Conformationally rigid amphetamine analogs as inhibitors of monoamine uptake by brain synaptosomes.

Four 3-phenyl-2-amino-trans-decalin isomers were synthesized in order to obtain derivatives of phenylethylamine with a rigid conformation between the phenyl ring and the amino function. The stereoisomers were tested as inhibitors of catecholamine uptake by rat brain synaptosomes, and their potency was compared with that of amphetamine. The most potent inhibitor of catecholamine uptake was the diaxial 2(a)-amino-3(a)-phenyl-trans-decalin, which was one-fourth to one-third as potent as (+/)-amphetamine. As a dopamine uptake inhibitor in the stiatum, this compound was competitive. The results differ from those obtained earlier with similar analogs with a norepinephrine moiety incorporated into the decalin structure, since a gauche derivative [2(a)-amino-3(e)-3,4-dihydroxyphenyl-3-trans-decalol] was then the most potent and over 20 times as potent as the diaxial anti derivative. It remains to be seen whether this indicates that the mode of binding of phenylethylamines is different from that of catecholamines.     

The inhibition of monoamine uptake into rat brain synaptosomes by selected bicyclo-octanes and an analogous bicyclo-octene

The ability of a cis bicyclo-octane (LR5182), its optical isomere (LR5866) and (LR5953), a trans bicyclo-octane LR5309 and a bicyclo-octane LR5659 to inhibit the uptake of noradrenaline, dopamine and 5-hydroxy tryptamine was studied in an in vitro synaptosomal preparation from rat brain areas. The activity of the novel compounds in the system was compared to that of known uptake inhibitors and anti-depressants. Only LR5309 showed significant activity against 5HT having an IC₅₀ of 0.59 μM compared to that of imipramine of 0.44 μM. All the novel compounds were active at inhibiting noradrenaline and dopamine uptake. The most interesting compounds were LR5182 and its two optical isomers since these compounds had a similar IC₅₀ to that of desipramine (DMI) in blocking noradrenaline uptake (DMI IC₅₀ = 0.27 mM, 5182 IC₅₀ = 0.11 mM) and were the most active compounds so far reported in blocking dopamine uptake (5182 IC₅₀ = 0.069 μM, nomifensin IC₅₀ = 0.23 μM). Since 5182 and its isomers are as active at blocking noradrenaline uptake as known antidepressants, and in addition are very potent at blocking dopamine uptake, these compounds may provide a useful treatment for certain categories of depressed patients.     

Effects of mazindol on rat brain synaptosomal monoamine uptake

The effects of the anorectic drug mazindol on the uptake of [³H]NA and (³H]5HT by rat hypothalamic synaptosomes and the uptake of [³H]DA by rat striatal synaptosomes were investigated. In in vitro studies drugs were added to the incubation medium. In ex vivo experiments drugs were injected i.p. at various times prior to death and synaptosomal [³H] monoamine uptake subsequently determined. Two other anorectics (d-amphetamine and dl-fenfluramine) and two inhibitors of monoamine uptake (chlorimipramine and desipramine) were included for comparative purposes. Mazindol was a potent inhibitor of [³H]NA and [³H]DA uptake in vitro being approx. 0.5 times as potent as desipramine and d-amphetamine respectively. The abilities of mazindol, fenfluramine and desipramine to block the in vitro uptake of [³H]5HT were comparable and all three drugs were appreciably less potent than chlorimipramine. Following 1hr pretreatment, d-amphetamine was the most potent of the five drugs at inhibiting synaptosomal [³H]NA and [³H]DA uptake. Mazindol was approx. 2.5 times more potent than desipramine at blocking [³H]NA uptake. In contrast to the other drugs, pretreatment with large doses of mazindol had essentially no effect on hypothalamic synaptosomal [³H]5HT uptake. Results of ex vivo studies thus confirm in vivo findings that mazindol is a selective inhibitor of rat brain catecholamine uptake.     

Neurocatin-induced inhibition of monoamine oxidase A in rat brain synaptosomes.

Neurocatin is a small (about 2000 Da) neuroregulator isolated from mammalian brain. Earlier it was shown that addition of nanomolar concentrations of neurocatin to synaptosomes isolated from rat brain increased levels of serotonin and decreased catabolism of serotonin to 5-hydroxyindoleacetic acid (Fernandez-Novoa L and Pastuszko A. Neurosci Lett 122: 83-86, 1991). In the present study, we report that neurocatin addition resulted in a striking inhibition of monoamine oxidase A activity. This inhibition became statistically significant at a neurocatin concentration of approximately 5 nM and was significant at all higher neurocatin concentrations. Neurocatin at approximately 50 nM inhibited monoamine oxidase A activity by about 90%. The inhibitory effect of neurocatin on monoamine oxidase required its incubation with intact synaptosomes since addition after breaking the synaptosomes by hypotonic buffer or lysis by Triton X-100 almost completely blocked the inhibitory effect. Measurements of the kinetic parameters of the enzyme in lysates prepared from synaptosomes incubated with neurocatin showed a decrease in Vmax with no change in Km for the substrate (serotonin) compared to controls. Incubation of the synaptosomes with approximately 25 nM neurocatin resulted in an 80% decrease in the Vmax of monoamine oxidase A. Evidence that neurocatin is a powerful endogenous modulator of monoamine oxidase activity is particularly intriguing. This enzyme plays a major role in catabolism of the biogenic amines and is believed to contribute to several important neurological disorders.     

Effects of lithium on adrenergic amine uptake in rat brain synaptosomes

The effects of lithium on the uptake and retention of (?)-erythro-metaraminol (MA) and (?)-m-octopamine (OA) by synaptosomes from rat whole brain were examined after (a) addition of lithium chloride (1 mmol/l.) to synaptosomal fractions in vitro and (b) administration of lithium chloride (1 mmol/kg) i.p. to rats twice daily for 10 days prior to isolation of synaptosomes. Under control conditions, apparent K_m concentrations (5 min incubation) for MA and OA were 33 and 3 μM respectively. As incubation times were increased to 15 min, retention of MA increased and OA decreased. Retention of both amines was reduced by low temperature. Short term application of lithium to synaptosomes in vitro did not alter the uptake and retention of either amine during 5 or 10 min incubation periods. Chronic pretreatment of rats with lithium resulted in greater uptake and retention of low concentrations of MA when compared with synaptosomes from control (saline-treated) animals. Chronic lithium pretreatment did not alter retention of OA. The data support the hypothesis that lithium acts to increase synaptosomal membrane uptake of aromatic amines and decrease binding of accumulated amine.     

Halothane inhibits 5-hydroxytryptamine uptake by synaptosomes from rat brain

The activity of 5-hydroxytryptamine (serotonin; 5-HT) in the central nervous system modulates sleep, the perception of pain and other functions of the body which might possibly relate to mechanisms of general anesthetic action. While administration of anesthetics has inconsistent effects on the content of 5-HT in brain, in vivo, accumulated data suggest that anesthetic drugs alter 5-HT homeostasis in the central nervous system. In an effort to identify one possible site of anesthetic action, the effect of halothane on the uptake of 5-HT was studied in synaptosomes isolated from the brain of rat. Established techniques were used to prepare the synaptosomal fractions and measure high affinity transport of radiolabelled 5-HT. Halothane inhibited synaptosomal accumulation of 5HT in a concentration-dependent manner, but had little effect on the passive or spontaneous release of the accumulated 5-HT. Rates of uptake of 5HT were inhibited by 43% in the presence of 1 mM halothane and by 75% of control in the presence of 5 mM halothane; the apparent I50 for halothane was 1.0 +/- 0.1 mM and Lineweaver-Burk analysis indicated the inhibition to be competitive at concentrations around the I50.     

The effects of some slow channel blocking drugs on high affinity serotonin uptake by rat brain synaptosomes

The effects of some slow channel blocking drugs were investigated on high affinity serotonin uptake into crude rat brain synaptosomes. Serotonin uptake was sodium-dependent and competitively inhibited by imipramine (IC50 0.6 microM, Ki 0.26 microM). Bepridil, verapamil and diltiazem produced an apparent competitive inhibition of serotonin uptake with respective IC50 of 4.8, 5.2 and 308 microM. Nitrendipine and the sodium channel blocker, lidocaine, were without effect, even at 100 microM. The mechanism of the inhibitory effect is unknown but may involve an allosteric interaction with the sodium-dependent transporter.     

Effects of lipid peroxidation on neurotransmitters uptake by rat synaptosomes

This paper described the effect of in vitro peroxidation achieved by 60 s or 5 min exposure to 60 microM Fe2+ with 200 microM ascorbic acid, on selected properties of rat brain synaptosomes reflecting some steps of chemical neurotransmission. The studies have revealed dramatic differences between dopamine, GABA and choline high affinity uptake systems in response to peroxidation. The uptake of calcium by synaptosomes submitted to free radical oxidation, mostly its K+-depolarization-dependent portion, was significantly suppressed. In contrast, peroxidation appeared not to influence the transmembrane synaptosomal potential. It is concluded, that peroxidation of synaptic endings modifies the lipid content of synaptoplasmatic membranes and consequently leads to severe disturbances in the function of neurotransmitter uptake systems and depolarization-dependent calcium channels.     

Inhibition of sodium-dependent L-leucine uptake in rat brain synaptosomes

Synaptosomes isolated from adult rat cerebral cortices were used for studying the uptake of L-leucine by the Na(+)-dependent route. Three non-metabolizable amino acid analogues, which had been used previously to discriminate the Na(+)-dependent A-type uptake system of animal cells, were employed in this study. It was found that Na(+)-dependent uptake of leucine was insensitive to inhibition by 2-aminoisobutyric acid (AIB) and N-methylaminoisobutyric acid (MeAIB) whereas N-methylalanine (NMA) was markedly inhibitory. Inhibition by NMA was stereospecific--only the L-isomer had a pronounced effect. Na(+)-dependent uptake of leucine as well as its inhibition by L-NMA were rather insensitive to changes in pH from 6 to 9. Kinetic analysis of inhibition by L-NMA of Na(+)-dependent uptake revealed a non-competitive type of inhibition with a Ki value of approximately 0.5 mM.     

Modulation of gamma-aminobutyric acid uptake of rat brain synaptosomes by thyroid hormones

The effects of thyroid hormones on gamma-aminobutyric acid (GABA) uptake were compared in homogenates and purified synaptosomes from rat cerebral cortex. L-triiodothyronine (T3) and L-thyroxine (T4), but not reverse T3(rT3), blocked GABA uptake in homogenates when hormone and labeled GABA were added to the system simultaneously. Similar effects were obtained when homogenates were preincubated with hormone and washed to remove free hormone before addition of GABA. A competitive inhibitor of neuronal GABA uptake, diaminobutyric acid (DABA), blocked GABA uptake when added with GABA, but not after preincubation and washing. In purified synaptosomes, T3, T4, and rT3 blocked GABA uptake when added simultaneously with GABA and also after preincubation and washing before addition of GABA, thus indicating that binding of hormones had occurred. Other experiments suggested that hormone binding was not at the GABA recognition site and that the effect of T4 was not dependent on conversion to T3.     

Inhibition by neuroleptics of uptake of 3H-GABA into rat brain synaptosomes

Neuroleptics from various chemical groups and some amino acids were examined for GABA uptake-inhibiting properties in rat brain synaptosomes. The phenothiazine and thioxanthene neuroleptics inhibited GABA uptake by 50% in the concentration range of 10-30 micron. No difference in potency was found between the cis and the trans isomers of the thioxanthenes. Clozapine and particularly sulpiride were weak inhibitors of the GABA uptake process, whereas some of the butyrophenones examined exhibited rather potent effect. Secific GABA uptake inhibitors (nipecotic acid and guvacine) were as active as the most potent butyrophenones. As no significant correlation was obtained between GABA uptake-inhibiting effect of the neuroleptics and their clinical or pharmacological effects it is concluded that the influence on GABA uptake is not an important aspect of the neuroleptic action.     

Effects of antihistaminic drugs on feline brain monoamine metabolism.

Diphenhydramine and phenindamine were administered in various doses to cats and the time course of changes in monoamine metabolite levels in cerebrospinal fluid was determined. Diphenhydramine at a sedative dose did not alter 5-hydroxytryptamine or dopamine metabolism. Excitant doses of diphenhydramine elevated 5-hydroxyindolacetic acid levels, while homovanillic acid levels remained unchanged. At a convulsant dose, diphenhydramine lowered rectal temperature while elevating both 5-hydroxyindolacetic acid and homovanillic acid levels. Phenindamine, which reportedly produces only central nervous system (CNS) excitation and convulsions, caused excitation, tremor and stereotypy, while elevating 5-hydroxyindolacetic acid and homovanillic acid levels in cerebrospinal fluid. These data suggest that antihistaminic-induced sedation is not due to an alteration in brain 5-hydroxytryptamine or dopamine metabolism and that only 5-hydroxytryptamine metabolism is increased during CNS excitation. Stereotypic behavior after phenindamine may occur through a dopaminergic system as reflected by elevated levels of homovanillic acid in cerebrospinal fluid.     

Ginseng saponins: influence on neurotransmitter uptake in rat brain synaptosomes.

The total ginsenoside ( N-butanol extract) fraction inhibited the uptake of radioactive gamma-aminobutyrate (GABA), glutamate (Glu), dopamine (DA), noradrenaline (NA), serotonin (5-HT), but not the uptake of 2-deoxy- D-glucose (2-DG) and leucine (Leu) into rat brain Synaptosomes. Among the ginsenoside fractions investigated, Fraction III which contains mainly R (d) was most effective in reducing the uptake of neurotransmitters. The inhibition was in the order of GABA = NA > DA > Glu > 5-HT, and this effect was concentration-dependent. This inhibitory effect appears to be specific as this fraction, at several concentrations, failed to influence the uptake of 2-DG and Leu significantly. Similar studies indicated that ginsenoside R (c) could affect neurotransmitter uptake specifically as well. Since the GABA uptake was most sensitive to the inhibitory action of these ginsenosides, it is postulated that ginseng may exert its action(s) in the central nervous system by affecting the removal of neurotransmitter substances in synaptic regions and that the GABA-nergic neurons may be one of the major sites of action.     

Inhibition of monoamine uptake in synaptosomes by tetrahydroharmane and tetrahydroisoquinoline compounds

Summary The effect of some tetrahydroharmane derivatives on the synaptosomal uptake of monoamines was studied. 1,2,3,4-Tetrahydroharmane (TH), which can be viewed as an analog of tryptamine, inhibited 5-hydroxytryptamine (5-HT), noradrenaline (NA) and dopamine (DA) uptake, but TH was about 10 times less potent than tryptamine. 6-Hydroxy-1,2,3,4-tetrahydroharmane (6-HTH), a 5-HT analog, was slightly more potent an inhibitor of 5-HT uptake than TH, but as catecholamine uptake inhibitors these compounds were equipotent. Harmane-1,2,3,4-tetrahydro-3-carboxylic acid had no effect. Salsolinol and salsolidine, tetrahydroisoquinoline derivatives, were less potent than the harmane derivatives. It is to be concluded that also tetrahydroharmane derivatives have an affinity to the amine pump similarly to that of the tetrahydroisoquinolines, which are the corresponding analogs of catecholamines. These kinds of compounds are important because of the possibility that they may be formed in physiological conditions as condensation products of biogenic amines and aldehydes, e.g. acetaldehyde, which is an intermediate of ethanol metabolism.     

Effects of calcium antagonists on KCl-evoked calcium uptake by rat cortical synaptosomes

A series of calcium antagonists were used to study their blocking effect on high potassium-induced calcium uptake into rat cortical synaptosomes; these antagonists were classified into five groups: dihydropyridine group (i.e. nifedipine and nitrendipine), benzothiazepine group (i.e. diltiazem), phenylalkylamine group (i.e. verapamil and D600), phenothiazine group (i.e. trifluoperazine) and diphenylpiperazine group (i.e. flunarizine and cinnarizine). Voltage-dependent 45Ca2+-uptake into this fraction was measured after 20 sec KCl-induced depolarization. The ID30 values of the above-mentioned antagonists affecting 45Ca2+-uptake were calculated to be nitrendipine (80 microM), nifedipine (100 microM), verapamil (50 microM), D600 (15 microM), diltiazem (70 microM), trifluoperazine (7 microM), cinnarizine (1.2 microM) and flunarizine (0.7 microM). Our results reveal that in rat brain synaptosomal fractions, calcium influx via the voltage-gated calcium channel appears to be more sensitive to diphenylpiperazine and phenothiazine groups; whereas, phenylalkylamine, benzothiazepine and dihydropyridine groups were relatively insensitive. This contrasts with the well known data obtained from vascular smooth muscle, in which the dihydropyridine group is the most sensitive of all the groups studied. Our results suggest that calcium channels in neuronal tissue are most likely different from those in non-neuronal tissue.