DT-n-PROPYLACETATE AND GABA DEGRADATION. PREFERENTIAL INHIBITION OF SUCCINIC SEMIALDEHYDE DEHYDROGENASE AND INDIRECT INHIBITION OF GABA-TRANSAMINASE

The kinetic constants for 4-aminobutyrate: 2-oxoglutarate aminotransferase (GABA-trans-aminase) and succinate-semialdehyde: NAD⁺ oxidoreductase (SSA-DH) have been determined using rat brain homogenate. The Michaelis constants for GABA-T at saturated substrate concentrations were calculated to be K_gaba= 1.5 mM, K_2-OG= 0.25 mM, K_GLU= 620 μM, and K_SSA= 87 μm. The V_max for the reaction using GABA and 2-oxoglutarate (2-OG) as substrates (forward reaction) was found to be 35.2 μmol/ These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/gh and 167 pmol/g These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g/h in the brain and spinal cord respectively were calculated. The kinetics of GABA-T have been shown to be consistent with a Ping Pong Bi Bi mechanism. Substrate inhibition of the forward reaction, through formation of a dead-end complex, was found to occur with 2-OG (K_i 3.3 mM), whereas GABA was found to be a product inhibitor of the reverse reaction (K_i= 0.6 mM). Using the appropriate Haldane relationship, a K_eq of 0.04 for GGBA-T was found, indicating that the reaction was strongly biased towards GABA. For SSA-DH, the K_m of SSA was determined (9.1 μM) and the V_max was 27.5 μmol/ These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g/h in the brain and spinal cord respectively were calculated. h. The effect of di-n-propylacetate (DPA) on both GABA-T and SSA-DH was measured. DPA inhibited SSA-DH competitively with respect to SSA, giving a K_i of 0.5 mM. GABA-T was only slightly inhibited. The K_i of DPA for the forward reaction was 23.2 mM with respect to GABA, which was 40-50 times higher than that for SSA-DH. For the reverse reaction the K_i of DPA was found to be nearly the same (15.2 mM with respect to Glu and 22.9 mM with respect to SSA). These results suggest that GABA accumulation in the brain, after administration of DPA in vivo, is caused by SSA-DH inhibition. Two mechanisms are indicated by the data. (1) The higher level of SSA, which results from inhibition of SSA-DH, initiates the reverse reaction of GABA-T, thus increasing the level of GABA via conversion of SSA. (2) The degradation of GABA is inhibited by SSA, since SSA has a strong inhibitory effect on the forward reaction, as calculated from the present data.     

ON THE ORIGIN OF VANILLYLMANDELIC ACID AND 3-METHOXY-4-HYDROXY-PHENYLGLYCOL IN THE RAT BRAIN

Abstract— The effects of electrothermic destruction and electrical stimulation of the locus coeruleus on the brain levels of vanillylmandelic acid (VMA), 3-methoxy-4-hydroxyphenylglycol (MOPEG) and noradrenaline (NA) were studied in the rat. Fourteen days after destruction of the locus coeruleus, the content of NA in the hippocampus and that of MOPEG in the rest of the brain were decreased by more than 70% and 50% respectively.
Stimulation of the locus coeruleus induced a decrease in hippocampal levels of NA of 38%, while MOPEG levels were found to be increased more than 3-fold After intraventricular injection of 1 μg of adrenaline (A), the levels of MOPEG were Substantially increased In none of these experiments was any variation in VMA levels found, These results suggest that in the rat brain endogenous VMA is not a metabolite of either NA or A The formation of MOPEG from A as well as from NA appears to be possible.     

A method for the assay of conjugated 3,4-dihydroxyphenylglycol,a major noradrenaline metabolite in the rat brain

We present the first published procedure for the measurement of endogenous conjugated 3,4-dihydroxyphenylglycol (DOPEG) in the rat brain. Conjugated DOPEG is estimated from brain extracts after enzymic hydrolysis, isolation of hydrolysed DOPEG on alumina, methylation of DOPEG to 3-methoxy-4-hydroxyphenylglycol (MOPEG) and gas chromatographic quantification of MOPEG. The level of conjugated DOPEG in the CNS of rats (65.7 +/- 0.7 ng/g whole brain tissue corrected for recovery) almost equals the level of conjugated MOPEG. The sensitivity of the method is about 6 ng/g brain tissue. After inhibition of monoamine oxidase with clorgyline (30 mg/kg) conjugated DOPEG and MOPEG both disappeared from the brain with a half-life of about 1 h. Turnover calculations indicate that conjugated DOPEG and MOPEG are the two major noradrenaline end-metabolites in the rat brain. The method of estimating conjugated DOPEG also allows the measurement of noradrenaline, dopamine and total MOPEG in an extract from one half of a rat brain.     

FACTORS INFLUENCING BRAIN AND TISSUE LEVELS OF TRYPTAMINE: SPECIES, DRUGS AND LESIONS

With a modification of the spectrophotofluorometric (SPF) method of HESS & UDENFRIEND (1959) (J. Pharmac. exp. Ther. 127 , 175-177), brain tryptamine levels in the rat (20.9 ng/g) and guinea-pig (20.7 ng/g) were found to be less than those in the dog (32.1 ng/g) and cat (52.2 ng/g). Regional distribution studies in the dog and cat showed that tryptamine was present in all major brain regions with highest concentrations in the spinal cord. Blood levels of tryptamine in the guinea-pig, dog and cat (6-7 ng/ml) were lower than brain levels. Pargyline significantly increased brain tryptamine in both the dog and cat; whereas, isocarboxazid (after 4 h) increased brain tryptamine levels in the dog but decreased brain levels in the cat. Reserpine (0.5-1.0 mg/kg per day for 1-4 days) did not significantly decrease brain, spinal cord or blood tryptamine levels in the dog. Spinal cord transection did not decrease tryptamine levels below the lesion in the chronic spinal dog.     

BIOSYNTHESIS OF PSYCHOSINE AND LEVELS OF CEREBROSIDES IN THE CENTRAL AND PERIPHERAL NERVOUS SYSTEMS OF QUAKING MICE

Abstract— The levels of cerebrosides in neural tissues of adult mice were determined by densitometry of cerebroside spots on charred thin-layer chromatograms of washed total lipid extracts. Values for brain, spinal cord and peripheral nerves were 9·2, 33·0 and 36·9 mg/g of tissue, respectively. In adult Quaking mice these values were 6·4, 24 and 35 % of normal, respectively. Normal levels in brain, spinal cord and peripheral nerve of 21-day-old mice were 3·10, 13·5 and 17·8 mg/g, respectively. In 21-day-old Quaking mice the levels were reduced to 16,21 and 57% of normal, respectively. Biosynthesis of psychosine (galacto-sylsphingosine) by homogenates of Quaking brain, spinal cord and peripheral nerve, respectively, was 18, 24 and 42% of the normal rates at 21 days after birth and 16, 66 and 60% of the normal rates at 94 days. Our results suggest a quantitative relationship between the rate of formation of psychosine in vitro and the rate of accumulation of cerebrosides. in vivo. Biosynthesis of lactosylceramide was not reduced in homogenates of brain and spinal cord from Quaking mice. Cerebroside levels in normal and Quaking spinal cord and in normal brain increased 2- to 3-fold after 21 days of age, but in Quaking brain there was little or no increase.     

COMPARATIVE STUDIES ON THE DEGRADATION OF GABA and TAURINE IN THE BRAIN

Abstract— The degradation of taurine and GABA in mammalian brain was studied in vivo and in vitro. Small amounts of [³⁵S]isethionate (10–20 pmol/g brain wet weight) and [³⁵S]sulphate (about 2 pmol/g) were detected in mouse brain after intramuscular injection of [³⁵S]taurine. Taurine also produced isethionate in rat brain homogenates (about 20 nmol/h/g protein) and subcellular fractions (about 40 nmol/h/g protein in synaptosomes and about 300 nmol/h/g in mitochondria), but the reaction was not stimulated either by external electrical pulses or by the addition of various cofactors (NAD and NADP in both oxidized and reduced forms, riboflavin, glutathione. pyridoxal-5'-phosphate, ATP) to the incubation medium. [¹⁴C]GABA was readily metabolized to [¹⁴C]succinate both in vivo and in vitro. Isethionate formation activity was concentrated in the mitochondrial fraction, as was also GABA-T activity. Partially purified GABA-T from calf brain also slightly catalysed the formation of [³⁵S]isethionate (about 1.3 μmol/min/g protein) from [³⁵S]taurine. It appears that the slight formation of isethionate from taurine is coupled to GABA-T activity. The formation of isethionate from taurine is so small, that it apparently has no role in the control of the brain taurine pool.     

STEADY STATE MAINTENANCE OF ELECTROLYTES IN THE SPINAL CORD OF THE FROG

Isolated frog spinal cords equilibrated from 3 to 24 h in Ringer's solution maintained steady state conditions with regard to electrolyte composition. Total sodium and chloride contents measured on the same spinal cords were found to be nearly equivalent (Na = 46.6 ± 1.4μmol/g wet wt vs Cl = 46.2 ± 1.2μmol/g wet wt). Calcium and magnesium contents were 3.0 ± 0.5 and 4.8 ± 0.2pμol/g wet wt respectively for fresh spinal cords and 2.1 ± 0.4 and 5.5 ± 0.9pmol/g wet wt respectively for spinal cords equilibrated for 24 h. Zinc content was 0.29 ± 0.01 μmol/g wet wt. Insulin space was found to be smaller than sucrose space (0.24 ml/g vs 0.37 ml/g). Sodium and chloride spaces were slightly higher than sucrose space. Sodium and chloride in the non-sucrose space was 4.8 and 9.5 μmol/g wet wt. Residual radioactive sodium, or sodium content of spinal cords washed out for 180 min in non-radioactive Ringer's solution or in choline or lithium Ringer's solution, was 4.2 ± 0.4μmol/g wet wt (n = 9). The agreement between residual sodium content and sodium in the non-sucrose space suggests that the mean intracellular sodium content of the spinal cord neurons is low.     

Concentration and molecular forms of brain natriuretic peptide in rat plasma and spinal cord

To characterize the biological functions of rat brain (B-type) natriuretic peptide (BNP), which has been shown to be present mainly in the heart and only faintly in the spinal cord, the concentration and molecular forms of BNP in plasma and spinal cord were determined. The concentration of immunoreactive (ir-) BNP was 2.00 fmol/ml in normal rat and 13.29 fmol/ml in morphine-treated rat, being respectively about 1/20 and 1/80 those of ir-atrial (A-type) natriuretic peptide (ANP). In morphine-treated rats, ir-BNP was shown to circulate mainly as BNP-45, which is identical to a major storage form found in cardiac atrium. In the spinal cord, BNP was also shown to be present as BNP-45, but its concentration was only 0.057 pmol/g, being about 1/60 that of spinal cord ANP. These results confirm that BNP mainly functions as a circulating hormone in the molecular form of BNP-45. Morphine stimulates secretion of ANP and BNP but by different ratios, suggesting different regulation systems for storage and secretion of ANP and BNP.     

ACCUMULATION AND DISAPPEARANCE OF NORADRENALINE AND ITS MAJOR METABOLITES SYNTHESIZED FROM INTRAVENTRICULARLY INJECTED [3H]DOPAMINE IN THE RAT BRAIN

Abstract— A new combined ion-exchange and thin-layer-chromatographic procedure is described which separates and measures quantitatively, after intraventricular injection of [³H]dopamine (DA), the rat brain content of labelled noradrenaline (NA) and the following labelled noradrenaline metabolites: free 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG), conjugated MOPEG, free plus conjugated dihydroxyphenylethyleneglycol (DOPEG), vanillic mandelic acid (VMA) and normetanephrine (NM). Labelled dopamine and its metabolites were also measured. The time-course study performed from 5 min to 24 h after [³H]DA showed that MOPEG and DOPEG, mainly as conjugates, are major NA metabolites whereas VMA is a very insignificant NA metabolite in the rat brain. A very rapid initial increase of [³H]NM, free MOPEG and conjugated MOPEG was found during the time interval where the [³H]NA biosynthesis is very high (0–15 min). This combined with the finding that these metabolites stabilize at lower levels during the [³H]NA ‘storage phase’ (9–24 h) provides a strong indication that newly synthesized NA preferentially is metabolized. Our measurements of endogenous NA, free MOPEG and conjugated MOPEG provide additional support. The injections of various decreasing doses of [³H]DA (3·08–0·0010 μg) showed that the proportions of total [³H]MOPEG and total [³H]DOPEG to [³H]NA were constant after all [³H]DA doses investigated. This finding indicates that the [³H]NA synthesized in situ behaves as a tracer, even after injections of non-tracer doses of [³H]DA. The results seem thus to indicate that the present technique provides a powerful tool for the investigations on central noradrenaline metabolism.     

DISTRIBUTION AND TURNOVER RATE OF VANILLYL-MANDELIC ACID AND 3-METHOXY- 4- HYDROXYPHENYLGLYCOL IN RAT BRAIN

Abstract— Vanillylmandelic acid (VMA) and 3-methoxy-4- hydroxyphenylglycol (MHPG) were measured in rat brain by a mass fragmentographic procedure. The concentration of VMA and MHPG in whole brain is 11 and 533 pmol/g, respectively. Both compounds were found in all areas of brain studied with VMA, as a percentage of both metabolites, ranging between about 1 and 8%. From the decline of the compounds after pargyline. 75 mg/kg i.p., we calculated that the rate of formation of VMA is 15 and for MHPG 202 pmol/g per h. The fractional rate of elimination of VMA and MHPG is 1.4 and 0.38 h⁻¹, respectively. The rapid rate of loss of VMA suggests that it is transported from brain. However, we were unable to block the elimination of VMA from brain by treatment with probenecid. In contrast, the elimination of MHPG could be blocked by treatment with probenecid. Our study adds support to the notion that MHPG is a major whereas VMA is a minor product of norepinephrine metabolism in brain.     

THE DISTRIBUTION OF ACETYL-CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO-ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE1

A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl-CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl-CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane-chloroform wash followed by an ether extraction. In the acetyl-CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl-CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of [Me-¹⁴C]choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The [¹⁴C]ACh formed from acetyl-CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the [¹⁴C]ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl-CoA levels in rat whole brain when killed by the near-freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl-CoA was the same whether the rats were killed by the near-freezing method or by total freezing in liquid nitrogen. The levels of acetyl-CoA did not change with time after death when the tissue was maintained at a temperature of ?10°C. In the same tissue extracts from rat whole brain killed by the near-freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl-CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl-CoA in heart, liver and kidney are presented.     

GABA CONTENT OF DISCRETE BRATN NUCLEI AND SPINAL CORD OF THE RAT

Abstract– The GABA content of the spinal cord and of approx 70 discrete rat brain nuclei is measured with a simple rapid semi-automated fluorimetric assay, after prevention of post-mortem effects with 3-mercaptopropionic acid. We found that microwave irradiation produced decreases in the GABA contents of the microdissected zona reticulata of the substantia nigra, indicating that microwave fixation is not suitable to measure GABA levels in microdissected brain nuclei. In approx 70% of the nuclei in the anterior half of the brain the GABA concentration was found to be between 41 and 90nmol GABA/mg protein. The GABA content varied from 11 to 40 nmol GABA/mg protein in the posterior half of the brain. High GABA levels were found in some hypothalamic nuclei, the globus pallidus and eminentia mediana. An extremely high GABA level was found in the zona reticulata of the substantia nigra. GABA is unevenly distributed in the striatum. The highest concentration was found in the caudal part and in the ventral region at any level of the striatum. In the spinal cord the highest concentration of GABA was in the sacral region.     

LOCALIZATION OF THE THY-1 ANTIGEN IN RAT BRAIN AND SPINAL CORD BY IMMUNOFLUORESCENCE

Abstract— The Thy-1 antigen of rat brain is a membrane glycoprotein of molecular weight 17,500. It was localized in sections of brain and spinal cord by indirect immunofluorescence using rabbit antisera raised against purified Thy-1 and fluorescein conjugated purified sheep F(ab')₂, anti-(rabbit IgG) antibody fragments. The specificity of the anti-(Thy-1) sera was tested by a quantitative indirect radioactive binding assay which is particularly useful for ascertaining the specificity of reagents used in immunohistochemical studies. Purified Thy-1 was used to absorb the anti-(Thy-1) sera for controls in the immunofluorescence experiments. Strong specific fluorescence was found throughout the gray matter of brain and spinal cord with lesser amounts in white matter. The nuclei of all neural cells and also myelin lacked fluorescence. Some of the large neurons contained weak cytoplasmic fluorescence, but the majority of the immunofluorescence was located in the neuropil of the brain and spinal cord. There was an indication that Thy-1 was associated with synaptic knobs due to its presence in synaptic glomeruli and its granular appearance around some neurons. An additional association with glial membranes could not be excluded.     

SIMULTANEOUS ISOLATION OF PURIFIED MICROSOMAL and MYELIN FRACTIONS FROM RAT SPINAL CORD

Abstract— The fraction that sediments between 2 × 10⁵ g -min and 6 × 10⁶ g -min from dilute dispersions of rat brain in 0.32 m -sucrose is a microsomal fraction with very little contamination by myelin. A crude microsomal fraction prepared in the same way from rat spinal cord contains more myelin than microsomes. Centrifugation of the crude microsomal fraction in 0.85 m -sucrose gave a floating fraction, an infranatant fraction (purified microsomes) and a small pellet. The purified microsomes contained very little myelin as judged by electron microscopy and polyacrylamide gel electrophoresis. The lipid composition resembled that of spinal cord myelin except that the purified microsomes contained relatively less cholesterol and ethanolamine plasmalogens. The content of galactolipids was much greater in spinal cord microsomes than in brain microsomes. The spinal cord CDP-ethanol-amine:diglyceride ethanolaminephosphotransferase activity (EC 2.7.8.1) was concentrated in the purified microsomes.
A spinal cord myelin fraction isolated from the 2 × 10⁵ g -min pellet was quite pure as judged by electron microscopy, enzyme activities and polyacrylamide gel electrophoresis. No NADPH-cyto-chrome c reductase activity (EC 1.6.2.3) could be detected in the purified myelin. The ethanolaminephosphotransferase specific activity was about 5% of that found in the purified microsomal fraction. The protein content was 25% by weight for spinal cord myelin and 31% for brain myelin. Of the total spinal cord 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, 16% was lost from the crude myelin during purification, 21% was recovered in the purified myelin, and 11% was found in the floating fraction from the crude microsomes. The purified myelin and microsomal fractions from spinal cord were relatively pure. Additional myelin was recovered in the floating fraction from the crude microsomes.     

人和大鼠脊髓内的心钠素样物质

应用特异的心钠素免疫金银染色和放射免疫测定法,证明在人和大鼠脊髓内亦存在有心钠素样物质。心钠素免疫金银染色发现在人脊髓各段均有心钠素免疫反应阳性的神经元广泛分布。这些神经元主要位于脊髓腹角,同时脊髓背角和侧角亦有少量分布。应用对照吸收试验,其心钠素免疫反应阳性颗粒便消失或明显减少。心钠素放射免疫测定发现,从大鼠颈髓到胸、腰、骶髓均有心钠素样物质存在,其中以骶髓含量最高,为21.9±4.48ng/g组织;腰髓次之,为3.78±0.74ng/g组织;颈、胸髓含量最低,分别为0.58±0.14和0.46±0.21ng/g组织。应用凝胶过滤和高压液相层析证明,大鼠脊髓中心钠素亦以多分子形式存在,但以28个氨基酸的大鼠心房利纳多肽(rANP)为主。此外,对在体大鼠脊髓蛛网膜下腔灌流研究发现,高钾去极化刺激可使大鼠脊髓心钠素样物质释放。     

EFFECTS OF 5,6-DIHYDROXYTRYPTAMINE ON MONOAMINERGIC NEURONES IN THE CENTRAL NERVOUS SYSTEM OF THE RAT

—The injection of 50 μg of 5,6-dihydroxytryptamine (5,6-HT) into a lateral ventricle of the rat depleted the spinal cord and various regions of the brain of indoleamines (presumably 5-HT) and 5-hydroxyindole acetic acid. The concentrations of 5-HT were measured by two different methods: the formation of a fluorescent derivative with o-phthalaldehyde, and the native fluorescence in hydrochloric acid. When the results of both methods were compared on the pons and medulla 4 days after injecting 5,6-HT, the loss in indoleamine appeared to be greater when o-phthalaldehyde was used. This suggests that the two methods may be measuring different compounds. According to both methods, the loss of 5-HT persisted for several days after the injection of 5,6-HT, but by 2 months 5-HT concentrations (measured only by the native fluorescence procedure), had recovered to near-normal values. The depletion of 5-HT was most pronounced in regions adjacent to the ventricular system and in the spinal cord. Initially, caudate and septum were more affected on the side of the injection, and later showed some permanent atrophy. The injection of up to 50 μg of 5,6-HT did not lead to any significant loss of noradrenaline or dopamine from the brain, or to any reduction in the activity of the enzyme tyrosine hydroxylase. The drug was a potent inhibitor of the uptake of [³H]5-HT by brain slices, but was less effective in inhibiting catecholamine uptake systems. These observations suggest a preferential action on tryptaminergic neurones. Larger doses of 5,6-HT caused a loss of catecholamines and tyrosine hydroxylase from the brain, and were severely toxic.     

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.     

吗啡降低大鼠脊髓内cAMP的含量

有资料表明,吗啡或脑啡肽可影响脑内 cAMP 与 cGMP 的含量,但对脊髓内 cAMP 与cGMP 的含量有何影响,未见报道。本实验采用放射免疫分析(RIA)方法研究的结果表明,吗啡可使大鼠离体与在体脊髓内 cAMP 的含量明显降低;而对脊髓内 cGMP 的含量则无明显影响;纳洛酮可特异阻断吗啡对脊髓内 cAMP 含量的抑制效应。提示:吗啡的上述作用是通过大鼠脊髓内阿片受体所介导。脑和脊髓内 cAMP 含量的变化可能部分介导了吗啡作用的中枢机制。     

DIFFERENTIATION OF DOPAMINERGIC AND NORADRENERGIC NEURONS IN RAT SPINAL CORD

Abstract— Norepinephrine (NE), dopamine (DM) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) content have been measured in different parts of rat spinal cord and cerebellum by a gas chromatographic mass spectrometric method. In cerebellum, which does not contain dopaminergic neurons, the ratio of NE to DA content was 47, whereas in parts of the spinal cord this ratio varied between 11 and 19. In the cord after desipramine (25 mg/kg, i.p.) plus 6-hydroxydopamine (6-HDA, 100/jg intracisternally), there was a significant depletion of DM but not of NE. Conversely, after benztropine (25 mg/kg, i.p.) plus 6-HDA there was a significant depletion of NE but not of DM. Chlorpromazine (10 mg/kg, i.p.) or clozapine (25 mg/kg, i.p.) caused a significant increase in spinal cord HVA concentration 1 h after treatment. Evidence is presented which suggests that the increased HVA measured in the cord did not originate in the brain. After electrolytic lesion of the locus coeruleus there was a significant reduction of NE but not of DM. Spinal cord DM and NE were depleted by reserpine in a dose-dependent manner, the threshold dose for DM depletion being less than that for NE depletion. Seven days after cord transection at T₁₀ spinal cord DM was significantly reduced in the lumbar region. These results suggest that dopaminergic neurons exist in rat spinal cord independently of noradrenergic neurons and that the DM is likely to be present in the terminals of descending axons.     

N-Acetylsuccinimidylglutamate, a Cyclic Imide Form of the Peptide N-Acetylaspartylglutamate, Is Present in Low Micromolar Concentrations in Murine and Bovine CNS

Abstract: N -Acetylsuccinimidylglutamate [(asu)NAAG], a cyclic form of the peptide N -acetylaspartylglutamate (NAAG) in which the aspartyl residue is linked to glutamate via the α- and β-carboxylates, was identified and quantified by HPLC in the murine and bovine CNS. In the rat, the highest concentrations of (asu)NAAG were detected in the spinal cord (1.83 ± 0.15 pmol/mg of wet tissue weight) and brainstem (1.16 ± 0.08 pmol/mg wet weight), whereas the levels were below the limit of detection in cerebellum, hippocampus, and cerebral cortex. (Asu)NAAG was also detected in significant amounts in the superior colliculus and lateral genicutale nucleus (1.17 ± 0.05 and 0.82 ± 0.13 pmol/mg wet weight, respectively). Although the tissue content of (asu)NAAG was about three orders of magnitude lower than that of NAAG, levels of both peptides were positively correlated among different CNS regions ( r = 0.74, p < 0.003). In the rat spinal cord, (asu)NAAG levels progressively increased from week 2 to month 12 after birth. In bovine spinal cord, the contents of (asu)NAAG and NAAG were comparable in gray and white matter as well as in the dorsal and ventral horns. These results suggest that NAAG and (asu)-NAAG are closely related metabolically and raise the question of the physiological significance of such a cyclic peptide.