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At least 19 recordsLinked to original sources
PubMed1977

Differential effects of reserpine and tetrabenazine on rat striatal synaptosomal dopamine biosynthesis and synaptosomal dopamine pools.

Rat striatal dopamine (DA) levels and synaptosomal DA synthesis were determined after the administration of the catecholamine-depleting agents reserpine (RES) and tetrabenazine (TBZ). Striatal synaptosomal DA synthesis remained unchanged from control levels after Res administration over a wide range of doses (0.5-5 mg/kg) and times (up to 48 hour). In contrast, after TBZ administration, DA synthesis rapidly increased to values greater than 200% of control values, then returned to control levels. The changes in DA synthesis inversely paralleled the depletion of and recovery of striatal DA levels. The increased levels of DA synthesis did not appear to originate with alterations either in the kinetic properties of tyrosine hydroxylase or in the availability of exogenous or endogenous tyrosine. To assess the contribution of synaptosomal DA pools to the regulation of DA synthesis in tissue preparations from RES-or TBZ-pretreated animals, synaptosomal DA synthesis was assessed in the presence of DA releasing agents and compared with analogous experimental manipulations on tissue preparations from animals pretreated with alpha-methyltyrosine or the monoamine oxidase inhibitor, clorgyline. The data are consistent with a differential in vivo interaction of RES and TBZ with a nerve ending pool of DA which participates in end-product inhibition of tyrosine hydroxylase.

Animals↗
PubMed1979

Effect of fenfluramine administration on synaptosomal uptake of some neurotransmitters and on synaptosomal enzymes which metabolise GABA.

Fenfluramine has been considered to deplete neuronal 5-hydroxytryptamine (5-HT). This compound is able to reduce the synaptosomal uptake of 5-HT and other neurotransmitters such as GABA and glutamic acid (Glu). The effects of fenfluramine on these three compounds considered as neurotransmitters are different. The inhibition is of competitive type for 5-HT and non-competitive for GABA and Glu. Concerning the enzymes involved in GABA synthesis and degradation, Fenfluramine increases Glutamic acid decarboxylase activity and decreases GABA-trasaminase activity in synaptosomes. Decreased synaptosomal GABA levels could be attributed to a lower uptake. An enzymatic regulating system may be responsible in restoring the GABA level. A similar mechanism concerning serotonin has been previously suggested (Costa et al., 1971).

4-Aminobutyrate Transaminase↗
PubMed1975

Membrane potentials in pinched-off presynaptic nerve ternimals monitored with a fluorescent probe: evidence that synaptosomes have potassium diffusion potentials.

1. Some physiological properties of tissue fractions from rat brain homogenates have been examined. Of the three fractions studied (presynaptic nerve terminals, mitochondria and fragmented membranes), only the nerve terminals (synaptosomes) have the ability to accumulate 42K from physiological salt solutions. 2. The ability to accumulate and retain K is lost if synaptosomes are exposed to very hypotonic solutions. The K uptake and total K content is reduced by ouabain and by inhibitors of glycolysis and oxidative phosphorylation. 3. These results suggest that synaptosomes in physiological saline accumulate K against a concentration gradient, and may have K diffusion potentials across their surface membranes. The voltage-sensitive fluorescent probe, 3,3'-dipentyl 2,2'-oxacarbocyanine (CC5), was used to test this possibility. 4. In the squid axon, the fluorescent emission of CC5 is directly proportional to membrane potential; depolarization causes an increase in fluorescence. 5. The fluorescence of synaptosomes ('synaptosome fluorescence') treated with CC5 is increased when [K]o is increased or [K]o is reduced; replacement of external Na by Li or choline has little effect on the synaptosome fluorescence. In quantitative terms, synaptosome fluorescence is proportional to log ([K]o plus 0-05[Na]o). Rb is about as effective as K in enhancing synaptosome fluorescence; Cs is about 1/4 as effective. The effect of increased [K]o is reversible. 6. The fluorescence data provide corroborative evidence that there is normally a large K gradient ([K]o smaller than [I]i) across the synaptosome surface membrane. The data suggest the [K]i may be in excess of 100 mM. 7. Replacement of Cl- by methylsulphate did not significantly affect the relationship between synaptosome fluorescence and [K]o, nor did removal of external Ca. 8. The fluorescence of CC5-treated mitochondria, membrane fragmnets, or lysed synaptosomes is unaffected by changes in the K concentration of the medium. 9. Veratridine and gramicidin D, both of which enhance Na permeability (PNa) in some intact tissues, increase synaptosome fluorescence when added to the standard medium. The increment is greatly reduced or abolished when external Na is replaced by choline. 10. If synaptosomes are first Na-loaded (by pre-treatment with cyanide + iodoacetate), and then placed in a choline medium, addition of gramicidin D significantly decreases fluorescence. This effect could be explained if, with [Na]o smaller than [Na]i, the increase in PNa causes the synaptosomes to hyperpolarize. 11. The veratridine-induced increase in synaptosome fluorescence was prevented by 3 times 10- minus 7M tetrodotoxin, which also blocks the depolarizing effect of veratridine in intact neurones. 12. The main conclusion is that synaptosomes may retain resting membrane potentials and the ability to increase Na permeability.

Alkenes↗
PubMed1978

Inhibition of synaptosomal uptake of norepinephrine and dopamine by conformationally restricted sympathomimetic amines.

The conformationally restricted cis and trans isomer of substituted cyclobutanes were examined for their ability to inhibit 3H-norepinephrine and 3H-dopamine accumulation by synaptosomes prepared from the cortex and corpus striatum, respectively. The drugs were more effective in preventing the accumulation of 3H-norepinephrine by cortical synaptosomes than 3H-dopamine by striatal synaptosomes. However, in the synaptosomes isolated from both regions, the trans isomers were more potent inhibitors of accumulation than the cis isomers. The greatest stereoselectivity was exhibited by the isomers of 2-amino-1-phenylcyclobutanol. The accumulation of 3H-norepinephrine by cortical synaptosomes and the accumulation of 3H-dopamine by striatal synaptosomes were inhibited 50% by concentrations of the trans isomer of 7.4 X 10(-6) M and 1.7 X 10(-4) M, respectively. The cis isomer was inactive. In separate experiments, the releasing capabilities of the restricted analogs were determined by superfusing cortical and striatal synaptosomes labelled in vitro with 3H-catecholamines. The trans and cis isomers elicited a trivial release of 3H-norepinephrine and 3H-dopamine from cortical and striatal synaptosomes, respectively. The results indicate that the decreased synaptosomal accumulation of 3H-catecholamines caused by the analogs was due mainly to inhibition of uptake. The influence of dihydral angle between phenyl--NH2 on the inhibition of uptake is discussed. It is concluded that the anti conformation of sympathomimetic amines is the preferred conformation at the noradrenergic amine pump.

Aminophenols↗
PubMed1977

Net uptake of gamma-aminobutyric acid by a high affinity synaptosomal transport system.

Reuptake of gamma-aminobutyric acid (GABA) by a high affinity transport system in nerve endings in the central nervous system is thought to terminate the action of this postulated neurotransmitter. This hypothesis has been challenged since the demonstration of exchange between synaptosomal and exogenous GABA (G. Levi and M. Raiteri, Nature 250: 735, 1974). In our studies, rat cortical synaptosomes were incubated (25 degrees C) in various media containing 10 muM 14C-GABA. After the synaptosomes were removed by centrifugation, 14C and total GABA (fluorometric assay) in the resulting supernatant were measured. Uptake of labeled GABA, detected by a decrease in medium radioactivity, is Na+- and K+-dependent. Net GABA uptake, however, does not parallel 14C-GABA translocation. Exchange accounts for 20 to 70% of radiolabeled GABA accumulation depending upon the experimental conditions. On the other hand, GABA-deficient synaptosomes (prepared by treatment with 56 mM KCl and 1 mM CaCl2) show equivalent net and radiolabeled GABA uptake in Ringer's solution containing 1 to 4 mM KCl and 60 to 150 mM NaCl (average 4.6 nmol of GABA accumulated per mg of synaptosomal protein). Net and 14C-GABA uptake by GABA-deficient synaptosomes are identical at various pH values (6.0-8.5), synaptosomal protein concentrations (0.4-3.5 mg/ml) and temperatures (5-37 degrees C). Although GABA homoexchange may contribute significantly to radiolabel accumulation by synaptosomes containing higher GABA levels (9.5-9.9 nmol/mg), homoexchange is limited in GABA-depleted synaptosomes. Our results are consistent with the proposal that presynaptic GABA capture by a high affinity system in vivo may terminate the action of this neuroactive amino acid.

Aminobutyrates↗
PubMed1977

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.

Animals↗
PubMed1976

Effects of neonatal hypothyroidism on cerebral and cerebellar synaptosome development.

The effect of hypothyroidism on cerebral and cerebellar synaptosome development has been studied. Neonatal hypothyroidism was induced following addition of 0.3% propylthiouracil to the diet of nursing mothers. Maturation profiles of total synaptosome fraction and specific activities of lactate dehydrogenase, Na+K ATPase, cytochrome c oxidase, and protein were obtained from days 6 to 32 on synaptosomes isolated from Ficoll-sucrose gradients. The greatest changes were found in the total activities of enzymes isolated from the cerebellum. Hypothyroidism induced a retardation of LDH and cytochrome c oxidase in cerebellar synaptosomes, but no change in corresponding specific activities. Maximum rates of 14C-leucine incorporation into cerebellar synaptosome protein was found at 16-20 days, after which a rapid decline occurred to adult levels at 32 days. In neonatal hypothyroidism, synthesis was significantly reduced at 8 and 14 days, but reached control levels or above at 21--32 days. In the cerebrum, maximum rates of 14C-leucine incorporation into synaptosome protein were identified at 8--12 days in normal with a rapid drop to adult levels at approximately 20 days. In neonatal hypothyroidism, peak activities were identified at 14 days and increased activities over control were noted at 14, 20 and 30 days. These observations demonstrate the sensitivity of the developing cerebellar synaptic apparatus to neonatal hypothyroidism, with a protraction in the peak levels of synaptosome protein synthesis in cerebrum and cerebellum.

Adenosine Triphosphatases↗
PubMed1976

Carrier-mediated sodium-dependent and calcium-dependent calcium efflux from pinched-off presynaptic nerve terminals (synaptosomes) in vitro.

The influence of external cations on 45Ca2+ efflux from Ca2+ loaded synaptosomes has been examined. The synaptosomes were pre-loaded with 45Ca2+ by incubating the suspensions in potassium-rich media for 2 min. The suspensions were then diluted into "efflux" media containing a "normal" (5mM) K+ concentration; the content of Na+ and Ca2+ was varied, as noted below. Efflux of 45Ca2+ was measured for a 2-min period (except for "zero-time" samples), and was terminated by filtering the suspensions on 0.3 mum cellulose acetate filters. 45Ca2+ retained on the filters was determined by liquid scintillation spectroscopy. The difference between the 45Ca2+ in the "zero-time" samples (="Ca2+ load") and in the samples incubated for 2 min was taken as the 45Ca2+ efflux. 45Ca2+ loss into Ca2+ -free efflux media containing ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) was markedly influenced by the Na+ concentration: nearly 80% of the 45Ca2+ was lost from the synaptosomes if the media contained 132 mM Na+, but only about 7% was lost in 2 min if 97% of the Na+ was replaced mol-for-mol by choline. In media containing 1.2 mM Ca2+ and 132 mM Na+, the 45Ca2+ uptake by synaptosomes previously loaded with 40Ca2+ was significantly less than 45Ca2+ loss from synaptosomes loaded with 45Ca2+. Thus there was a net efflux of Ca2+ from the Ca2+ -loaded synaptosomes; this efflux was, presumably, Na+ dependent. In media containing 1.2 mM 40Ca2+ and only 4 mM Na+, the 45Ca2+ efflux from 45Ca2+ -loaded synaptosomes was significantly greater if most of the external Na+ (128 mM) was replaced isomotically by Li+ rather than by choline, guanidine or glucose. This observation may be evidence for a Ca2+ -Ca2+ exchange which is promoted by Li+. Both the Na+ -dependent and the Ca2+ -dependent Ca2+ effluxes were inhibited by Mn2+. The data are consistent with a Ca2+ carrier mechanism which can extrude Ca2+ in exchange for Na+ or for Ca2+, the latter being activated by Li+. These properties bear a striking resemblance to those of a Ca2+ efflux mechanism which has been characterized in squid axons. This mechanism may there fore have evolved fairly early on in the history of the animal kingdom.

Animals↗
PubMed1979

Large-scale preparation of synaptosomes from bovine brain using a zonal rotor technique.

A zonal rotor technique for the preparation of synaptosomes in bulk from bovine brain frontal cortex based on an impirical transformation of a small-volume discontinuous sucrose density gradient arrangement is presented in detail. The procedure yields new information concerning synaptosomes prepared in sucrose gradients. Cerebroside analysis and electron microscopy show myelin contamination to be restricted to the leading, less dense edge of the synaptosomal profile, free mitochondria to the trailing, more dense edge. Exclusion of fringe areas yields a highly purified synaptosome preparation which entirely enters the next dense layer beyond the 0.8 : 1.2 M sucrose interface. This interface collects most of the oubain-sensitive (Na+, K+) adenosine triphosphatase activity. The purified synaptosomes display very high intrinsic sialidase activity and are rich in di-, tri-, and tetrasialogangliosides, the preferred substrates for the enzyme. Up to 90% of the cholinesterase activity in the zonal rotor synaptosome preparation is specific acetylcholinesterase.

Adenosine Triphosphatases↗
PubMed1979

Choline and ethanolamine glycerophospholipid synthesis in isolated synaptosomes of rat brain.

Substantial activities of cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found with lysed synaptosomes but not with intact synaptosomes isolated from adult rat brains. Synaptosomal and non-synaptosomal microsomal transferases were similar in kinetic properties. Substantial activities of synaptosomal transferases have not been described previously. Part of the glycerophospholipids in synaptosomal membranes may be synthesized in the nerve ending in addition to the glycerophospholipids supplied by axonal transport. The synthesis of the alkylacyl type of choline and ethanolamine glycerophospholipids was moderately inhibited by 1 mM ATP and 1 microM cyclic AMP. This synthesis was also inhibited by more than 50% by 1 mM norepinephrine and to a lesser extent by 5 mM hydroxytryptamine and 1 mM acetylcholine. Cyclic AMP may mediate the effects of biogenic amines. The relative synthesis of different glycerophospholipid classes and the relative proportion of alkylacyl type (plasmalogen precursors) and diacyl type of glycerophospholipids may be influenced by the levels of adenine nucleotides and/or biogenic amines. Elevated cyclic AMP levels will decrease the synthesis of plasmalogen precursors.

Adenine Nucleotides↗
PubMed1979

Presynaptic noradrenergic alpha-receptors and modulation of 3H-noradrenaline release from rat brain synaptosomes.

The depolarization (15 mM K+)-induced release of 3H-NA from superfused rat brain synaptosomes and the effects of alpha-noradrenergic drugs thereon were studied. Noradrenaline (NA; in the presence of the uptake inhibitor desipramine) reduced synaptosomal 3H-NA release. Reduction of the concentration of calcium ions in the medium during K+ stimulation greatly enhanced the sensitivity of 3H-NA release to alpha-receptor-mediated inhibition. Under these conditions NA dose-dependently inhibited 3H-NA release from synaptosomes obtained from cortex or hypothalamus, but did not affect 3H-NA release from striatal (i.e dopaminergic) synaptosomes. Adrenaline, clonidine and oxymetazoline potently inhibited 3H-NA release from cortex synaptosomes at concentrations in the nanomolar range. Phentolamine by itself did not affect synaptosomal 3H-NA release, but antagonized the inhibitory effects of both noradrenaline and adrenaline. The data obtained further substantiate the hypothesis that the alpha-receptors mediating a local negative feedback control of NA release are localized on the varicosities of central noradrenergic neurons, Furthermore, noradrenergic nerve terminals in the hypothalamus appear to be less senstive to alpha-receptor-mediated presynaptic inhibition than those in the cortex.

Adrenergic alpha-Agonists↗
PubMed1978

Calcium-activated hydrolysis of phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-bisphosphate in guinea-pig synaptosomes.

1. Addition of the bivalent ionophore A23187 to synaptosomes isolated from guinea-pig brain cortex and labelled with [(32)P]phosphate in vitro or in vivo caused a marked loss of radioactivity from phosphatidyl-myo-inositol 4-phosphate (diphosphoinositide) and phosphatidyl-myo-inositol 4,5-bisphosphate (triphosphoinositide) and stimulated labelling of phosphatidate. No change occurred in the labelling of other phospholipids. 2. In conditions that minimized changes in internal Mg(2+) concentrations, the effect of ionophore A23187 on labelling of synaptosomal di- and tri-phosphoinositide was dependent on Ca(2+) and was apparent at Ca(2+) concentrations in the medium as low as 10(-5)m. 3. An increase in internal Mg(2+) concentration stimulated incorporation of [(32)P]phosphate into di- and tri-phosphoinositide, whereas lowering internal Mg(2+) decreased labelling. 4. Increased labelling of phosphatidate was independent of medium Mg(2+) concentration and apparently only partly dependent on medium Ca(2+) concentration. 5. The loss of label from di- and tri-phosphoinositide caused by ionophore A23187 was accompanied by losses in the amounts of both lipids. 6. Addition of excess of EGTA to synaptosomes treated with ionophore A23187 in the presence of Ca(2+) caused a rapid resynthesis of di- and tri-phosphoinositide and a further stimulation of phosphatidate labelling. 7. Addition of ionophore A23187 to synaptosomes labelled in vivo with [(3)H]inositol caused a significant loss of label from di- and tri-phosphoinositide, but not from phosphatidylinositol. There was a considerable rise in labelling of inositol diphosphate, a small increase in that of inositol phosphate, but no significant production of inositol triphosphate. 8. (32)P-labelled di- and tri-phosphoinositides appeared to be located in the synaptosomal plasma membrane. 9. The results indicate that increased Ca(2+) influx into synaptosomes markedly activates triphosphoinositide phosphatase and diphosphoinositide phosphodiesterase, but has little or no effect on phosphatidylinositol phosphodiesterase.

Animals↗