Endogenous nitric oxide blocks calcium influx induced by glutamate in neurons containing NADPH diaphorase

Neuroscience Letters, 158 (1993) 193-196

193

© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940193l$ 06.00 NSL 09724

Endogenous nitric oxide blocks calcium influx induced by glutamate in neurons containing NADPH diaphorase Junichi Ikeda, Keiko Ochiai, Ikuo Morita and Sei-itsu M u r o t a Department of Physiological Chemistry, Graduate School Tokyo Medical and Dental University, Tokyo (Japan) (Received 9 February 1993; Revised version received 6 May 1993; Accepted 6 May 1993)

Key words." Glutamate neurotoxicity; NADPH diaphorase; Cortical neuron; Nitric oxide; Calcium Change in cytosolic calcium ion level ([Ca2÷]) after glutamate exposure was evaluated using fluo-3 on rat cortical neurons. The result showed that neurons that contain nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) were capable of blocking glutamate-induced rise in [Ca2÷]. However, with the inhibitor of nitric oxide synthase, NADPH-d-positive cells lost their ability to regulate [Ca2÷], suggesting a possible role of nitric oxide in protecting this distinct class of neurons from glutamate neurotoxicity by inhibiting glutamate-induced calcium influx.

Glutamate receptor-mediated neurotoxicity contributes to the neuronal loss seen in several acute neurological diseases including stroke, epilepsy, and traumatic head injury and in chronic neurodegenerative diseases as well [5]. The precise mechanisms by which the activation of the glutamate receptor can lead to neuronal cell injury are largely unknown; however, calcium influx through the N M D A subtype of receptor and a subsequent sustained elevation of the intracellular calcium level have been suggested to trigger the catastrophic cell degeneration [14]. Recent purification, cloning of the cDNA, and expression of nitric oxide synthase (NOS) has revealed that one of these enzymes contained in neurons catalyzes the synthesis of nitric oxide (NO) in response to an increase in [Ca z+] [3]. NO, which was first identified as the active principle of endothelium-derived relaxing factors, is known to be synthesized in neuronal cells [4]. Besides its physiological role, NO has been implicated to modulate neuronal cell injury [9, 13]. For example, Koh et al. reported that NADPH-d-containing cells are spared in N M D A neurotoxicity [10]. NADPH-d and NOS are known to be identical in brain and peripheral nervous tissue [8]. These facts raise the possibility of modulatory effect of NO on calcium influx through the NMDA receptor. The findings that NO-producing Correspondence address: J. Ikeda, Department of Physiological Chemistry, Graduate School Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan. Fax: (81) (3) 5684-3929.

agents block the NMDA-induced increase in [Ca 2+] [12] support this possibility. In the present study, we examined the [Ca 2+] of cortical neurons in vitro to test the hypothesis that NADPH-d-positive neurons can modulate glutamate-induced calcium influx by releasing their endogenous NO. Cortical cell cultures were prepared based on the methods described by Banker and Cowan [2] with slight modifications. After removal of the meninges, the cortices were dissected and incubated with trypsin (0.25%) and DNase (0.02%) at 37°C for 15 min. The cells were then washed in Dulbeccos's modified Eagle's medium (DMEM) supplemented with 10% FBS and seeded onto 8 to 10 polylysine-coated cover slips that had been placed on a cortical glial monolayer (5 x 106/ml). After 4 days in vitro, non-neuronal cell division was halted by 3-days' exposure to 5 x 10 -6 M cytosine arabinoside. Cortical neurons grown on coverslips for 10-12 days (Fig. 1A) were loaded with 5 p M fluo-3/AM (Wako Chemical) for 30 min, rinsed, and allowed to sit for 15 min in DMEM. In some experiments, cells were preincubated with an inhibitor of NOS, L-N-monomethyl-Larginine (L-NMMA, Boehringer Mannheim) for 30 min. The coverslips were then mounted in a chamber and placed on the scanning stage of an ACAS 570 interactive laser cytometer (Merdian Instruments, Inc, Okemos, MI) equipped with a 5 W argon laser, an inverted phasecontrast microscope, and a 16-bit microcomputer. The chamber was continuously superfused with a gas mixture of 95% air plus 5% C02, and the temperature was kept

194 A

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Fig. 1. A: phase-contrastmicrographof neurons on a polylysine-coated coverglass that had been placed on an astroglial monolayer.Cells were cultured for 12 days. B: a magnified phase-contrast micrograph after incubation with NBT and NADPH showing a NADPH-d-positivecell.

between 36.5°C and 37.5°C. With the phase-contrast optics of the ACAS, the area containing 40-50 cells was selected from one cover slip. Fluorescence intensity of fluo-3 was measured at 530 nm with excitation at 488 nm. After a base line image had been taken, the medium on the cover slip was aspirated and 50/11 of 100 ~tM glutamate (in D M E M , pH 7.4) was applied to it for 5 min, followed by 2 washes with D M E M . Images were taken every 2 min for the first 8 min and every 4 min thereafter. After the calcium images had been taken, the coverslip was once washed with PBS, followed by fixation of the cells with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 20 min. For demonstration of the N A D P H - d reaction, the coverslip was incubated in PBS containing 0.3% Triton X-100, 0.1 mg/ml nitroblue tetrazolium, and 1.0 mg/ml fl-NADPH (all from Boehringer Mannheim) at 37°C for 30 min [18]. Cells having N A D P H - d activity were stained black when viewed by a phase-contrast microscopy (Fig. 1B). The time course of the fluorescence intensity of fluo-3 was compared among one N A D P H - d positive and four negative neurons randomly picked in each experiment. Neurons were identified as phase-bright cells with one to three long, tapering processes. For estimation of [Ca2+],

the base line value (F0) was subtracted from each value of the experimental record to produce the fluorescence difference value (AF). Finally, the difference, AF was normalized to P0 and converted to a percentage (%AF/FO) [7]. The magnitude of the [Ca 2+] response to glutamate was evaluated by the area under the [Ca:*] response curve and the peak height of the response. Finally, the data were expressed as a percentage of the control and analysis of variance followed by unpaired t-tests were used for statistical comparisons. Virtually all neurons showed considerable increases in fluorescence intensity when they were challenged with 100/IM glutamate. In many cases, the increased intensity declined prior to the termination of the 5-min exposure to glutamate towards the base-line level, a finding consistent with previous reports [14]; however, after washing out of the glutamate, the level of [Ca 2+] was sustained at one higher than the basal. We also observed that the [Ca 2+] remained elevated and did not decline from the initial increase when the pH and temperature were not controlled (data not shown). This result is in agreement with the report indicating that acidosis reduces glutamate neurotoxicity by inhibiting the accumulation of calcium [16]. Thirteen NADPH-d-positive cells were evaluated in total (n = 7 without L-NMMA, n -- 6 with L-NMMA). In the absence of the inhibitor, the [Ca R+] response of NADPH-d-positive cells to glutamate varied between each cell. Namely, some cells showed a transient response to glutamate followed by a decline of level to below or nearly 0% after several minutes, which level remained low thereafter. In some cells, we did not observe any response to glutamate, although we might have missed an earlier transient peak that might have occurred immediately after the glutamate exposure. In general, however, the response to glutamate was smaller in NADPH-d-positive cells than in NADPH-d-negative ones (Fig. 2A-C). To determine if the lower response to glutamate in the NADPH-d-positive neurons is due to endogenous NO, we evaluated the [Ca 2+] in the presence of a NOS inhibitor, c-NMMA. The inhibitor appeared to abolish the ability of these neurons to inhibit glutamate-induced calcium influx, suggesting the involvement of endogenous NO in this phenomenon (Fig. 3A,B). However, we cannot exclude the possible involvement of other factors that inhibit the rise in [Ca 2+] in response to glutamate, because two cells still showed a small [Ca 2+] response even in the presence of D N M M A (data not shown). In a recent study, NADPH-d and NOS were shown to be identical in brain and peripheral tissues [8]. Regarding the involvement of NO in NMDA-induced neurotoxicity, Dawson et al. were the first to report that N M D A

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as nitroglycerin and sodium nitroprusside ameliorate neuronal injury by inhibiting the calcium response mediated by the N M D A subtype of the glutamate receptor [11]. It has been already demonstrated that oxidation of the N M D A receptor attenuates N M D A receptor-mediated neurotoxicity [1, 15]. Therefore, Lei et al. suggested that nitric oxide, an unstable free radical, might oxidize a redox modulating site of the receptor, resulting in a relatively persistent inhibition of the NMDA-evoked response [11]. To know whether it is endogenous NO that protects NADPH-d-positive neurons from the lethal increase in [Ca 2÷] after glutamate exposure, we examined their response to glutamate in the presence of NMMA, an inhibitor of NOS. The result showed that the increase in [Ca 2÷] was of larger amplitude when they were exposed to glutamate in the presence than in the absence of the inhibitor. These data indicate that the attenuated change in [Ca 2÷] influx elicited by glutamate in NADPH-d-positive neurons is not due to a decrease in the number of N M D A receptors [17], but to the change in the activity of the receptor.

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Fig. 2. A: the time course curve of changes in intracellular calcium concentration induced by glutamate. The data are mean + S.E. of 7 NADPH-d-positive cells (solid line) and 28 NADPH-d-negative cells (dashed line). Glutamate (100 p M ) was applied for 5 min (horizontal bar). B: the response to glutamate (area under the curve) is smaller in NADPH-d-positive cells (n = 7) than in NADPH-d-negative (control) cells (n = 28). C: the peak magnitude o f the [Ca 2÷] response to glutamate is also smaller in NADPH-d-positive cells (n = 7) than in control cells (n = 28). Data are mean + S.E. **P < 0.01 vs. control (B and C).

receptor-mediated neuronal injury is blocked by nitroarginine, a selective inhibitor of NOS, and by arginine depletion from the incubation medium [6]. In view of these data, they concluded that NO mediated glutamate neurotoxicity. They also claimed that the source of the NO was NADPH-d-positive cells. In contrast, other investigators have reported that NO may be an inhibitory factor in glutamate neurotoxicity [12]. For example, Lei et al. reported that nitric oxide-synthesizing agents such

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Fig. 3. In the presence of L-NMMA (10/2M), the response to glutamate (A: area under the time course curve of [Ca2+], B: the peak height) of NADPH-d-positive cells (n = 6) is not significantly different from that of the control (NADPH-d-negative) cells (n = 24).

196 In s u m m a r y , to the e x t e n t t h a t excessive l o a d i n g o f c a l c i u m is a final c o m m o n

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findings d e m o n s t r a t e a p r o t e c t i v e effect o f N O o n g l u t a m a t e n e u r o t o x i c i t y , f u r t h e r p r o v i d i n g a p o s s i b l e clinical a p p r o a c h t o w a r d several n e u r o l o g i c a l diseases. 1 Aizenman, E., Lipton, S.A. and Loring, R.H., Selective modulation of NMDA responses by reduction and oxidation, Neuron, 2 (1989) 1257-1263. 2 Banker, G.A. and Cowan, W.M., Rat hippocampal neurons in dispersed cell culture, Brain Res., 126 (1977) 397425. 3 Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed, R.R. and Snyder, S.H., Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase, Nature, 351 (1991) 714-718. 4 Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770. 5 Cboi, D.W., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1 (1988) 623 634. 6 Dawson, V.L., Dawson, T.M., London, E.D., Bredt, D.S. and Snyder, S.H., Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures, Proc. Natl. Acad. Sci. USA, 88 (1991) 63686371. 7 Finkbeiner, S., Calcium waves in astrocytes: filling in the gaps, Neuron, 8 (1992) 1101-1108. 8 Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811-2814. 9 Izumi, Y., Benz, A.M., Clifford, D.B. and Zorumski, C.F., Nitric oxide inhibitors attenuate N-methyl-D-aspartate excitotoxicity in rat hippocampal slices, Neurosci. Lett., 135 (1992) 227-230.

10 Koh, J.-Y., Peters, S. and Choi, D.W., Neurons containing NADPH-diaphorase are selectively resistant to quinolinate toxicity, Science, 234 (1986) 73 76. 11 Lei, S.Z., Pan, Z.-H., Aggarwal, S.K., Chen, H.-S.V., Hartman, J., Sucher, N.J. and Lipton, S.A., Effect of nitric oxide production on the redox modulatory site of the NMDA receptor-channel complex, Neuron, 8 (1992) 1087-1099. 12 Manzoni, O., Prezeau, L., Marin, P., Deshager, S., Bockaert, J. and Fagni, L., Nitric oxide-induced blockade of NMDA receptors, Neuron, 8 (1992) 653-662. 13 Nowicki, J.P., Duval, D., Poignet, D.H. and Scatton, B., Nitric oxide mediates neuronal death after cerebral iscbemia in the mouse, Eur. J. Pharmacol., 204 (1991) 339-340. 14 Randall, R.D. and Thayer, S.A., Glutamate-induced calcium transient triggers delayed calcium overload and neurotoxicity in rat hippocampal neurons, J. Neurosci., 12 (1992) 1882 1895. 15 Sucher, N.J. and Lipton, S.A., Redox modulatory site of the NMDA receptor-channel complex:regulation by oxidized glutathione, J. Neurosci. Res., 30 (1991) 582 591. 16 Takadera, T., Shimada, Y. and Mohri, T., Extracellular pH modulates N-methyl-D-aspartate receptor-mediated neurotoxicity and calcium accumulation in rat cortical cultures, Brain Res., 572 (1992) 126 131. 17 Uemura, Y., Kowall, N.W. and Beal, M.F., Selective sparing of NADPH-diaphorase-somatostatin-neuropeptide Y neurons in ischemic gerbil striatum, Ann. Neurol., 27 (1990) 620-625. 18 Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755-784.