The subcellular distribution of [N-Me-3H]-acetylcholine synthesized by brain in vivo

1. Three forms of acetylcholine occur in subcellular fractions of brain tissue: free acetylcholine, present in the high-speed supernatant from eserinized sucrose homogenates; stable bound acetylcholine, present in synaptic vesicles; and labile bound acetylcholine, present in the cytoplasm of synaptosomes (detached presynaptic nerve terminals). 2. The relationship between these forms has been investigated by isolating the subcellular fractions from the cortical tissue of cats and guinea pigs excised 1hr. after infiltration of [N-Me-(3)H]choline into the cortex in vivo. 3. Since choline is a ubiquitous metabolite, means were devised for isolating the radioactive acetylcholine on columns of the weak acid ion-exchange resin IRF-97; control experiments with samples of extracts treated with acetylcholinesterase showed that the radioactivity attributed to acetylcholine migrated to the choline peak after cholinesterase treatment. 4. The specific radioactivities of the various forms of acetylcholine were different: labile bound (synaptosomal cytoplasmic) acetylcholine had the highest, stable bound (vesicular) acetylcholine the next highest, and the high-speed-supernatant form the lowest. 5. It is concluded that the various forms of acetylcholine could not have arisen during fractionation from a single pre-existing pool of acetylcholine.     

Phylogenetic distribution of [3H]cyclohexyladenosine binding sites in nervous tissue

The specific binding of the A1 adenosine receptor ligand, [3H]CHA, was investigated in membrane fractions prepared from brains of eleven vertebrate species and ganglia of four invertebrate species. Substantial amounts of specific [3H]CHA binding sites were demonstrated in brain membranes of all vertebrate species examined; however, [3H]CHA binding sites were not detectable in nervous tissue of the invertebrate species studied. The densities of [3H]CHA binding sites in vertebrate brains increase in higher vertebrates. Moreover, the pharmacological characteristics of the site labeled by [3H]CHA in two divergent classes of vertebrates were similar. The broad phylogenetic distribution of A1 adenosine receptors in primitive as well as advanced vertebrate species suggests a fundamental role for adenosine in neuronal modulation.     

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.     

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

The subcellular localization of enzymes of dolichol metabolism in rat liver

Dolichyl phosphate is an intermediate in the glycosylation of N-glycosamidic linked glycoproteins in mammalian systems, and its availability may be a limiting factor in glycoprotein biosynthesis. The basic kinetics and subcellular distribution of enzymes which may influence the concentration of dolichyl phosphate in rat liver have therefore been investigated. These include dolichyl phosphate phosphatase, dolichol phosphokinase, dolichyl fatty acyl ester synthetase, GDP-mannose dolichyl phosphate mannosyl transferase, and UDP-glucose dolichyl phosphate glucosyl transferase. The specific activity of the enzymes was highest in the microsomes, except for dolichyl phosphate phosphatase and dolichyl fatty acyl ester synthetase, which were most concentrated in the plasma membrane and the cytosol fraction, respectively. The nuclei contained all of the enzyme activities while the mitochondria and cytoplasm were generally less active. The presence of both dolichol phosphokinase and dolichyl phosphate phosphatase in microsomes and nuclei, which contain the highest glycosyl transferase activities, may provide a means for direct enzymatic control of levels of dolichyl phosphate.     

Comparison of high-affinity binding of [3H]GABA to subcellular particles of rat brain and liver

The binding of [³H]GABA and retention of [¹⁴C]sucrose have been studied in freshly prepared synaptosomal-mitochondrial (P₂) fractions of rat cerebral cortex and liver using bicarbonate-buffered medium (containing 147 mEq/liter of N⁺), and in frozen/thawed crude membrane fractions of rat whole brain and liver using Na⁺-free Tris HCl medium. GABA-sensitive sites (GSS) and bicucul-line-methiodide-(BMI-) sensitive sites (BMI-SS) were defined as those amounts of [³H]GABA that were sensitive to the displacement by 10^–3 M unlabeled GABA or BMI. In the presence of added Na⁺, two high-affinity GABA-binding processes were detected in the P₂fraction of cerebral cortex. The lower-affinity process (likely related mainly to uptake sites) hadK _B10^–5 M,B _max for GSS3 nmol/mg protein, andB _max for BMI-SS0.5 nmol/mg protein, whereas the higher-affinity process (likely related to synaptic GABA receptors) hadK _B10^–7 M,B _max for GSS43 pmol/mg protein, andB _max for BMI-SS2 pmol/mg protein. Only the higher-affinity process was detected in the liver P₂ fraction and it hadK _B3.7×10^–8 M,B _max for GSS0.48 pmol/mg protein, andB _max for BMI-SS0.1 pmol/mg protein (i.e., about 1/100 and 1/20 the receptiveB _max values of cerebral cortex). This binding process of the liver P₂ fraction could represent sites involved in mitochondrial GABA transport. In Na⁺-free Tris HCl medium, high-affinity [³H]GABA binding appeared to exist in frozen/thawed membrane preparations of both brain and liver when data were expressed on a protein basis. However, this binding to liver membranes was not displaceable by 10^–3 M unlabeled GABA, and when these data were expressed on a weight basis and corrected for [³H]GABA present in trapped supernatant fluid of the pellets, no [³H]GABA binding was detected in the liver preparation.     

The subsynaptosomal distribution and release of [3H]acetylcholine synthesized by rat cerebral cortical synaptosomes

Synaptosomes were prepared from rat cerebral cortex and incubated in [³H]choline for periods ranging from 1 to 90 min. The [³H]ACh synthesized during this period was found only in the cytoplasm and in a membrane-associated fraction. A negligible amount of the newly formed [³H]ACh was recovered in the vesicular fraction despite concerted efforts to protect a hypothetical population of labile vesicles. The specific activity of the membrane-associated component, accounting for 21% of the total [³H]ACh, was by far the highest. This membrane-associated fraction was not released by hypotonic shock or homogenization and apparently was not in association with the monodisperse synaptic vesicles. The [³H]ACh was released in a calcium dependent manner. This investigation has determined that the ACh synthesized by synaptosomes is localized in only two fractions, cytoplasmic and membrane-associated; that this newly synthesized ACh can be released from synaptosomes by a process consistent with physiological release; and that at least part of the ACh released was originally present in the cytoplasm.     

Inhibition of the nuclear localization of [3H]estradiol in rat uterine tissue in vitro

The temperature coefficient (Q10) for the nuclear-cytoplasmic intracellular distribution of specifically-bound [3H]estradiol is approximately 1.0 over the interval 10-30 degrees C. However, this value increases to 3.19 for the temperature influence upon the nuclear-cytoplasmic localization of hormone between 30 degrees and 37 degrees C. A Q10 value of this magnitude is indicative of a biological, rather than physical, translocation event. In assessment of a biological basis for translocation, several antimicrotubular/antimicrofilamentous agents were used alone and in combination to ascertain their effects upon in vitro nuclear localization of labeled estradiol in the uterus. The incubation of uterine tissue in D2O-Locke-Ringer's solution containing 10(-4) M colchicine or vinblastine significantly reduced the nuclear localization of [3H]estradiol to nonspecific retention. Tissue uptake of the hormone, cytoplasmic binding and retention of estrogen, and the nucleophilic property of the receptor-estrogen complex (REC) were unaffected. Other drug treatments were without effect upon nuclear occupancy of the REC. The apparent inhibition of translocation by the above regimen could be due to an alteration in cellular architecture incompatible with hormone movement or the result of a direct effect upon cellular components which impede the dynamic interactions of REC in nuclei of whole tissue. Although these results do not necessarily imply that a functional cytoskeleton is required for translocation, we suggest that the estrogen-mediated nuclear occupancy of REC is a biological process susceptible to disruption.     

Multiple binding sites for [3H]clonidine and [3H]WB-4101 in rat brain

Binding of the alpha-adrenergic agonist [3H]clonidine and the alpha-adrenergic antagonist [3H]WB-4101 exhibited multiple binding site characteristics in both rat frontal cortex and cerebellum. Kinetic analysis of the dissociation of both radioligands in rat frontal cortex suggests two high affinity sites for each ligand. Competition of various noradrenergic agonists and antagonists for [3H]WB-4101 binding yielded shallow competition curves, with Hill coefficients ranging from 0.45 to 0.7. This further suggests multiplicity in [3H]WB-4101 binding. In the rat cerebellum, competition of various noradrenergic drugs for [3H]clonidine binding yielded biphasic competition curves. Furthermore Scatchard analysis of [3H]clonidine binding in rat cerebellum showed two high affinity sites with KD = 0.5 nM and 1.9 nM, respectively. Competition of various noradrenergic drugs for [3H]WB-4101 binding in the rat cerebellum yielded biphasic competition curves. Lesioning of the dorsal bundle with 6-hydroxydopamine did not significantly affect the binding of either [3H]clonidine or [3H]WB-4101. These findings for both [3H]clonidine and [3H]WB-4101 binding in rat frontal cortex and cerebellum can be explained by the existence of postsynaptic binding sites for both 3H ligands.     

Uptake and subcellular distribution of [3H]arachidonic acid in murine fibrosarcoma cells measured by electron microscope autoradiography

We have used quantitative electron microscope autoradiography to study uptake and distribution of arachidonate in HSDM1C1 murine fibrosarcoma cells and in EPU-1B, a mutant HSDM1C1 line defective in high affinity arachidonate uptake. Cells were labeled with [3H]arachidonate for 15 min, 40 min, 2 h, or 24 h. Label was found almost exclusively in cellular phospholipids; 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. All incorporated radioactivity was found to be associated with cellular membranes. Endoplasmic reticulum (ER) contained the bulk of [3H]arachidonate at all time points in both cell types, while mitochondria, which contain a large portion of cellular membrane, were labeled slowly and to substantially lower specific activity. Plasma membrane (PM) also labeled slowly, achieving a specific activity only one-sixth that of ER at 15 min in HSDM1C1 cells (6% of total label) and one-third of ER in EPU-1B (10% of total label). Nuclear membrane (NM) exhibited the highest specific activity of labeling at 15 min in HSDM1C1 cells (twice that of ER) but was not preferentially labeled in the mutant. Over 24 h, PM label intensity increased to that of ER in both cell lines. However, NM activity diminished in HSDM1C1 cells by 24 h to a small fraction of that in ER. In response to agonists, HSDM1C1 cells release labeled arachidonate for eicosanoid synthesis most readily when they have been labeled for short times. Our results therefore suggest that NM and ER, sites of cyclooxygenase in murine fibroblasts, are probably sources for release of [3H]arachidonate, whereas PM and mitochondria are unlikely to be major sources of eicosanoid precursors.     

Spontaneous and evoked release of [3H]taurine from a P2 subcellular fraction of the rat retina

The effects of spontaneous and evoked [³H]taurine release from a P₂ fraction prepared from rat retinas were studied. The P₂ fraction was preloaded with [³H]taurine under conditions of high-affinity uptake and then examined for [³H]taurine efflux utilizing superfusion techniques. Exposure of the P₂ fraction to high K⁺ (56 mM) evoked a Ca²⁺-independent release of [³H]taurine. Li⁺ (56 mM) and veratridine (100 M) had significantly less effect (8–15% and 15–30%, respectively) on releasing [³H]taurine compared to the K⁺-evoked release. 4-Aminopyridine (1 mM) had no effect on the release of [³H]taurine. The spontaneous release of [³H]taurine was also Ca²⁺-independent. When Na⁺ was omitted from the incubation medium K⁺-evoked [³H]taurine release was inhibited by approximately 40% at the first 5 minute depolarization period but was not affected at a second subsequent 5 minute depolarization period. The spontaneous release of [³H]taurine was inhibited by 60% in the absence of Na⁺. Substitution of Br^– for Cl^– had no effect on the release of either spontaneous or K⁺-evoked [³H]taurine release. However, substitution of the Cl^– with acetate, isethionate, or gluconate decreased K⁺-evoked [³H]taurine release. Addition of taurine to the superfusion medium (homoexchange) resulted in no significant increase in [³H]taurine efflux. The taurine-transport inhibitor guanidinoethanesulfonic acid increased the spontaneous release of [³H]taurine by approximately 40%. These results suggest that the taurine release of [³H]taurine is not simply a reversal of the carrier-mediated uptake system. It also appears that taurine is not released from vesicles within the synaptosomes but does not rule out the possibility that taurine is a neurotransmitter. The data involving chloride substitution with permeant and impermeant anions support the concept that the major portion of [³H]taurine release is due to an osmoregulatory action of taurine while depolarization accounts for only a small portion of [³H]taurine release.     

In vivo conversion of [3H]myoinositol to [3H]chiroinositol in rat tissues.

We report here the in vivo conversion of [3H]myoinositol to [3H]chiroinositol. After labeling intraperitoneally with [3H]myoinositol for 3 days to reach radioisotope equilibrium in urine, [3H]chiroinositol was isolated from tissues and purified after 6 N HCl hydrolysis by two sequential paper chromatographies and high performance liquid chromatography (HPLC). Percent conversion of [3H]myoinositol to [3H]chiroinositol was highest in urine (36%), liver (8.8%), muscle (8.8%), and blood (7.6%) with intestine, brain, kidney, spleen, and heart decreasing in percentage from 2.8 to 0.7%. Labeling of other inositol isomers including scyllo-, neo-, and epi-, and mucoinositol was minimal, approximately 0.06% of [3H]myoinositol. Glucose was unlabeled, but glucuronate, the product of myoinositol oxidation, was labeled up to 1.5% of the [3H] myoinositol. Acid hydrolysates of combined inositol-containing phospholipids contain significant labeled chiroinositol. [3H]Phosphatidylinositols and [3H]glycosylphosphatidylinositols were extracted from liver, muscle, and blood, isolated by thin layer chromatography, and inositols purified by HPLC after acid hydrolysis. Percent conversion of [3H]myoinositol to [3H] chiroinositol was highest in blood (60.4%) followed by muscle (7.7%) and liver (2.2%).     

Uptake and metabolism ofl-[3H]glutamate andl-[3H]glutamine in adult rat cerebellar slices

Using very low concentrations (1 mumol range) of L-2-3-[3H]glutamate, (3H-Glu) or L-2-3-[3H]glutamine (3H-Gln), we have previously shown by autoradiography that these amino acids were preferentially taken up in the molecular layer of the cerebellar cortex. Furthermore, the accumulation of 3H-Glu was essentially glial in these conditions. We report here experiments in which uptake and metabolism of either (3H-Glu) or (3H-Gln) were studied in adult rat cerebellar slices. Both amino acids were rapidly converted into other metabolic compounds: after seven minutes of incubation in the presence of exogenous 3H-Glu, 70% of the tissue accumulated radioactivity was found to be in compounds other than glutamate. The main metabolites were Gln (42%), alpha-ketoglutarate (25%) and GABA (1,4%). In the presence of exogenous 3H-Gln the rate of metabolism was slightly slower (50% after seven minutes of incubation) and the metabolites were also Glu (29%), alpha-ketoglutarate (15%) and GABA (5%). Using depolarizing conditions (56 mM KCl) with either exogenous 3H-Glu or 3H-Gln, the radioactivity was preferentially accumulated in glutamate compared to control. From these results we conclude: i) there are two cellular compartments for the neurotransmission-glutamate-glutamine cycle; one is glial, the other neuronal; ii) these two cellular compartments contain both Gln and Glu; iii) transmitter glutamate is always in equilibrium with the so-called "metabolic" pool of glutamate; iv) the regulation of the glutamate-glutamine cycle occurs at least at two different levels: the uptake of glutamate and the enzymatic activity of the neuronal glutaminase.     

An autoradiographic analysis of [3H]alpha-bungarotoxin distribution in the rat brain after intraventricular injection

Purified alpha-bungarotoxin was isolated by chromatography and made radioactive with tritium ([3H]acetamidino-alpha-bungarotoxin). Infusions of [3H]alpha-bungarotoxin alone or preceded by tubocurarine or atropine were given into the third ventricle. 2. 12, or 24 h after injection the brains were prepared for autoradiography. Injections of alpha-bungarotoxin (radioinert) in buffer, or of [3H]parathyroid hormone in buffer, served as controls. The various patterns of labeling suggest the presence of nicotinic-cholinergic neurons within the arcuate and basolateral regions of the hypothalamus including the supraoptic and suprachiasmatic nuclei and, in addition, the central nucleus of the amygdala.     

Distribution of [3H]dexamethasone in rat subcutaneous tissue after delivery from osmotic pumps

Inflammation surrounding implantable glucose sensors may be controlled through local release of dexamethasone at the site of implantation. In the present study, we evaluated the distribution of dexamethasone in rat subcutaneous tissue during the first 2.5 days after local release. Osmotic pumps containing [3H]dexamethasone were implanted into the subcutaneous tissue of rats. Digital autoradiography was used to measure the distribution of the [3H]dexamethasone within the subcutaneous tissue at 6, 24, and 60 h after implantation. Measured concentration profiles, near the catheter tip through which the agent was released, were compared to mathematical models of drug diffusion and elimination. The results demonstrate that the majority of the [3H]dexamethasone delivered into the subcutaneous tissue was found within a 3 mm region surrounding the catheter tip. There was good agreement between the experimental data and the mathematical model. The diffusion coefficient for dexamethasone in subcutaneous tissue was found to be D = 4.11 +/- 1.77 x 10(-10) m2/s, and the elimination rate constant was found to be k = 3.65 +/- 2.24 x 10(-5) s(-1). The diffusion coefficient and elimination rate constants for dexamethasone in subcutaneous tissue have not been previously reported. The use of a mathematical model may be useful in predicting the effectiveness of local delivery of dexamethasone around implantable glucose sensors.     

The distribution of [3H]kainate binding sites in primate hippocampus is similar to the distribution of both Ca2+-sensitive and Ca2+-insensitive [3H]kainate binding sites in rat hippocampus

The distribution of [³H]kainate binding sites was determined by quantitative autoradiography in three vertebrate species: rat, monkey, and human. These animals displayed a similar pattern of binding site density in the hippocampus. Highest levels were found within the stratum lucidum and moderate levels in the inner portion of the dentate gyrus molecular layer. Although the distribution is similar, there is a lower density of binding sites in the stratum lucidum of primates than in rodents. Experiments using rat brain synaptic plasma membrane fractions indicated that inclusion of Ca²⁺ ions results in a selective reduction in binding at the high affinity sites. The Ca²⁺-inhibited and Ca²⁺-insensitive binding sites in the rat hippocampus exhibited a similar distribution. Together, these results suggest that in a variety of mammalian species kainate receptors exhibit similar regional distributions, and that the high anf loe affinity kainate binding sites also exhibit similar regional distributions.Special Issue dedicated to Prof. Eduardo De Robertis.