Rat Brain Microsomes Research Articles

Glucuronidation of apomorphine

Apomorphine, a dopaminergic receptor agonist, is largely used in the therapy of Parkinson's disease. In this study, we characterized the glucuronidation of apomorphine and other catechols in rat liver and brain microsomes, using UDP-[U- 14C]glucuronic acid and separation of the glucuronides formed by a thin layer chromatographic method. rat liver microsomes glucuronidate apomorphine at a significant rate, that was increased in the presence of dithiothreitol. Two apomorphine glucuronides were separated by high pressure liquid chromatography. We showed by electrospray mass spectrometry that both products were monoglucuronides. Other catechols were also glucuronidated in liver microsomes at various rates, and among them, 4-nitrocatechol was the most efficiently conjugated. in rat brain microsomes, only 4-nitrocatechol was significantly glucuroni-dated, suggesting that in the liver, several uridine-diphosphate glucuronosyltransferase (UGT) isoforms participate to the conjugation of catechols. To determine which isoforms catalyze apomorphine glucuronidation, two recombinant enzymes expressed in V79 cells were used. The isoform UGT1A6 was unable to glucuronidate apomorphine, but we observed a significant activity catalyzed by the isoform UGT2B1. These results provide, to our knowledge, the first demonstration of apomorphine conjugation by recombinant UGT2B1, and the first evidence of the lack of apomorphine glucuronidation in the rat brain.

Separate entry pathways for phosphate and oxalate in rat brain microsomes.

ATP-dependent (45)Ca uptake in rat brain microsomes was measured in intracellular-like media containing different concentrations of PO(4) and oxalate. In the absence of divalent anions, there was a transient (45)Ca accumulation, lasting only a few minutes. Addition of PO(4) did not change the initial accumulation but added a second stage that increased with PO(4) concentration. Accumulation during the second stage was inhibited by the following anion transport inhibitors: niflumic acid (50 microM), 4,4'-dinitrostilbene-2, 2'-disulfonic acid (DNDS; 250 microM), and DIDS (3-5 microM); accumulation during the initial stage was unaffected. Higher concentrations of DIDS (100 microM), however, inhibited the initial stage as well. Uptake was unaffected by 20 mM Na, an activator, or 1 mM arsenate, an inhibitor of Na-PO(4) cotransport. An oxalate-supported (45)Ca uptake was larger, less sensitive to DIDS, and enhanced by the catalytic subunit of protein kinase A (40 U/ml). Combinations of PO(4) and oxalate had activating and inhibitory effects that could be explained by PO(4) inhibition of an oxalate-dependent pathway, but not vice versa. These results support the existence of separate transport pathways for oxalate and PO(4) in brain endoplasmic reticulum.

The role of cyclic nucleotides and calcium in the relaxation produced by amrinone in rat aorta

(1) The vasorelaxation produced by the phosphodiesterase 3 (PDE3) inhibitor, amrinone was investigated in isolated rat aorta denuded of endothelium. In the presence of extracellular Ca 2+, amrinone, milrinone and 3-isobutyl-1-methylxanthine (IBMX), relaxed endothelium-denuded rat aortic rings constricted with phenylephrine. While the actions of milrinone and IBMX were inhibited by the protein kinase G (PKG) inhibitor, Rp-8-Bromo guanosine-3′,5′ monophosphothioate (Rp-8-Br-cGMPS; 0.5 mM), that of amrinone was only slightly affected; whereas the protein kinase A (PKA) inhibitor, Rp-adenosine-3′,5′ cyclic monophosphothioate (Rp-cAMPS; 0.5 mM) had no effect on any agent. (2) Amrinone (100 μM) inhibited 45Ca 2+ influx through receptor- or store-operated Ca 2+ channels following stimulation with phenylephrine (1 μM) or thapsigargin (1 μM). In contrast, amrinone had no effect on KCl (120 mM)-stimulated Ca 2+ influx. (3) In the absence of extracellular Ca 2+, amrinone (30 μM) inhibited the constriction produced by phenylephrine, 5-hydroxytryptamine (5HT) and U46619, and this effect was not affected by Rp-cAMPS or Rp-8-Br-cGMPS. (4) The intracellular mechanism of action of amrinone may involve the phospholipase C (PLC)-inositol 1,4,5 trisphosphate (IP 3)-intracellular Ca 2+ signal transduction pathway. However, amrinone (100 μM) had no effect on either basal- or noradrenaline (100 μM)-stimulated PLC activity. Similarly, IP 3 stimulated a concentration-dependent release of Ca 2+ from rat brain microsomes that was not affected by amrinone (30 and 100 μM). (5) In conclusion, the vasorelaxant action of amrinone does not involve adenosine 3′,5′ cyclic monophosphate (cAMP) or involve guanosine 3′,5′ cyclic monophosphate (cGMP) but may include an inhibition of Ca 2+ influx through receptor- or store-operated Ca 2+ channels, although it does not directly affect intracellular Ca 2+ release.

Cytochrome P-450 activities in human and rat brain microsomes

The role of cytochrome P450 in the metabolism of dextromethorphan, amitriptyline, midazolam, S-mephenytoin, citalopram, fluoxetine and sertraline was investigated in rat and human brain microsomes. Depending on the parameters, the limit of quantification using gas chromatography–mass spectrometry methods was between 1.6 and 20 pmol per incubation, which generally contained 1500 μg protein. Amitriptyline was shown to be demethylated to nortriptyline by both rat and human microsomes. Inhibition studies using ketoconazole, furafylline, sulfaphenazole, omeprazole and quinidine suggested that CYP3A4 is the isoform responsible for this reaction whereas CYP1A2, CYP2C9, CYP2C19 and CYP2D6 do not seem to be involved. This result was confirmed by using a monoclonal antibody against CYP3A4. Dextromethorphan was metabolized to dextrorphan in rat brain microsomes and was inhibited by quinidine and by a polyclonal antibody against CYP2D6. Only the addition of exogenous reductase allowed the measurement of this activity in human brain microsomes. Metabolites of the other substrates could not be detected, possibly due to an insufficiently sensitive method. It is concluded that cytochrome P450 activity in the brain is very low, but that psychotropic drugs could undergo a local cerebral metabolism which could have pharmacological and/or toxicological consequences.

Properties and regulation of microsomal PAF-synthesizing enzymes in rat brain cortex.

Platelet-activating factor (PAF) is a phospholipid mediator of long-term potentiation, synaptic plasticity and memory formation as well as of the development of brain damage. In brain, PAF is synthesized by two distinct pathways but their relative contribution to its productions, in various physiological and pathological conditions, is not established. We have further investigated on the properties of the two enzymes that catalyze the last step of the de novo or remodeling pathways in rat brain microsomes, PAF-synthesizing phosphocholinetransferase (PAF-PCT) and lysoPAF acetyltransferase (lysoPAF-AT), respectively. The latter enzyme is fully active at microM Ca2+ concentration, inhibited by MgATP and activated by phosphorylation. Because the reversibility of the reaction catalyzed by PAF-PCT, its direction depends on the ratio [CDP-choline]/[CMP], which is related to the energy charge of the cell. These and other properties indicate that the de novo pathway should mainly contribute to PAF synthesis for maintaining its basal levels under physiological conditions. The remodeling pathway should be more involved in the production of PAF during ischemia. During reperfusion, the overproduction of PAF should be the result of the concomitant activation of both pathways.

Roles of A beta and carboxyl terminal peptide fragments of amyloid precursor protein in Alzheimer disease.

Several lines of evidence indicate that A beta may play an important role in the pathogenesis of AD. However, there are several discrepancies between the production of A beta and the development of the disease. Thus, A beta may not be the sole active fragment of beta-amyloid precursor protein (betaAPP) in the neurotoxicity assiciated with AD. We focused on the amyloidegenic carboxyl terminal fragments of betaAPP containing the full length of A beta (CT105). We synthesized a recombinant carboxyl-terminal 105 amino acid fragment of betaAPP and examined the effects of CT105 and A beta on cultured neurons, Ca++ uptake into rat brain microsomes, Na+-Ca++ exchange activity, ion channel forming activity in lipid bilayers and passive avoidance performance of mice. Our results suggest that the cytotoxic and channel inducing effects of CT105 are much more potent than that of A beta and toxic mechanisms of CT105 are different from those of A beta. Taken together, these lines of evidence postulate that CT is an alternative toxic element important in the generation of the symptoms common to AD.

Inhibition of rat microsomal lipid peroxidation by the oral administration of D002.

The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS). When D002 (5-100 mg/kg body weight) was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46%) occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg) also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40%) and brain (28-44%) microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg) for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

Testosterone metabolism in rat brain is differentially enhanced by phenytoin-inducible cytochrome P450 isoforms.

Many cytochrome P450 (P450) isoforms are selectively inducible by xenobiotics, e.g. pharmaceuticals like the anti-epileptic drug phenytoin. Some of these P450 enzymes are involved in the metabolism of gonadal hormones and are of great importance, especially in early brain development. In this study, the hydroxylation of testosterone by rat brain microsomes from control and phenytoin-induced animals was examined by use of high performance liquid chromatography (HPLC) provided with a photodiode array detector (PDA). In control rats, testosterone is converted by cytochrome(s) P450 to 6alpha-hydroxytestosterone (OHT) as the main metabolite and 6beta-OHT as well as androstenedione as minor metabolites. After phenytoin treatment, brain microsomes showed a strong increase of testosterone metabolism to 2alpha-, 6beta-, 16alpha-, 16beta-OHT and androstenedione, whereby 16alpha-OHT was the main degradation product. These metabolites indicated the action of isoforms of the P450 subfamilies CYP2B, CYP2C and CYP3A. Inhibition experiments with antibodies against CYP2B1/2 and with the CYP2B specific inhibitor orphenadrine indicated the occurrence of members of this subfamily which are known to catalyse the oxidation of testosterone to 16alpha-OHT, 16beta-OHT and androstenedione. Western blots revealed the phenytoin-inducible expression of CYP2B1 and the constitutive expression of CYP3A. The latter is involved in the 6beta-hydroxylation of testosterone which was found correspondingly in control microsomes. Distinct CYP2C isoforms involved in the hydroxylation of testosterone in phenytoin-induced microsomes are not yet identified. The highly increased testosterone metabolism by phenytoin-dependent induction of specific cytochrome P450 isoforms in adult rat brain illustrates the potential influence of exogenous substances on internal regulative and metabolic pathways in the brain.

N-acylphosphatidylethanolamine-hydrolysing phospholipase D lacks the ability to transphosphatidylate

The N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) generates N-acylethanolamines, including N-arachidonoyl-ethanolamine (anandamide), that may be neuroprotective and analgesic. The properties of NAPE-PLD from rat heart and brain microsomes are investigated and compared to those of other PLDs. NAPE-PLD is inhibited by the fatty acid aminohydrolase inhibitor MAFP in high concentrations (≥100 μM) while PMSF in high concentrations (10 mM) tends to stabilise NAPE-PLD activity. Oleate inhibits NAPE-PLD but the enzyme is not affected by PIP 2, α-synuclein or mastoparan. Furthermore, it is for the first time reported that NAPE-PLD is not capable of catalysing a transphosphatidylation reaction like most other known PLDs.

Nicotinic acid adenine dinucleotide phosphate triggers Ca2+ release from brain microsomes

Mobilization of Ca2+ from intracellular stores is an important mechanism for generating cytoplasmic Ca2+ signals [1]. Two families of intracellular Ca2+-release channels – the inositol-1,4,5-trisphosphate (IP3) receptors and the ryanodine receptors (RyRs) – have been described in mammalian tissues [2]. Recently, nicotinic acid adenine dinucleotide phosphate (NAADP), a molecule derived from NADP+ , has been shown to trigger Ca2+ release from intracellular stores in invertebrate eggs [3–6] and pancreatic acinar cells [7]. The nature of NAADP-induced Ca2+ release is unknown but it is clearly distinct from the IP3- and cyclic ADP ribose (cADPR)-sensitive mechanisms in eggs (reviewed in [8,9]). Furthermore, mammalian cells can synthesize and degrade NAADP, suggesting that NAADP-induced Ca2+ release may be widespread and thus contribute to the complexity of Ca2+ signalling [10,11]. Here, we show for the first time that NAADP evokes Ca2+ release from rat brain microsomes by a mechanism that is distinct from those sensitive to IP3 or cADPR, and has a remarkably similar pharmacology to the action of NAADP in sea urchin eggs [12]. Membranes prepared from the same rat brain tissues are able to support the synthesis and degradation of NAADP. We therefore suggest that NAADP-mediated Ca2+ signalling could play an important role in neuronal Ca2+ signalling.

Dehydration of 3-hydroxyacyl-CoA in brain very-long-chain fatty acid synthesis

Rat brain microsomes actively dehydrate 3-hydroxyacyl-CoAs. Using chemically synthesized [1- 14C] (R,S) 3-hydroxyeicosanoyl-CoA, we investigated the biochemical characteristics of the dehydration and reduction steps of stearoyl-CoA elongation. The reaction products, separated and identified as trans2,3-enoyl-CoAs and, in the presence of NADPH, as saturated acyl-CoAs, were released from the enzyme as thioesters which were partly hydrolysed. A kinetic analysis of the two coupled reactions showed that the 3-hydroxyacyl-CoA dehydrase catalysed a reversible reaction with kinetic constants of about 0.045 min −1 for forward reaction (dehydration) and 0.025 min −1 for reverse reaction (hydration); V max of the dehydration reached 20 nmoles/min/mg and the apparent Km was 44 μM. In the presence of NADPH, the kinetic constants for the dehydrase were unchanged and that for the trans2,3-enoyl-CoA reductase was 0.025 min −1. The relative proportion of trans2,3-enoyl-CoA and saturated acyl-CoA depended on the protein amount. An inhibition of the reduction step was observed for substrate concentrations above 15 μM. The 3-hydroxyacyl-CoA dehydrase used (R) rather than (S) 3-hydroxyacyl-CoA. Furthermore, the elongation of (R) 3-hydroxyeicosanoyl-CoA yielded saturated very-long-chain acyl-CoA. These results demonstrated that 3-hydroxyacyl-CoAs entered the elongating complex exclusively at the level of the dehydrase and not of the condensing enzyme.

Expression of calcium-binding protein regucalcin and microsomal Ca2+-ATPase regulation in rat brain: attenuation with increasing age.

The expression of calcium-binding protein regucalcin and its effect on the microsomal Ca2+-ATPase activity in rat brain tissues was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in brain tissues using rat regucalcin-specific primers. Regucalcin concentration in the brain tissues was about 5 x 10(-9)) M as measured using enzyme-linked immunoadsorbent assay (ELISA), and this level was lowered with increasing age (50 weeks old). The presence of regucalcin (10(-9) to 10(-7) M) in the enzyme reaction mixture caused a significant decrease in Ca2+-ATPase activity in the brain microsomes of young rats (5 weeks old). Meanwhile, the enzyme activity was not significantly altered by the addition of calmodulin (1 or 50 microg/ml), calbindin (1 or 10 microg/ml), and S-100 A protein (5 or 25 microg/ml), which are other Ca2+-binding proteins in rat brain. The effect of regucalcin to inhibit microsomal Ca2+-ATPase activity was weakened in the brain of rats with increasing age (50 weeks old). The present study demonstrates that regucalcin is expressed in the brain, and that it can uniquely inhibit Ca2+-ATPase activity in the brain microsomes of rats. The findings suggest that regucalcin plays a role in the regulation of microsomal Ca2+-ATPase activity in rat brain tissues.

In vitro study of fenoprofen chiral inversion in rat: comparison of brain versus liver

1. The extent and the overall stereoselectivity of the combined steps involved in the chiral inversion of fenoprofen, a non-steroidal anti-inflammatory drug, was investigated in rat brain microsomes and cytosol. Results were compared with those obtained with the same liver subcellular compartments. 2. Brain microsomes catalysed the stereoselective activation of the R(-)-enantiomer to its coenzyme A thioester with a specific activity ~10-fold less than that obtained with liver microsomes. 3. Rat brain microsomes and cytosol mediated the racemization and hydrolysis of both R(-)- and S(+)-fenoprofenoyl-CoA. In brain fractions the epimerase activity was lower than in liver, whereas the hydrolysis process appeared more efficient. 4. Thus, the data indicated that the three-step mechanism occurred in brain subcellular compartments leading to a minor chiral inversion of fenoprofen compared with that in liver.

Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufins in rat brain microsomes.

Characterization of xenobiotic metabolizing cytochrome P450s (P450s) was carried out in rat brain microsomes using the specific substrates, 7-pentoxy- and 7-ethoxyresorufin (PR and ER), metabolized in the liver by P450 2B1/2B2 and 1A1/1A2 respectively and 7-benzyloxyresorufin (BR), a substrate for both the isoenzymes. Brain microsomes catalysed the O-dealkylation of PR, BR and ER in the presence of NADPH. The ability to dealkylate alkoxyresorufins varied in different regions of the brain. Microsomes from the olfactory lobes exhibited maximum pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD) and ethoxyresorufin-O-dealkylase (EROD) activities. The dealkylation was found to be inducer selective. While pretreatment with phenobarbital (PB; 80 mg/kg; i.p. x 5 days) resulted in significant induction in PROD (3-4 fold) and BROD (4-5 fold) activities, 3-methylcholanthrene (MC; 30 mg/kg; i.p. x 5 days) had no effect on the activity of PROD and only a slight effect on that of BROD (1.4 fold). MC pretreatment significantly induced the activity of EROD (3 fold) while PB had no effect on it. Kinetic studies have shown that this increase in the activities following pretreatment with P450 inducers was associated with a significant increase in the velocity of the reaction (Vmax) of O-dealkylation. In vitro studies using organic inhibitors and antibodies have further provided evidence that the O-dealkylation of alkoxyresorufins is isoenzyme specific. While in vitro addition of alpha-naphthoflavone (ANF), an inhibitor of P450 1A1/1A2 catalysed reactions and antibody for hepatic P450 1A1/1A2 isoenzymes produced a concentration-dependent inhibition of EROD activity, metyrapone, an inhibitor of P450 2B1/2B2 and antibody for hepatic P450 2B1/2B2 significantly inhibited the activity of PROD and BROD in vitro. The data suggest that, as in the case of liver, dealkylation of alkoxyresorufins can be used as a biochemical tool to characterise the xenobiotic metabolising P450s and substrate selectivity of P450 isoenzymes in rat brain microsomes.

Scyphostatin, a neutral sphingomyelinase inhibitor from a discomycete, Trichopeziza mollissima: taxonomy of the producing organism, fermentation, isolation, and physico-chemical properties.

We performed experiments to screen for neutral sphingomyelinase inhibitors using rat brain microsomes as an enzyme source. Among more than 10,000 microbial extracts tested, a mycelial extract of Trichopeziza mollissima SANK 13892 exhibited potent inhibitory activity. The active compound, scyphostatin, was purified by a series of chromatographies. Scyphostatin inhibited the enzyme with an IC50 value of 1.0 microM.

C-terminal fragment of amyloid precursor protein inhibits calcium uptake into rat brain microsomes by Mg2+-Ca2+ ATPase

Numerous lines of evidence suggest that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid beta peptide (A beta). However, the possible role of other cleaved products of APP is less clear. In this study, the effects of a recombinant carboxy terminal 105 amino acid (CT105) fragment of APP on the calcium uptake by endoplasmic reticulum Mg2+-Ca2+ ATPase, the major mechanism for sequestering calcium in this organelle, were investigated. We found that CT 105 is a potent inhibitor of Mg2+-Ca2+ ATPase of endoplasmic reticulum, whereas A beta shows no effect. These results demonstrate that CT 105 inhibits the ability of brain microsomes to sequester calcium and suggest that this inhibitory effect of CT 105 may contribute to disruption of intracellular calcium concentration, possibly being involved in inducing the neural toxicity characteristic of AD.

Isolation of Partially Purified P450 2D18 and Characterization of Activity toward the Tricyclic Antidepressants Imipramine and Desipramine

Previous reports have shown that rat brain microsomes are capable of metabolizing tricyclic antidepressants such as imipramine. Subsequent studies have shown that the protein products of several clones isolated from rat brain cDNA libraries are capable of metabolizing imipramine to both its active metabolite, desipramine, and its inactive hydroxylated metabolites. We report here the overexpression and partial purification of P450 2D18 using the baculovirus expression system and the incorporation of a C-terminal [His]4tag. P450 2D18 was partially purified to a specific content of 4.8 nmol/mg protein and shown to be electrophoretically pure. The apparentKMvalues for P450 2D18 toward imipramine and desipramine were 374 and 314 μM, respectively. While apparentKMvalues were similar, P450 2D18 was shown to have a fivefold increasedVmax(2.2 nmol/min/nmol P450) for imipramine compared to desipramine (0.44 nmol/min/nmol P450), suggesting a primary involvement in the activation of imipramine to desipramine. We also examined the effect of theCYP2D6 inhibitor quinidine, theCYP3A inhibitor ketoconazole, and the dopamine reuptake inhibitor GBR-12935 for their ability to inhibit P450 2D18-mediated metabolism of imipramine. These results, when compared with previous studies using rat brain microsomes, suggest that P450 2D18 may play an important role in the conversion of imipramine to its active metabolite desipramine in the rat brain.

Immunochemical and Biochemical Evidence for Expression of Phenobarbital-and 3-Methylcholanthrene-Inducible Isoenzymes of Cytochrome P450 in Rat Brain

Expression of P450 1A1l 1A2 and 2 B1l 2B2 isoenzymes in rat brain was studied by Western blotting, using polyclonal antibodies raised against hepatic P450 1A1l 1A2 and 2B1l 2B2 isoenzymes. In addition, biochemical characterizations of the catalytic activities, pen toxyresorufin O-dealkylation (PROD) and ethoxyre-sorufin O-deethylation (EROD), selective for P450 2B1l 2B2 (PROD) and P450 1A1l 1A2 (EROD), were performed with rat brain microsomes. Control rat brain microsomes did not crossreact with either of the antibodies, whereas microsomes obtained from 3-methylcholanthrene (MC)-pretreated rats revealed significant immunoreactivity with anti-P450 1A1l 1A2. Similar results were observed with phenobarbital (PB)-pretreated rats, with the brain microsomes exhibiting significant immunoreactivity with anti-P450 2B1l 2B2. The induction in the P450 isoenzymes after PB or MC pretreatment was much less in the brain in comparison to the liver. Enzymatic studies indicated that the activities of PROD and EROD were induced in brain 3—4 fold by PB and MC pretreatment, respectively, and were almost completely inhibited on in vitro addition of anti-P450 2B1l 2B2 and 1A1l 1A2. These data demonstrate the expression of P4501A1l 1A2 and 2B1l 2B2 isoenzymes in the brain and indicate that, as in liver, these isoenzymes catalyze EROD and PROD, respectively, in the rat brain.

Increase of Ca 2+-ATPase activity in the brain microsomes of rats with increasing ages: Involvement of protein kinase C

A possible mechanism of aging-induced increase in brain microsomal Ca 2+-adenosine triphosphatase (ATPase) activity of rats was investigated. Calcium content in the brain tissues and Ca 2+-ATPase activity in the brain microsomes of aging rats (50 weeks of age) increased significantly as compared with those of young rats (5 weeks of age). Brain microsomal Ca 2+-ATPase activity in aging rats was decreased significantly by treatment of ethyleneglycol-bis-(aminoethylether) N,N,N′,N′-tetraacetic acid (EGTA) (2.7 mM) or digitonin (10 −3%), while such decrease was not seen in the enzyme activity of young rats. Microsomal Ca 2+-ATPase activity in aging rats was markedly decreased by the presence of staurosporine (10 −8 and 10 −7 M), an inhibitor of protein kinase C, in the enzyme reaction mixture, although the enzyme activity of young rats was not inhibited. Meanwhile, dibucaine (10 −6 and 10 −5 M), an inhibitor of Ca 2+/calmodulin-dependent protein kinase, did not have an effect on Ca 2+-ATPase activity in the brain microsomes of young and aging rats. The addition of protein kinase C (100 and 200 mU/ml) in the reaction mixture caused a significant increase in brain microsomal Ca 2+-ATPase activity of young rats. These results suggest that protein kinase C is partly involved in the elevation of brain microsomal Ca 2+-ATPase activity in rats with increasing ages.

Occurrence of an Unusual Phospholipid, Phosphatidyl-l-threonine, in Cultured Hippocampal Neurons: EXOGENOUS l-SERINE IS REQUIRED FOR THE SYNTHESIS OF NEURONAL PHOSPHATIDYL-l-SERINE AND SPHINGOLIPIDS

We have recently reported that L-serine released from astroglial cells supports the survival and neuritogenesis of hippocampal neurons under a serum- and glia-free culture condition (Mitoma, J., Furuya, S., and Hirabayashi, Y. (1998) Neurosci. Res. 30, 195-199). In this study, we show that exogenous L-serine is required for the synthesis of phosphatidyl-L-serine (PS) and sphingolipids in hippocampal neurons. When hippocampal neurons were maintained under an astroglial cell-free condition, the levels of sphingolipids and phosphatidyl-L-serine in the neurons were greatly reduced in the absence of external L-serine or glycine. Instead, a novel phospholipid appeared just ahead of PS on TLC. This novel lipid was determined to be phosphatidyl-L-threonine by TLC blotting/negative secondary ion mass spectrometry and amino acid analysis. Biochemical studies on rat brain microsomes have indicated that phosphatidyl-L-threonine is synthesized by the base exchange enzyme that is involved in PS synthesis with much lower affinity, that is, approximately (1)/(150) of L-serine. Addition of L-serine or glycine to the culture medium restored the synthesis of PS and sphingolipids in the neurons. These observations show that hippocampal neurons require exogenous L-serine for the synthesis of PS and sphingolipids in the absence of astroglial cells and suggested that astroglial cells contribute to neuronal lipid synthesis through the supply of L-serine.