Iptakalim attenuates self-administration and acquired goal-tracking behavior controlled by nicotine.

Iptakalim is an ATP-sensitive potassium channel opener, as well as an α4ß2-containing nicotinic acetylcholine receptor (nAChR) antagonist. Pretreatment with iptakalim diminishes nicotine-induced dopamine (DA) and glutamate release in the nucleus accumbens. This neuropharmacological profile suggests that iptakalim may be useful for treatment of nicotine dependence. Thus, we examined the effects of iptakalim in two preclinical models. First, the impact of iptakalim on the interoceptive stimulus effect of nicotine was evaluated by training rats in a discriminated goal-tracking task that included intermixed nicotine (0.4 mg/kg, SC) and saline sessions. Sucrose was intermittently presented in a response-independent manner only on nicotine sessions. On intervening test days, rats were pretreated with iptakalim (10, 30, 60 mg/kg, IP). Results revealed that iptakalim attenuated nicotine-evoked responding controlled by the nicotine stimulus in a dose-dependent manner. In a separate study, the impact of iptakalim on the reinforcing effects of nicotine was investigated by training rats to lever-press to self-administer nicotine (0.01 mg/kg/infusion) [Dosage error corrected]. Results revealed that pretreatment with iptakalim (1, 3, 6 mg/kg, IV) decreased nicotine intake (i.e., less active lever responding). Neither behavioral effect was due to a non-specific motor effect of iptakalim, nor to an ability of iptakalim to inhibit DA transporter (DAT) or serotonin transporter (SERT) function. Together, these finding support the notion that iptakalim may be an effective pharmacotherapy for increasing smoking cessation and a better understanding of its action could contribute to medication development.

Cocaine-induced increases in vesicular dopamine uptake: role of dopamine receptors.

The vesicular monoamine transporter-2 is the sole transporter responsible for sequestration of monoamines, including dopamine (DA), into synaptic vesicles. Previous studies demonstrate that agents that inhibit DA transporter function, such as cocaine, increase vesicular [(3)H]DA uptake and binding of the ligand [(3)H]dihydrotetrabenazine ([(3)H]DHTBZ), as assessed in vesicles prepared from treated rats. The present studies examine the role of DA receptors in these cocaine-induced effects. Results demonstrate that administration of the D(2) DA receptor antagonist, eticlopride, but not the D(1) DA receptor antagonist, SCH23390, inhibited these cocaine-induced increases. Similar to the effects of cocaine, treatment with the D(2) agonist, quinpirole, increased both vesicular [(3)H]DA uptake and [(3)H]DHTBZ binding. In contrast, administration of the D(1) agonist, SKF81297, was without effect on vesicular [(3)H]DA uptake or [(3)H]DHTBZ binding. Finally, coadministration of quinpirole and cocaine did not further increase vesicular [(3)H]DA uptake or [(3)H]DHTBZ binding when compared with treatment with either agent alone. These data suggest that cocaine-induced increases in vesicular DA uptake and DHTBZ binding are mediated by a D(2) receptor-mediated pathway. Furthermore, results indicate that D(2) receptor activation, per se, is sufficient to increase vesicular DA uptake.

Methamphetamine-induced rapid and reversible changes in dopamine transporter function: an in vitro model.

This laboratory has demonstrated that a single methamphetamine (METH) injection rapidly and reversibly decreases the activity of the dopamine transporter (DAT), as assessed ex vivo in synaptosomes prepared from treated rats. This decrease does not occur because of residual drug introduced by the original injection or nor is it associated with a change in binding of the DAT ligand WIN35428. The purpose of this study was to elucidate the mechanism or mechanisms of this METH effect by determining whether direct application of this stimulant to synaptosomes causes changes in DAT similar to those observed ex vivo. Similar to the ex vivo effect, incubation of striatal synaptosomes with METH decreased DAT activity, but not WIN35428 binding: the effect on activity was not eliminated by repeated washing of synaptosomes. Also, as observed ex vivo, incubation with 3,4-methylenedioxymethamphetamine, but not cocaine or methylphenidate, caused a METH-like reduction in DAT function. The rapid and reversible METH-induced diminution in DAT activity did not occur because of a change in membrane potential, as assessed in vitro and ex vivo by [(3)H]tetraphenylphosphonium accumulation. However, the METH-related decline in DAT function may be attributed to phosphorylation because NPC15437, a protein kinase C inhibitor, attenuated the METH-induced decline in DAT function. Similarities between previously reported effects ex vivo of a single METH injection on serotonin and norepinephrine transporter function and effects of direct METH application in vitro were also found. Together, these data demonstrate that the in vitro incubation model mimics the rapid and reversible effects observed after a single METH injection.

Regulation of the vesicular monoamine transporter-2: a novel mechanism for cocaine and other psychostimulants.

The plasmalemmal dopamine (DA) transporter (DAT) is a principal site of action for cocaine. This report presents the novel finding that in addition to inhibiting DAT function, cocaine administration rapidly alters vesicular DA transport. Specifically, cocaine treatment abruptly and reversibly increased both the V(max) of DA uptake and the B(max) of vesicular monoamine transporter-2 (VMAT-2) ligand (dihydrotetrabenazine) binding, as assessed ex vivo in purified rat striatal synaptic vesicles. Selective inhibitors of the DAT (amfonelic acid and GBR12935), but not the plasmalemmal serotonin transporter (fluoxetine), also increased vesicular DA uptake. Moreover, DA depletion resulting from administration of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine had cocaine-like effects. Conversely, administration of the DA-releasing agent methamphetamine rapidly decreased vesicular uptake. Taken together, these data demonstrate for the first time ex vivo that cocaine treatment rapidly alters vesicular monoamine transport, and suggest that alterations in cytoplasmic DA concentrations contribute to stimulant-induced changes in vesicular DA uptake. Hence, the VMAT-2 may be an important target for developing strategies to treat not only cocaine addiction but also other disorders involving alterations in neuronal DA disposition, including Parkinson's disease.

Methamphetamine decreases mouse striatal dopamine transporter activity: roles of hyperthermia and dopamine.

Multiple methamphetamine administrations rapidly decrease rat striatal dopamine transporter activity. To determine the species specificity of this phenomenon, the present studies examined effects of this stimulant on the dopamine transporter in mice. As in rats, multiple methamphetamine injections rapidly reduced striatal dopamine transporter activity; a decrease that was partially reversed 24 h later. Moreover, methamphetamine decreased binding of the dopamine transporter ligand, WIN35428, but to a lesser degree than the change in dopamine transporter function. These decreases did not appear to result from residual methamphetamine introduced by the original drug treatment. As in rats, hyperthermia contributed to this phenomenon. Unlike in rats, a role for dopamine was not observed in mice as dopamine depletion, resulting from alpha-methyl-p-tyrosine pretreatment, did not prevent this decrease. In addition, unlike in rats, pretreatment with either a dopamine D1 or D2 receptor antagonist (SCH23390 or eticlopride, respectively) did not attenuate the methamphetamine-induced reduction in dopamine uptake. These findings demonstrate both similarities and differences in the acute effects of methamphetamine on dopamine transporter function in mice and rats, and suggest the mouse as an additional model for assessing the acute effects of methamphetamine on the dopamine transporter.

Methamphetamine-induced rapid decrease in dopamine transporter function: role of dopamine and hyperthermia.

Single and multiple high-dose administrations of methamphetamine (METH) differentially decrease dopamine (DA) transporter (DAT) function, as assessed by measuring [(3)H]DA uptake into rat striatal synaptosomes prepared 1 h after treatment. Prevention of METH-induced hyperthermia attenuated the decrease in DAT activity induced by multiple injections of the stimulant. Likewise, this decrease was attenuated by previous depletion of striatal DA levels using alpha-methyl-p-tyrosine (alphaMT) or pretreatment with the D1 and D2 antagonists SCH-23390 and eticlopride, respectively. However, METH-induced hyperthermia was also blocked by alphaMT and eticlopride. Reinstatement of hyperthermia to alphaMT- or eticlopride-pretreated rats partially restored the METH-induced decrease in DAT activity. In contrast, neither prevention of METH-induced hyperthermia depletion of DA, nor DA antagonists altered the decrease in DAT function induced by a single administration of METH. Pretreatment with the antioxidant N-t-butyl-alpha-phenylnitrone prevented part of the decrease in DAT function associated with multiple, but not a single, METH injections. Although not tested directly, additional data presented here suggest that the reduction in DAT activity induced by a single METH administration constitutes a part of the total reduction observed immediately after multiple administrations. Taken together, the results indicate that DA, hyperthermia, and oxygen radicals contribute to a component of the rapid decrease in DAT function induced by multiple injections of METH but do not appear to be associated with the reduction induced by a single administration of the stimulant.

Age-dependent differential responses of monoaminergic systems to high doses of methamphetamine.

Abuse of methamphetamine (METH) by adolescents is a major public health issue in the U.S.A. Because of the neurotoxic potential of METH, we examined the response of CNS monoaminergic systems in young (adolescent) animals [postnatal day (PND) 40] to high-dose treatments (10 mg/kg, four injections, 2-h intervals) of this drug and contrasted these effects to those seen in older (young adult) rats (PND 90). Consistent with previous reports, we observed that PND 40 animals did not manifest the long-term (7-day) deficits in extrapyramidal dopamine (DA) parameters observed in PND 90 rats. In contrast, METH-induced rapid (1-h) reduction in the activity of striatal DA transporters occurred in both age groups. In addition, both persistent (7-day) and rapid (1-h) deficits in serotonergic systems (measured as reductions in tryptophan hydroxylase activity) were observed in PND 40 and 90 rats. Age-related differences in METH-induced hyperthermia did not appear to be a principal cause for our observations; however, age-dependent pharmacokinetics of this drug might have contributed to the differential METH monoaminergic responses by PND 40 and 90 animals.

The effects of methamphetamine on serotonin transporter activity: role of dopamine and hyperthermia.

Multiple administrations of methamphetamine (METH) rapidly decreased serotonin (5HT) transporter (SERT) function in rat striatum and hippocampus. The purpose of this study was to identify the mechanisms/ factors contributing to this METH-induced decrease in SERT function. Multiple high-dose METH injections rapidly decreased 5HT uptake without altering binding of the 5HT transporter ligand paroxetine. Hyperthermia contributed to this deficit in transporter function in striatum and hippocampus, as prevention of METH-induced hyperthermia attenuated this decrease. A role for dopamine (DA) was suggested by findings that pretreatment with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine, the D1 antagonist SCH-23390, or the D2 antagonist eticlopride attenuated the METH-induced decrease in striatal, but not hippocampal, SERT activity. These effects were independent of the ability of these DA-antagonizing drugs to prevent METH-induced hyperthermia. These results suggest that DA contributes to the decrease in SERT function caused by multiple METH injections in the striatum, but not hippocampus, and that hyperthermia facilitates these deficits in SERT function in both brain regions. In contrast, the response of SERT to a single administration of METH was DA and hyperthermia independent. These findings suggest that the mechanisms/ factors involved in decreasing SERT activity after a single administration of METH are distinct from that caused by multiple administrations.

Differential effects of stimulants on monoaminergic transporters: pharmacological consequences and implications for neurotoxicity.

Many psychostimulants alter plasmalemmal monoaminergic transporter function. Some, such as cocaine, prevent the reuptake of newly released dopamine, serotonin or norepinephrine into their associated neurons. Others, such as the amphetamines, facilitate release of these transmitters into the extraneuronal space by causing a reversal of function of these carrier proteins. An understanding of how psychostimulants regulate the function of not only plasmalemmal, but also vesicular monoamine transporter function is important to appreciate the pharmacological and sometimes neurotoxic consequences of administering these drugs, as well as the physiological regulation of these carrier proteins. Hence, this review will describe recent ex vivo studies investigating the rapid and differential affects of several stimulants on both plasmalemmal and vesicular monoamine transporter function.

Methamphetamine rapidly decreases vesicular dopamine uptake.

Vesicular sequestration is important in the regulation of cytoplasmic concentrations of monoamines such as dopamine. Moreover, recent evidence suggests that increases in cytoplasmic dopamine levels, perhaps attributable to changes in vesicular monoamine transporter function, contribute to methamphetamine-induced dopaminergic deficits. Hence, we examined whether striatal vesicular uptake is altered following methamphetamine treatment. Multiple administrations of methamphetamine rapidly (within 1 h) decreased vesicular dopamine uptake and dihydrotetrabenazine binding, an effect that (a) persisted at least 24 h, (b) was associated with dopamine and not serotonin neurons, and (c) was unrelated to residual drug introduced by the original methamphetamine treatment. These data suggest that methamphetamine rapidly decreases vesicular monoamine transporter function in dopaminergic neurons, a phenomenon that may be associated with the long-term damage caused by this stimulant.

Differential effects of methamphetamine on Na(+)/Cl(-)-dependent transporters.

It has been demonstrated that methamphetamine (METH) administration affects Na(+)/Cl(-)-dependent transporters; for example, METH treatment rapidly and reversibly decreases dopamine (DA) and serotonin (5HT) transporter function in rat striatum in vivo, as assessed in synaptosomes prepared from METH-treated rats. Because acute effects of METH on other transporters within this family have been less studied, the responses of norepinephrine (NE) and gamma-aminobutyric acid (GABA) transporters to METH administration(s) were determined. Both single and multiple METH administrations inhibited hippocampal NE uptake 1 h after METH treatment(s). In contrast, striatal GABA uptake was not affected by either treatment paradigm. The effects observed after both single and multiple METH administrations on NE transporters were attributable to increases in K(m,) with no changes in V(max); effects that were eliminated by repeated washing of the synaptosomes. These 'washout' data suggest that residual METH introduced by the in vivo subcutaneous injection(s) directly reduced NE transporter activity in the in vitro assay and that, unlike DA and 5HT transporters, METH did not indirectly alter NE transporter function. Taken together, these data demonstrate differences in the responses of NE, GABA, DA, and 5HT transporters to METH treatment.

Prolactin regulation of tuberoinfundibular dopaminergic neurons: immunoneutralization studies.

The purpose of this study was to determine the effects of acute hypoprolactinemia on tuberoinfundibular dopamine (DA) neurons using a rabbit anti-rat prolactin antiserum (PRL-AB) to immunoneutralize circulating prolactin under basal conditions and at various times after haloperidol-induced hyperprolactinemia. The specificity of PRL-AB for prolactin was determined by examining the ability of unlabelled hormone to displace binding of 125I-labelled prolactin to PRL-AB. Tuberoinfundibular DA neuronal activity was estimated by measuring the concentrations of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence which contains terminals of these neurons. Systemic (i.v.) administration of 200 microl of PRL-AB decreased plasma prolactin concentrations below detectable levels for at least 4 h, and this was accompanied by a pronounced decrease in DOPAC concentrations in the median eminence of females, but not males. Central (i.c.v.) administration of 2 microl PRL-AB diluted up to 1:100 mimicked the inhibitory effect of systemic administration of PRL-AB on median eminence DOPAC concentrations suggesting that the tonic stimulatory effect of prolactin on the basal activity of tuberoinfundibular DA neurons in females occurs via a central site of action. In male rats, blockade of anterior pituitary DA receptors with haloperidol (1 mg/kg; s.c.) caused an prompt (by 1 h) increase in plasma prolactin concentrations which was maintained for at least 12 h. Haloperidol-induced hyperprolactinemia also caused a delayed (at 6 and 12 h) increase in median eminence DOPAC concentrations in these animals which was blocked by PRL-AB. Exposure of rats to initial priming periods of endogenous hyperprolactinemia of up to 6 h duration (followed by 6 h or more of PRL-AB-induced hypoprolactinemia) failed to alter median eminence DOPAC concentrations unless prolactin exposure was reinstated by an i.c.v. injection of prolactin. These results confirm that prolactin mediates the stimulatory effects of haloperidol on tuberoinfundibular DA neurons, and reveal that delayed induced activation of these neurons by prolactin is dependent upon a priming period of sustained hyperprolactinemia longer than 3 h for initiation and maintenance of this response.

Differential effects of psychostimulants and related agents on dopaminergic and serotonergic transporter function.

High-dose administrations of amphetamine, methamphetamine, cathinone, methcathinone or methylenedioxymethamphetamine rapidly decrease dopamine and serotonin transporter function in vivo, as assessed in striatal synaptosomes obtained from drug-treated rats. In contrast, high-dose injections of fenfluramine, cocaine or methylphenidate had little or no effect on the activity of these transporters. Interestingly, the capacity of these agents to directly alter dopamine and serotonin uptake, as assessed in vitro by direct application to rat striatal synaptosomes, did not predict their potential to modulate transporter activity following in vivo administration. These findings demonstrate heretofore-unreported differences in the effects of these agents on monoamine transporter function, and a distinction between drug effects after direct application in vitro vs. administration in vivo.

Oxygen radicals differentially affect Na+/Cl(-)-dependent transporters.

Incubation with the oxygen radical-generating enzyme, xanthine oxidase, dramatically reduced striatal dopamine transporter activity, but was unexpectedly without effect on rat hippocampal norepinephrine uptake. To determine whether environmental differences between the striatum and hippocampus contributed to this lack of oxidative effect on norepinephrine transporters, synaptosomal gamma-aminobutyric acid (GABA) uptake was assessed in both regions. Xanthine oxidase similarly decreased [3H]GABA uptake in both the striatum and hippocampus, supporting the conclusion that environmental differences did not account for the lack of effect on norepinephrine transport. These data suggest that norepinephrine transporters are less vulnerable than other Na+/Cl(-)-dependent transporters to oxidative inactivation.

Differential regional effects of methamphetamine on the activities of tryptophan and tyrosine hydroxylase.

Administration of high doses of methamphetamine (METH) produces both short- and long-term enzymatic deficits in central monoaminergic systems. To determine whether a correlative relationship exists between these acute and long-term consequences of METH treatment, in the present study we examined the regional effects of METH on tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH) activities in various regions of the caudate nucleus, nucleus accumbens, and globus pallidus. A single METH administration decreased TPH activity 1 h after treatment in the globus pallidus, in the nucleus accumbens, and throughout the caudate; in the anterior caudate, the ventral-medial was more affected than the dorsal-lateral region. In contrast, TH activity was not decreased in either the caudate or the globus pallidus after a single METH administration; however, it was altered in the nucleus accumbens. Seven days after multiple METH administrations, TH and TPH activities were decreased in most caudate regions but not in the nucleus accumbens or globus pallidus. These data demonstrate that (1) the effects of METH on TPH and TH vary regionally; and (2) the short-term and long-term regional responses of TPH to METH in the caudate and globus pallidus correlated. In contrast, METH-induced acute TH responses did not predict the long-term changes in TH activity.

Nature of methamphetamine-induced rapid and reversible changes in dopamine transporters.

The nature of methamphetamine-induced rapid and transient decreases in dopamine transporter activity was investigated. Regional specificity was demonstrated, since [3H]dopamine uptake was decreased in synaptosomes prepared from the striatum, but not nucleus accumbens, of methamphetamine-treated rats. Differences among effects on dopamine transporter activity and ligand binding were also observed, since a single methamphetamine administration decreased [3H]dopamine uptake without altering [3H]WIN35428 ([3H](-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate) binding in synaptosomes prepared 1 h after injection. Moreover, multiple methamphetamine injections caused a greater decrease in [3H]dopamine uptake than [3H]WIN35428 binding in synaptosomes prepared I h after dosing. Finally, decreases in [3H]dopamine uptake, but not [3H]WIN35428 binding, were partially reversed 24 h after multiple methamphetamine injections. Western blotting indicated that saline- and methamphetamine-affected dopamine transporters co-migrated on sodium dodecyl sulfate (SDS) gels at approximately 80 kDa, and that acute, methamphetamine-induced decreases in [3H]dopamine uptake were not due to loss of dopamine transporter protein. These findings demonstrate heretofore-uncharacterized features of the acute effect of methamphetamine on dopamine transporters.

Methamphetamine-induced rapid and reversible reduction in the activities of tryptophan hydroxylase and dopamine transporters: oxidative consequences?

Treatment with high doses of methamphetamine (METH) results in dramatic changes in extrapyramidal monoaminergic systems. Elevated concentrations of extracellular dopamine (DA), caused by METH administration, are thought to contribute to these effects due to the oxidative potential of this reactive catecholamine. According to this hypothesis monoaminergic cellular elements, which are vulnerable to oxidative modification, may be especially sensitive to high-dose METH treatments. We confirmed this possibility by observing that both tryptophan hydroxylase (the synthesizing enzyme for serotonin) and the DA transporter, proteins particularly susceptible to oxidative modification, were rapidly (within 30 min), but reversibly (returned to control levels by 36 hr) inactivated by a single administration of METH. These findings suggest that there also may be other cellular elements similarly altered by METH treatment due to oxidative mechanisms.

Methamphetamine treatment rapidly inhibits serotonin, but not glutamate, transporters in rat brain.

Previous studies have demonstrated that multiple methamphetamine (METH) administrations rapidly and reversibly decrease dopamine transporter activity assessed in striatal synaptosomes. A role for reactive oxygen species was suggested by findings that: (1) METH treatment increases the formation of oxygen radicals in vivo; and (2) oxygen radicals, generated by the enzyme xanthine oxidase, attenuate dopamine uptake in vitro. To test the selectivity of transporter responses, the present study examined effects of METH and xanthine oxidase on [3H]serotonin ([3H]5HT) and [3H]glutamate transport into striatal synaptosomes. Multiple doses of METH, or incubation with xanthine oxidase, rapidly attenuated [3H]5HT transport; an effect attributable to a decrease in Vmax. The METH-induced decrease in transport activity completely recovered by 24 h, but was decreased again 1 week later. In contrast, [3H]glutamate transport was essentially unchanged after METH treatment or incubation with xanthine oxidase. These findings indicate that: (1) METH causes a rapid and reversible decrease in 5HT transporter activity; and (2) glutamate transporters are less susceptible than 5HT transporters to effects of reactive species or METH treatment.

3-4-Methylenedioxymethamphetamine-induced acute changes in dopamine transporter function.

The acute effects of the amphetamine designer drug, 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy'), on dopamine transporter function in rat striatum were investigated and compared to other psychostimulants known to influence monoaminergic systems. A single MDMA injection (10-20 mg/kg; s.c.) caused a dose-related decrease in [3H]dopamine uptake into striatal synaptosomes prepared 1 h after MDMA administration. This rapid effect on [3H]dopamine uptake returned to control levels 24 h after treatment. A single administration of other amphetamine analogs, such as methamphetamine (15 mg/kg; s.c.), p-chloroamphetamine (10 mg/kg; i.p.) or methcathinone (30 mg/kg; s.c.), also rapidly decreased striatal [3H]dopamine uptake. In contrast, a single or multiple administrations of cocaine (30 mg/kg; i.p.) had no effect on [3H]dopamine transport into striatal synaptosomes. These changes in dopamine transporter activity by the amphetamine analogs may occur via reactive oxygen species-mediated mechanisms.

Oxygen radicals diminish dopamine transporter function in rat striatum.

Incubation of striatal synaptosomes with the oxygen radical generating enzyme, xanthine oxidase, decreased [3H]dopamine uptake: an effect attributable to a decreased Vmax. Concurrent incubation with the superoxide radical scavenger, superoxide dismutase, abolished the xanthine oxidase-induced decrease. These results indicate that, like methamphetamine administration in vivo, reactive oxygen species diminish dopamine transporter function in vitro. The significance of these findings to mechanisms responsible for effects of methamphetamine is discussed.