Valproate uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: relevance to valproate's efficacy against bipolar disorder

Abstract

Background: The ability of chronic valproate (VPA) to reduce arachidonic acid (AA) turnover in brain phospholipids of unanesthetized rats has been ascribed to its inhibition of acyl-CoA synthetase (Acsl)-mediated activation of AA to AA-CoA. Our aim was to identify a rat Acsl isoenzyme that could be inhibited by VPA in vitro.

Methods: Rat Acsl3-, Acsl6v1- and Acsl6v2-, and Acsl4-flag proteins were expressed in E. coli, and the ability of VPA to inhibit their activation of long-chain fatty acids to acyl-CoA was estimated using Michaelis-Menten kinetics.

Results: VPA uncompetitively inhibited Acsl4-mediated conversion of AA and of docosahexaenoic (DHA) but not of palmitic acid to acyl-CoA, but did not affect AA conversion by Acsl3, Acsl6v1 or Acsl6v2. Acsl4-mediated conversion of AA to AA-CoA showed substrate inhibition and had a 10-times higher catalytic efficiency than did conversion of DHA to DHA-CoA. Butyrate, octanoate, or lithium did not inhibit AA activation by Acsl4.

Conclusions: VPA's ability to inhibit Acsl4 activation of AA and of DHA to their respective acyl-CoAs, when related to the higher catalytic efficiency of AA than DHA conversion, may account for VPA's selective reduction of AA turnover in rat brain phospholipids, and contribute to VPA's efficacy against bipolar disorder.

Conflict of interest statement

Disclosure/Conflict of interest.

No author has a financial or other conflict of interest related to this work.

Copyright © 2010 Elsevier B.V. All rights reserved.

Figures

Figure 1

Recombinant rat Acsl3, Acsl4, Acsl6v1 and Acsl6v2-Flag proteins expressed in E. Coli were analyzed by Western blotting with an anti-Flag M2 monoclonal antibody. Protein-antibody complexes were visualized by chemiluminescence detection of horseradish peroxidase linked to goat anti-mouse IgG. The molecular mass of the recombinant Acsl isoenzymes was 74-kDa. The same E. Coli strain containing the identical plasmid without the gene encoding for Acsl was used as control.

Figure 2

Initial reaction velocity (V, nmol/min/mg protein) of (2a) Acsl3, (2b) Acsl6v1 and (2c) Acsl6v2, plotted against increasing AA concentration [S] in the presence 0, 30, or 60 mM VPA.

Figure 3

(3a) Initial reaction velocity (V, nmol/min/mg protein) of Acsl4 plotted against increasing AA concentration [S] in the presence of 0, 10, 30, 60 or 90 mM VPA, or of 30 mM LiCl [I]. (3b) Lineweaver-Burk plots for the reciprocal of enzyme activity (1/V) against the inverse of substrate concentration, 1/[S] (1/[AA]).

Figure 4

Initial reaction velocity V of Acsl4 plotted against increasing palmitic acid concentration [S] in the presence of 0, 30, 60 or 90 mM VPA.

Figure 5

(5a) Initial reaction velocity V of Acsl4 plotted against increasing DHA concentration [S] in the presence of 0, 30, 60 or 90 mM VPA. (5b) Lineweaver-Burk plots of reciprocal of enzyme activity (1/V) against 1/[S] (1/[DHA]).

Figure 6

Initial reaction velocity V of Acsl4 plotted against increasing AA concentration [S] in control solution, 60 mM sodium butyrate, or 60 mM sodium octanoate.