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Research Report

Antihistamine terfenadine potentiates NMDA receptor-mediated

calcium influx, oxygen radical formation, and neuronal death

a a,b , c d

´

*

´

Ramon Diaz-Trelles , Antonello Novelli

, Jose A. Vega , Ann Marini ,

´

M. Teresa Fernandez-Sanchez

Department of Biochemistry and Molecular Biology, School of Medicine, University of Oviedo, 33071 Oviedo, Spain

Department of Psychology, Faculty of Psycology, University of Oviedo, 33071 Oviedo, Spain

Department of Morphology and Cellular Biology, Faculty of Medicine, University of Oviedo, 33071 Oviedo, Spain

Department of Neurology. Uniformed Services University of the Health Sciences. 4301 Jones Bridge Road, Bethesda, MD 20814, USA Accepted 27 June 2000

Abstract

We previously reported that the histamine H1 receptor antagonist terfenadine enhances the excitotoxic response to N-methyl-D-aspartate

(NMDA) receptor agonists in cerebellar neurons. Here we investigated whether this unexpected action of terfenadine relates to its antihistamine activity, and which specific events in the signal cascade coupled to NMDA receptors are affected by terfenadine. Low concentrations of NMDA (100mM) or glutamate (15mM) that were only slightly (,20%) toxic when added alone, caused extensive cell death in cultures pre-exposed to terfenadine (5mM) for 5 h. Terfenadine potentiation of NMDA receptor response was mimicked by other H1 antagonists, including chlorpheniramine (25mM), oxatomide (20 mM), and triprolidine (50 mM), was prevented by histamine (1 mM), and did not require RNA synthesis. Terfenadine increased NMDA-mediated intracellular calcium and cGMP synthesis by approximately 2.4 and 4 fold respectively. NMDA receptor-induced cell death in terfenadine-treated neurons was associated with a massive production of hydrogen peroxides, and was significantly inhibited by the application of either (1)-alpha-tocopherol (200mM) or the endogenous antioxidant melatonin (200mM) 15 min before or up to 30 min after receptor stimulation. This operational time window suggests that an enduring production of reactive oxygen species is critical for terfenadine-induced NMDA receptor-mediated neurodegeneration, and strengthens the importance of antioxidants for the treatment of excitotoxic injury. Our results also provide direct evidence for antihistamine drugs enhancing the transduction signaling activated by NMDA receptors in cerebellar neurons.  2000 Elsevier Science B.V. All rights reserved.

Theme: Neurotransmitters, modulators, transporters and receptors

Topic: Interactions between neurotransmitters

Keywords: Terfenadine; excitatory aminoacid receptors; histamine; reactive oxygen species; intracellular calcium; cultured cerebellar neurons.

1. Introduction explored. Among the antihistamines that penetrate poorly into the central nervous system (CNS), terfenadine is Several lines of evidence indicate that histaminergic widely used as a prototype of non-sedating histamine H1 fibers could have a role in the modulation of the neuro- receptor antagonist. Although terfenadine is generally transmission mediated by glutamate [3,38], and a role for devoid of CNS depressant activity, reported adverse effects histamine in NMDA-mediated excitotoxic death has been include sedation, drowsiness, fatigue and weakness [30], reported in a rat model of Wernicke’s encephalopathy [24]; suggesting that therapeutic concentrations of this drug may however, the effects of antihistamine compounds on also act at the CNS. In addition to its antihistamine neuronal survival and function have been much less activity, terfenadine has been shown to possess a complex pharmacological profile that includes the ability to block voltage dependent ion channels [11,26,42], and to reverse

*Corresponding author. Tel.: 134-985-104-210; fax: 1

34-985-103-drug resistance in a variety of cell types via its interaction

157.

E-mail address: [email protected] (A. Novelli). with P glycoprotein [45]. Very limited data exist about the

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effects of this drug on glutamate mediated neurotrans- medium with glucose every 4 days and compensating for

mission and excitotoxicity. lost amounts of water, due to evaporation.

Cerebellar granule neurons in culture represent a

neuro-nal model that has proved very useful in the study of the 2.2. Neuronal treatment and survival biochemical events coupled to excitatory amino acid

neurotransmission, and the conditions controlling ex- Neurons were used between 14 and 20 days in culture. citotoxicity [29,32–35]. They express ionotropic NMDA As a general rule, drugs were dissolved in distilled water glutamate receptors coupled with the synthesis of cGMP and pH was adjusted at approximately 7.4. Antihistamines [32], as well as H1 histamine receptors [20], which are were dissolved in ethanol and acidulated (5 mM HCl) associated with inositol phospholipid hydrolysis and cal- water (1:1) and diluted at least 200 times in the culture cium mobilization from intracellular stores [19]. We have medium. Vehicle controls were conducted containing the recently reported [10] that terfenadine significantly poten- same amount of ethanol and HCl. All drugs were added to tiates the excitotoxic response elicited by NMDA in the growth medium for the indicated times, after which cultured cerebellar neurons. The aim of the present study growth medium was removed and cultures were incubated was to extend these observations, specifically investigating for 5 min. with 1 ml incubation buffer containing 154 mM (1) whether the potentiating effects of terfenadine relate to NaCl, 5.6 mM glucose, 8.6 mM HEPES, 1 mM MgCl , 2.32

its antihistamine activity; for this purpose we tested other mM CaCl , pH 7.4, to which the vital stain fluorescein2

H1 histamine antagonists and performed competition diacetate (5mg / ml) was added. The staining mixture was studies using different concentrations of histamine; (2) then aspirated, replaced with incubation buffer, and cul-which specific events of the cascades triggered by NMDA tures were examined for neurotoxicity. Under fluorescent receptor agonists in cultured cerebellar neurons are modu- light, live neurons showed a bright green color in the cell lated by terfenadine and leading to excitotoxicity. In body and neurites, while dead neurons did not retain any particular we focused on the NMDA-mediated influx of fluorescein diacetate, and their nuclei could be stained in calcium, considered to be the key initial step in the red by 1 min exposure to 50 mg / ml ethidium bromide. biochemical processes following activation of NMDA Photographs of three randomly selected culture fields were receptors and evoking both NMDA receptor-mediated taken, and live and dead neurons were counted. Total neuronal plasticity and neurotoxicity [5,8]. We also ex- number of neurons per dish was calculated considering the amined the formation of oxygen radicals, as glutamate ratio between the area of the dish and the area of the exposure has been reported to be associated with an picture (|3000).

enhanced generation of reactive oxygen species [7,23]. The

role of neosynthesized RNA in the potentiating effects of 2.3. cGMP determination NMDA excitotoxicity induced by terfenadine in cultured

cerebellar granule neurons has been also investigated. Intracellular cGMP concentration was determined as previously reported [35] with minor modifications. Briefly, glutamate receptor agonists were added to the culture medium for 1 min. Terfenadine was added 5 h before 2. Materials and Methods agonists. MK-801 was added 1 min before agonists. Incubation was stopped by aspiration of the growth

2.1. Cell cultures medium and addition of 1 ml HClO4 (0.4 N). After

neutralizing the perchlorate extract, cGMP content was Primary cultures of rat cerebellar neurons were prepared determined by radioimmunoassay. Protein content was as previously described [34]. Briefly, cerebella from 8-day- determined on the membrane pellet from the same sample. old pups were dissected, cells were dissociated and

sus-pended in basal Eagle’s medium with 25 mM KCl, 2 mM 2.4. Confocal microscopy glutamine, 100 mg / ml gentamycin and 10% fetal calf

serum. Cells were seeded in poly-L-Lysine coated (5 mg / For intracellular calcium determination, neuronal

cul-5 2

ml) 35 mm dishes at 2.5310 cells / cm and incubated at tures were loaded for 20–30 min with 5 mM Fluo-3-AM 378C in a 5% CO2, 95% humidity, atmosphere. Cytosine ester in an incubation buffer containing (in mM): 154 arabinoside (10mM) was added after 20–24 h of culture to NaCl, 5.6 KCl, 5.6 glucose, 8.6 HEPES, 1 MgCl2, 2.3 inhibit the replication of non-neuronal cells. After 8 days CaCl2, pH 7.4. At the moment of the record the dye was in vitro, morphologically identified granule cells accounted removed and the indicated drugs were added. Fluo-3 for more than 95% of the neuronal population, the emission (.515 nm) was recorded in a Bio-Rad confocal remaining 5% being essentially GABAergic neurons. microscope with a krypton–argon laser excitation source Astrocytes did not exceed 3% of the overall number of (488 nm). Signals were digitized using Bio-Rad interface cells in culture. Cerebellar neurons were kept alive for and analyzed by NIH Image (1.61). Concentrations of

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function of Fluo-3 intensity (F ) using the calibration 5,10-imine hydrogen maleate (MK-801) was a generous procedure described previously [40], and according to the gift from Dr. G.J. Kaczarowski (Merck Sharp and Dohme

following equation: Laboratories, NJ, USA). Fluo-3 AM (F1241) and

carboxy-21

[Ca ]i5Kd (F 2Fmin) /(Fmax2 F ) where the dis- 29,79-dichlorodihydrofluoresceine diacetate (carboxy-sociation constant K has been estimated at 400 nM ford H DCFDA, C-400) were from Molecular Probes. All other2

Fluo-3 at vertebrate ionic strength [21], and the mean chemicals were from Sigma. values obtained for the camera signal Fmin and for the

maximum fluorescence Fmax were 6 and 230 respectively.

Oxygen radical formation was detected with carboxy- 3. Results 29,79-dichlorodihydrofluoresceine diacetate

(carboxy-H DCFDA). Following uptake, the carboxy-(carboxy-H DCFDA is₂ ₂ 3.1. Potentiation by terfenadine of NMDA receptor-converted by endogenous esterases to carboxy-H DCF,₂ mediated neuronal death is prevented by histamine, does which upon exposure to hydroperoxides is oxidized to the not require RNA synthesis, and is mimicked by other fluorescent probe carboxy-DCF. Neuronal cultures were histamine H1-receptor antagonists.

treated with the indicated drugs and loaded with 20 mM

carboxy-H DCFDA in the culture medium for 1 h, after2 We first investigated whether the effects of terfenadine which the dye was removed and cultures were washed on NMDA receptor mediated neurotoxicity were related to twice with a buffer containing (in mM): 154 NaCl, 5.6 its antihistamine activity. As previously reported [10] and KCl, 5.6 glucose, 8.6 HEPES, 1 MgCl2, 2.3 CaCl2, pH shown in Table 1, preincubation of neurons with ter-7.4. Carboxy-DCF fluorescence was recorded in a Bio-Rad fenadine (5 mM, 5 h) significantly potentiated neuro-confocal microscope with a krypton–argon laser excitation toxicity by 100mM NMDA, concentration which was not source (488 nm), and signals were digitized using Bio-Rad toxic when NMDA was added alone. Furthermore,

ter-interface and analyzed by NIH Image (1.61). fenadine reduced by approximately 75% the number of

surviving neurons after 12 h exposure to subtoxic

con-2.5. Inmunohistochemistry centrations of glutamate (15 mM) compared to cultures

exposed to glutamate alone (Table 1). To test the time To ascertain the presence of mast cells in the culture, an dependence of exposure to terfenadine, cultures were immunohistochemical method was used. Cultures were exposed to terfenadine for 15 min, 1 h, 3 h and 4 h prior to fixed in 4% formaldehyde and washed in 0.05 M TBS (pH application of 100 mM NMDA or 15mM glutamate. The 7.5) containing 0.1% bovine serum albumin, 0.2% fetal number of surviving neurons was significantly reduced calf serum, and 0.2% Triton-X. Samples were incubated (56%) in cultures pretreated with terfenadine for 4 h but overnight at 48C with a rabbit anti-histamine polyclonal not in cultures pretreated for shorter times (data not antibody (Chemicon, Temacula, CA, USA) diluted 1:100.

Table 1

Then, dishes were rinsed in the same buffer, and incubated

Potentiation by terfenadine of NMDA receptor-induced neurotoxicity is

with peroxidase-labelled anti-rabbit IgG (Amersham, a

prevented by histamine

Buckinganshire, UK) diluted 1:100, for 1 h at room

Treatment Neuronal Survival (%)

temperature. Finally, after washing, dishes were incubated

for 3 min in 3,39diaminobenzidine (0.5 mg / ml in TBS 0.2 2HIS 1HIS

M, pH 7.5) containing 0.01% H O . Number of histamine-2 2 _None _TEF _None _TEF

positive cells, morphologically identified as mast cells

2 None 8569 8168 n.d. 8462

[39], was estimated to be about 40–80 mast cells / cm . b d

GLU 81615 1963 7765 6069

MK-8011 GLU 8467 6066 n.d. n.d.

b d

2.6. Data presentation and analysis NMDA 80612 51610 8264 8066

MK-8011NMDA 8565 7565 n.d n.d

Results are presented as indicated in each figure. For Cerebellar neurons in primary culture were used at 14–18 days in

statistical analysis the one-way or the two-way analysis of culture and exposed to vehicle (None) or to the indicated drugs. Terfenadine (TEF, 5mM) was added 5 h before glutamate (GLU, 15mM)

variance (ANOVA) was used to identify overall treatment

or N-methyl-D-aspartate (NMDA, 100mM). Histamine (HIS, 5 mM) was

effects, followed by the unpaired two-tailed Student’s t-test

added 15 min before TEF. NMDA receptor antagonist MK-801 (1mM)

for selective comparison of individual data groups. Only _{was added 2 min before agonists. Neuronal viability was determined 12 h} significances relevant for the discussion of the data are after application of GLU or NMDA by staining of cultures with

indicated in each figure. fluorescein diacetate and ethidium bromide. Values are expressed as

percentage of live neurons and are from three independent experiments. Statistical significance was determined by two-way analysis of variance

2.6.1. Materials

(ANOVA) and two-tailed unpaired Student’s t-test.

Terfenadine and oxatomide were provided by Dr. J.R. b

P,0.01 vs. None in the same line in the absence of HIS.

Fernandez (Astur-Pharma, Llanera, Asturias, Spain). (1)- P,0.01 vs. the same treatment in the absence of MK-801.

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shown). NMDA- and glutamate-induced excitotoxicity in in the effects of terfenadine and for that purpose, we tested terfenadine-treated cultures were confirmed to be spe- the effect of terfenadine on NMDA receptor-mediated cifically due to NMDA receptor stimulation by using the excitotoxicity in the presence of actinomycin D. We found NMDA receptor antagonist (1)-10,11-dihydro-5-methyl- no significant differences due to the presence of ac-5H-dibenzo-[a,d]-cyclohepten-5,10-imine hydrogen tinomycin D. Thus, exposure of cultures to terfenadine (5 maleate (MK-801), and in both cases neuronal death could mM, 4 h) and then to NMDA (100mM) induced after 24 h be effectively prevented by the application of high con- a dramatic reduction in the number of surviving neurons centrations of histamine (5 mM) (Table 1). To further from 9761% to 1065% and to 1167% live neurons in the check the nature of this antagonism we constructed dose- absence and in the presence of actinomycin D respectively. response experiments using concentrations of histamine Exposure of neurons to terfenadine alone had no effect on from 500mM to 10 mM. As shown in Fig. 1, the ability of cell survival, and the concentration of NMDA (100 mM) terfenadine (5mM) to potentiate excitotoxicity by subtoxic used in these experiments induced very little toxicity (from concentrations of glutamate (15 mM) was prevented by 9761% to 7165% live neurons) when added alone. histamine in a concentration dependent manner. Although To exclude the possibility that potentiation of excitotox-a significexcitotox-ant increexcitotox-ase in neuronexcitotox-al survivexcitotox-al wexcitotox-as excitotox-alreexcitotox-ady icity by NMDA receptor agonists could be just a ter-observed at 500mM histamine, a concentration of at least fenadine-related effect, we tested the ability of other 1 mM histamine was required to achieve maximum histamine H1 receptor antagonists to increase the

ex-protection. citotoxic response to glutamate in cerebellar granule

The long exposures necessary to potentiate the ex- neurons. Pretreatment of cultures for 5 h with (6 )-chlor-citotoxic response to NMDA led us to consider the pheniramine (25mM) or oxatomide (20mM), significantly possibility that newly synthesized proteins were involved increased cell death in cultures exposed to subtoxic concentrations (15 mM) of glutamate (Fig. 2). A smaller but still significant potentiation of the excitotoxic response to glutamate was also observed for trans-triprolidine (50

mM) using the same exposure conditions.

3.2. Terfenadine increases the intracellular calcium response induced by NMDA

Calcium imaging experiments showed that in cultured cerebellar neurons stimulation of NMDA receptors elicits

Fig. 1. Potentiation by terfenadine of NMDA receptor-mediated ex-citotoxicity is prevented by histamine in a concentration-dependent manner. Neurons were exposed to 5mM terfenadine (TEF) for 5 h and then treated with a subtoxic concentration (15mM) of glutamate (GLU) in the absence (inset figure and first point of the curve) or in the presence of the indicated concentrations of histamine. Histamine was added 10 min

before TEF. Neuronal survival was determined 24 h after the addition of Fig. 2. Histamine H1 receptor antagonists potentiate NMDA receptor-GLU by staining live neurons with fluorescein diacetate (5 min) and dead mediated excitotoxicity. Neuronal survival after 24 h exposure to 15mM neurons nuclei with ethidium bromide (1 min), and compared with glutamate (GLU) was determined in vehicle-pretreated neurons (None) survival of neurons in sister cultures treated with vehicle (None) or with and in neurons pretreated for 5 h with 20mM oxatomide (OXA), 25mM TEF or GLU alone (see inset figure). Data represent the mean6S.D. of chlorpheniramine (CHP) or 50mM trans-triprolidine (TRP). Represented triplicate values from three separate experiments. *P,0.01 vs. 0 mM values are the mean6S.D. (n56). *P,0.05 vs. None within the same histamine; **P,0.001 vs. TEF or GLU. treatment.

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an intracellular calcium signal that can be significantly NMDA resulted in a rapid and a strong rise in fluorescence increased by terfenadine (Fig. 3). Thus, application of 25 intensity in most cell bodies (Figs. 3C and D), indicating a

mM NMDA induced a rather small increase in cellular large influx of extracellular calcium into cells. Quantifica-fluorescence intensity (Figs. 3A and B), consistent with a tion of the fluorescence intensity in about 20–30 neurons limited influx of calcium into neurons, while stimulation of per field revealed an NMDA-induced mean increase from terfenadine-treated neurons with this low concentration of 55618 to 127616 in untreated and from 62616 to

Fig. 3. Terfenadine increases intracellular calcium upon stimulation of NMDA receptors. Neuronal cultures were loaded for 20–30 min with 5mM Fluo-3-AM ester and then examined under a laser confocal microscope. (A–D) Representative images taken before (A,C) or 30 s after (B,D) the addition of 100mM NMDA in the absence (A,B) or in the presence (C,D) of 5mM terfenadine (TEF) for 5 h. Scale color bar spans from black (minimum) to red (maximum). (E) Images as the ones represented in A–D were used to estimate intracellular calcium concentrations before (None) or after application of NMDA (100mM) to cultures treated with vehicle (2TEF) or with terfenadine (5mM) for 5 h (1TEF). Concentrations of intracellular calcium were calculated from the fluorescence intensity averaged for 20–30 neurons per field as previously described [40]. Intracellular calcium after application of NMDA in the presence of the NMDA receptor antagonist MK-801 is also represented. Results represent the mean6S.D. of two independent experiments (n550–60). *P,0.01 vs. NMDA in the absence of TEF.

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171618 fluorescence units (f.u.) in terfenadine-treated in the presence of terfenadine (Fig. 4D). Radical formation neurons respectively within 30 s, and in both cases it could could be specifically abolished by the NMDA receptor be abolished by MK-801 (Fig. 3 E). The increase in the antagonist MK-801 (Fig. 4E), and was not observed in intracellular concentration of calcium induced by NMDA neurons exposed to terfenadine alone (Fig. 4B), indicating was estimated [40] (see Materials and Methods) to be from that activation of NMDA receptors was necessary. Similar approximately 112634 to 470660 nM and from approxi- results were obtained in experiments in which NMDA was mately 133630 to 11206125 nM in the absence and in the used as NMDA-receptor agonist (not shown). These

find-presence of terfenadine respectively. ings suggested a key role for hydrogen peroxides as

To investigate the effect of terfenadine on calcium- mediators of the neurodegeneration coupled to overactiva-dependent signal transduction we measured the intracellu- tion of NMDA receptors in cultured cerebellar neurons. lar formation of cGMP stimulated by NMDA. As shown in Indeed, stimulation of neurons with 50 mM glutamate or Table 2, preincubation of neurons with terfenadine (5mM, 500mM NMDA induced extensive formation of hydrogen 4 h) resulted in an approximately 4 fold increase in cGMP peroxides and resulted in over 80% neurotoxicity, due to formation induced by the selective stimulation of NMDA excessive stimulation of NMDA receptors as it could be receptors with 1 mM NMDA, and also increased by completely abolished by MK-801 (data not shown). approximately 2 fold the cGMP formation induced by 100

mM NMDA. The effect was specific for NMDA receptors 3.4. (1)-alpha-tocopherol and melatonin protect from in that it was specifically blocked by the NMDA receptor terfenadine potentiation of NMDA toxicity

antagonist MK-801, and that terfenadine had no effect on

cGMP formation stimulated by the non-NMDA receptor The role of oxygen radical formation in the pathway agonists AMPA or domoic acid (data not shown). mediating toxicity by NMDA receptors in the presence of terfenadine was further evaluated by testing the protective 3.3. Terfenadine potentiates the formation of oxygen effects of two different antioxidant compounds: the pineal radicals following stimulation of NMDA receptors secretory product melatonin, and (1)-alpha-tocopherol (vitamin E). In particular, we investigated whether To test for the participation of oxidative stress in the melatonin and (1)-alpha-tocopherol were able to prevent potentiation by terfenadine of NMDA receptor-mediated cell death produced by low concentrations of NMDA in the response we used the specific probe dichlorodihydrofl- presence of terfenadine, and whether addition of these uorescein diacetate (H DCFDA) to measure the generation2 antioxidant drugs after the excitotoxic insult could rescue of reactive oxygen species, and in particular hydrogen neurons. Both melatonin (200 mM) and (1 )-alpha-peroxides, in cultures exposed to subtoxic concentrations tocopherol (200mM) markedly inhibited NMDA-mediated (15 mM) of glutamate both in the presence and in the cell death in terfenadine treated neurons when added 15 absence of terfenadine. As shown in Fig. 4, stimulation of min prior to NMDA application (Fig. 5A), reducing neurons with this low concentration of glutamate did not neuronal death by approximately 70%. To determine result in a significant generation of hydrogen peroxides whether addition of these drugs after initiation of the (Fig. 4C) compared to unstimulated cultures (Fig. 4A). In excitotoxic insult exerted neuroprotective effects, we per-contrast, a massive formation of oxygen radicals occurred formed experiments in which melatonin or (1 )-alpha-in cultures exposed to this low concentration of glutamate tocopherol were added 0, 5, 15, and 30 min following stimulation with NMDA. As shown in Fig. 5B, addition of melatonin 0, 10 or 15 min after exposure to 100 mM

Table 2

Terfenadine potentiates cGMP formation induced by NMDA NMDA reduced neuronal death significantly, while no protection was observed in cultures to which melatonin

Treatment cGMP (pmol / mg protein)

had been added 30 min after NMDA. As for (1

)-alpha-None TEF

tocopherol, this compound showed a greater effectiveness,

Unstimulated 0.5860.3 0.5160.1 _{and significant protection from NMDA in terfenadine}

b b,c

NMDA (100mM) 3.360.3 6.1660.2

treated neurons was observed in cultures to which (1

)-b b,c

NMDA (1 mM) 1762 7068

alpha-tocopherol was added up to 30 min after NMDA

Cerebellar neurons in primary culture were used at 12–14 days in _{(Fig. 5B).} culture and vehicle (None) or the indicated drugs were added to the

growth medium. Terfenadine (TEF, 5mM) was added 5 h before NMDA. After 1 min exposure to NMDA culture medium was aspirated and 1 ml

HCLO (0.4 N) was added. The perchlorate extract was neutralized with4 4. Discussion NaOH and the cGMP content was determined by radioimmunoassay.

Data are from triplicate values from two independent experiments. _{In the present study we have investigated the} potentia-Statistical significance was determined by two-way analysis of variance

tion by terfenadine of the excitotoxic response to NMDA

(ANOVA) and two-tailed Student’s t-test.

b _{receptor agonists in cultured cerebellar neurons. We found}

P,0.01 vs. unstimulated in the same column.

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Fig. 4. Terfenadine enhances the formation of hydrogen peroxides induced by stimulation of NMDA receptors. Neuronal cultures untreated (A) or exposed to 5mM terfenadine (B), glutamate (C), and to terfenadine plus glutamate in the absence (D) or in the presence of MK-801 (E), were loaded with 20mM carboxy-H DCFDA in the culture medium for 1 h. Cultures were washed twice with a buffer containing (in mM): 154 NaCl, 5.6 KCl, 5.6 glucose, 8.62

HEPES, 1 MgCl2, 2.3 CaCl2, pH 7.4, and the carboxy-DCF fluorescence was recorded in a Bio-Rad confocal microscope with a kryptonc–argon laser excitation source (488 nm). Signals were digitized using Bio-Rad interface and analyzed by NIH Image (1.61). Scale color spans from white (minimum) to black (maximum). Terfenadine was added 5 h before glutamate and carboxy-H DCFDA was added 30 min after glutamate.2

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Fig. 5. Antioxidants melatonin and (1)-alpha-tocopherol prevent potentiation by terfenadine of NMDA excitotoxicity. Neurons at 14–17 DIC were either untreated (None) or exposed to the indicated drugs in the growth medium, and neuronal survival was determined 24 h later by staining of live neurons with fluorescein diacetate and dead neurons nuclei with ethidium bromide, and observation of cultures under a fluorescence light. Terfenadine (TEF) was added 5 h before NMDA. Melatonin (MEL) and (1)-alpha-tocopherol (a-TCF) were added 10 min before TEF (Panel A) or at the indicated times after application of NMDA (Panel B). Concentrations were: TEF, 5mM; NMDA, 100mM; MEL, 200mM;a-TCF, 200mM. Data represent the mean6S.D. of duplicate values from four independent experiments. *P,0.001 vs. NMDA in panel A and *P,0.01 vs. TEF1NMDA in panel B.

neurotoxicity appear to be related to its anti-histamine Interestingly, constitutive activity of wild-type human H1 activity for they can be effectively antagonized by high receptors and inverse agonistic activity of several his-concentrations of histamine in the growth medium (see tamine H1 antagonists has been recently reported [2]. Table 1) and mimicked by other histamine H1 antagonists Although taken together our findings suggest the specific (Fig. 2). These observations support a specific relationship involvement of histamine H1 receptors, non-selective between terfenadine activity as an histamine receptor cross-reactivity with other histamine receptor subtypes antagonist and the effects of this drug on excitotoxicity. An cannot be excluded completely given the relatively high aggravation of ischemic neuronal damage in the rat concentrations of terfenadine, as well as of other histamine hippocampus has been described in rats in which his- H1 receptor antagonists, needed to achieve potentiation of taminergic neurotransmission was impaired with the his- NMDA excitotoxicity.

tidine decarboxylase inhibitor alpha-fluoromethylhistidine Our results show that the increase in toxicity induced by [1]. Our results would be consistent with the hypothesis terfenadine in neurons stimulated with subtoxic concen-that terfenadine inhibited a tonic stimulation of H1 re- trations of NMDA or glutamate is associated with a greater ceptors. Approximately 50% of the histamine content of (about 2.5 fold increase) influx of extracellular calcium rat brain may derive from mast cells [43], and the presence into neurons (Fig. 3 and data not shown). The inhibitory in the cerebellum of meninges, difficult to remove com- effect of MK-801 indicates that calcium entry occurs via pletely, may allow for a certain amount of these cells in the NMDA receptor channels (Fig. 3). Although the mecha-cultures. By inmunohistochemistry experiments using a nisms underlying terfenadine potentiation of calcium influx polyclonal antihistamine antibody we have estimated the upon stimulation of NMDA receptors remain to be estab-extent of mast cell contamination in our cultures to be lished, the observation that it required at least 4 h exposure about 1–2 mast cell / 2500 neurons (see section 2.5 of of neurons to terfenadine before NMDA application (data Materials and Methods), while cerebellar neurons were not shown) suggests an intracellular regulation of NMDA histamine-negative (not shown). So, it cannot be excluded channels permeability rather than an extracellular inter-that the presence of this reduced number of mast cells may action of terfenadine with NMDA receptors. The simplest allow for a low amount of histamine in the culture mechanistic explanation may be related to a progressive medium. Alternatively, it is also possible that by acting as depolarization consequent to terfenadine blockade of sev-an inverse agonist terfenadine may be blocking sponta- eral types of K1 currents [4], and leading to a reduction of

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excitotoxicity. Against this possibility however, it should antihistamine drug of the transduction signaling activated be considered that the concomitant block of voltage by NMDA receptors in cerebellar neurons. The increase in sensitive sodium channels by terfenadine [11,27] may slow NMDA receptor-mediated formation of oxygen radicals down such depolarization process, and allow for progres- observed in terfenadine-treated cultures (Fig. 4) further sive use-dependent inactivation of the NMDA channel by support this idea. Reactive oxygen species have been released endogenous glutamate [13,28], resulting in neuro- demonstrated to play a pivotal role in the pathogenesis of protection. Furthermore, a reversible inactivation of excitotoxic death [7,36,41], and in cultured cerebellar NMDA channels by intracellular calcium has been shown granule cells, NMDA receptor stimulation has been shown in hippocampal neurons [25,31,44] as well as in recombi- to produce marked elevations in superoxide anion and nant NMDA receptors [22]. Neither ligand binding nor hydroxy radicals due to the activation by calcium of channel opening was required to elicit this calcium-depen- phospholipase A2 and the metabolism of arachidonic acid dent inactivation [25], that could be prevented by blocking [23]. Our results further confirm the generation of oxygen voltage sensitive calcium channels (VSCC) [31]. His- radicals, and in particular hydrogen peroxides, as a key tamine H1 receptors are coupled to a guanylnucleotide- event in the occurrence of NMDA receptor-mediated sensitive G protein and the phosphoinositide hydrolysis. neurotoxicity. Indeed, formation of radicals preceded Thus, by reducing tonic stimulation or spontaneous activity neuronal death of cultures exposed to terfenadine plus of H1-receptors terfenadine may decrease cytosolic cal- glutamate (Fig. 4D) but it was not detectable in neurons cium levels due to both the impairment of the release from exposed to terfenadine alone (Fig. 4B), and only a slight intracellular stores and the inhibition of the inositol-1,4,5- formation of hydrogen peroxides was observed in cultures triphosphate (IP3)-induced calcium influx via VSCC [9]. exposed to subtoxic concentrations of glutamate (Fig. 4C). Such a decrease in unstimulated levels of cytosolic calcium It should be noted that terfenadine and glutamate alone had cannot be visualized in experiments as the one represented no significant effect on neuronal viability (see Table 1). in Fig. 3, in which neurons were maintained in a physio- Moreover, the ability of free radical scavengers such as the logic solution containing 5 mM KCl to avoid toxicity due pineal hormone melatonin and (1)-alpha-tocopherol in to the activation of NMDA receptors by endogenously protecting neurons from death by NMDA in terfenadine-released glutamate. However, a substantial decrease in treated cultures (Fig. 5) further supports a prominent role basal intracellular calcium was observed in cells pretreated for oxygen free radicals in excitotoxicity. Melatonin has with terfenadine for 5 h compared to untreated neurons been reported to be neuroprotective ‘in vivo’ against kainic when a solution containing 25 mM KCl and MK-801 was acid-induced lesions [6,18], and ‘in vitro’ from cell death used during the calcium imaging experiment (data not of cultured cerebellar neurons following exposure to kainic

shown). acid [17]. In contrast, melatonin treatment was suggested

A direct action of terfenadine on VSCC may be also to be ineffective in protecting neurons from toxicity considered. Terfenadine is structurally related to induced by exposure of neurons to NMDA [17]. A possible

diphenylalkylamine L-type Ca channel blockers, and in explanation for the discrepancy between the latter

observa-21

Ca channel affinity studies using rat cortical membranes tion and the protective effect of melatonin we describe terfenadine showed a pKd of about 6.4 [46]. An inhibition here may be that our experimental paradigm relies on of calcium currents possibly via L-type channels by neurotoxicity precipitated by enhancing the excitatory concentrations of terfenadine in the low micromolar range signal induced by a subtoxic concentration of NMDA. has been reported in ventricular myocytes [26]. Moreover, Indeed, melatonin has been shown to prevent death of we have shown that terfenadine is also effective in hippocampal neurons induced by enhanced excitatory reducing the depolarization-induced influx of calcium via neurotransmission via NMDA receptors [41]. Melatonin is VSCC in cerebellar neurons [12]. Therefore, an inhibition believed to work via electron donation to directly detoxify of VSCC currents by terfenadine could result in partial free radicals including the highly toxic hydroxyl radical recovering of NMDA receptors from inactivation as cyto- and peroxynitrite [16], and it has been also shown to solic calcium levels decrease, and lead to a higher number inhibit cerebellar NO synthase [37] and to maintain the of ‘normal’ (not inactivated) NMDA receptors on the cellular homeostasis of reduced glutathione, a key com-neuronal membrane. According to this hypothesis, the ponent of the cellular defense cascade against injury ability of histamine to antagonize the effects of terfenadine caused by reactive oxygen species [15]. Moreover, (Fig. 1) could be associated to a compensatory increase in melatonin and vitamin E inhibited NO-induced lipid the release of calcium from intracellular stores due to the peroxidation in rat brain homogenates [14]. Further studies stimulation of histamine H1 receptors. Experiments are are necessary to establish the specific contribution of these currently in progress to investigate this possibility. pathways to the neuroprotective action of melatonin and

Our observations that terfenadine significantly poten- alpha-tocopherol we report here.

tiates the NMDA-dependent synthesis of cGMP (Table 2), Cerebellar granule cells have been frequently used to which depends upon extracellular calcium influx [32,35], study the signaling pathways triggered by excitatory amino provide for the first time evidence for the modulation by an acids [29,32–35]. In the present study we have used this

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´ ´ ´

[10] R. Dıaz-Trelles, A. Novelli, M.T. Fernandez-Sanchez, Terfenadine

primary cell culture system to investigate the molecular

induces toxicity in cultured cerebellar neurons: a role for glutamate

mechanism by which the antihistamine terfenadine

poten-receptors, Amino Acids 16 (1999) 59–70.

tiates the excitotoxic response elicited by NMDA receptor _{[11] R. Dıaz-Trelles, A. Novelli, G. Puia, M. T Fernandez-Sanchez,}_´ _´ _´ agonists. We have shown that this novel action of ter- Terfenadine prevents NMDA receptor-dependent and -independent

fenadine involves an increase in the influx of calcium via toxicity following sodium channel activation, Brain Res. 842 (1999) 478–481.

NMDA receptors and the NMDA receptor mediated

in-´ ´ ´

[12] R. Dıaz-Trelles, A. Novelli, M.T. Fernandez-Sanchez, Modulation

tracellular synthesis of cGMP, and does not depend upon

of excitatory neurotransmission by terfenadine, Soc. Neurosci.

RNA synthesis. Terfenadine potentiation of NMDA re- _{Abstr. 25 (1999) 2254.}

ceptor-induced cell death was histamine sensitive, associ- _{[13] R. Dingledine, K. Borges, D. Bowie, S.F. Traynelis, The Glutamate} ated with a massive production of hydrogen peroxides, and receptor ion channels, Pharmacol. Rev. 51 (1999) 7–61.

[14] G. Escames, J.M. Guerrero, R.J. Reiter, J.J. Garcia, A.

Munoz-was prevented by the antioxidants vitamin E and

Hoyos, G.G. Ortiz, C.S. Oh, Melatonin and vitamin E limit nitric

melatonin. Our findings provide for the first time direct

oxide-induced lipid peroxidation in rat brain homogenates, Neurosci.

evidence for antihistamine drugs enhancing the transduc- _{Lett. 230 (1997) 147–150.}

tion signaling activated by NMDA receptors, and _{[15] M. Floreani, S.D. Skaper, L. Facci, M. Lipartiti, P. Giusti, Melatonin} strengthen the importance of antioxidants for the treatment maintains glutathione homeostasis in kainic acid-exposed rat brain

tissues, FASEB J. 11 (1997) 1309–1315.

of excitotoxic injury.

´ [16] E. Gilad, S. Cuzzocrea, B. Zingarelli, A.L. Salzman, C. Szabo,

Melatonin is a scavenger of peroxynitrite, Life Sci. 60 (1997) PL169–PL174.

Acknowledgements [17] P. Giusti, M. Gusella, M. Lipartiti, D. Milani, W. Zhu, S. Vicini, H. Manev, Melatonin protects primary cultures of cerebellar granule neurons from kainate but not from N-methyl-D-aspartate

excitotox-´ ´

We thank Dr. J.R. Fernandez Gonzalez for the generous

icity, Exp. Neurol. 131 (1995) 39–46.

gift of some of the drugs used in this study. We also thank

[18] P. Giusti, M. Lipartiti, D. Franceschini, N. Schiavo, M. Floreani, H.

Dr. A. Sampedro and Dr. A.M. Nistal from the Image _{Manev, Neuroprotection by melatonin from kainate-induced} ex-Process Service of the University of Oviedo for the use of citotoxicity in rats, FASEB J. 10 (1996) 891–896.

the laser confocal microscope. This work was supported by [19] S.J. Hill, Distribution, properties and functional characteristics of three classes of histamine receptor, Pharmacol. Rev. 42 (1990)

CICYT, Grant SAF98-0171, FICYT, Grant PA-PG195-01,

45–83.

and University of Oviedo, Grant NP-00-530-27. During

¨ ¨

[20] E. Hosli, L. Hosli, Autoradiographic localization of binding sites for

part of this work R.D.T was supported by FICYT-Astur- 3

[ H] histamine and H - and H -antagonists on cultured neurons and1 2

pharma, Grant PA-PG195-01. glial cells, Neuroscience 13 (1984) 863–870.

[21] J.P.Y. Kao, A.T. Harootunian, R.Y. Tsien, Photochemically generated cytosolic calcium pulses and their detection by Fluo-3, J. Biol. Chem. 264 (1989) 8179–8184.

References _{[22] J.J. Krupp, B. Vissel, S.F. Heinemann, G.L. Westbrook,} Calcium-dependent inactivation of recombinant N-methyl-D-aspartate recep-[1] N. Adachi, R. Oishi, Y. Itano, T. Yamada, M. Hirakawa, K. Saeki, tors is NR2 subunit specific, Mol. Pharmacol. 50 (1996) 1680–1688. Aggravation of ischemic neuronal damage in the rat hippocampus [23] M. Lafon-Cazal, S. Pietri, M. Culcasi, J. Bockaert, NMDA-depen-by impairment of histaminergic neurotransmission, Brain Res. 602 dent superoxide production and neurotoxicity, Nature 364 (1993)

(1993) 165–168. 535–537.

[2] R.A. Bakker, K. Wieland, H. Timmerman, R. Leurs, Constitutive [24] P.J. Langlais, S.X. Zhang, G. Weilersbacher, L.B. Hough, K.E. activity of the histamine H1 receptor reveals inverse agonism of Barke, Histamine-mediated neuronal death in a rat model of histamine H1 receptor antagonists, Eur. J. Pharmacol. 387 (2000) Wernicke’s encephalopathy, J. Neurosci. Res. 38 (1994) 565–574. R5–R7. [25] P. Legendre, C. Rosenmund, G.L. Westbrook, Inactivation of NMDA [3] J.M. Bekkers, Enhancement by histamine of NMDA-mediated channels in cultured hippocampal neurons by intracellular calcium,

synaptic transmission in the hippocampus, Science 261 (1993) J. Neurosci. 13 (1993) 674–684.

21 104–106. [26] S. Liu, R.B. Melchert, R.H. Kennedy, Inhibition of L-type Ca [4] C.I. Berul, M. Morad, Regulation of potassium channels by non- channel current in rat ventricular myocytes by terfenadine, Cir. Res.

sedating antihistamines, Circulation 91 (1995) 2220–2225. 81 (1997) 202–210.

[5] T.V.P. Bliss, G.L. Collingridge, A synaptic model of memory: [27] Y. Lu, Z.J. Wang, Terfenadine block of sodium current in canine long-term potentiation in the hippocampus, Nature 361 (1993) atrial myocytes, Cardiovasc. Pharmacol. 33 (1999) 507–513. 31–39. [28] A. Marini, A. Novelli,DL-threo-3-hydroxyaspartate reduces NMDA [6] S.T. Chen, J.I. Chuang, The antioxidant melatonin reduces cortical receptor activation by glutamate in cultured neurons, Eur. J.

neuronal death after intrastriatal injection of kainate in the rat, Exp. Pharmacol. 194 (1991) 131–132.

Brain. Res. 124 (1999) 241–247. [29] P.P. McCaslin, W.W. Morgan, Cultured cerebellar cells as an in vitro [7] J.T. Coyle, P. Puttfarcken, Oxidative stress, glutamate, and neurode- model of excitatory amino acid receptor function, Brain Res. 417

generative disorders, Science 262 (1993) 689–695. (1987) 380–384.

[8] D.W. Choi, S.M. Rothman, The role of glutamate neurotoxicity in [30] D. McTavish, K.L. Goa, M. Ferrill, Terfenadine: An updated review hypoxic-ischemic neuronal death, Annu. Rev. Neurosci. 13 (1990) of its pharmacological properties and therapeutic efficacy, Drugs 39

171–182. (1990) 552–574.

[9] M. De Waard, M. Seagar, A. Feltz, F. Couraud, Inositol phosphate [31] I. Medina, X. Leinekugel, Y. Ben-Ari, Calcium-dependent inactiva-regulation of voltage-dependent calcium channels in cerebellar tion of the monosynaptic NMDA EPSCs in rat hippocampal neurons granule neurons, Neuron 9 (1992) 497–503. in culture, Eur. J. Neurosci. 11 (1999) 2422–2430.

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[32] A. Novelli, F. Nicoletti, J.T. Wroblewski, H. Alho, E. Costa, A. [40] M. Segal, D. Manor, Confocal microscopic imaging of [Ca21]i in Guidotti, Excitatory amino acid receptors coupled with guanylate cultured rat hippocampal neurons following exposure to N-methyl-cyclase in primary cultures of cerebellar granule cells, J. Neurosci. 7 D-aspartate, J. Physiol. 448 (1997) 655–676.

(1987) 40–47. [41] S.D. Skaper, B. Ancona, L. Facci, D. Franceschini, P. Giusti, [33] A. Novelli, J.A. Reilly, P.G. Lypsko, R.C. Henneberry, Glutamate Melatonin prevents the delayed death of hippocampal neurons becomes neurotoxic via the NMDA receptor when intracellular induced by enhanced excitatory neurotransmission and the ni-energy levels are reduced, Brain Res. 451 (1988) 205–212. tridergic pathway, FASEB J. 12 (1998) 725–731.

´ ´

[34] A. Novelli, J. Kispert, M.T. Fernandez-Sanchez, A. Torreblanca, V. [42] S. Suessbrich, F. Waldegger, A.E. Lang, A.E. Busch, Blockade of Zitko, Domoic acid-containing toxic mussels produce neurotoxicity HERG channels expressed in Xenopus oocytes by the histamine in neuronal cultures through a synergism between excitatory amino receptor antagonists terfenadine and astemizole, FEBS Lett. 385 acids, Brain Res. 577 (1992) 41–48. (1996) 77–80.

[35] A. Novelli, R.C. Henneberry, cGMP synthesis in cultured cerebellar [43] K. Sugimoto, K. Maeyama, K. Alam, E. Sakurai, H. Onoue, T.

neurons is stimulated by glutamate via a Ca -mediated, differen- Kasugai, Y. Kitamura, T. Watanabe, Brain histaminergic system in tiation-dependent mechanism, Dev. Brain. Res. 34 (1987) 307–310. mast cell deficient (Ws / Ws) rats: histamine content, histidine [36] M. Patel, B.J. Day, J.D. Crapo, I. Fridovich, J.O. McNamara, decarboxylase activity, and effects of (S )

alpha-fluoromethylhis-Requirement for superoxide in excitotoxic cell death, Neuron 16 tidine, J. Neurochem. 65 (1995) 791–797.

(1996) 345–355. [44] L. Vyklicky Jr., Calcium-mediated modulation of N-methyl-D -aspar-[37] D. Pozo, R.J. Reiter, J.R. Calvo, J.M. Guerrero, Inhibition of tate (NMDA) responses in cultured hippocampal neurones, J.

cerebellar nitric oxide synthase and cyclic GMP production by Physiol. 470 (1993) 575–600.

melatonin via complex formation with calmodulin, J. Cell. Biochem. [45] J.-M. Yang, S. Goldenberg, M.M. Gottesman, W.N. Hait, Charac-65 (1997) 430–442. teristics of P388 / VMDRC.04, a simple, sensitive model for

study-´

´ ´

[38] F.J. Rodrıguez, M. lluch, J. Dot, I. Blanco, J. Rodrıguez-Alvarez, ing P-glycoprotein antagonists, Cancer Res. 54 (1994) 730–737. Histamine modulation of glutamate release from hippocampal [46] M.Q. Zhang, P. Caldirola, H. Timmerman, Calcium antagonism and synaptosomes, Eur. J. Pharmacol. 323 (1997) 283–286. structure-affinity relationships of terfenadine, a histamine H1 an-[39] J.J. Rozniecki, V. Dimitriadou, M. Lambracht-Hall, X. Pang, T.C. tagonist, and some related compounds, J. Pharm. Pharmacol. 45

Theoharides, Morphological and functional demonstration of rat (1993) 63–66. dura mater mast cell-neuron interactions in vitro and in vivo, Brain

(1)

was estimated [40] (see Materials and Methods) to be from that activation of NMDA receptors was necessary. Similar approximately 112634 to 470660 nM and from approxi- results were obtained in experiments in which NMDA was mately 133630 to 11206125 nM in the absence and in the used as NMDA-receptor agonist (not shown). These find-presence of terfenadine respectively. ings suggested a key role for hydrogen peroxides as To investigate the effect of terfenadine on calcium- mediators of the neurodegeneration coupled to overactiva-dependent signal transduction we measured the intracellu- tion of NMDA receptors in cultured cerebellar neurons. lar formation of cGMP stimulated by NMDA. As shown in Indeed, stimulation of neurons with 50 mM glutamate or Table 2, preincubation of neurons with terfenadine (5mM, 500mM NMDA induced extensive formation of hydrogen 4 h) resulted in an approximately 4 fold increase in cGMP peroxides and resulted in over 80% neurotoxicity, due to formation induced by the selective stimulation of NMDA excessive stimulation of NMDA receptors as it could be receptors with 1 mM NMDA, and also increased by completely abolished by MK-801 (data not shown). approximately 2 fold the cGMP formation induced by 100

antagonist MK-801, and that terfenadine had no effect on

radicals following stimulation of NMDA receptors secretory product melatonin, and (1)-alpha-tocopherol (vitamin E). In particular, we investigated whether To test for the participation of oxidative stress in the melatonin and (1)-alpha-tocopherol were able to prevent potentiation by terfenadine of NMDA receptor-mediated cell death produced by low concentrations of NMDA in the response we used the specific probe dichlorodihydrofl- presence of terfenadine, and whether addition of these uorescein diacetate (H DCFDA) to measure the generation2 antioxidant drugs after the excitotoxic insult could rescue of reactive oxygen species, and in particular hydrogen neurons. Both melatonin (200 mM) and (1 )-alpha-peroxides, in cultures exposed to subtoxic concentrations tocopherol (200mM) markedly inhibited NMDA-mediated (15 mM) of glutamate both in the presence and in the cell death in terfenadine treated neurons when added 15 absence of terfenadine. As shown in Fig. 4, stimulation of min prior to NMDA application (Fig. 5A), reducing neurons with this low concentration of glutamate did not neuronal death by approximately 70%. To determine result in a significant generation of hydrogen peroxides whether addition of these drugs after initiation of the (Fig. 4C) compared to unstimulated cultures (Fig. 4A). In excitotoxic insult exerted neuroprotective effects, we per-contrast, a massive formation of oxygen radicals occurred formed experiments in which melatonin or (1 )-alpha-in cultures exposed to this low concentration of glutamate tocopherol were added 0, 5, 15, and 30 min following stimulation with NMDA. As shown in Fig. 5B, addition of melatonin 0, 10 or 15 min after exposure to 100 mM

Table 2

Terfenadine potentiates cGMP formation induced by NMDA NMDA reduced neuronal death significantly, while no protection was observed in cultures to which melatonin

Treatment cGMP (pmol / mg protein)

had been added 30 min after NMDA. As for (1

)-alpha-None TEF

tocopherol, this compound showed a greater effectiveness,

Unstimulated 0.5860.3 0.5160.1 _{and significant protection from NMDA in terfenadine}

b b,c

NMDA (100mM) 3.360.3 6.1660.2

treated neurons was observed in cultures to which (1

)-b b,c

NMDA (1 mM) 1762 7068

alpha-tocopherol was added up to 30 min after NMDA

Cerebellar neurons in primary culture were used at 12–14 days in _{(Fig. 5B).} culture and vehicle (None) or the indicated drugs were added to the

growth medium. Terfenadine (TEF, 5mM) was added 5 h before NMDA. After 1 min exposure to NMDA culture medium was aspirated and 1 ml

HCLO (0.4 N) was added. The perchlorate extract was neutralized with4 4. Discussion

NaOH and the cGMP content was determined by radioimmunoassay.

Data are from triplicate values from two independent experiments. _{In the present study we have investigated the} potentia-Statistical significance was determined by two-way analysis of variance

tion by terfenadine of the excitotoxic response to NMDA

(ANOVA) and two-tailed Student’s t-test.

b _{receptor agonists in cultured cerebellar neurons. We found}

P,0.01 vs. unstimulated in the same column.

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(3)

Fig. 5. Antioxidants melatonin and (1)-alpha-tocopherol prevent potentiation by terfenadine of NMDA excitotoxicity. Neurons at 14–17 DIC were either untreated (None) or exposed to the indicated drugs in the growth medium, and neuronal survival was determined 24 h later by staining of live neurons with fluorescein diacetate and dead neurons nuclei with ethidium bromide, and observation of cultures under a fluorescence light. Terfenadine (TEF) was added 5 h before NMDA. Melatonin (MEL) and (1)-alpha-tocopherol (a-TCF) were added 10 min before TEF (Panel A) or at the indicated times after application of NMDA (Panel B). Concentrations were: TEF, 5mM; NMDA, 100mM; MEL, 200mM;a-TCF, 200mM. Data represent the mean6S.D. of duplicate values from four independent experiments. *P,0.001 vs. NMDA in panel A and *P,0.01 vs. TEF1NMDA in panel B.

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shown). acid [17]. In contrast, melatonin treatment was suggested

diphenylalkylamine L-type Ca channel blockers, and in explanation for the discrepancy between the latter observa-21

Our observations that terfenadine significantly poten- alpha-tocopherol we report here.

(5)

fenadine involves an increase in the influx of calcium via toxicity following sodium channel activation, Brain Res. 842 (1999) 478–481.

NMDA receptors and the NMDA receptor mediated

in-´ ´ ´

[12] R. Dıaz-Trelles, A. Novelli, M.T. Fernandez-Sanchez, Modulation

tracellular synthesis of cGMP, and does not depend upon

of excitatory neurotransmission by terfenadine, Soc. Neurosci.

RNA synthesis. Terfenadine potentiation of NMDA re- _{Abstr. 25 (1999) 2254.}

[14] G. Escames, J.M. Guerrero, R.J. Reiter, J.J. Garcia, A.

Munoz-was prevented by the antioxidants vitamin E and

Hoyos, G.G. Ortiz, C.S. Oh, Melatonin and vitamin E limit nitric

melatonin. Our findings provide for the first time direct

oxide-induced lipid peroxidation in rat brain homogenates, Neurosci.

evidence for antihistamine drugs enhancing the transduc- _{Lett. 230 (1997) 147–150.}

tissues, FASEB J. 11 (1997) 1309–1315.

of excitotoxic injury.

´ [16] E. Gilad, S. Cuzzocrea, B. Zingarelli, A.L. Salzman, C. Szabo,

Melatonin is a scavenger of peroxynitrite, Life Sci. 60 (1997) PL169–PL174.

excitotox-´ ´

We thank Dr. J.R. Fernandez Gonzalez for the generous

icity, Exp. Neurol. 131 (1995) 39–46.

gift of some of the drugs used in this study. We also thank

[18] P. Giusti, M. Lipartiti, D. Franceschini, N. Schiavo, M. Floreani, H.

the laser confocal microscope. This work was supported by [19] S.J. Hill, Distribution, properties and functional characteristics of three classes of histamine receptor, Pharmacol. Rev. 42 (1990)

CICYT, Grant SAF98-0171, FICYT, Grant PA-PG195-01,

45–83.

and University of Oviedo, Grant NP-00-530-27. During

¨ ¨

[20] E. Hosli, L. Hosli, Autoradiographic localization of binding sites for

part of this work R.D.T was supported by FICYT-Astur- 3

[ H] histamine and H - and H -antagonists on cultured neurons and1 2

pharma, Grant PA-PG195-01. glial cells, Neuroscience 13 (1984) 863–870.

[21] J.P.Y. Kao, A.T. Harootunian, R.Y. Tsien, Photochemically generated cytosolic calcium pulses and their detection by Fluo-3, J. Biol. Chem. 264 (1989) 8179–8184.

References _{[22] J.J. Krupp, B. Vissel, S.F. Heinemann, G.L. Westbrook,}

Calcium-dependent inactivation of recombinant N-methyl-D-aspartate recep-[1] N. Adachi, R. Oishi, Y. Itano, T. Yamada, M. Hirakawa, K. Saeki, tors is NR2 subunit specific, Mol. Pharmacol. 50 (1996) 1680–1688. Aggravation of ischemic neuronal damage in the rat hippocampus [23] M. Lafon-Cazal, S. Pietri, M. Culcasi, J. Bockaert, NMDA-depen-by impairment of histaminergic neurotransmission, Brain Res. 602 dent superoxide production and neurotoxicity, Nature 364 (1993)

(1993) 165–168. 535–537.

R5–R7. [25] P. Legendre, C. Rosenmund, G.L. Westbrook, Inactivation of NMDA

[3] J.M. Bekkers, Enhancement by histamine of NMDA-mediated channels in cultured hippocampal neurons by intracellular calcium, synaptic transmission in the hippocampus, Science 261 (1993) J. Neurosci. 13 (1993) 674–684.

104–106. [26] S. Liu, R.B. Melchert, R.H. Kennedy, Inhibition of L-type Ca

[4] C.I. Berul, M. Morad, Regulation of potassium channels by non- channel current in rat ventricular myocytes by terfenadine, Cir. Res. sedating antihistamines, Circulation 91 (1995) 2220–2225. 81 (1997) 202–210.

31–39. [28] A. Marini, A. Novelli,DL-threo-3-hydroxyaspartate reduces NMDA

[6] S.T. Chen, J.I. Chuang, The antioxidant melatonin reduces cortical receptor activation by glutamate in cultured neurons, Eur. J. neuronal death after intrastriatal injection of kainate in the rat, Exp. Pharmacol. 194 (1991) 131–132.

generative disorders, Science 262 (1993) 689–695. (1987) 380–384.

171–182. (1990) 552–574.

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(1987) 40–47. [41] S.D. Skaper, B. Ancona, L. Facci, D. Franceschini, P. Giusti,

[33] A. Novelli, J.A. Reilly, P.G. Lypsko, R.C. Henneberry, Glutamate Melatonin prevents the delayed death of hippocampal neurons becomes neurotoxic via the NMDA receptor when intracellular induced by enhanced excitatory neurotransmission and the ni-energy levels are reduced, Brain Res. 451 (1988) 205–212. tridergic pathway, FASEB J. 12 (1998) 725–731.

´ ´

acids, Brain Res. 577 (1992) 41–48. (1996) 77–80.

[35] A. Novelli, R.C. Henneberry, cGMP synthesis in cultured cerebellar [43] K. Sugimoto, K. Maeyama, K. Alam, E. Sakurai, H. Onoue, T.

alpha-fluoromethylhis-Requirement for superoxide in excitotoxic cell death, Neuron 16 tidine, J. Neurochem. 65 (1995) 791–797.

(1996) 345–355. [44] L. Vyklicky Jr., Calcium-mediated modulation of N-methyl-D

-aspar-[37] D. Pozo, R.J. Reiter, J.R. Calvo, J.M. Guerrero, Inhibition of tate (NMDA) responses in cultured hippocampal neurones, J. cerebellar nitric oxide synthase and cyclic GMP production by Physiol. 470 (1993) 575–600.

melatonin via complex formation with calmodulin, J. Cell. Biochem. [45] J.-M. Yang, S. Goldenberg, M.M. Gottesman, W.N. Hait,

Charac-65 (1997) 430–442. teristics of P388 / VMDRC.04, a simple, sensitive model for

study-´

´ ´

Theoharides, Morphological and functional demonstration of rat (1993) 63–66. dura mater mast cell-neuron interactions in vitro and in vivo, Brain

Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol880.Issue1-2.2000:

Research Report

Antihistamine terfenadine potentiates NMDA receptor-mediated

calcium influx, oxygen radical formation, and neuronal death

´

*

´

Ramon Diaz-Trelles , Antonello Novelli

, Jose A. Vega , Ann Marini ,

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Dukungan

Links

Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol880.Issue1-2.2000:

Research Report

Antihistamine terfenadine potentiates NMDA receptor-mediated

calcium influx, oxygen radical formation, and neuronal death

´

*

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Ramon Diaz-Trelles , Antonello Novelli

, Jose A. Vega , Ann Marini ,

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PERENCANAAN PORTAL KELUAR KAMPUS III UMM

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