Cerebrocortical Synaptosomes Research Articles

5-(2-Cyclohexylideneethyl)-5-ethyl barbituric acid (CHEB): Correlation of hypnotic and convulsant properties with alterations of synaptosomal 45Ca 2+ influx

Male ICR mice (20–35 g) were given either 5-(2-cyclohexylideneethyl)-5-ethyl barbituric acid (CHEB) alone (10–15 mg/kg i.p.) or CHEB (25–75 mg/kg i.p.) after a 1 h pretreatment with phenobarbital (75 mg/kg i.p.). CHEB alone (10 mg/kg) produced excitatory behavior but not convulsive seizures. Higher doses (11–15 mg/kg) produced convulsive seizures resulting in death. Pretreatment with phenobarbital prevented seizure activity. Following phenobarbital pretreatment, CHEB in doses of 50 and 75, but not 25 mg/kg, resulted in hypnosis of 53 ± 16 and 64 ± 9 min duration, respectively. In vitro, CHEB (10–200 μM) significantly inhibited ‘fast-phase’ (3 s) K +-stimulated 45Ca 2+ uptake into cerebrocortical synaptosomes. CHEB (10 and 100 μM) also significantly increased basal 45Ca 2+ uptake. The addition of CHEB (50 and 100 μM) or pentobarbital (100 μM) to striatal synaptosomes inhibited ‘fast-phase’ K +-stimulated 45Ca 2+ uptake and endogenous dopamine release. CHEB (10–200 μM), but not pentobarbital (100 μM), produced a time- and dose-dependent increase in the resting release of endogenous dopamine from striatal synaptosomes. The results of this study show that CHEB possesses hypnotic activity if its lethal convulsant actions are blocked. The hypnotic actions of CHEB appear to correlate with inhibition of voltage-dependent calcium channels in brain synaptosomes.

Structural requirements for cocaine congeners to interact with [3H]batrachotoxinin A 20-alpha-benzoate binding sites on sodium channels in mouse brain synaptosomes.

The present study examines the possible role of sodium channels in the behavioral effects of cocaine. Cocaine congeners are apparent competitive inhibitors of the scorpion toxin-enhanced binding of [3H]batrachotoxinin A 20-alpha-benzoate to sodium channels in mouse cerebrocortical synaptosomes. However, in agreement with the allosteric model for heterotropic cooperative interactions, the compounds produce a concentration-dependent increase in the rate of dissociation of binding. Concentrations that give a 2-fold increase of k-1 are close to Ki values for inhibiting equilibrium binding of [3H]bactrachotoxinin A 20-alpha-benzoate, suggesting that the inhibitory effect on binding results mostly from an increase of the apparent dissociation rate constant. The ester linkage between the tropane and benzoyl ring of cocaine is not essential for the inhibitory potency, and for both the C-2 and C-3 substituents the equatorial position results in a higher potency than the axial position. There is reasonable agreement between the rank order of potencies in blocking the sodium channel and in inhibiting locomotor behavior. The present results do not support a relationship between the capability of cocaine congeners in blocking sodium flux and in inhibiting uptake of dopamine into striatal synaptosomes. However, peak levels of cocaine in the brain of cocaine addicts could be high enough to interfere with sodium channel functioning, possibly contributing to some of cocaine's actions.

On the Specificity of Verapamil as a Calcium Channel-Blocker

Abstract The stimulated uptake of 45 Ca 2+ into incubated cerebrocortical synaptosomes caused by veratrine (75 μM) was blocked by low concentrations of verapamil (0.5–30 μM) which did not prevent or reduce depolarization as judged by efflux of potassium (K + ). However, verapamil did not prevent amino acid neutrotransmitter release at these low concentrations and this is discussed in terms of mobilization of internal calcium (Ca 2+ ) stores. At higher concentrations (30–200 μM) verapamil appeared to act additionally at sodium (Na − ) channels since both depolarization-induced K + efflux and neurotransmitter release were reduced or prevented. When K + , at a high concentration (56 mM), was used as the depolarizing agent, both 45 Ca 2+ influx and neurotransmitter release were prevented by verapamil across a wide concentration range (0.5–200 μM). The data are discussed in terms of the specificity of action of verapamil on Ca 2+ channels.

新抗うつ薬，ＭＯ‐８２８２の薬理学的性質 ＩＩ

We investigated the pharmacological characteristics of MO-8282 as a novel antidepressant. MO-8282 inhibited the specific binding of 3H-clonidine to cerebro-cortical membrane fractions from rats about five times more potently than mianserin, and it competed with clonidine in the twitch response of the isolated guinea-pig ileum under field stimulation. The results indicated that MO-8282 possessed alpha 2-adrenergic receptor blocking activity. MO-8282 in a dose of 30 mg/kg (p.o.) showed no inhibition against the uptake of noradrenaline, dopamine and serotonin in the rat brain, whereas mianserin inhibited the uptake of serotonin specifically. MO-8282, similar to mianserin, had no effect on spontaneous release of 3H-noradrenaline and slightly stimulated the release of 3H-serotonin from the rat cerebrocortical synaptosome. The turnover rate of noradrenaline in rat brain was accelerated by administration of MO-8282 (30 mg/kg) for 15 days; however, that of dopamine and serotonin was not affected. The above findings indicate that MO-8282, unlike tricyclic antidepressants, mainly exerts alpha 2-adrenoceptor blocking action on the central noradrenergic system, similar to mianserin. In addition, the fact that MO-8282 unlike mianserin showed no inhibition against uptake of serotonin in brain suggests that the alpha 2-adrenoceptor blocking of MO-8282 is more specific and potent than that of mianserin.

Botulinum neurotoxin type B. Its purification, radioiodination and interaction with rat-brain synaptosomal membranes.

Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein. 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially. Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.

On the specificity of verapamil as a calcium channel-blocker

The stimulated uptake of 45Ca 2+ into incubated cerebrocortical synaptosomes caused by veratrine (75 μM) was blocked by low concentrations of verapamil (0.5–30 μM) which did not prevent or reduce depolarization as judged by efflux of potassium (K +). However, verapamil did not prevent amino acid neutrotransmitter release at these low concentrations and this is discussed in terms of mobilization of internal calcium (Ca 2+) stores. At higher concentrations (30–200 μM) verapamil appeared to act additionally at sodium (Na −) channels since both depolarization-induced K + efflux and neurotransmitter release were reduced or prevented. When K +, at a high concentration (56 mM), was used as the depolarizing agent, both 45Ca 2+ influx and neurotransmitter release were prevented by verapamil across a wide concentration range (0.5–200 μM). The data are discussed in terms of the specificity of action of verapamil on Ca 2+ channels.

Synaptosomal T3 binding sites in rat brain: their localization on synaptic membrane and regional distribution.

Previous studies have shown the existence of specific high affinity T3 binding sites in rat cerebrocortical synaptosomes. In this study, to define the localization of the binding sites, T3 binding to disrupted synaptic membrane fractions was compared with the binding to intact synaptosomes obtained from the rat cerebral cortex. Scatchard analysis revealed two orders of T3 binding sites with almost identical apparent dissociation constant (Kd) in synaptosomes and synaptic membrane fractions. However, the maximal binding capacity (MBC) of the higher affinity binding sites for synaptic membrane fractions was significantly greater than that for intact synaptosomes (4.2 +/- 0.2 vs 3.0 +/- 0.3 pg T3/mg protein). Regional distribution of the synaptosomal T3 binding sites in various areas of the rat brain was also studied. The Kd values were not significantly different in discrete brain regions. On the other hand, the MBCs of the higher affinity binding sites were greater in the cerebral cortex and hypothalamus (72.8 +/- 1.4 and 64.8 +/- 9.5 pg T3/g tissue) than in the cerebellum (22.1 +/- 1.6 pg T3/g tissue). Similar difference was also observed in the lower affinity sites. These results indicate that the specific T3 binding sites in brain synaptosomes are localized mainly on synaptic membranes and that the MBC is different in discrete brain regions.

Amino acid neurotransmitter release from cerebrocortical synaptosomes of sheep with severe ryegrass staggers in New Zealand

Enhanced unstimulated release of the excitatory amino acid neurotransmitters aspartic acid and glutamic acid was measured in cerebrocortical synaptosomes prepared from sheep showing severe symptoms of ryegrass staggers. A similar pattern of deviation from neurologically normal animals was seen whether the sheep had contracted the disorder naturally or whether they had been deliberately caused to develop the disorder by grazing specific pastures rich in endophyte infected ryegrass. Deranged neurotransmitter release may reasonably be involved in the chemical pathology of ryegrass staggers and the findings are in accord with the putative synonymy of lolitrem toxicosis and ryegrass staggers.

Synaptic binding sites in brain for [ 3H]β-bungarotoxin — A specific probe that perturbs transmitter release

A neurotoxic, [ 3H]labelled derivative of β-bungarotoxin, known to inhibit neurotransmitter release and to be free of phospholipase activity, was used to demonstrate autoradiographically the distribution and ultrastructural location of its saturable binding component in brain. Light-microscope autoradiography of rat cerebellum and hippocampus showed that it resides primarily in synaptic-rich areas, with much lower densities of sites in other regions containing cell bodies; also, little binding was associated with myelinated tracts. Ultrastructural localisation and sub-fractionation studies on purified cerebrocortical synaptosomes showed that [ 3H]toxin binding sites are located predominantly on brain synaptosomal membranes, consistent with their possible association with transmitter release.

Tritiation of β-bungarotoxin and its saturable binding to membranes of cerebrocortical synaptosomes

Tritiation of β-bungarotoxin and its saturable binding to membranes of cerebrocortical synaptosomes

Biochemical and electrophysiological demonstrations of the actions of beta-bungarotoxin on synapses in brain.

Homogeneous beta-bungarotoxin interacts irreversibly with rat olfactory cortex and produced permanent inhibition of neurotransmission (half-time of blockade for 230 nM toxin in 25 min). Binding occurs in the absence of divalent cations, but the rate of synaptic blockade is increased by Ca2+, which activates the intrinsic phospholipase A2 activity of the toxin. Other observable actions of the toxin, seen with rat cerebrocortical synaptosomes, are an increase in the release of acetylcholine, glutamate and gamma-aminobutyrate and impairment of transmitter uptake, which are all insensitive to tetrodotoxin. Inactivation of the toxin's phospholipase activity by chemical modification with p-bromophenacyl bromide diminishes the observed concomitant efflux of the neurotransmitters and lactate dehydrogenase. Collectively, the results support the idea that the toxin binds specifically and irreversibly to component(s) on nerve terminals and this together with the resultant phospholipolysis leads eventually to synaptic blockade. Such a proposal would account for the unique toxicity of the protein relative to phospholipase A2 enzymes.

Localization of sites for 125I-labelled botulinum neurotoxin at murine neuromuscular junction and its binding to rat brain synaptosomes

Botulinum neurotoxin, purified to homogeneity from Clostridium botulinum (Type A), was found to be highly neurotoxic (>8 x 10 7 mouse ld 50/mg protein). Labelling of this pure neurotoxin with 125I-iodine to high specific radioactivity was achieved without appreciable loss of biological activity. This was used to demonstrate saturable binding sites for this toxin at the neuromuscular junction, following in vivo administration into mice. A demonstrable inhibitory effect of the neurotoxin on release of acetylcholine from rat cerebrocortical synaptosomes indicates that it affects synapses in the central nervous system. Kinetic studies on the binding of 125I-labelled neurotoxin to brain synaptosomes yielded an association rate constant of 2.3 x 10 5M −1s −1; dissociation plots were biphasic and the predominant species showed a rate constant of 1.2 x 10 −4s −1. The saturable binding component is heatsensitive and inactivated by trypsin. Preliminary studies showed that botulinum neurotoxin associates with plasma membrane fractions of synaptosomes and that binding does not result in any gross structural changes, at least in the majority of the toxin molecules.

Actions of tremorgenic fungal toxins on neurotransmitter release.

The neurochemical effects of the tremorgenic mycotoxins Verruculogen and Penitrem A, which produce a neurotoxic syndrome characterised by sustained tremors, were studied using sheep and rat synaptosomes. The toxins were administered in vivo, either by chronic feeding (sheep) or intraperitoneal injection 45 min prior to killing (rat), and synaptosomes were subsequently prepared from cerebrocortical and spinal cord/medullary regions of rat, and corpus striatum of sheep. Penitrem A (400 mg mycelium/kg) increased the spontaneous release of endogenous glutamate, GABA (gamma-aminobutyric acid), and aspartate by 213%, 455%, and 277%, respectively, from cerebrocortical synaptosomes. Verruculogen (400 mg mycelium/kg) increased the spontaneous release of glutamate and aspartate by 1300% and 1200%, respectively, but not that of GABA from cerebrocortical synaptosomes. The spontaneous release of the transmitter amino acids or other amino acids was not increased by the tremorgens in spinal cord/medullary synaptosomes. Penitrem A pretreatment reduced the veratrine (75 microM) stimulated release of glutamate, aspartate, and GABA from cerebrocortical synaptosomes by 33%, 46%, and 11%, respectively, and the stimulated release of glycine and GABA from spinal cord/medulla synaptosomes by 67% and 32% respectively. Verruculogen pretreatment did not alter the veratrine-induced release of transmitter amino acids from cerebrocortex and spinal cord/medulla synaptosomes. Penitrem A pretreatment increased the spontaneous release of aspartate, glutamate, and GABA by 68%, 62%, and 100%, respectively, from sheep corpus striatum synaptosomes but did not alter the synthesis and release of dopamine in this tissue. Verruculogen was shown to cause a substantial increase (300-400%) in the miniature-end-plate potential (m.e.p.p.) frequency at the locust neuromuscular junction. The response was detectable within 1 min, rose to a maximum within 5-7 min, and declined to the control rate over a similar period. No change in the amplitude of the m.e.p.p.'s was observed. These effects of the tremorgens on transmitter release are interpreted in terms of their mode of action.