PRELIMINARY COMMUNICATION REDUCED POTENCY OF ZINC TO INTERACT WITH NMDA RECEPTORS IN HIPPOCAMPAL TISSUE OF SUICIDE VICTIMS

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1 Copyright 2003 by Institute of Pharmacology Polish Academy of Sciences Polish Journal of Pharmacology Pol. J. Pharmacol., 2003, 55, ISSN PRELIMINARY COMMUNICATION REDUCED POTENCY OF ZINC TO INTERACT WITH NMDA RECEPTORS IN HIPPOCAMPAL TISSUE OF SUICIDE VICTIMS Gabriel Nowak 1,2,#, Bernadeta Szewczyk 1, Krystyna Sadlik 3, Wojciech Piekoszewski 3,4, Franciszek Trela 5, Ewa Florek 7, Andrzej Pilc 1,6 Department of Neurobiology, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL Kraków, Poland; Department of Pharmacobiology, Collegium Medicum, Jagiellonian University, Medyczna 9, PL Kraków, Poland;! Institute of Forensic Research, Westerplatte 9, PL Kraków, Poland; " Department of Clinical and Industrial Toxicology, Collegium Medicum, Jagiellonian University, Z³otej Jesieni 1, PL Kraków, Poland; # Department of Forensic Medicine, Collegium Medicum, Jagiellonian University, Grzegórzecka 16, PL Kraków, Poland; $ Institute of Public Health, Collegium Medicum, Jagiellonian University, Micha³owskiego 12, PL Kraków, Poland; % Department of Toxicology, Karol Marcinkowski University of Medical Sciences, Dojazd 20, PL Poznañ, Poland Reduced potency of zinc to interact with NMDA receptors in hippocampal tissue of suicide victims. G. NOWAK, B. SZEWCZYK, K. SADLIK, W. PIEKOSZEWSKI, F. TRELA, E. FLOREK, A. PILC. Pol. J. Pharmacol., 2003, 55, Zinc is involved in both psychopathology and treatment of depression. Since a considerable percentage of suicide victims had suffered from depression, we hypothesized that alteration in zinc homeostasis might occur in their brain tissue. We now report that zinc content is not altered in the hippocampal or cortical tissue of suicide victims (n = 10) compared to age-matched controls (n = 10). However, there is a statistically significant 26% decrease in the potency (increase in the IC 50 value) of zinc to inhibit [ 3 H]MK-801 binding to NMDA receptors in the hippocampal but not cortical tissue of suicide subjects. The data represent the first demonstration that the alteration in zinc interaction with NMDA receptors may be involved in psychopathology underlying suicidal attempts. Key words: zinc, NMDA receptors, hippocampus, cortex, human, suicide correspondence

2 G. Nowak, B. Szewczyk, K. Sadlik, W. Piekoszewski, F. Trela, E. Florek, A. Pilc INTRODUCTION Zinc is a trace element, essential for function of over 300 enzymes. This metal plays catalytic, coactive and structural roles in these enzymes. Besides, zinc is necessary for DNA replication, transcription and protein synthesis [22]. The brain zinc pool is mostly localized in the hippocampus and cortex. Approximately 5% of the total brain zinc is stored in the synaptic vesicles in zinc-containing neurons. All zinc-containing neurons are glutamatergic, however, not all glutamatergic neurons contain this metal [1, 2]. The zinc-containing neurons accumulate, sequester and release zinc along with glutamate from their presynaptic vesicles. Vesicular zinc plays a role as an endogenous neuromodulator of glutamate ionotropic (NMDA, AMPA, kainate) and metabotropic receptors [3, 23, 25, 28, 32]. The main effect of zinc is connected with inhibition of NMDA receptors [3, 20]. Zinc is involved in the pathophysiology and therapy of depression (for review see [12, 17]). Clinical observations demonstrated a reduced blood zinc level in depressed patients, which was normalized by effective antidepressant therapy [7 10, 18, 24]. Moreover, the severity of the illness (assessed according to Hamilton Depression Rating Scale) was negatively correlated with serum level of this ion [8, 18, 24]. All the data suggest that serum zinc concentrations may be a sensitive and specific marker of depression. Furthermore, zinc exhibits antidepressant-like activity in the forced swim test, and olfactory bulbectomy, chronic mild stress and chronic unpredictable stress rat models of depression ([5, 6], unpublished data). Since a considerable percentage of suicide victims had been affected by depression, we hypothesized that alteration in zinc homeostasis might occur in the brain tissue. In the present paper, we report a preliminary data on examination of zinc content and zinc potency to inhibit [ 3 H]MK-801 binding to NMDA receptors in the hippocampus and frontal cortex of suicide victims and age-matched sudden death control subjects. MATERIALS and METHODS Tissue collection Brain tissue from 10 suicide victims and 10 sudden death controls (mean age ± SEM, 36.4 ± 5.5 years for suicide and 41.4 ± 8.0 years for controls) was obtained as discarded tissue at the time of autopsy at the Department of Forensic Medicine, Collegium Medicum, Jagiellonian University. Study subjects comprised 2 females and 18 males. The causes of death included: 7 hanging, 2 jumping, 1 drug overdose, for suicide group; and 2 homicide, 3 road accident, 1 myocardial infarction, 2 alcohol/ drug overdose, 1 diabetes, 1 drown, for control group. At autopsy, blocks (approximately 2 2 2cm)of frontal cortex (Brodman Area 10) and hippocampus were dissected, frozen and stored at 80 C. Before analysis, each sample was divided into two parts (weight g). One part was used for zinc determination and the second for radioligand assay. Zinc determination Samples were wet-digested with nitric acid and hydrogen peroxide (microwave digestion, Milestone MLS-1200 Mega Microwave Digestion System). Determination of zinc was carried out by flame atomic absorption spectrometry. A Pye Unicam SP AA Spectrophotometer with deuterium background correction (air flow 4.5 l/min, acetylene flow 1.1 l/min, analytical wavelength nm). Relative standard deviation (RSD) of the method (the whole analytical procedure: digestion + zinc determination) did not exceed 2.4%. Mean recovery of zinc was 99% (SD 0.78). Radioligand binding assay Radioligand binding assays were conducted as previously described [27]. The tissue was thawed in 50 volumes of ice-cold 5mM HEPES/4.5mM Tris (HTS) buffer (ph 7.4), homogenized and centrifuged at g for 20 min. Pellet was subsequently reconstituted in HTS containing 1mM EDTA and then centrifuged at g for 20 min. The obtained tissue pellet was reconstituted once more in 20 volumes of HTS containing 1mM EDTA and then centrifuged at 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 g for 20 min. [ 3 H] MK-801 binding was assayed in HTS buffer under nonequilibrium conditions by incubating membranes (0.05 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 ) or buffer to a final volu- 456 Pol. J. Pharmacol., 2003, 55,

3 ZINC AND NMDA INTERACTION IN SUICIDE VICTIMS me 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/Prism, ver. 3.0). Group differences were assessed using unpaired Student s t-test. Data were deemed significant when p < RESULTS and DISCUSSION The previous reports demonstrated alterations in glutamate sites with no changes in glycine or dizocilpine sites of the NMDA receptor complex in the cerebral cortex of suicide victims [4, 13, 19]. Also zinc interaction with NMDA receptor was not altered in the frontal or parietal cortex [19]. The present data demonstrate a 26% increase in the IC 50 value of zinc inhibition of [ 3 H]MK-801 binding to NMDA receptors in the hippocampus, but not in the frontal cortex (Tab. 1). There were no alterations in specific [ 3 H]MK-801 binding in both brain regions (data not shown). Thus, our data confirm report of Palmer et al. [19], who described no alterations in zinc potency to NMDA receptor in the cerebral cortex of suicide victims. The zinc content was not altered in both brain structures (frontal cortex and hippocampus, Tab. 2). The reduction in the potency of zinc to inhibit NMDA receptors (rise in the IC 50 value) in the hippocampus, in spite of unchanged zinc concentration, may indicate the enhanced function of the NMDA receptor complex in this brain area. The enhanced function of NMDA receptor in pathophysiology of depression might be predicted from animal experiments: increased potency of glycine (glutamate co-agonist) at NMDA receptor in the cortex of stressed rats (swim or chronic mild stress, [14, 15]). On the other hand, chronic unpredictable stress (more severe) and olfactory bulbectomy induced opposite results, namely, the decreased glycine potency at NMDA receptors [11, 14]. Also, alterations in the glutamate sites of the NMDA receptor complex in the frontal cortex of suicide subjects suggest reduced rather Table 1. Potency of zinc to inhibit [ 3 H]MK-801 binding in the hippocampus and frontal cortex of suicide victims and sudden death controls Hippocampus Frontal cortex Suicide ± 18.4* ± 56.6 Control ± ± 34.3 Data are expressed as IC 50 (mm) values and represent mean ± SEM of 10 subjects. *p<0.05vs.control Table 2. Concentration of zinc in the hippocampus and frontal cortex of suicide victims and sudden death controls Hippocampus Frontal cortex Suicide 10.4 ± ± 0.4 Control 10.4 ± ± 0.3 Data are expressed in mg Zn 2+ /g tissue and represent mean ± SEM of 10 subjects than enhanced function of the NMDA receptors [13]. Taking into consideration that in both studies suicide subjects were not diagnosed, and animal models of depression are far from satisfactory in resembling depression symptoms [30, 31], this issue needs further studies. Chronic treatment with electroconvulsive shock, imipramine or citalopram induce an increase in hippocampal zinc concentration in rats [16]. Using Timm s method for zinc staining, Vaidya et al. [29] demonstrated that chronic treatment with electroconvulsive shock induced hippocampal mossy fiber sprouting. These data suggest the increase in the vesicular zinc level in the hippocampus following electroconvulsive shock. Moreover, chronic treatment with imipramine increases the potency of zinc to inhibit the NMDA receptor complex in the cerebral cortex but not in the hippocampus in mice [27]. Such effect has not been demonstrated in rats, which suggests the existence of species-specific imipramine-induced adaptive mechanisms in the zinc sites of the NMDA receptor complex. The data may suggest diminished function of the NMDA receptor following antidepressant treatment. In fact, such functional (behavioral) NMDA receptor desensitization was observed after chronic treatment with antidepressants and electroconvulsive shock [21]. Zinc induced antidepressant-like effects in the forced swim test [5, 6]. Moreover, very low doses ISSN

4 G. Nowak, B. Szewczyk, K. Sadlik, W. Piekoszewski, F. Trela, E. Florek, A. Pilc of zinc administered together with low, ineffective doses of imipramine and citalopram enhanced antidepressant-like effect in this test [5, 26]. Zinc is also active in olfactory bulbectomy, chronic mild stress and chronic unpredictable stress animal models of depression (our unpublished data). All these animal data strongly suggest possible antidepressant activity of zinc in human depression. Summing up, 1) antidepressants induced reduction of the NMDA receptor function, in which zinc might play some role, 2) zinc exhibited antidepressant-like activity in animal experiments and 3) the role of the NMDA receptor function and zinc homeostasis in the pathophysiology of depression needs further studies. Nevertheless, this is the first demonstration of the reduction of zinc interaction with NMDA receptors in the suicide hippocampus, which suggests the involvement of this mechanism in the psychopathology of suicide disorder. Acknowledgments. This study was supported by the grant no. 6P05B from the State Committee for Scientific Research, Warszawa, Poland. B. Szewczyk is a grantee of the Foundation for Polish Science. REFERENCES 1. Frederickson C.J.: Neurobiology of zinc and zinccontaining neurons. Int. Rev. Neurobiol., 1989, 31, Frederickson Ch. J., Won Suh S., Silva D., Frederickson C.J., Thompson R.B.: Importance of zinc in the central nervous system: the zinc-containing neuron. J. Nutr., 2000, 130, 1471S 1483S. 3. Harrison N.L., Gibbsons S.J.: Zn: an endogenous modulator of ligand-and voltage-gated ion channels, Neuropharmacology, 1994, 33, Holemans S., De Paermentier F., Horton R.W., Crompton M.R., Katona C.L.E., Maloteaux J.-M.: NMDA glutamatergic receptors, labelled with [ 3 H]MK-801, in brain samples from drug-free depressed suicides. Brain Res., 1993, 616, Kroczka B., Brañski P., Pa³ucha A., Pilc A., Nowak G.: Antidepressant-like properties of zinc in rodent forced swim test. Brain Res. Bull., 2001, 55, Kroczka B., Ziêba A., Dudek D., Pilc A., Nowak G.: Zinc exhibits an antidepressant-like effect in the forced swimming test in mice. Pol. J. Pharmacol., 2000, 52, Maes M., De Vos N., Demedts P., Wauters A., Neels H.: Lower serum zinc in major depression in relation to changes in serum acute phase proteins. J. Affect. Disorders, 1999, 56, Maes M., D Haese P.C., Scharpe S., D Hondt P.D., Cosyns P., De Broe M.E.: Hypozincemia in depression. J. Affect. Disorders, 1994, 31, Maes M., Vandoolaeghe E., Neels H., Demedts P., Wauters A., Meltzer H.Y., Altamura C., Desnyder R.: Lower serum zinc in major depression is a sensitive marker of treatment resistance and of the immune/inflammatory response in that illness. Biol. Psychiat., 1997, 42, McLoughlin I.J., Hodge J.S.: Zinc in depressive disorder. Acta Psychiatr. Scand., 1990, 82, Nowak G.: Calcium antagonists in the olfactory bulbectomy animal model of depression: effect on the cortical NMDA receptor complex. Pol. J. Pharmacol., 1996, 48, Nowak G.: Does interaction between zinc and glutamate system play a significant role in the mechanism of antidepressant action? Acta Pol. Pharm.-Drug Res., 2001, 58, Nowak G., Ordway G., Paul I.A.: Alterations in the N-methyl-D-aspartate (NMDA) receptor complex in the frontal cortex of suicide victims. Brain Res., 1995, 675, Nowak G., Ossowska G., Jopek R., Papp M.: Strychnine-insensitive glycine sites are altered in two stress models of depression. Pol. J. Pharmacol., 1998, 50, Nowak G., Redmond A., McNamara M., Paul I.A.: Swim stress increases the potency of glycine at the N-methyl-D-aspartae receptor complex. J. Neurochem., 1995, 64, Nowak G., Schlegel-Zawadzka M.: Alterations in serum and brain trace element levels after antidepressant treatment. Part I. Zinc. Biol. Trace Elem. Res., 1999, 67, Nowak G., Szewczyk B.: Mechanisms contributing to antidepressant zinc actions. Pol. J. Pharmacol., 2002, 54, Nowak G., Ziêba A., Dudek D., Kroœniak M., Szymaczek M., Schlegel-Zawadzka M.: Serum trace elements in animal models and human depression. Part I. Zinc. Hum. Psychopharmacol. Clin. Exp., 1999, 14, Palmer A.M., Burns M.A., Arango V., Mann J.J.: Similar effects of glycine, zinc and oxidizing agent on [ 3 H]dizocilpine binding to the N-methyl-D-aspartate receptor in neocortical tissue from suicide victims and controls. J. Neural Transm., 1994, 96, Paoletti P., Ascher P., Neyton J.: High-affinity zinc inhibition of NMDA NR1-NR2 receptors. J. Neurosci., 1997, 17, Popik P., Wróbel M., Nowak G.: Chronic treatment with antidepressants affects glycine/nmda receptor function: behavioral evidence. Neuropharmacology, 2000, 39, Prasad A.S.: Zinc: an overview. Nutrition, 1995, 11, Rassendren F.A., Lory P., Pin J.P., Nargeot J.: Zinc has opposite effects on NMDA and non-nmda receptors 458 Pol. J. Pharmacol., 2003, 55,

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