Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Autonomic innervation of the arteriovenous anastomoses in the dog tongue

Summary The innervation of the arteriovenous anastomoses in the dog tongue has been investigated. At the lightmicroscopic level, the vessels were found to be densely supplied with adrenergic and AChE-positive nerve plexuses and less densely with the quinacrine-binding nerve plexus. At the electron-microscopic level, at least two apparently different types of axon profiles were identified: 1) Small vesicle-containing axons, characterized by many small granular vesicles, variable numbers of small clear vesicles and large granular vesicles. Storage of endogenous amines and uptake of exogenous amines into most small granular vesicles and many large granular vesicles was demonstrated. These axons stained only lightly with reaction products for AChE activity and thus seemed to be adrenergic in nature. Some axons contained numerous large granular vesicles, whose cores occasionally stained with uranyl ions; this suggests a co-localization of ATP or peptides as neurotransmitters. 2) Small granular vesicle-free axons, containing small clear vesicles and large granular vesicles in variable ratio. Most cores of these large granular vesicles were heavily stained with uranyl ions. No storage or uptake of amine into the synaptic vesicles was detected. Some axons appeared to be typically cholinergic, some, typically non-adrenergic, noncholinergic, and the rest, intermediate between the two. All axons stained heavily with reaction products for AChE activity, suggesting their cholinergic nature.     

A gradient of synaptic efficacy and its presynaptic basis in the cercal system of the cockroach

1. The cerci of the cockroach Periplaneta americana bear longitudinal columns of wind-sensitive receptors which provide excitatory inputs to the giant interneurons (GIs) of the abdominal nerve cord. By using sound stimuli, we showed that spikes were more easily induced in the GIs from the most proximal than from the most distal receptors of the same column. 2. This was not due to a greater responsiveness of proximal sensilla to tones but to stronger synaptic connections; for the 3 largest GIs, the amplitude of the monosynaptic unitary EPSP tended to be all the higher as the stimulated sensillum was more proximal in each column. 3. The differences in EPSP size were due, at least partly, to presynaptic factors: a statistical analysis of the amplitude fluctuations of single-fibre EPSPs, showed that the amount of transmitter released per presynaptic impulse was larger for proximal than for distal sensory neurons in each column. 4. These differences in synaptic strength were correlated with differences in the structure of the afferent terminals. The location, the size and the shape of the axonal arbors are nearly the same for all sensory neurons of the same column, but proximal neurons arborize more profusely, and the terminal arbor of distal neurons is generally characterized by dorsal clusters of varicosities. 5. During postembryonic development, a decrease in the connection strength of 2 identified cercal neurons was accompanied by a retraction of ramifications on the medial side of their axonal arbor. 6. Possible mechanisms involved in the genesis and the remodelling of the gradient of synaptic strength are discussed in the light of available data and hypotheses relative to the development of ordered afferent connections.     

A hemagglutinin specific for sialic acids in a rat brain synaptic vesicle-enriched fraction

A hemagglutinating activity was detected in a synaptic vesicle-enriched fraction prepared from adult rat brain, using trypsinized glutaraldehyde-fixed rabbit erythrocytes. The specific activity of the fraction, in two series of experiments, was 7.5 and 16-fold higher than in the other subcellular fractions. The activity was absent from the synaptosome cytosol. In a study using twenty-five different carbohydrates and glycoproteins, best inhibitors were N-acetylneuraminic acid and N-glycolylneuraminic acid, together with bovine submaxillary mucin and a glycopeptide fraction prepared from rabbit erythrocyte membranes. The activity was thermolabile and very sensitive to proteolytic enzymes (but insensitive to neuraminidase) indicating that a protein (agglutinin) is responsible for the activity. Experiments using detergents and high ionic strength showed that the agglutinin is tightly bound to membranes, inactivated by the so-called non denaturing detergents, and stable in diluted sodium dodecyl sulphate. Hypotheses are discussed on the possible function of the agglutinin.     

Tetanus Toxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline Release from Cultured Mouse Brain

The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P2) fraction of bovine brainstem. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 0.1 M NaCl at 0 degrees C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [3H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 microM GTP) in both preparations. These results were virtually identical to those of P2 membrane preparations and suggested that this high-affinity SP binding site belongs to the SP-P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a Bmax of 22.0 +/- 5.10 fmol/mg protein and a KD of 0.79 nM, and these values are almost the same as those obtained in the P2 fraction (Bmax = 31.3 +/- 3.56 fmol/mg protein, KD = 0.82 nM). Gel filtration analysis showed that the detergent-SP binding protein complex has two calculated molecular weights of greater than 1,000,000 and 55,000-60,000 (a corresponding Stokes radius of 35.5 nm).     

Kinetic Modulation by GABAergic Agents of High- and Low-Affinity Binding of [3H]Methyl β-Carboline-3-Carboxylate

The kinetics of dissociation of [3H]methyl beta-carboline-3-carboxylate (beta-CCM) binding was studied in a synaptosomal membrane preparation of rat cerebral cortex. Dissociation was biphasic: a faster phase (10-30% contribution) was followed by a slower phase. Picrotoxin pretreatment at 22 degrees C enhanced the equilibrium binding of [3H]beta-CCM. The half-life of the slower phase of beta-CCM dissociation (t1/2II) was increased by 60 muM picrotoxin from 1.7 min to 3.3 min. The dissociation of [3H]beta-CCM was identical when initiated by an excess of either diazepam or beta-CCM. Quasi-equilibrium Scatchard analysis of [3H]beta-CCM binding was performed by a kinetic separation of the rapid and slow phases of dissociation. The slow and rapid phases represented beta-CCM binding sites of high and low affinity, respectively. The dissociation of [3H]beta-CCM (control t1/2II = 2.0 min) was decelerated by the gamma-aminobutyric acid (GABA) antagonist 3-alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R 5135) (t1/2II = 2.5 min) and accelerated by GABA (t1/2II = 1.6 min). GABA inhibited both high- and low-affinity beta-CCM bindings.     

Host range differentiation of spore-positive and spore-negative strain types of Frankia in stands of Alnus glutinosa and Alnus incana in Finland

Host compatibility of different spore-positive (Sp⁺)and spore-negative (Sp^?) strain types of Frankia from alder stands in Finland was studied in Modulation tests with hydrocultures of Alnus glutinosa (L.) Gaertner, A. incana (L.) Moench and A. nitida Endl. Root nodules and soil samples from stands of A. incana (Lammi forest and Hämeenlinna forest) were dominated by Sp ⁺ types of Frankia (coded AiSp⁺ and AiSp⁺ H. respectively), which caused effective root nodules in test plants of A. incana, but failed to induce nodules in A. nitida. In A. glutinosa Frankia strain types AiSp ⁺ and AiSp ⁺ H caused small, ineffective root nodules with sporangia (coded Ineff ^?), which were recognized by the absence or near absence of vesicles in the nodule tissue. Ineffective nodules without sporangia (coded Ineff ^?) were induced on A. glutinosa with soil samples collected at Lammi swamp. The spore-negative strain type of Frankia was common in root nodules of A. glutinosa in Finland (Lammi swamp) and caused effective Sp^? type root nodules (coded AgSp ^?) in hydrocultures of A. incana, A. glutinosa and A. nitida. A different Sp ⁺ strain type of Frankia. coded AgSp⁺ Finland, was occasionally found in stands of A. glutinosa. It was clearly distinguished from strain type AiSp ⁺ by the ability to produce effective nodules on both A. glutinosa and A. incana. The nodulation capacities of soil and nodule samples were calculated from the nodulation response in hydrocutlure and served as a measure for the population density of infective Frankia particles. Sp ⁺ nodules from both strain types had equal and high nodulation capacities with compatible host species. The nodulation capacities of Sp type root nodules from A. glutinosa were consistently low. High frequencies of Frankia AiSp⁺ and AiSp⁺ H were found in the soil environment of dominant AiSp ⁺ nodule populations on A. incana. The numbers of infective particles of this strain type were insignificant in the soil environment of nearby Sp ^? nodule populations on A. glutinosa and in the former field at Hämeen-linna near the Sp⁺ nodule area in Hämeenlinna forest. Strain type AgSp^? had low undulation capacity in the soil environment of both A. incana and A. glutinosa stands, Explanations for the strong associations between Frankia strain types AiSp⁺ and AiSp ^? H and A. incana and between strain type AgSp^? and A. glutinosa are discussed in the light of host specificity and of some characteristics of population dynamics of both strain types. The possible need to adapt the concept of Frankia strain types Sp ⁺ and Sp ^? to strains with some variation in spore development was stressed by the low potentials of strain type AiSp ⁺ H to develop spores in symbioses with hydrocultures of A. incnna.     

Induction of reversible changes in cell-surface glycoconjugates and lung colonization potential by 13-cis retinoic acid

Murine squamous carcinoma cells (KLN205) grown in a medium supplemented with the retinoid, 13-cis retinoic acid (RA), had dose-dependent, selective increases in the expression of certain lectin receptors, which correlated with a dramatic decrease in the ability to form pulmonary colonies (P ?.0003) (Couch MJ, Pauli BU, Weinstein RS, Coon JS: JNCI, 78:971 ?977, 1987). These findings suggest a possible relationship between the RA-induced glycoconjugate alterations and the decreased experimental metastatic behavior. We further define the mechanism of RA's action. The finding that RA treatment (5 × 10^?6 M, 5 × 10^?7 M) did not perturb the cell cycle of KLN205 cells provides further proof that the decreased metastatic behavior is not attributable to any inhibition in the rate of growth or to alterations in the cell cycle. Furthermore, since stable subpopulations with variable lectin binding could not be detected, the mechanism of RA's action does not appear to be due to selection of variant tumor-cell subpopulations. Finally, in a scries of experiments designed to determine the reversibility of the RA treatment, the RA-induced decrease in metastatic behavior reverted back to a more metastatic state in the same time frame (3 days) as the reversion of the RA-induced changes in cell-surface glycoconjugate expression. This reversion provides further evidence for a close relationship between the RA-induced modulation of tumor cell-surface glycoconjugate expression and the decreased metastatic behavior; it suggests that transient, reversible modulation of the tumor cell surface may play a role in determining metastatic behavior.     

Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.     

Specific Binding of Glycosylated β-Galactosidase to Bovine Brain Synaptic Membrane

The binding of a series of glycosylated beta-galactosidases to a fraction rich in synaptic membrane of bovine brain was examined. beta-galactosidase modified with p-aminophenyl beta-D-galactopyranoside (beta-D-Gal beta-gal) was found the most effective in binding to synaptic membrane, followed by that modified with beta-D-glucopyranoside, whereas the enzyme modified with p-aminophenyl derivatives of alpha-D-galactopyranoside, alpha-D-glucopyranoside, and alpha- and beta-L-fucopyranoside were found not to bind to the membrane. The binding was dependent on time, temperature, and pH; the maximal binding was obtained within 15 min at 4 degrees C and the optimal pH was approximately 4.0. The binding of beta-D-Gal beta-gal was inhibited by free p-aminophenyl beta-D-galactopyranoside and by the treatment of synaptic membrane with trypsin or phospholipase A2 or C. The equilibrium dissociation constant and the maximal concentration of binding sites were determined by Scatchard analysis to be 470 +/- 35 nM and 27.5 +/- 3.1 pmol/mg protein (n = 1). The results suggest that a specific binding site for the specified carbohydrates exists in synaptic membrane and is involved in the internalization of glycoconjugates into nerve terminals.