Cardiovascular responses to combined treatment with selective monoamine oxidase type B inhibitors and L-DOPA in the rat

BACKGROUND AND PURPOSE: Postural hypotension is a common side-effect of L-DOPA treatment of Parkinson's disease, and may be potentiated when L-DOPA is combined with selegiline, a selective inhibitor of monoamine oxidase B (MAO-B). Rasagiline is a new, potent and selective MAO-B inhibitor, which does not possess the sympathomimetic effects of selegiline. We have studied the effects of these selective MAO inhibitors, L-DOPA and dopamine on the cardiovascular system of the rat. EXPERIMENTAL APPROACH: Blood pressure and heart rate was measured in conscious rats following acute or chronic administration of rasagiline, selegiline and L-DOPA, by comparison with the selective MAO-A inhibitor clorgyline, or the MAO-A/B inhibitor tranylcypromine. Cardiovascular responses, catecholamine release, and modification of pressor response to dopamine were studied in pithed rats. KEY RESULTS: In conscious rats neither rasagiline nor selegiline caused significant potentiation of the effects of L-DOPA (50, 100, 150 mg.kg(-1)) on blood pressure or heart rate at doses which selectively inhibited MAO-B, but L-DOPA responses were potentiated by clorgyline and tranylcypromine. In rats treated twice daily for 8 days with L-DOPA and carbidopa, selegiline (5 mg.kg(-1)) but not rasagiline (0.2 mg.kg(-1)) caused a significant hypotensive response to L-DOPA and carbidopa, although both drugs caused similar inhibition of MAO-A and MAO-B. In pithed rats, selegiline but not rasagiline increased catecholamine release and heart rate, and potentiated dopamine pressor response at MAO-B selective dose. CONCLUSIONS AND IMPLICATIONS: The different responses to the two MAO-B inhibitors may be explained by the amine releasing effect of amphetamine metabolites formed from selegiline.     

Effects of subchronic treatment with selegiline on L-DOPA-induced increase in extracellular dopamine level in rat striatum

Selegiline is used an adjunct to L-DOPA therapy. We investigated extracellular striatal dopamine (DA) level in awake rats treated with L-DOPA and/or selegiline using a microdialysis method. Rats given 10 mg/kg, i.p. per day selegiline for 7 days were administered with a single dose of 100 mg/kg, i.p. L-DOPA 0 (3 h), 1, 3, 7, 14, 21, or 28 days after the last selegiline treatment. Carbidopa was administered 0.5 h before L-DOPA administration. The significant increase in basal DA level before L-DOPA treatment persisted until 1 day after the last selegiline treatment, and the significant decrease in basal DOPAC level persisted for more than 28 days. Thus, selegiline affected DA catabolism for more than 28 days. Total monoamine oxidase (MAO) and MAO-B activities at day 0 decreased by 22% and 5.7%, respectively. The significant enhancement of L-DOPA-induced increase in DA level was observed until 3 days after the last selegiline treatment. Next, the effects of reducing L-DOPA dose by 25% were examined 3 h after the last selegiline treatment. A dose-dependent decrease in DA level was observed, indicating that DA level in selegiline-treated rats can be controlled by L-DOPA dose.     

Mitochondrial toxin inhibition of [(3)H]dopamine uptake into rat striatal synaptosomes

Administration of the mitochondrial inhibitors malonate and 3-nitropropionic acid (3-NP) to rats provides useful models of Huntington's disease. Exposure to these inhibitors has been shown to result in increased extracellular concentrations of striatal dopamine (DA), which is neurotoxic at high concentrations. The cause of this increase is unknown. The purpose of this study was to determine whether mitochondrial inhibition alters dopamine transporter (DAT) function. Striatal synaptosomes were incubated in the presence of several structurally unrelated inhibitors of mitochondrial Complexes I, II, and IV, and [(3)H]DA uptake was measured. Although all of the toxins inhibited [(3)H]DA uptake, there was a large variation in their inhibitory potencies, the rank order being rotenone>cyanide>azide>3-NP>malonate. Examination of the kinetic parameters of [(3)H]DA uptake revealed that inhibition was due to a reduction in maximum velocity (V(max)), with no change in affinity (K(m)). The addition of either ATP or of ADP plus P(i) to synaptosomes treated with 3-NP, or of the reactive oxygen species spin trap alpha-phenyl-N-tert-butyl nitrone to synaptosomes exposed to either malonate or cyanide failed to prevent mitochondrial toxin-induced inhibition of DAT function. The lack of effect of high energy substrates or of a free radical scavenger suggests that the mechanism by which extracellular DA is increased by several mitochondrial toxins involves factors other than mitochondrial ATP production or oxidative stress. Taken together, the results suggest that one mechanism whereby mitochondrial toxins increase extracellular concentrations of DA is via interaction with the DAT at a site other than the substrate site, i.e. noncompetitive inhibition of the DAT.     

Rapid delivery of the dopamine transporter to the plasmalemmal membrane upon amphetamine stimulation

The dopamine transporter, DAT, is a primary regulator of dopamine (DA) signaling at the synapse. Persistent stimulation with the substrate amphetamine (AMPH) promotes DAT internalization. AMPH rapidly elicits DA efflux, yet its effect on DAT trafficking at short times is unknown. We examined the rapid effect of AMPH on DAT trafficking in rat striatal synaptosomes using biotinylation to label surface DAT. Within 30s of treatment with 3 microM AMPH, synaptosomal DAT surface expression increased to 163% of control and remained elevated through at least 1 min before returning to control levels at 2.5 min. The increase in surface DAT was cocaine-sensitive but was not produced by DA itself. A 1-min preincubation with AMPH did not alter [(3)H]DA uptake, but did result in a higher basal DA efflux and efflux elicited in the presence of AMPH as compared to vehicle pretreatment. Reversible biotinylation experiments demonstrated that the AMPH-stimulated rise in surface DAT is due to an increase in the delivery of DAT to the plasmalemmal membrane rather than a reduction of the endocytic process. These studies suggest that AMPH has a biphasic effect on DAT trafficking and acts rapidly to regulate DAT in the plasmalemmal membrane.     

Effect of amphetamine-induced dopamine release on radiotracer binding to D1 and D2 receptors in rat brain striatal slices

The in vivo binding of positron emission tomography (PET) and single photon emission computer tomography (SPECT) radiotracers to dopamine D2 receptors in the striatum can be influenced by competition with endogenous dopamine. The present study was undertaken to determine if a similar inhibition of radiotracer binding to dopamine receptors could be observed following pharmacologically-evoked dopamine release in rat brain striatal slices. Striatal slices were incubated in a large volume of oxygenated Krebs saline and exposed to amphetamine or methamphetamine to evoke dopamine release within the slice. Amphetamine and methamphetamine, at concentrations up to 30 microM, reduced [3H]raclopride binding in the slices by 77% and 86%, respectively, with 50% inhibition at 1.6 microM amphetamine or 3.0 microM methamphetamine. Neither drug produced a significant effect on binding of [3H]SCH 23390 in the slices. This suggests that dopamine was able to interfere with radiotracer binding to D2 but not D1 receptors. The dopamine uptake blockers, cocaine and methylphenidate, had relatively little effect by themselves on [3H]raclopride binding but, by inhibiting amphetamine-induced dopamine release, significantly reduced inhibition of [3H]raclopride binding by a low (3 microM) amphetamine concentration. At a higher (30 microM) amphetamine concentration the inhibition of [3H]raclopride binding was not antagonized by uptake blockers and data obtained from homogenate binding experiments indicated a direct displacement of [3H]raclopride binding by amphetamine at this concentration. In conclusion the data obtained in the present study demonstrate that the effects of amphetamine on striatal radiotracer accumulation observed in PET and SPECT can also be observed in brain slices in vitro and, at least at low amphetamine concentrations, are mediated by competition with released dopamine.     

Clinical pharmacology of rasagiline: a novel, second-generation propargylamine for the treatment of Parkinson disease

Rasagiline is a novel second-generation propargylamine that irreversibly and selectively inhibits monoamine oxidase type B (MAO-B). For the management of Parkinson disease (PD), rasagiline is efficacious across the span of PD stages ranging from monotherapy in early disease to adjunctive treatment in patients with advancing disease and motor fluctuations. Rasagiline completely and selectively inhibits MAO-B with a potency 5 to 10 times greater than selegiline. Unlike the prototype propargylamine selegiline, which is metabolized to amphetamine derivatives, rasagiline is biotransformed to aminoindan, a non-amphetamine compound. Rasagiline is well tolerated with infrequent cardiovascular or psychiatric side effects, and at the recommended therapeutic dose of up to 1 mg once daily, tyramine restriction is unnecessary. In addition to MAO-B inhibition, the propargylamine chain also confers dose-related antioxidant and antiapoptotic effects, which have been associated with neuroprotection in multiple experimental models. Thus, in addition to symptomatic benefits, rasagiline offers the promise of clinically relevant neuroprotection.     

Rasagiline: A second-generation monoamine oxidase type-B inhibitor for the treatment of Parkinson's disease.

PURPOSE: The pharmacology, pharmacokinetics, clinical efficacy, and safety of rasagiline are reviewed. SUMMARY: Rasagiline is a novel, investigational propargylamine that irreversibly and selectively inhibits monoamine oxidase type B (MAO-B). Rasagiline demonstrates complete and selective inhibition of MAO-B and is at least five times more potent than selegiline. Unlike selegiline, which is metabolized to amphetamine derivatives, rasagiline is biotransformed to the nonamphetamine compound aminoindan. Clinical studies have revealed that rasagiline is associated with improved outcomes in patients with early Parkinson's disease (PD) and also reduces "off" time in patients with moderate to advanced PD with motor fluctuations. Rasagiline is rapidly absorbed by the gastrointestinal tract and readily crosses the blood-brain barrier. The optimal therapeutic dosage is 0.5-1 mg administered orally once daily. Rasagiline appears to be well tolerated, although elderly patients may be more prone to treatment-emergent adverse cardiovascular and psychiatric effects. At the recommended therapeutic dosage of up to 1 mg once daily, tyramine restriction is unnecessary. In addition to MAO-B inhibition, rasagiline has demonstrated neuroprotective properties in experimental laboratory models. The mechanisms whereby rasagiline exerts neuroprotective effects are multifactorial and include upregulation of cellular antioxidant activity and antiapoptotic factors. CONCLUSION: Rasagiline is an investigational selective and irreversible inhibitor of MAO-B that has demonstrated efficacy and safety for the treatment of PD. Whether rasagiline is associated with clinically significant neuroprotection is the subject of ongoing clinical trials.     

Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter

Although it is clear that amphetamine-induced dopamine (DA) release mediated by the dopamine transporter (DAT) is integral to the behavioral actions of this psychostimulant, the mechanism of this release is not clear. In this study, we explored the requirement for intracellular Ca(2+) in amphetamine-induced DA efflux and currents mediated by the human DAT. The patch-clamp technique in the whole-cell configuration was used on Na(+) and DA-preloaded human embryonic kidney 293 cells stably transfected with the human DAT (hDAT cells). Chelation of intracellular Ca(2+) by inclusion of 50 microM BAPTA in the whole-cell pipette reduced the voltage-dependent amphetamine-induced hDAT current, with the greatest effect seen at positive voltages. Likewise, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) reduced amphetamine-induced DA efflux as measured by amperometry. Furthermore, preincubation of the cells with 50 microM BAPTA acetoxy methyl ester (AM) or thapsigargin also blocked amphetamine-induced release of preloaded N-methyl-4-[(3)H]phenylpyridinium from superfused hDAT cells. BAPTA-AM also reduced DA release from striatal synaptosomes. Amphetamine also led to an increase in intracellular Ca(2+) that was blocked by prior treatment with 5 microM thapsigargin or 10 microM cocaine. These studies demonstrate that amphetamine-induced DAT-mediated currents and substrate efflux require internal Ca(2+) and that amphetamine can stimulate dopamine efflux by regulating cytoplasmic Ca(2+) levels through its interaction with DAT.     

Rasagiline [N-propargyl-1R(+)-aminoindan], a selective and potent inhibitor of mitochondrial monoamine oxidase B

1. Rasagiline [N-propargyl-1R(+)-aminoindan], was examined for its monoamine oxidase (MAO) A and B inhibitor activities in rats together with its S(-)-enantiomer (TVP 1022) and the racemic compound (AGN-1135) and compared to selegiline (1-deprenyl). The tissues that were studied for MAO inhibition were the brain, liver and small intestine. 2. While rasagiline and AGN1135 are highly potent selective irreversible inhibitors of MAO in vitro and in vivo, the S(-) enantiomer is relatively inactive in the tissues examined. 3. The in vitro IC(50) values for inhibition of rat brain MAO activity by rasagiline are 4.43+/-0.92 nM (type B), and 412+/-123 nM (type A). The ED(50) values for ex vivo inhibition of MAO in the brain and liver by a single dose of rasagiline are 0.1+/-0.01, 0.042+/-0.0045 mg kg(-1) respectively for MAO-B, and 6.48+/-0.81, 2.38+/-0.35 mg kg(-1) respectively for MAO-A. 4. Selective MAO-B inhibition in the liver and brain was maintained on chronic (21 days) oral dosage with ED(50) values of 0.014+/-0.002 and 0.013+/-0.001 mg kg(-1) respectively. 5. The degree of selectivity of rasagiline for inhibition of MAO-B as opposed to MAO-A was similar to that of selegiline. Rasagiline was three to 15 times more potent than selegiline for inhibition of MAO-B in rat brain and liver in vivo on acute and chronic administration, but had similar potency in vitro. 6. These data together with lack of tyramine sympathomimetic potentiation by rasagiline, at selective MAO-B inhibitory dosage, indicate that this inhibitor like selegiline may be a useful agent in the treatment of Parkinson's disease in either symptomatic or L-DOPA adjunct therapy, but lack of amphetamine-like metabolites could present a therapeutic advantage for rasagiline.     

Evidence that Formulations of the Selective MAO-B Inhibitor,Selegiline, which Bypass First-Pass Metabolism,also Inhibit MAO-A in the Human Brain

Selegiline (L-deprenyl) is a selective, irreversible inhibitor of monoamine oxidase B (MAO-B) at the conventional dose (10 mg/day oral) that is used in the treatment of Parkinson''s disease. However, controlled studies have demonstrated antidepressant activity for high doses of oral selegiline and for transdermal selegiline suggesting that when plasma levels of selegiline are elevated, brain MAO-A might also be inhibited. Zydis selegiline (Zelapar) is an orally disintegrating formulation of selegiline, which is absorbed through the buccal mucosa producing higher plasma levels of selegiline and reduced amphetamine metabolites compared with equal doses of conventional selegiline. Although there is indirect evidence that Zydis selegiline at high doses loses its selectivity for MAO-B, there is no direct evidence that it also inhibits brain MAO-A in humans. We measured brain MAO-A in 18 healthy men after a 28-day treatment with Zydis selegiline (2.5, 5.0, or 10 mg/day) and in 3 subjects receiving the selegiline transdermal system (Emsam patch, 6 mg/day) using positron emission tomography and the MAO-A radiotracer [¹¹C]clorgyline. We also measured dopamine transporter (DAT) availability in three subjects from the 10 mg group. The 10 mg Zydis selegiline dose significantly inhibited MAO-A (36.9±19.7%, range 11–70%, p<0.007)) but not DAT; and while Emsam also inhibited MAO-A (33.2±28.9 (range 9–68%) the difference did not reach significance (p=0.10)) presumably because of the small sample size. Our results provide the first direct evidence of brain MAO-A inhibition in humans by formulations of selegiline, which are currently postulated but not verified to target brain MAO-A in addition to MAO-B.     

High affinity binding of [3H]-tyramine in the central nervous system.

Optimum assay conditions for the association of [3H]-para-tyramine [( 3H]-pTA) with rat brain membranes were characterized, and a saturable, reversible, drug-specific, and high affinity binding mechanism for this trace amine was revealed. The binding capacity (Bmax) for [3H]-pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). The binding process of [3H]-pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine-rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]-pTA; (c) there was a severe loss in striatal binding capacity for [3H]-pTA and, reportedly, for [3H]-dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]-pTA and, reportedly, of [3H]-dopamine binding sites. In competition experiments [3H]-pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. The binding process of [3H]-pTA was both energy-dependent (ouabain-sensitive), and ATP-Mg2+-insensitive; furthermore, the potencies of various drugs in competing for [3H]-pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]-dopamine uptake into striatal synaptosomes. It is concluded that [3H]-pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine.     

Neuroprotection by rasagiline: a new therapeutic approach to Parkinson's disease?

Neuronal death in Parkinson's disease (PD) may originate from the reciprocal interactions of a restricted number of conditions, such as mitochondrial defects, oxidative stress and protein mishandling, which would favor a state of apoptotic cell death in the nigrostriatal pathway. The search for pharmacological treatments able to counteract the nigrostriatal degeneration, possibly by interfering with these phenomena, has recently raised considerable interest in rasagiline [R(+)-N-propargyl-1-aminoindan], a potent, selective, and irreversible inhibitor of monoamine oxidase B (MAO-B). Rasagiline, like selegiline, is a propargylamine, but is approximately 10 times more potent. Unlike selegiline, rasagiline is not metabolized to amphetamine and/or methamphetamine and is devoid of sympathomimetic activity. Numerous experimental studies, conducted both in vitro and in vivo, have shown that rasagiline possesses significant protective properties on neuronal populations. The pro-survival effects of the drug appear to be linked to its propargyl moiety, rather than to the inhibitory effect on MAO-B. Rasagiline's major metabolite, aminoindan--which possesses intrinsic neuroprotective activity--may also contribute to the beneficial effects of the parent compound. Rasagiline has been recently evaluated in early PD patients, with results that are consistent with slowing the progression of the disease. Therefore, the neuroprotective activity shown by the drug under experimental conditions may be reflected in the clinic, thus providing new perspectives for the treatment of PD.     

Translocation of dopamine and binding of WIN 35,428 measured under identical conditions in cells expressing the cloned human dopamine transporter

Translocation of [³H]dopamine and binding of [³H]WIN 35,428 were measured in intact C6 glioma cells expressing the cloned human dopamine transporter (hDAT) under identical conditions of assay buffer (phosphate-Krebs) and temperature (25°C) with uptake at initial velocity and binding at equilibrium. In the intact cells, [³H]dopamine uptake was a one-component process; in contrast, [³H]WIN 35,428 binding included both a high-affinity component, inhibitable by micromolar concentrations of dopamine, and a low-affinity component only partially inhibited by millimolar concentrations of dopamine. Binding (high-affinity) over uptake Ki ratios were on the average 2.3 for the inhibitors WIN 35,428, cocaine, GBR 12909, and BTCP. The potency of dopamine in inhibiting its own translocation was close to that in inhibiting [³H]WIN 35,428 binding consonant with a more rapid reorientation step of the DAT in the C6-hDAT system than in rat striatal synaptosomes. The similarity in turnover values of the DAT estimated in the current experiments with the C6-hDAT system and in our previous study on rat striatal synaptosomes, performed under comparable conditions, suggest that all DAT's inserted into the C6 cell membrane are functionally active.     

Sibutramine, a serotonin uptake inhibitor, increases dopamine concentrations in rat striatal and hypothalamic extracellular fluid

We measured, using microdialysis, the effects of sibutramine, given intraperitoneally, on brain dopamine and serotonin flux into striatal and hypothalamic dialysates of freely moving rats, and on the uptake of [(3)H]-DA into striatal synaptosomes. For microdialysis experiments, samples collected every 30 min were assayed by high-pressure liquid chromatography, in a single run. Administration of a low dose of sibutramine (2.0 mg/kg, i.p) had no effect on dopamine or serotonin concentrations in striatal dialysates but higher doses increased both: 5 mg/kg increased these concentrations to 196+/-24% (p<0.01) and 221+/-28% (p<0.01) of baseline, respectively; 10 mg/kg increased dopamine to 260+/-66% (p<0.01) and serotonin to 160+/-20% (p<0.05) of baseline. In hypothalamus, the 5 mg/kg sibutramine dose increased the dopamine concentration to 186+/-40% (p<0.05) and that of serotonin to 312+/-86% (p<0.01) of baseline, while the 10 mg/kg (i.p.) dose increased dopamine to 392+/-115% (p<0.01), and serotonin to 329+/-104% (p<0.01) of baseline. In vitro, sibutramine blocked [(3)H]-dopamine uptake into striatal synaptosomes, with an IC(50) value of 3.8 microM. These findings indicate that sibutramine has at least as great an effect on brain extracellular dopamine levels as on brain serotonin, and suggest that the drug's antiobesity action may result from the changes it produces in brain dopamine as well as serotonin metabolism.     

Sex and temporally-dependent effects of methamphetamine toxicity on dopamine markers and signaling pathways

Methamphetamine induces a greater neurodegenerative effect in male versus female mice. In order to investigate this sex difference we studied the involvement of Akt and extracellular signal-regulated kinase (ERK1/2) in methamphetamine toxicity as a function of time post-treatment (30 min, 1 and 3 days). Methamphetamine-induced decreases in dopamine concentrations and dopamine transporter (DAT) specific binding in the medial striatum were similar in female and male mice when evaluated 1 day post-methamphetamine (40 mg/kg). At 3 days post-methamphetamine, striatal dopamine concentration and DAT specific binding continued to decline in males, whereas females showed a recovery with increases in dopamine content and DAT specific binding in medial striatum at day 3 versus day 1 post-methamphetamine. The reduction in striatal vesicular monoamine transporter 2 specific binding observed at 1 and 3 days post-methamphetamine showed neither a sex- nor temporal-dependent effect. Under the present experimental conditions, methamphetamine treatments had modest effects on dopamine markers measured in the substantia nigra. Proteins assessed by Western blots showed similar reductions in both female and male mice for DAT proteins at 1 and 3 days post-methamphetamine. An increase in the phosphorylation of striatal Akt (after 1 day), glycogen synthase kinase 3β (at 1 and 3 days) and ERK1/2 (30 min post-methamphetamine) was only observed in females. Striatal glial fibrillary acidic protein levels were augmented in both females and males at 3 days post-methamphetamine. These results reveal some of the sex- and temporally-dependent effects of methamphetamine toxicity on dopaminergic markers and suggest some of the signaling pathways associated with these responses.     

The acute and subchronic effects of hemantane on [3H]-dopamine reuptake in rat striatal synaptosomes ex vivo

The ex vivo effects of the new antiparkinsonian drug (adamantane derivative) hemantane on the [3H]-dopamine reuptake rate (V) in striatum have been studied upon acute and subchronic administration of the drug in Wistar rats. The [3H]-dopamine reuptake was measured in striatal synaptosomes isolated from rats decapitated 1 hour after drug injection (40 mg/kg, i.p.), and in synaptosomes of rats treated for 7 days with hemantane (single daily dose, 20 mg/kg, i.p.). The acute experiment resulted in a significant (30%) decrease in the apparent Vmax of [3H]-dopamine reuptake, while the value of the apparent Km remained unchanged. The data obtained indicate that hemantane suppresses the DA transporter function within physiological concentration range via noncompetitive mode. In contrast, subchronic drug administration increases by 17% the apparent Vmax. These results demonstrate the active participation of presynaptic transport mechanism in dopamine-positive effects of hemantane.     

Reduced cardiovascular effects of methamphetamine following treatment with selegiline

Selegiline is a specific MAO-B inhibitor. As MAO-B has been shown to be significantly involved in the metabolism of dopamine in certain regions of the primate brain, selegiline has been proposed for use in the treatment of drug addiction. Selegiline is also metabolized in vivo to l-methamphetamine. Therefore, when given in combination with psychostimulants such as d-methamphetamine, there is the potential for adverse effects. To study this possibility, squirrel monkeys were treated with chronic selegiline and tested with two doses of d-methamphetamine (0.1 and 1.0 mg/kg, i.v.). Following at least 7 days of treatment with once daily 0.3 mg/kg i.m. selegiline, the effects of methamphetamine on blood pressure and heart rate were no different than the effects of methamphetamine observed prior to selegiline treatment. However, following at least 10 days of treatment with 1.0 mg/kg i.m. selegiline, the effects of methamphetamine on blood pressure and heart rate were significantly reduced. Both methamphetamine and amphetamine were detected in plasma following chronic selegiline treatment. When monkeys were given an acute selegiline injection prior to methamphetamine, reduced cardiovascular effects were also seen. These results indicate that selegiline can be used safely even in combination with methamphetamine, as the cardiovascular effects of the drug combination were no greater than either drug alone, and were actually reduced at the higher selegiline dose.     

Evidence for an inhibitory presynaptic component of neuroleptic drug action.

1 The action of five neuroleptic drugs (haloperidol, cis-flupenthixol, chlorpromazine, fluphenazine and thioridazine) was studied on the synthesis and release of dopamine from rat striatal synaptosomes. 2. In vitro application of the drugs induced an inhibition of synthesis of [14C]-dopamine from L-[U-14C]-tyrosine and a decrease in the tissue content of [14-C]-dopamine, with IC50 values for the latter effect ranging from 3.6 x 10(-7) to 5.9 x 10(-5) M. The rank of their potency was similar to the order of their clinical effectiveness: haloperidol greater than fluphenazine greater than cis-flupenthixol greater than chlorpromazine greater than thioridazine. Trans flupenthixol was without effect up to a concentration of 10(-4) M. 3 The tissue level and release of GABA were not affected by concentration of the neuroleptics up to 10(-4) M. 4 When the neuroleptics were administered in vivo, changes were also detected in the synthesis and release of [14C-]-dopamine from subsequently prepared synaptosomes. A marked inhibition of the K+-induced increase in [14C]-dopamine synthesis was seen following a dose of 2 mg/kg cis-flupenthixol and haloperidol. At this concentration, haloperidol also increased the control release of [14C]-dopamine and reduced the K+-induced increase in release of [14C]-dopamine. 5 Cis-flupenthixol at a dose of 20 mg/kg reduced the K+-induced release of [14C]-dopamine by 48% and to a lesser extent, that of gamma-aminobutyric acid (GABA, 25%). 6 An inhibitory mode of action is proposed for neuroleptics mediated through a presynaptic mechanism.     

Dopamine transporter activity mediates amphetamine-induced inhibition of Akt through a Ca2+/calmodulin-dependent kinase II-dependent mechanism

The primary mechanism for clearance of extracellular dopamine (DA) is uptake mediated by the dopamine transporter (DAT), which is governed, in part, by the number of functional DATs on the cell surface. Previous studies have shown that amphetamine (AMPH) decreases DAT cell surface expression, whereas insulin reverses this effect through the action of phosphatidylinositol 3-kinase (PI3K). Therefore, it is possible that AMPH causes DAT cell surface redistribution by inhibiting basal insulin signaling. Here, we show in a heterologous expression system and in murine striatal synaptosomes that AMPH causes a time-dependent decrease in the activity of Akt, a protein kinase immediately downstream of PI3K. This effect was blocked by the DAT inhibitor cocaine, suggesting that AMPH must interact with DAT to inhibit Akt. We also showed that AMPH is able to stimulate Ca2+/calmodulin-dependent kinase II (CaMKII) activity, both in the heterologous expression system as well as in murine striatal synaptosomes. The ability of AMPH to decrease Akt activity was blocked by the CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93), but not by its inactive analog 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN92). Furthermore, preincubation with KN93 prevented the AMPH-induced decrease in DAT cell surface expression. Thus, AMPH, but not cocaine, decreases Akt activity through a CaMKII-dependent pathway, thereby providing a novel mechanism by which AMPH regulates insulin signaling and DAT trafficking.     

<Emphasis Type="Italic">d</Emphasis>-Amphetamine responses in catechol-O-methyltransferase (COMT) disrupted mice

Rationale We have earlier found that 1) COMT inhibitors did not enhance amphetamine-induced dopamine efflux into striatal extracellular, that 2) they did not increase dopamine levels in striatal tissue and that 3) they did not potentiate amphetamine-induced turning behavior of hemiparkinsonian rats. Further, when COMT knockout mice were challenged with l-dopa or a dopamine transporter (DAT) inhibitor, an accumulation of dopamine occurred and the neurochemical and locomotor effects of l-dopa and GBR 12909 were modified accordingly.Objective Since DAT inhibitors and amphetamine apparently have different mechanisms of action, we were interested to see how COMT knockout mice would react to d-amphetamine treatment.Methods We measured the effects of d-amphetamine on locomotor activity and on the levels of catecholamines and their metabolites in striatal microdialysis fluid and in striatal, hypothalamic and cortical brain regions of COMT gene disrupted mice. Striatal dopamine receptor binding was also determined.Results After d-amphetamine administration, the DOPAC content in homozygous mice was 3-fold in the striatum, 17- to 18-fold in the cortex and 7- to 8-fold in the hypothalamus higher than in wild-type control mice, and there were no indications of genotype×sex interactions. However, the lack of COMT did not potentiate d-amphetamine-induced dopamine levels in brain tissue or in striatal extracellular fluid. d-Amphetamine-induced (10 mg/kg) hyperlocomotion was less suppressed in male COMT knockout mice than in their wild-type counterparts. Striatal dopamine D₁ and D₂ receptor levels in male mice were not altered by COMT gene disruption.Conclusions Changes in COMT activity modulates dopamine metabolism but the behavioral effects of d-amphetamine in male mice only to a small extent, and this action does not seem to depend on the actual extracellular dopamine concentration. Nor is it mediated through compensatory changes in dopamine D₁ and D₂ receptor levels. In dopaminergic neurons, the contribution of intracellular COMT remains secondary in conditions when dopamine is released by d-amphetamine.