DTNB Inhibits Calcium Response of Rat Brain Cortical Slices to Anozia of Various Duration
D. G. Semenov, M. O. Samoilov, and E. V. Lazarevich*
Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny , Vol. 131, No. 6, pp. 621-623, June, 2001 Original article submitted February 21, 2001
Key Words: hypoxia; calcium; NMDA receptor; DTNB
According to modern neuropathological concept, hyper- activation of glutamate NMDA receptors plays a key role in the development of acute brain injuries (ische- mia, trauma, and epilepsy) and chronic neurodegene- rative diseases. These conditions are accompanied by calcium overload of brain cells resulting in disorgani- zation of intracellular regulatory processes. In view of this, the search for new pharmacological preparations preventing hyperactivation of NMDA receptors is an important clinical problem. Ten-year experimental and clinical experience of using highly specific NMDA receptor antagonists as neuroprotectors during epi- lepsy and hypoxia showed that their positive effect (prevention of calcium overload) is often accompanied by impairment of physiological mechanisms under- lying neuronal synaptic plasticity, which is manifested in various neurological disorders. Elaboration of mild antagonists blocking only pathological hyperactivation of NMDA receptors is therefore of considerable im- portance. The preparations oxidizing sulfhydryl groups of NMDA channel subunits forming the so-called re- dox site of NMDA receptors hold much promise in this respect. Reducing compounds affecting this mo- dulatory complex promote opening of channels and
I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg; *Medical Research Center, Polish Academy of Sciences, Warsaw. Address for correspondence: [email protected]. Se- menov D. G.
entry of extracellular Ca2+. Oxidizing compounds, in- cluding 5-5′-dithio-bis(2-nitrobenzoic acid) (DTNB), produce opposite effects [6,7,12]. DTNB prevents in- juries during kainate-induced epilepsy in experimental animals [8-10]. Previous experiments showed that DTNB blocks long-term potentiation of synaptic trans- mission in hippocampal slices induced by short-term ischemia [6].
Here we evaluated the role of redox sites of NMDA receptor in 2 mechanisms underlying accumulation of intracellular Ca2+: pathological calcium overload and moderate adaptive increase in cytoplasmic Ca2+ con- centration (Ca2+cyt) induced by long-term and short- term anoxia, respectively [2]. These mechanisms were previously studied during measurements of Ca 2+ con- tent (Ca2+cyt and Ca2+ bound to hydrophobic intracel- lular domains, Ca-b) [3,5,11].
MATERIALS AND METHODS
Experiments were performed on 400-µ slices of the visual cortex from Kyoto-Wistar rats prepared on a 752 Vibroslice microtome (Campden Instruments). The slices were incubated in a standard oxygenated solution at 37.5oC [1]. The concentrations of Ca-b and Ca2+cyt were measured spectrofluorometrically using a LYuMAM KF contact fluorescent microscope and Hi- tachi F-2000 spectrofluorometer, respectively, in a 2- chamber incubation system [4]. Chlortetracycline fluorescence probe (CTC, Sigma) was used to produce fluorescence signals characterizing the dynamics of Ca-b content. We measured the quantum yield of fluor- escence at 520 nm after excitation with 410-nm light. The relative changes in Ca2+cyt content were recorded using a Fura-2/AM fluorochrome (Molecular Probes). The ratio between fluorescence signals with maximum of 510 nm after irradiation with 340 and 380 nm. Both methods were described previously [11]. Control mea- surements showed that DTNB solutions (200 µM) are characterized by intensive light absorption at 300-400 nm. Therefore, the presence of DTNB in the incuba- tion solution markedly hinders fluorometry under these regimens. However, modification of redox sites in NMDA receptors produced by oxidizers and reducers persists for a long time after washout [7,10,12]. In our experiments brain slices were perfused with DTNB for 30 min, without DTNB for 60 min, and then subjected to 2- or10-min anoxia followed by 70-min reoxygenation. The data are presented as M±SEM. The results were analyzed by ANOVA using the Dunnett test.
RESULTS
DTNB markedly changed the dynamics of Ca2+cyt con- tent in slices subjected to 10-min hypoxia: it inhibited the increase in Ca2+cyt content during anoxia and pre- vented its secondary rise after 25-min reoxygenation compared to the control (Fig. 1, a). DTNB had no effect on the dynamics of Ca-b content during anoxia, but 2-fold attenuated the posthypoxic increase in this parameter (Fig. 1, b). These changes in Ca2+cyt and Ca-b contents suggest that DTNB-induced oxidation of NMDA receptor redox sites blocks enter of extra- cellular Ca2+ during 10-min anoxia and after 25-min reoxygenation. Therefore, the contribution of Ca2+- binding intracellular sequesters into compensatory eli- mination of excessive Ca2+cyt decreases.
Fig. 1. Content of cytoplasmic Ca2+ (a, c) and calcium bound to hydrophobic intracellular domains ( b, d) during 10- (a, b) and 2-min anoxia (c, d) in control (1) and DTNB-treated slices (2). Negative values on abscissa correspond to anoxia; zero point: start of reoxygenation.
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