Effects of calcium chelators on intracellular calcium and excitotoxicity

Neuroscience Letters, 150 (1993) 129-132 © 1993ElsevierScientificPublishersIreland Ltd. All rights reserved0304-3940/93/$ 06.00

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NSL 09277

Effects of calcium chelators on intracellular calcium and excitotoxicity Janet M. D u b i n s k y Department of Physiology, University of Texas Health Science Center, San Antonio, TX 78284-7756 (USA)

(Received5 August1992;Revisedversionreceived 5 October 1992;Accepted26 October 1992) Key words: Excitotoxicity;Intracellularcalcium;Calciumchelator;Neurotoxicity;Glutamate;Fura-2

In an attemptto probe the relationshipbetweenexcitotoxicityand increasesin intracellularcalcium([Ca2÷]i),BAPTA-AMand its analogswere appliedto culturedhippocampalneurons.Chelationof [Ca2+]~depressedand prolongedtransientresponsesto glutamateand did not effectelevation of [Ca2÷]~by prolongedexposure.Thisexplainsthe inabilityof the chelatorsto prevent glutamate-inducedtoxicity.

Glutamate receptor overstimulation leads to excitotoxic neuronal death [2, 3, 9, 13, 14, 18, 21]. However, the intracellular mechanisms intermediate between receptor activation and eventual death remain largely unknown. Calcium is the leading candidate for an intracellular mediator since intracellular levels become transiently elevated [3, 13, 14, 18], presumably from influx through N-methyl-D-aspartate (NMDA) receptors [12], and removal of extracellular calcium is protective [2, 9, 21]. However, the immediate increase in cytoplasmic calcium concentrations and eventual neuronal death may be parallel events, both triggered by Glu receptor activation. A direct demonstration that excitotoxic neuronal death is preventable in the absence of the consistently observed increases in [Ca2÷]i following Glu receptor activation would greatly strengthen the case that calcium initiates the processes leading to eventual death. To manipulate the changes in [Ca2÷]i in hippocampal neurons during excitotoxic exposure, the calcium chelators, BAPTA, dimethylBAPTA, and dibromoBAPTA [26] were introduced into the intracellular compartment by exposing neurons to the membrane permeable acetylmethyl ester forms. By exogenous chelation of the calcium entering after Glu exposure, global elevations in calcium levels might be prevented and perhaps the toxic consequences avoided. Hippocampal neurons were dissociated and maintained in culture on glass substrates according to published procedures [5, 14]. After 15-17 days in vitro, the Correspondence: J.M. Dubinsky,Department of Physiology,Universityof TexasHealthScienceCenter,7703FloydCurl Drive, San Antonio, TX 78284-7756, USA.

cultures were exposed to 4 gM fura-2-AM in combination with either 10 gM 5,5'-dimethyl BAPTA-AM (MeBAPTA-AM), 50 g M BAPTA-AM, or 100 g M BAPTAAM for 60 min in their growing medium at 37°C. BAPTA-AM was dissolved in 10% pluronic acid in DMSO. Dishes were rinsed with a balanced salt solution [5] and placed upon the stage of a Nikon inverted microscope equipped with a heating stage, epifluorescence objectives, and appropriate filters for Fura-2 fluorescence ratioing, The photometer-based fluorescence acquisition system and calibration procedures have been previously described [5]. Data are presented here as the ratio values after background subtraction. Rmin, R . . . . and fl values for these experiments were 0.85, 27.8 and 14.7, respectively. Assuming a K D of 224 nM [8], ratio values of 1, 1.7, 3 and 5 correspond to [Ca2+]i of 18, 107,285 and 599 nM, respectively. Neuronal survival was assessed by counting neurons containing and excluding trypan-blue, 24 h after toxic exposure [5, 14]. Resting [Ca2÷]i were largely unaffected by the exogenous chelators, with the exception of the highest concentration of BAPTA-AM (Table I). Initially the calcium buffering activity of the internal chelators was assessed by monitoring Fura-2 fluorescence during a 100 ms pulse of 100 gM Glu delivered by pressure ejection from a nearby micropipet. In control dishes preincubated only with the calcium indicator dye, brief Glu pulses produced repeatable, transient increases in fluorescence that rapidly returned to basal levels (Fig. 1A). In dishes preincubated with 10 g M MeBAPTA-AM, the fluorescence ratio increases following Glu application were depressed in amplitude but were not abolished altogether (Fig. 1B). The recovery phase of the fluorescence transient was

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prolonged. Average values for the change in fluorescence ratio and time to recover baseline levels were significantly altered (Table II). Since low concentrations of the high affinity chelator MeBAPTA were not totally effective in preventing perturbations of [Ca2+]i in response to Glu, higher concentrations of the non-methylated chelator BAPTA were tried. In neurons preincubated with 50 /~M BAPTA-AM, responses to short Glu applications were similarly characterized by slightly lower amplitude but prolonged elevations of [Ca2÷]i. The ability of the exogenous buffers to prevent the larger, extended rise in [Ca2+]i following toxic Glu exposures was assessed by monitoring fluorescence ratios after direct addition of 500 ~tM Glu to the bathing solution. Fluorescence ratios were obtained intermittently to prevent bleaching of the dye during continuous exposures. In 6 out of 9 neurons pretreated with 100/IM BAPTA-AM Glu-induced rises in [Ca2+]~ were comparable to those of 6 control neurons (Fig. 1C). Before assessing the ability of exogenous calcium chelators to protect hippocampal neurons from Glu-induced cell death, control experiments were conducted testing the ability of neurons to survive exposure to such chelators. Even abbreviated incubation with high concentrations of BAPTA-AM caused a significant amount of neuronal death (Fig. 2A). Lower concentrations were not as toxic. Since the intracellular concentrations of the exogenous chelators were expected to exceed the concentration of the AM esters in the incubation medium [15], hippocampal cultures were preincubated with 4 /IM of either dimethylBAPTA-AM, BAPTA-AM, dibromoBAPTA-AM (BrBAPTA-AM), or Fura-2-AM for 60 min prior to a 5 min exposure of 500/IM Glu [4, 14]. For a 15 min preincubation, intracellular accumulation of Fura-2 has been previously measured to be 113 + 58/IM (mean + S.E.M.) [4]. For the 60 min preincubation employed here, the intracellular concentration of the calcium chelators would be expected to greatly exceed this value. However, the presence of exogenous calcium cheTABLE I INITIAL F L U O R E S C E N C E RATIOS INDICATIVE OF RESTING C A L C I U M LEVELS A M O N G HIPPOCAMPAL N E U R O N S TREATED WITH MEMBRANE-PERMEABLE CALCIUM CHELATORS Chelator

Mean

S.E.M.

n

none 10 JIM MeBAPTA-AM 50/xM BAPTA-AM 100/IM BAPTA-AM

1.69 1.63 1.22 0.90**

0.11 0.14 0.13 0.06

14 5 4 7

**Significantly different from control, P < 0.001, t-test with Bonferroni correction.

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lators was not able to protect hippocampal neurons from a toxic Glu exposure (Fig. 2B). Some toxicity of the exogenous chelators was also observed. BrBAPTA-AM toxicity may be related to the CNS effects of bromide intoxication [7]. BAPTA-AM toxicity could be attributable to lowering of the basal

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calcium levels (Table I) or competing calcium away from critical intracellular pools. Alternatively, the formaldehyde breakdown products of the acetylmethyl esters cleaved off of these exogenous molecules may reach toxic concentrations [6, 19, 27]. The inability of these exogenous calcium chelators to effectively buffer intracellular calcium and to prevent its elevation after both short and prolonged Glu exposure is surprising. Injection of BAPTA salts into squid giant synapse and dentate granule cells have successfully buffered intracellular calcium-mediated events [1, 17, 23]. However, an increasing number of other experiments attempting to utilize exogenous calcium chelators to prevent intracellular calcium-triggered events have also reported paradoxical findings consistent with an increased [Ca2+]i, rather than a decrease. BAPTA salts or MeBAPTA-AM, in hippocampal and cortical neurons and at the frog neuromuscular junction, have produced unexpected increases in evoked postsynaptic potential amplitudes, increased spike frequency adaptation, lowered tonic firing rates, and increased afterhyperpolarizations, all consistent with a transiently elevated supply of [Ca2+]i [17, 20, 24]. Such increases could be explained if the chelated calcium formed an additional reservoir of calcium that was slowly released into the cytoplasm, as in Fig. 1. Buffering effects consistent with this interpretation have been reported in adrenal chromaffin cells during Fura-2 loading [16]. In these cells, exogenous Fura-2 outcompetes the endogenous intracellular calcium buffering mechanism, reducing the amplitude of depolarizationinduced calcium influx and prolonging the recovery of baseline calcium levels. Since the endogenous buffering mechanisms appear to be of relatively low affinity, a small amount of exogenous buffer may dominate the TABLE II ALTERATION IN PEAK RESPONSE AND RECOVERY TIME FOLLOWING BRIEF APPLICATION OF 100/.tM Glu. Changes in fluorescence ratio (peak response minus initial values) and time to fully recover basal values are averaged for the indicated number of applications to 8 control neurons, 4 neurons preincubated in 50 p M BAPTA-AM and 5 neurons preincubated in 10 ~tM MeBAPTA-AM. Chelator

Control BAPTA-AM MeBAPTA-AM

change in peak ratio

recovery time (s)

Mean + S.E.M.

Mean _+ S.E.M.

n

2.10 + 0.32 0.30* + 0.07 0.86* + 0.11

13.4 + 1.1 82.4** + 7.9 141.3"* + 13.5

25 6 13

*P < 0.05 compared to control, t-test with Bonferroni correction. **P < 0.001.

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Fig. 2. A: hippocampal neuronal survial 24 h following exposure to either the potassium salt or the acetylmethyl ester of the calcium chelator, BAPTA. Both forms were tested at 4 and 100 p M concentrations for 15 or 60 min exposure times. Each bar represents mean + S.E.M. for 4 fields from 2 dishes. B: survival of hippocampal neurons preincubated in various exogenous calcium chelators prior to toxic glutamate exposure. Control bars indicate survival of cultures 24 h after pretreatment with chelators but receiving only solution changes instead of glutamate. DMSO bars refer to cultures exposed to the same final concentration of pluronic acid/DMSO vehicle (0.27%) as the dimethylBAPTA-AM (MeBAPTA-AM) and dibromoBAPTA-AM (BrBAPTA-AM) dishes. The concentrations of vehicle in the Fura-2AM and BAPTA-AM dishes were 0.04% and 0.004%, respectively. The chelators are arranged according to their relative affinities for calcium: MeBAPTA, 40 nM; BAPTA, 107 nM; Fura-2, 224 nM; BrBAPTA, 1580 nM [10]. Bars represent mean + S.E.M. for 3 cultures in each condition.

profile of [Ca2+]i behavior [16, 25]. Hence, exogenous buffers may prolong [Ca2*]i elevation rather than prevent it. By analogy, the similarity of the effects of exogenous chelators in hippocampal neurons and adrenal chromaffin cells [16] suggests that endogenous calcium buffering in hippocampal neurons is accomplished by a low affinity, immobile mechanism. Theoretical models of intracellular calcium buffering from two different groups [16, 22] have combined mobil,

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kinetically fast, low capacity, buffers with a slower, immobile, high capacity, cytoplasmic buffer. Both models predict that the presence of extrinsic faster buffering systems will slow the recovery of intracellular calcium transients before they will appreciably depress the peak response. Binding of calcium to these extremely fast buffers prevents the sequestration of calcium by the relatively slower, high capacity, endogenous systems. The mobility of the fast exogenous buffer serves to shunt calcium away from inside the cell membrane at the expense of prolonging the calcium rise in the cell interior [16, 22]. The calcium measurements made here in a photometer system that captures fluorescence over the entire cell soma are perhaps more indicative of calcium concentrations within the cell interior rather than just inside the lipid bilayer. Thus the presence of exogenous chelators may produce an effect opposite to the intended experimental goal, prolonging the general elevation of [Ca2+]i instead of preventing it. Higher concentrations might have been effective in reducing the overall calcium changes as seen in experiments in frog spinal cord where 60 min perfusion of 50 g M Quin-2-AM successfully protected against A23187 and Glu-induced motorneuron degeneration [11]. With the limited loading of exogenous buffers tolerated by the cultured hippocampal neurons, the lack of effect upon Glu-induced increases in [Ca2+]i would be expected. I would like to thank Dr. Steven M. Rothman for use of the photometer-based calcium ratioing system in his laboratory and Ms. Marta Fournier and Nancy Lancaster for preparation of the cultures. Support for these experiments was provided by NIH Grants NS19988 to S.M. Rothman and AG10034 to J.M.D. 1 Adler, E.M., Augustine, G.J., Duffy, S.N. and Charlton, M.P., Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse, J. Neurosci., 11 (1991) 1496-1507. 2 Choi, D.W., Ionic dependence of glutamate neurotoxicity, J. Neurosci., 7 (1987) 369-379. 3 Choi, D.W., Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage, Trends Neurosci., 10 (1988) 465-469. 4 Dubinsky, J.M., Intracellular calcium levels during the period of delayed excitotoxicity, J. Neurosci., in press. 5 Dubinsky, J.M. and-Rothman, S.M., Intracellular calcium concentrations during 'chemical hypoxia' and excitotoxic neuronal injury, J. Neurosci., 11 (1991) 2545-2551. 6 Garcia-Sancho, J., Pyruvate prevents the ATP depletion caused by formaldehyde or calcium-chelator esters in the human red cell, Biochim. Biophys. Acta, 813 (1985) 148-150. 7 Goodman, L.S. and Gilman, A., The Pharmacological Basis of Therapeutics, 4th edn., MacMillan, New York, 1970, 121 pp.

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