Effect of tetanus toxin on the accumulation of the permeant lipophilic cation tetraphenylphosphonium by guinea pig brain synaptosomes

Abstract

that mitochondria entrapped within the synaptosome but not free mitochondria make a large contribution to the TPP+ concentration gradients observed. Conditions are defined in which tetanus toxin binds specifically and immediately to synaptosomes in media used to measure IP'+ uptake. Under these conditions tetanus toxin induces dose-dependent changes in TPP+ uptake that are blocked by antitoxin and not mimicked by biologically inactivated toxin preparations. The effect of tetanus toxin on TPP+ uptake is not evident in the presence of 193 mM K+ or veratridme but remains under conditions known to abolish the mitochondrial Aui. Moreover, tetanus toxin has no effect on TPP+ uptake by isolated synaptosomal mitochondria. The results thus define an in vitro action of tetanus toxin on the synaptosomal membrane that can be correlated with biological potency in vivo and is consistent with the in vivo effects of tetanus toxin on neuronal transmission. The classic symptomatology of tetanus reflects a syndrome of dysinhibition in which tetanus toxin is thought to abolish neuronal transmission through certain inhibitory pathways in the central nervous system (see ref. 1 for a review). Studies carried out in vivo (2-4) suggest that the effect of the toxin is caused by its ability to inhibit the release of inhibitory neurotransmitters such as glycine at specific synaptic termini. However, tetanus toxin also has been shown to block conduction through peripheral cholinergic synapses with a concomitant decrease in acetylcholine release (5-7).

The estimation of free calcium within synaptosomes and mitochondria with fura-2; comparison to quin-2

“…In the absence of valinomycin, apparent synaptosomal charge was 147 ± 11 mV. Values obtained in the presence or absence of valinomycin compare well with those reported by Ramos et al (1979).…”

“…The determination of the electrical potential across the synaptosomal membrane (Iii/I) was carried out by the procedure of Ramos et al (1979). This method essentially consists of assay of the accumulation of a permeant lipophilic action (tetraphenylphosphonium, TPP+) by 1 ml of the final suspension of loaded synaptosomes used for the fluorometric studies.…”

Crosslinking of the receptors for immunoglobulin E depolarizes the plasma membrane of rat basophilic leukemia cells.

“…Studies with other cells (6, 7) and with synaptosomes (17,18) indicate that the uptake of Ph4P+ reflects both equilibration across the plasma membrane, governed by At, and accumulation into mitochondria (which are negative inside compared to the cytoplasm) (17,18). This also seems to be the case with RBL cells: blocking of the respiratory chain, which generates the mitochondrial AT with rotenone and blocking of the proton-translocating ATPase with oligomycin caused a 60% decrease of Ph4P+ uptake (Fig.…”

Effects of MPTP, MPP+ and paraquat on mitochondrial potential and oxidative stress

“…This method has been applied successfully to synaptosomes (23). Synaptosomes in HEPES buffer (125 mM NaCl, 5 mM KCl, 1.2 mM Na2HP04, 1.2 mM MgCl2, 5 mM NaHC03, 6 mM glucose, 1mMCaCl2,25 mM HEPES, pH 7.4) were incubated at 37°C for 20 minutes in a 1.0 ml volume containing 120 mg synaptosomal protein, 0.2 mCi of 3H-phenyl tetraphenylphosphonium (TPp+) (35.5 Ci/mmole) and sufficient TPp+ to ensure a final concentration of 2 mM was then added and incubation continued for a further 10 min.…”