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Deprenyl stimulates the efflux of monoamines from the rat hypothalamus in vitro
Brain Research Bulletin, Vol. 54, No. 6, pp. 675– 680, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/01/$–see front matter
PII S0361-9230(01)00481-6
Deprenyl stimulates the efflux of monoamines from the rat hypothalamus in vitro P. S. MohanKumar,* Sheba M. J. MohanKumar and S. K. Quadri Neuroendocrine Research Laboratory, Department of Veterinary Pathology, College of Veterinary Medicine, Michigan State University, E. Lansing, MI, USA [Received 14 February 2001; Accepted 2 March 2001] ABSTRACT: The direct effects of L-deprenyl, a monoamine oxidase inhibitor, on the hypothalamus of male Sprague-Dawley rats was investigated by measuring the efflux of norepinephrine (NE), dopamine (DA), serotonin (5-HT), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) using a combination of high performance liquid chromatography with electrochemical detection and an in vitro incubation system. After measuring basal efflux by incubating the hypothalami with Krebs-Ringers Henseleit (KRH) alone during the first incubation period, hypothalami were incubated either with the medium, KRH alone (0 mM), or KRH containing 0.1, 1, and 10 mM Ldeprenyl. During the third incubation period, hypothalami were again incubated with KRH alone to measure the residual effects if any. During the final incubation period, the hypothalami were stimulated with high Kⴙ KRH. Deprenyl produced a dose-dependent increase in the efflux of NE, DA, and 5-HT from the hypothalami. Neurotransmitter efflux returned to pretreatment levels when L-deprenyl was removed from the medium. In contrast to NE, DA, and 5-HT, the efflux of the metabolites DOPAC and 5-HIAA was inhibited in a dose-dependent fashion after incubation with L-deprenyl. Results from this study demonstrate that L-deprenyl is capable of stimulating the efflux of neurotransmitters in vitro by a direct action on the hypothalamus. © 2001 Elsevier Science Inc.
mary tumors and pituitary tumors in old acyclic rats [25]. We have also provided evidence that L-deprenyl can decrease serum prolactin levels in young and old rats [16,25]. However, the mechanism by which L-deprenyl produces these effects is not clear. Brain neurotransmitters, especially monoamines are known to be involved in various central and neuroendocrine effects, such as increases in sexual activity, life-span, reinitiation of estrous cycles, and decreasing serum prolactin concentrations [13–15]. More specifically, monoamine changes in the hypothalamus could play a role in integrating these effects because the hypothalamus houses all the releasing hormone neurons that regulate these functions. Therefore, we hypothesized that L-deprenyl could produce its central and neuroendocrine effects by affecting monoamine efflux from the hypothalamus. Interestingly, very little is known about the effects of L-deprenyl on the efflux of hypothalamic monoamines. In this study, we used a combination of high performance liquid chromatography with electrochemical detection (HPLC-EC) and a static in vitro incubation system to investigate the direct effects of L-deprenyl on hypothalamic monoamine efflux. MATERIALS AND METHODS Animals
KEY WORDS: Deprenyl, Dopamine, Norepinephrine, Serotonin, Hypothalamus, HPLC-EC.
Four- to six-month-old male Sprague-Dawley rats were obtained from Amitech (Omaha, NE, USA). These rats were housed in groups in air-conditioned (23 ⫾ 2°C) and light controlled (lights on from 0700 –1900 h) animal rooms. They were provided rat chow and water ad libitum. On the day of the experiment, the rats were sacrificed between 1200 –1300 h. The brains were removed immediately and the hypothalami were dissected out using the following boundaries: the posterior part of the optic chiasm as the anterior limit, the anterior part of the mammillary bodies as the posterior limit, and the lateral hypothalamic sulci as the lateral limits [18,19].
INTRODUCTION Deprenyl is an irreversible monoamine oxidase (MAO)-B inhibitor [3,4]. However, depending on the dose and duration of treatment it has also been shown to inhibit MAO-A at higher concentrations [3,31]. Like some other MAO inhibitors, L-deprenyl was also used originally as an antidepressant [3,4]. Recently, because of its ability to inhibit MAO and increase dopaminergic tone in the striatum, it is being used in the treatment of Parkinson’s disease [5,6,24]. Deprenyl has also been shown to have other novel biological actions, such as increasing life-span and sexual activity in male rats [8]. Recently, we reported that L-deprenyl also inhibits carcinogen-induced mammary tumor growth in rats and reinitiates estrous cycles and decreases the incidence of spontaneous mam-
In Vitro Incubation The hypothalami were weighed and divided into two halves along the sagittal plane and incubated at 37°C in a 12 ⫻ 75 mm glass tube containing 300 l of Krebs-Ringer Henseleit (KRH) solution in a static in vitro incubation system as described previ-
* Address for correspondence: Dr. P. S. MohanKumar, Neuroendocrine Research Laboratory, Department of Veterinary Pathology, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA. Fax: ⫹1-(517)-432-7480; E-mail: [email protected]
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ously [17–19]. The incubation medium, KRH, consisted of 117 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 24.8 mM NaHCO3, 11.1 mM glucose, 0.1 mg/ml ascorbic acid in 1 l of pyrogen-free water. During the experiment, the incubation medium was supplied a mixture of 95% oxygen and 5% carbon dioxide constantly at a slow and steady rate to avoid turbulence. To stabilize the efflux of monoamines, each hypothalamus was preincubated in KRH for 60 min. Following this, they were incubated for four 60-min periods as described below. During the first incubation period, the hypothalami were incubated in KRH for 60 min to measure the basal efflux of monoamines and their metabolites. During the second incubation period, the hypothalami were incubated in KRH containing 0, 0.1 mM, 1.0 mM, or 10 mM of L-deprenyl (n ⫽ 6 – 8/treatment group). After this, the hypothalami were rinsed and incubated in KRH alone (without L-deprenyl) during the third incubation period to measure the residual effects, if any, from the previous incubation period. In the final incubation period, the hypothalami were incubated with high K⫹ KRH, which had the same composition as the KRH except that it contained 60 mM KCl by withholding equimolar amount of NaCl, to check the viability of the tissue. Hypothalami were rinsed with KRH in between incubations. After each incubation period, incubation medium was removed and 0.1 M HClO4 was added to it at the ratio of 25:1 (v/v) and was stored at ⫺70°C until analysis for neurotransmitters by HPLC-EC. At the end of the treatment period, the hypothalami were transferred to microsample vials containing 0.1 M HClO4 and were stored at ⫺70°C. At the time of HPLC analysis the hypothalami were homogenized in KRH and centrifuged at 10,000 RPM for 15 min and the supernatant was used for the measurement of neurotransmitters. HPLC-EC The HPLC-EC system has been described before [18,19,25]. Briefly, it consisted of a LC-4B electrochemical detector (Bioanalytical Systems, West Lafayette, IN, USA); a phase II, 5 m ODS reverse phase, C-18 column; a glassy carbon electrode, a C-R6A Chromatopac integrator, a CTO-6A column oven, and a LC-6A pump (Shimadzu, Columbia, MD, USA). The mobile phase consisted of monochloroacetic acid (14.14 g/l), sodium hydroxide (4.675 g/l), octanesulfonic acid disodium salt (0.3 g/l), ethylenediaminetetraacetic acid (0.25 g/l), acetonitrile (3.5%), and tetrahydrofuran (1.4%). The mobile phase was made in pyrogen-free water and then filtered and degassed through a Milli-Q purification system (Millipore Co., Bedford, MA, USA). It was pumped through the HPLC system at a flow rate of 1.7 ml/min. The sensitivity of the detector was 1 nA full scale, and the potential of the working electrode was 0.65 V. The column and the electrodes were kept in a column oven maintained at 37°C. At the time of HPLC analysis, the incubation medium was diluted at the ratio of 1:4 with 0.1 M HClO4. Fifty microliters of the diluted incubation medium along with 25 l of the internal standard (0.05 M isoproterenol) was injected into the HPLC system. Statistical Analysis Differences in the efflux of neurotransmitters during various incubation periods in each group was analyzed by analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD). Differences in the contents of neurotransmitters in the hypothalamus among the various groups were analyzed by one way ANOVA followed by Fisher’s LSD.
FIG. 1. The effects of L-deprenyl (DEP) on the average efflux of norepinephrine (NE) from the hypothalamus in vitro (A) and on the average content of NE in the hypothalami at the end of the experiment (B). The hypothalami were incubated in Krebs-Ringers Henseleit (KRH) for four consecutive 60-min periods: with KRH alone in the first period, with KRH and L-deprenyl in the second period and again with KRH in the third period and with high K⫹ KRH in the fourth period (n ⫽ 6 – 8/treatment group). At the end of each period the incubation medium was removed for neurotransmitter analysis by high-performance liquid chromatography with electrochemical detection and the hypothalami were rinsed with KRH. At the end of the fourth incubation period the hypothalami were removed and stored in 0.1 M HClO4 and analyzed for neurotransmitter concentrations. (A) *Significantly different from NE levels in the corresponding group (10 mM) in the first incubation period (KRH) (p ⬍ 0.05); **significantly different from NE levels in the corresponding group in the first incubation period and also from other groups during the second incubation period (KRH⫹DEP) (p ⬍ 0.01). (B) *Significantly different from 0 and 0.1 mM (p ⬍ 0.05).
RESULTS Norepinephrine The effects of L-deprenyl on the efflux of norepinephrine (NE) are shown in Fig. 1A. The pretreatment NE efflux in various groups were not different from each other. In the control group (0 mM), NE efflux (Mean ⫾ SE; pg/mg hypothalamus;153.4 ⫾ 49) did not change significantly during the first 3 incubation periods but produced a marked increase of 497.9 ⫾ 76 when incubated with high potassium KRH (p ⬍ 0.05). In contrast to the control group, incubation with different doses of L-deprenyl produced a dose-dependent increase in NE efflux. When the hypothalami were incubated with 0.1 mM L-deprenyl, NE efflux seemed to increase from pretreatment levels of 196.9 ⫾ 43 to 252.3 ⫾ 48, but this increase was not significant. NE efflux returned to pretreatment
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levels (191.6 ⫾ 51) during the third incubation period. Once again challenge with high potassium KRH produced a robust increase in NE efflux (538.2 ⫾ 142; p ⬍ 0.05). Incubation with 1 mM L-deprenyl on the other hand, increased NE efflux significantly from 196.9 ⫾ 8 to 354.7 ⫾ 79 during the second incubation period (p ⬍ 0.05) and decreased to 267.7 ⫾ 28 during the third incubation period. High K⫹ KRH once again stimulated NE efflux (375.2 ⫾ 92) during the last incubation period (p ⬍ 0.05). Incubation with 10 mM L-deprenyl produced a robust increase in NE efflux during the second incubation period (794.3 ⫾ 181) when compared to the pretreatment incubation (153.9 ⫾ 67; p ⬍ 0.05) and then declined to159.8 ⫾ 41 during the third incubation period. However, incubation with high K⫹ KRH produced only a modest increase (222.4 ⫾ 57) which was not statistically significant. The content of NE in the hypothalami at the end of the experiment is shown in Fig. 1B. Hypothalami in the control and 0.1 mM L-deprenyl treated groups had significantly higher concentrations of NE (1104 ⫾ 588 and 743.3 ⫾ 179, respectively). These were nearly 3-4 fold higher than the NE concentrations in the 1 mM and 10 mM L-deprenyl treated groups (388.1 ⫾ 71 and 281.3 ⫾ 91, respectively; p ⬍ 0.05). Dopamine The effects of L-deprenyl on dopamine (DA) efflux are shown in Fig. 2A. In contrast to NE efflux, DA efflux seemed to decrease much rapidly over time. It decreased from 74.6 ⫾ 27 in the second incubation period in the control group to 16.1 ⫾ 7 in the third incubation period (p ⬍ 0.01). Incubation with high K⫹ KRH in this group produced a significant increase in DA efflux of 258.9 ⫾ 27 (p ⬍ 0.01). Similar to its effects on NE, L-deprenyl produced a dose-dependent increase in DA efflux. Incubation with 0.1 mM L-deprenyl seemed to increase DA efflux (125.1 ⫾ 49) but this was not significantly different from the pretreatment efflux rate. Treatment with 1 mM L-deprenyl on the other hand produced a sharp increase in DA efflux (154.8 ⫾ 59) when compared to pretreatment levels (42.9 ⫾ 6). In both these groups, removal of L-deprenyl from the medium during the third incubation period brought DA efflux to pretreatment levels and stimulation with high K⫹ KRH produced significant increases (293.1 ⫾ 109 and 106.8 ⫾ 27, respectively) in DA efflux. Incubation with 10 mM L-deprenyl produced a stark increase in DA efflux (197.7 ⫾ 63), removal of L-deprenyl from the medium returned DA efflux to basal levels as in the other groups. However, incubation with high potassium KRH did not produce a significant increase in DA efflux (85.3 ⫾ 40) compared to the pretreatment levels. DA content of the hypothalami in all the groups at the end of the experiment is shown in Fig. 2B. DA concentrations in the 1 mM and 10 mM groups were significantly less compared to the 0 mM group (p ⬍ 0.05). Dihydroxyphenylacetic Acid The dose-dependent inhibitory effects of L-deprenyl on dihydroxyphenylacetic acid (DOPAC) efflux are shown in Fig. 3A. DOPAC efflux decreased during the third incubation (205.3 ⫾ 38) when compared to the pretreatment level (329.3 ⫾ 70) in the control group but increased upon stimulation with high potassium KRH (467.3 ⫾ 84; p ⬍ 0.05). Incubation with 0.1 mM L-deprenyl did not produce any change in DOPAC efflux but incubation with 1 mM L-deprenyl produced a marked decrease in DOPAC efflux (139.6 ⫾ 19) when compared to pretreatment levels (267.7 ⫾ 46; p ⬍ 0.01). A more pronounced effect was observed after incubation with 10 mM L-deprenyl. DOPAC efflux decreased from pretreatment levels of 285.6 ⫾ 28 to 68.9 ⫾ 20 (p ⬍ 0.001). In
FIG. 2. The effects of L-deprenyl (DEP) on the average efflux of dopamine (DA) from the hypothalamus in vitro (A) and on the average content of DA in the hypothalami at the end of the experiment (B). (A) *Significantly different from DA levels in the corresponding groups (1 and 10 mM) in the first incubation period (KRH) (p ⬍ 0.05); (B) *significantly different from 0 mM (p ⬍ 0.05). See Fig. 1 legend for details.
both these groups, DOPAC efflux remained low even after removal of L-deprenyl from the medium. Moreover, stimulation with high potassium KRH did not produce any increase in DOPAC efflux in all the L-deprenyl-treated groups. The concentration of DOPAC in the hypothalamus at the end of the experiment (Fig. 3B) was markedly reduced in the 1 and 10 mM L-deprenyl treated groups (86.8 ⫾ 34 and 34.3 ⫾ 16, respectively) compared to control and 0.1mM L-deprenyl groups (416 ⫾ 176 and 290.0 ⫾ 110, respectively; p ⬍ 0.05). Serotonin The basal efflux of serotonin (5-HT) as observed in the control group decreased significantly with time (Fig. 4A). It decreased from 65.1 ⫾ 25 in the first incubation period to 41.0 ⫾ 21 and further declined to 23.2 ⫾ 12 during the third incubation period (p ⬍ 0.05). Upon incubation with different doses of L-deprenyl, a remarkable and dose-dependent increase in 5-HT efflux was observed (186.4 ⫾ 62, 438.4 ⫾ 85 and 670.3 ⫾ 199 with 0.1, 1 and 10 mM L-deprenyl, respectively). The efflux of 5-HT declined rapidly upon removal of L-deprenyl from the medium in the third incubation period. Stimulation with high potassium KRH produced a significant increase in 5-HT efflux in 0 mM and 0.1 mM groups. Similar to NE and DA, content of 5-HT at the end of the experiment was significantly reduced in 1 mM and 10 mM groups compared to the control group (Fig. 4B; p ⬍ 0.05).
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FIG. 3. The effects of 0, 0.1, 1, and 10 mM L-deprenyl (DEP; n ⫽ 6 – 8/treatment group) on the average efflux of dihydroxyphenylacetic acid (DOPAC) from the hypothalamus in vitro (A) and on the average content of DOPAC in the hypothalami at the end of the experiment (B). (A) *Significantly different from DOPAC levels in the corresponding group during the first incubation period (KRH); **significantly different from DOPAC levels in the corresponding group (10 mM) in the first incubation period (KRH) and also from other groups within the same incubation period (p ⬍ 0.01). (B) *Significantly different from 0 and 0.1 mM (p ⬍ 0.05). See Fig. 1 legend for details.
FIG. 4. The effects of L-deprenyl (DEP) on the average efflux of serotonin (5-HT) from the hypothalamus in vitro (A) and on the average content of 5-HT in the hypothalami at the end of the experiment (B). (A) *Significantly different from corresponding group in the first incubation period (KRH) and also from other groups within the same incubation period (KRH⫹DEP) (p ⬍ 0.05); **significantly different from corresponding groups in the first incubation period (KRH) and also from 0 and 0.1 mM groups within the same incubation period (KRH⫹DEP) (p ⬍ 0.05); (B) *significantly different from 0 and 0.1 mM (p ⬍ 0.05). See Fig. 1 legend for details.
5-Hydroxyindoleacetic Acid
with high potassium KRH produced a pronounced increase in the efflux of these neurotransmitters indicating that the tissues were viable. In contrast, the efflux of the metabolites, DOPAC, and 5-HIAA are inhibited upon incubation with L-deprenyl clearly indicating its ability to inhibit MAO. Results from this study are supported by another study in which L-deprenyl increased NE content in the teldiencephalon and the rest of the brain [34]. Recent studies from our laboratory have shown that L-deprenyl can increase the concentration of NE in the striatum and the mediobasal hypothalamus and stimulate the efflux of NE from the mediobasal hypothalamus in vivo [25,28]. The present study demonstrates that L-deprenyl stimulates NE efflux through its action on the hypothalamus. Deprenyl-induced increase in NE efflux from the hypothalamus could explain some of the novel effects of deprenyl. Deprenyl is known to increase sexual activity in male rats and reinitiate estrous cycles in old female rats. Both of these effects are known to involve luteinizing hormone (LH), the efflux of which from the pituitary is stimulated by L-deprenyl [17]. NE is one of the important neurotransmitters involved in the regulation of LH secretion. The fact that the synthesis, efflux, and content of NE declines in the hypothalamus of old animals is well established
In contrast to the efflux of 5-HT, the efflux of its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), showed a dose-dependent decrease when the hypothalami were incubated with different doses of L-deprenyl or KRH (Fig. 5A). The inhibition of 5-HIAA efflux observed over time continued even after stimulation with high potassium KRH. Moreover the content of 5-HIAA in the hypothalami at the end of the experiment (Fig. 5B) was significantly low in the L-deprenyl treated groups when compared to the control group (p ⬍ 0.05). DISCUSSION The results from this study clearly demonstrate for the first time that L-deprenyl stimulates the efflux of NE, DA, and 5-HT from the hypothalamus in vitro in a dose-dependent fashion. The efflux of these neurotransmitters either remains stable or decreases gradually as a function of time. This is a characteristic feature of the in vitro incubation system and has been observed in other studies before [16,19]. The consistent finding in all the groups was that L-deprenyl not only prevented this decrease but produced a marked increase in the efflux of NE, DA, and 5-HT. Stimulation
DEPRENYL AND HYPOTHALAMIC MONOAMINES
FIG. 5. The effects of L-deprenyl (DEP) on the average efflux of 5-hydroxyindoleacetic acid (5-HIAA) from the hypothalamus in vitro (A) and on the average content of 5-HIAA in the hypothalami at the end of the experiment (B). (A) *Significantly different from corresponding groups in other incubation periods and also from other groups within the same incubation period (p ⬍ 0.01); **significantly different from 5-HIAA levels in the first incubation period (KRH) (p ⬍ 0.05); (B) *significantly different from 0 and 0.1 mM (p ⬍ 0.05). See Fig. 1 legend for details.
[14,15]. Administration of compounds, which increase brain NE levels such as l-dopa or the MAO inhibitor iproniazid could reinitiate estrous cycles in old rats [20]. Recently, we have demonstrated that administration of L-deprenyl itself could reinitiate estrous cycles in old female rats [25] indicating that this effect of L-deprenyl is most probably mediated through an increase in NE activity. The current study provides the first direct evidence for this phenomenon. Incubation of the hypothalamus with L-deprenyl also increased DA efflux in a dose-dependent manner. This finding is supported by another study in which direct infusion of L-deprenyl into the hypothalamus stimulated the release of DA [28]. Other studies have also indicated that L-deprenyl increases the turnover rate and release of DA from the striatum [2, 6]. Other indirect lines of evidence suggest that L-deprenyl may indeed produce this effect in the hypothalamus. Deprenyl has been shown to inhibit prolactin secretion [17,25], which, could be mediated through stimulation of DA because prolactin is under the inhibitory control of DA [13]. Moreover, L-deprenyl has been shown to cause the regression of prolactin-dependent mammary tumors [25–27]. This effect is probably achieved through an increase in DA, which causes suppression of prolactin secretion, which ultimately results in the regression of the mammary tumors. Therefore, it is conceivable that
679 L-deprenyl may indeed stimulate DA activity to produce some of its beneficial effects. Apart from stimulating the efflux of NE and DA from the hypothalamus, the present study provides evidence that L-deprenyl stimulates the efflux of 5-HT in a dose-dependent fashion. Other studies from our laboratory and others have provided evidence that prolonged daily administration or intracranial administration of deprenyl could affect monoaminergic activity of the hypothalamus both in male and female rats [26 –29]. Like NE and DA, 5-HT is also involved in a number of neuroendocrine effects including the regulation of LH and prolactin [11,32]. Thus it is quite probable that at least some of the effects of L-deprenyl could be mediated through its effects on 5-HT in the hypothalamus. After incubation with the highest dose of L-deprenyl, the efflux of NE, DA, and 5-HT were unaltered upon stimulation with high K⫹ KRH. The reasons for the inability of high K⫹ KRH to stimulate neurotransmitters after treatment with the highest dose of L-deprenyl are not clear. It is possible that the highest dose of deprenyl because of its pronounced neurotransmitter releasing effects (several-fold increase compared to the control group) could have exhausted the available pool of neurotransmitters in the hypothalamic blocks. This possibility is supported by the observation that neurotransmitter content at the end of the treatment (10 mM of L-deprenyl) were significantly low compared to the control group. In contrast to its effect on NE, DA, and 5-HT, L-deprenyl decreased the levels of the metabolites, DOPAC, and 5-HIAA, in a dose-dependent fashion. In rats, the metabolism of monoamines is a complex process and is achieved by both forms of monoamine oxidases (MAO-A and MAO-B) [31,33]. While DA acts as a substrate for both MAO-A and B, 5-HT is metabolized by MAO-A [21,22]. Thus, L-deprenyl being a MAO-B inhibitor inhibited the metabolism of DA and decreased the levels of its metabolite significantly. The interesting finding in the present study was the capacity of L-deprenyl to also inhibit the levels of 5-HIAA, even at the lowest dose used. As alluded to before, L-deprenyl could act on both forms of MAO [1,3,30,31], hence, it is probable that L-deprenyl could simultaneously inhibit the efflux of both DOPAC and 5-HIAA. A similar decrease in DOPAC and 5-HIAA concentrations in the mediobasal hypothalamus and the striatum have been observed before after L-deprenyl treatment [25,27,29]. The inhibition observed in the present study was so great that high K⫹ KRH which was capable of stimulating the efflux of NE, DA, and 5-HT was still unable to stimulate the efflux of DOPAC and 5-HIAA. This could only mean that L-deprenyl totally inhibited the break-down of DA and 5-HT to its metabolites while simultaneously stimulating the efflux of these neurotransmitters from the hypothalamus. This is not surprising because L-deprenyl is an established monoamine oxidase inhibitor, which acts on the flavin binding site to irreversibly inhibit this enzyme [12]. The enzyme activity is believed to be reinstated only after the synthesis of new enzyme molecules [12] which is not possible in the in vitro model. This clearly explains the stark decrease in the efflux of DOPAC and 5-HIAA after L-deprenyl treatment. The mechanism by which L-deprenyl stimulates the efflux of NE, DA, 5-HT is not known. It has been suggested that it is an indirectly acting amine that affects the peripheral nervous system [23]. It has been hypothesized to enter the neuronal membrane through passive diffusion or act as a substrate for membrane pumps for neurotransmitters such as NE. Upon entering the neuron, it could then act to evoke the efflux of neurotransmitters [23]. The data supporting all these speculations deals exclusively with the peripheral nervous system and it is not clear whether Ldeprenyl could act in a similar fashion in the central nervous system.
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Deprenyl has been described as a catecholaminergic activity enhancer [7,35]. It has been shown to enhance catecholaminergic activity at much lower doses in other parts of the brain compared to the doses used in this study [7]. The present report investigated, for the first time, the ability of deprenyl to enhance the activity of catecholamines as well as serotonin from the hypothalamus in vitro at a higher dose range. The mechanisms behind these effects of deprenyl are unclear. There is evidence to indicate that deprenyl could primarily act as a potent stimulant of action potential-transmitter release coupling in catecholaminergic neurons [9]. Another compound namely, (⫺)1-(benzofuran-2-yl)-2-propylaminopentane [(⫺) BPAP], has been shown to be much more potent than deprenyl in its ability to enhance brain catecholaminergic activity at much lower doses [10]. It has been hypothesized that the catecholaminergic activity enhancing effect of compounds such as L-deprenyl, BPAP, could also be due to their effect on endogenous phenylethylamines or by activating hitherto unknown catecholaminergic activity enhancer substance in the brain [7]. Regardless of the mechanism, this report provides evidence for the first time that L-deprenyl is a potent enhancer of monoaminergic efflux from the hypothalamus, which, explain the novel actions of this drug. ACKNOWLEDGEMENTS
The authors would like to thank Somerset Pharmaceuticals Inc., FL, for the kind gift of L-deprenyl and Mr. Shawn Taylor, Kansas State University, for his technical assistance. This study was supported by NIH AG 05980.
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of the flavin site on the inhibited enzyme. In: Wolstenholme, G. E. W.; Knight, J., eds. Monoamine oxidase and its inhibitors. Amsterdam: Elsevier/North Holland; 1976:33– 47. Meites, J. Relation of prolactin to development of spontaneous mammary and pituitary tumor. Prog. Clin. Biol. Res. 75:1– 8; 1981. Meites, J. Anti-ageing interventions and their neuroendocrine aspects in mammals. J. Reprod. Fertil. 46(suppl.):1–9; 1993. Meites, J. Role of hypothalamic catecholamines in aging processes. Acta Endocrinol. (Copenh.) 125(suppl. 1):98 –103; 1991 MohanKumar, P. S.; Meites, J.; Quadri, S. K. Deprenyl reduces serum prolactin concentrations in rats. Life Sci. 54:841– 845; 1994. MohanKumar, P. S.; MohanKumar, S. M. J.; Quadri, S. K. Deprenyl stimulates the release of luteinizing hormone from the pituitary in vitro. Life Sci. 61:1783–1788; 1997. Palazzolo, D. L.; Quadri, S. K. Interleukin-1 stimulates catecholamine release from the hypothalamus. Life Sci. 47:2105–2109; 1990. Palazzolo, D. L.; Quadri, S. K. Interleukin-1 inhibits serotonin from the hypothalamus in vitro. Life Sci. 51:1797–1802; 1992. Quadri, S. K.; Kledzik, G. S.; Meites, J. Reinitiation of estrous cycles in old constant estrous rats by central acting drugs. Neuroendocrinology 11:248 –254; 1973. Salach, J. I.; Detmer, K.; Youdim, M. B. H. The reaction of bovine and rat liver monoamine oxidase with 14C-clorgyline and 14C-L-deprenyl. Mol. Pharmacol. 16:234 –241; 1979. Schoepp, D. D.; Azzaro, A. J. Role of type A and type B monoamine oxidase in the metabolism of released [H]Dopamine from rat striatal slices. Biochem. Pharmacol. 31:2961–2968; 1982. Simpson, L. L. Evidence that L-deprenyl a type B monoamine oxidase inhibitor is an indirectly acting sympathomimetic amine. Biochem. Pharmacol. 27:1591–1595; 1978. Tetrund, J. W.; Langston, J. W. The effect of L-deprenyl (selegiline) on the natural history of Parkinson’s disease. Science 245:519 –522; 1989. ThyagaRajan, S.; Meites, J.; Quadri, S. K. Deprenyl reinitiates estrous cycles, reduces serum prolactin, and decreases the incidence of mammary and pituitary tumors in old acyclic rats. Endocrinology 136: 1103–1110; 1995. ThyagaRajan, S.; Felten, S. Y.; Felten, D. L. Antitumor effect of l-L-deprenyl in rats with carcinogen-induced mammary tumors. Cancer Lett. 123:177–183; 1998. ThyagaRajan, S.; Quadri, S. K. L-deprenyl inhibits tumor growth, reduces serum prolactin, and suppresses brain monoamine metabolism in rats with carcinogen-induced mammary tumors. Endocrine 10:225– 232; 1999. ThyagaRajan, S.; Quadri, S. K. L-deprenyl stimulates the release of catecholamines in the rat medial basal hypothalamus in vivo. Neurosci. Lett. 270:79 – 82; 1999. ThyagaRajan, S.; Stevens, S. Y.; Felten, D. L. Region-specific alterations in the concentrations of catecholamines and indoleamines in the brains of young and old F344 rats after L-deprenyl treatment. Brain Res. Bull. 48:513–520; 1999. Waldmeier, P. C.; Feiner, A. E. Deprenyl: Loss of selectivity for inhibition of B-type MAO after repeated treatment. Biochem. Pharmacol. 27:801– 802; 1979. Yang, H.-Y. T.; Neff, N. H. The monoamine oxidases of brain: Selective inhibition with drugs and the consequences for the metabolism of the biogenic amines. J. Pharmacol. Exp. Ther.180:733–740; 1974. Yatham, L. N.; Steiner, M. Neuroendocrine probes of serotonergic function: A critical review. Life Sci. 53:447– 463; 1993. Youdim, M. B.; Riederer, P. Dopamine metabolism and neurotransmission in primate brain in relationship to monoamine oxidase A and B inhibition. J. Neural Transm. Gen. Sect. 91:181–195; 1993. Zsilla, G.; Knoll, J. The action of (⫺) L-deprenyl on monoamine turnover rate in rat brain. In: Costa, E.; Racagni, G., eds. Typical and atypical antidepressants: Molecular mechanisms. New York: Raven Press; 1982:211–217. Zsilla, G.; Foldi, P.; Held, G.; Szekely, A. M.; Knoll, J. The effect of repeated doses of L-deprenyl on the dynamics of monoaminergic transmission, comparison with clorgyline. Pol. J. Pharmacol. Pharm. 38:57– 67; 1986.
PII S0361-9230(01)00481-6
Deprenyl stimulates the efflux of monoamines from the rat hypothalamus in vitro P. S. MohanKumar,* Sheba M. J. MohanKumar and S. K. Quadri Neuroendocrine Research Laboratory, Department of Veterinary Pathology, College of Veterinary Medicine, Michigan State University, E. Lansing, MI, USA [Received 14 February 2001; Accepted 2 March 2001] ABSTRACT: The direct effects of L-deprenyl, a monoamine oxidase inhibitor, on the hypothalamus of male Sprague-Dawley rats was investigated by measuring the efflux of norepinephrine (NE), dopamine (DA), serotonin (5-HT), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) using a combination of high performance liquid chromatography with electrochemical detection and an in vitro incubation system. After measuring basal efflux by incubating the hypothalami with Krebs-Ringers Henseleit (KRH) alone during the first incubation period, hypothalami were incubated either with the medium, KRH alone (0 mM), or KRH containing 0.1, 1, and 10 mM Ldeprenyl. During the third incubation period, hypothalami were again incubated with KRH alone to measure the residual effects if any. During the final incubation period, the hypothalami were stimulated with high Kⴙ KRH. Deprenyl produced a dose-dependent increase in the efflux of NE, DA, and 5-HT from the hypothalami. Neurotransmitter efflux returned to pretreatment levels when L-deprenyl was removed from the medium. In contrast to NE, DA, and 5-HT, the efflux of the metabolites DOPAC and 5-HIAA was inhibited in a dose-dependent fashion after incubation with L-deprenyl. Results from this study demonstrate that L-deprenyl is capable of stimulating the efflux of neurotransmitters in vitro by a direct action on the hypothalamus. © 2001 Elsevier Science Inc.
mary tumors and pituitary tumors in old acyclic rats [25]. We have also provided evidence that L-deprenyl can decrease serum prolactin levels in young and old rats [16,25]. However, the mechanism by which L-deprenyl produces these effects is not clear. Brain neurotransmitters, especially monoamines are known to be involved in various central and neuroendocrine effects, such as increases in sexual activity, life-span, reinitiation of estrous cycles, and decreasing serum prolactin concentrations [13–15]. More specifically, monoamine changes in the hypothalamus could play a role in integrating these effects because the hypothalamus houses all the releasing hormone neurons that regulate these functions. Therefore, we hypothesized that L-deprenyl could produce its central and neuroendocrine effects by affecting monoamine efflux from the hypothalamus. Interestingly, very little is known about the effects of L-deprenyl on the efflux of hypothalamic monoamines. In this study, we used a combination of high performance liquid chromatography with electrochemical detection (HPLC-EC) and a static in vitro incubation system to investigate the direct effects of L-deprenyl on hypothalamic monoamine efflux. MATERIALS AND METHODS Animals
KEY WORDS: Deprenyl, Dopamine, Norepinephrine, Serotonin, Hypothalamus, HPLC-EC.
Four- to six-month-old male Sprague-Dawley rats were obtained from Amitech (Omaha, NE, USA). These rats were housed in groups in air-conditioned (23 ⫾ 2°C) and light controlled (lights on from 0700 –1900 h) animal rooms. They were provided rat chow and water ad libitum. On the day of the experiment, the rats were sacrificed between 1200 –1300 h. The brains were removed immediately and the hypothalami were dissected out using the following boundaries: the posterior part of the optic chiasm as the anterior limit, the anterior part of the mammillary bodies as the posterior limit, and the lateral hypothalamic sulci as the lateral limits [18,19].
INTRODUCTION Deprenyl is an irreversible monoamine oxidase (MAO)-B inhibitor [3,4]. However, depending on the dose and duration of treatment it has also been shown to inhibit MAO-A at higher concentrations [3,31]. Like some other MAO inhibitors, L-deprenyl was also used originally as an antidepressant [3,4]. Recently, because of its ability to inhibit MAO and increase dopaminergic tone in the striatum, it is being used in the treatment of Parkinson’s disease [5,6,24]. Deprenyl has also been shown to have other novel biological actions, such as increasing life-span and sexual activity in male rats [8]. Recently, we reported that L-deprenyl also inhibits carcinogen-induced mammary tumor growth in rats and reinitiates estrous cycles and decreases the incidence of spontaneous mam-
In Vitro Incubation The hypothalami were weighed and divided into two halves along the sagittal plane and incubated at 37°C in a 12 ⫻ 75 mm glass tube containing 300 l of Krebs-Ringer Henseleit (KRH) solution in a static in vitro incubation system as described previ-
* Address for correspondence: Dr. P. S. MohanKumar, Neuroendocrine Research Laboratory, Department of Veterinary Pathology, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA. Fax: ⫹1-(517)-432-7480; E-mail: [email protected]
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ously [17–19]. The incubation medium, KRH, consisted of 117 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 24.8 mM NaHCO3, 11.1 mM glucose, 0.1 mg/ml ascorbic acid in 1 l of pyrogen-free water. During the experiment, the incubation medium was supplied a mixture of 95% oxygen and 5% carbon dioxide constantly at a slow and steady rate to avoid turbulence. To stabilize the efflux of monoamines, each hypothalamus was preincubated in KRH for 60 min. Following this, they were incubated for four 60-min periods as described below. During the first incubation period, the hypothalami were incubated in KRH for 60 min to measure the basal efflux of monoamines and their metabolites. During the second incubation period, the hypothalami were incubated in KRH containing 0, 0.1 mM, 1.0 mM, or 10 mM of L-deprenyl (n ⫽ 6 – 8/treatment group). After this, the hypothalami were rinsed and incubated in KRH alone (without L-deprenyl) during the third incubation period to measure the residual effects, if any, from the previous incubation period. In the final incubation period, the hypothalami were incubated with high K⫹ KRH, which had the same composition as the KRH except that it contained 60 mM KCl by withholding equimolar amount of NaCl, to check the viability of the tissue. Hypothalami were rinsed with KRH in between incubations. After each incubation period, incubation medium was removed and 0.1 M HClO4 was added to it at the ratio of 25:1 (v/v) and was stored at ⫺70°C until analysis for neurotransmitters by HPLC-EC. At the end of the treatment period, the hypothalami were transferred to microsample vials containing 0.1 M HClO4 and were stored at ⫺70°C. At the time of HPLC analysis the hypothalami were homogenized in KRH and centrifuged at 10,000 RPM for 15 min and the supernatant was used for the measurement of neurotransmitters. HPLC-EC The HPLC-EC system has been described before [18,19,25]. Briefly, it consisted of a LC-4B electrochemical detector (Bioanalytical Systems, West Lafayette, IN, USA); a phase II, 5 m ODS reverse phase, C-18 column; a glassy carbon electrode, a C-R6A Chromatopac integrator, a CTO-6A column oven, and a LC-6A pump (Shimadzu, Columbia, MD, USA). The mobile phase consisted of monochloroacetic acid (14.14 g/l), sodium hydroxide (4.675 g/l), octanesulfonic acid disodium salt (0.3 g/l), ethylenediaminetetraacetic acid (0.25 g/l), acetonitrile (3.5%), and tetrahydrofuran (1.4%). The mobile phase was made in pyrogen-free water and then filtered and degassed through a Milli-Q purification system (Millipore Co., Bedford, MA, USA). It was pumped through the HPLC system at a flow rate of 1.7 ml/min. The sensitivity of the detector was 1 nA full scale, and the potential of the working electrode was 0.65 V. The column and the electrodes were kept in a column oven maintained at 37°C. At the time of HPLC analysis, the incubation medium was diluted at the ratio of 1:4 with 0.1 M HClO4. Fifty microliters of the diluted incubation medium along with 25 l of the internal standard (0.05 M isoproterenol) was injected into the HPLC system. Statistical Analysis Differences in the efflux of neurotransmitters during various incubation periods in each group was analyzed by analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD). Differences in the contents of neurotransmitters in the hypothalamus among the various groups were analyzed by one way ANOVA followed by Fisher’s LSD.
FIG. 1. The effects of L-deprenyl (DEP) on the average efflux of norepinephrine (NE) from the hypothalamus in vitro (A) and on the average content of NE in the hypothalami at the end of the experiment (B). The hypothalami were incubated in Krebs-Ringers Henseleit (KRH) for four consecutive 60-min periods: with KRH alone in the first period, with KRH and L-deprenyl in the second period and again with KRH in the third period and with high K⫹ KRH in the fourth period (n ⫽ 6 – 8/treatment group). At the end of each period the incubation medium was removed for neurotransmitter analysis by high-performance liquid chromatography with electrochemical detection and the hypothalami were rinsed with KRH. At the end of the fourth incubation period the hypothalami were removed and stored in 0.1 M HClO4 and analyzed for neurotransmitter concentrations. (A) *Significantly different from NE levels in the corresponding group (10 mM) in the first incubation period (KRH) (p ⬍ 0.05); **significantly different from NE levels in the corresponding group in the first incubation period and also from other groups during the second incubation period (KRH⫹DEP) (p ⬍ 0.01). (B) *Significantly different from 0 and 0.1 mM (p ⬍ 0.05).
RESULTS Norepinephrine The effects of L-deprenyl on the efflux of norepinephrine (NE) are shown in Fig. 1A. The pretreatment NE efflux in various groups were not different from each other. In the control group (0 mM), NE efflux (Mean ⫾ SE; pg/mg hypothalamus;153.4 ⫾ 49) did not change significantly during the first 3 incubation periods but produced a marked increase of 497.9 ⫾ 76 when incubated with high potassium KRH (p ⬍ 0.05). In contrast to the control group, incubation with different doses of L-deprenyl produced a dose-dependent increase in NE efflux. When the hypothalami were incubated with 0.1 mM L-deprenyl, NE efflux seemed to increase from pretreatment levels of 196.9 ⫾ 43 to 252.3 ⫾ 48, but this increase was not significant. NE efflux returned to pretreatment
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levels (191.6 ⫾ 51) during the third incubation period. Once again challenge with high potassium KRH produced a robust increase in NE efflux (538.2 ⫾ 142; p ⬍ 0.05). Incubation with 1 mM L-deprenyl on the other hand, increased NE efflux significantly from 196.9 ⫾ 8 to 354.7 ⫾ 79 during the second incubation period (p ⬍ 0.05) and decreased to 267.7 ⫾ 28 during the third incubation period. High K⫹ KRH once again stimulated NE efflux (375.2 ⫾ 92) during the last incubation period (p ⬍ 0.05). Incubation with 10 mM L-deprenyl produced a robust increase in NE efflux during the second incubation period (794.3 ⫾ 181) when compared to the pretreatment incubation (153.9 ⫾ 67; p ⬍ 0.05) and then declined to159.8 ⫾ 41 during the third incubation period. However, incubation with high K⫹ KRH produced only a modest increase (222.4 ⫾ 57) which was not statistically significant. The content of NE in the hypothalami at the end of the experiment is shown in Fig. 1B. Hypothalami in the control and 0.1 mM L-deprenyl treated groups had significantly higher concentrations of NE (1104 ⫾ 588 and 743.3 ⫾ 179, respectively). These were nearly 3-4 fold higher than the NE concentrations in the 1 mM and 10 mM L-deprenyl treated groups (388.1 ⫾ 71 and 281.3 ⫾ 91, respectively; p ⬍ 0.05). Dopamine The effects of L-deprenyl on dopamine (DA) efflux are shown in Fig. 2A. In contrast to NE efflux, DA efflux seemed to decrease much rapidly over time. It decreased from 74.6 ⫾ 27 in the second incubation period in the control group to 16.1 ⫾ 7 in the third incubation period (p ⬍ 0.01). Incubation with high K⫹ KRH in this group produced a significant increase in DA efflux of 258.9 ⫾ 27 (p ⬍ 0.01). Similar to its effects on NE, L-deprenyl produced a dose-dependent increase in DA efflux. Incubation with 0.1 mM L-deprenyl seemed to increase DA efflux (125.1 ⫾ 49) but this was not significantly different from the pretreatment efflux rate. Treatment with 1 mM L-deprenyl on the other hand produced a sharp increase in DA efflux (154.8 ⫾ 59) when compared to pretreatment levels (42.9 ⫾ 6). In both these groups, removal of L-deprenyl from the medium during the third incubation period brought DA efflux to pretreatment levels and stimulation with high K⫹ KRH produced significant increases (293.1 ⫾ 109 and 106.8 ⫾ 27, respectively) in DA efflux. Incubation with 10 mM L-deprenyl produced a stark increase in DA efflux (197.7 ⫾ 63), removal of L-deprenyl from the medium returned DA efflux to basal levels as in the other groups. However, incubation with high potassium KRH did not produce a significant increase in DA efflux (85.3 ⫾ 40) compared to the pretreatment levels. DA content of the hypothalami in all the groups at the end of the experiment is shown in Fig. 2B. DA concentrations in the 1 mM and 10 mM groups were significantly less compared to the 0 mM group (p ⬍ 0.05). Dihydroxyphenylacetic Acid The dose-dependent inhibitory effects of L-deprenyl on dihydroxyphenylacetic acid (DOPAC) efflux are shown in Fig. 3A. DOPAC efflux decreased during the third incubation (205.3 ⫾ 38) when compared to the pretreatment level (329.3 ⫾ 70) in the control group but increased upon stimulation with high potassium KRH (467.3 ⫾ 84; p ⬍ 0.05). Incubation with 0.1 mM L-deprenyl did not produce any change in DOPAC efflux but incubation with 1 mM L-deprenyl produced a marked decrease in DOPAC efflux (139.6 ⫾ 19) when compared to pretreatment levels (267.7 ⫾ 46; p ⬍ 0.01). A more pronounced effect was observed after incubation with 10 mM L-deprenyl. DOPAC efflux decreased from pretreatment levels of 285.6 ⫾ 28 to 68.9 ⫾ 20 (p ⬍ 0.001). In
FIG. 2. The effects of L-deprenyl (DEP) on the average efflux of dopamine (DA) from the hypothalamus in vitro (A) and on the average content of DA in the hypothalami at the end of the experiment (B). (A) *Significantly different from DA levels in the corresponding groups (1 and 10 mM) in the first incubation period (KRH) (p ⬍ 0.05); (B) *significantly different from 0 mM (p ⬍ 0.05). See Fig. 1 legend for details.
both these groups, DOPAC efflux remained low even after removal of L-deprenyl from the medium. Moreover, stimulation with high potassium KRH did not produce any increase in DOPAC efflux in all the L-deprenyl-treated groups. The concentration of DOPAC in the hypothalamus at the end of the experiment (Fig. 3B) was markedly reduced in the 1 and 10 mM L-deprenyl treated groups (86.8 ⫾ 34 and 34.3 ⫾ 16, respectively) compared to control and 0.1mM L-deprenyl groups (416 ⫾ 176 and 290.0 ⫾ 110, respectively; p ⬍ 0.05). Serotonin The basal efflux of serotonin (5-HT) as observed in the control group decreased significantly with time (Fig. 4A). It decreased from 65.1 ⫾ 25 in the first incubation period to 41.0 ⫾ 21 and further declined to 23.2 ⫾ 12 during the third incubation period (p ⬍ 0.05). Upon incubation with different doses of L-deprenyl, a remarkable and dose-dependent increase in 5-HT efflux was observed (186.4 ⫾ 62, 438.4 ⫾ 85 and 670.3 ⫾ 199 with 0.1, 1 and 10 mM L-deprenyl, respectively). The efflux of 5-HT declined rapidly upon removal of L-deprenyl from the medium in the third incubation period. Stimulation with high potassium KRH produced a significant increase in 5-HT efflux in 0 mM and 0.1 mM groups. Similar to NE and DA, content of 5-HT at the end of the experiment was significantly reduced in 1 mM and 10 mM groups compared to the control group (Fig. 4B; p ⬍ 0.05).
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FIG. 3. The effects of 0, 0.1, 1, and 10 mM L-deprenyl (DEP; n ⫽ 6 – 8/treatment group) on the average efflux of dihydroxyphenylacetic acid (DOPAC) from the hypothalamus in vitro (A) and on the average content of DOPAC in the hypothalami at the end of the experiment (B). (A) *Significantly different from DOPAC levels in the corresponding group during the first incubation period (KRH); **significantly different from DOPAC levels in the corresponding group (10 mM) in the first incubation period (KRH) and also from other groups within the same incubation period (p ⬍ 0.01). (B) *Significantly different from 0 and 0.1 mM (p ⬍ 0.05). See Fig. 1 legend for details.
FIG. 4. The effects of L-deprenyl (DEP) on the average efflux of serotonin (5-HT) from the hypothalamus in vitro (A) and on the average content of 5-HT in the hypothalami at the end of the experiment (B). (A) *Significantly different from corresponding group in the first incubation period (KRH) and also from other groups within the same incubation period (KRH⫹DEP) (p ⬍ 0.05); **significantly different from corresponding groups in the first incubation period (KRH) and also from 0 and 0.1 mM groups within the same incubation period (KRH⫹DEP) (p ⬍ 0.05); (B) *significantly different from 0 and 0.1 mM (p ⬍ 0.05). See Fig. 1 legend for details.
5-Hydroxyindoleacetic Acid
with high potassium KRH produced a pronounced increase in the efflux of these neurotransmitters indicating that the tissues were viable. In contrast, the efflux of the metabolites, DOPAC, and 5-HIAA are inhibited upon incubation with L-deprenyl clearly indicating its ability to inhibit MAO. Results from this study are supported by another study in which L-deprenyl increased NE content in the teldiencephalon and the rest of the brain [34]. Recent studies from our laboratory have shown that L-deprenyl can increase the concentration of NE in the striatum and the mediobasal hypothalamus and stimulate the efflux of NE from the mediobasal hypothalamus in vivo [25,28]. The present study demonstrates that L-deprenyl stimulates NE efflux through its action on the hypothalamus. Deprenyl-induced increase in NE efflux from the hypothalamus could explain some of the novel effects of deprenyl. Deprenyl is known to increase sexual activity in male rats and reinitiate estrous cycles in old female rats. Both of these effects are known to involve luteinizing hormone (LH), the efflux of which from the pituitary is stimulated by L-deprenyl [17]. NE is one of the important neurotransmitters involved in the regulation of LH secretion. The fact that the synthesis, efflux, and content of NE declines in the hypothalamus of old animals is well established
In contrast to the efflux of 5-HT, the efflux of its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), showed a dose-dependent decrease when the hypothalami were incubated with different doses of L-deprenyl or KRH (Fig. 5A). The inhibition of 5-HIAA efflux observed over time continued even after stimulation with high potassium KRH. Moreover the content of 5-HIAA in the hypothalami at the end of the experiment (Fig. 5B) was significantly low in the L-deprenyl treated groups when compared to the control group (p ⬍ 0.05). DISCUSSION The results from this study clearly demonstrate for the first time that L-deprenyl stimulates the efflux of NE, DA, and 5-HT from the hypothalamus in vitro in a dose-dependent fashion. The efflux of these neurotransmitters either remains stable or decreases gradually as a function of time. This is a characteristic feature of the in vitro incubation system and has been observed in other studies before [16,19]. The consistent finding in all the groups was that L-deprenyl not only prevented this decrease but produced a marked increase in the efflux of NE, DA, and 5-HT. Stimulation
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FIG. 5. The effects of L-deprenyl (DEP) on the average efflux of 5-hydroxyindoleacetic acid (5-HIAA) from the hypothalamus in vitro (A) and on the average content of 5-HIAA in the hypothalami at the end of the experiment (B). (A) *Significantly different from corresponding groups in other incubation periods and also from other groups within the same incubation period (p ⬍ 0.01); **significantly different from 5-HIAA levels in the first incubation period (KRH) (p ⬍ 0.05); (B) *significantly different from 0 and 0.1 mM (p ⬍ 0.05). See Fig. 1 legend for details.
[14,15]. Administration of compounds, which increase brain NE levels such as l-dopa or the MAO inhibitor iproniazid could reinitiate estrous cycles in old rats [20]. Recently, we have demonstrated that administration of L-deprenyl itself could reinitiate estrous cycles in old female rats [25] indicating that this effect of L-deprenyl is most probably mediated through an increase in NE activity. The current study provides the first direct evidence for this phenomenon. Incubation of the hypothalamus with L-deprenyl also increased DA efflux in a dose-dependent manner. This finding is supported by another study in which direct infusion of L-deprenyl into the hypothalamus stimulated the release of DA [28]. Other studies have also indicated that L-deprenyl increases the turnover rate and release of DA from the striatum [2, 6]. Other indirect lines of evidence suggest that L-deprenyl may indeed produce this effect in the hypothalamus. Deprenyl has been shown to inhibit prolactin secretion [17,25], which, could be mediated through stimulation of DA because prolactin is under the inhibitory control of DA [13]. Moreover, L-deprenyl has been shown to cause the regression of prolactin-dependent mammary tumors [25–27]. This effect is probably achieved through an increase in DA, which causes suppression of prolactin secretion, which ultimately results in the regression of the mammary tumors. Therefore, it is conceivable that
679 L-deprenyl may indeed stimulate DA activity to produce some of its beneficial effects. Apart from stimulating the efflux of NE and DA from the hypothalamus, the present study provides evidence that L-deprenyl stimulates the efflux of 5-HT in a dose-dependent fashion. Other studies from our laboratory and others have provided evidence that prolonged daily administration or intracranial administration of deprenyl could affect monoaminergic activity of the hypothalamus both in male and female rats [26 –29]. Like NE and DA, 5-HT is also involved in a number of neuroendocrine effects including the regulation of LH and prolactin [11,32]. Thus it is quite probable that at least some of the effects of L-deprenyl could be mediated through its effects on 5-HT in the hypothalamus. After incubation with the highest dose of L-deprenyl, the efflux of NE, DA, and 5-HT were unaltered upon stimulation with high K⫹ KRH. The reasons for the inability of high K⫹ KRH to stimulate neurotransmitters after treatment with the highest dose of L-deprenyl are not clear. It is possible that the highest dose of deprenyl because of its pronounced neurotransmitter releasing effects (several-fold increase compared to the control group) could have exhausted the available pool of neurotransmitters in the hypothalamic blocks. This possibility is supported by the observation that neurotransmitter content at the end of the treatment (10 mM of L-deprenyl) were significantly low compared to the control group. In contrast to its effect on NE, DA, and 5-HT, L-deprenyl decreased the levels of the metabolites, DOPAC, and 5-HIAA, in a dose-dependent fashion. In rats, the metabolism of monoamines is a complex process and is achieved by both forms of monoamine oxidases (MAO-A and MAO-B) [31,33]. While DA acts as a substrate for both MAO-A and B, 5-HT is metabolized by MAO-A [21,22]. Thus, L-deprenyl being a MAO-B inhibitor inhibited the metabolism of DA and decreased the levels of its metabolite significantly. The interesting finding in the present study was the capacity of L-deprenyl to also inhibit the levels of 5-HIAA, even at the lowest dose used. As alluded to before, L-deprenyl could act on both forms of MAO [1,3,30,31], hence, it is probable that L-deprenyl could simultaneously inhibit the efflux of both DOPAC and 5-HIAA. A similar decrease in DOPAC and 5-HIAA concentrations in the mediobasal hypothalamus and the striatum have been observed before after L-deprenyl treatment [25,27,29]. The inhibition observed in the present study was so great that high K⫹ KRH which was capable of stimulating the efflux of NE, DA, and 5-HT was still unable to stimulate the efflux of DOPAC and 5-HIAA. This could only mean that L-deprenyl totally inhibited the break-down of DA and 5-HT to its metabolites while simultaneously stimulating the efflux of these neurotransmitters from the hypothalamus. This is not surprising because L-deprenyl is an established monoamine oxidase inhibitor, which acts on the flavin binding site to irreversibly inhibit this enzyme [12]. The enzyme activity is believed to be reinstated only after the synthesis of new enzyme molecules [12] which is not possible in the in vitro model. This clearly explains the stark decrease in the efflux of DOPAC and 5-HIAA after L-deprenyl treatment. The mechanism by which L-deprenyl stimulates the efflux of NE, DA, 5-HT is not known. It has been suggested that it is an indirectly acting amine that affects the peripheral nervous system [23]. It has been hypothesized to enter the neuronal membrane through passive diffusion or act as a substrate for membrane pumps for neurotransmitters such as NE. Upon entering the neuron, it could then act to evoke the efflux of neurotransmitters [23]. The data supporting all these speculations deals exclusively with the peripheral nervous system and it is not clear whether Ldeprenyl could act in a similar fashion in the central nervous system.
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Deprenyl has been described as a catecholaminergic activity enhancer [7,35]. It has been shown to enhance catecholaminergic activity at much lower doses in other parts of the brain compared to the doses used in this study [7]. The present report investigated, for the first time, the ability of deprenyl to enhance the activity of catecholamines as well as serotonin from the hypothalamus in vitro at a higher dose range. The mechanisms behind these effects of deprenyl are unclear. There is evidence to indicate that deprenyl could primarily act as a potent stimulant of action potential-transmitter release coupling in catecholaminergic neurons [9]. Another compound namely, (⫺)1-(benzofuran-2-yl)-2-propylaminopentane [(⫺) BPAP], has been shown to be much more potent than deprenyl in its ability to enhance brain catecholaminergic activity at much lower doses [10]. It has been hypothesized that the catecholaminergic activity enhancing effect of compounds such as L-deprenyl, BPAP, could also be due to their effect on endogenous phenylethylamines or by activating hitherto unknown catecholaminergic activity enhancer substance in the brain [7]. Regardless of the mechanism, this report provides evidence for the first time that L-deprenyl is a potent enhancer of monoaminergic efflux from the hypothalamus, which, explain the novel actions of this drug. ACKNOWLEDGEMENTS
The authors would like to thank Somerset Pharmaceuticals Inc., FL, for the kind gift of L-deprenyl and Mr. Shawn Taylor, Kansas State University, for his technical assistance. This study was supported by NIH AG 05980.
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