N-acetyl-l-glutamate in brain: Assay,levels, and regional and subcellular distribution

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200–300 times lower than the levels of N-acetyl-l-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 g/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-l-aspartyl-l-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.Special issue dedicated to Dr. Santiago Grisolía     

Simultaneous determination of N-acetylaspartylglutamate and N-acetylaspartate in rat brain homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization

Simultaneous determination method of N-acetyl-l-aspartyl-l-glutamate (NAAG), an endogenous agonist at type 3 metabotropic glutamate receptor, and its degradation product, N-acetyl-l-aspartate (NAA) was developed by using reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole. The detection limits of NAAG and NAA were approximately 12 and 34 fmol on the column, respectively (signal to noise ratio 3). The proposed HPLC method was applied to determine NAAG and NAA simultaneously in the rat brain homogenate. Both concentrations of NAAG and NAA in the male rat cerebrum (13 weeks old) were 5.7+/-0.30 and 2.1 x 10(2)+/-9.2 nmol/mg protein, respectively (n=6), while those in the hippocampus were 6.8+/-0.48 and 1.9 x 10(2)+/-8.5 nmol/mg protein, respectively (n=5). Hippocampal NAA concentration was significantly increased in the ketamine-treated rats as compared to the control rats (p<0.01).     

N-Acetyl-Aspartyl-Glutamate: Regional Levels in Rat Brain and the Effects of Brain Lesions as Determined by a New HPLC Method

Abstract: An isocratic HPLC method to measure endogenous N -acetyl-aspartyl-glutamate (NAAG) and N -acetyl-aspartate (NAA) is described. After removal of primary amines by passage of tissue extracts over AG-50 resin, the eluate was subject to HPLC anion-exchange analysis and eluted with phosphate buffer with absorbance monitored at 214 nm. The retention time for NAA was 5.6 min and for NAAG 11.4 min with a limit sensitivity of 0.1 nmol. The levels of NAA and NAAG were measured in 16 regions of rat brain and in heart and liver. NAAG was undetectable in heart and liver and exhibited 10-fold variation in concentration among brain regions; the highest levels were found in spinal cord. In contrast, low concentrations of NAA were detectable in heart and liver, and the regional distribution of NAA in brain varied only twofold. The regional distribution of NAA and NAAG correlated poorly. To assess the neuronal localization of these two compounds, the effects of selective brain lesions on their levels were examined. Decortication caused a 28% decrease in NAAG levels in the ipsi-lateral striatum while NAA decreased 38%. Kainate lesion of the striatum resulted in a 31% decrease in NAAG in the ipsilateral striatum, whereas NAA fell by 58%. Kainate lesion of the hippocampus resulted in significant decrements in NAAG and NAA in the hippocampus and septum. Transection of the spinal cord at midthorax resulted in a 51% decrease in NAAG levels immediately caudal and a 40% decrease immediately rostral to the lesion; however, NAA decreased only 30% in these areas. These results are consistent with a neuronal localization of NAAG in brain. Combined with the fact that NAAG interacts with a subpopulation of glutamate receptors, these results suggest that NAAG may serve as an excitatory neurotransmitter.     

Postmortem Degradation of N-Acetyl Aspartate and N-Acetyl Aspartylglutamate: An HPLC Analysis of Different Rat CNS Regions

N-acetyl aspartate (NAA), a putative marker of neuronal injury, can be measured non-invasively in patients by magnetic resonance spectroscopy (MRS). Interpretation of in vivo MRS data, however, requires neuropathological correlates to NAA alterations using autopsy or biopsy material. Since detailed hydrolysis data is lacking, NAA and the related dipeptide N-acetyl aspartylglutamate (NAAG) were quantified by high performance liquid chromatography (HPLC) in different rat CNS regions over 24 h postmortem. Both molecules decreased rapidly 1-4 h postmortem, and subsequently slower with time. The average reduction at 24 h was 46% and 38% for NAA and NAAG respectively. The NAA reduction was proportionally smaller in cortical areas (34-37%) compared to more caudal regions (54-58%). An exception was the optic nerve, a pure white matter tract, where NAA and NAAG hydrolysis was slower. The NAA/NAAG ratio remained relatively constant, but exhibited marked regional differences. The data show a significant postmortem degradation of NAA and NAAG that needs to be considered when these compounds are studied ex-vivo.     

Competitive Inhibition of N-Acetylated-α-Linked Acidic Dipeptidase Activity by N-Acetyl-L-Aspartyl-β-Linked L-Glutamate

The endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) fulfills several criteria required to be accepted as a neurotransmitter. NAAG inactivation may proceed through enzymatic hydrolysis into N-acetyl-L-aspartate and glutamate by an N-acetylated-alpha-linked acidic dipeptidase (NAALADase). Therefore, some properties of NAALADase activity were investigated using crude membranes from the rat forebrain. Kinetic parameters of the hydrolysis of [Glu-3H]NAAG were determined first (Km = 0.40 +/- 0.05 microM; Vmax = 155 +/- 20 pmol/min/mg of protein). The enzymatic activity, i.e., NAALADase, was inhibited noncompetitively by the glutamatergic agonist quisqualate (Ki = 1.9 +/- 0.3 microM), and competitively by N-acetyl-L-aspartyl-beta-linked L-glutamate (beta-NAAG; Ki = 0.70 +/- 0.05 microM). To determine whether glutamate-containing dipeptides, such as NAAG, beta-NAAG, N-acetyl-L-aspartyl-D-glutamate, L-aspartyl-L-glutamate, L-alanyl-L-glutamate, L-glutamyl-L-glutamate, and L-glutamyl-gamma-linked L-glutamate, were substrates of NAALADase, rat brain membranes were immobilized on a C-8 column. Thus, endogenous trapped glutamate was washed away and formation of unlabelled glutamate could be estimated using an o-phthaldialdehyde/reverse-phase HPLC detection procedure. beta-NAAG was shown to be a nonhydrolyzable competitive inhibitor of NAALADase. L-Aspartyl-L-glutamate was hydrolyzed faster than NAAG, suggesting that the acetylated moiety is not essential for NAALADase specificity. Rat brain membranes also contained nonspecific peptidase activities (insensitive to both quisqualate and beta-NAAG), which, in the case of L-alanyl-L-glutamate, for instance, accounted for all observed hydrolysis.     

N-Acetylaspartylglutamate Selectively Inhibits Neuronal Responses to N-Methyl-d-Aspartic Acid In Vitro

Abstract: Canavan's disease is an autosomal recessive disorder characterized by a deficiency of aspartoacylase and accumulation of N -acetylaspartic acid (NAA), leading to a severe leukodystrophy and spongy degeneration of the brain. N -Acetylaspartylglutamate (NAAG), the presumed product of NAA, also accumulates in this disease. The endogenous dipeptide NAAG has been suggested to have low potency at NMDA receptors. Here we have tested the actions of NAAG and NAA on NMDA-evoked responses in cultured cerebellar granule cells. In differentiating granule cells grown in low-K⁺ medium, NAAG negated the survival-promoting effects of NMDA but not K⁺ depolarization. Neither NAAG nor NAA alone promoted cell survival in low-K⁺ medium. The modest trophic action of 50 µ M kainic acid in low-K⁺ medium was reinforced by the NMDA receptor antagonist dizocilpine maleate and by NAAG. In K⁺-differentiated granule cells, NAAG raised the threshold of NMDA neurotoxicity but not that of kainate. The observed activities of NAAG were overcome by excess NMDA and were not mimicked by NAA. These data raise the possibility that disruption of NMDA receptor processes by NAAG may be of pathophysiological relevance.     

Regulation of N-acetylaspartate and N-acetylaspartylglutamate biosynthesis by protein kinase activators

The neuronal dipeptide N-acetylaspartylglutamate (NAAG) is thought to be synthesized enzymatically from N-acetylaspartate (NAA) and glutamate. We used radiolabeled precursors to examine NAA and NAAG biosynthesis in SH-SY5Y human neuroblastoma cells stimulated with activators of protein kinase A (dbcAMP; N6,2'-O-dibutyryl cAMP) and protein kinase C (PMA; phorbol-12-myristate-13-acetate). Differentiation over the course of several days with dbcAMP resulted in increased endogenous NAA levels and NAAG synthesis from l-[(3)H]glutamine, whereas PMA-induced differentiation reduced both. Exogenously applied NAA caused dose dependent increases in intracellular NAA levels, and NAAG biosynthesis from l-[(3)H]glutamine, suggesting precursor-product and mass-action relationships between NAA and NAAG. Incorporation of l-[(3)H]aspartate into NAA and NAAG occurred sequentially, appearing in NAA by 1 h, but not in NAAG until between 6 and 24 h. Synthesis of NAAG from l-[(3)H]aspartate was increased by dbcAMP and decreased by PMA at 24 h. The effects of PMA on l-[(3)H]aspartate incorporation into NAA were temporally biphasic. Using short incubation times (1 and 6 h), PMA increased l-[(3)H]aspartate incorporation into NAA, but with longer incubation (24 h), incorporation was significantly reduced. These results suggest that, while the neuronal production of NAA and NAAG are biochemically related, significant differences exist in the regulatory mechanisms controlling their biosynthesis.     

N-Acetylaspartate (NAA) and N-Acetylaspartylglutamate (NAAG) Promote Growth and Inhibit Differentiation of Glioma Stem-like Cells

Metabolic reprogramming is a pathological feature of cancer and a driver of tumor cell transformation. N-Acetylaspartate (NAA) is one of the most abundant amino acid derivatives in the brain and serves as a source of metabolic acetate for oligodendrocyte myelination and protein/histone acetylation or a precursor for the synthesis of the neurotransmitter N-acetylaspartylglutamate (NAAG). NAA and NAAG as well as aspartoacylase (ASPA), the enzyme responsible for NAA degradation, are significantly reduced in glioma tumors, suggesting a possible role for decreased acetate metabolism in tumorigenesis. This study sought to examine the effects of NAA and NAAG on primary tumor-derived glioma stem-like cells (GSCs) from oligodendroglioma as well as proneural and mesenchymal glioblastoma, relative to oligodendrocyte progenitor cells (Oli-Neu). Although the NAA dicarboxylate transporter NaDC3 is primarily thought to be expressed by astrocytes, all cell lines expressed NaDC3 and, thus, are capable of NAA up-take. Treatment with NAA or NAAG significantly increased GSC growth and suppressed differentiation of Oli-Neu cells and proneural GSCs. Interestingly, ASPA was expressed in both the cytosol and nuclei of GSCs and exhibited greatest nuclear immunoreactivity in differentiation-resistant GSCs. Both NAA and NAAG elicited the expression of a novel immunoreactive ASPA species in select GSC nuclei, suggesting differential ASPA regulation in response to these metabolites. Therefore, this study highlights a potential role for nuclear ASPA expression in GSC malignancy and suggests that the use of NAA or NAAG is not an appropriate therapeutic approach to increase acetate bioavailability in glioma. Thus, an alternative acetate source is required.     

Production and characterization of monoclonal antibodies to N-acetyl-aspartyl-glutamate

N-acetyl-aspartyl-glutamate (NAAG) is a putative neuromodulator/neurotransmitter in the mammalian nervous system. Immunohistochemical studies with polyclonal NAAG antisera have revealed immunoreactive neurons and processes in several brain regions. However, these antisera crossreact to some degree with N-acetyl-aspartate (NAA), which is present in mM concentrations in brain, prompting the development of monoclonal antibodies (MAb) more specific for NAAG. By fusing spleen lymphocytes obtained from BALB/c mice pre-immunized with NAAG covalently linked to bovine serum albumin by carbodiimide with SP2/0-Ag 14 mouse myeloma cells, we produced three IgG2a (kappa) MAb which specifically reacted with NAAG. These MAb exhibited negligible crossreactivity with NAA or with structurally similar peptides, as shown by solid-phase radioimmunoassay. Antibody activity was absorbed out selectively by both NAAG-thyroglobulin conjugate and free NAAG. These MAb stained many nuclei of the medulla-pons and midbrain, mitral cells in the olfactory bulb, pyramidal neurons in sensorimotor cortex, locus ceruleus, and several cholinergic cranial nuclei. The staining pattern strongly correlated with NAAG levels determined by HPLC. Monoclonal antibodies significantly enhanced sensitivity of staining, allowing visualization of dorsal horn neurons in spinal cord, which were not readily detectable with polyclonal antiserum. Availability of these MAb now facilitates further clarification of the role of NAAG in the brain.     

Evidence that the tri-cellular metabolism of N-acetylaspartate functions as the brain’s “operating system”: how NAA metabolism supports meaningful intercellular frequency-encoded communications

N-acetylaspartate (NAA), an acetylated derivative of l-aspartate (Asp), and N-acetylaspartylglutamate (NAAG), a derivative of NAA and l-glutamate (Glu), are synthesized by neurons in brain. However, neurons cannot catabolize either of these substances, and so their metabolism requires the participation of two other cell types. Neurons release both NAA and NAAG to extra-cellular fluid (ECF) upon stimulation, where astrocytes, the target cells for NAAG, hydrolyze it releasing NAA back into ECF, and oligodendrocytes, the target cells for NAA, hydrolyze it releasing Asp to ECF for recycling to neurons. This sequence is unique as it is the only known amino acid metabolic cycle in brain that requires three cell types for its completion. The results of this cycling are two-fold. First, neuronal metabolic water is transported to ECF for its removal from brain. Second, the rate of neuronal activity is coupled with focal hyperemia, providing stimulated neurons with the energy required for transmission of meaningful frequency-encoded messages. In this paper, it is proposed that the tri-cellular metabolism of NAA functions as the “operating system” of the brain, and is essential for normal cognitive and motor activities. Evidence in support of this hypothesis is provided by the outcomes of two human inborn errors in NAA metabolism.     

Biosynthesis of NAAG by an enzyme-mediated process in rat central nervous system neurons and glia

The peptide transmitter N-acetylaspartylglutamate (NAAG) is present in millimolar concentrations in mammalian spinal cord. Data from the rat peripheral nervous system suggest that this peptide is synthesized enzymatically, a process that would be unique for mammalian neuropeptides. To test this hypothesis in the mammalian CNS, rat spinal cords were acutely isolated and used to study the incorporation of radiolabeled amino acids into NAAG. Consistent with the action of a NAAG synthetase, inhibition of protein synthesis did not affect radiolabel incorporation into NAAG. Depolarization of spinal cords stimulated incorporation of radiolabel. Biosynthesis of NAAG by cortical astrocytes in cell culture was demonstrated by tracing incorporation of [3H]-glutamate by astrocytes. In the first test of the hypothesis that NAA is an immediate precursor in NAAG biosynthesis, [3H]-NAA was incorporated into NAAG by isolated spinal cords and by cell cultures of cortical astrocytes. Data from cerebellar neurons and glia in primary culture confirmed the predominance of neuronal synthesis and glial uptake of NAA, leading to the hypothesis that while neurons synthesize NAA for NAAG biosynthesis, glia may take it up from the extracellular space. However, cortical astrocytes in serum-free low-density cell culture incorporated [3H]-aspartate into NAAG, a result indicating that under some conditions these cells may also synthesize NAA. Pre-incubation of isolated spinal cords and cultures of rat cortical astrocytes with unlabeled NAA increased [3H]-glutamate incorporation into NAAG. In contrast, [3H]-glutamine incorporation in spinal cord was not stimulated by unlabeled NAA. These results are consistent with the glutamate-glutamine cycle greatly favoring uptake of glutamine into neurons and glutamate by glia and suggest that NAA availability may be rate-limiting in the synthesis of NAAG by glia under some conditions.     

Functions of N-acetyl-L-aspartate and N-acetyl-L-aspartylglutamate in the vertebrate brain: role in glial cell-specific signaling

N-Acetyl-L-aspartate (NAA) and its derivative N-acetylaspartylglutamate (NAAG) are major osmolytes present in the vertebrate brain. Although they are synthesized primarily in neurons, their function in these cells is unclear. In the brain, these substances undergo intercompartmental cycles in which they are released by neurons in a regulated fashion and are then rapidly hydrolyzed by catabolic enzymes associated with glial cells. Recently, the catabolic enzyme for NAA hydrolysis has been found to be expressed only in oligodendrocytes, and the catabolic enzyme for NAAG expressed only in astrocytes. These results indicate an unusual tricellular metabolic sequence for the synthesis and hydrolysis of NAAG wherein it is synthesized in neurons from NAA and L-glutamate, hydrolyzed to NAA and L-glutamate by astrocytes, and further hydrolyzed to L-aspartate and acetate by oligodendrocytes. Since the discovery that the NAA and NAAG anabolic products of neurons are specifically targeted to oligodendrocytes and astrocytes, respectively, this unique metabolic compartmentalization also suggests that these substances may play an important role in cell-specific glial signaling. In this review, it is hypothesized that a key function of NAA and NAAG in the vertebrate brain is in cell signaling and that these substances are important in the regulation of interactions of brain cells and in the establishment and maintenance of the nervous system.     

Antipsychotic drugs increase N-acetylaspartate and N-acetylaspartylglutamate in SH-SY5Y human neuroblastoma cells

N -Acetylaspartate (NAA) and N -acetylaspartylglutamate (NAAG) are related neuronal metabolites associated with the diagnosis and treatment of schizophrenia. NAA is a valuable marker of neuronal viability in magnetic resonance spectroscopy, a technique which has consistently shown NAA levels to be modestly decreased in the brains of schizophrenia patients. However, there are conflicting reports on the changes in brain NAA levels after treatment with antipsychotic drugs, which exert their therapeutic effects in part by blocking dopamine D₂ receptors. NAAG is reported to be an agonist of the metabotropic glutamate 2/3 receptor, which is linked to neurotransmitter release modulation, including glutamate release. Alterations in NAAG metabolism have been implicated in the development of schizophrenia possibly via dysregulation of glutamate neurotransmission. In the present study we have used high performance liquid chromatography to determine the effects of the antipsychotic drugs haloperidol and clozapine on NAA and NAAG levels in SH-SY5Y human neuroblastoma cells, a model system used to test the responses of dopaminergic neurons in vitro . The results indicate that the antipsychotic drugs haloperidol and clozapine increase both NAA and NAAG levels in SH-SY5Y cells in a dose and time dependant manner, providing evidence that NAA and NAAG metabolism in neurons is responsive to antipsychotic drug treatment.     

Changes in the concentration and fatty acid composition of phosphoinositides induced by hormones in hepatocytes

The hormonal regulation of phosphoinositide levels in isolated hepatocytes was studied using chemical means. Extracted inositol phospholipids were adsorbed to neomycin-coated glass beads and then eluted and quantitated by charring after separation by thin layer chromatography on silica gel. The amounts (in nanograms/mg wet weight) of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) were 20 +/- 1, 16 +/- 1, and 1790 +/- 140, respectively). Incubation of the cells with 100 nM vasopressin decreased the value for PIP2 to 10 +/- 0.2 at 15 s, 12 +/- 1.5 at 1 min, and 14 +/- 2.1 at 5 and 30 min. In contrast, the hormone increased 1,2-diacylglycerol plus phosphatidate by over 200 ng/mg wet weight at 5 min under similar conditions (Bocckino, S. B., Blackmore, P. F., Wilson, P. B., and Exton, J. H. (1987) J. Biol. Chem. 262, 15309-15315). PIP2 was also significantly decreased at 15 s by angiotensin II (100 nM), ATP (100 microM), and epinephrine (1 microM). In contrast, PIP was not significantly changed, and PI was significantly decreased (by approximately 15%) at later times (15 and 30 min). The changes in phosphoinositide mass were well correlated with changes in labeled phosphoinositides in hepatocytes previously incubated with [3H]inositol for 90 min. The amounts of inositol phospholipids in liver plasma membranes (in micrograms/mg protein) were 2.1 +/- 0.2 for PIP2, 0.24 +/- 0.03 for PIP, and 23 +/- 4 for PI. Comparison of these values with those for whole cells suggests that PIP2 is enriched in the plasma membrane, whereas PIP is present elsewhere in the cell. The fatty acid composition of whole cell PIP2 showed significant differences from that of PI. The percentages of palmitic, stearic, linoleic, and arachidonic acids were, respectively, 14, 41, 10, and 25 for PIP2 and 10, 34, 7, and 37 for PI. Vasopressin treatment for 15 s did not alter the fatty acid composition of PIP2. The corresponding fatty acid percentages for liver plasma membranes were 13, 41, 11, and 21 for PIP2 and 8, 34, 0, and 40 for PI. The fatty acid composition of PIP in whole cells and plasma membranes resembled that of PIP2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of carbon monoxide (CO) in rodent tissue: effect of heme administration and environmental CO exposure

Carbon monoxide (CO), produced endogenously during heme degradation, is considered a messenger molecule in vascular and neurologic tissues. To study this role, it is important to determine CO concentration in target tissues pre- and post-perturbations. Here, we describe a sensitive and reproducible method, which is linear and accurate, and provide some examples of its application for quantitation of CO concentrations in tissues pre- and post-perturbations. Tissues from adult rats and mice were sonicated (20% w/w), and volumes representing 0.04-8 mg fresh weight (FW) were incubated at 0 degrees C for 30 min with sulfosalicylic acid. CO liberated into the headspace was quantitated by gas chromatography. Tissue CO concentrations (mean+/-SD, pmol CO/mg FW) were as follows: blood (47+/-10, 45+/-5), muscle (4+/-4, 10+/-1), kidney (5+/-2, 7+/-2), heart (6+/-3, 6+/-1), spleen (11+/-3, 6+/-1), liver (4+/-1, 5+/-1), intestine (2+/-1, 4+/-2), lung (2+/-1, 3+/-1), testes (1+/-1, 2+/-1), and brain (2+/-1, 2+/-0) in untreated rat (n=3) and mouse (n=5), respectively. Between the rat and the mouse, only CO concentrations in the muscle and spleen were significantly different (p0.05). Endogenous CO generation, after administration of heme arginate to mice (n=3), increased CO concentrations by 0-43 pmol/mg FW. Exposure of mice (n=3) to 500 ppm CO for 30 min yielded significantly elevated CO concentrations by 4-2603 pmol/mg FW in all tissues over the native state. While blood had the highest CO concentration for all conditions, muscle, kidney, heart, spleen, and liver, all rich in hemoglobin and/or other CO-binding hemoproteins, also contained substantial CO concentrations. Intestine, lung, testes, and brain contained the lowest CO concentrations.     

Molecular Characterization of N-Acetylaspartylglutamate Synthetase

The dipeptide N-acetylaspartyl-glutamate (NAAG) is an abundant neuropeptide in the mammalian brain. Despite this fact, its physiological role is poorly understood. NAAG is synthesized by a NAAG synthetase catalyzing the ATP-dependent condensation of N-acetylaspartate and glutamate. In vitro NAAG synthetase activity has not been described, and the enzyme has not been purified. Using a bioinformatics approach we identified a putative dipeptide synthetase specifically expressed in the nervous system. Expression of the gene, which we named NAAGS (for NAAG synthetase) was sufficient to induce NAAG synthesis in primary astrocytes or CHO-K1 and HEK-293T cells when they coexpressed the NAA transporter NaDC3. Furthermore, coexpression of NAAGS and the recently identified N-acetylaspartate (NAA) synthase, Nat8l, in CHO-K1 or HEK-293T cells was sufficient to enable these cells to synthesize NAAG. Identity of the reaction product of NAAGS was confirmed by HPLC and electrospray ionization tandem mass spectrometry (ESI-MS). High expression levels of NAAGS were restricted to the brain, spinal cord, and testis. Taken together our results strongly suggest that the identified gene encodes a NAAG synthetase. Its identification will enable further studies to examine the role of this abundant neuropeptide in the vertebrate nervous system.     

The effects of chronic trimetazidine treatment on mechanical function and fatty acid oxidation in diabetic rat hearts

Clinical and experimental evidence suggest that increased rates of fatty acid oxidation in the myocardium result in impaired contractile function in both normal and diabetic hearts. Glucose utilization is decreased in type 1 diabetes, and fatty acid oxidation dominates for energy production at the expense of an increase in oxygen requirement. The objective of this study was to examine the effect of chronic treatment with trimetazidine (TMZ) on cardiac mechanical function and fatty acid oxidation in streptozocin (STZ)-diabetic rats. Spontaneously beating hearts from male Sprague-Dawley rats were subjected to a 60-minute aerobic perfusion period with a recirculating Krebs-Henseleit solution containing 11 mmol/L glucose, 100 muU/mL insulin, and 0.8 mmol/L palmitate prebound to 3% bovine serum albumin (BSA). Mechanical function of the hearts, as cardiac output x heart rate (in (mL/min).(beats/min).10-2), was deteriorated in diabetic (73 +/- 4) and TMZ-treated diabetic (61 +/- 7) groups compared with control (119 +/- 3) and TMZ-treated controls (131 +/- 6). TMZ treatment increased coronary flow in TMZ-treated control (23 +/- 1 mL/min) hearts compared with untreated controls (18 +/- 1 mL/min). The mRNA expression of 3-ketoacyl-CoA thiolase (3-KAT) was increased in diabetic hearts. The inhibitory effect of TMZ on fatty acid oxidation was not detected at 0.8 mmol/L palmitate in the perfusate. Addition of 1 mumol/L TMZ 30 min into the perfusion did not affect fatty acid oxidation rates, cardiac work, or coronary flow. Our results suggest that higher expression of 3-KAT in diabetic rats might require increased concentrations of TMZ for the inhibitory effect on fatty acid oxidation. A detailed kinetic analysis of 3-KAT using different concentrations of fatty acid will determine the fatty acid inhibitory concentration of TMZ in diabetic state where plasma fatty acid levels are increased.     

A novel key–lock mechanism for inactivating amino acid neurotransmitters during transit across extracellular space

There are two kinds of neurotransmissions that occur in brain. One is neuron to neuron at synapses, and the other is neuron to glia via extracellular fluid (ECF), both of which are important for maintenance of proper neuronal functioning. For neuron to neuron communications, several potent amino acid neurotransmitters are used within the confines of synaptic space. However, their presence at elevated concentrations in extra-synaptic space could be detrimental to well organized neuronal functioning. The significance of the synthesis and release of N-acetylaspartylglutamate (NAAG) by neurons has long been a puzzle since glutamate (Glu) itself is the “key” that can interact with all Glu receptors on membranes of all cells. Nonetheless, neurons synthesize this acetylated dipeptide, which cannot be catabolized by neurons, and release it to ECF where its specific physiological target is the Glu metabotropic receptor 3 on the surface of astrocytes. Since Glu is excitotoxic at elevated concentrations, it is proposed that formation and release of NAAG by neurons allows large quantities of Glu to be transported in ECF without the risk of injurious excitotoxic effects. The metabolic mechanism used by neurons is a key–lock system to detoxify Glu during its intercellular transit. This is accomplished by first synthesizing N-acetylaspartate (NAA), and then joining this molecule via a peptide bond to Glu. In this paper, a hypothesis is presented that neurons synthesize a variety of relatively nontoxic peptides and peptide derivatives, including NAA, NAAG, homocarnosine (γ-aminobutyrylhistidine) and carnosine (β-alanylhistidine) from potent excitatory and inhibitory amino acids for the purpose of releasing them to ECF to function as cell-specific neuron-to-glia neurotransmitters.     

Establishment of the fatty acid profile of the brain of the king penguin (Aptenodytes patagonicus) at hatch: effects of a yolk that is naturally rich in n-3 polyunsaturates

Because the yolk lipids of the king penguin (Aptenodytes patagonicus) contain the highest concentrations of long-chain n-3 polyunsaturated fatty acids yet reported for an avian species, the consequences for the establishment of the brain's fatty acid profile in the embryo were investigated. To place the results in context, the fatty acid compositions of yolk lipid and brain phospholipid of the king penguin were compared with those from three other species of free-living birds. The proportions of docosahexaenoic acid (22:6n-3; DHA) in the total lipid of the initial yolks for the Canada goose (Branta canadensis), mallard (Anas platyrhynchos), moorhen (Gallinula chloropus), and king penguin were (% w/w of fatty acids) 1.0+/-0.1, 1.9+/-0.2, 3.3+/-0.1, and 5.9+/-0.2, respectively. The respective concentrations of DHA (% w/w of phospholipid fatty acids) in brains of the newly hatched chicks of these same species were 18.5+/-0.2, 19.6+/-0.7, 16.9+/-0.4, and 17.6+/-0.1. Thus, the natural interspecies diversity in yolk fatty acid profiles does not necessarily produce major differences in the DHA content of the developing brain. Only about 1% of the amount of DHA initially present in the yolk was recovered in the brain of the penguin at hatch. There was no preferential uptake of DHA from the yolk during development of the king penguin.