Protein Kinase Inhibition by
-3 Fatty Acids*
Banafsheh
Mirnikjoo
,
Sarah E.
Brown
,
H. Florence Seung
Kim§,
Lauren B.
Marangell§,
J. David
Sweatt
, and
Edwin J.
Weeber
¶
From the
Division of Neuroscience and the
§ Department of Psychiatry and Behavioral Sciences, Baylor
College of Medicine, Houston, Texas 77030
Received for publication, September 9, 2000, and in revised form, December 20, 2000
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ABSTRACT |
Recent data suggest that
-3 fatty acids may be
effective in epilepsy, cardiovascular disorders, arthritis, and
as mood stabilizers for bipolar disorder; however, the mechanism of
action of these compounds is unknown. Based on earlier studies
implicating
-3 fatty acids as inhibitors of protein kinase C
activity in intact cells, we hypothesized that
-3 fatty acids may
act through direct inhibition of second messenger-regulated kinases and
sought to determine whether the
-3 double bond might uniquely confer
pharmacologic efficacy and potency for fatty acids of this type. In our
studies we observed that
-3 fatty acids inhibited the in
vitro activities of cAMP-dependent protein kinase,
protein kinase C, Ca2+/calmodulin-dependent protein kinase
II, and the mitogen-activated protein kinase (MAPK). Our results
with a series of long-chain fatty acid structural homologs suggest an
important role for the
-3 double bond in conferring inhibitory
efficacy. To assess whether
-3 fatty acids were capable of
inhibiting protein kinases in living neurons, we evaluated their effect
on signal transduction pathways in the hippocampus. We found that
-3
fatty acids could prevent serotonin receptor-induced MAPK activation in
hippocampal slice preparations. In addition, we evaluated the effect of
-3 fatty acids on hippocampal long-term potentiation, a form of
synaptic plasticity known to be dependent on protein kinase activation. We observed that
-3 fatty acids blocked long-term
potentiation induction without inhibiting basal synaptic transmission.
Overall, our results from both in vitro and live
cell preparations suggest that inhibition of second messenger-regulated
protein kinases is one locus of action of
-3 fatty acids.
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INTRODUCTION |
There are twelve essential fatty acids
(EFAs)1 that are subdivided
into two structural categories depending on the saturation state of the
molecule. The designation omega-3 (
-3) or omega-6 (
-6),
respectively, indicates if the third or sixth carbon from the methyl
terminus is unsaturated. The
-3 and
-6 fatty acids are derived
from the dietary intake of the 18-carbon precursors
-linolenic acid
and linoleic acid, respectively. The
-3 fatty acids include:
-linolenic acid (18:3 ALA), stearidonic acid (18:4), eicosatetraenoic acid (20:4), eicosapentenoic acid (20:5 EPA), docosapentenoic acid (22:5), and docosaehexanoic acid (22:6 DHA).
Dietary provision of long-chain
-3 and
-6 fatty acids is
essential because mammals are incapable of synthesizing fatty acids with a double bond past the
-9 position; thus, dietary intake of
EFAs has far reaching consequences on membrane composition in all cells
in the body and may influence neural function as well. The elucidation
of the role for EFAs in neuronal function has followed two disparate
lines of investigation. One approach has focused on the effects of
dietary intake of EFAs on neuronal membrane composition and function.
The reduction of dietary
-3 fatty acids has been shown to have
deleterious effects on cAMP-dependent protein kinase A
(PKA) and protein kinase C (PKC) activities (1, 2), brain membrane
lipid composition (3), neurotransmission (4, 5), and learning ability
in rats (6). In addition, increasing dietary intake of
-3 fatty
acids can improve learning and memory tasks in young rats and overcome
deficits in long-term potentiation (LTP, Ref. 7), a robust form of
synaptic plasticity, in aged rats. These effects are theorized as being
because of physical alterations in membrane characteristics, changing
either the fluidity or rigidity of the membrane itself, and affecting neurotransmitter release as well as receptor and channel function (8-10).
An alternative investigative approach has focused on the effect
in vitro of direct application of
-3 fatty acids on
cellular biochemistry and synaptic transmission. Early reports showed
that a variety of fatty acids are capable of activating PKC (11-13); however, in other studies
-3 and
-6 fatty acids caused inhibition when PKC was activated with phosphatidylserine (PS) and diacylglycerol (DAG) (14). This latter effect may explain more recent reports that the
application of
-3 fatty acids to hippocampal slices can decrease
membrane excitability and block low frequency stimulation-induced long-term depression (LTD, Refs. 15-17). Overall these reports suggest
a multifaceted role for
-3 EFAs involving both direct and indirect
actions on neural function and suggest the hypothesis that
-3 fatty
acids may affect protein kinase activity.
Questions concerning the mechanisms of action of EFAs are not trivial,
as EFAs have been shown to have profound behavioral effects in humans
in vivo. For example, dietary EPA and DHA elicit beneficial
effects in certain neuropsychiatric disorders, such as bipolar disorder
and schizophrenia. New studies of patients with manic-depression have
shown significant reduction in reoccurrence of symptoms when the
standard lithium treatment is supplemented with
-3 fatty acids (18).
The exact cellular and biochemical mechanisms underlying the
mood-stabilizing effect of
-3 fatty acids are mysterious, making
this a necessary and attractive area for study.
To better understand the biochemical affects of
-3 fatty acids on
the CNS, we focused our attention on their effects on the activities of
prevalent protein kinases of the CNS, using kinase assays in
vitro. We found that DHA and EPA reduced the activity of protein
kinase C, cAMP-dependent protein kinase A,
mitogen-activated protein kinase (MAPK) (ERK1 and ERK2), and
Ca2+/calmodulin-dependent protein kinase II
(CaMKII) at low concentrations (IC50 2-36
µM). These effects appear to be dependent on the
-3 double bond within the fatty acid, as structurally similar compounds saturated at the
-3 position had little or no effect on kinase activity. We also used the hippocampal slice preparation to assess whether
-3 fatty acids were capable of affecting protein kinase activity in the living neuron. In one series of studies, we observed that DHA and EPA inhibited serotonin (5-HT) receptor-induced activation of MAPK. In another study, we capitalized on the fact that hippocampal LTP is known to require protein kinase activation. We observed that
perfusion of DHA or EPA onto hippocampal slices interfered with the
induction of LTP in area CA1, presumably through the reduced activity
of multiple protein kinases and disruption of the signaling pathways in
which they are involved. Thus, the
-3 fatty acids are surprisingly
potent and efficacious broad-spectrum protein kinase inhibitors,
suggesting that protein kinases may be a target of action for
-3
fatty acids in vivo.
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MATERIALS AND METHODS |
Chemicals--
[
-32P]ATP was obtained from
Amersham Pharmacia Biotech. The catalytic subunit of protein kinase C
from rat brain was purchased from CalBiochem-Novabiochem Corp., protein
kinase A catalytic subunit from Sigma Chemical Co.,
Ca2+/calmodulin-dependent protein kinase II
from New England BioLabs Inc., and activated MAPK from Stratagene.
General laboratory reagents were purchased from commercial sources and
were of analytical quality.
Fatty Acids--
Docosahexaenoic acid (DHA) [22:6(
-3)];
behenic acid (BA) [22:0];
cis-7,10,13-docosatetraenoic (DTEA) [22:4(
-6)];
eicosapentaenoic (EPA) [20:5(
-3)]; arachidic [20:0]; arachidonic
[20:4(
-6)]; cis-11,14,17-eicosatrienoic (ESEA)
[20:3(
-3)]; cis-7,10,13,16,19-docosapentaenoic (DPEA)
[22:5(
-3)]; and Valproic acids were all obtained from Sigma
Chemical Co. 2-ene-valproate was kindly provided by Dr. Wolfgang
Loescher and Dr. Danny D. Shen. Free fatty acids were made as 20 mM stock solutions in ethanol (vehicle) and stored under
nitrogen, in the dark at
20 °C until needed.
Protein Kinase Assays
PKC Assay--
Enzyme activity was measured by quantifying
incorporation of [32P]PO4 into a synthetic
peptide substrate, amino acids 28-43 of neurogranin, PKC selectide,
CalBiochem. Various concentrations of fatty acids, ranging from
0.4 to 400 µM were incubated with the catalytic domain of
protein kinase C from rat brain. The reaction mixtures contained 10 ng
of the PKC catalytic domain; reaction buffer (200 mM Tris,
5 mM EGTA, 10 mM EDTA, 20 mM
Na2PO4); 10 µM of peptide
substrate; [
-32P]ATP; 0.5 mM ATP and
H2O in a final assay volume of 50 µl. The reaction
samples were incubated at 37 °C for 20 min and terminated by
addition of 25 µl of stop solution (225 mM
H3PO4, 1 mM ATP). Two aliquots from
each sample were transferred onto Whatman P-81 paper. After a 2-min
incubation at room temperature, the filter papers were washed three
times for 10 min in 0.25 M H3PO4
and one time for 2 min in 95% (v/v) ethanol with gentle agitation. Chromatography papers were air dried prior to quantitation by liquid
scintillation counting.
PKA Assay--
Assays for PKA activity were performed as
described in Roberson and Sweatt (19) with the exception that PKC and
CaMKII inhibitors were absent from the reaction buffer, and the
chromatography papers were washed in 75 mM
H3PO4 three times for 10 min before rinsing in
methanol. Kemptide (100 µM) was used as the substrate for
PKA.
CaMKII Assay--
The assays were performed as described in
Roberson and Sweatt (19). Reaction conditions consisted of 20 mM Tris-HCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 0.1 mM Na2EDTA,
100 µM autocamtide, 25 units of CaMKII, 2.4 µM calmodulin, and 2 mM CaCl2 (to
activate the enzyme), 100 µM ATP and
-32P-labeled ATP to a final specific activity of 100 µCi/µmol. The mixture was incubated for 10 min at 30 °C before
stop solution was added.
MAPK Assay--
Reaction mixtures (prepared on ice) contained 20 ng of (1:5 dilution of 0.1 µg/µl MAPK) activated MAPK, 25 mM HEPES, pH 7.5, 10 mM magnesium acetate, 500 µM ATP; 0.5 µg/µl Phas-I, [
-32P]ATP
1.0 µCi/µl, and various concentrations of DHA and EPA (0.1-100 µM) in a final assay volume of 40 µl. The reaction
samples were incubated 30 °C for 30 min and terminated by placing
the reaction samples on ice with 5 µl of 0.25 M
H3PO4 (stop solution). Two aliquots of each
sample were transferred onto P-51 chromatography paper. After 2 min of
incubation at room temperature, the filter papers were washed four
times for 3 min each in 100 ml of 75 mM H3PO4 and then briefly in 95% (v/v) ethanol
with gentle agitation. Chromatography papers were air-dried before the
-32P incorporation was quantitated by liquid
scintillation counting.
Hippocampal MAPK Activation and Quantitation--
Hippocampal
slices (400 µm) were prepared from 8-12 week-old male mice as
described previously (19). Slices were allowed to recover in artificial
cerebral spinal fluid (ACSF: 125 mM NaCl, 2.5 mM KCl, 1.24 mM
NaH2PO4, 25 mM NaHCO3,
10 mM D-glucose, 2 mM
CaCl2, and 1 mM MgCl2) for 3 h
at 32 °C.
-3 fatty acids or control fatty acids (10 µM) were added to slices for 1 h prior to 5-HT
addition. 5-HT was added to slices at a 10 µM
concentration for 10 min at which time the slices were collected and
frozen on dry ice. Slices were then homogenized, and samples were
prepared as described previously (19). Each n represents the
pooled protein homogenate from three whole hippocampal slices. The
samples were electrophoresed on 10% SDS-polyacrylamide gels, and
transferred to immobilon-P membranes. Membranes were blocked in 5% dry
milk solution and 1 µM microcysteine for 1 h. All
blots were incubated at room temperature and washed in TTBS buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05%
Tween 20). Immunoreactivity was assessed using the enhanced
chemiluminescence method (Amersham Pharmacia Biotech). The bands
corresponding to phospho-p42 MAPK on each Western blot were quantified
by densitometry using a StudioScan desktop scanner and NIH Image software.
Hippocampal Slice Physiology--
Hippocampal slices (400 µm)
were prepared from 8-12 week-old male mice as described previously
(19). Slices were perfused (1 ml/min) with ACSF in an interface chamber
maintained at 25 °C. Field recordings of the Schaffer collateral
synapse were monitored for a minimum of 10 min before fatty acid
application to ensure a stable baseline. Responses are presented as an
average of 6 individual traces. Baseline stimulus intensities were
determined from the intensity that produced a field EPSP at 50% of the
maximal response. LTP was induced with two trains of 100-Hz stimulation for 1 s, separated by 20 s with the same stimulus intensity
used in baseline recordings. Slices were perfused with ACSF at 1 ml/min with either fatty acids diluted to 10 µM or ACSF with the
addition of an equal volume of vehicle. Fatty acids were perfused onto the slice 20 min prior to HFS and remained for 20 min after HFS delivery for a total of 40 min (see Fig. 7; B-D, black
bar).
Data Analysis--
The GraphPad Prism and Microsoft Power Point
software packages were used for curve-fitting and statistical analyses.
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RESULTS |
The therapeutically beneficial
-3 fatty acids (typically found
in fish oils) are long-chain, unbranched molecules. In the present
studies we used a series of structurally related long-chain fatty acids
to investigate the structure/function relationships for this category
of compounds, examining their efficacy as inhibitors of protein kinases
(Fig. 1). For the DHA family of fatty
acids we investigated three structurally similar compounds.
Docosahexaenoic acid [22:6 (
-3), DHA] is a twenty-two carbon
-3
fatty acid with a total of six double bonds. Compounds used as DHA
controls included Behenic acid [22:0, BA], the saturated form of DHA
lacking all double bonds including the
-3 double bond, and
cis-7,10,13,16-docosatetraenoic acid [22:4 (
-6), DTEA],
which lacks the
-3 double bond and the number four double bond (Fig.
1). Eicosapentaenoic acid [20:5 (
-3), EPA] is a twenty carbon
-3 fatty acid with a total of five double bonds. Arachidic acid
[20:0], the saturated form of EPA, and Arachidonic acid [20:4
(
-6), AA], lacking the
-3 double bond, were also investigated as
EPA controls (Fig. 1).

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Fig. 1.
Structures of -3
fatty acids and control isomers. A graphic representation of the
fatty acids used in these experiments is shown. DHA,
docosahexaenoic acid [22:6 ( -3)]; Behenic acid (saturated DHA)
[22:0]; DTEA, cis-7,10,13,16-docosatetraenoic
acid (DHA analog lacking the -3 double bond) [22:4 ( -6)];
DPEA, cis-7,10,13,16,19-docosapentaenoic acid
[22:5 ( -3)]; EPA, eicosapentaenoic acid [20:5
( -3)]; arachidic acid (the saturated form of EPA) [20:0];
AA, arachidonic acid (EPA isomer lacking the -3 double
bond) [20:4 ( -6)]; ESEA,
cis-11,14,17-eicosatrienoic acid; valproic acid [8:0];
2-ene-valproate [8:1 -3].
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DHA and EPA Inhibit the PKA Catalytic Subunit--
We first tested
if the parent
-3 compounds DHA and EPA caused an effect on PKA
catalytic subunit activity. To determine the potency of DHA and EPA as
PKA inhibitors, we used various concentrations of fatty acids (0.4-400
µM) in assays in vitro for PKA phosphorylation of Kemptide substrate. Both DHA and EPA significantly inhibited the
activity of the PKA catalytic subunit with an IC50 of 34 µM for DHA and 2 µM for EPA (Fig.
2, A-B). Compounds such as BA
and DTEA (Fig. 1) lacking the
-3 moiety were used as controls for DHA. BA, the saturated form of DHA, did not significantly inhibit PKA
activity (Fig. 2A). DTEA, which not only lacks the
-3
double bond but also lacks the double bond in the fourth position, had a substantially higher IC50 than DHA, with an
IC50 of 230 µM. These results demonstrate
that DHA is more potent in PKA inhibition than BA and DTEA and suggest
that the
-3 double bond is important in conferring potency and
efficacy for inhibition of PKA. As controls for EPA we evaluated
arachidic acid and AA. Arachidic acid, the saturated form of EPA
lacking the
-3 double bond, was not as potent in the inhibition of
PKA phosphotransferase activity as EPA. This was evident in the
calculated IC50 of >400 µM for arachidic acid. In addition, a calculated IC50 of 59 µM
for AA, which is structurally equivalent to EPA except for the lack of
the
-3 double bond, was 30-fold greater than the IC50
for EPA of 2 µM (Fig. 2B). Overall these data
indicate that both DHA and EPA effectively inhibit PKA activity
in vitro and suggest that the
-3 double bond plays an
important role in conferring inhibitory efficacy and potency for both
types of compounds.

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Fig. 2.
Inhibition of PKA by DHA and EPA.
In vitro measurement of PKA catalytic domain activity using
Kemptide as a substrate. A, DHA -3 fatty acid at
increasing concentrations (1-400 µM) inhibits PKA
catalytic subunit activity. Behenic acid (BA) and DTEA were
used as 22-carbon chain-length control fatty acids. The
IC50 for DHA is 34 µM; for BA >400
µM, and for DTEA, 230 µM. B, EPA
-3 fatty acid at increasing concentrations (1-400 µM)
to inhibit PKA catalytic subunit activity. Arachidic acid and AA were
used as 20-carbon chain-length control fatty acids. The
IC50 for EPA is 2.1 µM; for arachidic acid
>400 µM; and for AA, 59 µM. C,
ESEA and DPEA fatty acids (concentrations of 1-400 µM
and 0.1-400 µM, respectively) inhibited PKA catalytic
subunit activity. The IC50 of ESEA is 22 µM
and DPEA is 10 µM. The results shown are mean ± S.E. for three experiments in all cases.
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The
-3 Fatty Acid Moiety Is Important in Inhibiting PKA
Activity--
Given the results described above, we wanted to test if
the presence of the
-3 double bond was sufficient to confer
inhibitory efficacy. We therefore tested two other long-chain fatty
acids that have the
-3 double bond moiety incorporated into their
structure. Toward this end we evaluated
cis-11,14,17-eicosatrienoic acid (ESEA), a twenty-carbon
-3 fatty acid with three double bonds, and
cis-7,10,13,16,19-docosapentaenoic acid (DPEA), a twenty-two carbon
-3 fatty acid with a total of five double bonds (Fig. 1). We
examined whether ESEA or DPEA exhibited inhibitory effects by
evaluating the effect of increasing concentrations (1.0-400 µM for ESEA and 0.1-400 µM for DPEA) on
PKA phosphotransferase activity (Fig. 2C). We found that
both ESEA and DPEA blocked PKA catalytic subunit activity;
IC50 values were calculated to be 22 µM for
ESEA and 10 µM for DPEA. Once again these data suggest that the
-3 double bond plays an important role in the inhibition of
PKA by these long-chain fatty acids. This is illustrated quite nicely,
for example by comparing the potencies of DTEA versus DPEA
(Fig. 2, A versus C). These two
compounds are structurally identical with the exception of the
-3
double bond contained within DPEA, which showed much greater potency
than DTEA in PKA inhibition.
EPA Is Not a Competitive Inhibitor of ATP--
Most broad-spectrum
inhibitors of second messenger-regulated kinases are competitive with
ATP (20, 21), and we sought to determine whether this was the mechanism
of action of
-3 fatty acids. If
-3 fatty acids are competitive
with ATP, then higher ATP concentrations should cause a shift to a
higher IC50 for
-3 fatty acid inhibition of PKA. We
therefore compared the IC50 for EPA inhibition of PKA,
using two different final ATP concentrations: 100 and 500 µM. EPA reduced PKA activity indistinguishably under both
concentrations with no significant difference in inhibition of PKA
activity (IC50 for both ATP concentrations was ~2
µM, Fig. 3). These data
strongly suggest that EPA is not a competitive inhibitor of ATP binding
to PKA.

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Fig. 3.
EPA is not a competitive inhibitor of
ATP. Concentrations of 100 and 500 µM ATP were added
to the reaction assay to determine whether EPA inhibition of PKA was
caused by competition with ATP. The IC50 for EPA with 100 µM ATP is 2.1 µM and EPA with 500 µM ATP is 1.7 µM. The results shown are
mean ± S.E. for three experiments in both cases.
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-3 Fatty Acid Inhibition of PKC, CaMKII, and MAPK--
If fatty
acids unsaturated in the
-3 position have the capability of
inhibiting the catalytic subunit of PKA, then it is reasonable to
hypothesize that this effect may transcend to other protein kinases
with homologous catalytic domains. Therefore we examined the effect of
different long-chain fatty acids on two other kinases; PKC and CaMKII.
We tested EPA, DHA, and AA on PKC catalytic domain phosphotransferase
activity using a peptide substrate (in vitro) using
increasing fatty acid concentrations (Fig.
4A). We found that
-3 fatty
acids inhibited PKC and, similar to results for PKA, EPA was found to
be a more potent inhibitor of PKC activity than DHA (IC50
for EPA was 2 µM compared with 36 µM for
DHA). AA, which lacks the
-3 double bond, was used as a control and did not cause significant inhibition of PKC enzymatic activity (IC50 >400 µM). Similarly, the inhibitory
effect of EPA and DHA was also shown in assays using the catalytic
subunit of CaMKII (Fig. 4B). Both EPA and DHA inhibited
CaMKII activity at relatively low concentrations with a calculated
IC50 for EPA of 15 µM and for DHA of 36 µM. Thus similar to what was observed with PKC and PKA,
EPA and DHA inhibit CaMKII.

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Fig. 4.
EPA and DHA inhibition of PKC, MAPK, and
CaMKII. A, EPA and DHA inhibit PKC catalytic domain
activity. PKC activity was measured as described under "Materials and
Methods." The IC50 for EPA is 1.8 µM, DHA
is 36 µM, and AA >400 µM. AA inhibits PKC
activity only at high concentrations (400 µM). EPA
inhibits the PKC activity at lower concentrations than DHA.
B, EPA and DHA inhibition of CaMKII catalytic domain.
Measurement of CaMKII activity using autocamtide as a substrate was as
described under "Materials and Methods." The IC50 for
EPA is 15 µM and DHA is 36 µM. EPA inhibits
CaMKII activity at a lower concentration than DHA. C, EPA
and DHA inhibition of MAPK activity. EPA and DHA concentrations ranged
from 0.1 to 100 µM. Results shown are mean ± S.E.
for three experiments in all cases. Measurement of MAPK activity using
Phas-I as a substrate was as described under "Materials and
Methods." The IC50 for EPA is 23 µM and DHA
is 34 µM. Both EPA and DHA inhibit MAPK completely at
concentrations higher than 40 µM, but EPA is a more
potent inhibitor than DHA.
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The second messenger-regulated kinases PKA, PKC and CaMKII share
sequence homology within their catalytic domains. The ERK MAPKs are
regulated by phosphorylation and not second messengers, but
nevertheless exhibit structural homology to the PKA, PKC, and CaMKII
catalytic domains (22, 23). We therefore determined if
-3 fatty
acids inhibited MAPK activity. To determine the potency of EPA and DHA
at inhibiting MAPK activity, increasing concentrations of both fatty
acids (0.1-100 µM) were used (Fig. 4C). Both
-3 fatty acids inhibited MAPK activity, and EPA was a slightly more potent inhibitor of MAPK activity than DHA (IC50 for EPA
was 23 µM and for DHA, 34 µM). Overall
these data show that DHA and EPA act as broad-spectrum kinase
inhibitors, blocking not only the activity of PKA, PKC, and CaMKII, but
also the activity of MAPK.
VPA and 2-ene-VPA Exhibit No Effect on PKC or PKA--
Valproic
acid (VPA, 2-propylpentanoic acid), is a short chain length, eight
carbon, branched fatty acid (Fig. 1) and, like
-3 fatty acids, VPA
has recently been used for treatment of bipolar disorder (24, 25).
2-ene-VPA (2-ene-valproate, 2-propyl-2-pentenoic acid, Fig. 1) is the
major VPA metabolite in humans, which also contains the
-3 moiety.
Given that VPA and the
-3 fatty acids DHA and EPA exhibit similar
neurologic effects, we thought it an intriguing possibility that
2-ene-VPA might inhibit protein kinases in a fashion similar to the
long-chain fatty acids described above. Therefore we tested whether VPA
or 2-ene-VPA had the same inhibitory effect as long-chain
-3 fatty
acids on PKA and PKC activity in vitro. As expected, we
found a lack of an inhibitory effect of VPA on PKC or PKA
phosphotransferase activity at concentrations ranging from 1 to 200 µM (data not shown). Interestingly, there was also a lack
of inhibition of PKA and PKC activity with 2-ene-VPA using
concentrations of 100, 200, and 400 µM (Fig.
5). Thus, the
-3 moiety within the
2-ene-VPA does not confer significant inhibitory efficacy for PKC or
PKA activity in vitro. This lack of effect of 2-ene-VPA may
be because of its overall short chain length or the proximity of the
carboxylic acid in relation to the
-3 double bond. These data also
indicate that neither VPA nor its major metabolite act by directly
inhibiting PKA or PKC, suggesting that a different mechanism of action
exists in vivo for VPA versus
-3 long-chain
fatty acids.

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Fig. 5.
Lack of effect of 2-ene-VPA on PKC and PKA
activity. Data are presented as the percent change of PKA ( )
and PKC ( ) catalytic domain activity in the presence of 2-ene-VPA
compared with control assays. The in vitro action of
2-ene-VPA on PKA and PKC activities were determined using increasing
concentrations of 100, 200, and 400 µM. Results shown are
mean ± S.E. for n = 3.
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DHA and EPA Can Inhibit Serotonin-induced Activation of
MAPK--
Whereas the results thus far demonstrate that DHA and EPA
have effects on kinase activity in vitro, they do not
address whether the compounds are capable of inhibiting protein kinases
in the intact cell. For the next stage of our studies we sought to
develop a preparation that we could use to test the efficacy of
-3
fatty acids in inhibiting protein kinases in living cells. The
serotonergic signal transduction pathway can activate MAPK in a
PKA-dependent process in aplysia sensory neurons, but the
effects of 5-HT on MAPK in the mammalian brain have not been determined
(26). We reasoned that investigating serotonin-coupled kinase
activation was a desirable avenue of pursuit because the serotonergic
system is widely implicated in affective disorders for which
-3
fatty acids have beneficial effects. In our first studies, we found that 10 µM 5-HT caused MAPK activation in hippocampal
slices evaluated by the increased phosphorylation of p42 MAPK (Fig.
6), an effect blocked by EPA and DHA. The
control compounds behenic acid and arachidic acid showed no inhibition
of 5-HT receptor-induced MAPK activation. Application of
-3 fatty
acids (DHA and EPA) or control fatty acids (behenic acid and arachidic
acid) to hippocampal slices alone had no effect on basal MAPK
activation (data not shown). As an additional control we used the MEK
inhibitor U0126 on 5-HT receptor-induced Erk activation. The compound
U0126 completely blocked 5-HT-induced activation of MAPK in the
hippocampus. Taken together, these results suggest that
-3 fatty
acids are capable of blocking serotonin-induced kinase activation in
living neurons in the hippocampus.

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Fig. 6.
Effect of EPA and DHA on serotonergic signal
transduction. Serotonin activation of MAPK can be blocked by DHA
and EPA, but not behenic acid or arachidic acid. Effect of a 10-min
application of 5-HT on MAPK activation in mouse hippocampal slices
(lane 2) compared with 5-HT activation following a 1-h
incubation with DHA, EPA, behenic acid (BA), and arachidic
acid (lanes 4-8, respectively). Controls consisted of
untreated (lane 1) and slices treated with the MEK inhibitor
U0126 (lane 3). Top, representative immunoblot
for phospho-p42 and phospho-p44 MAPK from whole hippocampal
homogenates. Bands correspond to the labeled column directly
beneath. Bottom, quantitative Western analysis of
immunoblots that were probed for phospho-MAPK (p42) and standardized to
nontreated controls. Application of -3 fatty acids (DHA and EPA) or
control fatty acids (behenic acid and arachidic acid) to hippocampal
slices alone had no effect on basal MAPK activation (data not shown).
Columns and error bars represent the mean ± S.E. from n = 3 or 5 Western blot analyses (**,
p < 0.01).
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DHA and EPA Inhibit Induction of Hippocampal LTP in Area
CA1--
The very kinases shown here to be inhibited by
-3 fatty
acids have been shown previously to be necessary for mammalian synaptic plasticity, specifically LTP in the hippocampus. To determine the
effect of
-3 fatty acids on this kind of
kinase-dependent synaptic plasticity in intact living
neurons, we tested the ability of EPA and DHA to block the induction of
LTP in area CA1 of the hippocampus. The application of HFS (two trains
at 100 Hz for 1 s, separated by 20 s) is sufficient to induce
a long lasting potentiation at Schaffer collateral synapses (Fig.
7A). Application of DHA (10 µM) did not affect baseline synaptic transmission; however, perfusion prior to HFS completely blocks the induction of LTP
(Fig. 7C). Like DHA, EPA has little effect on baseline synaptic transmission and also blocks induction of LTP (Fig.
7D). As a control, we perfused slices with the long-chain
fatty acid AA prior to HFS. (Fig. 7B). Slices treated with
AA showed potentiation (>150% compared with baseline) that was not
different from untreated slices. Our results are consistent with the
observations of Itokazu et al. (27), who show that
intracerebroventricular injection of DHA inhibited the induction of LTP
in hippocampal area CA1, in vivo. Our studies examining the
effect of the
-3 moiety show that both EPA and DHA can disrupt LTP
in hippocampal slice preparations and that LTP is unaffected with the
structural analog AA that lacks the
-3 double bond. Interestingly,
the effects of DHA and EPA on tetanus-induced potentiation in
hippocampal area CA1 are typical of the effect of broad-spectrum kinase
inhibitors; blocking late stages of LTP whereas leaving a transient
potentiation intact. Overall, our studies suggest that DHA and EPA
inhibit LTP induction via the reduction of neural protein kinase
activity.

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Fig. 7.
Effect of DHA and EPA on Hippocampal LTP in
area CA1. LTP in area CA1 of the hippocampus can be induced by HFS
consisting of two trains of 100-Hz stimulation for 1 s separated
by a 20-s interval (indicated by an arrow). In control
slices, a stable 20-min baseline recording was followed by HFS
resulting in a long-lasting increase in synaptic potentiation
(A). Following a stable 20-min baseline recording, vehicle
(ETOH)(A), AA (B), DHA (C), or EPA
(D) (10 µM) was bath-applied (black
bar) for 20 min prior to HFS and maintained for another 20 min
following HFS (40 min total). All recordings are normalized to the mean
field excitatory postsynaptic potential (fEPSP) of the initial
20-min baseline recordings. Data points and error
bars represent the mean ± S.E. from n = 4 slice recordings.
|
|
 |
DISCUSSION |
The purpose of these studies was to investigate the mechanism of
action of
-3 fatty acids and to clarify the effects of
-3 fatty
acids on specific protein kinases established as playing important
roles in signal transduction, synaptic function and plasticity in the
mammalian CNS. In our in vitro experiments, EPA and DHA
significantly reduced the activity of PKA, PKC, MAPK, and CaMKII. This
effect was in contrast to control fatty acids of similar composition
that were saturated at the
-3 carbon (BA, DTEA, AA, arachidic acid),
which exhibited a greatly diminished potency in kinase inhibition. Our
in vitro observations that
-3 fatty acids may act as
nonspecific protein kinase inhibitors is an important first step in
understanding the potential molecular targets of free
-3 fatty acids
in the cell. To test our hypothesis that
-3 fatty acid effects on
protein kinases can have a physiologic effect in living tissue, we
evaluated DHA and EPA effects on mouse hippocampal slices. We found
that application of low concentrations of
-3 fatty acids blocked
5-HT-receptor induced kinase activation and blocked the induction of
LTP in area CA1 of the hippocampus. This indicates that
-3 fatty
acids block protein kinase activity in the intact cell as well as
in vitro, and suggests the interesting possibility that
neuronal kinase inhibition may underlie the known therapeutic effects
of
-3 fatty acids in affective disorders.
In our experiments, we measured the phosphotransferase activity of PKA,
PKC, MAPK, and CaMKII in the presence of the
-3 fatty acids DHA and
EPA and fatty acids of equal length with different degrees and sites of
saturation. Both DHA and EPA appear to reduce kinase activity at low
concentrations in vitro with EPA appearing to be slightly
more efficient in reducing kinase activity than DHA. Importantly, only
the catalytic domains of PKA, PKC and CaMKII were used in these assays.
Thus in our experiments
-3 fatty acid interactions occur at site(s)
other than the second messenger binding sites present in the
holoenzymes. However, the observed effect of
-3 fatty acids did not
appear to be due to competitive inhibition at the ATP binding domain,
as increasing the concentration of ATP in the kinase assay had no
effect on the efficacy of EPA inhibition. Recently, Radominska-Pandya
et al. (28) reported that direct binding of
all-trans-retinoic acid (atRA) to PKC can significantly
reduce its activity in vitro; however the enzyme region
responsible for this interaction has yet to be identified. Given that
atRA is also a long-chain unbranched fatty acid, it is an appealing
speculation that atRA and
-3 fatty acids share a similar locus of action.
We show that addition of 5-HT (10 µM) to hippocampal
slices can activate MAPK. This activation can be blocked by
-3 fatty acids, but not with structurally similar fatty acids lacking the
-3
double bond moiety. Our results showing the effect of
-3 fatty acids
on 5-HT-dependent signal transduction raises the
interesting idea that this pathway may be a locus for the
mood-stabilizing effects of
-3 fatty acids. This hypothesis is
supported by observations that alterations in serotonergic
neurotransmission have been implicated in the pathophysiology of major
depression and suicide. Moreover, treatment of depression often
involves the regulation of 5-HT-coupled intracellular signal
transduction pathways through the therapeutic action of 5-HT specific
reuptake inhibitors, which act to desensitize 5-HT receptors and
depress serotonergic processes. A normal function for
-3 fatty acids
in serotonergic function may exist, in light of studies showing that
chronic
-3 fatty acid deficiency in rats can alter serotonergic
neurotransmission (3), although it is unknown if these effects are
because of changes in membrane-associated
-3 fatty acids or the
availability of free
-3 fatty acids. Overall, our results support
the hypothesis that a means by which
-3 fatty acids may exert their
beneficial mood stabilizing actions is through the suppression of
5-HT-dependent signal transduction pathways.
We determined that perfusion of
-3 fatty acids prevents the
induction of LTP in area CA1 of the hippocampus. The necessity for PKA,
PKC, MAPK, and CaMKII in synaptic plasticity is well established, as
numerous studies have shown that inhibition of one or more of these
kinases, or pathways in which these kinases are involved, will disrupt
LTP induction (19, 29-31). Perfusion of hippocampal slices with low
concentrations (10 µM) DHA or EPA, but not arachidonic
acid, was sufficient to disrupt induction of LTP. This strongly
suggests that the inhibition of CA1 LTP is the result of DHA and
EPA-dependent reductions in the activities of several
kinases involved in LTP induction. These results may give a glimpse
into the ability of
-3 fatty acids to exert an effect on neuronal
function in vivo. The
-3 fatty acid concentrations we
used in our LTP studies caused only a modest kinase inhibition in our
in vitro studies, but were nonetheless able to block LTP induction. It is possible that in these experiments hippocampal slices
are concentrating the highly hydrophobic fatty acids, increasing the
concentration locally. An interesting alternative possibility is that
the effect is because of the kinase-dependent signal
integration and amplification that occurs during high frequency
stimulus-induced LTP induction. In this scenario, slight reductions in
the activity of CaMKII, PKA, PKC, and MAPK sum up to prevent the cell
from reaching an LTP induction threshold. Regardless, reducing kinase activity through
-3 fatty acid application does not appear to interrupt basic synaptic function because the application of EPA or DHA
appears to have no effect on normal CA1 synaptic transmission.
The present studies complement previous research into the action of
dietary
-3 fatty acids, specifically on membrane composition, and
the associated changes in synaptic function. Work by McGahon et
al. (5) has shown that increasing the dietary consumption of
-3
fatty acids has the ability to overcome age-related impairments in LTP.
This raises the interesting question of how dietary intake of
-3
fatty acids can rescue hippocampal LTP in light of studies by Young
et al. (16) showing that DHA can inhibit low
frequency-induced LTD and our studies showing that perfusion of
-3
fatty acids can disrupt high frequency-induced LTP. It may be that the
depletion of
-3 fatty acids, through dietary restriction or normal
aging, has a deleterious effect on CNS membranes, and thus the
supplementation of dietary fatty acids restores synaptic membrane
function. However, available data suggest that the idea that
-3
fatty acids have a role limited to maintaining membrane fluidity is
unlikely. Actions of free, unesterified DHA and EPA are suggested in
response to a variety of stimuli such as cortical injury (32),
modulation of calcium signaling in vascular smooth muscle (33) and in
proper retinal function (34, 35). In addition, it has long been known that release of
-3 fatty acids can occur following electroconvulsive shock and ischemia (36, 37) and, more recently, the identification of
specific sites for
-3 fatty acid accumulation at the synapse (38,
39) would appear to support the hypothesis that both free and
membrane-bound
-3 fatty acids may play diverse roles in synaptic function.
Whereas we have focused our studies on direct effects of
-3 fatty
acids on protein kinases, it is possible, even likely, that
-3 fatty
acids play multiple roles in neuronal function and regulation through
direct action upon membrane-bound proteins, alterations in physical
membrane characteristics, and direct action upon protein
kinases. However, we find it interesting to consider the possible role
for direct
-3 fatty acid-induced inhibition of protein kinases in
the CNS. Particularly intriguing is the possibility of
stimulus-provoked release of free fatty acids as a mechanism for
neuromodulation (Fig. 8). Specifically,
we propose that liberation of free
-3 fatty acids through the
receptor-mediated activation of phospholipases may lead to transient
local elevation of DHA and EPA and consequent inhibition of protein
kinase activity. This biochemical mechanism could serve as a means
whereby one receptor could dampen the effect of another simultaneously
activated receptor. This raises the possibility that the mood
stabilizing effects of increased dietary EPA and DHA resides in the
increased abundance of
-3 fatty acids available for lipase-mediated
release (40). The fact that EPA concentrations in the brain are
extremely low (<1.0% of total fatty acid, Refs. 41, 42) coupled with our results showing the potent inhibitory effect of very low
concentrations of EPA, would support this model.

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Fig. 8.
A model for EPA and DHA action on synaptic
function. This diagram depicts the possible actions of -3 fatty
acids within the CNS. DHA and EPA may play a role in at least two
cellular areas; the incorporation into the plasma membrane and the
presence of intracellular free -3 fatty acids. The integration of
-3 fatty acids may alter membrane dynamics, which subsequently can
affect function of postsynaptic receptors and membrane-bound enzymes,
as well as presynaptic glutamate release. Free cytoplasmic EPA and DHA
in the postsynaptic neuron may exert sufficient inhibitory action on
protein kinase activities to alter synaptic plasticity events,
dependent on gene expression and protein synthesis, or reduce overall
neuronal excitability by blocking modulation of potassium
channels.
|
|
In conclusion, our results illustrate that one mechanism by which
-3
fatty acids can affect cellular function is to reduce the activity of
several protein kinases and thereby down-regulate the signal
transduction pathways in which these kinases are involved. This
potential mechanism should be kept in mind when considering the
therapeutic benefits of
-3 fatty acids in heart disease, inflammation, metabolic disorders, cancer, and neuropsychiatric disorders.
 |
FOOTNOTES |
*
This work was supported by grants from the National
Institutes of Health, NARSAD, and the Texas Advanced Technology
Program.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. E-mail:
eweeber@cns.bcm.tmc.edu.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M008150200
 |
ABBREVIATIONS |
The abbreviations used are:
EFA, essential fatty
acid;
PKA, cAMP-dependent protein kinase A;
PKC, protein
kinase C;
CaMKII, Ca2+/calmodulin-dependent
protein kinase II;
MAPK, mitogen-activated protein kinase;
HFS, high
frequency stimulation;
LTP, long-term potentiation;
LTD, long-term
depression;
DHA, docosahexaenoic acid;
BA, behenic acid;
DTEA, cis-7,10,13-docosatetraenoic;
EPA, eicosapentaenoic acid;
AA, arachidonic acid;
ESEA, cis-11,14,17-eicosatrienoic
acid;
DPEA, cis-7,10,13,16,19-docosapentaenoic acid;
VPA, valproic acid;
2-ene-VPA, 2-ene-valproate.
 |
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