REGIONAL DIFFERENCES IN HIGH AFFINITY CHOLINE TRANSPORT VELOCITY IN GUINEA-PIG BRAIN

The kinetic parameters of choline accumulation by synaptosomes prepared from different regions of guinea-pig brain (cortex, brainstem, and cerebellum) were determined. Choline was accumulated by a high affinity transport process for all regions tested and the apparent Michaelis constants were similar. However, the apparent maximal velocities of choline accumulation for the brain regions differed; the differences were related to the amount of acetylcholine formed by the respective regions. The results suggested that the maximal velocity of the high affinity transport process may reflect the density of cholinergic nerve endings within brain regions.     

MULTIPLE FORMS OF CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN BRAIN OF DEVELOPING AND ADULT RATS

The multiple molecular forms of choline acetyltransferase (ChAT) were analysed during the postnatal development of rat brain. Changes in the sodium-dependent, high affinity uptake of [³H]choline (HAUC) and in the efficiency of conversion of labelled choline into ACh in vitro were also examined. Both mature and 7-day old brain contained three molecular forms of ChAT, with isoelectric points of pH 7.3, 7.9 and 8.3, but the immature brain appeared to contain smaller concentrations of the most basic form of the enzyme (pI = 8.3). Of the total choline uptake measured in slices of frontal cortex, adult samples exhibited a greater proportion of HAUC than 7-day samples and appeared to acetylate more efficiently the [³H]choline accumulated by high affinity uptake. This evidence suggests a basic molecular form of ChAT, appearing in rat brain during postnatal development, might be responsible for the efficient coupling of the high affinity uptake and subsequent acetylation of choline in cholinergic nerve terminals.     

A MODEL OF HIGH AFFINITY CHOLINE TRANSPORT IN RAT CORTICAL SYNAPTOSOMES

Abstract— Initial velocity of choline uptake by cortical synaptosomes from the Long-Evans rat has been measured as a function of both choline and sodium concentration. These data were then fitted to the rate equation for each of several possible models which characterize the participation of sodium in the transport process, and the models giving best fit were identified. Although one cannot unequivocally distinguish between a model including a high affinity carrier component plus diffusion and one including both high affinity and low affinity carriers, the conclusions concerning the high affinity component are the same in both cases. The major conclusions from the model are as follows: (1) The carrier may first combine with either choline or sodium; if the first reaction is with sodium, then there is an obligatory reaction with a second sodium before choline can interact with the carrier. (2) Translocation may occur as either CS or CNa₂S (C= carrier; S= choline; CS= carrier-substrate complex). (3) The apparent maximal velocity (Va) is dependent on the sodium concentration. (4) K₁, the choline concentration giving Va/2. is also dependent on the sodium concentration. K₁ increases with [Na] from 0 to 38.41 mm ; above 38.41 mm -[Na]. K₁ declines with [Na]. (5) There is a sigmoidal relationship between velocity of uptake and [Na]; however, uptake is not zero at [Na] = 0. (6) Jm. uptake at a given [choline] and infinite [Na]. is hyperbolically related to the choline concentration, but changes slowly over the range of 0.5–5.0 ± 10^-6m . (7) K_Na, the sodium concentration giving a velocity equal to Jm/2, is related to the choline concentration by a quadratic equation, and was found to be greater than physiological [Na] at choline concentrations of 0.5, 0.6, or 1.0 ± 10^-6m . but less than physiological [Na] at choline concentrations of 2.0 or 5.0 ± 10^-6m . The best fit model for the high affinity uptake of choline is very similar to what has been found in previous studies for the high affinity uptake of glutamic acid and GABA, thus raising the question of whether or not all high affinity synaptosomal mechanisms may be variations of a common model.     

CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN CORPUS STRIATUM OF RESERPINISED RATS1

Rats treated with reserpine show increased V_max for the high affinity uptake of choline into small slices of corpus striatum. The choline acetyltransferase activity of whole homogenates of striatum is also increased. These changes are consistent with increased cholinergic neuronal activity in the striatum and seem likely to be adaptations mediating increased rates of synthesis of acetylcholine. The maximal increases found occurred concurrently, consistent with coupling of the high affinity uptake of choline and its acetylation in cholinergic nerve terminals of the rat. That increased high affinity uptake is accompanied by increased choline acetyltransferase activity, suggests the input of choline is not the sole determinant of rates of synthesis of acetylcholine, in spite of the large Vmas for striatal choline acetyltransferase, compared with that for high affinity uptake. These results seem best explained by kinetic coupling, in the rat, of the high affinity uptake of choline with a limited pool of choline acetyltransferase preferentially localised at the nerve terminal plasma membrane.     

EFFECT OF NOREPINEPHRINE ON 32P INCORPORATION INTO INDIVIDUAL PHOSPHATIDES IN SLICES FROM DIFFERENT AREAS OF THE GUINEA PIG BRAIN1

Abstract—

• 1 Incorporation of ³²P into phosphatidic acid was significantly increased in the presence of an appropriate concentration of norepinephrine, either 0.1 mm , or 1 mm , or both, in slices of guinea pig cerebellar cortex, olfactory bulbs, cerebral cortex, hypothalamus and thalamus; it was not significantly affected at either concentration of norepinephrine in slices of corpus striatum and the posterior and anterior colliculi.
• 2 Incorporation of ³²P into phosphatidylinositol was significantly increased in the presence of norepinephrine in a concentration of either 0.1 mm , or 1 mm , or both, in three of the areas studied—the olfactory bulbs, cerebral cortex, and thalamus.
• 3 There was no significant effect of 0.1 mm -norepinephrine on ³²P incorporation into phosphatidylcholine or phosphatidylethanolamine plus phosphatidylserine in any of the areas studied. Norepinephrine (1 mm ) significantly inhibited ³²P incorporation into phosphatidylcholine in cerebral cortex, hypothalamus, corpus striatum and anterior colliculus; ³²P incorporation into phosphatidylethanolamine plus phosphatidylserine was inhibited by 1 mm -norepinephrine in cerebral cortex, hypothalamus, and posterior and anterior colliculi.
• 4 In slices of cerebellar cortex, 0.1 mm and 1 mm -norpinephrine did not significantly affect either the specific activity or the level of nucleotide ～ P in the tissue.
• 5 The physiological significance of changes in phosphatide metabolism in response to neurotransmitters in the central nervous system is discussed.

     

ONTOGENESIS AND REGIONAL DISTRIBUTION OF HISTAMINE AND HISTAMINE-N-METHYLTRANSFERASE IN THE GUINEA PIG BRAIN1

The ontogenetic development and the regional distribution of histamine (HA) and histamine-N-methyltransferase (HMT, EC 2.1.1.8) in guinea pig brain and pituitary gland were studied. The samples were taken every fourth day beginning on the 28th foetal day. The HA concentration in the brains of the youngest foetuses was almost undetectable. A significant increase in HA concentrations occurred between days 40 and 44, which coincides with the period of rapid growth of nerve cell processes in this species. After this, a steep increase continued to the end of gestation in the hypothalamus, and to a lesser degree in the medulla-midbrain and in the forebrain. In all parts of the brain the adult HA levels were reached by the time of birth. The HMT activity increased 15-fold from the 28th foetal day to the adult and reached ca. 80% of the adult activity by the time of birth. The HMT activity developed earlier in the midbrain than in the forebrain or in the cerebellum, but after the birth the regional distribution of HMT was fairly even. In the pituitary gland the HA concentration and HMT activity increased hundredfold and tenfold, respectively. The developmental patterns of HA and HMT in the guinea pig brain give support to the concept that HA might be a neurotransmitter in the brain.     

THE CORRELATION BETWEEN CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE ACTIVITY IN DIFFERENT AREAS OF THE CEREBELLUM OF RAT AND GUINEA PIG

Abstract— Choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) levels were measured quantitatively in samples from the archi- and paleocerebellar vermis (Larsell's Lobules IX c,d,-X, and Lobules VII-VIII, respectively) and from the cerebellar peduncles, nuclei and white matter of rat and guinea pig. Lesions to isolate archi- or paleocerebellar areas were made in some rats and the effect on enzyme levels and ultrastructure were studied. In the rat there was a striking correlation between the activity of ChAc and AChE in the different areas; thus in the archicerebellar cortex the levels of both enzymes were 3–4 times those in the paleocortex. Deafferentation caused a fall in ChAc and this practically paralleled the fall in AChE in the same area. The reduction in both enzymes was more pronounced in the archi- than in the paleocerebellar cortex. In the guinea pig the results were very different. The ChAc activity was much lower than in the rat and was equal in the archi- and paleocerebellum. The AChE activity was also uniform in the different areas but, in contrast to ChAc, was higher than in the rat.     

POST-MORTEM CHANGES IN HIGH AFFINITY CHOLINE UPTAKE

Abstract— When animals were killed by decapitation and the heads kept refrigerated at 2–4°C. high affinity choline uptake was maintained up to 3 days post-mortem. At 5 days post-mortem, there was a significant reduction in uptake. In tissues kept at 2–4°C for 1 day, the ionic dependence, drug sensitivity and kinetic parameters of uptake were identical to that of control tissues. At 3 days post-mortem, intact synaptosomal profiles, although with features characteristic of degenerating neuronal tissues, were observed in electronmicroscopic studies. In tissues maintained at room temperature, however, the uptake activity was nearly completely gone by 1 day. It is concluded that high affinity choline uptake is maintained for days, a surprisingly long time, in tissues kept in the cold immediately after death.
When pentylenetetrazol or pentobarbital were administered to rats in order to activate or depress the choline uptake, it was found that the activity-related changes in choline uptake undergo a reversal in post-mortem tissues. The changes in uptake were significantly lost by 10min post-mortem and totally absent by 30min post-mortem. In vitro studies with whole hippocampi indicate that the postmortem reversal in activity-related changes in uptake is temperature-dependent. It is concluded that because of post-mortem reversals in activity-related states of uptake the true magnitude of these activity-related changes in uptake may be underestimated by existing methods of assay. Acetylcholine levels in synaptosomal preparations did not clearly correlate with levels of choline uptake.     

去甲肾上腺素对豚鼠回肠纵肌标本中脑啡肽能神经抑制效应的加强作用

已有许多实验表明,脑啡肽能抑制肾上腺素能神经的作用。还有资料证明,10Hz电场刺激豚鼠回肠纵肌标本产生的抑制效应是脑啡肽能神经的作用。本工作以此模型作为实验对象,观察到去甲肾上腺素能加强脑啡肽能神经的抑制效应。并且,在一定范围内,这种加强作用随去甲肾上腺素的剂量加大而增强。将本实验工作与他人的结果联系起来,可形成一个概念,即去甲肾上腺素可加强脑啡肽能神经的作用,反之脑啡肽又能抑制肾上腺素能神经的作用。两者紧密联系,相互制约。本工作还提示,去甲肾上腺素的这种加强作用是既通过激活突触前膜又通过激活突触后膜上的α-肾上腺素能受体实现的。本文对去甲肾上腺素的这种加强作用是否具有普遍意义等问题进行了讨论。     

METHYLHISTAMINE IN GUINEA PIG BRAIN1

The concentration of methylhistamine in whole brain of guinea pig is 72 ng (about 0-5 mμ mole/g). The greatest portion is found in the crude mitochondrial fraction.     

肿瘤抑制基因APCmRNA在豚鼠脑中的分布

APC基因是1991年被发现的一类肿瘤抑制基因,它被定位于人第5号染色体5q21处。APC基因如发生缺失或突变,则易患直肠肿瘤,并伴有部分先天痴呆的病例。本工作在孟帆已获得的APC基因在豚鼠中的同源cDNA的基础上,完成了对它的亚克隆,并利用原位杂交和RNA酶保护分析的方法,对它在脑中的分布进行了研究。发现APCmRNA主要在海马、大脑和小脑中表达;嗅球中杂交信号稍弱,脑干中最弱。海马中阳性细胞主要是锥体细胞,小脑中则主要是内层颗粒细胞。在一个月大的豚鼠胚胎的脑中也观察到相似的表达型式。进一步的研究有助于我们更好地了解神经发育和先天痴呆发生的分子机制。     

THE EFFECT OF DEVELOPMENT ON THE GANGLIOSIDES OF RAT AND PIG BRAIN

Abstract— The ganglioside content of the forebrain, brain stem and cerebellum have been studied, in the rat at various ages from 1 day to 27 months, and in the pig at various ages from 93 days gestation to 30 months. Each part of the brain was analysed for total ganglioside NANA and for four major gangliosides (G_Ml, G_D1a, G_Dlb and G_T1 in the nomenclature of S vennerholm , 1963). In the rat forebrain, the concentration of ganglioside NANA rose rapidly between 1 and 21 days after birth, fell to 3 months and subsequently rose to a mature value at 6 months. In the rat cerebellum, the peak concentration was reached at 2 months and the lower adult value at 9 months, whilst in the brain stern, the concentration rose more slowly and had a broad peak from 15 days to 2 months. Values are also given for the changes in the total amounts in each brain part. The changes in the concentrations and total amounts of ganglioside NANA, in the three parts of the pig brain were, on the whole, similar to those in rat brain except that the percentage distribution of the major gangliosides had almost attained the mature pattern at birth. In the forebrain of both species, the disialoganglioside, G_D1a, accounted for the highest percentage of the total gangliosides. The results are discussed with respect to their possible structural significance.     

EFFECT OF PRETREATMENT UNDER VARIOUS CATIONIC CONDITIONS ON ACETYLCHOLINE CONTENT AND CHOLINE TRANSPORT IN RAT WHOLE BRAIN SYNAPTOSOMES

The effects of different ionic environments were measured on the concentration of acetyl-choline (ACh) from synaptosomes and their effect on subsequent high affinity choline (Ch) transport and ACh synthesis after resuspension of the synaptosomes in the normal Krebs medium. KCl (40 mM) was used to induce ACh release and reduce synaptosomal ACh content. The effects of Na⁺ omission, Ca²⁺ omission, and high Mg²⁺ on spontaneous (KC1: 4.75 mM) and potassium induced (KC1: 40 mM) ACh release and other cholinergic parameters are presented. The high affinity transport of Ch was more highly correlated with the reciprocal of the ACh level (r= 0.934, P= 9.7 × 10^-4) than with the ACh release rate during preincubation (r= 0.792, P= 3.4 × 10^-2). The results are consistent with the view that the consequences of the various ionic conditions on Ch transport and ACh synthesis are dependent on their effects on intrasynaptosomal ACh levels and only secondarily on synaptosomal ACh release.     

SODIUM-DEPENDENT HIGH AFFINITY CHOLINE UPTAKE: A REGULATORY STEP IN THE SYNTHESIS OF ACETYLCHOLINE

The sodium-dependent high affinity choline uptake into synaptosomes from rat brain has been studied after in vivo treatments which would alter the activity of cholinergic neurons. We utilized a number of treatments to reduce the activity of cholinergc neurons in the brain. Administration of pentobarbital (65 mg/kg), chloral hydrate (40 mg/kg) and γbutyrelactone (750 mg/kg) caused a 50-80% reduction in sodium-dependent high affinity choline uptake in several brain regions (30 min). This depression was not found 24 h after injection. Interruption of the cholinergic septal-hippocampal or habenuleinterpeduncular tracts by lesions (10 min-1 h) also caused a similar, large reduction in sodium-dependent high affinity choline uptake in the hippocampus and the interpeduncular nucleus respectively. We reversed the inactivity after pentobarbital administration by direct electrical stimulation of the cholinergic septal-hippocampal tract. Stimulation (40 Hz) for 10-15 min completely reversed the depression in sodium-dependent high affinity choline uptake. Stimulation at lower frequencies or for shorter times caused a partial reversal. Administration of pentylenetetrazol (75 mg/kg), a convulsant, was utilized to increase the activity of central cholinergic neurons. After drug administration, we found a large (60%) increase in sodium-de-pendent high affinity choline uptake. This increase was not found in the hippocampus when cholinergic afferents were interrupted by septal lesion prior to drug administration. We also examined the uptake after administration of cholinergic drugs. Oxotremorine (0.75 mg/kg), a muscarinic agonist which reduces acetylcholine release and turnover, caused a reduction in uptake. On the other hand, administration of scopolamine (5 mg/kg), a cholinergic antagonist which increases acetylcholine turnover, caused an increase in sodium-dependent high affinity choline uptake. Addition of any drug utilized, drectly to uptake samples, did not alter uptake. We examined the conversion of [³H]choline to [³H]acetylcholine in hippocampal synaptosomes after septal lesion, pentylenetetrazol administration and in untreated controls. In all cases, 60-70% of the total sodium-dependent tritium content was present as [³H]acetylcholine. Evidence was presented that homoexchange is not or is less involved in choline uptake than in GABA uptake. A kinetic analysis of sodium-dependent high affinity choline uptake was performed after all treatments. We found changes in V_max, after all treatments, which were consistently in the same direction as the alterations in activity. The proposal is made that the sodium-dependent high affinity choline uptake is coupled to cholinergic activity in such a way as to regulate the entry of choline for the maintenance of acetylcholine synthesis. The findings also lead us to propose that sodium-dependent high affinity choline uptake in vitro be utilized as a rapid, relative measure of the activity of cholinergic nerve terminals in vivo.     

POST MORTEM CHANGES IN EXTRACTABLE PROTEIN AND IN TISSUE pH IN THE GUINEA PIG BRAIN

—Guinea pigs were killed by asphyxiation with nitrogen and the soluble proteins were extracted from the brain at various times post mortem. The quantity of extractable brain protein decreased by 21 per cent when the animals remained at room temperature for 2 h post mortem. This decrease was not a consequence of extensive proteolysis or variations in blood volume but was probably a result of precipitation. After death, the pH of the brain fell rapidly to a minimum of ～6CE4 within about 35 min. Examination of the patterns of brain proteins after acrylamide gel electrophoresis showed a concomitant decrease in the content of several protein bands.     

RESPIRATORY ACTIVITY OF GUINEA PIG BRAIN NUCLEI

Abstract— Preparations of guinea pig brain nuclei, obtained by discontinuous gradient centrifugation in sucrose solutions of pH 6.7–6.8, containing 3 mM-MgCl₂ and phosphate exhibited steady and reproducible oxygen uptake. Oxygen uptake was stimulated 60–70 per cent by glucose, pyruvate, oxalacetate or α-ketoglutarate and 267 per cent by succinate. This respiratory activity was unaffected by the relative sodium or potassium ion content of the medium and by variations in the concentration of inorganic phosphate. Agents known to inhibit citric acid cycle oxidation, oxidative phosphorylation and glycolysis diminished oxygen uptake, but antibiotics inhibiting nucleic acid or protein synthesis did not. Treatment of the nuclear preparation with DNase decreased respiratory capacity, which was partially restored by the addition of polyacrylic acid.     

HIGH AFFINITY CHOLINE UPTAKE: IONIC AND ENERGY REQUIREMENTS

Abstract— High affinity choline uptake into rat hippocampal synaptosomes was examined at 37°C when various ions were deleted from normal Kreb's-Ringer media. When sodium chloride was replaced by sucrose, lithium chloride, cesium chloride or rubidium chloride, choline uptake was markedly reduced. When the sodium concentrations of the Kreb's media were gradually reduced to zero, the uptake was gradually reduced in parallel. A kinetic analysis performed at low and normal sodium concentrations revealed changes in K_m and V^max values. When several non-chloride sodium salts were utilized, the uptake was reduced in all cases suggesting also a chloride-dependence in addition to the sodium-dependence. Omission of calcium chloride or magnesium sulfate from the media did not alter uptake. Sodium-dependent choline uptake was examined over a range of potassium concentrations (0–35 DIM). It was found that uptake was maximal between potassium concentrations of 0.35–4.8 mm but was reduced at both lower and higher potassium concentrations. The kinetics of uptake were examined under varying potassium concentrations, and at low potassium, only a change in V_max was observed while at high potassium concentrations, there were changes in both K^m and V^max values. Preincubation and incubation of synaptosomes with 0.1 m -ouabain, 0.1 mm -2,4-dinitrophenol and 1 mm -KCN caused a reduction in sodium-dependent uptake. When dextrose was omitted from the preincubation and incubation media there was also a reduction in sodium-dependent uptake. By contrast, the sodium-independent uptake was unaffected by the metabolic inhibitors or omission of dextrose, and had a very low Q₁₀. When various incubation temperatures were utilized in uptake experiments, the Q¹⁰ for the interval 37-27°C was 2.7 and the activation energy was 22.7 kcal/mol. Slightly different ionic dependences were observed when animals pretreated with pentobarbital of oentylenetetrazol were utilized as the source of synaptosomes.     

AXOPLASMIC TRANSPORT OF CHOLINE ACETYLTRANSFERASE ACTIVITY IN MICE: EFFECT OF AGE AND NEUROTOMY

—We studied the axoplasmic transport of choline acetyltransferase (CAT) activity in sciatic nerves of normal mice of various ages. For at least 3 days after unilateral ligation of sciatic nerves of 6 and 30-week-old mice, the CAT activity in the ligated nerve increased as a linear function of time and the increase was confined to the 3 mm length of nerve immediately proximal to the ligature. The rate of increase of CAT activity in the ligated nerves of the 30-week-old mice was only 45 ± 6% that of the 6-week-old mice, whereas the CAT activity of non-ligated sciatic nerves of the older mice was 87 ± 6% more than that of the younger mice (n = 18, P < 0·001). The average velocity of axoplasmic transport of CAT activity was five times greater in the younger mice (1·5 ± 0·2 mm/day vs 0·3 ± 0·1 mm/day, n = 6, P < 0·01). Even greater differences were observed between still younger and older animals: the av velocity of axoplasmic transport of 2-week-old mice (3·5 ± 0·2 mm/day) was 17·5 times greater than that of 36-week-old mice (0·2 ± 0·1 mm/day). We also studied the axoplasmic transport of CAT activity in 6-week-old mice after unilateral section of the sciatic nerve. For at least 3 months after the operation, there were no differences between the sectioned nerves and the intact contralateral nerves with respect to the increase in CAT activity immediately proximal to a ligature placed at various times after neurotomy and one day before sacrifice. On the other hand, there was a reduction in the CAT activity of more proximal segments of the sectioned nerves. The reduction of CAT activity was maximal (52 ± 3%) 3 weeks after the operation when the maximum increase (2·5-fold) in the av velocity of axoplasmic transport of CAT activity was recorded (n = 6, P < 0·001). The inclusion of purified (100-fold) mouse brain CAT activity in the assays for the CAT activity of nerve segments demonstrated that the differences in content and rate of transport were not due to the presence of activators or inhibitors of CAT activity. These differences probably reflect physiologic changes in the axoplasmic transport of cholinergic neurons during development and regeneration.