and 144% for cerebral hemisphere and cerebellum, respectively. Purified Ga-transferrin was also taken up into both brain regions examined in the presence of OX-26. These results indicate that the transport of non-transferrin bound gallium is an important mechanism for gallium uptake into brain.