Acute glutamate toxicity and its potentiation by serum albumin are determined by the Ca2+ concentration

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Neuroscience Letters, 130 (1991) 125-127 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 A DONIS 030439409100494T NSL 08012

Acute glutamate toxicity and its potentiation by serum albumin are determined by the Ca 2 + concentration Sara Eimerl and Michael Schramm Department of Biological Chemistry and The Otto Loevi Centerfor Neurobiology, The Hebrew University of Jerusalem, Jerusalem (lsrael) (Received 8 May 1991; Revised version received 7 June 1991; Accepted 7 June 1991)

Key words: NMDA toxicity; Cerebellar granule cell; Toxicity potentiation in cultured neurons Two different processes, mediated by the N-methyl-D-aspartate receptor, appear to cause acute cell death in cultured cerebellar granule cells. A Ca2+-independent process takes place at zero and very low concentration of the added cation. Under these conditions, the known destabilization of excitable membranes at low extracellular Ca 2÷ probably plays a major role. A Ca2+-dependent process becomes dominant as its concentration is increased above 1.0 mM. The remarkable potentation of glutamate toxicity by serum albumin is a calcium-dependent reaction.

Excessive exposure of brain neurons to glutamate, which results in cell death within approximately 2 h is termed acute toxicity. Damage which develops more slowly, causing cell death within 24 h, is termed delayed toxicity. Ion substitution experiments suggested that acute glutamate toxicity is Ca2+-independent [5, 15] while delayed toxicity is Ca2+-dependent [5, 12, 16]. Studying postnatal cerebellar slices it was, however, shown that acute NMDA toxicity does require Ca 2+ and it was proposed that cell death in absence of Ca 2÷, in dispersed cell cultures, might be due to excessive swelling [7]. The seemingly simple task of drawing conclusions about the requirement of an ion by withholding it from the medium is, in the case of glutamate toxicity, greatly complicated by the following considerations. Elimination of an ion may entirely change the properties of the cell; specifically, omission of Ca 2+ destabilizes excitable membranes [9]; cell death may result from different causes, each prevailing under a different ionic composition; Na + is usually required for depolarization which removes the Mg 2+ block of the NMDA receptor channel [1]; choline, used as a substitute for Na + in ion requirement studies, apparently blocks the N M D A receptor [2]; depolarization by high K +, employed to assess the role of depolarization per se in cell death, can in fact cause release of toxic doses of glutamate [17]. Because of the above considerations it was decided to study in detail the effect of graded changes in Ca 2+ conCorrespondence: M. Schramm, Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.

centration on acute glutamate toxicity mediated by the NMDA receptor in cultured cerebellar granule cells. The findings suggest that acute toxicity is Ca2+-independent in the absence of this cation, and Ca2+-mediated in its presence. Furthermore, the strong potentiation of toxicity by serum albumin only takes place if Ca 2+ is present at a concentration of at least 1 mM. Granule cells were cultured 9 ___1 days in BME-25 mM KCI-10% fetal calf serum (5 x 105 cells in 0.5 ml) [11, 17]. All toxicity experiments were conducted at 25°C in 0.5 ml Locke medium containing (mM): NaCI 124, KCI 25, MgCI2 1.0, NaHCO3 4, HEPES 10 (pH 7.5), CaCI2 2.3, glucose 5. Changes in CaC12 concentration were compensated for by adjustment of NaC1 to preserve constant ionic strength. Cells were washed once with Locke of the composition to be used in the subsequent experiment. For each Ca 2+ concentration, two experimental systems were set up: one received no further additions and served to measure toxicity due to release of endogenous glutamate and other causes; the other system received 50~M glutamate plus 10/zM glycine. After 10 min the medium was removed and replaced by Locke containing 2.3 mM CaCI: and 160 nM MK-801. After 1 h, the per cent of cells killed was determined by the tetrazolium reduction procedure [3, 4]. Each experiment was also visually checked by phase contrast microscopy. All experiments were conducted in duplicate. Experiments were repeated at least 3 times. The average of all experiments ___S.E.M. are presented. Fig. 1 demonstrates extreme toxicity at 0 mM and at 20 mM Ca 2+ in absence of added glutamate. The toxicity

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is apparently due to release of endogenous glutamate [13, 17, 18] since it is prevented by the blocker of the N M D A receptor channel, MK-801. Transmitter release, as well as the toxicity at zero added Ca 2+, are putatively ascribed to the destabilization of the excitable membranes [9] in absence of this cation, resulting apparently, in osmotic lysis [15]. Mechanisms of transmitter release induced by Ca 2+ removal have been discussed with regard to y-aminobutyric acid (GABA) in hippocampus slices [14]. The extreme toxicity at 20 mM Ca 2÷ is ascribed to a Ca 2+ dependent glutamate release. At high Ca 2÷ concentration, enough of the cation might enter through spontaneously opening channels to start a process which feeds on itself. It should be noted that within the range of 0.25-5 mM Ca 2+ only ~< 12% of the cells are killed, which is not significantly different from 0%. The range of Ca 2+ concentrations 0.25-10 mM was tested for toxicity caused by added glutamate (Fig. 2). At 0.25 mM Ca 2+ high toxicity is evident. As Ca 2+ is being added, two antipodal processes come into play: stabilization of the cell membrane, resulting in a decrease in glutamate induced cell death, and a gradual increase in a Ca2+-dependent glutamate toxicity. Up to 1.0 mM Ca 2+, stabilization has the upper hand and cell death decreases to a mere 8%. However, further increases in Ca 2+ lead to steadily increasing glutamate induced cell death. It should be emphasized that the low toxicity of 8% at 1 mM Ca 2+ has only relative meaning; longer incubation

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Fig. 2. Toxicity caused by added glutamate as a function of Ca 2~ concentration, in absence and in presence of serum albumin. The per cent cells killed by glutamate was calculated at each Ca `'+ concentration relative to the amount of surviving cells in the system without glutamate (Fig. l) which was taken as 100% live cells. Experimental detail is given in the text. Bovine serum albumin (Sigma, A-4503) and MK801 (RBI) were added at concentrations of 4.0 mg/ml and 160 nM, respectively. MK-801 inhibited glutamate toxicity in presence and absence of BSA to the same extent ( × ). The potentiation by BSA at 1 mM Ca 2÷ was highly significant. Wilcoxon test: * * P < 0.005.

times, higher glutamate concentrations, older cultures which are more sensitive, would produce a high toxicity even in presence of 1 mM Ca 2÷ (not shown). Over the entire range of Ca 2÷ concentrations shown in Figs. 1 and 2, toxicity appears dependent on activation of the N M D A receptor as indicated by the inhibition with MK-801 [see also ref. 17]. No less revealing is the effect of Ca 2+ on the potentiation of glutamate toxicity by bovine serum albumin (BSA). A potentiating activity of serum was first found in our earlier study [17]. It was subsequently attributed to serum albumin [6]. Potentiation was demonstrated in presence or absence of Mg 2 +, with N M D A or glutamate as the agonist. Fig. 2 shows that potentiation by BSA is dependent on the presence of Ca 2+ at a concentration of at least 1 mM. The maximal effect is achieved at about 1.6 mM, boosting toxicity from 10% to 62% cells killed. BSA, in absence of glutamate caused no toxicity at any of the Ca 2+ concentrations. The current findings lead to the conclusion that acute toxicity in presence of Ca 2+ has a Ca2+-dependent component. Thus there may not be much difference between acute and delayed toxicity, except for the rate at which cells die. The similarity becomes even more evident if it is considered that delayed toxicity is largely due to the

127 c o n t i n u o u s e x p o s u r e to e n d o g e n o u s g l u t a m a t e which is being released a n d acts in a m e d i u m c o n t a i n i n g p h y s i o logical c o n c e n t r a t i o n s o f all the ions [8, 16]. In a d d i t i o n , d e l a y e d toxicity, like acute toxicity, c o u l d a p p a r e n t l y be initiated to a certain extent also in absence o f C a 2+ [Fig. 3 in ref. 5]. T h e r e m a r k a b l e p o t e n t i a t i o n by B S A a p p e a r s t o t a l l y d e p e n d e n t o n the presence o f C a 2 +. Its m e c h a n i s m o f action is c u r r e n t l y being studied. Since the a m o u n t o f C a 2+ entering the cell m a y be the limiting f a c t o r in N - m e t h y l D - a s p a r t a t e ( N M D A ) toxicity [12], it seems possible t h a t B S A facilitates this step. The c o n c e n t r a t i o n o f s e r u m a l b u m i n in c e r e b r o s p i n a l fluid is low b u t increases u n d e r p a t h o l o g i c a l c o n d i t i o n s [10]. It m i g h t therefore p l a y an i m p o r t a n t role in p o t e n t i a t i n g events o f g l u t a m a t e toxicity which w o u l d otherwise have been o f m i n o r significance. T h e a u t h o r s are m o s t grateful to Ms. A v i v a Petcho for the p r e p a r a t i o n o f the cerebellar g r a n u l e cells a n d assistance in the experiments. This w o r k was s u p p o r t e d b y a g r a n t f r o m the D e u t s c h e F o r s c h u n g s G e m e i n s c h a f t .

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