Effects of pH changes and charge characteristics in the uptake of norepinephrine by synaptosomes of rat brain

The pH dependence of the initial uptake of norepinephrine by rat whole brain synaptosomes was studied short incubation times at 37°C in order to examine the possible involvement of the phenolic OH group. The pH vs. uptake profile exhibits a maximum near pH 8.2 in H₂O medium. When the medium was changed to ²H₂O, the profile showed a shift of maximum corresponding to the pK_a change of the phenolic OH group. The pH vs. uptake profile of tyramine was quite different from that of norepinephrine. These pH effects on uptake were explained as manifestations of the involvement of the phenolic OH group in the process.The amine and phenolic hydroxyl groups in norepinephrine were studied separately by employing two series of compounds structurally related to catecholamines, amphetamine-like and catechol0like, for their inhibitory effects on the uptake. The inhibitions were affected by changes in pH with changes in opposite directions found for the two series indicating the need for a positive charge in the side chain and suggesting an effect of the negative charge on the ring. These charge characteristics agreed with the pH profile observed in uptake. Consequently, the two groups with opposite charge characteristics in norepinephrine both appear to function in the uptake process.     

Effects of pH changes and charge characteristics in the uptake of norepinephrine by synaptosomes of rat brain.

The pH dependence of the initial uptake of norepinephrine by rat whole brain synaptosomes was studied using short incubation times at 37 degrees C in order to examine the possible involvement of the phenolic OH group. The pH vs. uptake profile exhibits a maximum near pH 8.2 in H2O medium. When the medium was changed to 2H2O, the profile showed a shift of maximum corresponding to the pKa change of the phenolic OH group. The pH vs. uptake profile of tyramine was quite different from that of norepinephrine. These pH effects on uptake were explained as manifestations of the involvement of the phenolic OH group in the process. The amine and phenolic hydroxyl groups in norepinephrine were studied separately by employing two series of compounds structurally related to catecholamines, amphetamine-like and catechol-like, for their inhibitory effects on the uptake. The inhibitions were affected by changes in pH with changes in opposite directions found for the two series indicating the need for a positive charge in the side chain and suggesting an effect of the negative charge on the ring. These charge characteristics agreed with the pH profile observed in uptake. Consequently, the two groups with opposite charge characteristics in norepinephrine both appear to function in the uptake process.     

Effects of pressure on uptake and release of calcium by brain synaptosomes

Uptake of radioactive calcium from guinea pig brain fractions enriched in synaptosomes could be significantly and reproducibly decreased by exposure to high pressure. Calcium efflux from preloaded synaptosomes was unaffected by pressure exposure. It was hypothesized that the development of pressure-induced encephalopathy may be related to an effect of pressure on the central nervous system calcium transport system.     

d-glucosamine uptake by rat brain synaptosomes

Uptake of d-glucosamine by rat brain synaptosomes occurs via a saturable transport process (K_m 2.1 mM, V 3.0 nmol/mg per min) which was clearly distinguishable from simple diffusion. This transport process is highly sensitive to cytochalasin (K_i = 7 · 10^?5 mM. d-Glucose competitively inhibits d-glucosamine uptake with a K_i value of 8 · 10^?1 mM.     

Clostridium neurotoxins influence serotonin uptake and release differently in rat brain synaptosomes

Clostridium neurotoxins produce inhibition of both basal and K(+)-evoked serotonin release in rat brain synaptosomes. To produce these effects, tetanus toxin (TeTx), as well as botulinum neurotoxin type A (BoNT/A), added to brain synaptosomes, must be incubated at 37 degrees C over a long interval (hours). This serotonin exocytosis inhibition was abolished with previous treatment with specific Zn2(+)-metalloprotease inhibitors. Nevertheless, a short incubation time produces different behavior of the indicated neurotoxins: TeTx significantly blocks the sodium-dependent, high-affinity serotonin uptake, whereas a small increase of this uptake was found with BoNT/A. Both Zn2(+)-metalloprotease active fragments, light chains of TeTx and BoNT/A, are unable to reproduce the block of the serotonin uptake, whereas the C-terminal portion of the TeTx heavy chain (Hc-TeTx), which binds specifically to the target tissue, inhibited the serotonin uptake in a dose-dependent manner. The IC50 of Hc-TeTx ranges from 0.62 to 2.08 nM. Binding of [3H]imipramine and [3H]serotonin did not change after toxin treatments, which indicates that these clostridium neurotoxins do not act on the serotonin high-affinity site at the serotonin transporter or at other serotonin high-affinity sites. These results could indicate that TeTx and Hc-TeTx bind to different targets than BoNT/A in the plasma membrane.     

Effect of lithium on dopamine uptake by brain synaptosomes

Lithium chloride exerts two opposite effects on dopamine uptake by synaptosomes isolated from rat caudate nucleus. Added in vitro, it inhibits dopamine uptake; whereas administered chronically in vivo, it enhances dopamine uptake in vitro. Thus, in vitro, 1, 2.5, 5 and 10 meqiv.l-1 of lithium chloride decrease [3H]dopamine uptake by 13, 17, 25 and 31%, respectively. Synaptosomes isolated from rats treated with lithium chloride for 20 days, show a 23% increase in [3H]dopamine uptake with respect to synaptosomes isolated from control rats. It is suggested that chronic lithium treatment stimulates a compensatory mechanism which overcomes its direct inhibitory effect on [3H]dopamine uptake.     

Normal serotonin uptake by blood platelets and brain synaptosomes but selective impairment of platelet serotonin storage in mice with Chediack-Higashi syndrome

The Chediack-Higashi syndrome (CHS) is an autosomal recessive disorder reported in man and in several animal species including the "beige mice" (bg/bg). Among several manifestations of this genetic trait, deficiency of secretable substances - including serotonin - normally stored in platelet dense granules is a characteristic feature. The animal model of Chediak-Higashi syndrome used in the present study provides a unique opportunity to compare the kinetics of serotonin (5-hydroxytryptamine, 5-HT) uptake in platelets and brain synaptosomes in conditions of selective reduction of 5HT concentration in the platelets. The kinetics of 5HT uptake, as measured in the present study, was normal in synaptosomes and platelets from the same animals. The lower intraplatelet 5HT levels in bg/bg animals as compared to normal synaptosomes levels in the presence of normal uptake offer an indirect proof that the 5HT defect described in the CHS is due to an impaired 5HT storage mechanism. This is supported by the observation that spontaneous release of 5HT was markedly increased in platelets from CH5 mice but was normal in synaptosomes from the same animals. Thus platelets are a reliable model to study 5HT uptake, but not 5HT storage and release in brain synaptosomes.     

The effect of phospholipases on the active uptake of norepinephrine by synaptosomes isolated from the cerebral cortex of guinea pig

—Phospholipase A (EC 3.1.1.4) and phospholipase C (EC 3.1.4.3) were used for studying the role of phospholipid of synaptosomal membrane on norepinephrine uptake activity. Synaptosomes were isolated from cerebral cortex of guinea pigs and treated with phospholipase A or phospholipase C before the uptake experiments. Treatment of synaptosomes with phospholipase A has resulted in severe inhibition of norepinephrine-uptake. Under similar conditions, the activity of synaptosomal (Na + K)-ATPase (EC 3.6.1.4) was also inhibited by phospholipase A treatment whereas the activity of synaptosomal acetylcholinesterase (EC 3.1.1.8) was not affected. On the other hand, the norepinephrine-uptake was not influenced by phospholipase C treatment. The inhibition of norepinephrine-uptake after phospholipase A treatment may be due to the liberation of lyso-components of phospholipids since a low concentration of lysolecithin as well as other detergents (deoxycholate and sodium dodecyl sulphate) also inhibit the uptake activity. However, electron microscopic examination indicated that the synaptosomal particles still maintain their morphological features after phospholipase A treatment. It is possible that the active uptake of norepinephrine depends upon the fine arrangement of phospholipids present at the active sites of the synaptosomal membrane.     

Kainate-induced uptake of calcium by synaptosomes from rat brain

Kainic acid induces a rapid increase in 45Ca2+ uptake by crude synaptosomal fractions isolated from rat brain. This enhanced Ca2+ permeability occurs with a half-time of approx. 1 s, similar to the fast phase of depolarization-induced calcium uptake. The depolarization-induced uptake of calcium is inhibited 85% by 3 mM CoCl2, 80% by 100 microM quinacrine and 50% by 15 microM trifluoperazine while these agents had little effect on the kainate-induced uptake. It is proposed that kainate induces receptor-mediated opening of a class of calcium channels with properties different from those of the voltage-dependent channels.     

The subcellular distribution of endogenous and exogenous serotonin in brain tissue: comparison of synaptosomes storing serotonin, norepinephrine, and gamma-aminobutyric acid

Abstract— We have studied the subcellular distribution of exogenous and endogenous serotinin in slices from the hypothalamus and midbrain of several species. In a procedure which appears to label the endogenous pools, tissue slices were incubated with low concentrations of [³H]5-HT (5 × 10^-8 M), for 45 min, when there was apparent equilibrium between [³H]5-HT of tissue and medium. After the tissue slices were homogenized in 0-32 M-sucrose and subjected to differential centrifugation, the distribution of exogenous and endogenous 5-HT in pellets and supernatant fluid was similar. In some experiments, the crude mitochondrial pellets were resuspended in 0-32 M-sucrose, layered on linear, continuous density gradients of sucrose (1 -5-0-32 M), and centrifuged for short times (incomplete equilibrium centrifugation). The subcellular distribution of particulate tritium, total tritium, and particulate endogenous 5-HT was the same in portions of the gradients containing synaptosomes. The peak distribution of [³H]5-HT in sucrose gradients was separable from the peak for [¹⁴C]GABA by four to five fractions; potassium (a marker for cytoplasm occluded within synaptosomes) occurred in the regions of the gradients containing most of the labelled compounds. The distribution of monoamine oxidase activity (a mitochondrial marker) overlapped the distribution of [³H]5-HT after a 15 min centrifugation but appeared in denser regions of the gradient after centrifuging for 2 h. Particles containing [3H]5-HT and [I4C]NE were slightly but consistently separable in synaptosomal fractions isolated from the hypothalamus or midbrain of rat, guinea pig and hamster.     

Inhibition of serotonin uptake in synaptosomes and glia cells by some pharmacological substances

The uptake of serotonin -14 C by glial cells and synaptosomes of the rabbit brain cortex was studied. The Km value of the uptake of serotonin -14 C proved to be equal (0.83 + 0.02 microM) both for synaptosomes and glial cells. Synaptosomes of the rabbit brain cortex take up serotonin -14 C twice as fast as glial cells (uptake rates were compared from protein). Among psychotropic drugs studied the tricyclic antidepressant imipramine and psychostimulant cocaine turned out the most active inhibitors of both synaptosomal and glial uptake of serotonin -14 C. The drugs in 50 microM concentration inhibit the uptake of serotonin -14 C in synaptosomes and glial cells by 90 and 75-80%, respectively.     

Effects of pinealectomy and melatonin treatments on serotonin uptake and release from synaptosomes of rat hypothalamic regions

This study examined the effects induced by long-term pinealectomy, daily melatonin treatment to pinealectomized and intact rats, and a single melatonin injection on [¹⁴C]-serotonin (5-HT) uptake and release from synaptosomes obtained of hypothalamic regions. Pinealectomy inhibited the accumulation of labeled 5-HT by synaptosomes of the preoptic area-anterior hypothalamus (POA-AH), but it failed to alter the [K⁺]-evoked 5-HT release. Melatonin treatment for 10 consecutive days to pinealectomized rats restored 5-HT uptake in POA-AH, and also increased 5-HT release in medial and posterior hypothalamus. These results suggest that pineal melatonin plays a stimulatory role on the serotoninergic terminals of the hypothalamus. Moreover, when daily melatonin treatment was administered to intact rats a significant increase in 5-HT uptake activity by synaptosomes of all the hypothalamic regions was observed, but 5-HT release was unaffected. In contrast, a single melatonin injection induced a significant decrease in 5-HT release from synaptosomes of the POA-AH was observed. The results suggest the existence of a differential sensitivity in the mechanisms mediating melatonin actions on 5-HT uptake/release, which depends on the presence of the pineal gland in the animals and on the frequency of the treatments with the pineal hormone.     

Developmental and other characteristics of lysine uptake by rat brain synaptosomes

Synaptosomes isolated from adult or newborn rat cerebrum take up l-lysine by two saturable systems, one with a high affinity low capacity and the other with a low affinity high capacity. Initial rate of uptake for low lysine concentrations is more rapid in newborn, but for high concentrations the rate is greater in adult tissue. Analysis of kinetic data indicates that synaptosomes of the newborn have a higher $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ than those of the adult for high affinity system but adult synaptosomes have a higher $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ than newborn for low affinity system. At a physiological lysine concentration of 0.5 mM, the calculated contributions of two systems indicate that the adult uptake occurs for about 71% by low affinity system but the newborn utilizes both systems to the same extent. The uptake is sodium independent but pH dependent. Lysine uptake is inhibited by other dibasic amino acids, arginine and ornithine but not cystine. Kinetic analysis indicates that arginine specifically inhibits the high affinity, low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ system for lysine uptake.