Brain Research 880 2000 17–27 www.elsevier.com locate bres
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 ,
a
´ ´
M. Teresa Fernandez-Sanchez
a
Department of Biochemistry and Molecular Biology , School of Medicine, University of Oviedo, 33071 Oviedo, Spain
b
Department of Psychology , Faculty of Psycology, University of Oviedo, 33071 Oviedo, Spain
c
Department of Morphology and Cellular Biology , Faculty of Medicine, University of Oviedo, 33071 Oviedo, Spain
d
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 100 mM or glutamate 15 mM that were only slightly ,20 toxic when added alone, caused extensive cell
death in cultures pre-exposed to terfenadine 5 mM for 5 h. Terfenadine potentiation of NMDA receptor response was mimicked by other H1 antagonists, including chlorpheniramine 25 mM, 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 200 mM or the endogenous antioxidant melatonin 200 mM 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: 134-985-103-
drug resistance in a variety of cell types via its interaction
157. E-mail address
: neurolabsauron.quimica.uniovi.es A. Novelli.
with P glycoprotein [45]. Very limited data exist about the
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 6 2 7 - 5
18 R
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 , 2.3
2
its antihistamine activity; for this purpose we tested other mM CaCl , pH 7.4, to which the vital stain fluorescein
2
H1 histamine antagonists and performed competition diacetate 5 mg 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.
