Introduction of macromolecules into synaptosomes using electroporation.
Synaptic terminals are sites of high metabolic activity and thus are particularly vulnerable to oxidative stress. Oxidative damage to proteins can be toxic to neurons and may cause irreversible cell damage and neurodegeneration. A neuroprotective mechanism used by cells to combat oxidative damage is to selectively degrade damaged proteins. Therefore, it is of interest to study the mechanism of degradation of oxidatively damaged proteins in synaptosomes. One way of oxidizing synaptosomal proteins in vitro is by incubating intact synaptosomes in the presence of an oxidizing agent. A problem with this approach is that it may also cause oxidative damage to the machinery required to recognize and degrade oxidized proteins. We have, therefore, introduced a fluorescent macromolecule into synaptosomes to assess the feasibility of using this technique to study how oxidized proteins are degraded and removed from synaptic terminals. Synaptosomes were subjected to electroporation in the presence of FITC labelled-dextran with an average molecular weight of 70000 (FD-70) and non-specific binding was determined by running parallel experiments in lysed synaptosomes. Following extensive washing, synaptosomes were assayed for the presence of intra-synaptosomal FD-70 by measuring fluorescence in a microplate fluorescence reader. Significant differences in fluorescence were found between intact and lysed synaptosomes with maximal uptake at 100 V/ 1500 microF (approx. 36 pmol/mg protein). To determine if membrane transport was compromised by electroporation, uptake of 3H-arginine was compared in control and electroporated synaptosomes. While untreated electroporated synaptosomes showed a loss of 22% in the ability to transport arginine, preincubation in the presence of 1 mM ATP resulted in a complete restoration of arginine transport. These results show that electroporation is a potentially useful technique for introducing a specific oxidized protein, into synaptic terminals so its metabolic fate can be examined.
Ramanathan
M
M
Department of Pharmacology and Physiology, New Jersey Medical School UMDNJ, 185 South Orange Avenue, Newark, NJ 07103, USA.
Kuo
H R
HR
Lambert
C W
CW
Ingoglia
N A
NA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000493:pharmacokinetics; D001692:Biological Transport / Q000187:drug effects; D002021:Buffers; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D003911:Dextrans / Q000737:chemistry / Q000493:pharmacokinetics*; D004553:Electric Conductivity; D018274:Electroporation / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics; D008297:Male; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000378:metabolism*; D014316:Tritium
D002021:Buffers; D003911:Dextrans; C015219:fluorescein isothiocyanate dextran; D014316:Tritium; D000255:Adenosine Triphosphate; D001120:Arginine; D016650:Fluorescein-5-isothiocyanate