Characterization of the Participation of Sodium Channels on the Rise in Na⁺Induced by 4-Aminopyridine (4-AP) in Synaptosomes

Abstract

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.

Lysophospholipids and ATP Mutually Suppress Maturation and Release of IL-1β in Mouse Microglial Cells Using a Rho-Dependent Pathway

“…[K ϩ ] i was measured using PBFI fluorescence as described previously (28). In brief, potassium-binding benzofuran isophtalate acetomethylester was added to the cell suspension (10 M) and the cells were loaded for 1 h at 37°C.…”

“…These data suggest that the inhibitory mechanisms in mIL-1␤ release by LPC and SPC on P2X7R-mediated response may not be related to K ϩ efflux. To further assess the effect of LPC and SPC on [K ϩ ] i , the change in [K ϩ ] i was monitored using PBFI fluorescence (28). We observed that 5 mM ATP induced a sustained decrease in the …”

Antiepileptic Drugs Targeting Cerebral Presynaptic Ion Channels Reduce Cerebral Excitability Decreasing Glutamate Release

“…The involvement of K + channels in the mode of action of 4-aminopyridine at the presynaptic brain level, first suggested by the changes on 86 Rb + fluxes in brain nerve endings (Sitges et al, 1986), was confirmed later using the K + selective indicator dye, PBFI (Galindo & Sitges 2004). In summary, in cerebral isolated nerve endings 4-aminopyridine increases Na + channels permeability (Galván and Sitges, 2004), Ca 2+ channels permeability (Tibbs et al, 1989;Heemskerk et al, 1991;Galván & Sitges, 2004;, and decreases K + channels permeability (Sitges et al, 1986;Galván & Sitges, 2004). Therefore, the changes that may occur in cerebral nerve endings under the excitatory conditions that t a k e p l a c e d u r i n g s e i z u r e s s e e m t o b e m o re closely resembled by 4-aminopyridine; although its mechanism of action is complicated.…”

“…More recently, we also test the effects of antiepileptic drugs in the cerebral nerve endings in vitro using 4-aminopyridine as depolarizing strategy. Because 4-aminopyridine exposure may more closely mimic some of the changes that may take place in the epileptic tissue, since in cerebral nerve endings 4-aminopyridine besides increasing the permeability of Na + and Ca 2+ channels, also decreases the permeability of some K + channels, and by this mean arrests indirectly the Na + /K + ATPase (Galván & Sitges, 2004), making even more difficult the limitation of neuronal excitability. Fig.…”

“…Although the rise in the internal concentration of Ca 2+ induced by 4-aminopyridine was not resolved using 45 Ca 2+ (Tapia et al, 1985), it became evident when the more sensitive fura-2 technique was used in cerebral isolated nerve endings. The role of voltage sensitive sodium channels in the mode of action of 4-aminopyridine, first suggested by the sensitivity of the Ca 2+ response induced by 4-aminopyridine to the Na + channel blocker, tetrodotoxin (Tibbs et al, 1989;Heemskerk et al, 1991) was later demonstrated in cerebral isolated nerve endings using the Na + selective indicator dye, SBFI (Galván & Sitges 2004). The involvement of K + channels in the mode of action of 4-aminopyridine at the presynaptic brain level, first suggested by the changes on 86 Rb + fluxes in brain nerve endings (Sitges et al, 1986), was confirmed later using the K + selective indicator dye, PBFI (Galindo & Sitges 2004).…”