Copyright 2001 by Institute of Pharmacology Polish Academy of Sciences Polish Journal of Pharmacology Pol. J. Pharmacol., 2001, 53, 641 645 ISSN 1230-6002 SHORT COMMUNICATION RISE IN ZINC AFFINITY FOR THE NMDA RECEPTOR EVOKED BY CHRONIC IMIPRAMINE IS SPECIES-SPECIFIC Bernadeta Szewczyk 1,#, Renata Kata 2, Gabriel Nowak 1,2 Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland, Laboratory of Radioligand Research, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland Rise in zinc affinity for the NMDA receptor evoked by chronic imipramine is species-specific. B. SZEWCZYK, R. KATA, G. NOWAK. Pol. J. Pharmacol., 2000, 53, 641 645. Zinc and magnesium are potent inhibitors of the NMDA receptor complex. Previous reports demonstrated that both zinc and magnesium, like other NMDA receptor antagonists, exhibit antidepressant-like effects in rodent screening tests. Moreover, chronic treatment with antidepressants and electroconvulsive shock increase zinc concentration in the rat hippocampus. The present study examined the effect of tricyclic antidepressant, imipramine, on the potency of zinc and magnesium to inhibit [! H]MK-801 binding in the mouse and rat cerebral cortex and hippocampus. Chronic treatment with imipramine produced statistically significant increase in the potency of zinc to inhibit [! H]MK-801 binding in the mouse cerebral cortex but not in the hippocampus. However, this treatment neither influenced the zinc affinity in rat tissue nor magnesium affinity in tissue of both species. The present data indicate that, although imipramine-induced rise in zinc affinity to the NMDA receptor complex is in agreement with previously reported antidepressant-induced reduction of the NMDA receptor function, this effect is species-specific. Key words: imipramine, NMDA receptor, zinc, magnesium, cortex, hippocampus, mice, rats correspondence; former name: Kroczka
B. Szewczyk, R. Kata, G. Nowak INTRODUCTION N-methyl-D-aspartate (NMDA)/glutamate receptor is implicated in many pathological and physiological processes [24]. Recent preclinical data have suggested that NMDA/glutamate receptor may be involved in the mechanism of antidepressant treatments [20, 21]. Chronic administration of a wide range of clinically effective antidepressants produced a significant reduction in the potency of glycine to inhibit the [ 3 H]5,7-dichlorokynurenic acid binding to glycine receptors. Also such chronic treatment with antidepressants caused a decrease in the proportion of high-affinity glycine sites inhibiting the binding of the NMDA antagonist [ 3 H]CGP- -39653 [14, 16, 17]. Several studies have demonstrated that NMDA antagonists exhibit antidepressant-like actions in animal test and models, and that combined administration of NMDA antagonists with antidepressants has a synergistic effect [6, 21, 22]. This interaction between NMDA receptors and antidepressants suggest possible role of glutamatergic system in the pathophysiology of depression and in the mechanism of antidepressant drugs. Zinc and magnesium play an important role in function of the central nervous system. Recent studies indicated that zinc and magnesium might be involved in the mechanism of antidepressant therapy and/or pathophysiology of depression. Clinical observations demonstrated significant reduction in serum zinc concentration in the depressed patients [5, 19]. The data concerning the serum magnesium concentration in the depressed patients are inconsistent, since both increases and decreases have been observed [25]. However zinc and magnesium exhibit an antidepressant-like effect in the forced swim test in rodents [2 4]. In the present study, we have used the selective non-competitive NMDA receptor antagonist, radioligand [ 3 H]MK-801, to examine the effect of chronic treatment with imipramine on the potency of zinc and magnesium to inhibit NMDA receptor in the mouse and rat cerebral cortex and hippocampus. MATERIALS and METHODS Animals The experiments were carried out on male Swiss-Webster mice (20 25 g) and male Wistar rats (200 g). The animals were housed under a 12/12 h light/dark cycle with free access to food and water. Whole procedure was conducted according to NIH Animal Care and Use Committee guidelines, and was approved by the Ethics Committee of the Institute of Pharmacology, Kraków. Imipramine (15 mg/kg) or vehicle (saline) was administered ip once a day for 14 days. Animals were sacrificed by decapitation 24 h after the last injection. Their brains were removed and cortices (neocortex) and hippocampi were dissected and rapidly frozen over solid CO 2. Tissues were stored at 80 C for 2 4 weeks until they were assayed. Radioligand binding assay Radioligand binding assays were conducted essentially as previously described [14]. The tissue was thawed in 50 volumes of ice-cold 5 mm HEPES/ 4.5 mm Tris (HTS) buffer (ph 7.4), homogenized and centrifuged at 20.000 g for 20 min. Pellet was subsequently reconstituted in HTS containing 1 mm EDTA and then centrifuged at 20.000 g for 20 min. The obtained tissue pellet was reconstituted once more in 20 volumes of HTS containing 1mM EDTA and then centrifuged at 20.000 g for 20 min. Following centrifugation the pellet was reconstituted in 5 volumes of fresh HTS and stored at 70 C for at least 3 days before assay. On the day of the assay, the frozen aliquots were thawed in 20 volumes of HTS and centrifuged at 20.000 g for 20 min. [ 3 H] MK-801 binding was assayed in HTS buffer under nonequilibrium conditions by incubating membranes (0.5 mg of protein) for 2 h at 25 C in the presence of 5 nm [ 3 H]MK-801 (28.8 Ci/mmol; NEN), zinc (10 3 to 10 7 ); magnesium (10 3 to 10 7 ) or buffer to a final volume of 0.3 ml. Nonspecific binding was assessed using 100 M phencyclidine. The incubation was terminated by vacuum filtration through Whatman GF/C filters. The filters were then washed twice with 0.1 ml of ice-cold HTS placed in scintillation vials with 4 ml of liquid scintillation cocktail and bound radioactivity was measured in Beckman, LS-6500 scintillation counter. Data analysis and statistics Radioligand binding data were analyzed using iterative curve fitting routines (GraphPAD/InPlot Version 2.0). A one-site model was assumed unless employing a two-site model significantly reduced the sum of squares of the model. Group differences were assessed using unpaired Student s t-test. Data were deemed significant when p < 0.05. 642 Pol. J. Pharmacol., 2001, 53, 641 645
SPECIES-SPECIFIC EFFECT OF IMIPRAMINE IN ZINC AFFINITY FOR NMDA RECEPTOR RESULTS Chronic treatment with imipramine (15 mg/kg/ day) induced statistically significant increase in the potency of zinc to inhibit [ 3 H]MK-801 binding to NMDA receptor ionophore in the mouse cortex. The IC 50 value of zinc inhibition was decreased by ca. 53% in tissue of imipramine-treated relative to saline-treated mice (Tab. 1). In contrast, chronic imipramine treatment did not significantly affect the potency of zinc to inhibit [ 3 H]MK-801 binding in the mouse hippocampus (Tab. 1). Moreover, zinc affinity was not influenced by chronic imipramine treatment in rats (Tab. 1). Chronic treatment with imipramine had no effect on the potency of magnesium to inhibit [ 3 H]MK-801 binding neither in the cortex nor in the hippocampus (Tab. 1). Maximal inhibition induced by the examined metals was not affected by chronic imipramine treatment (data not shown). Table 1. Effect of chronic treatment with imipramine on zinc and magnesium-induced inhibition of [! H] MK-801 binding to the mouse and rat cortex and hippocampus MICE Zn IC # 90.0 ±20.5 Mg IC # 232.8 ±43.9 RATS Zn IC # 128.9 ±19.8 Mg IC # 180.1 ±84.0 Cortex Hippocampus Control Imipramine Control Imipramine 48.0 ± 3.0* 193.5 ±39.4 125.3 ±24.0 163.6 ±36.8 105.0 ±18.5 143.2 ±13.8 542.6 ±82.0 303.8 ±12.3 156.5 ±21.5 164.7 ±17.7 491.3 ±27.0 272.5 ±30.0 Data are expressed as IC # ( M) and represent the mean ± SEM of 5 (mice) or 3 (rats) subjects per group. *p<0.05 DISCUSSION Chronic treatment with various antidepressants alters the NMDA receptor complex [20, 21]. This treatment reduces the potency of glycine to inhibit [ 3 H]5,7-dichlorokynurenic acid binding to strychnine-insensitive glycine receptors [14, 16, 17] and, moreover, it induces a decrease in the proportion of high-affinity glycine-displaceable sites inhibiting [ 3 H]CGP 39653 binding to NMDA receptors [10, 11, 14, 16]. These effects were observed in the cerebral cortex, but not in the hippocampus, basal forebrain or striatum [11, 14]. These alterations are reflected also at the NMDA receptor subunits gene expression [20]. All these adaptive changes may lead to the reduction of the NMDA receptor function demonstrated behaviorally [18] and biochemically [7]. Moreover, functional antagonists of the NMDA receptor complex exhibit an antidepressant-like effect in animal tests (models of depression [21]). Involvement of zinc, a very potent inhibitor of the NMDA receptor complex, in the mechanism of antidepressant drug action has been suggested by previous studies. A lower zinc serum concentration was demonstrated in depressed patients, which was normalized only after successful antidepressant therapy [5, 14, 15, 19]. Moreover, electroconvulsive shock (ECS) treatment increased zinc concentrations in the hippocampus with a similar, although less significant effect in the cortex and cerebellum [12, 23]. Repeated treatment with imipramine or citalopram was shown to redistribute rat brain zinc concentration favoring hippocampus [8, 9, 12, 23]. We have demonstrated also that zinc induces an antidepressant-like effect and enhances the effect of imipramine in the forced swim test [3, 4]. The present data showing, that imipramine induces the increase in the potency of zinc to inhibit the NMDA receptor-coupled ionophore, support the notion of the critical role of the interaction between zinc and NMDA receptor in the mechanism of antidepressant drug action. On the other hand, lack of such effect in rats suggests that different imipramine-induced adaptive mechanisms (in respects to zinc sites of the NMDA receptor complex) exist in these two species. Similarly, some other antidepressant-induced adaptive changes are also species-specific (e.g. dopamine D-1 receptor down-regulation [13]). Moreover, a number of recent studies indicate that NMDA receptor- -channel complex exists in multiple forms which have different physiological and pharmacological properties and are differentially distributed throughout brain [1]. Based on this studies, we propose that the differences which we have observed in our study (concerning the potency of zinc to inhibit [ 3 H]MK-801 in the cortex and hippocampus) could reflect region-specific subunit composition of the NMDA receptor complex. ISSN 1230-6002 643
B. Szewczyk, R. Kata, G. Nowak As mentioned in the introduction, magnesium might be also involved in the pathophysiology and therapy of depression. Several clinical studies have examined serum magnesium in depression. However, the data are inconsistent and both increases and decreases in the serum magnesium have been observed in depressed patients [25]. Our unpublished data suggest that chronic antidepressant treatment increases magnesium concentration in the rat brain. Furthermore, in the forced swimming test, magnesium salts reduce the immobility time in a way similar to imipramine [2]. The present study shows no effect of chronic treatment with imipramine on the potency of magnesium to interact with the NMDA receptor complex. This finding suggests that magnesium regulatory site of this receptor complex is not modified by chronic imipramine treatment. 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