In vivo and in vitro effects of acrylamide on synaptosomal neurotransmitter uptake and release.
Evidence suggests acrylamide (ACR) neurotoxicity is mediated by impaired presynaptic transmission. To assess the effects of ACR on nerve terminal function, [3H] glutamate release and uptake were determined in brain synaptosomes isolated from intoxicated rats (50mg/kg per day x 8 days, i.p. or 21 mg/kg per day x 21 days, p.o.). Regardless of ACR dose-rate, a significant reduction in synaptosomal K(+)-stimulated, Ca(2+)-dependent release was detected, whereas kinetic analysis of Na(+)-dependent uptake did not reveal consistent changes. Immunoblot analysis showed normal protein levels (e.g. SNAP-25) in dysfunctional synaptosomes isolated from ACR-intoxicated rats. This suggests that defective release does not involve changes in protein synthesis and/or anterograde delivery of presynaptic constituents. To identify potential targets, synaptosomes were exposed in vitro to [ 14C ]-ACR and radiolabeled proteins were separated by gel electrophoresis and detected by autoradiography. [14C]-ACR labeling of distinct synaptosomal protein bands (10.5-154000 kDa) was blocked by the sulfhydryl alkylating agent, N-ethylmaleimide (NEM; 4mM) but not by the non-neurotoxic structural analog propionamide (10mM). In vitro characterization of synaptosomal [3H] glutamate uptake and release showed that ACR, NEM and iodoacetic acid (IAA) produced concentration-dependent decreases in each parameter that were highly correlated to reductions in free sulfhydryl content. All three chemicals were equiefficacious with respect to reducing sulfhydryl content and neurotransmitter uptake/release, although the relative potencies differed; [3H]. Kinetic analysis of uptake showed that in vitro exposure to ACR, IAA or NEM at their respective IC(50)'s caused similar reductions in V(max). These data suggest that ACR-induced synaptic dysfunction involves adduction of presynaptic protein thiol groups and subsequent reduction in neurotransmitter release.
LoPachin
Richard M
RM
Department of Anesthesiology, Albert Einstein College of Medicine, Montefiore Medical Center, Moses Research Tower-7, 111 E 210th Street, Bronx, NY 10467, USA. lopachin@aecom.yu.edu
Schwarcz
Aron I
AI
Gaughan
Christopher L
CL
Mansukhani
Shirley
S
Das
Soma
S
D020106:Acrylamide / Q000493:pharmacokinetics* / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000378:metabolism*; D008297:Male; D018377:Neurotransmitter Agents / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*
D018377:Neurotransmitter Agents; D020106:Acrylamide; D018698:Glutamic Acid