The metabolism of histamine in rat hypothalamus and cortex after reserpine treatment

Neurochemistry International 85-86 (2015) 31–39

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Neurochemistry International j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n c i

The metabolism of histamine in rat hypothalamus and cortex after reserpine treatment Martin Maldonado *, Kazutaka Maeyama Department of Pharmacology, School of Medicine, Ehime University, Shigenobu, Ehime 791-02, Japan

A R T I C L E

I N F O

Article history: Received 14 October 2014 Received in revised form 13 April 2015 Accepted 21 April 2015 Available online 29 April 2015 Keywords: Histamine Nτ-methylhistamine Reserpine Catecholamines Ws/Ws rats Histaminergic-neurons

A B S T R A C T

The effect of reserpine on histamine (HA) and tele-methylhistamine (Nτ-MHA) in hypothalamus and cortex of rats was analyzed and compared to catecholamines. IP injection of reserpine (5 mg/kg) confirmed the effectiveness of reserpine treatment on noradrenaline and dopamine levels. Our in-vitro experiment with synaptosomal/crude mitochondrial fraction from hypothalamus and cortex confirmed that while mono amine oxidase (MAO) is an efficient metabolic enzyme for catecholamines, HA is not significantly affected by its enzymatic action. HMT activity after reserpine, pargyline and L-histidine treatment showed no differences compared to the control values. However HDC was significantly increased in both hypothalamus and cortex. In this study, Ws/Ws rats with deficiency of mast cells were used to clarify aspects of HA metabolism in HAergic neurons by eliminating the contribution of mast cells. The irreversible MAO-B inhibitor Pargyline (65 mg/kg) failed to accumulate Nτ-MHA in the hypothalamus. However, when animals treated with reserpine and pargyline/reserpine were compared, the last group showed higher Nτ-MHA values (p < 0.01). Moreover, the precursor of HA, L-histidine (1 g/kg), produced an increase of HA in the hypothalamus to 166% and the cortex to 348%. In conclusion, our results suggest that the effect of reserpine on the HA pools in the brain might be different. The neuronal HA pools are more resistant to reserpine as compared to those of catecholamine. Moreover, the HAergic pool appears to be more resistant to depletion than mast cells’ pool, and thus HDC/HMT activity and its localization may play a key role in the understanding of HA metabolism in brain after reserpine treatment. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Histamine (HA), a heterocyclic primary amine, is synthesized from the decarboxylation of the amino acid histidine by the enzyme L-histidine decarboxylase (HDC, E.C.4.1.1.22) (Watanabe et al., 1979). HA participates in local immune response (Hébert et al., 1980), circadian rhythm (Chu et al., 2004; Tuomisto, 1991), feeding behavior (Sakata, 1991; Yoshimatsu, 2008), motion sickness and locomotor activity (Clapham and Kilpatrick, 1994; Matsunaga and Takeda, 1991), memory formation (Blandina et al., 1996), cardiovascular control (Imamura et al., 1996), food and water intake (Lecklin et al., 1998) and gastric secretion (Furtani et al., 2003; MacIntosh, 1938), among others. HA has also been reported to act as a neurotransmitter in mammalian brain (Prell and Green, 1986; Schwartz, 1975; Wada et al., 1991). Two different pools containing HA in the brain have been identified – histaminergic (HAergic) neurons and mast cells (Garbarg et al., 1976; Panula, 1986; Watanabe et al., 1983).

* Corresponding author. Research Center of Reproductive Medicine, Shantou University, Medical College. 22 Xinling Road, Shantou, Guangdong 515041, China. Tel.: +86 13501415490; fax: +86 75488900497. E-mail address: [email protected] (M. Maldonado). http://dx.doi.org/10.1016/j.neuint.2015.04.005 0197-0186/© 2015 Elsevier Ltd. All rights reserved.

The somata of HAergic neurons originate within the hypothalamic tuberomammillary nuclei, sending out their axons and innervating practically the entire brain and parts of the spinal cord (Oishi et al., 1983; Watanabe et al., 1984). Two enzymes are known to participate in the degradation of HA: diamine oxidase (DAO; E.C. 1.4.3.6), within the digestive tract, to imidazole acetaldehyde, responsible for scavenging extracellular histamine after mediator release (Agúndez et al., 2012; Garcia-Martin et al., 2009); and histamine N-metyltransferase (HMT, E.C. 2.1.1.8) to Nτ-methylhistamine (Nτ-MHA), which is responsible for inactivating histamine in brain. Therefore, the fluctuation of HA and NτMHA levels may provide an accurate information of HA turnover and HAergic neurons activity (Agúndez et al., 2012; Garcia-Martin et al., 2009; Oishi et al., 1983; Sugimoto et al., 1995). HA has been thought to accumulate in secretory vesicles in neurons and granules of enterochromaffin-like cells in stomach and mast cells through the action of vesicle monoamine transporter VMAT, a member of the vesicular neurotransmitter transporter family, responsible for the translocation of monoamines (serotonin, dopamine, norepinephrine, and HA). Two isoforms, VMAT1 and VMAT2, have been identified in mammals; however, VMAT2 is the only isoform expressed in neuronal cells (Erickson et al., 1992; Liu et al., 1992).

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Fig. 1. Chemical structure of the neurotransmitters and reserpine. From left to right: The precursor of HA; HA; the metabolite of HA; catecholamines; reserpine.

In mammalian brain, monoamines are transported from the cytoplasm of the pre-synaptic nerves, via a proton electrochemical gradient generated by the vacuolar type H+ -adenosine triphosphatase, into vesicles for storage and posterior release into the synaptic cleft via VMAT2 (Yelin and Schuldiner, 2002). Reserpine, an indole alkaloid with antihypertensive and antipsychotic properties (Frize, 1954), interferes with the transport of monoamines by irreversibly inhibiting VMAT2 (Henry and Scherman, 1989). Therefore, its blocking action leads to an accumulation of amines in the cytosol and subsequent degradation by enzymes like MAO (monoamine O2 oxidoreductase, EC 1.4.3.4). Brain and peripheral sympathetic nerves undergo a marked depletion of monoamines, resulting in symptoms of depression (Monoamine hypothesis) (Schildkuart, 1965) among others. Because of its numerous adverseeffects, nowadays reserpine is rarely used in human treatments. However, it has been proven to be a useful tool to study the mechanism of neurotransmitter transport into synaptic vesicles and represents a valuable instrument to understand the pathways of histamine metabolism in animal models. In the present study, the effect of reserpine on HA and Nτ-MHA levels in hypothalamus and cortex of rats were investigated and compared to those of catecholamines (dopamine and norepinephrine). The chemical structure of reserpine and the neurotransmitters studied in this work are shown in Fig. 1. The effects of pargyline, clorgyline, deprenyl (L-deprenyl) and L-histidine, which all affect the synthesis and/or catabolism of HA and Nτ-MHA, were analyzed to reinforce our findings.

2. Materials and methods 2.1. Chromatographic instrumentation HPLC experiments were performed according to Maldonado and Maeyama (2013). The HPLC system consists of a model LC-100 AS micro LC pump (BAS, Tokyo, Japan), a vacuum degasser Shodex Degas (Showa Denko, Japan), a Model 7125 injector (Reodyne, Cotati, CA, USA), a reversed phase analytical column (TSK-gel ODS 80Ts,5 μm, 4.6 mm i.dx 150 mm; Tosoh, Tokyo, Japan), and a potentiostat ALS/CHI, Electrochemical Analyzer Model 800 (BAS, Tokyo, Japan) with a glassy carbon electrode. The column was eluted with a 30% methanol in 50 mM potassium phosphate buffer (pH 6.50) containing 0.5 mM Na2-EDTA at a flow rate of 500 μl/min and the detector output current was monitored at a potential of +0.75 V. Peak areas (expressed as charges) and sample concentrations were collected with an ALS800a EC MFC (CH Instruments, Inc. USA).

2.2. Reagents and materials HA phosphate, OPA, sodium borate (Na2B4O7), Na2SO3-anhydrous, dopamine, norepinephrine and L-histidine hydrochloride monohydrate were purchased from Wako Pure Chemicals (Osaka, Japan). Nτ-MHA, pargyline hydrochloride, deprenyl (Selegiline), clorgyline, propionic acid and reserpine were purchased from Sigma-Aldrich (St Louis, MO, USA). Viva pure sulphonic acid (S) R-CH2-SO3-Na+ strong acidic cation exchange (high binding capacity) spin columns were purchased from Sartorious Stedim Biotech. OPA- Na2SO3 derivatization solution was prepared combining 25 μl of 0.2 M OPA in methanol and 25 μl of 1 M Na2SO3 with 950 μl of 0.1 M sodium borate buffer (pH 9.65). 2.3. Animals Male Wistar and Ws/Ws rats (220–250 g) were used in this study. The mast cell deficient Ws/Ws rat model is characterized by a homozygosis for a 12 base deletion in the tyrosine kinase domain of the c-kit receptor gene, commonly known as the white-spotting locus. The c-kit receptor induces mast cell proliferation and differentiation when activated by stem cell factors (Tsujimura et al., 1991). The animals were housed at a constant temperature of 22 ± 2 °C with an automatically controlled 12:12-h light-dark cycle (light on at 7:00 am). Food and water were available ad libitum. The experimental protocols were carried out in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was reviewed and approved by the Committee on the Ethics of Animal Studies of Ehime University. Surgeries were performed under IP injection of sodium pentobarbital (Nembutal) 50 mg/kg, and all efforts were made to minimize suffering. 2.4. Animal drug pre-treatment Drugs were administrated by IP injection. Reserpine, 5 mg/kg, was injected 24 hours before euthanasia; pargyline, 65 mg/kg, was injected 90 min before euthanasia; and L-histidine, 1 g/kg, was injected 3 hours before euthanasia. Euthanasia was carried out by decapitation under previous deep sedation with Nembutal. 2.5. Pre-purification of HA, Nτ-MHA and catecholamines from biological samples HA and Nτ-MHA were purified using a Vivapure column. This Vivapure mini H filter with 3–5 μm pore size was washed with 0.5 ml of 2 M HCl and 0.5 ml of 1 M NaOH, twice and then equilibrated with

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four times of 0.5 ml of 5 mM EDTA-Na2 (pH 6.5). Briefly, the brain cortex and hypothalamus of Wistar and Ws/Ws rats were isolated, weighed, and homogenized in five volumes (v/w) of 3% perchloric acid solution containing 0.5 mM EDTA with a Polytron homogenizer (Kinematica, Luzern, Switzerland). The homogenized product was centrifuged at 5500 g for 15 minutes at 4 °C. The supernatants of hypothalamus and cortex were treated with 10% of 5 M KOH/ 0.2 M KH 2 PO 4 , centrifuged at 900 g for 2 min and 400 μl of supernatant was transferred into the Vivapure tube containing the cation exchange column. The Vivapure tube containing the sample was centrifuged at 900 g for 3 min and washed with 200 μl of 5 mM EDTA-Na2 at 2000 g for 3 min. Thereafter, 200 μl of reagent was applied into the Vivapure column and centrifuged at 300, 600, 900, 1200 and 2000 g for a duration of 1 min each step. The reaction of the reagent with the amines took place during these centrifugation steps. Finally, 100 μl (half of the eluted volume) was injected into the HPLC-ECD. For catecholamine measurement, the HPLC-ECD buffer was replaced with Li-propionate and the TSK-gel ODS 80Ts column, substituted with a 5 C18 column (25 cm–2 mm) at a flow rate of 200 μl/min. Hypothalamus and cortex tissues were treated with 10% of 2 M KOH/1 M KH2PO4 and the resulting supernatant (pH 6.5) was directly applied via injection into the HPLC-ECD without Vivapure column pre-treatment.

2.6. In-vitro experiment of synaptosomal fraction of mitochondria extract from hypothalamus and cortex: HA, Nτ-MHA, dopamine and nor-epinephrine substrate Preparation of synaptosomal fractions of mitochondria was performed according to Rendon and Masmoudi (1985), with minor modifications. Briefly, the brain hypothalamus and cortex of Wistar rats were isolated and rapidly washed in buffer (0.32 M sucrose, 1 mM EDTA K+, 10 mMTris-HCl, pH 7.4) to remove blood debris. A 20% (w/v) homogenate in isolation buffer was made in a motordriven Potter homogenizer with 10 up and down strokes. The homogenate was then diluted to 10% (w/v) with isolation buffer and centrifuged at 1100 g for 5 min. The supernatant was centrifuged at 17,000 g for 10 min to yield the crude mitochondrial pellet containing synaptosomes. One hundred μM of HA, Nτ-MHA, dopamine and norepinephrine substrates was incubated separately, with the mitochondria extract (MAO) enzyme and with the inhibitors pargyline, clorgyline and deprenyl, 1 mM for 120 min and then analyzed by HPLC-ECD (Maldonado and Maeyama, 2013).

2.7. HMT assay procedure in hypothalamus and cortex of Wistar rats after drug treatment Hypothalamus and cortex of Wistar rats were homogenized in 5 volumes of ice-cold histamine HMT buffer solution with a Polytron homogenizer. The buffer solution was prepared with 50 mM potassium phosphate buffer (pH 6.8); 100 μg/ml of phenylmethylsulfonyl fluoride, as a protease inhibitor; and 1% (w/v) of polyethylene glycol (average molecular weight 300). The homogenate was centrifuged at 5500 g for 15 minutes, and the supernatant was dialyzed three times against 50 volumes of HMT buffer solution. The enzyme solution, combined with 100 μM HA and 250 μM S-adenosyl-methionine, as substrates, was incubated for 4 hours in 500 μl of HMT buffer solution at 37 °C. Forty μl of the reaction mixture was added with 160 μl of 100% methanol for deproteinization. The Nτ-MHA produced was then measured by HPLC-ECD as previously described. The protein content was measured with a Bio-Rad protein assay kit with bovine serum albumin as a standard.

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2.8. HDC activity in hypothalamus and cortex of Wistar rats after drug treatment The animals were starved for 12 h before the commencement of the experiment in order to exclude the effect of intrinsic histidine derived from food. HDC activity was assayed according to Watanabe et al. (1981), with minor modifications. Briefly, hypothalamus and cortex samples of Wistar rats were homogenized in 4 vol (wt/vol) of solution A (0.1 M potassium phosphate, pH 6.8/0.2 mM dithiothreitol/0.01 mM pyridoxal ‘-phosphate/1.0% polyethylene glycol [average Mr 300] containing protease inhibitors, at 2 ug/ml each) in a Polytron (Kinematica) in an ice bath. The homogenate was centrifuged at 10,000 × g for 20 min at 40 °C. One-tenth of the supernatant was added to 9 vol of cold 0.5 M perchloric acid/5 mM EDTA for histamine analysis. The remaining 90% of the supernatant were dialyzed overnight against 50 vol of solution A with two changes of the outer solution for measurement of HDC activity and protein. Protein was measured by the method of Lowry et al. (1951), using bovine serum albumin as a standard. For HDC activity, each assay was carried out in parallel with a mixture containing HA stock solution as an internal standard and a mixture containing no histidine, as a blank. Histamine formed during the reaction was analyzed with an HPLC-ECD (Maldonado and Maeyama, 2013). 2.9. Statistical analysis The data from animal experiments were evaluated with Microsoft Excel 2002 and expressed as means ± standard deviation (SD). The significance of the data was analyzed by ANOVA followed by the two-tailed paired Student’s t-test. We considered a difference to be significant when p < 0.05. 3. Results Male Wistar and Ws/Ws rats were IP treated with 5 mg/kg of reserpine. Twenty four hours after treatment, the animals showed symptoms of physical deterioration, a disabling side effect of lethargy due to a strong inhibition of sympathetic activity (Frize, 1954), and other visible symptoms such as hypersensitivity, crusts formation and diarrhea. 3.1. Control values of HA and Nτ-MHA levels in hypothalamus and cortex of Wistar and Ws/Ws rats The control levels of HA and its metabolite, before drug treatment, are shown in Table 1. Although no statistic differences were found on HA levels in hypothalamus and cortex between the two strains, Nτ-MHA levels of Ws/Ws rats were significantly lower than those of Wistar rats (p < 0.05).

Table 1 Control values of HA and Nτ-MHA levels in hypothalamus and cortex of Wistar and Ws/Ws rats.

Wistar HA Ws/Ws HA Wistar Nτ−ΜΗΑ Ws/Ws Nτ−ΜΗΑ

Hypothalamus control

Cortex control

4.58 ± 0.9 4.98 ± 1.64 1.36 ± 0.36 0.82 ± 0.07

0.29 ± 0.03 0.23 ± 0.07 0.19 ± 0.04 0.13 ± 0.02

*

*

Note. Data are means ± SD (n = 6) values for HA and Nτ-MHA contents, in nmol/g wet weight. *p < 0.05.

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Fig. 2. Effect of reserpine on catecholamine levels in hypothalamus and cortex of Wistar and Ws/Ws rats. Data are means ± SD (n = 6) values for noradrenaline and dopamine contents, in nmol/g wet weight. Rats were euthanized 24 h after intraperitoneal administration of reserpine (5 mg/kg). *p < 0.05; **p < 0.01.

3.2. Effect of reserpine on catecholamine levels in hypothalamus and cortex of Wistar and Ws/Ws rats To confirm the effectiveness of reserpine treatment, noradrenaline and dopamine levels of Ws/Ws and Wistar rats were analyzed (Fig. 2). As expected, these monoamines were significantly reduced. Noradrenaline values were found to be 9.85% in hypothalamus and 21.6% in cortex of Ws/Ws rats compared to the control values. Dopamine levels were also significantly reduced to 36.8% and 2.86% of the control group respectively. Moreover noradrenaline and dopamine from cortex of Wistar rats were significantly reduced to 10.3% and 10.1% respectively.

3.3. In-vitro experiment of synaptosomal fraction of mitochondria extract from hypothalamus and cortex with dopamine and norepinephrine substrate MAO is the enzyme responsible for the oxidative deamination of a wide range of biogenic and xenobiotic amines, including dopamine, noradrenaline, adrenaline, tyramine, serotonin, β-phenylethylamine, Nτ-MHA, benzylamine, and methoxy metabolites of the parent amines, such as metanephrine and normetanephrine (Youdim and Finberg, 1983). Our experiments have confirmed that MAO is an efficient metabolic enzyme for catecholamines. Figure 3 shows that 120 min after incubation of MAO enzyme, dopamine and norepinephrine substrates from synaptosomal fractions of mitochondria of hypothalamus and cortex of Wistar rats were significantly decreased (p < 0.01). Pargyline (MAO-B inhibitor), clorgiline (MAO-A inhibitor) and deprenile (MAO-A/-B inhibitor) demonstrated their effectiveness by showing no significant reduction in the level of neurotransmitters in comparison to the “No E No Inh” group values.

3.4. In-vitro experiment of synaptosomal fraction of mitochondria extract from hypothalamus and cortex with HA and Nτ-MHA substrate When MAO enzyme was incubated for 120 min with HA and NτMHA as substrates, HA values apparently decreased; however, the differences were not significant. These results confirm that the monoamine oxidase is an inefficient metabolic enzyme for HA substrate. Moreover, Nτ-MHA was significantly reduced by MAO enzyme in the hypothalamus (Fig. 4B).

Fig. 3. In-vitro experiment of synaptosomal fraction of mitochondria extract from hypothalamus and cortex. dopamine and norepinephrine substrates. Data are means ± SD (n = 6). Dopamine and norepinephrine substrates with no enzyme and inhibitor (No E No Inh) after incubation for 120 min with MAO enzyme of mitochondria extract from hypothalamus and cortex of Wistar rats. Dopamine and norepinephrine substrates were significantly decreased as expected. Pargyline, 1 mM (MAO-B inhibitor), clorgyline, 1 mM (MAO-A inhibitor) and deprenyl, 1 mM (MAOA/B inhibitor) confirmed its effectiveness by showing no significant differences compared to the “No E No Inh” group values. Peak areas (expressed as charges) were collected with the data system of an ALS800a EC MFC instrument. **p < 0.01.

3.5. HMT activity in hypothalamus and cortex of Wistar rats after drug treatment The supernatant of homogenate tissues was dialyzed three times against 50 volumes of HMT buffer solution and incubated with HA and S-adenosyl-methionine substrates for 4 hours. Nτ-MHA, the metabolite of HA produced by the action of HMT, was then measured by HPLC-ECD and the protein content, by Bio-Rad assay kit. Our results showed that the activity of HMT, the enzyme that catabolizes most of the HA in brain, was unchanged after treatment with reserpine, pargyline and L-histidine (Fig. 5, left). 3.6. HDC activity in hypothalamus and cortex of Wistar rats after drug treatment HDC is the only enzyme involved in the formation of HA from the precursor amino acid, L-histidine. HDC activity, after treatment with reserpine, pargyline and L-histidine in hypothalamus and cortex of Wistar rats, is shown in Fig. 5, right. HDC activity in hypothalamus (0.63 pmoles/min/mg protein) was found to be 3 to 5 folds higher (p < 0.01) than cortex values (0.23 pmoles/min/mg protein). After reserpine, pargyline and L-histidine treatment, the enzyme activity in hypothalamus increased significantly to 207.6% (p < 0.01), 132.1% (p < 0.05) and 134.6% (p < 0.01) respectively, compared to the control values; and produced 1.32, 0.83 and 0.85 pmoles/ min/mg protein respectively. The enzyme activity in cortex was also increased (p < 0.01) to 142.1%, 137.8% and 154.7% respectively, compared to the control group; and produced 0.33, 0.32 and 0.36 pmoles/ min/mg protein respectively. 3.7. Effect of reserpine on HA and Nτ-MHA levels in hypothalamus and cortex of Wistar and Ws/Ws rats Twenty four hours after IP injection of 5 mg/kg of reserpine, the control levels of HA in hypothalamus (4.58 ± 0.9 nmol/g) of Wistar rats significantly decreased (p < 0.05) to 60% (2.73 ± 0.65 nmol/g) of the control values. However, no significant differences were found

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were compared, the last group showed higher Nτ-MHA values (p < 0.01), suggesting that HA is continually produced and used rather than stored in hypothalamus. In the cortex region, pargyline induced a significant increase of Nτ-MHA levels to 384% (0.73 ± 0.13 nmol/g) compared to the control group (0.19 ± 0.04 nmol/g). This result may suggest a slow HA turn-over rate and the presence of storing cells such as mast cells in this region. Moreover, reserpine effectively decreased (p < 0.05) Nτ-MHA levels in hypothalamus (from 1.48 ± 0.14 to 0.98 ± 0.23 nmol/g) to 67% and cortex (p < 0.01) to 37% (from 0.73 ± 0.13 to 0.27 ± 0.07 nmol/g) of the animals treated with pargyline (Par/Res group) (Fig. 7). 3.9. Effect of reserpine on HA increase induced by L-histidine in hypothalamus and cortex of Wistar rats The precursor of HA and also carnosine, L-histidine (1 g/kg i.p.), produced an increase (p < 0.01) of HA (4.57 ± 0.98 nmol/g) in hypothalamus to 166% (7.58 ± 0.91 nmol/g) and cortex (0.29 ± 0.03 nmol/g) to 348% (1.01 ± 0.09 nmol/g). Reserpine decreased the levels of HA in hypothalamus and cortex (p < 0.05) to 70 and 78% of the L-histidine treated groups. HA in the cortex/L-histidine/reserpine group was found to be significantly higher than its corresponding control values (Fig. 8). 4. Discussion

Fig. 4. In-vitro experiment of synaptosomal fraction of mitochondria extract from hypothalamus and cortex. HA and Nτ-MHA substrates. Data are means ± SD (n = 5). HA and Nτ-MHA substrates with no enzyme and inhibitor (No E No Inh) after incubation for 120 min with MAO enzyme of mitochondria extracted from hypothalamus and cortex of Wistar rats. HA (A) showed no significant differences after 120 min of incubation with MAO enzyme. Nτ-MHA from hypothalamus (B) was significantly reduced by the effect of MAO. Pargyline, 1 mM and deprenyl, 1 mM effectively inhibited the effect of MAO enzyme. The peak areas (expressed as charges) were collected with the data system of an ALS800a EC MFC instrument. *p < 0.05.

inWs/Ws rats. In Cortex, HA also remained unchanged after treatment, in the two different strains. On the other hand, the control levels of Nτ-MHA in hypothalamus of Wistar rats (1.36 ± 0.36 nmol/g) were significantly decreased (0.3 ± 0.07 nmol/g) after reserpine treatment (p < 0.05) and remained unchanged in Ws/Ws rats. In cortex, Nτ-MHA levels increased in Wistar (from 0.19 ± 0.04 to 0.31 ± 0.03 nmol/g) and remained unchanged in Ws/Ws rats (Fig. 6). 3.8. Effect of reserpine on pargyline-induced accumulation of Nτ-MHA in hypothalamus and cortex of Wistar rats Although the effect of pargyline on HA and Nτ-MHA are presented in Fig. 7, only the results of Nτ-MHA will be discussed in this work. The irreversible MAO-B inhibitor pargyline (65 mg/kg i.p.) by itself failed to accumulate Nτ-MHA in the hypothalamus region. However, when the animals treated with reserpine and pargyline/reserpine

It has been reported that reserpine decreases brain HA levels in cats (Adam and Hye, 1966) and mice (Atack, 1971; Taylor and Snyder, 1972) but not in rats (Green and Erickson, 1964; Pollard et al., 1973; Taylor and Snyder, 1971). However, other authors have shown contrasting results in mice (Muroi et al., 1991). These fluctuations of reserpine effect are not well elucidated and need further investigation. It has been demonstrated that VMAT2 has a consistently high affinity for monoamine substrates, particularly histamine (Zheng et al., 2006). It has also been reported that HA levels in rat brain are somewhat lower than that of catecholamines; however, its turnover rate is considerably faster (Dismukes and Synder, 1974; Pollard et al., 1974). MAO, the mitochondrial enzyme largely responsible for the oxidative deamination of biogenic amines in neural and non-neural tissues, is directly related to the turn-over rate of HA and it has been demonstrated to exist in at least two isoforms (MAO-A and MAOB) (Holzbauer and Youdim, 1977; Owen et al., 1977; Tipton et al., 1976). The oxidative deamination of monoamines by MAO is accompanied by the reduction of molecular oxygen to a toxic product called hydrogen peroxide (Cohen and Kesler, 1999). Therefore, maintenance of low cytoplasmic concentrations of neurotransmitters by their reuptake into synaptic vesicles for storage is important to minimize their inherent toxicity (Liu and Edwards, 1997). Although MAO-A appears to be the main enzyme for metabolizing the neurotransmitters serotonin and noradrenaline, it can also metabolize dopamine. MAO-B is responsible for Nτ-MHA and dopamine metabolism; however, its function is not well understood (Nagatsu, 2004). In contrast to the human brain, a much higher proportion of MAO-A than MAO-B contributes to the metabolism in rodent brain (Oreland et al., 1983). Another study in rat brain shows that MAO-A mRNA synthesis is widespread in many catecholaminergic and serotonergic cell groups, whereas MAO-B mRNA synthesis is far more discrete and limited. The different expression patterns of MAO-A and MAO-B suggest that they may also have different physiological functions (Jahng et al., 1997). In addition, it has been suggested that when MAO-A is not functioning, MAO-B may oxidize those substrates that are usually oxidized by MAO-A (Berry et al., 1994). We hypothesize that a reverse situation may also occur when MAO-B is inhibited, for example by the action of the irreversible MAO-B

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Fig. 5. HMT and HDC activity in hypothalamus and cortex of Wistar rats after drug treatment. Data are means ± SD (n = 6) values for HMT and HDC activity contents, expressed in pmoles/min/mg protein. For HMT assay, the enzyme solution, combined with 100 μM histamine and 250 μM S-adenosyl-methionine, as substrates, was incubated for 4 hours in 500 μl of HMT buffer solution at 37 °C. The protein content was measured with a Bio-Rad protein assay kit with bovine serum albumin as a standard. For HDC activity, the protein level was measured using bovine serum albumin as a standard. HA and Nτ-MHA formed during HMT and HDC reactions were analyzed by HPLC-ECD. *p < 0.05; **p < 0.01.

inhibitor pargyline. In such situation, MAO-A may help with the oxidation of those substrates that are usually oxidized by MAO-B. In this study, reserpine produced a marked reduction of catecholamine levels (Fig. 2) due to the action of the MAO-A/-B enzyme. MAO, mostly located in the outer mitochondrial membrane on the cytosol of the presynaptic catecholamine containing neurons, rapidly catabolizes these amines. Due to the low turn-over of dopamine and norepinephrine, its biosynthesis in the cytosol takes a considerable time before normal levels are restored. Our in-vitro experiment with synaptosomal/crude mitochondrial fraction extract from hypothalamus and cortex (Fig. 3) confirmed that while MAO (containing the two isoforms, -A and -B) is an efficient metabolic enzyme for catecholamines, HA is not a good substrate of the monoamine oxidase (Fig. 4A). We hypothesize that after VMAT2 is blocked by the effect of reserpine, there is an accumulation of HA in the cytosol of the two different HA existing pools in brain, but MAO enzyme (-A and-B isoforms) cannot contribute

to the process of degrading HA. HMT (EC 2.1.1.8) is necessary to inactivate HA to Nτ-MHA; Nτ-MHA is then further deaminated by MAO-B into Nτ-methylimidazole acetic acid. It has been demonstrated that HMT distribution in brain is not homogeneous (Bischoff and Korf, 1978; Nishibori et al., 2000) and thus, the effect of reserpine on HA catabolism in hypothalamus and cortex of rats might depend on the distribution and activity of HMT. Our results with regard to HMT (Fig. 5 left) suggested that reserpine, pargyline and L-histidine treatments have no effect on the basal levels of HA and Nτ-MHA in the tissues analyzed. These results are in agreement with Muroi et al. It is interesting to note that since a high-affinity reuptake system for HA has not been demonstrated in the brain (Baudry et al., 1973), the process of methylation may play a key role in the inactivation of HA in the CNS. Bischoff and Korf (1978) suggested that a major part of HMT in brain is not localized in neurons containing HDC, the HA synthesizing enzyme. Nishibori et al. (2000) have

Fig. 6. Effect of reserpine on HA and Nτ-MHA levels in hypothalamus and cortex of Wistar and Ws/Ws rats. Data are means ± SD (n = 6) values for HA and Nτ-MHA contents, in nmol/g wet weight. Rats were euthanized 24 hrs after intra-peritoneal administration of reserpine (5 mg/kg). *p < 0.05; **p < 0.01.

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Fig. 7. Effect of reserpine on pargyline-induced accumulation of Nτ-MHA in hypothalamus and cortex of Wistar rats. Data are means ± SD (n = 6) values for HA and Nτ-MHA contents, in nmol/g wet weight. Rats were euthanized 24 hrs after reserpine (5 mg/kg). Pargyline (65 mg/kg) was administrated intra-peritoneally 90 min before decapitation. *p < 0.05; **p < 0.01.

concluded that very few amounts of HMT are located in the posterior hypothalamus where the cell bodies of HAergic neurons are localized. This pattern is in agreement with the high levels of HDC in this region (Baudry et al., 1973) and suggests that HAergic cell bodies do not contain much HMT and that cells other than HAergic neurons, such as mast cells, show large levels of HMT activity as estimated in the rat brain (Bischoff and Korf, 1978; Schwartz et al., 1991). Kuhar et al. (1971) have found high levels of HMT activity in the hypothalamus of rat brain. These, along with Nishibori et al.’s (2000) findings, suggest that high amounts of HMT from hypothalamus of normal rats come from mast cells, mainly localized in thalamus and hypothalamus (Pang et al., 1996). Our results on HMT activity are in good agreement with Kuhar et al. (1971) and show not only high amounts of HMT activity in hypothalamus of Wistar rats but also no difference in HMT activity compared with the cortex in the control groups, suggesting that the regional distribution of HMT/HDC may vary between different strains and species. Nishibori et al. have found that HA turnover in HAergic neurons is faster than HA from mast cells and it is correlated with the higher amounts of HDC. HA in HAergic neurons is constantly released and

Fig. 8. Effect of reserpine on HA-increase induced by L-histidine in hypothalamus and cortex of Wistar rats. Data are means ± SD (n = 6) values for HA contents, in nmol/g wet weight. Rats were euthanized 24 h after intra-peritoneal administration of reserpine (5 mg/kg). L-histidine (1 g/kg) was injected 3 hours before decapitation. *p < 0.05; **p < 0.01.

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probably used rather than stored. When HA from mast cell stores are depleted, it may take weeks before concentrations return to normal levels (Skidgel & Erdb’s, 2001). Our results showed higher HDC activity in the hypothalamus compared to the cortex in the control groups; results that were expected. However, unlike Muroi et al. (1991), our findings showed a significant increase in HDC activity after treatment with reserpine, pargyline and L-histidine in both cortex and hypothalamus. It is also interesting to note that this increase on HDC activity was correlated with the higher HA values due to L-histidine treatment in hypothalamus and Nτ-MHA values in cortex of Wistar rats (Fig. 7) but not with HA or Nτ-MHA after reserpine, or pargyline treatment, suggesting the possibility that higher values of HDC activity might be the result of auto-immune mechanisms due to the presence of exogenous substances. The adoption of real time in-vivo HDC techniques may provide a more realistic point of view of the reserpine-HDC processes after drug administration. In this study, the analysis of Ws/Ws rats, with deficiency of mast cells (due to the impaired signal transduction of the c-kit receptor (Tsujimura et al., 1991)), was useful to clarify aspects of HA metabolism in HAergic neurons, by eliminating the contribution of mast cells. As stated earlier, there are two main different pools of HA in the brain, HAergic neurons and mast cells. One is thought to be sensitive while the other seems to be resistant to reserpine (Garbarg et al., 1976; Panula, 1986; Taylor and Snyder, 1972; Watanabe et al., 1983). Sugimoto et al. (1995) have suggested that approximately half of the HA content in rat brain is derived from mast cells and the other half, from non-mast cell sources, such as HAergic neurons. Based on the fact that no mast cells are present in the brain of Ws/Ws rats, we expected lower control levels of HA in the brain of these animals; however, HA showed no significant differences compared to those of Wistar rats, and suggest a compensatory rate of HA synthesis and a single high turn-over/high HDC/Low HMT system characteristic of HAergic neurons which in turn correlates with the low levels of Nτ-MHA found in hypothalamus and cortex of these Ws/Ws rats. When Ws/Ws rats were treated with reserpine, the unaffected levels of HA in hypothalamus and cortex suggest that HAergic neurons are more resistant to depletion than mast cells, probably due to the constant HA synthesis of HAergic neurons. Furthermore, the significant decrease on HA levels in hypothalamus and cortex of Wistar rats confirm the possibility of the HA mast cell pool being more sensitive to the effect of reserpine due to its low turn-over rate and the presence of mast cells containing high amounts of HMT. In this study, the accumulation of Nτ-MHA induced by pargyline was thought to be a useful estimator of HA turn-over. Ninety min after IP injection of pargyline, Nτ-MHA increased in the cortex – a result that was expected; however, it remained unchanged in the hypothalamus. When the groups treated with reserpine and pargyline/reserpine were compared, the last group showed higher values (*p < 0.05) of Nτ-MHA in the hypothalamus, suggesting that the release of HA into the synaptic cleft via VMAT2 is interrupted and the accumulation of Nτ-MHA in the cytosol occurs as a result of the N-methylation of the imidazole ring into Nτ-MHA through the action of HMT and the irreversible inhibition of MAO-B mediated by pargyline. The cortex of the group treated with pargyline/ reserpine did not show any significant difference as found in the hypothalamus; results that are in agreement with the regional localization of HMT, which is required to convert HA to Nτ-MHA. In contrast to other biogenic amines, it has been suggested that the synthesis rate of HA seems to depend on the bio-availability of its precursor L-histidine (Haas et al., 2008). The essential aminoacid, precursor of HA synthesis, L-histidine (1 g/kg i.p.), increased HA levels in hypothalamus and cortex as expected. These results were in agreement with Taylor and Snyder (1972). Reserpine is shown to be effective on L-histidine treated groups by lowering

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histamine values in both hypothalamus and cortex (*p < 0.05). However, the effect of reserpine was not able to bring back the elevated HA, due to L-histidine administration, to its basal levels. As additional information, we would like to comment that there may be other factors involved in the metabolism of HA, after VMAT2 is blocked by reserpine: A lick of HA, probably by dissipating proton gradients across the membranes of synaptic vesicles, and mechanisms that regulate monoamine neurotransmitters, such as OCT2/3 and HMT translocation (Busch et al., 1998). Although all these mechanisms may be triggered under the effects of reserpine, it is unknown whether its influence may have a significant impact on HA metabolism. A recent study shows that PMAT, OCT3 and HMT in human astrocytes play a role in the regulation of extra neuronal histamine concentration and the activities of HAergic neurons (Yoshikawa et al., 2013). It would be of interest to know how these mechanisms keep the same pattern in the rat brain and how they are affected by reserpine treatment. Moreover, it has also been documented that reserpine binds to VMAT2 at the same site where HA binds to reserpine. HA exhibits about 30 folds higher affinity to VMAT2 compared to serotonin while catecholamines exhibit a 3 fold higher affinity (Erikson et al., 1996). These facts may also have a certain degree of influence on the effect of reserpine on these neurotransmitters. Although our results appear to be in agreement with the strength by which these amines bind to VMAT2, further studies are needed to confirm this hypothesis. We would like to add that the understanding of the regulation of HA metabolism in brain remains as a challenge, and due to the high complexity of the processes involved in the dynamics of neurotransmitters in the brain, further and extensive research is necessary. In conclusion, the results from our present study suggest that the effect of reserpine on the HA pools in the brain may vary. The neuronal HA pools are more resistant to reserpine compared to those of catecholamine. Moreover, the HAergic neuronal pool appears to be more resistant to depletion than mast cells pool, and thus the HDC/HMT activity and localization may play a crucial role in the understanding of the pathways of HA metabolism in the brain after reserpine treatment. Acknowledgements This research was supported by the Department of Pharmacology, Informational Biomedicine, Ehime University Graduate School of Medicine, Japan. The authors would like to thank Dr. Fritzie Celino for her contribution to the last stages of this work and Dr. Sima Sarvari on the final editing of the manuscript for publication. References Adam, H.M., Hye, H.K.A., 1966. Concentration of histamine in different partsof brain and hypophysis of cat and its modification by drugs. Br. J. Pharmacol. Chemother. 28, 137–152. Agúndez, J.A., Ayuso, P., et al., 2012. The diamine oxidase gene is associated with hypersensitivity response to non-steroidal anti-inflammatory drugs. PLoS One 7 (11), e47571. Atack, C., 1971. Reduction of histamine in mouse brain by N’-(D,L-seryl)-N2-(2,3,4trihydroxybenzyl) hydrazine and reserpine. J. Pharm. Pharmacol. 23, 992–993. Baudry, M., Martres, M.P., et al., 1973. The subcellular localization of histidine decarboxylase in various regions of rat brain. J. Neurochem. 21, 1301–1309. Berry, M.D., Juorio, A.V., et al., 1994. The functional role of mono-amine oxidase A and B in the mammalian central nervous system. Prog. Neurobiol. 42, 375– 391. Bischoff, S., Korf, J., 1978. Differential localization of histidine decarboxylase and histamine-N-methyltransferase in rat brain. Brain Res. 141, 375–379. Blandina, P., Giorgetti, M., et al., 1996. Inhibition of cortical acetylcholine release and cognitive performance by histamine h3 receptor activation in rats. Br. J. Pharmacol. 119, 1656–1664. Busch, A.E., Karbach, U., et al., 1998. Human neurons express the polyspecificcation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine. Mol. Pharm. 54, 342–352.

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