Mechanisms of sodium azide-induced changes in intracellular calcium concentration in rat primary cortical neurons.
An intracellular calcium ([Ca(2+)](i)) increase is involved in sodium azide (NaN(3))-induced neurotoxicity, an in vitro model of brain ischemia. In this study the questions of possible additional sources of calcium influx, besides glutamate receptor activation, and of the time-course of NaN(3) effects have been addressed by measuring [Ca(2+)](i) in rat primary cortical cultures with the FURA-2 method. Basal [Ca(2+)](i) of neuronal populations was concentration-dependently increased 30 min, but not 24h, after a 10-min NaN(3) (3-30 mM) treatment; conversely, the net increase induced by electrical stimulation (10Hz, 10s) was consistently reduced. All the above effects depended on glutamate release and consequent NMDA receptor activation, since the NMDA antagonist MK-801 (1 microM) prevented them, and the spontaneous efflux of [(3)H]-d-aspartate from superfused neurons was concentration-dependently increased by NaN(3). In single neuronal cells, NaN(3) application progressively and concentration-dependently increased [Ca(2+)](i) (to 177+/-5% and 249+/-7% of the controls, 4 and 12 min after a 10mM-treatment, respectively). EGTA (5mM) pretreatment reduced the effect of 10mM NaN(3) (to 118+/-5% at 4 min, and to 148+/-10% at 12 min, respectively), while 1 microM cyclosporin A did not. Both MK-801 and CNQX (a non-NMDA glutamate antagonist, 10 microM) prevented NaN(3) effect at 4 min (to 147+/-8% and 153+/-5%, respectively), but not at 12 min after NaN(3) treatment. Conversely, 10 microM verapamil and 0.1 microM omega-conotoxin (L- and N-type calcium channel blockers, respectively) significantly attenuated NaN(3) effects at 12 min (to 198+/-8% and 164+/-5%, respectively), but not at 4 min; the P/Q-type calcium channel blocker, agatoxin, 0.3 microM, was ineffective. These findings show that the predominant source of calcium increase induced by NaN(3) is extracellular, involving glutamate receptor activation in a first step and calcium channel (mainly of the N-type) opening in a second step.
Marino
Silvia
S
Department of Clinical and Experimental Medicine, Section of Pharmacology, University of Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy.
Marani
Luca
L
Nazzaro
Cristiano
C
Beani
Lorenzo
L
Siniscalchi
Anna
A
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000465:Algorithms; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000187:drug effects / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology* / Q000187:drug effects; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004558:Electric Stimulation; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects*; D018377:Neurotransmitter Agents / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists / Q000378:metabolism; D019810:Sodium Azide / Q000633:toxicity*
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D018691:Excitatory Amino Acid Antagonists; D018377:Neurotransmitter Agents; D016194:Receptors, N-Methyl-D-Aspartate; D001224:Aspartic Acid; D016291:Dizocilpine Maleate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D019810:Sodium Azide; D002118:Calcium