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Effects of haloperidol metabolites on neurotransmitter uptake and release: possible role in neurotoxicity and tardive dyskinesia
Brain Research 788 Ž1998. 215–222
Research report
Effects of haloperidol metabolites on neurotransmitter uptake and release: possible role in neurotoxicity and tardive dyskinesia Alesia M. Wright, Jeffrey Bempong, Michael L. Kirby, Rebecca L. Barlow, Jeffrey R. Bloomquist
)
Department of Entomology, Neurotoxicology Laboratory, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061, USA Accepted 23 December 1997
Abstract This research explored the effects of haloperidol ŽHP. metabolites on biogenic amine uptake and release, and compared them to those of MPTP and its toxic metabolite, MPPq. In synaptosome preparations from mouse striatum and cortex, the HP metabolites haloperidol pyridinium ŽHPPq., reduced haloperidol pyridinium ŽRHPPq., and haloperidol tetrahydropyridine ŽHPTP. inhibited the presynaptic uptake of dopamine and serotonin, with greater affinity for the serotonin transporter. HPPq was the most potent inhibitor of dopamine uptake, and HPTP of serotonin uptake, both with IC 50 values in the low micromolar range. RHPPq was less active than the other metabolites, but was more active than the parent compound, HP. Inhibition of uptake was reversed when free drug was removed by centrifugation and then resuspension of the synaptosomes in fresh buffer, suggesting that inhibition of uptake was due to interaction with the transporters and was not due to irreversible cytotoxicity. HPPq showed noncompetitive inhibition of both serotonin and dopamine uptake, suggesting that it has a relatively slow dissociation rate for its interaction with the transporter proteins. In experiments on amine release, HPPq and HPTP were four-fold less potent than MPPq for releasing preloaded dopamine from striatal synaptosomes, and only MPPq-dependent release was antagonized by the uptake blocker, mazindol. In contrast, RHPPq displayed little ability to release either amine neurotransmitter. HPTP was about two-fold more potent than MPPq for releasing serotonin from cortical synaptosomes, whereas HPPq was less active than MPPq. The specific serotonin transport blocker fluoxetine was only able to antagonize release induced by MPPq. These results suggest that HP metabolites bind to the transporters for dopamine and serotonin, but are not transporter substrates. In contrast to their potent effects on amine release, HPPq and HPTP were unable to release preloaded GABA from cortical synaptosomes. The implications of these results concerning a possible role of HP metabolites in the development of tardive dyskinesia are discussed. q 1998 Elsevier Science B.V. Keywords: Schizophrenia; Parkinsonism; Haloperidol pyridinium; Reduced haloperidol pyridinium; Haloperidol tetrahydropyridine
1. Introduction Tardive dyskinesia ŽTD. is a well known side effect of haloperidol ŽHP, Fig. 1. treatment that is slow to develop and often irreversible w14x. TD has been attributed to the supersensitivity of dopamine receptors w7x, but this mechanism is not consistent with a number of factors documented in TD patients w14x. Rollema et al. w20x provided evidence that a key metabolite of HP, haloperidol pyridinium ŽHPPq, Fig. 1., shared some structural similarity and toxic actions with MPPq and suggested that HPPq
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0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 5 5 1 - 5
Fig. 1. Structures of the compounds used in this study, along with established or hypothesized metabolic reactions of haloperidol responsible for the three metabolites. The butyrophenone group ŽR. is unchanged in HP, HPTP, and HPPq, but is reduced in RHPPq.
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may induce TD by causing neuronal injury via nerve terminal transport and inhibition of mitochondrial respiration. In a related study, Bloomquist et al. w3x demonstrated that HPPq was, in fact, cytotoxic to cultured dopaminergic and serotonergic mesencephalic neurons. In addition, MPPq and HPPq blocked the uptake of both serotonin and dopamine into mouse brain synaptosomes, suggesting an interaction with the neuronal transporters for these biogenic amines w3x. These findings were confirmed and extended in subsequent studies w10–12x. Thus, the available data suggest that HPPq has a mode of action similar to that of MPPq, and that the MPTPrMPPq model of neurotoxicity may be valid for assessing the role of HP metabolites in the development of TD. An important component of the action of MPPq is its specific uptake by dopaminergic nerve terminals w16x. Thus, comparative studies were initiated on the inhibitory potency, reversibility, and kinetic nature of the interaction of HPPq and other HP metabolites ŽHPTP and RHPPq, Fig. 1. with the dopamine and serotonin transporters of mouse brain synaptosomes. We also assessed the ability of HP metabolites to release w 3 Hxdopamine and w 3 Hxserotonin from preloaded synaptosomes in the presence and absence of the uptake blockers mazindol and fluoxetine, in order to determine the role of transporter-mediated uptake in the actions of these compounds. Additional studies tested the ability of these compounds to release preloaded GABA from cortical synaptosomes as a measure of the specificity of their effects. Preliminary versions of these results have appeared w2,4x.
2. Materials and methods 2.1. Chemicals and animals Fluoxetine, mazindol, HP, HPTP, HPPq, RHPPq, MPTP, MPPq and pargyline were kindly provided by Neal Castagnoli, Jr., Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA. Veratridine ŽVTD., rotenone ŽROT., bovine serum albumin Žfraction V., Coomassie brilliant blue G250, and phosphoric acid were purchased from Sigma Chemical ŽSt. Louis, MO.. Samples of w 3 Hxdopamine Ž20 Cirmmol. and w 3 HxGABA Ž88 Cirmmol. were purchased from New England Nuclear ŽDuPont, Wilmington, DE.. w 3 HxSerotonin Ž11 Cirmmol. was purchased from Amersham ŽArlington Heights, IL.. Male ICR mice were obtained from Harlan– Sprague–Dawley ŽRaleigh-Durham, NC.. 2.2. Preparation of synaptosomes Procedures for isolating and incubating synaptosomes were essentially those described by Bloomquist et al. w3x.
Mice were sacrificed by rapid cervical dislocation and the brain dissected to yield cortical or striatal tissue for studies on the uptakerrelease of serotonin or dopamine, respectively. Following dissection, the tissue was homogenized in 4 ml of sucrose buffer Ž0.32 M sucrose and 2 mM HEPES, pH 7.4.. The homogenate was then centrifuged for 10 min at 2000 = g. After the initial spin, the supernatant was removed and centrifuged for 10–30 min at 12,000 = g. The final pellet containing the synaptosomes was rinsed gently and resuspended in buffer containing ŽmM.: NaCl Ž125., KCl Ž5., CaCl 2 Ž2., MgCl 2 Ž1., sucrose Ž10., pargyline Ž0.05., ascorbate Ž0.1., Tris–HCl Ž50. at pH 7.4. For calcium-free buffers, CaCl 2 was omitted from the saline, with no substitutions, as the small drop in osmolarity was considered insignificant. 2.3. Inhibition of dopamine and serotonin uptake For these studies, experimental conditions were adjusted to optimize, as much as possible, measurement of the inhibition of uptake resulting from interactions of drugs with the transporters, and not loss of uptake due to enhanced transmitter release or cytotoxic effects w18x. Incubations were done at room temperature in order to reduce the specific transport of MPPq or other compounds into the nerve terminal and thereby minimize any effects on mitochondrial function, since the thermodynamic properties of the transporter results in less efficient uptake of substrates at room temperature w5x. In addition, calcium-free buffer was also used in these experiments to minimize any contributions of calcium-dependent leakage. Synaptosomes were exposed to drug dissolved in saline, DMSO, or a DMSOrsaline mixture. Control incubates received vehicle alone. All drug treatments were incubated for at least 10 min at room temperature, which was sufficient time for the compounds to come to equilibrium w3x. Treated synaptosomes were then exposed to w 3 Hxserotonin Ž1 m Ci, 90 nM. or w 3 Hxdopamine Ž1 m Ci, 20 nM. for a 5 min incubation at 378C. Uptake was terminated by the addition of 3 ml of ice-cold buffer, the incubate was poured onto a Whatman GFrB glass fiber filter under vacuum, and then washed twice with 3 ml of ice-cold wash buffer Žcalcium-free uptake buffer without pargyline or ascorbate.. The filters were air dried, scintillation fluid added ŽScintiVerse, Fisher Scientific., and the radioactivity trapped in the synaptosomes counted by liquid scintillation spectrometry. Treatments were typically replicated three times on different synaptosomal preparations with three determinations in each replicate. Inhibition curves were plotted and analyzed by computer ŽInPlot 4.0 or Prism 2.0a, both from GraphPad Software, San Diego, CA.. Curves were compared for the maximum level of inhibition, as well as inhibitory potency as given by IC 50 values and the 95% confidence intervals of the IC 50 , when fit to a standard sigmoidal four parameter logistic equation.
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2.4. Recentrifugation protocol for assessing reÕersibility of transport inhibition
Table 1 Inhibition of serotonin and dopamine uptake under standard and drug removal Žrecentrifugation. conditions
A recentrifugation protocol was designed to confirm the extent to which inhibition of uptake involved interaction with the neurotransmitter transporters, as opposed to irreversible poisoning of the synaptosomes. Synaptosomes in calcium-free buffer were batch-exposed to compounds for 10 min at room temperature and each treatment group was then recentrifuged at 12,000 = g for 10 min. The supernatant containing unbound drug was discarded and the tissue was resuspended in drug-free uptake buffer. Aliquots of synaptosomes were then challenged with w 3 Hxserotonin or w 3 Hxdopamine and were incubated, filtered, washed, counted, and analyzed as described above.
Compound
2.5. Assay for competitiÕer noncompetitiÕe interaction of HPP q with amine transporters
min at 378C, and then centrifuged at 12,000 = g for 10 min to pellet the synaptosomes. The supernatant was discarded and the synaptosomes resuspended in fresh buffer with or without calcium ions. The vesicles were aliquoted into tubes, treated with drugs Žcontrols received vehicle alone., and incubated at 378C for 10 min. Each incubate then received 3 ml of wash buffer at 378C, was filtered under vacuum, and washed an additional 3 times with 378C buffer. For quantitation of label remaining in the synaptosomes, vacuum filtration and liquid scintillation spectrometry were employed as previously described. Extent of release and EC 50 values were determined from an iterative fit to a sigmoidal, four parameter logistic equation, as described above.
The kinetic nature of the interaction of HPPq with aminergic transporters was investigated by incubating cortical synaptosomes with 10 m M HPPq and striatal synaptosomes with 60 m M HPPq along with increasing amounts of w 3 Hxserotonin or w 3 Hxdopamine, respectively. Aliquots of labeled transmitter with and without HPPq were added to synaptosomes in calcium-free buffer and incubated at 378C. Ice-cold buffer Ž3 ml. was then added to stop uptake and the synaptosomes were filtered, washed, and counted as described previously. For calculation of uptake rates on a per mg protein basis, the protein content of the membrane preparation was determined using the method of Bradford w6x. Uptake curves with and without HPPq were compared with regard to Vmax and K m values following iterative fit to the same sigmoidal, four parameter logistic equation used for inhibition studies. 2.6. Stimulated release of preloaded transmitters Synaptosomes from cortex and striata were prepared as previously described. Aliquots of vesicles were incubated with 90 nM w 3 Hxserotonin or 20 nM w 3 Hxdopamine for 5
Dopamine
Serotonin
IC 50 , mM
IC 50 , mM
MPTP 39.25 MPPq 3.98 MPPq Žrecentrifugation. 55.63 HPTP 258.4 HPPq 22.4 HPPq Žrecentrifugation. y RHPPq 65.96 Fluoxetine y
95% C.L.)
95% C.L.)
30.30–50.83 0.29 0.21–0.40 3.05–5.19 3.04 1.45–6.38 41.50–74.59 60.45 6.3–583 147.5–452.6 1.19 0.83–1.72 11.31–43.21 4.43 3.33–5.88 y 44.60 22.60–88.02 63.83–68.16 10.01 1.65–60.6 y 0.16 0.09–0.27
)95% C.I.s95% confidence limits.
3. Results 3.1. Inhibition of dopamine and serotonin uptake Synaptosomes prepared from striatal tissue were used to evaluate inhibitory effects of MPTP, MPPq, HP, HPTP, HPPq and RHPPq on dopamine uptake. In nearly all cases, compounds showed inhibition of about 90% at maximally effective concentrations, where the residual
Fig. 2. Dose–response relationships for the inhibition of dopamine ŽA. and serotonin ŽB. transport. Each data point represents the mean Ž"S.E.M.. of triplicate replications with three determinations in each replicate. Lack of error bars indicates that the S.E.M. resides within the size of the symbol.
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Fig. 3. Dose–response curves under standard and recentrifugation conditions for MPPq-dependent inhibition of dopamine uptake ŽA. and HPPq-dependent inhibition of serotonin uptake ŽB.. Each data point represents the mean Ž"S.E.M. of triplicate replications with three determinations in each replicate. Lack of error bars indicates that the S.E.M. resides within the size of the symbol.
probably represents nonspecific binding of label to the synaptosomes. MPPq ŽFig. 2A. was the most potent and effective blocker of dopamine uptake, showing 91% maximal inhibition and an IC 50 value of about 4 m M ŽTable 1.. For comparison, MPTP had an inhibitory potency nearly 10-fold less than that of MPPq. The HP derivatives were less potent inhibitors of dopamine uptake ŽFig. 2A, Table 1., with a rank order of potency of HPPq) RHPPq) HPTP) HP. HPPq was over five-fold less active than MPPq and RHPPq was about three-fold less active for inhibiting dopamine uptake than HPPq. HPTP and HP were the least active compounds. Only a partial inhibition curve could be generated for HPTP, and it showed a maximal effect of 47% inhibition ŽFig. 2A.. HP was virtually inactive, showing only 15% inhibition at 300 m M ŽFig. 2A.. All the compounds were more potent inhibitors of serotonin uptake ŽFig. 2B.. MPTP was nearly as active as the standard uptake inhibitor, fluoxetine, and was about 10-fold more active than the pyridinium metabolite, MPPq ŽTable 1.. Similarly, among the HP metabolites, HPTP was the most potent inhibitor of serotonin uptake ŽTable 1. and was over 200-fold more active against serotonin uptake than dopamine uptake. The potency of HPPq for inhibiting
serotonin uptake was four-fold less than HPTP, but was comparable to that of MPPq in this system. The reduced pyridinium, RHPPq, was about three-fold less active than HPPq and MPPq, but was over six-fold more effective as a blocker of serotonin uptake than dopamine uptake ŽTable 1.. In addition, RHPPq showed a maximal inhibition of serotonin transport of 77%, which was slightly less than the other compounds ŽFig. 2B.. Again, HP was the least active compound, but was more effective as an inhibitor of serotonin uptake than dopamine uptake, showing about 50% inhibition at 300 m M ŽFig. 2B.. 3.2. Interaction with the transporter assessed by recentrifugation To determine whether the inhibitory actions of these compounds occurred via binding to the transporter, as opposed to irreversible cytotoxicity, each sample was preincubated with drug, and then recentrifuged and resuspended in fresh buffer to remove free drug from the system. For MPPq, removal of free drug following a 40 min incubation at room temperature caused a large shift to the right in the potency curve for inhibition of dopamine uptake ŽFig. 3A.. Nonlinear regression analysis found that
Fig. 4. Noncompetitive inhibition of dopamine and serotonin high affinity uptake by HPPq. Each data point represents the mean Ž"S.E.M.. of triplicate determinations measured in one or more experiments. Lack of error bars indicates that the S.E.M. resides within the size of the symbol. Inset: kinetic values for uptake in the presence or absence of HPPq. Numbers in parentheses beneath the values are the corresponding 95% confidence limits. The asterisk next to HPPq Vma x values denotes a statistically significant decrease Ž p - 0.05. compared to control values, due to nonoverlap of the 95% confidence limits.
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Fig. 5. Survey experiments on release of dopamine ŽA. and serotonin ŽB. under calcium free conditions. Data are expressed as the mean % retention of label Ž"S.E.M.., where the control is normalized to 100%. MPPq and the haloperidol metabolites were incubated at the concentrations given below the bars. Identical drug incubations were performed in the additional presence of 10 m M mazindol ŽMaz. or 100 m M fluoxetine ŽFluox. as labeled at the top of the bars.
the potency of MPPq was reduced about 20-fold ŽTable 1.. Preincubation times of 10 and 20 min also shifted the curve to the right to about the same extent Ždata not shown.. In other experiments, both the HPPq and MPTP inhibition curves for dopamine uptake also displayed shifts to the right Ždata not shown., suggesting that these compounds are also directly interacting with the transporter under these conditions. Similar effects were observed in recentrifugation experiments applied to serotonin uptake. Removal of free HPPq by recentrifugation produced a parallel shift to the right in the dose–response curve ŽFig. 3B. and an IC 50 value that was 10-fold higher than under standard conditions ŽTable 1.. Similarly, the MPPq dose– response curve also displayed a parallel shift to the right and an IC 50 that was 14-fold higher than that obtained under standard conditions ŽTable 1.. 3.3. NoncompetitiÕe interaction of HPP q with aminergic transporters Dose–response curves for serotonin and dopamine uptake were run in the absence and presence of HPPq to determine if the inhibition was competitive ŽFig. 4A,B.. These studies employed concentrations of HPPq that were approximately three times the IC 50 value of this compound for inhibiting dopamine and serotonin uptake. HPPq Ž60
m M. was a noncompetitive inhibitor of dopamine transport ŽFig. 4A., causing a statistically significant reduction Ž37%. in Vmax with virtually no effect on K m ŽFig. 4A, inset.. HPPq Ž10 m M. displayed similar noncompetitive effects on serotonin uptake ŽFig. 4B., significantly reducing maximal uptake Ž Vmax . of serotonin about 37%, with little change in the K m ŽFig. 4B, inset.. 3.4. Release of labeled transmitters from preloaded synaptosomes Survey experiments on the ability of HP metabolites and MPPq to release preloaded dopamine and serotonin are shown in Fig. 5. MPPq released virtually all the dopamine following incubation at 10 m M, and this release was blocked by the dopamine uptake antagonist, mazindol. HPTP was about as active as MPPq for releasing dopamine, while HPPq was slightly less effective and RHPPq was inactive. With the HP metabolites, however, preincubation with mazindol had no effect on release. MPPq released about 70% of preloaded serotonin and this effect was also reversed by the specific uptake antagonist fluoxetine ŽFig. 5.. The metabolites HPTP and HPPq were less active, with 30–50% release that was not blocked by fluoxetine. As was observed with dopamine, there was no stimulation of serotonin release by RHPPq.
Fig. 6. Drug-induced release of preloaded dopamine ŽA. or serotonin ŽB. in buffer containing calcium. Each data point represents the mean Ž"S.E.M.. of triplicate replications with three determinations in each replicate. Lack of error bars indicates that the S.E.M. resides within the size of the symbol.
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Table 2 Release of preloaded serotonin and dopamine from cortical and striatal synaptosomes, respectively Compound
Dopamine
Serotonin
EC 50 , m M
95% C.L.)
EC 50 , m M
95% C.L.)
MPPq HPPq HPTP
1.08 4.7 4.1
0.62–1.87 0.85–25.46 4.05–4.18
14.5 y 6.6
10.9–19.2 y 0.2–260
)95% C.I.s95% confidence limits.
Fig. 7. Survey experiments on release of preloaded GABA from cortical synaptosomes under calcium free conditions. Data are expressed as the mean % retention of label Ž"S.E.M.., where the control is normalized to 100%. All compounds were incubated at 10 m M, except for veratridine ŽVTD., which was applied at 100 m M.
Dose–response studies of release were performed for HP Ždopamine only., MPPq, HPPq, and HPTP. For dopamine, release was essentially complete for MPPq, HPPq, and HPTP, whereas HP was less effective ŽFig. 6A.. MPPq was the most potent compound, with HPPq and HPTP about four-fold less active ŽTable 2.. All the compounds released somewhat less serotonin at maximal concentrations ŽFig. 6B., and were less potent in their effect. In this system, HPTP was the most potent compound, with MPPq about two-fold less active ŽTable 2.. The reduced effectiveness of HPPq in this system ŽFig. 6B. precluded calculation of a meaningful EC 50 value. Additional studies assessed the ability of HPTP and HPPq to release w 3 HxGABA from cortical synaptosomes to test whether the effects of these compounds were specific for aminergic neurotransmitters. As shown in Fig. 7, the HP metabolites were completely inactive in this assay. MPPq and the mitochondrial inhibitor rotenone had some activity, but were not as effective as they were for releasing biogenic amines. The sodium channel activator veratridine served as a positive control for GABA release and this compound released about 80% of label at 100 m M ŽFig. 7..
4. Discussion For blocking the uptake of serotonin and dopamine, there was considerably more activity present for the metabolites of HP than for the parent compound, confirm-
ing that conversion of HP to HPTP, HPPq, and RHPPq was bioactivating metabolism. The tetrahydropyridines, MPTP and HPTP, were the most potent blockers of serotonin uptake in their respective structural series. The greater potency of MPTP than MPPq in this regard is consistent with previous studies w16,17x. In contrast, the tetrahydropyridines were among the least active compounds against dopamine uptake ŽFig. 1A.. The extremely low potency of HPTP for inhibiting dopamine uptake in striatal synaptosomes ŽIC 50 of 258 m M, Table 1. stands in contrast to the IC 50 of 1.8 m M measured for HPTP in blocking uptake of dopamine into rat striatal slices w10x. The large difference in potency between slices and synaptosomes may be related to the presence of neuronal electrical activity present in brain slices. Alternatively, HPTP-mediated release might have been the primary mechanism for reducing dopamine uptake in the study of Fang and Yu w10x, a possibility that they explicitly discussed in their paper. If so, our potency determination of 4 m M for releasing dopamine from preloaded synaptosomes would be in good agreement with their findings. The pyridinium metabolites, HPPq and MPPq, had nearly identical IC 50 s for the inhibition of serotonin uptake and slightly different IC 50 s for inhibiting dopamine uptake. With regard to structure–activity relationships, it is clear that potent interactions with the serotonin transport protein in these structural series do not require a positively charged molecule, whereas this moiety is important for binding to the dopamine transporter and for translocation into the nerve terminal. This latter conclusion is predicated on the observation that w 3 HxMPPq, but not w 3 HxMPTP, is taken up by rat brain striatal or cortical synaptosomes w15x. RHPPq was less active than HPPq for blocking uptake in both systems, demonstrating that affinity for the transporters is attenuated by reduction of the butyrophenone carbonyl group present in HP. The finding that HPPq was a noncompetitive inhibitor of both dopamine and serotonin uptake was at odds with the reversible effects observed in recentrifugation experiments. The recentrifugation data would predict competitive inhibition, but for both transporters the predominant effect of HPPq was on maximal uptake, with little effect on K m . This finding suggests that HPPq does not rapidly dissociate once bound to the transporter, at least in the presence of free ligand. This consideration is relevant to the recentrifugation experiments, where washing-induced dissociation of HPPq occurred before challenge with w 3 Hxserotonin, whereas in the competition studies the two compounds were applied together. Studies on the ability of these compounds to release preloaded dopamine and serotonin from nerve terminals were used to test for interactions with the transporters and the calcium dependence of release. These studies revealed that the HP metabolites are not substrates for the transporters. Neither of the specific transporter antagonists, mazindol nor fluoxetine, were able to inhibit release stimulated by HPPq and HPTP. However, both inhibitors were
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able to block the release stimulated by MPPq, confirming that in this case release was dependent upon transportermediated uptake into the nerve terminals. These findings are consistent with those of Fang et al. w11x, who observed that toxicity to cell cultures from HPPq exposure was not increased when the cell line was transfected with a gene coding for the dopamine transporter. We also observed that HPPq- or HPTP-induced release was not affected by the absence of calcium ions, which is evident in comparisons of the magnitude of release at the single concentrations given in Fig. 5 compared to those of Fig. 6. Calcium-independent release was previously described for MPPq w21x and strengthens the evidence for a common set of actions for these compounds. The lack of effect of RHPPq on amine release suggests that reduction of the carbonyl moiety of HPPq constitutes a detoxication step in HP metabolism. The reduced effectiveness for releasing GABA observed with HPPq, HPTP, and MPPq demonstrated that these compounds possess a significant degree of specificity for aminergic nerve terminals. Reduced activity for releasing GABA was expected for MPPq, since it was assumed that it would be a poor substrate for the GABA transporter. On the other hand, the lack of transporter-mediated uptake of the HP metabolites suggested that they might have relatively nonspecific effects on nerve terminals, and therefore would release GABA to a greater extent than MPPq. The reduced effectiveness of the HP metabolites for releasing GABA demonstrates a specificity for aminergic terminals that occurs via a transporter-independent mechanism. We also observed that rotenone possessed some ability to release GABA ŽFig. 7., but like the other compounds, was more active against dopamine release Ždata not shown.. These latter results are similar to those of Marey-Semper et al. w19x, who found that rotenone was more effective for blocking dopamine uptake than GABA uptake into striatal synaptosomes or cultured mesencephalic neurons. These authors concluded that dopaminergic neurons contain a constitutive metabolic deficiency that would account for their greater sensitivity to metabolic inhibitors. Nonetheless, the complete lack of effect of the HP metabolites is surprising, if their primary biochemical action is mitochondrial inhibition. Moreover, in this regard, it is worth noting that HPPq is over 10-fold more potent as a mitochondrial poison in vitro than MPPq w20x. Thus, other modes of action may be involved in determining which neurotransmitter systems are affected by HPPq. It was recently observed that rotenone potently blocks w 3 Hxtyramine transport into synaptic vesicle preparations Ž K i s 9.9 m M. via interaction with the vesicular monoamine transporter w22x. A similar effect might contribute to dopamine and serotonin release by the HP metabolites. Abundant evidence documents the neurotoxic potential of HPPq w3,11,12x, which appears to be greater than that of HPTP or RHPPq. Postmortem analyses indicate that HPPq and RHPPq are present in the brains of patients
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given HP w8x. Moreover, both in vitro liver microsomal w9x and in vivo rodent studies w23x have suggested the possibility that HPTP is an intermediate in the formation of HPPq, although it may be only transiently present after treatment with HP w1x. The results of the present study show that HPPq and HPTP interact with biogenic amine transporters and induce amine release, often at concentrations similar to those observed for the established neurotoxin MPPq. Blockage of uptake and augmented release would increase synaptic levels of serotonin and dopamine, and there is evidence that high levels of dopamine itself can be neurotoxic w13x. Therefore, it is possible that neurotoxicity from other HP metabolites, as well as HPPq, may play a role in the extrapyramidal side effects observed in patients treated with HP. However, the low abundance or transient nature of HPTP in humans w1x, as well as the low potency of RHPPq in our assays, argue against their significant participation in the development of TD. Because the HP metabolites, including HPPq, are not transporter substrates, future studies should address the specificity of action of HPPq as it relates to the neurotransmitter systems involved in TD, as well as other possible biochemical targets besides mitochondrial inhibition.
Acknowledgements The authors would like to thank Drs. N. Castagnoli and C. Van der Schyf for their review of the manuscript. Financial support was provided by a National Institute of Health Biomedical Research Support Grant Žto J.R.B.. and a Virginia Commonwealth predoctoral fellowship Žto A.M.W...
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uptake of the metabolite n-methyl-4-phenyl-pyridine by dopamine neurons explains selective toxicity, Proc. Natl. Acad. Sci. U.S.A. 82 Ž1985. 2173–2177. G. Jonsson, E. Nwanae, J. Luthman, E. Sundstrom, Effect of MPTP and its pyridinium metabolites on monoamine uptake and on central catecholamine neurons in mice, Acta Physiol. Scand. 128 Ž1986. 187–194. B.K. Krueger, Kinetics and block of dopamine uptake in synaptosomes from rat caudate nucleus, J. Neurochem. 5 Ž1990. 260–267. I. Marey-Semper, M. Gelman, M. Levi-Strauss, The high sensitivity to rotenone of striatal dopamine uptake suggests the existence of a constitutive metabolic deficiency in dopamine neurons from the substantia nigra, Eur. J. Neurosci. 5 Ž1993. 1029–1034. H. Rollema, M. Skolnik, J. D’engelbronner, K. Igarashi, E. Usuki, N. Castagnoli Jr., MPPq-like neurotoxicity of a pyridinium metabolite derived from haloperidol: in vivo microdialysis and in vitro mitochondrial studies, J. Pharmacol. Exp. Ther. 268 Ž1994. 380–387. D. Sirinathsinghji, R. Heavens, C. McBride, Dopamine-releasing action of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ŽMPTP. and 1-methyl-4-phenylpyridine ŽMPPq. in the neostriatum of the rat as demonstrated in vivo by the push–pull perfusion technique: dependence on sodium but not calcium ions, Brain Res. 443 Ž1988. 101–116. A. Vaccari, P. Saba, The tyramine-labelled vesicular transporter for dopamine: a putative target of pesticides and neurotoxins, Eur. J. Pharmacol. 292 Ž1995. 309–314. C. Van der Schyf, K. Castagnoli, E. Usuki, H. Fouda, J. Rimoldi, N. Castagnoli Jr., Metabolic studies on haloperidol and its tetrahydropyridine analog in C57BLr6 mice, Chem. Res. Toxicol. 7 Ž1994. 281–285.
Research report
Effects of haloperidol metabolites on neurotransmitter uptake and release: possible role in neurotoxicity and tardive dyskinesia Alesia M. Wright, Jeffrey Bempong, Michael L. Kirby, Rebecca L. Barlow, Jeffrey R. Bloomquist
)
Department of Entomology, Neurotoxicology Laboratory, Virginia Polytechnic Institute and State UniÕersity, Blacksburg, VA 24061, USA Accepted 23 December 1997
Abstract This research explored the effects of haloperidol ŽHP. metabolites on biogenic amine uptake and release, and compared them to those of MPTP and its toxic metabolite, MPPq. In synaptosome preparations from mouse striatum and cortex, the HP metabolites haloperidol pyridinium ŽHPPq., reduced haloperidol pyridinium ŽRHPPq., and haloperidol tetrahydropyridine ŽHPTP. inhibited the presynaptic uptake of dopamine and serotonin, with greater affinity for the serotonin transporter. HPPq was the most potent inhibitor of dopamine uptake, and HPTP of serotonin uptake, both with IC 50 values in the low micromolar range. RHPPq was less active than the other metabolites, but was more active than the parent compound, HP. Inhibition of uptake was reversed when free drug was removed by centrifugation and then resuspension of the synaptosomes in fresh buffer, suggesting that inhibition of uptake was due to interaction with the transporters and was not due to irreversible cytotoxicity. HPPq showed noncompetitive inhibition of both serotonin and dopamine uptake, suggesting that it has a relatively slow dissociation rate for its interaction with the transporter proteins. In experiments on amine release, HPPq and HPTP were four-fold less potent than MPPq for releasing preloaded dopamine from striatal synaptosomes, and only MPPq-dependent release was antagonized by the uptake blocker, mazindol. In contrast, RHPPq displayed little ability to release either amine neurotransmitter. HPTP was about two-fold more potent than MPPq for releasing serotonin from cortical synaptosomes, whereas HPPq was less active than MPPq. The specific serotonin transport blocker fluoxetine was only able to antagonize release induced by MPPq. These results suggest that HP metabolites bind to the transporters for dopamine and serotonin, but are not transporter substrates. In contrast to their potent effects on amine release, HPPq and HPTP were unable to release preloaded GABA from cortical synaptosomes. The implications of these results concerning a possible role of HP metabolites in the development of tardive dyskinesia are discussed. q 1998 Elsevier Science B.V. Keywords: Schizophrenia; Parkinsonism; Haloperidol pyridinium; Reduced haloperidol pyridinium; Haloperidol tetrahydropyridine
1. Introduction Tardive dyskinesia ŽTD. is a well known side effect of haloperidol ŽHP, Fig. 1. treatment that is slow to develop and often irreversible w14x. TD has been attributed to the supersensitivity of dopamine receptors w7x, but this mechanism is not consistent with a number of factors documented in TD patients w14x. Rollema et al. w20x provided evidence that a key metabolite of HP, haloperidol pyridinium ŽHPPq, Fig. 1., shared some structural similarity and toxic actions with MPPq and suggested that HPPq
) Corresponding [email protected]
author.
Fax:
q 1-540-231-9131;
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 5 5 1 - 5
Fig. 1. Structures of the compounds used in this study, along with established or hypothesized metabolic reactions of haloperidol responsible for the three metabolites. The butyrophenone group ŽR. is unchanged in HP, HPTP, and HPPq, but is reduced in RHPPq.
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may induce TD by causing neuronal injury via nerve terminal transport and inhibition of mitochondrial respiration. In a related study, Bloomquist et al. w3x demonstrated that HPPq was, in fact, cytotoxic to cultured dopaminergic and serotonergic mesencephalic neurons. In addition, MPPq and HPPq blocked the uptake of both serotonin and dopamine into mouse brain synaptosomes, suggesting an interaction with the neuronal transporters for these biogenic amines w3x. These findings were confirmed and extended in subsequent studies w10–12x. Thus, the available data suggest that HPPq has a mode of action similar to that of MPPq, and that the MPTPrMPPq model of neurotoxicity may be valid for assessing the role of HP metabolites in the development of TD. An important component of the action of MPPq is its specific uptake by dopaminergic nerve terminals w16x. Thus, comparative studies were initiated on the inhibitory potency, reversibility, and kinetic nature of the interaction of HPPq and other HP metabolites ŽHPTP and RHPPq, Fig. 1. with the dopamine and serotonin transporters of mouse brain synaptosomes. We also assessed the ability of HP metabolites to release w 3 Hxdopamine and w 3 Hxserotonin from preloaded synaptosomes in the presence and absence of the uptake blockers mazindol and fluoxetine, in order to determine the role of transporter-mediated uptake in the actions of these compounds. Additional studies tested the ability of these compounds to release preloaded GABA from cortical synaptosomes as a measure of the specificity of their effects. Preliminary versions of these results have appeared w2,4x.
2. Materials and methods 2.1. Chemicals and animals Fluoxetine, mazindol, HP, HPTP, HPPq, RHPPq, MPTP, MPPq and pargyline were kindly provided by Neal Castagnoli, Jr., Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA. Veratridine ŽVTD., rotenone ŽROT., bovine serum albumin Žfraction V., Coomassie brilliant blue G250, and phosphoric acid were purchased from Sigma Chemical ŽSt. Louis, MO.. Samples of w 3 Hxdopamine Ž20 Cirmmol. and w 3 HxGABA Ž88 Cirmmol. were purchased from New England Nuclear ŽDuPont, Wilmington, DE.. w 3 HxSerotonin Ž11 Cirmmol. was purchased from Amersham ŽArlington Heights, IL.. Male ICR mice were obtained from Harlan– Sprague–Dawley ŽRaleigh-Durham, NC.. 2.2. Preparation of synaptosomes Procedures for isolating and incubating synaptosomes were essentially those described by Bloomquist et al. w3x.
Mice were sacrificed by rapid cervical dislocation and the brain dissected to yield cortical or striatal tissue for studies on the uptakerrelease of serotonin or dopamine, respectively. Following dissection, the tissue was homogenized in 4 ml of sucrose buffer Ž0.32 M sucrose and 2 mM HEPES, pH 7.4.. The homogenate was then centrifuged for 10 min at 2000 = g. After the initial spin, the supernatant was removed and centrifuged for 10–30 min at 12,000 = g. The final pellet containing the synaptosomes was rinsed gently and resuspended in buffer containing ŽmM.: NaCl Ž125., KCl Ž5., CaCl 2 Ž2., MgCl 2 Ž1., sucrose Ž10., pargyline Ž0.05., ascorbate Ž0.1., Tris–HCl Ž50. at pH 7.4. For calcium-free buffers, CaCl 2 was omitted from the saline, with no substitutions, as the small drop in osmolarity was considered insignificant. 2.3. Inhibition of dopamine and serotonin uptake For these studies, experimental conditions were adjusted to optimize, as much as possible, measurement of the inhibition of uptake resulting from interactions of drugs with the transporters, and not loss of uptake due to enhanced transmitter release or cytotoxic effects w18x. Incubations were done at room temperature in order to reduce the specific transport of MPPq or other compounds into the nerve terminal and thereby minimize any effects on mitochondrial function, since the thermodynamic properties of the transporter results in less efficient uptake of substrates at room temperature w5x. In addition, calcium-free buffer was also used in these experiments to minimize any contributions of calcium-dependent leakage. Synaptosomes were exposed to drug dissolved in saline, DMSO, or a DMSOrsaline mixture. Control incubates received vehicle alone. All drug treatments were incubated for at least 10 min at room temperature, which was sufficient time for the compounds to come to equilibrium w3x. Treated synaptosomes were then exposed to w 3 Hxserotonin Ž1 m Ci, 90 nM. or w 3 Hxdopamine Ž1 m Ci, 20 nM. for a 5 min incubation at 378C. Uptake was terminated by the addition of 3 ml of ice-cold buffer, the incubate was poured onto a Whatman GFrB glass fiber filter under vacuum, and then washed twice with 3 ml of ice-cold wash buffer Žcalcium-free uptake buffer without pargyline or ascorbate.. The filters were air dried, scintillation fluid added ŽScintiVerse, Fisher Scientific., and the radioactivity trapped in the synaptosomes counted by liquid scintillation spectrometry. Treatments were typically replicated three times on different synaptosomal preparations with three determinations in each replicate. Inhibition curves were plotted and analyzed by computer ŽInPlot 4.0 or Prism 2.0a, both from GraphPad Software, San Diego, CA.. Curves were compared for the maximum level of inhibition, as well as inhibitory potency as given by IC 50 values and the 95% confidence intervals of the IC 50 , when fit to a standard sigmoidal four parameter logistic equation.
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2.4. Recentrifugation protocol for assessing reÕersibility of transport inhibition
Table 1 Inhibition of serotonin and dopamine uptake under standard and drug removal Žrecentrifugation. conditions
A recentrifugation protocol was designed to confirm the extent to which inhibition of uptake involved interaction with the neurotransmitter transporters, as opposed to irreversible poisoning of the synaptosomes. Synaptosomes in calcium-free buffer were batch-exposed to compounds for 10 min at room temperature and each treatment group was then recentrifuged at 12,000 = g for 10 min. The supernatant containing unbound drug was discarded and the tissue was resuspended in drug-free uptake buffer. Aliquots of synaptosomes were then challenged with w 3 Hxserotonin or w 3 Hxdopamine and were incubated, filtered, washed, counted, and analyzed as described above.
Compound
2.5. Assay for competitiÕer noncompetitiÕe interaction of HPP q with amine transporters
min at 378C, and then centrifuged at 12,000 = g for 10 min to pellet the synaptosomes. The supernatant was discarded and the synaptosomes resuspended in fresh buffer with or without calcium ions. The vesicles were aliquoted into tubes, treated with drugs Žcontrols received vehicle alone., and incubated at 378C for 10 min. Each incubate then received 3 ml of wash buffer at 378C, was filtered under vacuum, and washed an additional 3 times with 378C buffer. For quantitation of label remaining in the synaptosomes, vacuum filtration and liquid scintillation spectrometry were employed as previously described. Extent of release and EC 50 values were determined from an iterative fit to a sigmoidal, four parameter logistic equation, as described above.
The kinetic nature of the interaction of HPPq with aminergic transporters was investigated by incubating cortical synaptosomes with 10 m M HPPq and striatal synaptosomes with 60 m M HPPq along with increasing amounts of w 3 Hxserotonin or w 3 Hxdopamine, respectively. Aliquots of labeled transmitter with and without HPPq were added to synaptosomes in calcium-free buffer and incubated at 378C. Ice-cold buffer Ž3 ml. was then added to stop uptake and the synaptosomes were filtered, washed, and counted as described previously. For calculation of uptake rates on a per mg protein basis, the protein content of the membrane preparation was determined using the method of Bradford w6x. Uptake curves with and without HPPq were compared with regard to Vmax and K m values following iterative fit to the same sigmoidal, four parameter logistic equation used for inhibition studies. 2.6. Stimulated release of preloaded transmitters Synaptosomes from cortex and striata were prepared as previously described. Aliquots of vesicles were incubated with 90 nM w 3 Hxserotonin or 20 nM w 3 Hxdopamine for 5
Dopamine
Serotonin
IC 50 , mM
IC 50 , mM
MPTP 39.25 MPPq 3.98 MPPq Žrecentrifugation. 55.63 HPTP 258.4 HPPq 22.4 HPPq Žrecentrifugation. y RHPPq 65.96 Fluoxetine y
95% C.L.)
95% C.L.)
30.30–50.83 0.29 0.21–0.40 3.05–5.19 3.04 1.45–6.38 41.50–74.59 60.45 6.3–583 147.5–452.6 1.19 0.83–1.72 11.31–43.21 4.43 3.33–5.88 y 44.60 22.60–88.02 63.83–68.16 10.01 1.65–60.6 y 0.16 0.09–0.27
)95% C.I.s95% confidence limits.
3. Results 3.1. Inhibition of dopamine and serotonin uptake Synaptosomes prepared from striatal tissue were used to evaluate inhibitory effects of MPTP, MPPq, HP, HPTP, HPPq and RHPPq on dopamine uptake. In nearly all cases, compounds showed inhibition of about 90% at maximally effective concentrations, where the residual
Fig. 2. Dose–response relationships for the inhibition of dopamine ŽA. and serotonin ŽB. transport. Each data point represents the mean Ž"S.E.M.. of triplicate replications with three determinations in each replicate. Lack of error bars indicates that the S.E.M. resides within the size of the symbol.
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Fig. 3. Dose–response curves under standard and recentrifugation conditions for MPPq-dependent inhibition of dopamine uptake ŽA. and HPPq-dependent inhibition of serotonin uptake ŽB.. Each data point represents the mean Ž"S.E.M. of triplicate replications with three determinations in each replicate. Lack of error bars indicates that the S.E.M. resides within the size of the symbol.
probably represents nonspecific binding of label to the synaptosomes. MPPq ŽFig. 2A. was the most potent and effective blocker of dopamine uptake, showing 91% maximal inhibition and an IC 50 value of about 4 m M ŽTable 1.. For comparison, MPTP had an inhibitory potency nearly 10-fold less than that of MPPq. The HP derivatives were less potent inhibitors of dopamine uptake ŽFig. 2A, Table 1., with a rank order of potency of HPPq) RHPPq) HPTP) HP. HPPq was over five-fold less active than MPPq and RHPPq was about three-fold less active for inhibiting dopamine uptake than HPPq. HPTP and HP were the least active compounds. Only a partial inhibition curve could be generated for HPTP, and it showed a maximal effect of 47% inhibition ŽFig. 2A.. HP was virtually inactive, showing only 15% inhibition at 300 m M ŽFig. 2A.. All the compounds were more potent inhibitors of serotonin uptake ŽFig. 2B.. MPTP was nearly as active as the standard uptake inhibitor, fluoxetine, and was about 10-fold more active than the pyridinium metabolite, MPPq ŽTable 1.. Similarly, among the HP metabolites, HPTP was the most potent inhibitor of serotonin uptake ŽTable 1. and was over 200-fold more active against serotonin uptake than dopamine uptake. The potency of HPPq for inhibiting
serotonin uptake was four-fold less than HPTP, but was comparable to that of MPPq in this system. The reduced pyridinium, RHPPq, was about three-fold less active than HPPq and MPPq, but was over six-fold more effective as a blocker of serotonin uptake than dopamine uptake ŽTable 1.. In addition, RHPPq showed a maximal inhibition of serotonin transport of 77%, which was slightly less than the other compounds ŽFig. 2B.. Again, HP was the least active compound, but was more effective as an inhibitor of serotonin uptake than dopamine uptake, showing about 50% inhibition at 300 m M ŽFig. 2B.. 3.2. Interaction with the transporter assessed by recentrifugation To determine whether the inhibitory actions of these compounds occurred via binding to the transporter, as opposed to irreversible cytotoxicity, each sample was preincubated with drug, and then recentrifuged and resuspended in fresh buffer to remove free drug from the system. For MPPq, removal of free drug following a 40 min incubation at room temperature caused a large shift to the right in the potency curve for inhibition of dopamine uptake ŽFig. 3A.. Nonlinear regression analysis found that
Fig. 4. Noncompetitive inhibition of dopamine and serotonin high affinity uptake by HPPq. Each data point represents the mean Ž"S.E.M.. of triplicate determinations measured in one or more experiments. Lack of error bars indicates that the S.E.M. resides within the size of the symbol. Inset: kinetic values for uptake in the presence or absence of HPPq. Numbers in parentheses beneath the values are the corresponding 95% confidence limits. The asterisk next to HPPq Vma x values denotes a statistically significant decrease Ž p - 0.05. compared to control values, due to nonoverlap of the 95% confidence limits.
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Fig. 5. Survey experiments on release of dopamine ŽA. and serotonin ŽB. under calcium free conditions. Data are expressed as the mean % retention of label Ž"S.E.M.., where the control is normalized to 100%. MPPq and the haloperidol metabolites were incubated at the concentrations given below the bars. Identical drug incubations were performed in the additional presence of 10 m M mazindol ŽMaz. or 100 m M fluoxetine ŽFluox. as labeled at the top of the bars.
the potency of MPPq was reduced about 20-fold ŽTable 1.. Preincubation times of 10 and 20 min also shifted the curve to the right to about the same extent Ždata not shown.. In other experiments, both the HPPq and MPTP inhibition curves for dopamine uptake also displayed shifts to the right Ždata not shown., suggesting that these compounds are also directly interacting with the transporter under these conditions. Similar effects were observed in recentrifugation experiments applied to serotonin uptake. Removal of free HPPq by recentrifugation produced a parallel shift to the right in the dose–response curve ŽFig. 3B. and an IC 50 value that was 10-fold higher than under standard conditions ŽTable 1.. Similarly, the MPPq dose– response curve also displayed a parallel shift to the right and an IC 50 that was 14-fold higher than that obtained under standard conditions ŽTable 1.. 3.3. NoncompetitiÕe interaction of HPP q with aminergic transporters Dose–response curves for serotonin and dopamine uptake were run in the absence and presence of HPPq to determine if the inhibition was competitive ŽFig. 4A,B.. These studies employed concentrations of HPPq that were approximately three times the IC 50 value of this compound for inhibiting dopamine and serotonin uptake. HPPq Ž60
m M. was a noncompetitive inhibitor of dopamine transport ŽFig. 4A., causing a statistically significant reduction Ž37%. in Vmax with virtually no effect on K m ŽFig. 4A, inset.. HPPq Ž10 m M. displayed similar noncompetitive effects on serotonin uptake ŽFig. 4B., significantly reducing maximal uptake Ž Vmax . of serotonin about 37%, with little change in the K m ŽFig. 4B, inset.. 3.4. Release of labeled transmitters from preloaded synaptosomes Survey experiments on the ability of HP metabolites and MPPq to release preloaded dopamine and serotonin are shown in Fig. 5. MPPq released virtually all the dopamine following incubation at 10 m M, and this release was blocked by the dopamine uptake antagonist, mazindol. HPTP was about as active as MPPq for releasing dopamine, while HPPq was slightly less effective and RHPPq was inactive. With the HP metabolites, however, preincubation with mazindol had no effect on release. MPPq released about 70% of preloaded serotonin and this effect was also reversed by the specific uptake antagonist fluoxetine ŽFig. 5.. The metabolites HPTP and HPPq were less active, with 30–50% release that was not blocked by fluoxetine. As was observed with dopamine, there was no stimulation of serotonin release by RHPPq.
Fig. 6. Drug-induced release of preloaded dopamine ŽA. or serotonin ŽB. in buffer containing calcium. Each data point represents the mean Ž"S.E.M.. of triplicate replications with three determinations in each replicate. Lack of error bars indicates that the S.E.M. resides within the size of the symbol.
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Table 2 Release of preloaded serotonin and dopamine from cortical and striatal synaptosomes, respectively Compound
Dopamine
Serotonin
EC 50 , m M
95% C.L.)
EC 50 , m M
95% C.L.)
MPPq HPPq HPTP
1.08 4.7 4.1
0.62–1.87 0.85–25.46 4.05–4.18
14.5 y 6.6
10.9–19.2 y 0.2–260
)95% C.I.s95% confidence limits.
Fig. 7. Survey experiments on release of preloaded GABA from cortical synaptosomes under calcium free conditions. Data are expressed as the mean % retention of label Ž"S.E.M.., where the control is normalized to 100%. All compounds were incubated at 10 m M, except for veratridine ŽVTD., which was applied at 100 m M.
Dose–response studies of release were performed for HP Ždopamine only., MPPq, HPPq, and HPTP. For dopamine, release was essentially complete for MPPq, HPPq, and HPTP, whereas HP was less effective ŽFig. 6A.. MPPq was the most potent compound, with HPPq and HPTP about four-fold less active ŽTable 2.. All the compounds released somewhat less serotonin at maximal concentrations ŽFig. 6B., and were less potent in their effect. In this system, HPTP was the most potent compound, with MPPq about two-fold less active ŽTable 2.. The reduced effectiveness of HPPq in this system ŽFig. 6B. precluded calculation of a meaningful EC 50 value. Additional studies assessed the ability of HPTP and HPPq to release w 3 HxGABA from cortical synaptosomes to test whether the effects of these compounds were specific for aminergic neurotransmitters. As shown in Fig. 7, the HP metabolites were completely inactive in this assay. MPPq and the mitochondrial inhibitor rotenone had some activity, but were not as effective as they were for releasing biogenic amines. The sodium channel activator veratridine served as a positive control for GABA release and this compound released about 80% of label at 100 m M ŽFig. 7..
4. Discussion For blocking the uptake of serotonin and dopamine, there was considerably more activity present for the metabolites of HP than for the parent compound, confirm-
ing that conversion of HP to HPTP, HPPq, and RHPPq was bioactivating metabolism. The tetrahydropyridines, MPTP and HPTP, were the most potent blockers of serotonin uptake in their respective structural series. The greater potency of MPTP than MPPq in this regard is consistent with previous studies w16,17x. In contrast, the tetrahydropyridines were among the least active compounds against dopamine uptake ŽFig. 1A.. The extremely low potency of HPTP for inhibiting dopamine uptake in striatal synaptosomes ŽIC 50 of 258 m M, Table 1. stands in contrast to the IC 50 of 1.8 m M measured for HPTP in blocking uptake of dopamine into rat striatal slices w10x. The large difference in potency between slices and synaptosomes may be related to the presence of neuronal electrical activity present in brain slices. Alternatively, HPTP-mediated release might have been the primary mechanism for reducing dopamine uptake in the study of Fang and Yu w10x, a possibility that they explicitly discussed in their paper. If so, our potency determination of 4 m M for releasing dopamine from preloaded synaptosomes would be in good agreement with their findings. The pyridinium metabolites, HPPq and MPPq, had nearly identical IC 50 s for the inhibition of serotonin uptake and slightly different IC 50 s for inhibiting dopamine uptake. With regard to structure–activity relationships, it is clear that potent interactions with the serotonin transport protein in these structural series do not require a positively charged molecule, whereas this moiety is important for binding to the dopamine transporter and for translocation into the nerve terminal. This latter conclusion is predicated on the observation that w 3 HxMPPq, but not w 3 HxMPTP, is taken up by rat brain striatal or cortical synaptosomes w15x. RHPPq was less active than HPPq for blocking uptake in both systems, demonstrating that affinity for the transporters is attenuated by reduction of the butyrophenone carbonyl group present in HP. The finding that HPPq was a noncompetitive inhibitor of both dopamine and serotonin uptake was at odds with the reversible effects observed in recentrifugation experiments. The recentrifugation data would predict competitive inhibition, but for both transporters the predominant effect of HPPq was on maximal uptake, with little effect on K m . This finding suggests that HPPq does not rapidly dissociate once bound to the transporter, at least in the presence of free ligand. This consideration is relevant to the recentrifugation experiments, where washing-induced dissociation of HPPq occurred before challenge with w 3 Hxserotonin, whereas in the competition studies the two compounds were applied together. Studies on the ability of these compounds to release preloaded dopamine and serotonin from nerve terminals were used to test for interactions with the transporters and the calcium dependence of release. These studies revealed that the HP metabolites are not substrates for the transporters. Neither of the specific transporter antagonists, mazindol nor fluoxetine, were able to inhibit release stimulated by HPPq and HPTP. However, both inhibitors were
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able to block the release stimulated by MPPq, confirming that in this case release was dependent upon transportermediated uptake into the nerve terminals. These findings are consistent with those of Fang et al. w11x, who observed that toxicity to cell cultures from HPPq exposure was not increased when the cell line was transfected with a gene coding for the dopamine transporter. We also observed that HPPq- or HPTP-induced release was not affected by the absence of calcium ions, which is evident in comparisons of the magnitude of release at the single concentrations given in Fig. 5 compared to those of Fig. 6. Calcium-independent release was previously described for MPPq w21x and strengthens the evidence for a common set of actions for these compounds. The lack of effect of RHPPq on amine release suggests that reduction of the carbonyl moiety of HPPq constitutes a detoxication step in HP metabolism. The reduced effectiveness for releasing GABA observed with HPPq, HPTP, and MPPq demonstrated that these compounds possess a significant degree of specificity for aminergic nerve terminals. Reduced activity for releasing GABA was expected for MPPq, since it was assumed that it would be a poor substrate for the GABA transporter. On the other hand, the lack of transporter-mediated uptake of the HP metabolites suggested that they might have relatively nonspecific effects on nerve terminals, and therefore would release GABA to a greater extent than MPPq. The reduced effectiveness of the HP metabolites for releasing GABA demonstrates a specificity for aminergic terminals that occurs via a transporter-independent mechanism. We also observed that rotenone possessed some ability to release GABA ŽFig. 7., but like the other compounds, was more active against dopamine release Ždata not shown.. These latter results are similar to those of Marey-Semper et al. w19x, who found that rotenone was more effective for blocking dopamine uptake than GABA uptake into striatal synaptosomes or cultured mesencephalic neurons. These authors concluded that dopaminergic neurons contain a constitutive metabolic deficiency that would account for their greater sensitivity to metabolic inhibitors. Nonetheless, the complete lack of effect of the HP metabolites is surprising, if their primary biochemical action is mitochondrial inhibition. Moreover, in this regard, it is worth noting that HPPq is over 10-fold more potent as a mitochondrial poison in vitro than MPPq w20x. Thus, other modes of action may be involved in determining which neurotransmitter systems are affected by HPPq. It was recently observed that rotenone potently blocks w 3 Hxtyramine transport into synaptic vesicle preparations Ž K i s 9.9 m M. via interaction with the vesicular monoamine transporter w22x. A similar effect might contribute to dopamine and serotonin release by the HP metabolites. Abundant evidence documents the neurotoxic potential of HPPq w3,11,12x, which appears to be greater than that of HPTP or RHPPq. Postmortem analyses indicate that HPPq and RHPPq are present in the brains of patients
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given HP w8x. Moreover, both in vitro liver microsomal w9x and in vivo rodent studies w23x have suggested the possibility that HPTP is an intermediate in the formation of HPPq, although it may be only transiently present after treatment with HP w1x. The results of the present study show that HPPq and HPTP interact with biogenic amine transporters and induce amine release, often at concentrations similar to those observed for the established neurotoxin MPPq. Blockage of uptake and augmented release would increase synaptic levels of serotonin and dopamine, and there is evidence that high levels of dopamine itself can be neurotoxic w13x. Therefore, it is possible that neurotoxicity from other HP metabolites, as well as HPPq, may play a role in the extrapyramidal side effects observed in patients treated with HP. However, the low abundance or transient nature of HPTP in humans w1x, as well as the low potency of RHPPq in our assays, argue against their significant participation in the development of TD. Because the HP metabolites, including HPPq, are not transporter substrates, future studies should address the specificity of action of HPPq as it relates to the neurotransmitter systems involved in TD, as well as other possible biochemical targets besides mitochondrial inhibition.
Acknowledgements The authors would like to thank Drs. N. Castagnoli and C. Van der Schyf for their review of the manuscript. Financial support was provided by a National Institute of Health Biomedical Research Support Grant Žto J.R.B.. and a Virginia Commonwealth predoctoral fellowship Žto A.M.W...
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