Uliginosin B, a natural phloroglucinol derivative with antidepressant-like activity, increases Na⁺,K⁺-ATPase activity in mice cerebral cortex

ABSTRACT

Uliginosin B, a phloroglucinol isolated from Hypericum polyanthemum Klotzsch ex Reichardt, Hypericaceae, has antidepressant-like effect in the forced swimming test in rodents and inhibits monoamines neuronal reuptake without binding to their neuronal carriers. Studies showed the involvement of Na⁺,K⁺-ATPase brain activity in depressive disorders, as well as the dependence of neuronal monoamine transport from Na⁺ gradient generated by Na⁺,K⁺-ATPase. This study aimed at evaluating the effect of uliginosin B on Na⁺,K⁺-ATPase activity in mice cerebral cortex and hippocampus (1 and 3 h after the last administration) as well as the influence of veratrine, a Na⁺ channel opener, on the antidepressant-like effect of uliginosin B. Mice were treated (p.o.) with uliginosin B single (10 mg/kg) or repeated doses (10 mg/kg/day, 3 days). Acute administration reduced the immobility in the forced swimming test and tail suspension test and increased Na⁺,K⁺-ATPase activity in cerebral cortex 1 h after treating, whereas the repeated treatment induced the antidepressant-like effect and increased the Na⁺,K⁺-ATPase activity at both times evaluated. None treatment affected the hippocampus enzyme activity. Veratrine pretreatment prevented uliginosin B antidepressant-like effect in the forced swimming test, suggesting the involvement of Na⁺ balance regulation on this effect. Altogether, these data indicate that uliginosin B reduces the monoamine uptake by altering Na⁺ gradient.

Keywords:
Uliginosin B; Antidepressant-like activity; Na+,K+-ATPase; Veratrine; Voltage-gated sodium channel

Introduction

Based on the well-known efficacy of Hypericum perforatum (St. John's wort herb) for the treatment of mild to moderate depression (Linde, 2009) our group has been studying chemical and pharmacological features of South Brazilian Hypericum species (Daudt et al., 2000; Ferraz et al., 2002; Viana, 2007; Viana et al., 2003, 2005, 2006, 2008; Stein et al., 2012). Hypericum polyanthemum Klotzsch ex Reichardt, Hypericaceae, extracts have shown antinociceptive (Viana et al., 2003; Haas et al., 2010) and antidepressant-like effects in rats and mice (Stein et al., 2012). Its major chemical constituents are three benzopyrans, named HP1 (6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran), HP2 (hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran) and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran), and a phloroglucinol derivative, uliginosin B (Von Poser et al., 2006). Uliginosin B (1) has a dimeric structure consisting of phloroglucinol and filicinic acid moieties (Rocha et al., 1995; Nör et al., 2004; Duarte et al., 2014), and seems to be responsible for the antidepressant-like effects observed in animals behavioral tests (Stein et al., 2012). This compound showed antidepressant-like effect in the forced swimming test in mice, which was prevented by the impairment of the monoaminergic neurotransmission in vivo; it also inhibited the sinaptossomal uptake of dopamine, noradrenaline and serotonin, but interestingly it did not interact with their respective site on neuronal carriers (Stein et al., 2012). These findings suggest that uliginosin B acts by a distinct mechanism than the classical antidepressant drugs, which act by blocking monoamine transporters.

Monoamine transporters are located in the plasma membrane and are driven by the electrochemical gradient of Na⁺ generated by Na⁺,K⁺-ATPase (Nelson and Lill, 1994). It has been shown that some antidepressants, such as amitriptyline, nortriptyline, imipramine and desipramine, inhibit the Na⁺,K⁺-ATPase activity (Carfagna and Muhoberac, 1993; Sanganahalli et al., 2000; Viola and Arnaiz, 2007). On the other hand, dopamine, noradrenaline and serotonin are able to stimulate the activity of this enzyme (Viola and Arnaiz, 2007).

Na⁺,K⁺-ATPase activity is essential to the brain normal function, since the flow of Na⁺ and K⁺ ions across the membrane is necessary to neuronal excitability, regulation of osmotic balance, cell volume and intracellular transport of molecules linked to the co-transport of Na⁺, such as glucose, amino acids and neurotransmitters (Kaplan, 2002; Jorgensen et al., 2003). Several experimental studies have described the involvement of Na⁺,K⁺-ATPase brain activity in the depressive disorders (Zanatta et al., 2001; Gamaro et al., 2003; Vasconcellos et al., 2005; Acker et al., 2009). Mania and bipolar depression have been associated with increased intracellular Na⁺ concentrations (Li and El-Mallakh, 2004). Rats exposed to chronic variable stress (CVS) developed despair-like endophenotypes and had decreased hippocampal and amygdalar Na⁺,K⁺-ATPase activity (Crema et al., 2010). The antidepressant fluoxetine and the mood stabilizer lithium simultaneously prevented despair induced by CVS and prevented the decrease in Na⁺,K⁺-ATPase activity (Vasconcellos et al., 2005).

The observation that several neuroactive drugs act on Na⁺ channels also indicates the importance of Na⁺ gradient in brain disorders neurobiology. Sodium channels are voltage-dependent transmembrane proteins responsible for the increase of permeability to sodium, which initiates and propagates the action potential in excitable cells (Cestèle and Catterall, 2000; Bourin et al., 2009). The Na⁺ channels are targets to different classes of drugs such as anticonvulsants, local anesthetics and antiarrhythmics (Rasgdale et al., 1996). Some anticonvulsants (carbamazepine, lamotrigine, phenytoin, topiramate, valproate sodium) are also used to treat bipolar disorder (Reinares et al., 2012) and lamotrigine has been used specially to treat bipolar depression (Leng et al., 2013).

In this context, the aim of the present study was to evaluate the effect of the acute and sub-acute administration of uliginosin B on Na⁺,K⁺-ATPase activity in mice cerebral cortex and hippocampus and the involvement of sodium channels in uliginosin B antidepressant-like effect.

Materials and methods

Plant material

Hypericum polyanthemum Klotzsch ex Reichardt, Hypericaceae aerial parts were collected at Caçapava do Sul, in the state of Rio Grande do Sul – Brazil (October, 2008). The voucher specimens were deposited at the herbarium of the Federal University of Rio Grande do Sul (ICN Bordignon, 3118 Herbário do Departamento de Botânica – Instituto de Biociências – UFRGS). The plant collection was authorized by IBAMA (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis) (nº 003/2008; Protocol 02000.001717/2008 – 60).

Preparation of extract

H. polyanthemum lipophilic extract was obtained from dried and powdered plant material (300 g) extracted with cyclohexane (plant/solvent ratio 1:10, w/v) by static maceration for 48 h. The extract was evaporated to dryness under reduced pressure at 45 ºC to eliminate the solvent, yielding a free solvent extract termed POL (3.5%). Then, the extract was treated with acetone to remove the waxes, according to Rocha et al. (1994), producing an insoluble residue, which was eliminated by filtering with paper filter.

Characterization of the extract by HPLC and uliginosin B isolation

The extract was dissolved in HPLC grade methanol (2 mg/ml), filtered (0.22 µm pore size, Merck) and analyzed by high performance liquid chromatography Water HPLC system (Milford, MA, USA). Uliginosin B (1) determination was carried out with an isocratic solvent condition (95% CH₃CN, 5% H₂O, 0.01% TFA) through a Waters Nova-Pack C18 column (4 µm, 3.9 mm × 150 mm) adapted to a guard column Waters Nova-Pack C18 60A (3.9 mm × 20 mm), flow rate of 1 ml min^-1 and UV detection at 220 nm, according to the method previously described (Nunes et al., 2009). Uliginosin B concentration was determined as 16%, isolated by means of preparative thin layer and column chromatography and finally identified by ¹H–¹³C NMR as described elsewhere (Rocha et al., 1995; Ferraz et al., 2002; Nör et al., 2004).

Animals

Behavioral and biochemical tests were carried out with male CF1 mice (25–30 g) purchased from Fundação Estadual de Produção e Pesquisa em Saúde, RS (Brazil). The animals were housed by five in plastic cages (17 cm × 28 cm × 13 cm) and were kept under a 12 h light/dark cycle (lights on at 7 a.m.) at constant temperature of 23 ± 1 ºC with free access to standard certified rodent diet and tap water. All experimental protocols were approved by The Animal Care Local Ethical Committee (CEUA UFRGS; Protocol 18518), and performed according to Brazilian law (Brazil, 2008), which are in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and International Guiding Principles for Biomedical Research Involving Animals (Bankowski, 1985).

Experimental design

Mice (n = 8 per group) were treated by gavage (10 ml/kg) with uliginosin B (1) acutely (10 mg/kg, p.o.) or sub-acutely (10 mg/kg, p.o., once a day, during 3 days), based on previous results of our group (Viana et al., 2008). Control groups were performed for both treatment regimens and the animals were treated with vehicle (saline + polisorbate 80 2.0%). Different groups were used for biochemical and behavioral experiments (tail suspension test, TST – and forced swimming test, FST).

Time-course of uliginosin B antidepressant-like effect on the TST and FST was evaluated by testing independent groups of mice which were acutely or repeatedly treated with uliginosin B (10 mg/kg, p.o.) or vehicle. The animals were evaluated in the behavioral assays only once after treatment. Measurement of Na⁺,K⁺-ATPase activity was performed using animals that were not submitted to any behavioral test: different groups (acutely or repeatedly treated) were euthanized by decapitation 1 or 3 h after receiving the last drug administration and brain structures were removed immediately.

In another set of experiments, we investigated the possible contribution of sodium channels to uliginosin B antidepressant-like effect. We evaluated the influence of the pre-treatment with veratrine, a Na⁺ channel opener, on the FST. Initially, dose-response experiments were performed in the FST and locomotor activity in order to determine the dose of veratrine to be used: veratrine (0.06, 0.125 and 0.5 mg/kg, i.p.) was administered 45 min before the test. The sub effective dose was defined as the dose not able to reduce immobility in the FST and with no effect on locomotor activity (Centurião et al., 2014). The association of veratrine and uliginosin B consisted of the pretreatment with veratrine (0.06 mg/kg) or vehicle, i.p., 45 min before the FST, plus uliginosin B or vehicle, p.o., 30 min before the test.

Assays

Tissue preparation

Cerebral cortex and hippocampus were homogenized in 10 volumes (1:10, w/v) of 0.32 mM sucrose solution containing 5 mM HEPES and 1 mM EDTA, pH 7.5. The homogenates were centrifuged at 1000 × g for 10 min and the supernatants were removed for Na⁺,K⁺-ATPase activity determination.

Na⁺,K⁺-ATPase activity assay

The reaction mixture for Na⁺,K⁺-ATPase assay contained 5 mM MgCl₂, 80 mM NaCl, 20 mM KCl, and 40 mM Tris–HCl, pH 7.4, in a final volume of 200 µl. After 10 min of pre-incubation at 37 ºC, the reaction was initiated by the addition of ATP to a final concentration of 3 mM, and incubated for 20 min. Controls were carried out under the same conditions with the addition of 1 mM ouabain. Na⁺,K⁺-ATPase activity was calculated by the difference between the two assays according to the method described by Wyse et al. (2000). Released inorganic phosphate (Pi) was measured by the method of Cham et al. (1986). Specific activity of the enzyme was expressed as nmol Pi released per min per mg of protein.

Protein determination

Protein concentration was determined by the Bradford method (1976) using bovine serum albumin as standard.

Behavioral experiments

Tail suspension test (TST)

The TST was conducted according to Steru et al. (1985) with minor modifications (Müller et al., 2012). Mice were adapted to the laboratory conditions 1 h before the experiment. Animals were suspended by tail 60 cm above the floor using adhesive tape (1 cm from the tip of the end) in a dim light room. Immobility time was recorded (in seconds) by a blind to treatment observer during 6 min. Mice were considered immobile when they hung passively and completely motionless.

Forced swimming test (FST)

The FST was carried out according to Porsolt et al. (1978) with minor modifications standardized and validated in our laboratory (Viana et al., 2005). Mice were adapted to the laboratory conditions 1 h before being exposed to the FST. The animals were individually forced to swim in a cylinder pool (10 cm diameter, 13 cm high, water at 22 ± 1 ºC) and the total time of immobility during 6 min was scored (in seconds). Immobility time was recorded when the mouse remained floating motionless or making only the movements necessary to keep its head above water.

Locomotor activity

The spontaneous locomotor activity was performed in the open-field (Viana et al., 2005). Forty-five minutes after the administration, mice were placed in a transparent acrylic box measuring 45 cm × 30 cm × 30 cm with a dark bottom divided into 24 equal quadrants. They were evaluated during 10 min, after 5 min habituation. The number of crossings was recorded by an observer blinded to treatments.

Statistical analysis

Data were expressed as mean + SEM of the mean. Data from biochemical analysis were analyzed by Student's t test. Behavioral experiments data were analyzed by one-way or two-way ANOVA followed by Student–Newman–Keuls. Differences were considered statistically significant at p < 0.05. The statistical procedures were performed using the Sigma Stat software 2.03 (Jandel Scientific Corporation).

Results

Acute administration of uliginosin B increased Na⁺,K⁺-ATPase activity in cerebral cortex 1 h after treatment [t (9) = 2.447, p < 0.05] (Fig. 1A), but not after 3 h [t (8) = 0.323, p = 0.755] (Fig. 1B). In the hippocampus, Na⁺,K⁺-ATPase activity was not altered at both times studied: 1 h [t (9) = 0.898, p = 0.393] (Fig. 1C) and 3 h [t (8) = 1.155, p = 0.281] (Fig. 1D).

Fig. 1
Effect of the acute administration of uliginosin B on brain Na⁺,K⁺-ATPase activity. Mice were acutely treated with uliginosin B (10 mg/kg, p.o.) and euthanized by decapitation 1 or 3 h after the last administration; immediately cerebral cortex and hippocampus were removed to measure enzyme activity. Student t test; values are expressed as mean + SEM (n = 5–6). Difference from Control *p < 0.05.

Sub-acute administration of uliginosin B increased Na⁺,K⁺-ATPase activity in cerebral cortex 1 h after the last administration [t (10) = 3.300, p < 0.01] (Fig. 2A), as well as 3 h after [t (9) = 2.518, p < 0.05] (Fig. 2B). No alterations were verified in the hippocampus at the two different times: 1 h [t (10) = 1.494, p = 0.166] (Fig. 2C) and 3 h [t (8) = 0.788, p = 0.449] (Fig. 2D).

Fig. 2
Effect of the sub-acute treatment of uliginosin B on brain Na⁺,K⁺-ATPase activity. Mice were repeatedly treated with uliginosin B (10 mg/kg/day, 3 days, p.o.) and euthanized by decapitation 1 or 3 h after the last administration; immediately cerebral cortex and hippocampus were removed to measure enzyme activity. Student t test; values are expressed as mean + SEM (n = 6). Difference from Control *p < 0.05.

The acute administration of uliginosin B (10 mg/kg, p.o.) in the TST and FST are shown in Fig. 3. One-way ANOVA showed a significant effect of uliginosin B administration in the TST [F(2,25) = 4.411; p < 0.05]; post hoc analysis indicated a reduction on the immobility time at 1 h (p < 0.05), but not at 3 h after administration (p = 0.079) when compared to the control group (Fig. 3A). The same effect was observed when animals were submitted to the FST [F(2,30) = 11.136; p < 0.001]: post hoc analysis indicated a significant decrease in the immobility time 1 h after administration (p = 0.076), but not after 3 h (p > 0.05) (Fig. 3B).

Fig. 3
Effect of the acute and sub-acute treatment of uliginosin B (10 mg/kg, p.o.) on the immobility time. Mice were acutely treated with uliginosin B and submitted to test, 1 or 3 h after administration: tail suspension test (TST, panel A) and forced swimming test (FST, panel B); mice were treated for 3 days, once a day and submitted to FST 1 or 3 h after administration (FST, panel C). One-way ANOVA followed by Student–Newman–Keuls; values are expressed as mean + SEM (n = 8–10). Difference from Control *p < 0.05, **p < 0.01.

The effect of sub-acute administration of uliginosin B (10 mg/kg/day, p.o.) is shown in Fig. 3C. One-way ANOVA revealed a significant effect of uliginosin B [F(2,21) = 6.360; p < 0.01] and post hoc analysis indicated a significant anti-immobility effect at 1 h (p < 0.01) and 3 h (p < 0.05) after the last administration when compared to control group.

The anti-immobility effect of uliginosin B (10 mg/kg, p.o.) was prevented by the pretreatment with veratrine (0.06 mg/kg, i.p.) [two-way ANOVA: F_{pre-treatment × treatment}(1,31) = 6,205, p = 0.019] (Fig. 4). The co-administration of veratrine and uliginosin B has no effect when compared to the uliginosin B and vehicle (Tween), thus veratrine affect the antidepressant-like effect of uliginosin B in mouse FST.

Fig. 4
Effect of the pretreatment of mice with veratrine (0.06, mg/kg i.p.) on the anti-immobility effect of uliginosin B (10 mg/kg, p.o.) in the FST. Independent groups of mice were pretreated (i.p.) with vehicle (saline + polisorbate 80 2% = tween) and treated (p.o.) with vehicle (Tween – Control group) or uliginosin B (Tween – Uliginosin B group); or pretreated (i.p.) with veratrine and treated (p.o.) with vehicle (Veratrine – Control group) or uliginosin B (Veratrine – Uliginosin B group). Values expressed as mean + SEM (n = 8–10). Two-way ANOVA followed by Student–Newman–Keuls. * Difference from Tween – Uliginosin B versus Tween – Control, p < 0.05. @Difference from Tween – Uliginosin B versus Veratrine – Uliginosin B, p < 0.05.

Discussion

In the present study, we demonstrated that uliginosin B, the main phloroglucinol derivative from H. polyanthemum, increases activity of Na⁺,K⁺-ATPase in mice cerebral cortex, but not in hippocampus. This effect was treatment regimen dependent, being sustainable only in animals that were repeatedly treated. The same temporal activity profile was observed in the TST and FST suggesting that the uliginosin B effect is, at least in part, due to its action on Na⁺,K⁺-ATPase. Furthermore, the pretreatment with veratrine, a Na⁺ channel opener, prevented the uliginosin B antidepressant-like effect, reinforcing that the activity of uliginosin B involves the regulation of Na⁺ balance.

Depression is defined clinically as a pathological complex of psychological, neuroendocrine and somatic symptoms that cannot be reproduced in animals. However, in mice, specific measurable behaviors' can be assayed such as FST and TST which are a good screening tools with good reliability and predictive validity (Petit-Demouliere et al., 2005; Cryan et al., 2005; Castagné et al., 2009). The effect of uliginosin B in reducing immobility time in the TST and FST reinforces our previous results that have already demonstrated the antidepressant-like effect of this phloroglucinol derivative (Stein et al., 2012). In addition, in this study we moved on the possible mode of action of uliginosin B by studying its effect on the enzyme Na⁺,K⁺-ATPase. Clinical (Goldstein et al., 2006, 2009; Tochigi et al., 2008) and preclinical studies (Acker et al., 2009; Gamaro et al., 2003; Vasconcellos et al., 2005; Crema et al., 2010; Kirshenbaum et al., 2011) reported that Na⁺,K⁺-ATPase is diminished in depressive disorders. Gamaro et al. (2003) demonstrated that the Na⁺,K⁺-ATPase activity decreased in rats hippocampus subjected to chronic stress model of depression, effect that was reversed by the repeated treatment with fluoxetine and lithium (Vasconcellos et al., 2005), whereas tricyclic antidepressants inhibited the enzyme activity (Sanganahalli et al., 2000).

Although the animals were not subjected to any depression model, the results showed that uliginosin B increased Na⁺,K⁺-ATPase activity in cerebral cortex about 18% 1 h after the last administration, but it did not alter the enzyme activity after 3 h. In the sub-acute treatment, uliginosin B was able to increase in 20% the enzyme activity 1 and 3 h after the last administration, suggesting a longstanding effect of the repeated treatment (for 3 days). These findings are consistent with Zanatta et al. (2001), who demonstrated that the chronic administration of fluoxetine (14 days) increases the enzyme activity, fact that can contribute to fluoxetine therapeutic efficacy.

On the other hand, none of the uliginosin B treatment regimens (acute and sub-acute) have changed the enzyme activity in the hippocampus. Morphological and neurochemical alterations have been reported in the hippocampus of depressed patients (Sheline et al., 2003). Thus, the lack of the effect of uliginosin B on the Na⁺,K⁺-ATPase activity in mice hippocampus may be due the fact that the animals were not submitted to any experimental model of chronic depression. This finding also suggests a selective effect of uliginison B on cortical Na⁺,K⁺-ATPase, which could be particularly relevant, since this brain structure is involved in depression neurobiology (Price and Drevets, 2010) and post mortem studies have demonstrated alterations on Na⁺,K⁺-ATPase activity in the cortex of depressed patients (Goldstein et al., 2006). Noteworthy, in a previous study our group has found similar results (Müller et al., 2015), where the Na⁺,K⁺-ATPase activity was increased in the cortex, but not in the hippocampus, of animals treated with valepotriates. We speculate that this profile could be related to the irregular distribution of Na⁺,K⁺-ATPase isoenzymes. Three α isoforms are abundant in the brain: α1, expressed by neurons and glia; α2, predominant in glia; and α3, neuronal (McGrail et al., 1991). Tochigi et al. (2008) studied the gene expression pattern in postmortem brains of subjects with major depression and found that Na⁺,K⁺-ATPase α3 gene expression is decreased in prefrontal cortex of subjects with major and bipolar depression. Another study with Na⁺,K⁺-ATPase α3 heterozygous mice showed a reduction of 15% in neuronal Na⁺,K⁺-ATPase activity. We speculate that this profile could be related to the irregular distribution of Na⁺,K⁺-ATPase isoenzymes. Three α isoforms are abundant in the brain: α1, expressed by neurons and glia; α2, predominant in glia; and α3, neuronal (McGrail et al., 1991). Tochigi et al. (2008) studied the gene expression pattern in postmortem brains of subjects with major depression and found that Na⁺,K⁺-ATPase α3 gene expression is decreased in prefrontal cortex of subjects with major and bipolar depression. Another study with Na⁺,K⁺-ATPase α3 heterozygous mice showed a reduction of 15% in neuronal Na⁺,K⁺-ATPase activity. Furthermore, these animals were vulnerable to develop increased depression-like endophenotypes in a chronic variable stress model (Kirshenbaum et al., 2011). These data suggest that Na⁺,K⁺-ATPase α3 in cerebral cortex could be a target to new antidepressant drugs and to study the pathophysiology of depressive disorders.

The pre-treatment with veratrine prevented the anti-immobility effect of uliginosin B in the FST, effect that is in line with the literature, which demonstrated a similar profile to hyperbrasilol B (Centurião et al., 2014), hyperforin (Codagnone et al., 2007) and lamotrigine (Calabrese et al., 2008; Prica et al., 2008; Bourin et al., 2009). A recent study from our group showed that hyperbrasilol B, a natural dimeric phloroglucinol derivative from H. caprifoliatum, also had its anti-immobility effect prevented by veratrine, and it was able to increase Na⁺,K⁺-ATPase activity in mice hippocampus, but not cerebral cortex (Centurião et al., 2014). Hyperforin, a phloroglucinol derivative from H. perforatum, which is an European species worldwide used for depression, seems to exert its antidepressant action by mechanisms dependent on Na⁺ channels, without altering the activity of Na⁺,K⁺-ATPase (Chatterjee et al., 1998a,b). In addition, hyperforin inhibits the reuptake of monoamines without binding to their transporters (Wonnemann et al., 2000). Several authors demonstrated the presence of a carrier-mediated monoamine transport mechanism, responsible for the entrance of dopamine, norepinephrine and serotonin in the nerve terminal, accompanied by Na⁺ ions (Xhaard et al., 2008; Kristensen et al., 2011) mechanism abolished in the absence of Na⁺ (Krueger, 1990; Gu et al., 1994; Pifl et al., 1997). Lamotrigine, in turn, is an anti-epiletic agent used at bipolar depression to maintenance treatment. It acts by stabilizing the presynaptic membrane through the blockade of voltage-gated Na⁺ channels (Codagnone et al., 2007). Vitezic et al. (2008) demonstrated a partial protective effect of lamotrigine on the inhibition of Na⁺,K⁺-ATPase activity induced by kainic acid in rats prefrontal cortex and hippocampus. Also, Southam et al. (1998) demonstrated in vitro that lamotrigine inhibits the reuptake of serotonin, norepinephrine and dopamine, reinforcing the involvement of sodium gradient (Xhaard et al., 2008).

Considering the effect of uliginosin B on the activity of Na⁺,K⁺-ATPase and on Na⁺ channels together with previous studies of our group demonstrating that uliginosin B inhibits the reuptake of monoamines in a different manner from most antidepressants (Stein et al., 2012) we can speculate that uliginosin B reduces the monoamine uptake by altering Na⁺ gradient.

In conclusion, the present study represents one step ahead in the elucidation of the mechanism of action of the antidepressant-like activity of uliginosin B, a natural phloroglucinol derivative, suggesting that involvement of uliginosin B in regulation of Na⁺ balance may occur through increased of Na⁺,K⁺-ATPase activity. The regulatory mechanism involving Na⁺ may be regarded an important property for antidepressant-like activity of this phloroglucinol derivative.

• Ethical disclosures
  Protection of human and animal subjects. The authors declare that the all experimental protocols were approved by The Animal Care Local Ethical Committee (CEUA UFRGS; Protocol 18518), and performed according to Brazilian law (Brazil, 2008), which are in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and International Guiding Principles for Biomedical Research Involving Animals (Bankowski, 1985). All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques.
  Confidentiality of data. The authors declare that no patient data appear in this article.
  Right to privacy and informed consent. The authors declare that no patient data appear in this article.

Acknowledgments

This work was supported by CAPES-COFECUB [grant number 656/09], CNPq fellowships and Programa de Pós-graduação em Ciências Farmacêuticas (PPGCF-UFRGS).

References

• Acker, C., Luchese, C., Prigol, M., Nogueira, C.W., 2009. Antidepressant-like effect of a diphenyl diselenide on rats exposed to malation: involvement of Na⁺,K⁺-ATPase activity. Neurosci. Lett. 455, 168-172.
• Bankowski, Z., 1985. CIOMS. Council for International Organizations of Medical Sciences International Guiding Principles for Biomedical Research Involving Animals.
• Bourin, M., Chenu, F., Hascoët, M., 2009. The role of sodium channels in the mechanism of action of antidepressants and mood stabilizers. Curr. Drug Targets 10, 1052-1060.
• Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein-die-binding. Anal. Biochem. 72, 248-254.
• Brazil. Congresso Nacional. Lei 11794; Brasília, 8 de outubro de 2008.
• Calabrese, J.R., Huffman, R.F., White, R.L., Edwards, S., Thompson, T.R., Ascher, J.A., Monaghan, E.T., Leadbetter, R.A., 2008. Lamotrigine in the acute treatment of bipolar depression: results of five double-blind, placebo controlled clinical trials. Bipolar Disord. 10, 323-333.
• Carfagna, M.A., Muhoberac, B.B., 1993. Interaction of tryciclic drugs analogs with synaptic plasma membranes: structure-mechanism relationships in inhibition of neuronal Na⁺/K⁺-ATPase activity. Mol. Pharmacol. 44, 129-141.
• Castagné, V., Porsolt, R.D., Moser, P., 2009. Use of latency to immobility improves detection of antidepressant-like activity in the behavioral despair test in the mouse. Eur. J. Pharmacol. 616, 128-133.
• Centurião, F.B., Sakamoto, S., Stein, A.C., Müller, L.G., Chagas, P.M., Von Poser, G., Nogueira, C.W., Rates, S.M.K., 2014. The antidepressant-like effect of hyperbrasilol B, a natural dimeric phloroglucinol derivative, is prevented by veratrine, a sensitive-voltage Na⁺ channel opener. Eur. J. Med. Plants 4, 1268-1281.
• Cestèle, S., Catterall, W.A., 2000. Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. Biochimie 82, 883-892.
• Cham, K.M., Delfert, D., Junger, K.D., 1986. A direct colorimetric assay for Ca²⁺-stimulated activity. Anal. Biochem. 220, 375-380.
• Chatterjee, S.S., Nöldner, M., Koch, E., Erdelmeier, C., 1998a. Antidepressant activity of Hypericum perforatum and hyperforin: the neglected possibility. Pharmacopsychiatry 31, 7-15.
• Chatterjee, S.S., Bhattacharya, S.K., Wonnemann, M., Singer, A., Müller, W.E., 1998b. Hyperforin as a possible antidepressant component of Hypericum extracts. Life Sci. 63, 499-510.
• Codagnone, F.T., Consoni, A.L.S., Rodrigues, M.A.B.F., Andreatini, V.R., 2007. Veratrine blocks the lamotrigine-induced swimming increase and immobility decrease in the modified forced swimming test. Prog. Neuropsychopharmacol. Biol. Psychiatry 31, 1307-1311.
• Crema, L., Schlabitz, M., Tagliari, B., Cunha, A., Simão, F., Krolow, R., Pettenuzzo, L., Salbego, C., Vendite, D., Wyse, A.T.S., Dalmaz, C., 2010. Na⁺,K⁺-ATPase activity is reduced in amygdala of rats with chronic stress-induced anxiety-like behavior. Neurochem. Res. 35, 1787-1795.
• Cryan, J.F., Mombereau, C., Vassout, A., 2005. The tail suspension test as a mode for assessing antidepressant activity: review of pharmacological and genetic studies in mice. Neurosci. Biobehav. Rev. 29, 571-625.
• Daudt, R., von Poser, G.L., Neves, G., Rates, S.M.K., 2000. Screening for the antidepressant activity of some species of Hypericum from South Brazil. Phytother. Res. 15, 344-346.
• Duarte, M.O., Lunardelli, S., Kiekow, C.J., Stein, A.C., Müller, L., Stolz, E.D., Rates, S.M.K., Gosmann, G., 2014. Phloroglucinol derivatives present an antidepressant-like effect in the mice tail suspension test. Nat. Prod. Commun. 9, 671-674.
• Ferraz, A.B.F., Schripsema, J., Pohlmann, A.R., von Poser, G.L., 2002. Uliginosin B from Hypericum myrianthum Cham. & Schltdl. Biochem. Syst. Ecol. 30, 989-991.
• Gamaro, G.D., Streck, E.L., Matté, C., Prediger, M.E., Wyse, A.T.S., Dalmaz, C., 2003. Reduction of hippocampal Na⁺,K⁺-ATPase activity in rats subjected to an experimental model of depression. Neurochem. Res. 28, 1339-1344.
• Goldstein, I., Levy, T., Galili, D., Ovadia, H., Yirmiya, R., Rosem, H., Lichtstein, D., 2006. Involvement of Na⁺, K⁺-ATPase and endogenous digitalis-like compounds in depressive disorders. Biol. Psychiatry 60, 491-499.
• Goldstein, I., Lerer, E., Laiba, E., Mallet, J., Muyahed, M., Laurent, C., Rosen, H., Ebstein, R.P., Lichtstein, D., 2009. Association between sodium-and-potassium-activated adenosine triphosphatase α isoforms and bipolar disorders. Biol. Psychiatry 65, 985-991.
• Gu, H., Wall, S.C., Rudnick, G., 1994. Stable expression of biogenic amine transporters reveals differences in inhibitors sensitivity, kinetics and ion dependence. J. Biol. Chem. 269, 7124-7130.
• Haas, J.S., Viana, A.F., Heckler, A.P.M., von Poser, G.L., Rates, S.M.K., 2010. The antinociceptive effect of a benzopyran (HP1) isolated from Hypericum polyanthemum in mice hot-plate test is blocked by naloxone. Planta Med. 76, 1419-1423.
• Jorgensen, P.L., Hakansson, K.O., Karlish, S.J.D., 2003. Structure and mechanism of Na⁺,K⁺-ATPase: functional sites and their interactions. Annu. Rev. Physiol. 65, 817-849.
• Kaplan, J.H., 2002. Biochemistry of Na⁺,K⁺-ATPase. Annu. Rev. Biochem. 71, 511-535.
• Kirshenbaum, G.S., Saltzman, K., Rose, B., Petersen, J., Vilsen, B., Roder, J.C., 2011. Decreased neuronal Na⁺,K⁺-ATPase activity in Atp1a3 heterozygous mice increases susceptibility to depression-like endophenotypes by chronic variable stress. Genes Brain. Behav. 10, 542-550.
• Kristensen, A.S., Andersen, J., Jorgensen, T.N., Sorensen, L., Eriksen, J., Loland, C.J., Stromgaard, K., Gether, U., 2011. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol. Rev. 63, 585-640.
• Krueger, B.K., 1990. Kinetics and block of dopamine uptake in synaptosomes from rat caudate nucleus. J. Neurochem. 55, 260-267.
• Leng, Y., Fessler, E.B., Chuang, D.M., 2013. Neuroprotective effects of the mood stabilizer lamotrigine against glutamate excitotoxicity: roles of chromatin remodelling and Bcl-2 induction. Int. J. Neuropsychopharmacol. 16, 607-620.
• Li, R., El-Mallakh, R.S., 2004. Differential response of bipolar and normal control lymphoblastoid cell sodium pump to ethacrynic acid. J. Affect. Disord. 80, 11-17.
• Linde, K., 2009. St. John's Wort – an overview. Forsch. Komplementmed. 16, 146-155.
• McGrail, K.M., Phillips, J.M., Sweadner, K.J., 1991. Immunofluorescent localization of three Na⁺,K⁺-ATPase isozymes in the rat central nervous system: both neurons and glia can express more than one Na⁺,K⁺-ATPase. J. Neurosci. 11, 381-391.
• Müller, L.G., Salles, L.A., Stein, A.C., Betti, A.H., Sakamoto, S., Cassel, E., Vargas, R.F., von Poser, G.L., Rates, S.M.K., 2012. Antidepressant-like effect of Valeriana glechomifolia Meyer (Valerianaceae) in mice. Prog. Neuropsychopharmacol. Biol. Psychiatry 36, 101-109.
• Müller, L.G., Salles, L., Lins, H.A., Feijó, P.R., Cassel, E., Varga, R., von Poser, G.L., Noël, F., Quintas, L.E., Rates, S.M., 2015. Effects of diene valepotriates from Valeriana glechomifolia on Na⁺/K⁺-ATPase activity in the cortex and hippocampus of mice. Planta Med. 81, 200-207.
• Nelson, N., Lill, H., 1994. Porters and neurotransmitter transporters. J. Exp. Biol. 196, 213-228.
• Nör, C., Albring, D., Ferraz, A.B.F., Schripsema, J., Pires, V., Sonnet, P., Guillaume, D., von Poser, G.L., 2004. Phloroglucinol derivatives from four Hypericum species belonging to the Trigynobrathys section. Biochem. Syst. Ecol. 32, 517-519.
• Nunes, J.M., Pinhatti, A.V., von Poser, G.L., Rech, S.B., 2009. Promotive effects of long-term fertilization on growth of tissue culture-derived Hypericum polyanthemum plants during acclimatization. Ind. Crops Prod. 30, 329-332.
• Petit-Demouliere, B., Chenu, F., Bourin, M., 2005. Forced swimming test in mice: a review of antidepressant activity. Psychopharmacology (Berlin) 177, 245-255.
• Pifl, A., Drobny, H., Reither, H., Singer, E.A., 1997. Introduction by low Na⁺ or Cl^- of cocaine sensitive carrier-mediate efflux of amines from cells transfected with cloned human catecholamine transporters. Br. J. Pharmacol. 121, 205-212.
• Porsolt, R.D., Anton, G., Blavet, N., Jafre, M., 1978. Behavioral despair in rats: a new model sensitive to antidepressant treatments. Eur. J. Pharmacol. 47, 379-391.
• Prica, C., Hascoet, M., Bourin, M., 2008. Antidepressant-like effect of lamotrigine is reversed by veratrine: a possible role of sodium channels in bipolar depression. Behav. Brain Res. 191, 49-54.
• Price, J.L., Drevets, W.C., 2010. Neurocircuitry of mood disorders. Neuropsychopharmacology 35, 192-216.
• Rasgdale, D.S., Mcphee, J.C., Scheuer, T., Catterall, W.A., 1996. Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na⁺ channels. Proc. Natl. Acad. Sci. U. S. A. 93, 9270-9275.
• Reinares, M., Rosa, A.R., Franco, C., Goikolea, J.M., Fountoulakis, K., Siamouli, M., Gonda, X., Frangou, S., Vieta, E., 2012. A systematic review on the role of anticonvulsants in the treatment of acute bipolar depression. Int. J. Neuropsychopharmacol. 10, 1-12.
• Rocha, L., Marston, A., Kaplan, M.A.C., Stoeckli-Evans, H., Thull, U., Testa, B., Hostettmann, K., 1994. An antifungal gamma-pyrone and xanthones wit monoamine oxidase inhibitory activity from Hypericum brasiliense Phytochemistry 36, 1381-1385.
• Rocha, L., Marston, A., Potterat, O., Kaplan, M.A.C., Stoeckli-Evans, H., Hostettmann, K., 1995. Antibacterial phloroglucinols and flavonoids from Hypericum brasiliense Phytochemistry 40, 1447-1452.
• Sanganahalli, B.G., Joshi, P.G., Joshi, N.B., 2000. Differential effects of tricyclic antidepressant drugs on membrane dynamics – a fluorescence spectroscopic study. Life Sci. 68, 81-90.
• Sheline, Y.I., Gado, M.H., Kraemer, H.C., 2003. Untreated depression and hippocampal volume loss. Am. J. Psychiatry 160, 1516-1518.
• Southam, E., Kirkby, D., Higgins, G.A., Hagan, R.M., 1998. Lamotrigine inhibits monoamine uptake in vitro and modulates 5-hydroxytryptamine uptake in rats. Eur. J. Pharmacol. 358, 19-24.
• Stein, A.C., Viana, A.F., Müller, L.G., Nunes, J.M., Stolz, E.D., Do Rego, J.C., Constentin, J., von Poser, G.L., Rates, S.M.K., 2012. Uliginosin B, a phloroglucinol derivative from Hypericum polyanthemum: a promising new molecular pattern for the development of antidepressant drugs. Behav. Brain. Res. 228, 66-73.
• Steru, L., Chermat, R., Thierry, B., Simon, P., 1985. The tail suspension test: a new method for screening antidepressants in mice. Psychopharmacology (Berlin) 85, 367-370.
• Tochigi, M., Iwamoto, K., Bundo, M., Sasaki, T., Kato, N., Kato, T., 2008. Gene expression profiling of major depression and suicide in the prefrontal cortex of postmortem brains. Neurosci. Res. 60, 184-191.
• Vasconcellos, A.P.S., Zugno, A.I., Dos Santos, A.H., Nietto, F.B., Crema, L.M., Gonçalves, M., Franzon, R., Wyse, A.T.S., Rocha, E.R., Dalmaz, C., 2005. Na⁺,K⁺-ATPase activity is reduced in hippocampus of rats submitted to an experimental model of depression: effect of chronic lithium treatment and possible involvement in learning deficits. Neurobiol. Learn. Mem. 84, 102-110.
• Viana, A.F., (PhD Thesis) 2007. Estudo de moléculas potencialmente antidepressivas e analgésicas de espécies de Hypericum nativas do RS. Porto Alegre. Universidade Federal do Rio Grande do Sul/Université de Rouen, 220 p.
• Viana, A.F., Heckler, A.P., Fenner, R., Rates, S.M.K., 2003. Antinociceptive activity of Hypericum caprifoliatum and Hypericum polyanthemum (Guttiferae). Braz. J. Med. Biol. Res. 36, 631-634.
• Viana, A.F., Do Rego, J.C., von Poser, G., Ferraz, A., Heckler, A.P., Constentin, J., Rates, S.M.K., 2005. The antidepressant-like effect of Hypericum caprifoliatum Cham & Schlecht (Guttiferae) on forced swimming test results from an inhibition of neuronal monoamine uptake. Neuropharmacology 49, 1042-1052.
• Viana, A., Do Rego, J.C., Munari, L., Dourmap, N., Heckler, A.P., Dalla Costa, T., von Poser, G.L., Costentin, J., Rates, S.M.K., 2006. Hypericum caprifoliatum (Guttiferae) Cham. & Schltdl.: a species native to South Brazil with antidepressant-like activity. Fundam. Clin. Pharmacol. 20, 507-514.
• Viana, A.F., Rates, S.M.K., Naudin, B., Janin, F., Costentin, J., Do Rego, J.C., 2008. Effects of acute or 3-day treatments of Hypericum caprifoliatum Cham. & Schltdt. (Guttifereae) extract or of two established antidepressants on basal and stress-induced increase in serum and brain corticosterone levels. J. Psychopharmacol. 22, 681-690.
• Viola, M.S., Arnaiz, G.R.L., 2007. Brain Na⁺, K⁺-ATPase isoforms: different hypothalamus and mesencephalon response to acute desipramine treatment. Life Sci. 81, 228-233.
• Vitezic, D., Pelcic, J.M., Zupan, E., Vitezic, M., Ljulicic, D., Simonic, A., 2008. Na⁺, K⁺-ATPase activity in the brain of the rats with kainic acid-induced seizures: influence of lamotrigine. Psychiatr. Danub. 20, 270-277.
• Von Poser, G.L., Rech, S.B., Rates, S.M.K., 2006. Chemical and pharmacological aspects of southern Brazilian Hypericum species. In: Teixeira da Silva, J.A. (org.). Floriculture, Ornamental and Plant Biotechnology Ikenobe: Global Science Books, pp. 510–516.
• Wonnemann, M.S., Singer, M.S., Müller, W.E., 2000. Inhibition of synaptosomal uptake of hyperforin, a major constituent of St. John's Wort: the role of amiloride sensitive sodium conductive pathways. Neuropsychopharmacology 23, 188-197.
• Wyse, A.T.S., Streck, E.L., Worm, P., Wajner, A., Ritter, F., Netto, C.A., 2000. Precognition prevents the inhibition of Na⁺,K⁺-ATPase activity after brain ischemia. Neurochem. Res. 25, 917-975.
• Xhaard, H., Backström, V., Denessiouk, K., Johnson, M.S., 2008. Coordination of Na(+) by monoamine ligands in dopamine, norepinephrine, and serotonin transporters. J. Chem. Inf. Model. 48, 1423-1437.
• Zanatta, I.M., Nascimento, F.C., Barros, S.V.T., Silva, G.R.R.S., Zugno, A.L., Netto, C.A., Wyse, A.T.S., 2001. In vivo and in vitro effect of imipramine and fluoxetine on Na,K-ATPase activity in synaptic plasma membranes froma the cerebral cortex of rats. Braz. J. Med. Biol. Res. 34, 1265-1269.

Publication Dates

• Publication in this collection
  Sep-Oct 2016

History

• Received
  23 Dec 2015
• Accepted
  12 Apr 2016