Interaction of the central analgesic, tramadol, with the uptake and release of 5-hydroxytryptamine in the rat brain in vitro.

1. Tramadol is a centrally acting analgesic with low opioid receptor affinity and therefore presumably other mechanisms of analgesic action. Tramadol inhibits noradrenaline uptake but since 5-hydroxytryptamine (5-HT) is also involved in the modulation of pain perception, we tested the effects of tramadol on 5-HT uptake and release in vitro. 2. Tramadol inhibited the uptake of [3H]-5-HT into purified rat frontal cortex synaptosomes with an IC50 of 3.1 microM. The (+)-enantiomer was about four times more potent than the (-)-enantiomer; the main metabolite of tramadol, O-desmethyltramadol, was about ten times less potent. 3. Rat frontal cortex slices were preincubated with [3H]-5-HT, then superfused and stimulated electrically. Tramadol facilitated the basal outflow of [3H]-5-HT, at concentrations greater than 1 microM, while the uptake inhibitor 5-nitroquipazine enhanced both basal and stimulation-evoked overflow. Effects of the (+)-enantiomer were more potent than either the racemate, the (-)-enantiomer or the principal metabolite. 4. The effects of tramadol on the basal outflow of [3H]-5-HT were almost completely abolished when the superfusion medium contained a high concentration of the selective 5-HT uptake blocker, 6-nitroquipazine. 5. The results provide evidence for an interaction of tramadol with the neuronal 5-HT transporter. An intact uptake system is necessary for the enhancement of extraneuronal 5-HT concentrations by tramadol indicating an intraneuronal site of action.     

Effects of the central analgesic tramadol and its main metabolite, O-desmethyltramadol, on rat locus coeruleus neurones.

1. Tramadol is a centrally acting analgesic with low opioid receptor affinity and, therefore, presumably additional mechanisms of analgesic action. Tramadol and its main metabolite O-desmethyltramadol were tested on rat central noradrenergic neurones of the nucleus locus coeruleus (LC), which are involved in the modulation of nociceptive afferent stimuli. 2. In pontine slices of the rat brain the spontaneous discharge of action potentials of LC cells was recorded extracellularly. (-)-Tramadol (0.1-100 microM), (+)-tramadol (0.1-100 microM), (-)-O-desmethyl-tramadol (0.1-100 microM) and (+)-O-desmethyltramadol (0.01-1 microM) inhibited the firing rate in a concentration-dependent manner. (+)-O-desmethyltramadol had the highest potency, while all other agonists were active at a similar range of concentrations. 3. (-)-Tramadol (10, 100 microM) was less inhibitory in brain slices of rats pretreated with reserpine (5 mg kg-1, 5 h before decapitation) than in controls. 4. The effect of (-)-tramadol (10 microM) was abolished in the presence of the alpha 2-adrenoceptor antagonist, rauwolscine (1 microM), whilst that of (+)-O-desmethyltramadol (0.3 microM) virtually disappeared in the presence of the opioid antagonist, naloxone (0.1 microM). (+)-Tramadol (30 microM) and (-)-O-desmethyl-tramadol (10 microM) became inactive only in the combined presence of naloxone (0.1 microM) and rauwolscine (1 microM). 5. In another series of experiments, the membrane potential of LC neurones was determined with intracellular microelectrodes. (-)-Tramadol (100 microM) inhibited the spontaneous firing and hyper-polarized the cells; this effect was abolished by rauwolscine (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of phentolamine on noradrenaline uptake and release

Summary The influence of phentolamine on the uptake of exogenous noradrenaline infused into the aortic cannula and on the overflow of endogenous noradrenaline caused by sympathetic nerve stimulation was investigated in the isolated perfused rabbit heart. 10^–6 M phentolamine doubled the overflow of endogenous noradrenaline, but did not change noradrenaline uptake. 10^–5 M phentolamine increased the stimulation-induced overflow of noradrenaline 4-fold and inhibited amine uptake by about 50%. 10^–4 M phentolamine elevated the overflow of noradrenaline less than 10^–5 and 3×10^–5 M did. The augmentation of transmitter overflow was only partly reversed by 13 min perfusion with drug-free medium.Pretreatment of hearts with 1.5×10^–5 M cocaine or with 10^–7 or 10^–6 M desipramine did not change the effect of phentolamine on the overflow of noradrenaline evoked by nerve impulses. Pretreatment of hearts with 10^–5 M, but not with 10^–6 M, phentolamine prevented the increase of transmitter overflow by cocaine.It is concluded that low concentrations of phentolamine potentiate the overflow of noradrenaline during nerve stimulation by a mechanism different from that of cocaine, i.e. different from inhibition of neuronal re-uptake. The nature of this mechanism is discussed.This work was supported by the Deutsche Forsehungsgemeinschaft. We have the pleasure to thank Mrs. Ch. Arts, Miss B. Piel and Mr. E. Hagelskamp for skilful technical assistance.     

Effects of intermittent and continuous cocaine administration on dopamine release and uptake regulation in the striatum: in vitro voltammetric assessment

Chronic daily injections of cocaine induce behavioral sensitization to subsequent cocaine challenge, while continuous infusion induces tolerance. Following a 7-day withdrawal period, we examined the effects of these two dosing regimens on: (1) baseline dopamine efflux and uptake following single-pulse electrical stimulation, (2) inhibition of uptake by cocaine; and (3) inhibition of efflux by autoreceptor activation. Cocaine (40 mg/kg per day) was administered to rats for 14 days either continuously by osmotic minipumps or intermittently by once-a-day injections. Minipumps containing saline were implanted in the control group. After 7 days of withdrawal, dopamine kinetics in the caudate was examined using in vitro fast-scan cyclic voltammetry. This technique provides very rapid measurements of dopamine in the extracellular space. Thus, when combined with endogenous dopamine efflux evoked by single-pulse, electrical stimulations, it was possible directly to measure the release and uptake components of the efflux. In the absence of pharmacological agents, no group differences were found in the amount of baseline dopamine released or in the uptake kinetics; the potency of bath-applied cocaine (0.03–60 µM) in inhibiting the uptake was also unaltered in either group. In contrast, the potency of quinpirole (an autoreceptor agonist, 5–250 nM) was significantly decreased and increased in the cocaine injection and pump groups, respectively. Thus, the cocaine administration regimen which produces sensitization results in a functional subsensitivity of release-modulating autoreceptors, while the tolerance-producing regimen results in autoreceptor supersensitivity.     

Effects of 4-aminopyridine on mechanical activity and noradrenaline release in the rat portal vein in vitro.

4-Aminopyridine (4-AP) increased the spontaneous mechanical activity of the isolated rat portal vein. Since denervation and adrenergic receptor blockade failed to prevent this effect of 4-AP it is suggested that the drug enhances the electrical excitability of the muscle membrane. 4-AP significantly increased the response of the muscle to electrical nerve stimulation in most experiments but had little effect on the response to applied noradrenaline (NA). Both spontaneous and evoked release of 3H-activity, following preincubation in 3H-noradrenaline, were increased in the presence of 4-AP (10(-3) M). The present results with 4-AP can be explained by its known ability to block the transient potassium conductance which accompanies the action potential in excitable tissues.     

Effects of methylene blue on the uptake, release and metabolism of noradrenaline in mesenteric arterial vessels

Methylene blue (3, 10 and 30 microM) increased the spontaneous outflow of endogenous dopamine and noradrenaline from sympathetic nerves supplying the dog mesenteric artery and drastically reduced the formation of endogenous dihydroxyphenylglycol (DOPEG). In addition, it decreased the accumulation of [3H]noradrenaline in the tissue, reduced the formation of [3H]DOPEG and [3H]normetanephrine, without altering the formation of [3H]dihydroxymandelic acid. In tissue homogenates of the same blood vessel, methylene blue 30 and 100 microM produced a significant reduction in the deamination of 5-hydroxytryptamine (5-HT), beta-phenylethylamine (beta-PEA) and tyramine. Methylene blue increased the accumulation of [3H]isoprenaline in the tissue, and markedly reduced the formation of [3H]O-methylisoprenaline ([3H]OMI). These results show that methylene blue alters the storage and disposition of the adrenergic transmitter.     

Adrenergic receptor blocking agents: Effects on central noradrenaline and dopamine receptors and on motor activity

Phenoxybenzamine, but not phentolamine or propranolol, blocked central noradrenaline receptors in flexor reflex experiments on the rat spinal cord using the selective noradrenaline receptor stimulating agent clonidine. None of these three drugs blocked dopamine receptors in experiments on turning of unilaterally striatectomized rats induced by the dopamine receptor stimulating agent apomorphine. The -methyltyrosine-induced disappearance of noradrenaline in the central nervous system of rats and mice was accelerated by phenoxybenzamine at doses related to the functional changes, whereas the other drugs were inefficient. The disappearance of dopamine was decelerated by phenoxybenzamine, but not by phentolamine or propranolol.The effects of the adrenergic receptor blocking agents on motor activity were studied in a model in which clonidine potentiated the activation induced by apomorphine in reserpine-treated mice. Phentolamine (20 mg/kg) and propranolol (10 mg/kg) reduced the stimulation seen both after apomorphine alone and in combination with clonidine, indicating nonspecific sedative effects. Phentolamine (10 mg/kg) blocked the peripheral effects of clonidine but did not markedly diminish the activation induced by the receptor stimulants. Phenoxybenzamine (20 mg/kg) blocked the clonidine-induced potentiation without interfering with the apomorphine-induced stimulation and can thus be used as a blocking agent of central and peripheral noradrenaline receptors in behavioural experiments.     

Effects of amphetamine isomers, methylphenidate and atomoxetine on synaptosomal and synaptic vesicle accumulation and release of dopamine and noradrenaline in vitro in the rat brain

D- and L-amphetamine sulphate isomers, methylphenidate and atomoxetine, are effective treatments for attention-deficit hyperactivity disorder (ADHD). This study provides a detailed comparison of their effects on the synaptosomal and vesicular accumulation of dopamine (DA) and noradrenaline (NA) and release in vitro in rat prefrontal cortex and striatum. D-amphetamine was more potent than L-amphetamine at inhibiting accumulation of DA or NA in synaptosomes and vesicles. All drugs were weaker at inhibiting the accumulation of vesicular DA and NA compared to synaptosomal accumulation and more potently inhibited NA accumulation than DA. Methylphenidate was weak at inhibiting vesicular accumulation of DA and NA compared to its potent synaptosomal effects. The D-isomer had greater potency than the L-isomer on basal and electrically stimulated striatal DA release; however the L-isomer was 2-fold more potent than the D-isomer on basal fronto-cortical NA release. The selective DA reuptake inhibitor, GBR-12909 and NA reuptake inhibitors, maprotiline and atomoxetine, had different release profiles both on the potency and magnitude of basal and stimulated DA and NA release compared to the amphetamine isomers. These results identify distinct pharmacological action by the amphetamine isomers on dopaminergic and noradrenergic neurotransmission, which may impact on their therapeutic effects in the treatment of ADHD.     

Neurotransmitter effects on in vitro prolactin and growth hormone release from fowl pituitary glands: dopamine and noradrenaline

Pituitary glands and hypothalami from broiler fowl heads were incubated alone or together with dopamine, noradrenaline or monoaminergic drugs (apomorphine, pimozide, phentolamine, isoproterenol and propranolol). The basal release of both prolactin and growth hormone was not affected by any of these amines or aminergic drugs. The co-incubation of pituitary glands with whole hypothalami consistently stimulated prolactin and growth hormone secretion. Apomorphine inhibited hypothalamus-induced prolactin and growth hormone release and the response was blocked by pimozide. Noradrenaline markedly stimulated hypothalamus-induced prolactin release, an effect antagonised by phentolamine. Thyrotrophin-releasing hormone (TRH) stimulated the release of prolactin and this stimulation was reversed, in a dose-related manner, by dopamine. TRH also increased the release of growth hormone, in the presence or absence of dopamine. These results demonstrate inhibitory dopaminergic effects on prolactin and growth hormone secretion and stimulatory noradrenergic effects on prolactin release. These aminergic effects may be mediated at pituitary and/or hypothalamic sites.     

Comparative action of fenfluramine on the uptake and release of serotonin and dopamine.

The anorectic agent, fenfluramine, proves to be a good inhibitor of serotonin uptake in vitro, in synaptosomes from rat whole brain (IC50 = 8.5 +/- 0.6 X 10(-7) M). After administration in vivo, its inhibitory activity in vitro equals that of chlorimipramine and in contrast to the latter, its effect is of long duration. Fenfluramine is also effective in promoting the release of serotonin from pre-loaded synaptosomes. In comparison, the structurally related compound, amphetamine, has little activity with respect to these serotonin mechanisms. It is, however, active both in inhibiting the uptake of dopamine and in promoting its release, whereas fenfluramine is inactive. The implication of these mechanisms in the serotonin-depleting capacity as well as in the anorectic activity of fenfluramine is discussed.     

Effects of amphetamine derivatives on brain dopamine and noradrenaline

1. Intracisternally administered metaraminol, α-methyl-octopamine, α-methyl-m-tyramine, and α-methyl tyramine were found to lower brain noradrenaline without having an effect on brain dopamine.

2. Amphetamine, mephentermine, and norephedrine had no effect on brain catecholamines after intracisternal injection.

3. There was no reduction in brain dopamine content after intracisternal injection of α-methyl-m-tyramine, yet the resulting brain concentration of α-methyl-m-tyramine was several times higher than after intraperitoneal injection of α-methyl-m-tyrosine, which decreased brain dopamine.

4. The decreased synthesis of labelled catecholamines from ¹⁴C-tyrosine after α-methyl-m-tyrosine suggested that this compound inhibits tyrosine hydroxylase in addition to its action of displacing brain amines.

     

Inhibition of uptake1 by (+)-oxaprotiline reveals a differential central regulation of noradrenaline and adrenaline release

Summary Inhibition of uptake, in the central nervous system leads to a decrease of sympathetic outflow to many tissues; central a2-adrenoceptors are involved in this decrease. The aim of the present study was to compare the effects of the selective uptake, inhibitor (+)-oxaprotiline on the plasma kinetics of noradrenaline and adrenaline in anaesthetized and in conscious rabbits. [³H]Noradrenaline and [^3H]adrenaline were infused iv. The arterial plasma concentrations of endogenous and radiolabelled noradrenaline and adrenaline were measured, and the clearance from and spillover into the plasma of noradrenaline and adrenaline were calculated.Results obtained in conscious and anaesthetized rabbits were similar. (+)-Oxaprotiline 0.2, 0.6 and 1.8 mg kg^–1 iv. dose-dependently reduced the clearance of [³H]noradrenaline from the plasma. The clearance of [³H]adrenaline was reduced less. The spillover of endogenous noradrenaline was decreased by up to 35%. In contrast, the spillover of adrenaline tended to be enhanced. Prazosin 0.1 and 1 mg kg^–1 was injected iv. in a second part of each experiment. It lowered the blood pressure and caused a marked increase in noradrenaline spillover but no increase or even a decrease in adrenaline spillover.The results are compatible with the following hypothesis. The sympathetic outflow from the central nervous system is subject to a twofold a-adrenoceptor-mediated modulation: -adrenoceptor-mediated inhibition and ₁-adrenoceptor-mediated excitation. In the control of the sympathetic outflow to many extra-adrenal tissues, the ₂-adrenergic inhibition prevails. Uptake₁ inhibitors depress sympathetic outflow to such tissues by enhancing the ₂-adrenergic inhibition. In the regulation of the sympathetic outflow to the adrenal medulla, in contrast, ₂-adrenergic inhibition and ₁-adrenergic excitation have a similar impact. Uptake, inhibitors, hence, cause little change in adrenaline release: the two opposing influences cancel out. Prazosin produces an increase in noradrenaline but not adrenaline release because the loss of the central ₁ sympathoexcitation attenuates at best slightly the baroreflex to most extra-adrenal tissues but dampens markedly the baroreflex to the adrenal medulla. Correspondence to B. Szabo at the above address     

Biphasic actions of L-DOPA on the release of endogenous noradrenaline and dopamine from rat hypothalamic slices.

Effects of L-DOPA on the release of endogenous noradrenaline and dopamine from rat hypothalamic slices evoked by electrical field stimulation at 5 Hz were investigated in the absence and presence of p-bromobenzyloxyamine (NSD-1055), a DOPA-decarboxylase inhibitor. In the absence of NSD-1055, L-DOPA produced a facilitation of impulse-evoked release of noradrenaline at 0.1 microM but not at 1 and 10 microM, and had no effect on the spontaneous release. On the other hand, L-DOPA 0.1 to 10 microM dose-dependently increased the spontaneous release of dopamine and the highest concentration only increased the evoked release and tissue content of dopamine. In the presence of NSD-1055 10 microM, the increase in the spontaneous release of dopamine was prevented and L-DOPA produced biphasic regulatory effects on the evoked release of noradrenaline and dopamine, a facilitation at 0.1 microM and an inhibition at 1 microM. The facilitation was antagonized by (-)-propranolol 0.1 microM, but not by the (+)-isomer, whereas the inhibition was antagonized by S-sulpiride 1 nM, but not by the R-isomer. In conclusion, L-DOPA appears to produce biphasic actions on the release of endogenous noradrenaline and dopamine from rat hypothalamic slices, not through its conversion to dopamine but through presynaptic regulatory mechanisms, an inhibition via dopamine receptors at a micromolar concentration and a facilitation via beta-adrenoceptors at the lower concentration.