Vol. 171, No. 3, 1990 September 28, 1990
BIOCHEMICAL
REGULATION
OF ION UPTAKE RAT BRAIN
Lucien Laboratory
Bettendorff, of General
August
IN MEMBRANE
BY THIAMINE
Pierre
VESICLES
FROM
COMPOUNDS
Wins,
and Ernest
and Comparative
Liege, Received
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1137-1144
Schoffeniels
Biochemistry,
B-4020 Liege,
University
of
Belgium
7, 1990
We examined the effects of thiamine derivatives on ion uptake in rat brain membrane vesicles. Thiamine triphosphate (1 mM) and pyrithiamine (0.1 mM) increase chloride uptake. Preincubation of crude homogenate with thiamine or pyrithiamine increases chloride uptake while oxythiamine has the reverse effect. Thiamine and oxythiamine also affect 22Na+ and g6Rb+ uptake in the same way as for 36Cl- but to a lesser extent. Thiamine-dependent 36Cluptake is activated by sodium bicarbonate (10 mM) and partially inhibited by bumetanide (0.1 mM) and 2,4-dinitrophenol (0.1 mM) . Prelncubation with thiamine increases the thiamine triphosphate content of the vesicles. The hypothesis that TTP is the activator of a particular chloride uptake mechanism is discussed. O1990 Academic Pre?.s,
Inc.
Although phosphate system
there
is much
derivatives,
has
independent
of
diphosphate
(TDP).
still
unidentified
was
remain based
thiamine Itokawa
on
this
the
that
a specific
the
known
molecular [1,2].
the
hypothesis
triphosphate
(TTP).
thiamine, function
targets Our own that This
thiamine highly
electricus in rat specialized
or some of its in
coenzyme
the
for those key
of thiamine
to this
problem
is played
by
first
suggested
by
by the finding
organ,
TTP, which
brain,
makes
up 90% of total also
effects
role
possibility,
We
nervous
special
electric
tissue.
the
role
approach
and Cooper [3], was substantiated
Eiectrop~orus of total
evidence
[4] that
accounts
in
for 1 X
thiamine
in
characterized
a
ABBREVIATIONS: DlDS, 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid; TMP, thiamine monophosphate; TDP, thiamine diphosphate; TTP. thiamine triphosphate. 0006-291X/90
1137
All
$1.50
Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
membrane-associated thiamine triphosphatase in this electric tissue. The enzyme is activated by anions and irreversibly inhibited by 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS), an anion transport inhibitor [5,6]. Although DIDS may react unspecifically with amino groups of the active site, we did observe that anions protected against DIDS inhibition. This led us to suspect that TTP could be involved in some kind of anion transport mechanism. The data reported here are the first piece of evidence in favor of this hypothesis. Experimental
Procedures
Materials: TTP was a gift from Dr Yamazaki from the New Lead Laboratories (Sankyo Co., Ltd., Tokyo, Japan). Pyrithiamine, oxythiamine, TDP,TMP, thiamine, DIDS, bumetanide were purchased from Sigma (St. Louis, MO, U. S. A. ) . [%l]NaCl and [86Rb] RbCl were from Amersham International (Buckinghamshire, England) and [22Na]NaCl was from New England Nuclear. M mb e le D euaration: The procedure . descri:d by”Harriy:z Alla: [7]. Female Wistar rats (150-&l $ were decapitated and the brain homogenized by hand (6 to 8 strokes in a glass-teflon homogenizer) in 15 ml ice-cold incubation buffer (145 mM NaCl, 5 mM KCl, 1 mM MgC12, 1 mM CaCl2, 10 mM D-glucose and 10 mM HEPES-Tris buffer at pH 7.5). The homogenate was filtered through a double layer of gauze. In some experiments aliquots of the homogenate were incubated (15 min at 37°C) in the absence or in the presence of 1 mM thiamine, oxythiamine or pyrithiamine and processed as follows. The homogenate, incubated or not, was centrifuged at 900 g for 15 minutes> the pellet suspended in 10 ml of buffer and again centrifuged for 15 minutes at 900 g. The final pellet was suspended in the incubation buffer at a protein concentration [8] of about 8 mg/ml. Attempts to fractionate the preparation further did not yield better results. Measurement of 36ainflux: This method is as described by Harris and Allan [7) for GABA-mediated chloride uptake into brain membrane vesicles with slight modifications. 75 Pl of the membrane suspension were incubated at 57°C in the presence of thiamine compounds at the concentrations indicated. After 10 minutes 25 ~1 of a buffer solution containing 36~ (0.5 &ml) and NaHC% (40 mM, PH 7.5) were added. Ten seconds after the addition of 3%X’, influx was stopped by addition of 4 ml ice-cold incubation buffer and rapid flltratlon through 2.4 cm Whatman SF/C filters. The filters were rapidly washed with 8 ml of ice-cold buffer and the radioactivity of the filters was counted by liquid scintillation spectrometry.
Thiamine and its phosphorylated compounds were determined using a HPLC procedure exactly as described previously [9,4] . Prior
Vol.
171, No. 3, 1990
to injection thiochromes
BlOCHEMlCAL
Although
a role
trying
using
on a preparation
previously
been characterized chloride
carried
out
derivatives
on
Preliminary results incubation
%l-
medium.
demonstrate
We can
10 9%while
have
no
TTP
and while
(1 mM)
TDP, TMP,
significant
pyrithiamine
15 mM
Our first effect
which
has
of
NaHQ
more
thiamine (Table
1).
reproducible
is present
obtained
of
experiments
vesicles
that
were
to examine
in
the
for ion uptake
10 or 20 sec.
see that
about
when
shown
uptake
used for the study
membrane
have
TTP
of any
ion
vesicles
a direct
by
aware
We decided
[7,10,11,12].
The best results
lasting
not
by direct
and intensively
uptake
obtained
measurements
effects:
to
we are
tracers.
uptake
fluorescent
and especially
of sealed membrane
experiments are
thiamine
hypothesis
radioactive
this point
GABA-mediated
that
this
into
Discussion
in ion permeability
to elucidate
measurements
were
and
it is acknowledged
play
studies
RESEARCH COMMUNICATIONS
thiamine derivatives were transformed for fluorescence detection. Results
could
AND BIOPHYSICAL
thiamine
effect. (5.1
chloride
uptake
and sodium
tripolyphosphate
thiamine
antimetabolites,
Two
oxythiamine,
pyrithiamine
increases
were
found
mM)
slightly,
to
have but
opposite
significantly
Table 1: Chloride uptake (mean f SD) in membrane vesicles in the presence of several thiamine derivatives and GABA Compound tested Control TTP (1 mM) TDP (1 mM) TMP (1 mM) Thiamine (1 mM) Sodium tripolyphosphate Pyrithiamine (100 PM) Oxythiamine (1 mM) GABA (30 PM)
number of experiments
(1 mM)
63 31 39 12 27 8 66 25 10
Chloride uptake (nmol/mg/lO set) 38.6 f 42.6 f 39.4 f 38.6 f 37.6 f 36.6 f 40.0 f 35.4 f 63.4 f
2.2 2.6” 2.6 1.6 2.2 2.4 1.0s 2.4** 5.6**
The experiments were carried out as described in Materials and Methods. 3%1- influx was measured for 10 seconds. The treatments are compared to the control group using analysis of variance (p < 0.05) followed by the Dunnett test (*, p < 0.05; %*, p -z 0.01)
1139
by
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
increases chloride uptake, oxythiamine (1 m&l) is inhibitory. As a control, the effect of GABA was also tested. As expected GABA (30 PM) increases chloride uptake to the same extent as previously found [lo] _ The stimulation of chloride uptake in the presence of 1 mM TTP is about 27 X of that observed in the presence of GABA. to obtain accurate Due to the small effects, it is difficult dose-response curves, but half-maximum activation is obtained at a TTP concentration around 0.5 mM. Pyrithiamine acts at lower concentrations, the maximum effect being obtained at 0.1 mM. Under these conditions, no effect of TTP or pyrithiamine on 22Na+ or 86Rb+ uptake was evidenced but oxythiamine slightly decreased the uptake of 22Na+ and 86Rb+ (not shown). High TTP concentrations and a 10 mfn incubation before adding 36~11 are necessary for full activation. These observations suggest that activation is limited either by permeability barriers that this highly charged molecule has to cross or by a chemical reaction. Fox and Duppel [13] have shown that thiamine derivatives (TTP > TDP > thiamine) prevent the exponential decline of the Na+ and K+ current at the node of Ranvier during long lasting voltage-clamp experiments. They conclude that TDP or TTP are the active compounds and that their site of action is located at the internal surface of the membrane, which would fit wfth ou results. We introduced a slight sophistication of our experimental procedure: instead of adding thiamine compounds just before tracer addition, the compounds -namely thiamine, oxythiamine and pyrithiaminewere added to the crude homogenate and the mixture was preincubated 10 min at 37 ‘C. Then, the vesicles were sedimented and washed as described with saline containing no thiamine compound. Ion uptake was then measured, again in the absence of added thiamine compounds. At this stage, 98 % of the compounds added to the homogenate had been lost during the preparative process. While thiamine had no direct effect on 3%luptake (Table l), preincubation with thiamine in the presence of glucose induces a significant activating effect (Table 2). In contrast, pyrithiamine and oxythiamine have about the same effect whether they are added at the preincubation or the incubation stage. When 1140
Vol. 171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 2 : Effect of preincubation of homogenates with thiamine derivatives on %I-, zzNa+ and s6Rb+ uptake in rat brain membrane vesicles 22Nat
86Rb+
117.5 f 11.1 124.3 f 15.8=
0.40 f 0.04 0.43 f 0.03%
36c1-
Control Thiamine Pyrithiamine Oxythiamine
40.7 44.0 43.2 37.0
f f f It
2.5 5.0** 3.0* 2.2**
Rat brain homogenates were incubated (10 min at 37’C) in the absence (control) or in the presence of thiamine, pyrithiamine or oxythiamine at 1 mM. Then microvesicles were prepared and 36Cl(0.5 &i/ml), 22Na+ (25 &i/ml) or *6Rb+ (IO&ml) uptake was measured as described in Materials and Methods and expressed in nmol. mg-1. 10 set-1. The results are the mean f SD for 5 rats. For each rat 8 uptake measurements were realized for each condition. For 36~ uptake, the significance of the results was tested by analysis of variance for repeated measures followed by the Dunnett test for the comparison with the control values. For 22Nat and s6Rb+ a paired Student t test was done (*, p < 0.05; **p +z0.01).
was replaced
%l-
by 22Na+ or *6Rb+,
uptake
thiamine-dependent
interpretation are bound slowly.
of these to a high
However
also
but significant
observed.
A
data
would
be to consider
that
affinity
site,
the compounds
dissociate
this is
seems
unlikely
totally
uneffective
concerned
since
it
incubation
stage.
It is more
&ble
was
a slighter
probable
as that
far
as
when
simple
once they very
thiamine
added
at
TTP, and not thiamine,
3: Thiamine derivatives in rat brain microvesicles after preincubation with thiamine or oxythiamine
TTR
Thiamine (pmoV:g,
Control Thiamine Oxythiamine
0.35 f 0.03 0.8 f 0.2* 0.32 f 0.05
mean l%,
92 f 9 96 f 8 90 f 15
n=5)
4.9 f 1.6 8.9 f 2.1** 8.1 f 1.7**
2.5 f 0.1 2100 f 500** 8.6 f 0.6**
Rat brain homogenates were incubated (10 min at 37°C) in the absence (control) or in the presence of 1 mM thiamine, or oxythiamine. Then microvesicles were prepared as described in Materials and Methods. Aliquots of 400 ~1 were sampled, precipitated by addition of 100 ~1 trichloroacetic acid (60 %) and processed further for the determination of thiamine derivatives (Bettendorff et al., 1986) Statistical analysis (5 rats) was done by analysis of variance followed by the Dunnett test (*, p < 0.05; **, p -z 0.01) for comparison of the thiamine and oxythiamine treatments to the control.
1141
is the
Vol.
171, No. 3, 1990
iS the
BIOCHEMICAL
active
species
binding
affinity.
We have
previously
binding
site
TTP
for
~P’CtrOphOfiiS
more
(Kd
stage would
is not
enzymes
content;
the inhibitory
to impairement
against
Pyrithiamine
this.
increase
in the fluorescence
homogenates that
with
the
spectrum
are identical probably
it is known
that
not
significantly
alter
derivatization
[17].
pyrithiamine
respective shown
that
It
in different
least oxythiamine
is worth
mentioning
leads to symptoms [19]. the
This derivative compound
hyperpolarization the
synaptic
miniature
[ZO]. transmission
potentials
that
(Table
On the by
thiamine
other
method.
[21]. 1142
The
derivatives
which
of their
would
affect
2). It has indeed
been
[18]. of pyrithiamine
to increase to obliterate hand,
does with
of Wernicke’s
potentiation
and
preincubation
administration
and
to the
after
that
those
430.
optimum
be phosphorylated
potential
crude
phosphorylation
derivatives
was also shown
action
not
thiamine
may
resembling
of
leads to the synthesis
ways
an
to TTP, TMP
for
emission
possible
and tri-phosphate
ion permeabilities
Indeed
corresponding
times
fluorescence
or oxythiamine
mono-
a fluorescent
at
our chromatographic
is thus
argument
was
with
the It
an
that
however
retention
for thiamine,
to
We noticed
The
at least
TTP
observation
preincubation
at 460 nm
with
However,
oxythiamine.
emission
but
of
is not likely
into
shown).
the
decrease
is also
hold for the corresponding
and
either
(not
fluorescence
of pyrichrome.
same will
not after
pyrithiamine
of thiochrome,
pyrithiamine
[14,15].
of the peaks corresponding
observed
optimum
characteristic
but
at
associated
The
effect
of
the case. TDP
of oxythiamine
inhibitory [US])
was
is mostly
synthesis.
organ
the synthesis
this is indeed it
high affinity
thiamine
can be transformed
(pyrichrome,
thiamine
effect
a direct
compound and
of
does not significantly
of TTP
has
electric
if it allows
since
oxythiamine
oxythiamine
the
effects
with
of a high
and has a slow turnover
with
be due
in
be explained
preincubation thus
PM)
The
affected
receptor
the existence
Table 3 show that
much
TDP-dependent
a specific
= 0.5 [6].
TTP. Data from
content
to shown
e~ectf~cus
preincubation
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
encephalopathy the the
oxythiamine of
the
amplitude
of
post-tetanic affected postsynaptic
Vol.
171,
No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
Among
compounds
tested,
various
2,4-dinitrophenol
(0.1
mM)
RESEARCH COMMUNICATIONS
bumetanide
significantly
(0.1 mM)
decreased
and
thiamine-
dependent chloride uptake by 55 f 37 and 67 f 24 W respectively. The influence of HCO3- is puzzling. No significant response was obtained
in the
significant
absence
effect,
involved
[22].
suggesting
In most
lies between
is no more
specific. of a high
in rat. brain mM
t.hus
uptake.
inhibitor
of the chloride
an inhibitor
ion and
vesicles,
range
are without
channel and
chloride
but the exact target
the
site has a Kd of 0.2 this
picrotoxinin with
compound
on
(0,l
an
mM,
the GABA receptor
anthracene-9-carboxylate chloride
effect
shown
site for bumetanide
IC50 for
associated
is not
this compound
f24] have
binding
mM),
channels
(1 mM, [25,26,27])
and
(not shown).
we can state
especially
system
for (Na-K-Cl)
At 0.1 mM
et al.
as the
(0.2
(1OpM)
As a conclusion, affect
Babila
of voltage-dependent
azide (1 mM)
exchange
This low affinity
Ouabain
valinomycine
PM [23].
and a low affinity
same
was without
the X50 of bumetanide
0.05-5
synaptosomes. in the
DIDS at 1 mM
the anion
Recently,
chloride [7]),
that
tissues
cotransport existence
of HC03-.
that
thiamine
uptake
and mechanism
derivatives
in rat
brain
remain
indeed
membrane unknown.
Acknowledgments : This work was supported by grants from the Fonds de la Recherche Collective (Belgium) to E. S. and from the Fonds National de la Recherche Scientifique (Belgium) to L.B. Dr. M. Yamazaki from the New Lead Laboratories (Sankyo Co.’ Ltd) is kindly acknowledged for the gift of TTP. P. W. is research associate at the Fonds National de la Recherche Scientifique. References 1. 2. 3. 4. 5. 6. 7.
Cooper J.R. and Pincus J.H. (1979) Neurochem. Res., 4, 223-239. Haas R. H. (1988) Ann. Rev. Nutr. 8, 483-515. Itokawa Y., and Cooper J.R. (1970) Biochim. Biophys. Acta, 196, 274-284. Bettendorff L., Michel-Cahay C., Grandfils Chr., De Rycker C., and Schoffeniels E. (1987) J. Neurochem. 49, 495-502. Bettendorff L., Wins P., and Schoffeniels E. (1988) B&hem. Biophys. Res. Commun. 154, 942-947. Bettendorff L., Grandfils Chr., Wins P., and Schoffeniels E. (1989) J. Neurochem. 53, 738-746. Harris R.A., and Allan A.M. (1985) Science 228, 1108-1110. 1143
Vol. 171, No. 3, 1990
8.
9. 10. 11. 12. 15. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Peterson G.L. (1977) Anal. Biochem., 85, 546-356. Bettendorff L., Grandfils Chr., De Rycker C., and Schoffenids E. (1986) J. Chromatogr. 382, 297-302. Allan A. M., and Harris R. A. (1986) Mol. Pharmacol. 29, 497-505. Schwartz R.D. I Skolnick P., Hollingsworth E.B., and Paul S.M. (1984) FEBS Lett. 175, 193-196. Obata T. and Yamamura H.I. (1986) Biochem. Biophys. Res. Commun. 141, l-6. Fox J. M., and Duppel W. (1975) Brain Res., 89, 287-302. Berman K., and Fishman R. A. (1975) J. Neurochem. 24, 457-465 Gaitonde M.K., and Evans G.M. (1983) Biochem. Sot. Trans. 11, 695-696. Airth R. L. 1 and Foerster G.E. (1970) Methods in Enzymology (D. B. McCormick, and L.D. Wright, eds) vol 18A, pp 81-86, Academic Press, Inc., New York. lshii K., Sarai K., Sanemori H., and Kawasaki T. (1979) Anal. B&hem. 97, 191-195. Rindi G., de Guiseppe L., and Ventura U. (1963) J. Nutr. 81 147-154. H&roux M, , and Butterworth R.F. (1988) J. Neurochem. 51, 1221-1226. Armett C.J., and Cooper J.R. (1965) J. Pharmacol. Exp. Ther. 148, 137-143. Eder L., Hirt L., and Dunant Y. (1976) Nature 264, 186-188. Hoffmann E.K. (1986) Biochim. Biophys. Acta 864, l-34. Haas M. (1989) Annu. Rev. Physiol. 51, 443-457. Babila T., Gottleb Y., Lutz R. A., and Lichtenstein D. (1989) Life Sci. 44, 1665-1675. Palade P. T., and Barchi R.L. (1977) J. Gen. Physiol. 69, 325-342. Horvath P. J., Ferriola P.C., Weiser M.M, and Duffey M.E. (1986) Am. J. Physiol. 250, G185-Gl90. Franciolini F. ) and Nonner W. (1987) J. Gen. Physiol. 90, 453-478.
1144
BIOCHEMICAL
REGULATION
OF ION UPTAKE RAT BRAIN
Lucien Laboratory
Bettendorff, of General
August
IN MEMBRANE
BY THIAMINE
Pierre
VESICLES
FROM
COMPOUNDS
Wins,
and Ernest
and Comparative
Liege, Received
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1137-1144
Schoffeniels
Biochemistry,
B-4020 Liege,
University
of
Belgium
7, 1990
We examined the effects of thiamine derivatives on ion uptake in rat brain membrane vesicles. Thiamine triphosphate (1 mM) and pyrithiamine (0.1 mM) increase chloride uptake. Preincubation of crude homogenate with thiamine or pyrithiamine increases chloride uptake while oxythiamine has the reverse effect. Thiamine and oxythiamine also affect 22Na+ and g6Rb+ uptake in the same way as for 36Cl- but to a lesser extent. Thiamine-dependent 36Cluptake is activated by sodium bicarbonate (10 mM) and partially inhibited by bumetanide (0.1 mM) and 2,4-dinitrophenol (0.1 mM) . Prelncubation with thiamine increases the thiamine triphosphate content of the vesicles. The hypothesis that TTP is the activator of a particular chloride uptake mechanism is discussed. O1990 Academic Pre?.s,
Inc.
Although phosphate system
there
is much
derivatives,
has
independent
of
diphosphate
(TDP).
still
unidentified
was
remain based
thiamine Itokawa
on
this
the
that
a specific
the
known
molecular [1,2].
the
hypothesis
triphosphate
(TTP).
thiamine, function
targets Our own that This
thiamine highly
electricus in rat specialized
or some of its in
coenzyme
the
for those key
of thiamine
to this
problem
is played
by
first
suggested
by
by the finding
organ,
TTP, which
brain,
makes
up 90% of total also
effects
role
possibility,
We
nervous
special
electric
tissue.
the
role
approach
and Cooper [3], was substantiated
Eiectrop~orus of total
evidence
[4] that
accounts
in
for 1 X
thiamine
in
characterized
a
ABBREVIATIONS: DlDS, 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid; TMP, thiamine monophosphate; TDP, thiamine diphosphate; TTP. thiamine triphosphate. 0006-291X/90
1137
All
$1.50
Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
membrane-associated thiamine triphosphatase in this electric tissue. The enzyme is activated by anions and irreversibly inhibited by 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS), an anion transport inhibitor [5,6]. Although DIDS may react unspecifically with amino groups of the active site, we did observe that anions protected against DIDS inhibition. This led us to suspect that TTP could be involved in some kind of anion transport mechanism. The data reported here are the first piece of evidence in favor of this hypothesis. Experimental
Procedures
Materials: TTP was a gift from Dr Yamazaki from the New Lead Laboratories (Sankyo Co., Ltd., Tokyo, Japan). Pyrithiamine, oxythiamine, TDP,TMP, thiamine, DIDS, bumetanide were purchased from Sigma (St. Louis, MO, U. S. A. ) . [%l]NaCl and [86Rb] RbCl were from Amersham International (Buckinghamshire, England) and [22Na]NaCl was from New England Nuclear. M mb e le D euaration: The procedure . descri:d by”Harriy:z Alla: [7]. Female Wistar rats (150-&l $ were decapitated and the brain homogenized by hand (6 to 8 strokes in a glass-teflon homogenizer) in 15 ml ice-cold incubation buffer (145 mM NaCl, 5 mM KCl, 1 mM MgC12, 1 mM CaCl2, 10 mM D-glucose and 10 mM HEPES-Tris buffer at pH 7.5). The homogenate was filtered through a double layer of gauze. In some experiments aliquots of the homogenate were incubated (15 min at 37°C) in the absence or in the presence of 1 mM thiamine, oxythiamine or pyrithiamine and processed as follows. The homogenate, incubated or not, was centrifuged at 900 g for 15 minutes> the pellet suspended in 10 ml of buffer and again centrifuged for 15 minutes at 900 g. The final pellet was suspended in the incubation buffer at a protein concentration [8] of about 8 mg/ml. Attempts to fractionate the preparation further did not yield better results. Measurement of 36ainflux: This method is as described by Harris and Allan [7) for GABA-mediated chloride uptake into brain membrane vesicles with slight modifications. 75 Pl of the membrane suspension were incubated at 57°C in the presence of thiamine compounds at the concentrations indicated. After 10 minutes 25 ~1 of a buffer solution containing 36~ (0.5 &ml) and NaHC% (40 mM, PH 7.5) were added. Ten seconds after the addition of 3%X’, influx was stopped by addition of 4 ml ice-cold incubation buffer and rapid flltratlon through 2.4 cm Whatman SF/C filters. The filters were rapidly washed with 8 ml of ice-cold buffer and the radioactivity of the filters was counted by liquid scintillation spectrometry.
Thiamine and its phosphorylated compounds were determined using a HPLC procedure exactly as described previously [9,4] . Prior
Vol.
171, No. 3, 1990
to injection thiochromes
BlOCHEMlCAL
Although
a role
trying
using
on a preparation
previously
been characterized chloride
carried
out
derivatives
on
Preliminary results incubation
%l-
medium.
demonstrate
We can
10 9%while
have
no
TTP
and while
(1 mM)
TDP, TMP,
significant
pyrithiamine
15 mM
Our first effect
which
has
of
NaHQ
more
thiamine (Table
1).
reproducible
is present
obtained
of
experiments
vesicles
that
were
to examine
in
the
for ion uptake
10 or 20 sec.
see that
about
when
shown
uptake
used for the study
membrane
have
TTP
of any
ion
vesicles
a direct
by
aware
We decided
[7,10,11,12].
The best results
lasting
not
by direct
and intensively
uptake
obtained
measurements
effects:
to
we are
tracers.
uptake
fluorescent
and especially
of sealed membrane
experiments are
thiamine
hypothesis
radioactive
this point
GABA-mediated
that
this
into
Discussion
in ion permeability
to elucidate
measurements
were
and
it is acknowledged
play
studies
RESEARCH COMMUNICATIONS
thiamine derivatives were transformed for fluorescence detection. Results
could
AND BIOPHYSICAL
thiamine
effect. (5.1
chloride
uptake
and sodium
tripolyphosphate
thiamine
antimetabolites,
Two
oxythiamine,
pyrithiamine
increases
were
found
mM)
slightly,
to
have but
opposite
significantly
Table 1: Chloride uptake (mean f SD) in membrane vesicles in the presence of several thiamine derivatives and GABA Compound tested Control TTP (1 mM) TDP (1 mM) TMP (1 mM) Thiamine (1 mM) Sodium tripolyphosphate Pyrithiamine (100 PM) Oxythiamine (1 mM) GABA (30 PM)
number of experiments
(1 mM)
63 31 39 12 27 8 66 25 10
Chloride uptake (nmol/mg/lO set) 38.6 f 42.6 f 39.4 f 38.6 f 37.6 f 36.6 f 40.0 f 35.4 f 63.4 f
2.2 2.6” 2.6 1.6 2.2 2.4 1.0s 2.4** 5.6**
The experiments were carried out as described in Materials and Methods. 3%1- influx was measured for 10 seconds. The treatments are compared to the control group using analysis of variance (p < 0.05) followed by the Dunnett test (*, p < 0.05; %*, p -z 0.01)
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BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
increases chloride uptake, oxythiamine (1 m&l) is inhibitory. As a control, the effect of GABA was also tested. As expected GABA (30 PM) increases chloride uptake to the same extent as previously found [lo] _ The stimulation of chloride uptake in the presence of 1 mM TTP is about 27 X of that observed in the presence of GABA. to obtain accurate Due to the small effects, it is difficult dose-response curves, but half-maximum activation is obtained at a TTP concentration around 0.5 mM. Pyrithiamine acts at lower concentrations, the maximum effect being obtained at 0.1 mM. Under these conditions, no effect of TTP or pyrithiamine on 22Na+ or 86Rb+ uptake was evidenced but oxythiamine slightly decreased the uptake of 22Na+ and 86Rb+ (not shown). High TTP concentrations and a 10 mfn incubation before adding 36~11 are necessary for full activation. These observations suggest that activation is limited either by permeability barriers that this highly charged molecule has to cross or by a chemical reaction. Fox and Duppel [13] have shown that thiamine derivatives (TTP > TDP > thiamine) prevent the exponential decline of the Na+ and K+ current at the node of Ranvier during long lasting voltage-clamp experiments. They conclude that TDP or TTP are the active compounds and that their site of action is located at the internal surface of the membrane, which would fit wfth ou results. We introduced a slight sophistication of our experimental procedure: instead of adding thiamine compounds just before tracer addition, the compounds -namely thiamine, oxythiamine and pyrithiaminewere added to the crude homogenate and the mixture was preincubated 10 min at 37 ‘C. Then, the vesicles were sedimented and washed as described with saline containing no thiamine compound. Ion uptake was then measured, again in the absence of added thiamine compounds. At this stage, 98 % of the compounds added to the homogenate had been lost during the preparative process. While thiamine had no direct effect on 3%luptake (Table l), preincubation with thiamine in the presence of glucose induces a significant activating effect (Table 2). In contrast, pyrithiamine and oxythiamine have about the same effect whether they are added at the preincubation or the incubation stage. When 1140
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BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 2 : Effect of preincubation of homogenates with thiamine derivatives on %I-, zzNa+ and s6Rb+ uptake in rat brain membrane vesicles 22Nat
86Rb+
117.5 f 11.1 124.3 f 15.8=
0.40 f 0.04 0.43 f 0.03%
36c1-
Control Thiamine Pyrithiamine Oxythiamine
40.7 44.0 43.2 37.0
f f f It
2.5 5.0** 3.0* 2.2**
Rat brain homogenates were incubated (10 min at 37’C) in the absence (control) or in the presence of thiamine, pyrithiamine or oxythiamine at 1 mM. Then microvesicles were prepared and 36Cl(0.5 &i/ml), 22Na+ (25 &i/ml) or *6Rb+ (IO&ml) uptake was measured as described in Materials and Methods and expressed in nmol. mg-1. 10 set-1. The results are the mean f SD for 5 rats. For each rat 8 uptake measurements were realized for each condition. For 36~ uptake, the significance of the results was tested by analysis of variance for repeated measures followed by the Dunnett test for the comparison with the control values. For 22Nat and s6Rb+ a paired Student t test was done (*, p < 0.05; **p +z0.01).
was replaced
%l-
by 22Na+ or *6Rb+,
uptake
thiamine-dependent
interpretation are bound slowly.
of these to a high
However
also
but significant
observed.
A
data
would
be to consider
that
affinity
site,
the compounds
dissociate
this is
seems
unlikely
totally
uneffective
concerned
since
it
incubation
stage.
It is more
&ble
was
a slighter
probable
as that
far
as
when
simple
once they very
thiamine
added
at
TTP, and not thiamine,
3: Thiamine derivatives in rat brain microvesicles after preincubation with thiamine or oxythiamine
TTR
Thiamine (pmoV:g,
Control Thiamine Oxythiamine
0.35 f 0.03 0.8 f 0.2* 0.32 f 0.05
mean l%,
92 f 9 96 f 8 90 f 15
n=5)
4.9 f 1.6 8.9 f 2.1** 8.1 f 1.7**
2.5 f 0.1 2100 f 500** 8.6 f 0.6**
Rat brain homogenates were incubated (10 min at 37°C) in the absence (control) or in the presence of 1 mM thiamine, or oxythiamine. Then microvesicles were prepared as described in Materials and Methods. Aliquots of 400 ~1 were sampled, precipitated by addition of 100 ~1 trichloroacetic acid (60 %) and processed further for the determination of thiamine derivatives (Bettendorff et al., 1986) Statistical analysis (5 rats) was done by analysis of variance followed by the Dunnett test (*, p < 0.05; **, p -z 0.01) for comparison of the thiamine and oxythiamine treatments to the control.
1141
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iS the
BIOCHEMICAL
active
species
binding
affinity.
We have
previously
binding
site
TTP
for
~P’CtrOphOfiiS
more
(Kd
stage would
is not
enzymes
content;
the inhibitory
to impairement
against
Pyrithiamine
this.
increase
in the fluorescence
homogenates that
with
the
spectrum
are identical probably
it is known
that
not
significantly
alter
derivatization
[17].
pyrithiamine
respective shown
that
It
in different
least oxythiamine
is worth
mentioning
leads to symptoms [19]. the
This derivative compound
hyperpolarization the
synaptic
miniature
[ZO]. transmission
potentials
that
(Table
On the by
thiamine
other
method.
[21]. 1142
The
derivatives
which
of their
would
affect
2). It has indeed
been
[18]. of pyrithiamine
to increase to obliterate hand,
does with
of Wernicke’s
potentiation
and
preincubation
administration
and
to the
after
that
those
430.
optimum
be phosphorylated
potential
crude
phosphorylation
derivatives
was also shown
action
not
thiamine
may
resembling
of
leads to the synthesis
ways
an
to TTP, TMP
for
emission
possible
and tri-phosphate
ion permeabilities
Indeed
corresponding
times
fluorescence
or oxythiamine
mono-
a fluorescent
at
our chromatographic
is thus
argument
was
with
the It
an
that
however
retention
for thiamine,
to
We noticed
The
at least
TTP
observation
preincubation
at 460 nm
with
However,
oxythiamine.
emission
but
of
is not likely
into
shown).
the
decrease
is also
hold for the corresponding
and
either
(not
fluorescence
of pyrichrome.
same will
not after
pyrithiamine
of thiochrome,
pyrithiamine
[14,15].
of the peaks corresponding
observed
optimum
characteristic
but
at
associated
The
effect
of
the case. TDP
of oxythiamine
inhibitory [US])
was
is mostly
synthesis.
organ
the synthesis
this is indeed it
high affinity
thiamine
can be transformed
(pyrichrome,
thiamine
effect
a direct
compound and
of
does not significantly
of TTP
has
electric
if it allows
since
oxythiamine
oxythiamine
the
effects
with
of a high
and has a slow turnover
with
be due
in
be explained
preincubation thus
PM)
The
affected
receptor
the existence
Table 3 show that
much
TDP-dependent
a specific
= 0.5 [6].
TTP. Data from
content
to shown
e~ectf~cus
preincubation
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
encephalopathy the the
oxythiamine of
the
amplitude
of
post-tetanic affected postsynaptic
Vol.
171,
No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
Among
compounds
tested,
various
2,4-dinitrophenol
(0.1
mM)
RESEARCH COMMUNICATIONS
bumetanide
significantly
(0.1 mM)
decreased
and
thiamine-
dependent chloride uptake by 55 f 37 and 67 f 24 W respectively. The influence of HCO3- is puzzling. No significant response was obtained
in the
significant
absence
effect,
involved
[22].
suggesting
In most
lies between
is no more
specific. of a high
in rat. brain mM
t.hus
uptake.
inhibitor
of the chloride
an inhibitor
ion and
vesicles,
range
are without
channel and
chloride
but the exact target
the
site has a Kd of 0.2 this
picrotoxinin with
compound
on
(0,l
an
mM,
the GABA receptor
anthracene-9-carboxylate chloride
effect
shown
site for bumetanide
IC50 for
associated
is not
this compound
f24] have
binding
mM),
channels
(1 mM, [25,26,27])
and
(not shown).
we can state
especially
system
for (Na-K-Cl)
At 0.1 mM
et al.
as the
(0.2
(1OpM)
As a conclusion, affect
Babila
of voltage-dependent
azide (1 mM)
exchange
This low affinity
Ouabain
valinomycine
PM [23].
and a low affinity
same
was without
the X50 of bumetanide
0.05-5
synaptosomes. in the
DIDS at 1 mM
the anion
Recently,
chloride [7]),
that
tissues
cotransport existence
of HC03-.
that
thiamine
uptake
and mechanism
derivatives
in rat
brain
remain
indeed
membrane unknown.
Acknowledgments : This work was supported by grants from the Fonds de la Recherche Collective (Belgium) to E. S. and from the Fonds National de la Recherche Scientifique (Belgium) to L.B. Dr. M. Yamazaki from the New Lead Laboratories (Sankyo Co.’ Ltd) is kindly acknowledged for the gift of TTP. P. W. is research associate at the Fonds National de la Recherche Scientifique. References 1. 2. 3. 4. 5. 6. 7.
Cooper J.R. and Pincus J.H. (1979) Neurochem. Res., 4, 223-239. Haas R. H. (1988) Ann. Rev. Nutr. 8, 483-515. Itokawa Y., and Cooper J.R. (1970) Biochim. Biophys. Acta, 196, 274-284. Bettendorff L., Michel-Cahay C., Grandfils Chr., De Rycker C., and Schoffeniels E. (1987) J. Neurochem. 49, 495-502. Bettendorff L., Wins P., and Schoffeniels E. (1988) B&hem. Biophys. Res. Commun. 154, 942-947. Bettendorff L., Grandfils Chr., Wins P., and Schoffeniels E. (1989) J. Neurochem. 53, 738-746. Harris R.A., and Allan A.M. (1985) Science 228, 1108-1110. 1143
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Peterson G.L. (1977) Anal. Biochem., 85, 546-356. Bettendorff L., Grandfils Chr., De Rycker C., and Schoffenids E. (1986) J. Chromatogr. 382, 297-302. Allan A. M., and Harris R. A. (1986) Mol. Pharmacol. 29, 497-505. Schwartz R.D. I Skolnick P., Hollingsworth E.B., and Paul S.M. (1984) FEBS Lett. 175, 193-196. Obata T. and Yamamura H.I. (1986) Biochem. Biophys. Res. Commun. 141, l-6. Fox J. M., and Duppel W. (1975) Brain Res., 89, 287-302. Berman K., and Fishman R. A. (1975) J. Neurochem. 24, 457-465 Gaitonde M.K., and Evans G.M. (1983) Biochem. Sot. Trans. 11, 695-696. Airth R. L. 1 and Foerster G.E. (1970) Methods in Enzymology (D. B. McCormick, and L.D. Wright, eds) vol 18A, pp 81-86, Academic Press, Inc., New York. lshii K., Sarai K., Sanemori H., and Kawasaki T. (1979) Anal. B&hem. 97, 191-195. Rindi G., de Guiseppe L., and Ventura U. (1963) J. Nutr. 81 147-154. H&roux M, , and Butterworth R.F. (1988) J. Neurochem. 51, 1221-1226. Armett C.J., and Cooper J.R. (1965) J. Pharmacol. Exp. Ther. 148, 137-143. Eder L., Hirt L., and Dunant Y. (1976) Nature 264, 186-188. Hoffmann E.K. (1986) Biochim. Biophys. Acta 864, l-34. Haas M. (1989) Annu. Rev. Physiol. 51, 443-457. Babila T., Gottleb Y., Lutz R. A., and Lichtenstein D. (1989) Life Sci. 44, 1665-1675. Palade P. T., and Barchi R.L. (1977) J. Gen. Physiol. 69, 325-342. Horvath P. J., Ferriola P.C., Weiser M.M, and Duffey M.E. (1986) Am. J. Physiol. 250, G185-Gl90. Franciolini F. ) and Nonner W. (1987) J. Gen. Physiol. 90, 453-478.
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