COMPARISON OF DNA RENEWAL IN GERM-FREE AND CONVENTIONAL MICE USING [125I]IODODEOXYURIDINE AND [3H]THYMIDINE

Germ-free (GF) and conventional (CV) C3H mice received a single injection of 1 μCi [³H]thymidine and 3 μCi [¹²⁵I]iododeoxyuridine to provide simultaneous labeling of DNA with the two precursors. Thymus, spleen, mesenteric lymph nodes, bone marrow (femora), small intestine, colon and skin were examined for total organ activity and rate of DNA renewal 1–8 days after injection. Precursor incorporation, assayed on day 1, was lower in the thymus, mesenteric lymph nodes and femora (and, to a lesser extent, in the spleen and colon) of GF mice as compared to CV animals. The opposite was observed in the small intestine and skin, i.e. total organ activity was higher in GF animals. Differences in precursor incorporation were partly due to differences in organ weights between the two groups of mice. In comparison to CV animals, DNA renewal rates were diminished in the mesenteric lymph nodes, bone marrow, colon (following a 3-day plateau) and spleen of GF mice. Little, if any, difference was observed between the two groups with respect to the rate of DNA turnover in the thymus and skin. Radioactivity of the small intestine remained constant for 2 days. Thereafter intestinal activity in GF mice declined at an initial slow rate between days 2 and 5 followed by a rapid decrease between days 5 and 8. In CV mice the first phase of activity loss was short with the rapid decline in intestinal activity beginning on day 3. From the slopes of the regression lines, the percentage thymidine reutilization was estimated. Reutilization varied from 0 to 63% in the various organs examined, with the greatest difference between GF and CV mice occurring in the mesenteric lymph nodes.     

ON THE RELATIONSHIP BETWEEN [3H]CHOLINE UPTAKE ACTIVATION AND [3H]ACETYLCHOLINE RELEASE

The depolarization-induced, calcium-dependent release of [³H]ACh from hippocampal synaptosomes was studied in a superfusion system. Release increased, with increasing depolarization. Barium and strontium effectively substituted for calcium during the depolarization, but magnesium inhibited the release. Releasable [³H]ACh is derived from the sodium-dependent component of the [³H]choline uptake which points out the physiologic importance of sodium-dependent choline transport. It is concluded that [³H]ACh release in this system has the same properties as neurotransmitter release in many other systems. Previous studies have shown that treatments which alter the activity of cholinergic neurons in vivo result in parallel changes in sodium-dependent choline uptake in vitro. When synaptosomes were utilized from animals treated to reduce cholinergic activity, there was a reduced release following the reduced uptake. Conversely, when synaptosomes were taken from animals treated to increase sodium-dependent choline uptake, there was an increase in the release. It is concluded that the changes in sodium-dependent choline uptake in vitro consequent to changes in neuronal activity in vivo result in parallel changes in releasable ACh. A comparison was made between the effect of a number of ions and agents on release and their effect on the in vitro, depolarization-induced activation of sodium-dependent choline uptake. Barium and strontium, ions which substitute for calcium in the release process, support the in vitro activation of uptake. Vinblastine and Bay a 1040, compounds which block release, prevented the in vitro activation of sodium-dependent choline uptake. However, magnesium blocked release in a dose-dependent manner, but did not block the activation of uptake in vitro. Rather, magnesium substituted for calcium and supported the activation of uptake in a dose-dependent fashion. It is concluded that acetylcholine release is not necessary for the activation of choline uptake.     

Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum

Abstract The binding of [³H]aspartate and [³H]glutamate to membranes prepared from frozen human cerebellar cortex was studied. The binding sites differed in their relative proportions, their inhibition by amino acids and analogues, and by the effects of cations. A proportion (about 30%) of [³H]glutamate binding was to sites similar to those labelled by [³H]aspartate. An additional component of [³H]gluta-mate binding (about 50%) was displaced by quisqualate and aL-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and may represent a “quisqualate-preferring” receptor. Neither N-methyl-d-aspartic acid-sensitive nor dl-2-amino-4-phosphonobutyric acid-sensitive [³H]glutamate binding was detected.     

Heterogeneity of [3H]Dipyridamole Binding to CNS Membranes: Correlation with [3H]Nitrobenzylthioinosine Binding and [3H]Uridine Influx Studies

The relationship between the nucleoside transport system and the nitrobenzylthioinosine-sensitive and -resistant [3H]dipyridamole binding sites was examined by comparing the characteristics of [3H]dipyridamole binding with those of [3H]nitrobenzylthioinosine binding and [3H]-uridine influx in rabbit and guinea pig cerebral cortical synaptosomes. Two distinct high-affinity synaptosomal membrane-associated [3H]dipyridamole binding sites, with different sensitivities to inhibition by nitrobenzylthioinosine, were characterized in the presence of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, 0.01%) to prevent [3H]dipyridamole binding to glass tubes and filters. The nitrobenzylthioinosine-resistant [3H]-dipyridamole binding sites represented a greater proportion of the total membrane sites in guinea pig than in rabbit (40 vs. 10% based on inhibition studies). In rabbit, nitrobenzylthioinosine-sensitive [3H]dipyridamole binding (KD = 1.4 +/- 0.2 nM) and [3H]nitrobenzylthioinosine binding (KD = 0.30 +/- 0.01 nM) appeared to involve the same membrane site associated with the nitrobenzylthioinosine-sensitive nucleoside transporter. By mass law analysis, [3H]-dipyridamole binding in guinea pig could be resolved into two components based on sensitivity to inhibition by 1 microM nitrobenzylthioinosine. The nitrobenzylthioinosine-resistant [3H]dipyridamole binding sites were relatively insensitive to inhibition by all of the nucleoside transport substrates and inhibitors tested, with the exception of dipyridamole itself. In guinea pig synaptosomes, 100 microM dilazep blocked nitrobenzylthioinosine-resistant [3H]uridine transport completely but inhibited the nitrobenzylthioinosine-resistant [3H]dipyridamole binding component by only 20%. Furthermore, a greater percentage of the [3H]dipyridamole binding was nitrobenzylthioinosine resistant in guinea pig compared with rabbit, yet both species had a similar percentage of nitrobenzylthioinosine-resistant [3H]uridine transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE SUBCELLULAR DISTRIBUTION OF [14C]GABA AND [3H]DOPAMINE IN THE RETINA

Abstract— Rabbit retinae were homogenized in isotonic sucrose and subjected to differential and density gradient centrifugation. Preliminary electron microscopic examination of some of the fractions indicated that in addition to the subcellular particles usually observed in brain homogenates, the photoreceptor cells gave rise to several characteristic fragments. These included fragmented outer limbs, aggregations of mitochondria from the inner segments, and photoreceptor terminals. Unlike the synaptosomes formed from the conventional type of synapses in the retina, these photoreceptor terminals appeared to sediment mainly in the low speed crude nuclear pellet (P1).
Retinae were incubated with low concentrations of [¹⁴C]GABA and/or [³H]dopamine prior to subcellular fractionation and in these experiments the P2 pellet was further fractionated on sucrose density gradients. Analysis of the radioactivity in the fractions showed that labelled GABA was accumulated by osmotically sensitive particles which had the sedimentation characteristics of synaptosomes. The panicles accumulating [³H]dopamine appeared to belong to a different, slightly lighter, population than those accumulating [¹⁴C]GABA. It is tentatively suggested that the particles accumulating labelled GABA were synaptosomes because the fractions containing these particles also possessed most of the GAD activity of the gradient. In contrast, GABA-T and MAO activity was found in the dense fractions of the gradients usually associated with mitochondria.
When retinae were incubated with a high concentration of labelled GABA a'lighter'population of particles seemed to accumulate the amino acid than when a low external GABA concentration was used. These results suggest that the high and low affinity uptake processes for GABA in the retina may have different cellular sites.     

ISOLATION OF [3H]ACETYLCHOLINE POOLS BY SUBCELLULAR FRACTIONATION OF CEREBRAL CORTEX SLICES INCUBATED WITH [3H]CHOLINE

Abstract— Subcellular fractions were isolated from tissue incubated in [³H]choline with or without the addition of 33 mM-KCl. Radioactive and bioassayable ACh were measured in the synaptosomes, synaptosomal cytoplasm and in the vesicles. After incubation with KCI the vesicles, as isolated, contained ACh of a lower specific activity than the cytoplasmic ACh. Therefore the vesicle fraction as isolated does not represent the source of the high specific activity ACh released upon K⁺ stimulation. However the vesicle fraction is heterogeneous. Most of the bioassayable ACh but little of the radioactive ACh in the vesicles passed through iso-osmotic Sephadex columns. These results raise the question of the existence of vesicles which contain highly radioactive ACh but which lose it during their isolation by current methods. Different possible forms of heterogeneity are discussed.     

[1251]2α-BUNGAROTOXIN AND [3H]QUINUCLIDINYLBENZILATE BINDING IN CENTRAL NERVOUS SYSTEMS OF DIFFERENT SPECIES

Abstract— [¹²⁵I]Diiodo α-bungarotoxin ([¹²⁵I]₂BuTx) and [³H]quinuclidinylbenzilate ([³H]QNB) binding sites were measured in post-nuclear membrane fractions prepared from whole brains or brain regions of several species. Species studied included Drosophila melanogaster (fruit fly), Torpedo californiea (electric ray), Carassius auratus (goldfish), Ram pipiens (grass frog), Kana cutesheiana (bullfrog), Rattus norvegicus (rat, Sprague-Dawley), Mus muscalus (mouse, Swiss random, C58/J, LG/J), Oryctolagus cuniculus (rabbit, New Zealand Whitc), and Bos (cow). Acetyl-CoA: choline O -acetyltransferase (EC 2.3.1.6) levels were also determined in the post nuclear supernatants and correlated with the number of binding sites.
All species and regions except Drosophila had 16–150 fold more [³H]QNB binding sites than [¹²⁵I]₂BuTx binding sites. Brain regions with the highest levels of [¹²⁵I]₂BuTx binding were Drosophila heads (300 fmol/mg), goldfish optic tectum (80fmol/mg), and rat and mouse hippocampus (3040 fmol/mg). The highest levels of [³H]QNB binding were seen in rat and mouse caudate (1.3–1.6 pmol/mg). Lowest levels of [³H]QNB and [¹²⁵I]₂BuTx binding were seen in cerebellum. The utility of [¹²⁵I]₂BuTx and [³H]QNB binding as quantitative measures of nicotinic and muscarinic acetylcholine receptors in CNS is discussed.     

CHEMICAL SYNTHESIS OF [3H]CEREBROSIDE [35S]SULFATE AND ITS IN VIVO METABOLISM IN RAT BRAIN

—Double-labeled sulfatide containing [3-³H]lignoceric acid and [³⁵S]sulfate was synthesized and injected intracerebrally into 28-day-old rats. The ³H-labeled sulfatide was synthesized by condensing (RS)-[3-³H]lignoceroyl chloride with lysosulfatide which had been obtained by saponification of sulfatide. The ³⁵S-labeled sulfatide was synthesized by using [³⁵S]sulfuric acid for sulfating 2′, 4′, 6′-tri-benzoyl-galactosyl N-fatty acyl, N-benzoyl-3-0-benzoyl-sphingosine, which had been obtained by per-benzoylation followed by solvolysis of calf brain nonhydroxycerebrosides. The perbenzoylated [³⁵S]sul-fatide was then subjected to mild alkaline saponification. Eight hours following the injection, the brain lipids contained various radioactive sphingolipids in addition to sulfatides. Fourteen per cent of the injected ³H was recovered in total lipids, and 26% of this was found in sulfatide. Nonhydroxy- and hydroxyceramides, nonhydroxy- and hydroxycerebrosides, and polar lipids contained 7, 1, 8, 3, and 22 per cent of the ³H found in total lipids, respectively. On the other hand, only 6% of the ³⁵S injected was recovered in total lipids; 63% of this was found in sulfatide, 5% in a mixture of seminolipid and cholesterol sulfate and 10% in a water-soluble material.     

THE EFFECT OF PHOSPHOLIPASE C, PHOSPHOLIPASE A2 AND NEURAMINIDASE ON THE UPTAKE OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN

Abstract— The uptake of [³H]norepinephrine ([³H]NE) and [³H]serotonin ([³H]5-HT) by rat brain synaptosomes is reduced as a result of pretreatment of the synaptosomes with phospholipase C (EC 3.1.4.3) or phospholipase A₂ (EC 3.1.1.4). This effect is not due to inhibition of the Na⁺-K⁺-ATPase but rather is caused by hydrolysis of neuronal membrane phospholipids, mainly phosphatidylcholine, which seem to be important to the uptake.     

Increased Binding of [3H]Muscimol and [3H]Flunitrazepam in the Rat Brain Under Hypoxia

We examined the effects of in vivo hypoxia (10% O2/90% N2) on the gamma-aminobutyric acid (GABA)/benzodiazepine receptors and on glutamic acid decarboxylase (GAD) activity in the rat brain. Male Wistar rats were exposed to a mixture of 10% O2 and 90% N2 in a chamber for various periods (3, 6, 12, and 24 h). The control rats were exposed to room air. The brain regions examined were the cerebral cortex, striatum, hippocampus, and cerebellum. GABA and benzodiazepine receptors were assessed using [3H]muscimol and [3H]flunitrazepam, respectively. Compared with control values, GAD activity was decreased significantly following a 6-h exposure to hypoxia in all four regions studied. On the other hand, the numbers of both [3H]muscimol and [3H]flunitrazepam binding sites were increased significantly. The increase in receptor number tended to return to control values after 24 h. Treatment of the membrane preparations with 0.05% Triton X-100 eliminated the increase in the binding capacity. These results may represent an up-regulation of postsynaptically located GABA/benzodiazepine receptors corresponding to the impaired presynaptic activity under hypoxia.     

CATECHOLAMINE METABOLISM IN THE DOG: COMPARISON OF INTRAVENOUSLY AND INTRAVENTRICULARLY ADMINISTERED [14C]DOPAMINE AND [3H]NOREPINEPHRINE

—The urinary excretion of labelled metabolites was measured in dogs which had been injected intravenously or intraventricularly with [³H]norepinephrine or [¹⁴C]dopamine. [³H]Norepinephrine injected by either route produced more labelled 3-methoxy-4-hydroxy-phenylglycol than 3-methoxy-4-hydroxymandelic acid, as did [¹⁴C]dopamine after intravenous administration. In contrast, following the intraventricular injection of [¹⁴C]dopamine, more [¹⁴C]3-methoxy-4-hydroxymandelic acid was formed than [¹⁴C]3-methoxy-4-hydroxyphenylglycol. These observations suggest that the metabolism of exogenously-administered and endogenously-formed norepinephrine may proceed through different routes and that the predominant metabolite of norepinephrine in canine brain may be 3-methoxy-4-hydroxymandelic acid rather than 3-methoxy-4-hydroxyphenylglycol.     

Phospholipid Methylation Increases [3H]Diazepam and [3H]GABA Binding in Membrane Preparations of Rat Cerebellum

The effect of phospholipid methylation on both [3H]diazepam and [3H]GABA ( [3H]gamma-aminobutyric acid) binding to crude synaptic plasma membrane from rat cerebellum has been studied. S-Adenosylmethionine (SAM) stimulates [3H]methyl group incorporation into membrane phospholipids and enhances [3H]diazepam binding by increasing the apparent Bmax. Conversely, inhibition of [3H]methyl group transfer from [3H]SAM to phospholipids by preincubation with SAM at 0 degrees C or with SAH abolishes the increase of binding. After preincubation with SAM, analysis of the GABA binding reveals the presence of binding sites with high affinity, a property absent in control membranes preincubated without SAM. Among the neurotransmitter bindings tested, only those of GABA and benzodiazepine in the cerebellum and beta-adrenergic ligands in the cerebral cortex are enhanced upon stimulation of phospholipid methyltransferase activity. [3H]Dihydromorphine, [3H]dihydro-alpha-ergokryptine and [3H]spiroperidol bindings are not affected by SAM. The present data suggest an involvement of phospholipid methylation in regulation of both [3H]GABA and [3H]-diazepam binding.     

Adenosine A2a Receptor-Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release

Abstract: The ability of adenosine agonists to modulate K⁺-evoked 4D†-[³H]aminobutyric acid ([³H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A_2a receptor-selective agonist CGS 21680 inhibited Ca²⁺-dependent [³H]GABA release evoked by 15 m M KCI with a maximal inhibition of 29 ± 4% (IC₅₀ of ∼4 ± 10 ⁻¹² M ). The relative order of potency of three agonists was CGS 21680 ± 5'- N -ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A_2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [³H]GABA was evoked by 30 mM KCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [³H]ACh evoked by 30 m M KCI, with a maximal stimulation of 26 ± 5% (IC₅₀ of ∼10⁻¹¹ M ). This effect was blocked by CP 66,713 but not by DPCPX. The A₁ agonist R -PIA inhibited [³H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A_2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively. Key Words : GABA—Acetylcholine—Adenosine receptors—Striatum.     

THE UPTAKE AND RELEASE OF [3H]DOPAMINE IN THE GOLDFISH RETINA

Abstract— The uptake and release of [³H]dopamine was studied in the goldfish retina with the following results: (1) when goldfish retinas were incubated with 2 ± 10^-7m -[³H]dopamine for less than 20min and processed for autoradiography. most of the label was associated with dopaminergic terminals that contact certain horizontal cells. Biochemical analysis showed that > 93% of this label was [³H]-dopamine. (2) [³H]dopamine uptake saturated with increasing dopamine concentration and followed Michaelis-Menten kinetics. This uptake could be explained by a single ‘high-affinity’ mechanism with a K_m of 2.61 ± 0.41 ± 10^-7m and a V_max of 66 ± 12 ± 10^-12 mol/min/mg protein. (3) [³H]dopamine uptake was temperature-dependent with a temperature coefficient of 1.7 and an energy of activation of 11.4 kcal/mol. (4) The initial rate of uptake was unaffected by the absence of Ca²⁺ or the presence of Co²⁺; however, more than 85, uptake was blocked in the absence of external Na⁺. (5) Neither 1 mm -cyanide nor 5 mm -iodoacetate blocked more than 30% of uptake individually; however, in combination > 70% of uptake was blocked. (6) Centrally acting drugs benztropine and diphenylpyraline inhibited at least 60–70% of [³H]dopamine uptake. (7) [³H]dopamine in the retina could be released by increasing the external K⁺ concentration. This release was Ca²⁺ -dependent and was blocked by 10mm -Co²⁺ or 2Omm -Mg²⁺. The amount of [³H]dopamine released was not affected by the presence of benztropine, diphenylpyraline or fluphenazine in the incubation medium. These studies add further support for dopamine as a neurotransmitter used by interplexiform cells of the goldfish retina.     

Comparison of [3H]Choline and d-[3H]Aspartate Efflux from Guinea Pig and Human Neocortex

The outflow of [3H]choline ([3H]Ch) evoked by electrical field stimulation and the efflux of D-[3H]Asp induced by 35 mM KCl and 1-10 microM ouabain were studied in human and guinea pig cortical slices, kept under identical experimental conditions. [3H]Ch outflow was significantly lower whereas D-[3H]Asp efflux was significantly higher in humans than in guinea pigs. This suggests a different proportion of the two neuronal systems in these two species. Blockade of muscarinic autoreceptors with atropine increased, whereas stimulation of alpha 2 receptors with norepinephrine (NE) reduced, the evoked [3H]Ch outflow to the same extent in human and guinea pig cortical slices. Conversely, NE did not affect ouabain-induced D-[3H]Asp efflux, suggesting that an alpha 2-mediated control is not operative in the glutamatergic cortical structures. Desmethylimipramine, 2-5 microM, was able to increase [3H]Ch outflow through atropine-like mechanisms only in the human. This drug at 20-50 microM inhibited [3H]Ch and D-[3H]Asp efflux in both species, through mechanisms unrelated to its monoamine reuptake blocking properties. Thus, similarities and differences can be detected between humans and guinea pigs with regard to (a) the relative potency of the cholinergic and acidic amino acidergic signals and (b) the modulation of neurotransmitter outflow by drugs acting on auto- and the heteroreceptors.     

Comparison of the Kinetics of [3H]Diazepam and [3H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Abstract: [³H]Diazepam and [³H]flunitrazepam ([³H]FNP) binding to washed and frozen synaptosomal membranes from rat cerebral cortex were compared. In Tris-citrate buffer, γ -aminobutyric acid (GABA) and NaCl both increased [³H]diazepam binding more than [³H]FNP binding. GABA and pentobarbital both enhanced this effect of NaCl. Because of the extremely rapid dissociation of [³H]diazepam in the absence of NaCl and GABA, the B_max (maximal binding capacity) was smaller by the filtration assay than by the centrifugation assay. [³H]FNP, which dissociates more slowly, had the same B_max in both assays. [³H]Diazepam association had two components, and was faster than [³H]FNP association. [³H]Diazepam dissociation, which also had two components, was faster than that of [³H]FNP, and also had a greater fraction of rapidly dissociating species. [³H]FNP dissociation was similar when initiated by diazepam, flunitrazepam, clonazepam, or Ro15-1788, which is a benzodiazepine antagonist. [³H]Diazepam dissociation with Ro15-1788, flunitrazepam, or clonazepam was slower than with diazepam. GABA and NaCl, but not pentobarbital, increased the percentage of slowly dissociating species. This effect of NaCl was potentiated by GABA and pentobarbital. The results support the cyclic model of benzodiazepine receptors existing in two interconvertible conformations, and suggest that, distinct from their binding affinity, some ligands (like flunitrazepam) are better than others (like diazepam) in inducing the conversion of the receptor to the higher-affinity state.     

POTASSIUM-INDUCED RELEASE OF [3H]GABA AND OF [3H]NORADRENALINE FROM NORMAL AND RESERPINIZED RAT BRAIN CORTEX SLICES. DIFFERENCES IN CALCIUMDEPENDENCY, AND IN SENSITIVITY TO POTASSIUM IONS

The release of [³H]GABA induced by elevated extracellular potassium (K)_o, from thin rat brain cortex slices, has been compared with that of [³H]noradrenaline ([³H]NA), released by the same procedures, both from normal slices, and from slices pre-treated with reserpine and nialamide, [³H]NA being predominantly a vesicular component in the former situation, and a soluble substance in the latter one. 46 mM-(K)_o released considerably more [³H]NA from normal than from drug-treated slices, while the release of GABA was about two thirds of the latter. When 4min ‘pulses’ of increasing concentrations of potassium were applied, it was observed that the release of GABA and of [³H]NA from drug-treated slices increased in proportion to (K)_o, up to 36-46 mM and then declined considerably with higher (K)_o. The dependency of potassium-induced release on the concentration of calcium in the medium, indicated that release of [³H]NA from normal slices was proportional to calcium up to 1.5-2 mM, while that of [³H]NA from drug-treated slices increased up to 0.5 mM-Calcium, and then declined with higher concentrations. GABA release also increased up to 0.5 mM-calcium, but no further changes were observed at higher concentrations. The calcium antagonist D-600 inhibited high (K)_o-induced release of [³H]NA from normal slices to a greater extent than that of [³H]GABA or of [³H]NA from drug-treated slices. These results, in which elevated (K)_o-induced release of [³H]GABA resembles considerably that of soluble NA, but differs from that of NA present in synaptic vesicles, suggest that release of [³H]GABA also occurs from the soluble cytoplasmic compartment, and that the partial calcium requirement that is found is unrelated to that of transmitter secretion. These findings are also a further indication of the lack of specificity of elevated (K)_o as a stimulus for inducing transmitter secretions.