Effects of reserpine on dopamine metabolite in the nucleus accumbens and locomotor activity in freely moving rats

The effect of reserpine (2 mg/kg i.p.) on both locomotor activity and the turnover of dopamine metabolite in the rat nucleus accumbens was estimated by using an activity monitor (Animex) and by in vivo brain microdialysis. Three to five hours after reserpine administration locomotor activity was reduced and there was a concomitant increase in the level of the dopamine metabolite, homovamillic acid. These findings suggest that depletion of dopamine from the nucleus accumbens may result in decreased locomotor activity. The data support the notion that dopamine in this tissue contributes to the control of locomotion.     

Increased acoplasmic transport of H3-dopamine in nigro-neostriatal neurons after reserpine

Recent evidence suggests that dopamine can undergo axoplasmic transport in nigro-neostriatal neurons by binding to amine storage granules. In the present experiments it was demonstrated that reserpine pretreatment (10 mg/kg) 24 hours before stereotaxic injections of ³H-DOPA or ³H-dopamine into the substantia nigra increases the amount of ³H-dopamine transported to the neostriatum by about 300 percent. The activity recovered from the substantia nigra was significantly reduced by reserpine pretreatment however. Stereotaxic injection of ¹⁴C-leucine into the substantia nigra indicated that neither fast nor slow axoplasmic transport of protein was influenced by reserpine pretreatment in these same neurons. The increased transport of dopamine appears therefore to be due to a relatively selective action of reserpine. The results suggest that reserpine either (i) increases the binding of dopamine to newly synthesized amine storage granules, (ii) increases the number of newly synthesized amine storage granules, or (iii) accelerates the rate of transport of amine storage granules. In addition, the results support the view that reserpine can increase the membrane permeability of adrenergic neurons to the outward movement of catecholamines.     

Release of dopamine, noradrenaline and dopamine B-hydroxylase from mouse neuroblastoma

The murine C1300 neuroblastoma tumor was found to secrete dopamine, noradrenaline and dopamine B-hydroxylase into the circulation of tumor-bearing A/J mice. The plasma levels of dopamine, noradrenaline and dopamine B-hydroxylase increased with the size of the tumor, and the increase in noradrenaline paralleled the increase in dopamine B-hydroxylase (r = 0.86). The vesicular storage of dopamine and noradrenaline in the tumor was evidenced by a decrease of the tissue content of dopamine and noradrenaline 24 hours after the administration of reserpine (5 micrograms/g) respectively to 17.6% and 7.8% of control values. A similar observation could be made for the levels of dopamine and noradrenaline in the plasma of reserpinized C1300 mice. The total activity of dopamine B-hydroxylase in the tumor and in plasma was unaffected by the reserpine treatment. Chronic administration of 6-hydroxydopamine (100 micrograms/g for 8 days) had no effect on the tissue contents of dopamine, noradrenaline or dopamine B-hydroxylase. The release of catecholamines and dopamine B-hydroxylase from the C1300 neuroblastoma was studied in vitro on superfused tumor slices. Stimulation of these slices with 56 mM KC1 or with 5.10(-5) M tyramine failed to induce the release of endogenous dopamine, noradrenaline or dopamine B-hydroxylase above the basal outflow levels. These results are suggestive for a non-exocytotic release of catecholamines and dopamine B-hydroxylase from the neuroblastoma tumor.     

Gastric mucosal lesions induced by complete dopamine system failure in rats. The effects of dopamine agents, ranitidine, atropine, omeprazole and pentadecapeptide BPC 157.

Up to now, for gastric lesions potentiation or induction, as well as determination of endogenous dopamine significance, dopamine antagonist or dopamine vesicle depletor were given separately. Therefore, without combination studies, the evidence for dopamine significance remains split on either blockade of dopamine post-synaptic receptor or inhibition of dopamine storage, essentially contrasting with endogenous circumstances, where both functions could be simultaneously disturbed. For this purpose, a co-administration of reserpine and haloperidol, a dopamine granule depletor combined with a dopamine antagonist with pronounced ulcerogenic effect, was tested, and the rats were sacrificed 24 h after injurious agent(s) administration. Haloperidol (5 mg x kg(-1) b.w. i.p.), given alone, produced the lesions in all rats. Reserpine (5 mg x kg(-1) b.w. i.p.), given separately, also produced lesions. When these agents were given together, the lesions were apparently larger than in the groups injured with separate administration of either haloperidol or reserpine alone. Along with our previous results, when beneficial agents were co-administered, all dopaminomimetics (bromocriptine 10 mg, apomophine 1 mg, amphetamine 20 mg x kg(-1) i.p.) apparently attenuated the otherwise consistent haloperidol-gastric lesions. Likewise, an apparent inhibition of the reserpine-lesions was noted as well. However, if they were given in rats injured with combination of haloperidol and reserpine, their otherwise prominent beneficial effects were absent. Ranitidine (10 mg), omeprazole (10 mg), atropine (10 mg), pentadecapeptide BPC 157 (Gly-Glu-Pro-Pro-Pro-Gly-Lys-Pro-Ala-Asp-Asp-Ala-Gly-Leu-Val) (10 microg or 10 ng x kg(-1) i.p.) evidently prevented both haloperidol-gastric lesions and reserpine-gastric lesions. Confronted with potentiated lesions following a combination of haloperidol and reserpine, these agents maintained their beneficial effects, noted in the rats treated with either haloperidol or reserpine alone. The failure of dopaminomimetics could be most likely due to more extensive inhibition of endogenous dopamine system activity, and need for remained endogenous dopamine for their salutary effect, whereas the beneficial activities of ranitidine, omeprazole, atropine, pentadecapeptide BPC 157 following dopamine system inhibition by haloperidol+reserpine suggest their corresponding systems parallel those of dopamine system, and they may function despite extensive inhibition of endogenous dopamine system activity.     

Differences in the effects of amphetamine and methylphenidate on brain dopamine turnover and serum prolactin concentration in reserpine-treated rats.

In the present study we compared the ability of amphetamine and methylphenidate to antagonize the elevation of serum prolactin produced by reserpine because of the differences in the actions of amphetamine and methylphenidate on brain dopamine turnover. Groups of male rats were treated with either methylphenidate (10 mg/kg) or amphetamine (5 mg/kg) alone or in combination with reserpine (5 mg/kg). The reserpine treatment was given 4 hours before methylphenidate or amphetamine, and the rats were killed 5 hours after reserpine. Neither amphetamine nor methylphenidate alone was able to suppress serum prolactin. Amphetamine but not methylphenidate was able to block the increase of serum prolactin in response to reserpine. Amphetamine lowered brain DOPAC in control and reserpine-treated rats, but methylphenidate elevated brain DOPAC in control rats and had no effect in reserpine-treated rats. These results indicate that the methylphenidate group of CNS stimulants can be differentiated on the basis of their neuroendocrine effects from the amphetamine group of stimulants.     

INHIBITION BY APOMORPHINE OF DOPAMINE DEAMINATION IN THE RAT BRAIN

Abstract— Apomorphine (A) inhibited dopamine deamination by rat brain mitochondria, but did not influence catechol- O -methyltransferase (COMT) activity by brain homogenates. The administration of apomorphine (10mg/kg i.p.) to normal rats increased brain dopamine (DA) by 34 per cent and decreased homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) by 60 per cent. In rats treated with reserpine 15 min prior to A, the latter prevented the rise of cerebral HVA and DOPAC and the depletion of DA produced by the former. Finally, A decreased the L-DOPA-induced accumulation of HVA and DOPAC in the rat basal ganglia. These results indicate that A inhibits DA deamination by monoamine oxidase.
This inhibition seems to be specific since apomorphine did not influence 5-HIAA levels in normal rats and prevented neither central 5-HT depletion nor 5-HIAA rise induced by reserpine.     

Stimulation of presynaptic dopamine autoreceptors by 4-(2-di-n-propylaminoethyl) indole (DPAI)

The dopaminergic activity of 4-(2-di-n-propylaminoethyl)indole (DPAI) was investigated. In animal models for postsynaptic dopaminergic activity DPAI showed only very weak or no effects. In rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal tract, very weak contralateral turning behavior was induced in 4/6 rats. DPAI did not induce stereotyped behavior but caused a pronounced reduction in locomotor activity. In male rats pretreated with reserpine, DPAI lowered serum prolactin levels. Levels of homovanillic acid (HVA) were monitored in the striatum of the chloral hydrate anesthetized rat by means of in vivo voltammetry. DPAI administration reduced the faradaic peak corresponding to HVA. In rats pretreated with the decarboxylase inhibitor, NSD-1015, DPAI blocked the accumulation of dopa in response to gamma-butyrolactone. The results of this study indicate that DPAI possesses a high degree of selectivity for presynaptic dopamine autoreceptors, and little or no effect on postsynaptic dopamine receptors.     

Differential Roles of Raphe Nuclei in the Regulation of Dopamine β-Hydroxylase in the Adrenal Gland of the Rat

The effect of reserpine on the activity of dopamine beta-hydroxylase (DBH) in the adrenal gland of the rat was determined following electrolytic lesion of the dorsal raphe nucleus (DRN) or medial raphe nucleus (MRN). In sham-operated rats, as well as in those with a lesion of the DRN, there was no significant modification of the action of reserpine on this enzyme. However, a lesion of MRN potentiated the inducing action of the drug. A specific role of MRN in the serotonergic regulation of adrenal DBH is suggested by this work.     

Reserpine induced intraneuronal dopamine oxidation: reversal by MPP+ action.

1-methyl-4-phenylpyridinium ion (MPP+) was tested for its effects upon dopamine level after incubating striatal synaptosomes in medium with and without reserpine. In the absence of reserpine, MPP+ enhanced the total incubation mixture dopamine level when tyrosine was present in the medium but that enhancing effect was considerably weaker when tyrosine was replaced by alpha methyl p-tyrosine. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) also had effects upon dopamine but likely due to MPP+, which was formed from MPTP by free mitochondrial MAO present in the tissue preparation. The incubation mixture dopamine level was drastically reduced by the addition of only reserpine and its presence in the medium markedly raised the ability of MPP+ to increase dopamine; the effects of MPTP in this medium were weaker than those of MPP+. Pargyline also raised dopamine levels under these conditions but only at concentrations much higher than those of MPP+. The particulate uptake of MPP+, at several medium concentrations, and the corresponding value of dopamine increase above the basal level were determined; the dopamine increase in p-moles was much greater than the p-moles of MPP+ uptake. These results indicate that, in the presence of reserpine, MPP+ has a potent action and that may lead to a release of intraneuronal free dopamine; this action is also likely to be independent of the countertransport from MPP+ uptake. The possibility of MPP+ being a potent inhibitor of intraneuronal MAO may have to be considered.     

Aromatic L-Amino Acid Decarboxylase Activity of Mouse Striatum Is Modulated via Dopamine Receptors

Abstract: Aromatic L-amino acid decarboxylase (AAAD) activity is enhanced in the striatum of control and MPTP-treated mice after administration of a single dose of the dopamine receptor antagonists haloperidol, sulpiride, and SCH 23390. MPTP-treated mice appear more sensitive to the antagonists, i.e., respond earlier and to lower doses of antagonists than control mice. The rise of AAAD activity induced by the antagonists is prevented by pretreatment with cycloheximide. The apparent K _m values for L-3,4-dihydroxyphenylalanine (L-DOPA) and pyridoxal 5-phosphate appear unchanged after treatment with the antagonists. Increased AAAD activity was observed also after subchronic administration of dopamine receptor antagonists or treatment with reserpine. A single dose of a selective dopamine receptor agonists had no effect on AAAD activity. In contrast, administration of L-DOPA, quinpirole, or SKF 23390 for 7 days lowers AAAD activity in the striatum. We conclude that AAAD is modulated in striatum via dopaminergic receptors.     

Reserpine pretreatment prevents increases in extracellular striatal dopamine following L-DOPA administration in rats with nigrostriatal denervation

The influence of L-DOPA and reserpine on extracellular dopamine (DA) levels in the striatum of intact and dopaminergic denervated rats was studied using the brain microdialysis technique. In intact rats, reserpine (5 mg/kg s.c.) reduced extracellular DA levels to 4% of basal values. L-DOPA (50 mg/kg i.p.) had no effect on extracellular DA levels in reserpine-pretreated rats. In rats with 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic system, basal levels of extracellular DA were low but markedly increased by L-DOPA (50 mg/kg i.p.). In 6-hydroxydopamine-lesioned rats, pretreatment with reserpine (5 mg/kg s.c.) diminished L-DOPA (50 mg/kg i.p.)-induced increases in extracellular DA levels to 16% of those obtained in denervated animals not pretreated with reserpine (p<0.01). These results suggest that in the intact striatum, extracellular DA stems mainly from vesicular storage sites and that in the striatum with dopaminergic denervation, a large part of the L-DOPA-derived extracellular DA is also derived from a vesicular pool that is released by an exocytosis mechanism.     

An investigation of the peroxidase activity of <Emphasis Type="Italic">Vitreoscilla</Emphasis> hemoglobin

In order to investigate the ability of the Vitreoscilla hemoglobin (VHb) to act as a peroxidase, the protein was overexpressed in Escerichia coli and purified using a 6xHis-tag. The peroxidase activity of VHb was studied using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferrocene carboxylic acid (FcCOOH) dopamine and l-dopa as substrates. The effects of external agents such as pH, salt concentration/ionic strength, and the thermal stability of VHb on the catalytic activity were assessed. The optimum pH for VHb using ABTS as a substrate was estimated to be 6–7. The VHb protein proved to be stable up to 80 °C, as judged by its peroxidase activity. Furthermore, NaCl concentrations up to 100 mM did not exert any significant effect on the activity. The catalytic activity against ABTS and FcCOOH was similar to that measured for horseradish peroxidase, whereas in the case of the phenolic substrates dopamine and l-dopa the activity was several orders of magnitude lower. The Michaelis constants, were in good agreement with the data for human and bovine hemoglobin. No activity could be detected for the negative controls lacking VHb. These results demonstrate that VHb exhibits peroxidase activity, a finding in line with the hypothesis that VHb has cellular functions beyond the role as an oxygen carrier. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Malin Kvist and Ekaterina S. Ryabova contributed equally to this work.     

The effects of nitric oxide on the oxidations of l-dopa and dopamine mediated by tyrosinase and peroxidase

The effects of nitric oxide (NO) on both tyrosinase/O(2)- and horseradish peroxidase/H(2)O(2)-mediated oxidations of dopamine and its o-dihydric phenol precursor l-dopa were compared with autoxidative processes and quantitatively assessed by oxidative and reductive electrochemical detection systems. In peroxidase/H(2)O(2)/NO-catalyzed reactions, significantly more substrate was oxidized than in the corresponding control incubations lacking NO. In tyrosinase/O(2)/NO-promoted reactions the total amounts of l-dopa and dopamine oxidized were significantly less than the amounts of the substrates oxidized by enzyme alone. These data indicate that the activity of the heme protein peroxidase was enhanced by NO, whereas tyrosinase, a copper-containing monoxygenase, was inhibited. The NO-mediated reduction of tyrosinase/O(2) activity may be attributed to the formation of an inhibitory copper.nitrosyl complex. An oxidized nitrodopamine derivative, considered to be either the quinone or semiquinone of 6-nitrosodopamine, was generated in peroxidase/H(2)O(2)/NO-mediated reactions with dopamine along with two oxidized melanin precursors, dopamine quinone and dopaminechrome. No corresponding nitroso compound was formed in reactions involving l-dopa or in any of the tyrosinase-mediated reactions. The formation of such a noncyclized nitrosodopamine represents an important alternative pathway in catecholamine metabolism, one that by-passes the formation of cytoprotective indole precursors of melanin. The results of this investigation suggest that cellular integrity and function can be adversely affected by NO-promoted oxidations of dopamine and other catechols, reactions that not only accelerate their conversion to reactive quinones but also form potentially cytotoxic noncyclized nitroso derivatives. Reduced levels of dopamine in the brain through NO-enhanced oxidation of the catecholamine will almost certainly be manifested by diminished levels of the dopamine-derived brain pigment neuromelanin.     

Developmental pattern of dopamine beta-hydroxylase induction in the adrenals of the rat. Influence of neonatal hypothyroidism

The extent of dopamine beta-hydroxylase induction elicited by reserpine was measured in young rats rendered hypothyroid from birth and in controls. Hypothyroidism impairs adrenal dopamine beta-hydroxylase induction in the young rat up to 50 days of age and also in the adult. In contrast, hypothyroidism has practically no effect on brainstem dopamine beta-hydroxylase induction.     

Selective regulation of carboxypeptidase peptide hormone-processing enzyme during enkephalin biosynthesis in cultured bovine adrenomedullary chromaffin cells

Bovine adrenomedullary chromaffin cells in culture were incubated with reserpine or forskolin, two agents acting through different mechanisms, which increase cellular [Met]enkephalin levels by 2-fold after 72 h. Cells were harvested and chromaffin granules were purified on a linear sucrose gradient. After reserpine treatment, carboxypeptidase-processing enzyme specific activity in chromaffin granule fractions was stimulated 1.9-fold, and Co2+-stimulated carboxypeptidase specific activity was stimulated 3-fold. The increase in enzyme activity was dependent on the time of reserpine treatment. Forskolin, on the other hand, had no significant effect on carboxypeptidase activity. The differential effects of reserpine and forskolin suggest that the carboxypeptidase-processing enzyme may be selectively regulated during periods of elevated enkephalin formation. Kinetic studies revealed that in cells exposed to reserpine, the Km value for [Met]enkephalin-Arg6 for the Co2+-stimulated carboxypeptidase activity was lowered to 0.136 from 0.447 mM, but there was no change in the Km values of the non-Co2+-stimulated carboxypeptidase activity from reserpine and control groups. Cellular levels of immunoreactive carboxypeptidase-processing enzyme, measured by a radioimmunoassay method, were not altered after reserpine treatment. These data suggest that while the total number of carboxypeptidase enzyme molecules remained constant, there may be a conversion of existing enzyme molecules to a more active form which displays a higher affinity for [Met]enkephalin-Arg6 in the presence of Co2+.     

The ultrastructure,catecholamine, and prolactin contents of the rostral pars distalis of the fish Mugil platanus after reserpine or 6-hydroxydopamine administration

The rostral pars distalis (RPD) of the teleost Mugil plantanus from animals pretreated with reserpine or 6-hydroxydopamine (6-HODA) were assayed for dopamine (DA) or noradrenaline (NA) or for prolactin hormone. Such determinations were coupled with electron microscopy. It was found that reserpine and 6-HODA produced a significant decrease in the content of DA, NA, and prolactin. Electron microscope studies revealed that prolactin cells became activated as judged by ultrastructural criteria. After 6-HODA treatment type "B" neurosecretory fibers entering the RPD became selectively destroyed. These observations lead us to suggest that prolactin secretion is under inhibitory control by type "B" neurosecretory fibers of adrenergic nature.     

Plasma gonadotropin,prolactin levels and hypothalamic tyrosine hydroxylase activity in rats during estrous cycle,after overiectomy and after blockade of catecholamine biosynthesis

Plasma gonadotropin, prolactin levels and hypothalamic tyrosine hydroxylase activity were evaluated at 0900, 1200 and 1700 h during diestrus, proestrus and estrus, ovariectomized and after systemic administration of reserpine or α-methyl p-tyrosine, which interfere with catecholamine biosynthesis, in rats. Gonadotropin and prolactin levels showed peak values during the afternoon of proestrus, while hypothalamic tyrosine hydroxylase activity was markedly lowered at 1200 on proestrus. Gonadotropin levels were slightly lowered whereas prolactin concentrations and hypothalamic tyrosine hydroxylase activity were significantly increased by reserpine. Depletion of hypothalamic dopamine by reserpine apparently resulted in significant elevation of prolactin levels which inturn induce tyrosine hydroxylase. Gonadotropin levels and hypothalamic tyrosine hydroxylase activity were significantly suppressed after the administration of α-methyl p-tyrosine. Prolactin levels, however, were elevated significantly. These results indicate that catecholamines are involved in the control of gonadotropin and prolactin release during estrous cycle and inhibition of catecholamines biosynthesis by α-methyl p-tyrosine could result in suppression of gonadotropin levels, whereas removal of tonic inhibition of hypothalamic dopamine by α-methyl-p-tyrosine elevate prolactin levels.     

Lithium attenuates dopamine depleting effects of reserpine and tetrabenazine but not that of alpha methyl-p-tyrosine

Concurrent administration of lithium (Li) significantly attenuates the dopamine (DA) depleting effects of reserpine and tetrabenazine, but does not change alpha-methyl-p-tyrosine (AMPT) induced DA depletion in rat brain. This effect of Li is probably mediated, in part, by inhibiting the magnesium-dependent binding of both reserpine and tetrabenazine to their specific receptor sites. Such interaction between these drugs may attenuate the beneficial effects of tetrabenazine and reserpine on patients with tardive dyskinesia or tardive dystonia who are treated concurrently with lithium for their psychiatric disorder.     

Acute changes in the state of dopamine receptors; in vitro monitoring with 3H-dopamine

Preincubation of homogenates of rat caudate nucleus at 37 degrees C induced a 3-fold increase in the saturable and stereospecific binding of 3H-dopamine in washed suspensions of the 20,000 × g pellet. EGTA prevented and CaCl2 or MgCl2 reinstated the increase in 3H-dopamine binding. Stereospecific binding was reduced by 53% in the caudate nuclei of animals given a single dose of reserpine which depleted brain dopamine. The addition of dopamine to depleted homogenates restored the effect of preincubation on 3H-dopamine binding. The binding of 3H-spiroperidol was unaffected by preincubation or by reserpine pretreatment. These results suggest that dopamine regulates the 3H-dopamine, but not the 3H-spiroperidol binding site in rat brain.     

Biochemistry of Somatodendritic Dopamine Release in Substantia Nigra: An In Vivo Comparison with Striatal Dopamine Release

Abstract: The somatodendritic release of dopamine in substantia nigra previously has been suggested to be nonvesicular in nature and thus to differ from the classical, exocytotic release of dopamine described for the dopaminergic nerve terminal in striatum. We have compared the effects of reserpine, a compound that disrupts vesicular sequestration of monoamines, on the storage and release of dopamine in substantia nigra and striatum of rats. Reserpine administration (5 mg/kg, i.p.) significantly decreased the tissue level of dopamine in substantia nigra pars reticulata, substantia nigra pars compacta, and striatum. In these brain areas, reserpine-induced reductions in tissue dopamine level occurred within 2 h and persisted at 24 h postdrug. In vivo measurements using microdialysis revealed that reserpine administration rapidly decreased the extracellular dopamine concentration to nondetectable levels in substantia nigra as well as in striatum. In both structures, it was observed that reserpine treatment significantly attenuated the release of dopamine evoked by a high dose of amphetamine (10 mg/kg, i.p.) given 2 h later. In contrast, dopamine efflux in response to a low dose of amphetamine (2 mg/kg, i.p.) was not altered by reserpine pretreatment either in substantia nigra or in striatum. The present data suggest the existence, both at the somatodendritic and at the nerve terminal level, of a vesicular pool of dopamine that is the primary site of transmitter storage and that can be displaced by high but not low doses of amphetamine. The physiological release of dopamine in substantia nigra and in striatum is dependent on the integrity of this vesicular store.