The Regional Distribution of N-Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System

Abstract: N -Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N -acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N -acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.     

Nitric Oxide Regulates Substance P Release from Rat Spinal Cord Synaptosomes

Abstract: In order to determine whether nitric oxide (NO) acts directly upon nerve terminals to regulate the synaptic transmission at the level of spinal cord, effects of NO-donors on release of substance P (SP) and glutamic acid (Glu) were investigated by superfusion of synaptosomes prepared from the rat spinal cord. Basal levels of endogenous SP and Glu release were 5.99 ± 2.50 fmol/min/mg of protein and 26.2 ± 4.8 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCI evoked 2.7- and 3.8-fold increases in SP and Glu release in a calcium-dependent manner, respectively. Sodium nitroprusside (NP) caused a reduction in the depolarization-evoked overflow of SP in a concentration-dependent manner without affecting its basal release, although it failed to affect either basal or evoked release of Glu. The reduction in SP overflow was also observed by the perfusion with S -nitroso- N -acetyl-penicillamine or membrane-permeable cyclic GMP, but not with cyclic AMP. NP caused the concentration-dependent increases in cyclic GMP levels in synaptosomes. Together with reports that excitatory amino acids stimulate NO synthase and release NO in the spinal cord, these data suggest that there may be an interaction between nerve terminals containing Glu and SP, and that NO may directly participate in the regulation of synaptic transmission in SP-containing nerve terminals, which may be mediated through the activation of guanylate cyclase and the increase in cyclic GMP levels.     

Neurochemical and Immunocytochemical Studies on the Distribution of N-Acetyl-Aspartylglutamate and N-Acetyl-Aspartate in Rat Spinal Cord and Some Peripheral Nervous Tissues

HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.     

Release of N-Acetylaspartylglutamate from Slices of Rat Cerebellum, Striatum, and Spinal Cord, and the Effect of Climbing Fiber Deprivation

Abstract: The release of endogenous N -acetylaspartylglutamate (NAAG) from slices of rat cerebellum, striatum, and spinal cord upon depolarization with 50 m M K⁺ was investigated. NAAG in superfusates was prepurified using an ion exchanger, esterified, and then quantified by gas chromatography-mass spectrometry. Deuterated NAAG was used as internal standard. A depolarization-induced release of NAAG was found in all three regions. The release was Ca²⁺ dependent to over 85% in cerebellum and striatum, but only to approximately 70% in spinal cord. In addition, the effect of lesions of the olivocerebellar pathway on the K⁺-induced release of NAAG was studied: Treatment of the animals with 3-acetylpyridine reduced the release of NAAG from cerebellar hemispheres significantly, by about 40% compared with controls. These results suggest that part of the NAAG released from cerebellar slices on depolarization is related to climbing fibers. Implications of these findings concerning possible physiological roles of NAAG in the three CNS regions are discussed.     

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.     

Characterisation and Molecular Identification of Adrenomedullin Binding Sites in the Rat Spinal Cord: A Comparison with Calcitonin Gene-Related Peptide Receptors

Abstract: Calcitonin gene-related peptide (CGRP) and its receptors are found in mammalian spinal cord. We show, for the first time, binding sites for the novel related peptide adrenomedullin in rat spinal cord microsomes. ¹²⁵I-Adrenomedullin binding showed high affinity ( K _D = 0.45 ± 0.06 n M ) and sites were abundant ( B _max = 723 ± 71 fmol/mg of protein). CGRP, amylin, and calcitonin did not compete at these sites ( K _i > 10 µ M ). High-affinity CGRP binding sites ( K _D = 0.18 ± 0.01 n M ) were much less numerous ( B _max = 17.7 ± 2.4 fmol/mg of protein) and showed competition by unlabeled adrenomedullin ( K _i = 34.6 ± 2.4 n M ). Chemical cross-linking revealed a major band for ¹²⁵I-adrenomedullin of M_r = 84,400 ± 1,200 and a minor band of M_r = 122,000 ± 8,700. ¹²⁵I-CGRP cross-linking showed bands of lower molecular weight (M_r = 74,500 ± 5,000 and 61,000 ± 2,200). Enzymic deglycosylation of the adrenomedullin binding site showed a considerable carbohydrate content. Neither adrenomedullin nor CGRP was able to increase cyclic AMP in spinal cord. Adrenomedullin mRNA was present in spinal cord, at one-third of its level in lung, and adrenomedullin immunoreactivity was present, at a low concentration (40 fmol/g of tissue). Thus, the presence of abundant binding sites and adrenomedullin mRNA and immunoreactivity anticipate an as yet undefined function for this peptide in spinal cord.     

High Levels of Ascorbic Acid, Not Glutathione, in the CNS of Anoxia-Tolerant Reptiles Contrasted with Levels in Anoxia-Intolerant Species

Abstract: Ascorbic acid and glutathione (GSH) are antioxidants and free radical scavengers that provide the first line of defense against oxidative damage in the CNS. Using HPLC with electrochemical detection, we determined tissue contents of these antioxidants in brain and spinal cord in species with varying abilities to tolerate anoxia, including anoxia-tolerant pond and box turtles, moderately tolerant garter snakes, anoxia-intolerant clawed frogs (Xenopus laevis), and intolerant Long-Evans hooded rats. These data were compared with ascorbate and GSH levels in selected regions of guinea pig CNS, human cortex, and values from the literature. Ascorbate levels in turtles were typically 100% higher than those in rat. Cortex, olfactory bulb, and dorsal ventricular ridge had the highest content in turtle, 5–6 µmol g^?1 of tissue wet weight, which was twice that in rat cortex (2.82 ± 0.05 µmol g^?1) and threefold greater than in guinea pig cortex (1.71 ± 0.03 µmol g^?1). Regionally distinct levels (2–4 µmol g^?1) were found in turtle cerebellum, optic lobe, brainstem, and spinal cord, with a decreasing anterior-to-posterior gradient. Ascorbate was lowest in white matter (optic nerve) in each species. Snake cortex and brainstem had significantly higher ascorbate levels than in rat or guinea pig, although other regions had comparable or lower levels. Frog ascorbate was generally in an intermediate range between that in rat and guinea pig. In contrast to ascorbate, GSH levels in anoxia-tolerant turtles, 2–3 µmol g^?1 of tissue wet weight, were similar to those in mammalian or amphibian brain, with no consistent pattern associated with anoxia tolerance. GSH levels in pond turtle CNS were significantly higher (by 10–20%) than in rat for several regions but were generally lower than in guinea pig or frog. GSH in box turtle and snake CNS were the same or lower than in rat or guinea pig. The distribution GSH in the CNS also had a decreasing anterior-to-posterior gradient but with less variability than ascorbate; levels were similar in optic nerve, brainstem, and spinal cord. The paradoxically high levels of ascorbate in turtle brain, which has a lower rate of oxidative metabolism than mammalian, suggest that ascorbate is an essential cerebral antioxidant. High levels may have evolved to protect cells from oxidative damage when aerobic metabolism resumes after a hypoxic dive.     

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.     

Regional concentration and chromatographic characterization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the brain of the bullfrog,Rana catesbeiana

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide which functions as a hypothalamic factor for pituitary hormone release, and as a neurotransmitter, neuromodulator and neurotrophic factor in both frogs and mammals. This study examined the quantitative distribution and chromatographic characterization of immunoreactive PACAP in the central nervous system (CNS) of the bullfrog, Rana catesbeiana, using an enzyme immunoassay (EIA), named avidin-biotin complex detectable EIA for PACAP, and high-performance liquid chromatographic (HPLC) analysis. The brain of adult bullfrogs contained relatively high levels of immunoreactive PACAP (344.63 pmol/g wet weight of tissue). The average concentrations of immunoreactive PACAP in the regions of the telencephalon, diencephalon, tectum, cerebellum, rhombencephalon, and spinal cord were 213.84, 767.14, 524.94, 192.71, 237.67, and 362.04 pmol/g wet weight of tissue, respectively. The concentrations of immunoreactive PACAP increased with the brain development during metamorphosis, and the concentration of immunoreactive PACAP in the brain of tadpoles at the end of metamorphosis was approximately 200 pmol/g wet weight of tissue. The predominant form of immunoreactive PACAP in the CNS of adult and tadpole was eluted closely with synthetic PACAP38, but another smaller immunoreactivity also appeared in a the fraction, which corresponded to the retention time of synthetic PACAP27, as analyzed by reverse-phase HPLC.     

Biochemical Mapping of Peptidyl-Glycine α-Amidation Activity in the Rat CNS

Peptidyl-glycine alpha-amidation enzyme activity has been measured in 36 nuclei or areas in the rat CNS and pituitary using D-Tyr-Phe-Gly as the substrate. The distribution of this enzyme is highly uneven, with highest activity levels (greater than 30 pmol/mg of protein/h) in hypothalamic nuclei, substantia grisea centralis, and nucleus ruber; moderate activity levels (10-30 pmol/mg of protein/h) in globus pallidus, septum, midbrain, pons, medulla oblongata, and cervical spinal cord; and low activity levels (1-10 pmol/mg of protein/h) in other telencephalic and thalamic structures. Almost no alpha-amidation activity (less than 0.5 pmol/mg of protein/h) was detected in cerebellar cortex. The Km values in several brain regions are of the same order.     

Characterisation of a Myristoyl CoA:Glycylpeptide N-Myristoyl Transferase Activity in Rat Brain: Subcellular and Regional Distribution

Abstract: An enzyme activity in rat brain, capable of catalysing the transfer of myristic acid from myristoyl CoA to the amino terminus of synthetic peptides, has been characterised. The synthetic peptides used as substrates were one based on the N-terminal eight amino acids of cyclic AMP-dependent protein kinase and another hexadecapeptide based on the N-terminal sequence of p60^src. This N -myristoyl transferase (NMT) activity, which is both peptide dependent and heat labile, occurs in rat brain at levels at least three times those found in other rat tissues. In the presence of both ATP and CoA the enzyme catalysed the transfer of myristic acid, but not palmitic acid, specifically to the N-terminal glycine of the peptides. Both peptide substrates exhibited Mi-chaelis-Menten kinetics yielding K _m values of 100 μ M and 60 μ M , and V_max values of 5 and 14.8 pmol/min/mg for the cyclic AMP-dependent protein kinase peptide and sre-derived peptides, respectively. The majority of the NMT activity was present in the cytosol of the brain homogenates, and there was evidence of an NMT inhibitory activity in both the particulate fraction of brain homogenates and in brain cytosol. NMT activity could also be demonstrated in the 100,000 g supernatant of lysed synaptosomes, and the synaptosomal membranes also exhibited an inhibitory activity on the soluble enzyme. Different brain areas exhibited different levels of the N -myristoyl transferase activity and there was a fivefold difference in the activity found in the most active area, the hippocampus, compared to spinal cord.     

MICROASSAY OF ADENOSINE-3′,5′-MONOPHOSPHATE (CYCLIC AMP) IN BRAIN AND OTHER TISSUES BY THE LUCIFERIN-LUCIFERASE SYSTEM1

Abstract— A simple, sensitive and specific method for assaying cyclic AMP in various tissues is reported. Cyclic AMP was isolated from contaminating nucleotides and was converted to ATP with a phosphodiesterase-myokinase-pyruvate kinase system. The ATP was determined enzymically in a liquid scintillation counter by the firefly luciferin-luciferase technique. This procedure was capable of detecting as little as 5 × 10^?14 mol of cyclic AMP and could therefore be used for analyses on less than 1 mg of brain. The assay was reproducible and linear over a wide range of tissue concentrations. In the rat, the highest levels of cyclic AMP (2.7–4.2 pmol/mg wet wt. of tissue) were present in the pineal, heart, pituitary, thyroid, cerebellar cortex, kidney, adrenal, liver and pyloric region of the stomach; intermediate levels (1.5–2.7 pmol/mg wet wt. of tissue) were found in testis, skin, aorta, intestine, submaxillary gland, spleen, muscle and cerebral cortex, moderately low levels (1.0–1.5 pmol/mg wet wt. of tissue) were found in lung, trachea and greater curvature of the stomach; whereas low levels (0.15–0.60 pmol/mg wet wt. of tissue) were found in adipose tissue.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).     

Effects evoked by pentamethylenetetrazol-induced seizures upon N-acetylaspartate and N-acetylaspartylglutamate levels in different regions of the rat neuraxis

We have examined the effects evoked by pentamethylenetetrazol (PTZ)-induced seizures upon the concentration of N-acetylaspartate (NAA) and of N-acetylaspartylglutamate (NAAG) in the forebrain, brainstem and spinal cord of rats. We observed a significant decrease of both NAA and NAAG in each one of the studied regions. These findings are consistent with an inhibitory role proposed in CNS for NAA and NAAG.     

Astrocytes from Forebrain, Cerebellum, and Spinal Cord Differ in Their Responses to Vasoactive Intestinal Peptide

Astrocytes from cortex, cerebellum, and spinal cord responded to isoproterenol and vasoactive intestinal peptide (VIP) with increases in intracellular cyclic AMP levels. The response to VIP was as great as that to isoproterenol in cortical astrocytes (180-fold and 185-fold, respectively), and the effect of VIP in combination with isoproterenol was partially additive. Spinal cord astrocytes also responded to VIP and isoproterenol with equal potency (seven- to ninefold and eight- to 13-fold, respectively), but the level of response was much smaller than in cortex. Spinal cord astrocytes were synergistic in their response to VIP and isoproterenol. The response to VIP was lowest in cerebellar astrocytes (only threefold), and no additivity was observed when VIP was added together with isoproterenol. A small response to alpha-melanocyte stimulating hormone (alpha-MSH) was also observed in cortex and cerebellum, but not in spinal cord. Somatostatin inhibited the response to isoproterenol in cortex and cerebellum, but had no effect in spinal cord. The results from the above study show that astrocytes obtained from these three regions of the rat CNS express quite different responses to VIP and alpha-MSH and further point to possible astrocyte heterogeneity.     

Localization and Transport of N-Acetylaspartylglutamate in Cells of Whole Murine Brain in Primary Culture

Abstract: N -Acetylaspartylglutamate (NAAG) is the most abundant neuropeptide in the mammalian nervous system. Considerable data support the hypothesis that NAAG is synaptically released in a manner consistent with neurotransmission. Primary murine brain cultures containing neurons and glia expressed 1.2-3.5 nmol of NAAG/mg of protein. In contrast to conclusions drawn from immunohistochemistry, pure glial cultures also expressed high levels of NAAG (0.6-2.11 nmol/mg of protein). These data suggest that although a subpopulation of neurons contains very high NAAG levels, micromolar concentrations of the peptide also are present in glia. Both culture types demonstrated robust extracellular peptidase activity when incubated with NAAG, as well as peptide transport. Uptake of [³H]NAAG was both temperature and sodium dependent, yet relatively insensitive to the presence of extracellular glutamate. These results indicate that synaptically released NAAG, as well as that which may be released from glia, is removed from the extracellular space by direct uptake as well as the robust enzymatic degradation of the peptide. A kinetic analysis of this NAAG transport (estimated K _m= 1.8 μ M ) suggests a high-affinity NAAG transport system. The balance of the two processes of direct peptide uptake and peptide hydrolysis would markedly influence the sequence of receptor-mediated events that follow NAAG release.     

DEVELOPMENTAL AND AGING CHANGES IN PROTEIN CONCENTRATION AND 2'',3''-CYCLIC NUCLEOSIDEMONOPHOSPHATE PHOSPHODIESTERASE ACTIVITY (EC 3.1.4.16) IN HUMAN CEREBRAL WHITE AND GRAY MATTER AND SPINAL CORD

Abstract— The concentration of protein as assayed by the Lowry method and the specific activity of 2′.3’-cyclic nucleosidemonophosphate phosphodiesterase (CNP), an enzyme characteristic of the myelin sheath, were determined in human CNS tissues obtained at autopsy from subjects ranging in age from 26 weeks gestation to 83 y. CNP activity in cerebral white matter samples was very low until approx 2 months of age when it increased rapidly, reaching near-adult levels by 2 y of age. CNP activity in adult (15–60 y) cerebral white matter was 8.1 ± 1.0 μmol/min/mg protein (mean ±s.d. ). The protein concentration of cerebral white matter increased from 64 mg/g wet tissue at 26 weeks gestation to adult levels (118.5 ± 10.0 mg/g wet tissue) by 16–18 months. CNP activity in cerebral gray matter was initially very low and showed only a small increase during development to adult values of approx 1.4 μmol/min/mg protein. In spinal cord, adult values (3.7 ± 0.56 μmol/min/mg protein) were found shortly after birth. The increase in CNP activity to near-adult values occurred earlier in cross-sections of cervical spinal cord than in cerebral white matter. The increase in spinal cord protein concentration showed a similar trend (adult values = 103.1 ± 9.5 mg/g wet tissue). The white matter protein concentration decreased significantly with age over the 15–83 y interval examined but the CNP specific activity in white matter did not. The protein concentration of the 61–83 y group was 8% lower than that of the 15–60 y group. The spinal cord protein concentration decreased significantly and the spinal cord CNP specific acitivity increased significantly with increasing time between death and sample freezing. The sex of the individual had no significant effect on any of the variables examined. The developmental curves obtained for these tissues are consistent with the hypothesis that CNP is an intrinsic myelin component in human CNS myelin. The marked increase in CNP activity in white matter coincides with the period of rapid myelin deposition as determined by other parameters. CNP activity may be useful as an index of myelination in human CNS tissues.     

Field desorption mass spectrometric measurement of picomole amounts of leucine enkephalin in canine spinal cord tissue

Leucine enkephalin is measured in canine spinal cord tissue in a structurally unambiguous manner. A rapid tissue procurement procedure minimizes enkephalin metabolism. High-performance liquid chromatography purification of brian neuropeptides is followed by field desorption mass spectrometric measurement of leucine enkephalin in spinal cord tissue extracts. Quantification is performed at the 70 ng (126 pmol) g-1 of wet weight tissue, or 70 parts per billion level. The higher homolog of leucine enkephalin, 2ala-leucine enkephalin, is utilized as internal standard. Straight-line statistics are obtained for a series of samples to which a peptide standard is added.     

The cloning and characterization of a second brain enzyme with NAAG peptidase activity

The peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non-overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar V(max) and K(m) values (1.4 micro m and 54 pmol/min/mg; 3.5 micro m and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG.     

COMPARISON OF THE EFFECTS OF DEPOLARIZING AGENTS AND NEUROTRANSMITTERS ON REGIONAL CNS CYCLIC GMP LEVELS IN VARIOUS''ANIMALS

Abstract— The effects of 121 m m -K⁺, 10 m m -glutamate, 5 m m -GABA, 1 m m -glycine, 0.1 m m -NE, and 1–10 μ m ACh on cyclic GMP levels in tissue slices prepared from cerebral cortex and cerebellum of mouse, rabbit, guinea-pig, cat, and rat were studied. Basal levels of cyclic GMP in the cerebella of mice, guinea-pigs and cats were 4–15 and 70 pmol/mg prot in rat, whereas in the cerebral cortex of the same animals, levels were only 0.6–2 pmol/mg prot. In contrast, basal levels of the cyclic nucleotide were 1–2 pmol/mg prot in both of these regions in rabbit brain. Only 121 m m -K⁺ was capable of increasing cyclic GMP levels in all the tissues studied. Elevations ranged from 30% in rat cerebral cortex to 2800% in mouse cerebellum. Glutamate produced a 30–1000% rise of cyclic GMP levels in all tissues except rabbit cerebellum. NE elevated levels of cyclic nucleotide 2- to 3-fold in slices of cerebellum from all species studied but had no effect in cerebral cortex. GABA and glycine had no effect in any tissue except mouse cerebellum. ACh had no consistent effect on levels of cyclic GMP in any brain region investigated. These results suggest that mechanisms regulating cyclic GMP levels in mammalian CNS vary among brain regions and among animal species.