Influence of the phosphodiesterase type 5inhibitor, sildenafil, on antidepressant-likeactivity of magnesium in the forced swim testin mice

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Short communication

Influence of the phosphodiesterase type 5 inhibitor, sildenafil, on antidepressant-like activity of magnesium in the forced swim test in mice

Katarzyna Soca³a¹, Dorota Nieoczym¹, Ewa Poleszak², Piotr WlaŸ¹

1Department of Animal Physiology, Institute of Biology and Biochemistry, Maria Curie-Sk³odowska University, Akademicka 19,

PL 20-033 Lublin, Poland

2Chair and Department of Applied Pharmacy, Medical University of Lublin, ChodŸki 1, PL 20-093 Lublin, Poland Correspondence: Piotr WlaŸ, e-mail: piotr.wlaz@umcs.lublin.pl

Abstract:

Magnesium, which acts as an antagonist of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, exerts antidepressant- like activity in animal models of depression. The present study was undertaken to elucidate the influence of sildenafil, a phosphodiesterase type 5 inhibitor, on the anti-immobility action of magnesium in the forced swim test in mice. Swim sessions were conducted by placing mice in glass cylinders filled with water for 6 min and the duration of the behavioral immobility during the last 4 min of the test was evaluated. Locomotor activity was measured with photoresistor actimeters. Serum and brain magnesium levels were as- sayed spectrophotometrically. Magnesium at a dose of 30 mg/kg, ip significantly decreased the immobility time while sildenafil (5, 10 and 20 mg/kg, ip) in a dose-dependent manner reduced the antidepressant-like activity of magnesium. The co-administration of magnesium with sildenafil at the highest dose entirely abolished the antidepressant-like effect of magnesium and caused a statistically significant increase in immobility duration as compared to the control group. Combination of magnesium with sildenafil resulted in a potent reduction (80%) of locomotor activity and pharmacokinetic studies showed a significant increase of magnesium concentration in serum (as compared to magnesium treatment alone) without changes within brain tissue in mice treated with magnesium and sildenafil. When given alone, sildenafil caused a significant increase in magnesium levels in both serum and brain. Our results indicate that a simultaneous treatment with magnesium and sildenafil results in hypermagnesemia in laboratory animals.

However, the mechanism underlying this effect remains elusive.

Key words:

phosphodiesterase 5, sildenafil, magnesium, depression, erectile dysfunction, antidepressant, forced swim test, mice

Introduction

Sildenafil, an active compound of Viagra, is the first- line oral treatment for erectile dysfunction of multiple etiologies. The mechanism of action of sildenafil in- volves the nitric oxide/cyclic guanosine 3´,5´-mono-

phosphate/phosphodiesterase type 5 (NO/cGMP/

PDE5) cell signaling pathway [6]. During sexual stimulation, corpus cavernosum nerves and endothe- lial cells release NO, which via guanylate cyclase ac- tivation promotes cyclic guanosine 3´,5´-monopho- sphate (cGMP) production. Cyclic GMP, in turn, al-

Pharmacological Reports 2012, 64, 205211 ISSN 1734-1140

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cyclic nucleotides. Sildenafil works as a selective inhibitor of PDE5. By inhibiting cGMP degradation, it improves the relaxation of smooth muscles in the cor- pus cavernosum and thus resulting in erection [16, 44]. Sildenafil exerts various effects on the central nervous system. The recent data show that it possesses neuroprotective properties, enhances neuro- genesis and improves memory [48].

The NO/cGMP pathway has also been implicated in the neurobiology of depression. A number of experiments indicate that inhibitors of this pathway ex- hibit antidepressant effects and enhance the efficacy of serotonin reuptake inhibitors [17, 18, 20]. Nitric oxide plays an extremely important role in the central nervous system where it acts as a neuronal messenger.

It is involved in neurotransmission, synaptic plastic- ity, perception of pain and learning [12]. The NO/

cGMP pathway affects i.a., excitatory neurotransmis- sion mediated through the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors [42]. Inter- actions between NO/cGMP pathway and glutamater- gic neurotransmission are bidirectional. On the one hand, stimulation of NMDA receptor results in Ca²⁺

influx which leads to NO synthase activation and NO production. On the other hand, NO and cGMP modulate NMDA receptor activity and may regulate release of glutamate by negative feedback mechanisms [18, 29, 42]. There is an increasing evidence for the involvement of NMDA-mediated neurotransmission in pathophysiology of depression and antidepressant- like activity of NMDA receptor antagonists in animal studies [19, 32, 33, 45]. Magnesium, which blocks the NMDA receptor channel in a voltage-dependent fash- ion, also exhibits antidepressant activity in animal studies and clinical trials [7, 38, 40]. It is also active in an animal model of mania [1]. This second most abundant intracellular cation plays a fundamental role in innumerable regulatory processes. It participates in over 325 different enzymatic reactions such as hy- drolysis of high-energy phosphate groups or nucleic acid and protein synthesis. It regulates conductivity of ion channels and is involved in hormone receptor binding, muscle contraction and neurotransmitters release [11, 13]. Therefore, magnesium depletion brings about dramatic clinical implications ranging from hypertension, arrhythmias and neuromuscular hyperex- citability to acute myocardial infarction or even sei-

patients’ general condition [14]. Numerous magnesium dietary supplements are available on the market and magnesium supplementation is an important issue in contemporary medicine. Moreover, it is one of the new emerging approaches in the treatment of depression [11].

Since both magnesium and sildenafil may alter glu- tamatergic neurotransmission and magnesium supplementation is of high probability in men taking sildenafil, the aim of the present study was to investigate the effect of sildenafil on the antidepressant-like activity of magnesium in the forced swim test in mice.

In order to evaluate the potential pharmacokinetic interactions between sildenafil and magnesium, total brain and serum concentrations of magnesium were determined.

Materials and Methods

Animals

The experiments were carried out on male Albino Swiss mice weighing 25–30 g, purchased from a li- censed breeder (Laboratory Animals Breeding, S³aboszów, Poland). The animals were housed in groups of up to 10 mice in polycarbonate cages under standard laboratory conditions (temperature main- tained at 23–25°C, humidity 50–60% and 12 h light/dark cycle, lights on at 06:00 h) with free access to food pellets (Murigran, Agropol S.J., Motycz, Po- land) and tap water. All experiments were performed following at least 7 days of acclimatization. The experimental protocols were approved by the Ethical Committee of the Medical University in Lublin (license number 69/2009) and all procedures were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Drugs

Magnesium hydroaspartate (Farmapol, Poznañ, Poland) and sildenafil citrate (Polpharma, Starogard Gdañski, Poland) were dissolved in saline and administered intraperitoneally (ip) at a volume of 10 ml/kg, 30 min

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before the respective test. The dosage of magnesium refers to pure magnesium ions. Control animals re- ceived an ip injection of saline. The doses and pre- treatment schedules were selected on the basis of those reported in the literature and previous experiments from our laboratory [29–31, 35–38].

Forced swim test

The test was conducted according to the method de- scribed by Porsolt et al. [41]. Mice were placed individually in glass cylinders (height 25 cm, diameter 10 cm). The cylinders contained 10 cm of water main- tained at 23–25°C. Animals were allowed to swim for 6 min. Total duration of immobility was recorded during the last 4 min of the test. The duration of immobility was defined as the time when the mouse remained floating passively, made no attempts to escape and showed only slow movements to keep its head above the water.

Locomotor activity

Locomotor activity of mice was determined by using actimeters (30 cm diameter, 12 cm high, MultiServ, Lublin, Poland) equipped with two perpendicular in- frared light beams located 1.5 cm above the floor.

Mice were placed individually in an actimeter for 5 min. After acclimatization, locomotor activity was recorded as the number of interruptions of light beams for the next 5 min.

Determination of serum and brain magnesium concentration

After drug pretreatment, mice were sacrificed by decapitation and the trunk blood was collected in poly- ethylene tubes. Serum was isolated 1 h after blood co- agulation by centrifugation at 5,000 × g for 10 min at 4°C and frozen at –30°C. The brains were rapidly re- moved after decapitation, washed in saline and ho- mogenized (PRP 200, PRO Scientific Inc., Connecti- cut, USA) in 0.1 M phosphate buffered saline (pH 7.4) at 26,000 rpm for 3 min at 4°C. The homogenates were centrifuged at 21,000 × g for 30 min at 4°C and the supernatant was frozen at –30°C.

Total magnesium concentration in serum and brain homogenates was determined by using xylidyl blue method. Ten microliters of thawed serum or brain supernatant was mixed with 1 ml of commercially avail-

able reagent (Liquick Cor-Mg, Cormay, Lublin, Po- land) and reaction mixture absorbance was measured at 520 nm employing a spectrophotometer (Hitachi U-3010, Tokyo, Japan). The magnesium concentrations were expressed in mg/100 ml for serum and µg/g of brain tissue.

Statistical analysis

All results are presented as the means ± standard error of the mean (SEM) and evaluated by using one-way analysis of variance (ANOVA) followed by Student- Newman-Keuls post-hoc test for multiple comparison or by unpaired Student’s t-test, where appropriate. A p value less than or equal to 0.05 was considered to be statistically significant.

Results

Forced swim test

The effect of magnesium, alone and in combination with sildenafil, on immobility time in the forced swim test in mice is shown in Figure 1 (one-way ANOVA:

F (4, 51) = 35.080; p < 0.001). Magnesium at a dose of 30 mg/kg significantly decreased immobility time from 193.80 ± 4.38 s in the control group to 132.00

± 5.69 s (p < 0.001). Sildenafil in a dose-dependent

Sildenafil and antidepressant-like activity of magnesium

Katarzyna Soca³a et al.

Fig. 1. The effect of sildenafil on the antidepressant-like activity of magnesium in the forced swim test in mice. Magnesium and sildenafil were administered ip 30 min before the test. Each experimental group consisted of 10–12 animals. Data are presented as the means

± SEM. ** p < 0.01, *** p < 0.001 vs. saline-treated group;^#p < 0.05,

###p < 0.001 vs. magnesium-treated group (one-way ANOVA with Student-Newman-Keuls post-hoc test)

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abolished the antidepressant-like effect of magnesium and caused a statistically significant increase in total duration of immobility as compared to saline-treated group (p = 0.006).

Locomotor activity

The effect of sildenafil and combined administration of sildenafil and magnesium on locomotor activity in

locomotor activity in mice. The combined administration of magnesium with sildenafil at the highest dose of 20 mg/kg significantly reduced the activity counts (p < 0.001 vs. the control group and magnesium- treated group).

Effect of sildenafil on serum and brain magnesium concentration

The effects of combined administration of magnesium and sildenafil on serum (one-way ANOVA: F (2, 30)

= 118.92; p < 0.001) and brain (one-way ANOVA:

F (2, 30) = 4.169; p = 0.025) magnesium concentrations in mice are shown in Table 2. The administration of magnesium at a dose of 30 mg/kg significantly increased the concentration of magnesium in serum (p < 0.001) as well as in the brain (p < 0.05) as com- pared to saline-treated group. Co-administration of magnesium with sildenafil (20 mg/kg) caused an additional increase in serum magnesium levels with no effect on magnesium concentration within the brain (p < 0.001 and p > 0.05 as compared to magne- sium-treated group, respectively).

The influence of sildenafil administered alone on serum (t-test p < 0.001) and brain (t-test p < 0.001) magnesium level is shown in Table 2. Sildenafil caused a statistically significant increase in magnesium concentrations in both serum and brain.

Discussion

In recent years, there has been an increased interest in the involvement of magnesium in the pathophysiology and treatment of depression. Magnesium deficiency is believed to contribute to mood disorders in spite of the fact that blood tests of depressed patients provide fre- quently contradictory results. Serum/plasma levels of magnesium ions in depressives may be elevated, decreased or may remain unchanged [10, 11, 24]. Nu- merous studies indicate that magnesium possesses potent antidepressant-like properties in the forced swim test in rodents and anxiolytic-like activity in the elevated plus maze test in mice. In addition, the anti- immobility action of imipramine, citalopram and tia-

Tab. 1. Effect of sildenafil and magnesium administered separately or jointly on spontaneous locomotor activity in mice

Treatment Activity counts/5 min

Saline + saline 68.90 ± 9.15

Sildenafil (5 mg/kg) + saline 65.50 ± 12.84 Sildenafil (10 mg/kg) + saline 75.30 ± 9.62 Sildenafil (20 mg/kg) + saline 39.60 ± 7.29 Magnesium (30 mg/kg) + saline 69.11 ± 6.55 Magnesium (30 mg/kg) + sildenafil (20 mg/kg) 13.10 ± 3.55^a,b

Magnesium and sildenafil were administered ip 30 min before the test. Each experimental group consisted of 9–10 animals. Data are presented as the means ± SEM.^ap < 0.001 vs. saline-treated group;

bp < 0.001 vs. magnesium-treated group (one-way ANOVA with Student-Newman-Keuls post-hoc test)

Tab. 2. Effect of magnesium and sildenafil administered separately or jointly on serum and brain magnesium concentrations in mice

Treatment Magnesium concentration Serum

(mg/100 ml)

Brain (µg/g) A. Saline + saline 1.87 ± 0.11 110.43 ± 2.60 Magnesium (30 mg/kg) + saline 3.70 ± 0.31^b 120.96 ± 3.04^a Magnesium (30 mg/kg)

+ sildenafil (20 mg/kg)

6.60 ± 0.11^b,c 122.29 ± 3.79^a

B. Saline 2.45 ± 0.05 97.03 ± 2.20

Sildenafil (20 mg/kg) 2.77 ± 0.07^a 116.53 ± 1.75^b

Magnesium and sildenafil were administered ip 30 min before de- capitation. Each experimental group consisted of 9–12 animals. Data are presented as the means ± SEM. A:^ap < 0.05,^bp < 0.001 vs.

saline-treated group; ^cp < 0.001 vs. magnesium-treated group (one-way ANOVA with Student-Newman-Keuls post-hoc test).

B:^ap < 0.01,^bp < 0.001 vs. saline-treated group (Student’s t-test)

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neptine as well as of NMDA receptor antagonists and glycine_Breceptor ligands is enhanced by joint administration of magnesium [7, 36, 37, 40]. Apart from data obtained in animal studies, magnesium supplementation was reported to be effective in the treatment of major depressive disorder [10], rapid cycling bipolar affective disorder [5] and mania [21]. The antidepressant- and anxiolytic-like effects of magnesium are most likely related to its antagonist properties to the NMDA/glutamate pathway.

The present study demonstrates that the anti- immobility action of magnesium in the forced swim test in mice was reduced by sildenafil in a dose- dependent manner. Interestingly, sildenafil, at the highest dose used, not only entirely abolished the magnesium-induced antidepressant effect but also prolonged the duration of immobility as compared to the control group. These results could imply that combined administration of magnesium and sildenafil leads to depressogenic effects. As the combination of magnesium with sildenafil resulted in a potent reduction (80%) of the locomotor activity, changes in the general motor function may have interfered with data obtained in the forced swim test.

Locomotion and exploratory behavior are regulated and mediated by various types of neurotransmitter systems, in particular mesolimbic dopaminergic pathways [3, 8, 25, 27]. Sildenafil, which acts through the NO/cGMP/PDE5 pathway, may affect both excitatory and inhibitory neurotransmission [42]. Likewise, magnesium is involved in signal transmission within the central nervous system. It acts as non-competitive antagonist of NMDA receptors [39], modulates the turnover of different neurotransmitters such as amino acids, neuropeptides, cytokines and nitric oxide [11]

and participates in binding of monoamines to their receptors [4]. A common site of action for sildenafil and magnesium may be the GABA_A receptor. Increased GABAergic transmission causes sedation and may lead to the reduction of locomotor activity [26].

Huang et al. [22] showed that increased cGMP concentration evoked by sildenafil treatment enhances the release of GABA, while Poleszak [35] suggests that anxiolytic-like effects of magnesium are related to its ability to activate GABA_A-gated chloride channels. In addition, synergistic interaction of magnesium with benzodiazepines (diazepam and chlordi- azepoxide) in the elevated plus maze test in mice ar- gue for the involvement of benzodiazepine/GABA_A system in the anxiolytic activity of magnesium [35].

Magnesium also activates the activity of guanylate cyclase which promotes cGMP formation [34]. Per- haps the influence of sildenafil and magnesium, administered alone, on the GABAergic neurotransmission was too weak to impair locomotor activity in mice and only when administered jointly they cause noticeable behavioral response. Data from pharmacokinetic studies revealed, nevertheless, that a distinct mechanism could be responsible for the reduction of spontaneous locomotor activity in mice after combined treatment with sildenafil and magnesium. Co- administration of these two substances resulted in a significant increase in magnesium serum level (by 78%), as compared with magnesium treatment alone, without changes within brain tissue. Magnesium overload occurs rarely, mainly because of excessive intake of magnesium or failure of its excretion. Hy- permagnesemia is manifested by hypotension, brady- cardia, hyporeflexia, neuromuscular hypoexcitability and sedation [9, 46, 47]. Clinical symptoms of magnesium overload may mimic central anesthetic effect.

But in fact, they are due to the peripheral action of magnesium [9]. The brain magnesium concentration is tightly regulated and the blood-brain barrier pro- tects against acute changes in the magnesium concentration. An acute increase in plasma magnesium concentration is usually not associated with an increase in brain intracellular magnesium concentration [11, 15, 40]. In most animals, serum magnesium level reaches 1.8–2.7 mg/100 ml and sedation becomes evident at serum magnesium level of 4–5 mg/100 ml [28]. It seems that sildenafil administered with magnesium leads to hypermagnesemia, as in the present study sildenafil elevated serum magnesium levels up to 6.5 mg/100 ml. Thus, sildenafil might have enhanced magnesium absorption from the peritoneal cavity and/or it could have impaired magnesium renal excretion. To establish the mechanism accountable for this effect, in a separate experiment we assayed serum and brain magnesium levels after single administration of sildenafil without magnesium pre-treatment. The increased serum magnesium concentration (by 13%) after sildenafil treatment implies that hypermagnesemia, observed in former experiment, was rather due to the malfunctioning of magnesium transport in kid- neys than the excessive magnesium intake from the peritoneal cavity.

Mammalian magnesium transport across biological membranes is mediated by two main systems: Na⁺- dependent and Na⁺-independent pathways. Although in-

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pathway in magnesium transport pathways. It is also claimed that the peripheral and central magnesium level may be regulated by different mechanisms [4].

The reabsorption of magnesium predominantly occurs in the loop of Henle and the distal tubule [2] and ele- ments of the NO/cGMP may regulate renal functions because NO as well as cGMP modulate many plasma membrane transporters [23]. Ikari and co-workers [23] showed that NO and cGMP up-regulate the Na⁺-dependent magnesium transport in renal epithelial cells and it was suggested that under pathological conditions, the increase in magnesium gradient with a concomitant increase in NO and cGMP levels activates magnesium transport system across the plasma membrane.

Unexpectedly, in our study, sildenafil, when administered alone, caused a 20% increase in magnesium level within the brain, which points at a redistri- bution of magnesium from blood to the brain and a failure of homeostatic regulations of magnesium concentration within the brain tissue. Because no changes in brain magnesium levels after joint administration of magnesium and sildenafil were noticed, it may be speculated that distinct mechanisms may be accountable for regulation of the brain magnesium concentration under physiological and abnormal conditions such as magnesium overload. However, the explanation is not highly convincing and the subject matter deserves further investigations. It would be ad- visable to determine changes in magnesium level in urine and to check whether sildenafil is able to change magnesium concentration in mice chronically treated with this bioelement.

In conclusion, our results indicate for the first time that sildenafil reverse the anti-immobility action of magnesium in the forced swim test in mice and that simultaneous treatment with magnesium and sildenafil results in hypermagnesemia in laboratory animals. Further studies are required to investigate the mechanisms underlying the observed phenomena.

Acknowledgments:

We gratefully acknowledge the technical assistance of Nina Kowalczyk. This study was supported by Funds for Statutory Activity of Maria Curie-Sk³odowska University, Lublin, Poland.

The authors wish to thank Polpharma S.A. (Starogard Gdañski, Poland) for generous gift of sildenafil.

Boerngen-Lacerda R, Andreatini R: Magnesium sulfate and sodium valproate block methylphenidate-induced hyperlocomotion, an animal model of mania. Pharmacol Rep, 2011, 63, 64–70.

2.Bastani B, Pandurangan G: Intraperitoneal route of magnesium sulphate supplementation in a patient with severe renal magnesium wasting. Nephrol Dial Transplant, 2001, 16, 2086–2089.

3.Cazalets J: Development ot the neural correlates of locomotion in rats. In: Handbook of brain and behaviour in hu- man development. Eds. Kalverboer A, Gramsbergen A, Kluwer Academic Publishing, Dordrecht, 2001, 447–467.

4.Chollet D, Franken P, Raffin Y, Malafosse A, Widmer J, Tafti M: Blood and brain magnesium in inbred mice and their correlation with sleep quality. Am J Physiol Regul Integr Comp Physiol, 2000, 279, R2173–R2178.

5.Chouinard G, Beauclair L, Geiser R, Etienne P: A pilot study of magnesium aspartate hydrochloride (Magnesio- card) as a mood stabilizer for rapid cycling bipolar affective disorder patients. Prog Neuropsychopharmacol Biol Psychiatry, 1990, 14, 171–180.

6.Corbin JD, Francis SH: Cyclic GMP phosphodiesterase-5:

target of sildenafil. J Biol Chem, 1999, 274, 13729–13732.

7.Decollogne S, Tomas A, Lecerf C, Adamowicz E, Seman M: NMDA receptor complex blockade by oral administration of magnesium: comparison with MK-801.

Pharmacol Biochem Behav, 1997, 58, 261–268.

8.Del Arco A, Segovia G, Mora F: Blockade of NMDA receptors in the prefrontal cortex increases dopamine and acetylcholine release in the nucleus accumbens and motor activity. Psychopharmacology (Berl), 2008, 201, 325–338.

9.Durlach J, Durlach V, Bac P, Bara M, Guiet-Bara A: Mag- nesium and therapeutics. Magnes Res, 1994, 7, 313–328.

10.Eby GA, Eby KL: Rapid recovery from major depression using magnesium treatment. Med Hypotheses, 2006, 67, 362–370.

11.Eby GA, III, Eby KL: Magnesium for treatment-resistant depression: a review and hypothesis. Med Hypotheses, 2010, 74, 649–660.

12.Esplugues JV: NO as a signalling molecule in the nervous system. Br J Pharmacol, 2002, 135, 1079–1095.

13.Fawcett WJ, Haxby EJ, Male DA: Magnesium: physiology and pharmacology. Br J Anaesth, 1999, 83, 302–320.

14.Fox C, Ramsoomair D, Carter C: Magnesium: its proven and potential clinical significance. South Med J, 2001, 94, 1195–1201.

15.Gee JB, Corbett RJ, Perlman JM, Laptook AR: Hypermag- nesemia does not increase brain intracellular magnesium in newborn swine. Pediatr Neurol, 2001, 25, 304–308.

16.Ghofrani HA, Osterloh IH, Grimminger F: Sildenafil: from angina to erectile dysfunction to pulmonary hypertension and beyond. Nat Rev Drug Discov, 2006, 5, 689–702.

17.Harkin A, Connor TJ, Burns MP, Kelly JP: Nitric oxide synthase inhibitors augment the effects of serotonin reuptake inhibitors in the forced swimming test. Eur Neu- ropsychopharmacol, 2004, 14, 274–281.

(7)

18.Harkin AJ, Bruce KH, Craft B, Paul IA: Nitric oxide synthase inhibitors have antidepressant-like properties in mice. 1. Acute treatments are active in the forced swim test. Eur J Pharmacol, 1999, 372, 207–213.

19.Hashimoto K: Emerging role of glutamate in the pathophysiology of major depressive disorder. Brain Res Rev, 2009, 61, 105–123.

20.Heiberg IL, Wegener G, Rosenberg R: Reduction of cGMP and nitric oxide has antidepressant-like effects in the forced swimming test in rats. Behav Brain Res, 2002, 134, 479–484.

21.Heiden A, Frey R, Presslich O, Blasbichler T, Smetana R, Kasper S: Treatment of severe mania with intravenous magnesium sulphate as a supplementary therapy. Psy- chiatry Res, 1999, 89, 239–246.

22.Huang LJ, Yoon MH, Choi JI, Kim WM, Lee HG, Kim YO: Effect of sildenafil on neuropathic pain and hemodynamics in rats. Yonsei Med J, 2010, 51, 82–87.

23.Ikari A, Nakajima K, Taki S, Suketa Y: Up-regulation of Na⁺-dependent Mg²⁺transport by nitric oxide and cyclic GMP pathway in renal epithelial cells. Eur J Pharmacol, 2002, 451, 133–139.

24.Imada Y, Yoshioka S, Ueda T, Katayama S, Kuno Y, Kawahara R: Relationships between serum magnesium levels and clinical background factors in patients with mood disorders. Psychiatry Clin Neurosci, 2002, 56, 509–514.

25.Kalivas PW, Duffy P, Eberhardt H: Modulation of A10 dopamine neurons by gamma-aminobutyric acid ago- nists. J Pharmacol Exp Ther, 1990, 253, 858–866.

26.Mohler H: GABA_Areceptors in central nervous system disease: anxiety, epilepsy, and insomnia. J Recept Signal Transduct Res, 2006, 26, 731–740.

27.Mora F, Segovia G, Del Arco A: Glutamate-dopamine- GABA interactions in the aging basal ganglia. Brain Res Rev, 2008, 58, 340–353.

28.Mordes JP, Wacker WE: Excess magnesium. Pharmacol Rev, 1977, 29, 273–300.

29.Nieoczym D, £uszczki JJ, Czuczwar SJ, WlaŸ P: Effect of sildenafil on the anticonvulsant action of classical and second-generation antiepileptic drugs in maximal electroshock-induced seizures in mice. Epilepsia, 2010, 51, 1552–1559.

30.Nieoczym D, Soca³a K, Rundfeldt C, WlaŸ P: Effects of sildenafil on pentylenetetrazol-induced convulsions in mice and amygdala-kindled seizures in rats. Pharmacol Rep, 2010, 62, 383–391.

31.Nieoczym D, Soca³a K, WlaŸ P: Lack of effect of sildenafil on cocaine-induced convulsions in mice. Pharmacol Rep, 2009, 61, 930–934.

32.Nowak G, Legutko B, Skolnick P, Popik P: Adaptation of cortical NMDA receptors by chronic treatment with specific serotonin reuptake inhibitors. Eur J Pharmacol, 1998, 342, 367–370.

33.Nowak G, Trullas R, Layer RT, Skolnick P, Paul IA:

Adaptive changes in the N-methyl-D-aspartate receptor complex after chronic treatment with imipramine and 1-

aminocyclopropanecarboxylic acid. J Pharmacol Exp Ther, 1993, 265, 1380–1386.

34.Padayatti PS, Pattanaik P, Ma X, van den Akker F: Struc- tural insights into the regulation and the activation mechanism of mammalian guanylyl cyclases. Pharmacol Ther, 2004, 104, 83–99.

35.Poleszak E: Benzodiazepine/GABA_Areceptors are involved in magnesium-induced anxiolytic-like behavior in mice. Pharmacol Rep, 2008, 60, 483–489.

36.Poleszak E: Modulation of antidepressant-like activity of magnesium by serotonergic system. J Neural Transm, 2007, 114, 1129–1134.

37.Poleszak E, WlaŸ P, Kêdzierska E, Nieoczym D, Wróbel A, Fidecka S, Pilc A et al.: NMDA/glutamate mechanism of antidepressant-like action of magnesium in forced swim test in mice. Pharmacol Biochem Behav, 2007, 88, 158–164.

38.Poleszak E, WlaŸ P, Kêdzierska E, Nieoczym D, Wyska E, Szymura-Oleksiak J, Fidecka S et al.: Immobility stress induces depression-like behavior in the forced swim test in mice: effect of magnesium and imipramine.

Pharmacol Rep, 2006, 58, 746–752.

39.Poleszak E, WlaŸ P, Kêdzierska E, Radziwoñ-Zaleska M, Pilc A, Fidecka S, Nowak G: Effects of acute and chronic treatment with magnesium in the forced swim test in rats. Pharmacol Rep, 2005, 57, 654–658.

40.Poleszak E, WlaŸ P, Szewczyk B, Kêdzierska E, Wyska E, Librowski T, Szymura-Oleksiak J et al.: Enhancement of antidepressant-like activity by joint administration of imipramine and magnesium in the forced swim test: Be- havioral and pharmacokinetic studies in mice. Pharmacol Biochem Behav, 2005, 81, 524–529.

41.Porsolt RD, Bertin A, Jalfre M: Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther, 1977, 229, 327–336.

42.Prast H, Philippu A: Nitric oxide as modulator of neuronal function. Prog Neurobiol, 2001, 64, 51–68.

43.Romani A: Regulation of magnesium homeostasis and transport in mammalian cells. Arch Biochem Biophys, 2007, 458, 90–102.

44.Rosen RC, McKenna KE: PDE-5 inhibition and sexual response: pharmacological mechanisms and clinical out- comes. Annu Rev Sex Res, 2002, 13, 36–88.

45.Skolnick P, Popik P, Trullas R: Glutamate-based antidepressants: 20 years on. Trends Pharmacol Sci, 2009, 30, 563–569.

46.Swaminathan R: Magnesium metabolism and its disorders. Clin Biochem Rev, 2003, 24, 47–66.

47.Topf JM, Murray PT: Hypomagnesemia and hypermagnesemia. Rev Endocr Metab Disord, 2003, 4, 195–206.

48.Uthayathas S, Karuppagounder SS, Thrash BM, Parameshwaran K, Suppiramaniam V, Dhanasekaran M:

Versatile effects of sildenafil: recent pharmacological ap- plications. Pharmacol Rep, 2007, 59, 150–163.

Received: July 6, 2011; accepted: September 1, 2011.