Indian Journal of Experimental Biology
Vol. 49, March 2011, pp. 169-176
Non-genomic effect of L-triiodothyronine on calmodulin-dependent synaptosomal
protein phosphorylation in adult rat cerebral cortex
Pradip K Sarkar1,2*, Jason J Morris2 & Joseph V Martin2,3
1
Department of Basic Sciences, Parker College of Chiropractic, 2500 Walnut Hill Lane, Dallas,Texas 75229, USA
Department of Biology, 3Center for Computational and Integrative Biology, Rutgers University, 315 Penn Street, Camden,
New Jersey 08102, USA
2
Received 30 June 2010; revised 5 December 2010
The present study was undertaken to examine calmodulin-dependent effect of thyroid hormones (THs) on synaptosomal
protein phosphorylation in mature rat brain. Effect of L-triiodothyronine (L-T3) on in vitro protein phosphorylation was
measured in a hypotonic lysate of synaptosomes prepared from adult male rat cerebral cortex, incubated in presence and
absence of calcium ion (Ca2+) and calmodulin. L-T3 significantly enhanced incorporation of 32P into synaptosomal proteins
as compared to basal level of phosphorylation in the presence of Ca2+ and calmodulin. Under these conditions, increase in
protein phosphorylation was 47, 74 and 52% for 10 nM, 100 nM and 1 µM L-T3, respectively. Chelation of Ca2+ using
ethylene glycol-bis (2-aminoethylether)-N, N, N’, N’-tetraacetic acid (EGTA) inhibited the effects of Ca2+/calmodulin on
TH-stimulated protein phosphorylation levels. This study suggests that a high proportion of L-T3-stimulated protein
phosphorylation involves Ca2+/calmodulin-dependent pathways in adult rat cerebrocortical synaptosomes.
Keywords: Adult rat brain, Brain, Ca2+/calmodulin, Protein phosphorylation, L-Triiodothyronine
Thyroid hormones (TH) immensely influence growth
and differentiation of developing mammalian brain by
a well-known classical mechanism mediated via gene
expression regulated by a specific nuclear receptor1.
Adult-onset dysthyroidism is accompanied by a
substantial number of neurological and psychological
manifestations in mature humans2. In contrast to
developing brain, most of the changes produced
during mature condition are reversible with an
appropriate adjustment of peripheral TH supply. The
emerging idea of direct nongenomic actions of TH in
certain types of cells3,4, including mature neurons, is
of particular relevance to the physiological basis of
signs and symptoms of thyroid dysfunction in
adults2,5.
Evidence
demonstrates
that
Ltriiodothyronine (L-T3) is taken up into brain tissue,
concentrated in nerve terminals in neuropil, and
metabolized and subsequently found in synaptosomal
preparation from adult rat cerebral cortex5,6. Although
adult-onset thyroid disorders manifest several
neuropsychiatric abnormalities, appreciative underlying
mechanisms for these remain unexplained. Recent
——————
*
Correspondent author
Telephone: 1-972-438-6932 Ext.: 7336
Fax: 214-902-2448
E-mail: [email protected]
reports on the nongenomic actions of thyroid
hormones (THs) are revealing new TH-regulated
signaling pathways in adult brain, including
the
hormone-related
regulation
of
protein
1
phosphorylation state .
Protein phosphorylation and dephosphorylation
mechanisms mediated through activation and
deactivation of several protein kinases and protein
phosphatases are vital regulatory mechanisms for
neuronal signal transduction. The principal second
messenger molecules that control these protein
kinases and protein phosphatases include, but are not
limited to, Ca2+, cyclic adenosine monophosphate,
cyclic guanidine monophosphate, nitric oxide,
inositide 3-phosphates, and diacylglycerol7,8. Most
nongenomic actions are rapid, within seconds to min.,
and are modulated by protein phosphorylation
/dephosphorylation mechanisms regulating intracellular
signal cascades9. In particular, TH-mediated Ca2+
entry in adult rat brain synaptosomes10-12, in
hypothyroid mouse cerebral cortex13, and in single rat
myocytes14 has been reported. A dose-dependent
increase has been reported in intracellular Ca2+ levels
within adult rat brain cerebrocortical synaptosomes
induced by brain physiological concentrations of
L-T3 (Ref. 15) in euthyroid and hypothyroid rats
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INDIAN J EXP BIOL, MARCH 2011
brain12. L-T3-induced protein phosphorylation of
several synaptosomal proteins in adult rat brain
synaptosomes has been reported5. Collectively, these
studies suggest a potential role of calcium-signaling
pathways in nongenomic mechanisms of action of TH
in adult brain.
Therefore, in the present study the relationship
between Ca2+ and/or calmodulin (CaM)-dependent
protein phosphorylation and effects of L-T3 in mature
rat brain were investigated.
Materials and Methods
Chemicals—Adenosine tri-phosphate di-sodium
(ATP), 3,5, 3’-L-triiodothyronine (L-T3), calmodulin
(CaM), bovine serum albumin (BSA), 2mercaptoethanol,
ethylene
glycol-bis
(2aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA),
N-(2-hydroxyehtyl) piperazine-N’-(2-ethanesulfonic
acid) (HEPES), sodium dodecyl sulfate (SDS),
sucrose, magnesium chloride (MgCl2), calcium
chloride (CaCl2), KN-62 and other reagents were
purchased from Sigma Chemical Co. (St. Louis, MO,
USA). [γ-32P]-ATP (10 Ci/mmol) was purchased from
Perkin Elmer Life and Analytical Sciences, Boston,
MA, USA. All other chemicals were of highest
reagent grade.
Experimental animals—Young adult male
Sprague-Dawley rats (~3 months old) were purchased
from Hilltop Lab Animals (Scottsdale, PA, USA).
The rats were housed at 25° ± 1°C in 12 h dark-12 h
light conditions (the light phase began at 8:00 AM)
and fed ad libitum with standard rat diet and water.
The animals were maintained in the Rutgers
University animal facilities according to the National
Institute of Health Guide for the Care and Use of
Laboratory Animals. The experimental protocol was
approved by the Institutional Animal Care and Use
Committee (IACUC) of Rutgers University, which
determined that the experiments appropriately
minimized the number of animals used.
Preparation of synaptosomes—Animals were
euthanized by quick decapitation, brains were
removed and cerebral cortices were dissected out and
maintained under ice-cold conditions. Synaptosomes
from cerebral cortex were prepared as described
previously16. Briefly, cerebral cortices were
homogenized in 10 volumes of 0.32 M sucrose
solution using 8 strokes of a Teflon-glass
homogenizer (15 sec). The resulting homogenate was
centrifuged at 1,000 × g for 10 min at 4ºC. The
supernatant was collected and re-centrifuged as
above. The supernatant obtained from second
centrifugation was layered over 1.2 M sucrose, and
centrifuged at 34,000 × g for 50 min at 4°C. The
fraction collected between 0.32 and 1.2 M sucrose
layers was diluted at 1:1.5 with ice-cold bi-distilled
water (to avoid shrinkage of the synaptosomes in the
0.8 M sucrose concentration and to maintain isoosmotic conditions), further layered on 0.8 M sucrose,
and centrifuged at 34,000 × g for 30 min. The pellet
thus obtained was washed by resuspension in fresh
0.32 M sucrose and re-pelleting at 20,000 × g for
20 min. The resulting pellet was washed again by
resuspension and centrifuging at 20,000 × g for
20 min. Finally, the purified synaptosomes were lysed
by hypotonic shock using ice-cold bi-distilled water,
and assayed for protein phosphorylation.
Protein phosphorylation—Protein phosphorylation
in synaptosomes from adult rat cerebral cortex was
determined by measuring incorporation of labeled
phosphate into endogenous proteins from the
membrane-containing synaptosomal lysate preparation.
The final reaction mixture contained 50 mM Hepes,
50 mM HCl, 10 mM MgCl2, 0.1 mM EGTA (to
chelate endogenously available Ca2+ ions), and 20 µM
[γ-32P]ATP (3 µCi) at pH 7.4, in a total volume of
50 µl. The final reaction mixture without [γ-32P]ATP
was pre-incubated for 5 min at 30°C for temperature
equilibration, and the reaction was initiated by
addition of [γ-32P]ATP. After incubating the reaction
mixture with a chosen concentration of synaptosomal
lysate for a selected time period in presence or
absence of various reagents as described in the
following sections, the reaction was terminated by the
addition of 0.4 M EDTA (10 µl). Aliquot (25 µl) of
reaction
product
was
then
spotted
on
phosphocellulose filter paper squares. Excess
unbound radioactivity was removed by washing with
75 mM phosphoric acid, the paper squares were dried
and the adsorbed radioactivity was counted in a liquid
scintillation spectrometer. This method was followed
in each of the experiments described below.
Optimization of protein concentration for
[32P]phosphate incorporation into synaptosomal
32
phosphoproteins—Relationship
between
P
incorporation and the amount of cerebrocortical
synaptosomal lysate was determined by incubating
increasing amounts of lysate protein (5 to 30 µg) in
the phosphorylation assay mixture. The final reaction
mixture without [γ-32P]ATP was pre-incubated for
SARKAR et al.: NON-GENOMIC EFFECT OF L-T3
5 min at 30°C for temperature equilibration, and
reaction was initiated by addition of [γ-32P]-ATP.
After 1 min, the reaction was terminated by addition
of 10 µl of 0.4 M EDTA.
Time course of incorporation of [32P]phosphate
into synaptosomal phosphoproteins—To determine
the temporal pattern of [32P]phosphate incorporation
into synaptosomal phosphoproteins following
addition of [γ-32P]ATP, a concentration of 10 µg of
synaptosomal protein/per assay was chosen. (The data
obtained in the previous experiment indicated that this
amount of lysate would give a substantial response in
the linear portion of a curve relating synaptosomal
protein
concentration
and
[32P]phosphate
incorporation.) As before, the reaction mixture
without [γ-32P]ATP was pre-incubated for 5 min at
30°C and the reaction was initiated by the addition of
[γ-32P]ATP. The reaction was terminated at various
time points (15 sec to 120 sec) by the addition of
10 µl of 0.4 M EDTA.
Effects of Ca2+ and calmodulin concentrations on
[γ-32P]phosphate incorporation into synaptosomal
phosphoproteins—Increasing concentrations of CaCl2
(0-2 mM) and calmodulin CaM (0-2 µM) were
incubated with synaptosomal lysates in the
phosphorylation assay mixture as described above. In
order to maintain a linear reaction rate, 10 µg
protein/assay was used and the incubation time with
ATP was 1 min, conditions determined by the
previous experiments to give optimal responses.
Rapid effect of L-T3 on [32P]phosphate
incorporation into synaptosomal phosphoproteins in
presence or absence of Ca2+ and calmodulin—L-T3
was dissolved in a minimum volume of 0.1 M NaOH,
serial dilutions were made using buffer and the pH
was adjusted to 7.4. To assure maximal Ca2+/CaMdependent protein phosphorylation, 2 µM CaM and
0.5 mM Ca2+ were selected, based on the results of the
earlier experiments. In order to maintain a linear
reaction rate, 10 µg protein/assay was used and ATP
was incubated with the mixture for 1 min. Incubation
of the synaptosomal fractions with L-T3 for 60 min.
at 0°C was chosen based on our previous observation
of maximal specific binding of L-T3 to the
synaptosomal fraction under these conditions17. A
control reaction mixture contained 0.1 mM EGTA. To
determine the effect of Ca2+ chelation on the Ca2+dependent phosphorylation reaction, some incubates
of synaptosomal lysates contained Ca2+, CaM, and
100 nM T3 with 1 mM EGTA added. Effect of
171
endogenous calmodulin was compensated for by
comparing L-T3 + Ca2+/CaM-treated group with its
respective Ca2+/CaM-treated group. Ca2+-treated
group was also compared with the basal
phosphorylation to indicate the effect of Ca2+ alone
and with Ca2+/CaM group to determine the effect of
CaM. The final reaction mixture without [γ-32P]ATP
was preincubated for 5 min at 30°C, and then the
reaction was initiated by addition of [γ-32P]ATP. After
1 min, reaction was terminated by addition of 10 µl of
0.4 M EDTA and 25 µl aliquots were spotted on
phosphocellulose paper squares. The squares were
washed with 75 mM phosphoric acid, dried and
counted radioactivity in a liquid scintillation Wallac
Winspectral™ 1414 liquid scintillation counter.
Incorporation of [32P]phosphate into synaptosomal
proteins was expressed as counts per min (CPM) in
initial experiments. Levels of Ca2+/CaM-dependent
protein phosphorylation were determined after
subtracting the incorporation of 32P in the absence of
Ca2+ and CaM into synaptosomal phosphoproteins
and was expressed as the pmol of 32P
incorporation/min/mg of synaptosomal protein.
Measurement of protein—Synaptosomal protein
content was measured using bovine serum albumin as
a standard18.
Statistical analysis—Results are expressed as mean
± SEM of 4 separate observations. Each observation
was made on pooled tissue from 6 rats. The statistical
analyses of the data for multiple groups were done by
one-way ANOVA followed by Student NewmanKeuls post-hoc comparisons, considering P<0.05 as
significant.
Results
Effect of protein concentrations, time of incubation,
and varying concentrations of Ca2+ and calmodulin
32
on
P
incorporation
into
synaptosomal
phosphoproteins —In vitro incorporation of 32Plabeled phosphate as a function of protein concentration in synaptosomal lysate increased with increasing
amount of protein concentrations (25 – 125 µg/mL).
The protein concentration range from 25 to 75 µg/ml
maintained a linear relationship with increasing
incorporation of 32P into synaptosomal proteins. A
concentration of 50 µg/mL of protein was chosen
from this linear range to be used in subsequent
phosphorylation reaction experiments. The data
showed some indications of saturation at higher
concentrations of synaptosomal lysate proteins.
Fitting the data to the equation for a sigmoid curve
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INDIAN J EXP BIOL, MARCH 2011
showed an apparent EC50 of 124 µg protein/ml and a
maximal rate of 32P incorporation of 8,300 CPM/min.
32
P incorporation increased linearly with time up
to roughly 100 sec (Fig.1B). 32P incorporation leveled
off thereafter, and the curve was fit to the equation for
pseudo-first order association kinetics, with a plateau
at 21,300 CPM/mg protein and an apparent rate
constant of 0.004737 sec-1.
Effect of varying concentrations of Ca2+ (0.125 mM
– 2 mM) on 32P incorporation into synaptosomal
proteins has been represented in Fig.1C. Ca2+ at a
concentration of 0.5 mM showed maximum
stimulation of 32P incorporation into synaptosomal
proteins (Fig. 1C). Thereafter the application of
higher concentrations of Ca2+ (1 -2 mM) in vitro, did
not result any further increase in the levels of protein
phosphorylation. Instead, it declined significantly
from the optimum value (0.5 mM final concentration).
A concentration of 0.5 mM Ca2+ was thus chosen for
further experiments.
Effect of increasing concentrations of CaM
(0.125 – 2 µM) on protein phosphorylation status in
the presence of Ca2+ has been represented in Fig.1D.
Incubation of synaptosomal lysates with 0.5 mM Ca2+
and increasing levels of CaM (0.125 to 2 µM)
amplified the 32P incorporation linearly up to 0.5 µM
of CaM; thereafter, with higher concentrations of
CaM, the incorporation of 32P plateaued. When the
curve was fit to the equation for a sigmoid curve,
EC50 calculated for calmodulin was 0.47 µM and the
Fig.1—Optimization of conditions for [32P]phosphate incorporation into synaptosomal phosphoproteins in adult rat cerebral cortex. [All
values are mean ± SEM of 4 independent experiments and vertical bars indicate SEM]. Effect of (A) protein concentrations [values are
significant at *P<0.05 as compared to the value for 25 µg protein/mL]; (B) incubation time. [Significant at *P<0.05 compared to initial
value and **P<0.05 as compared to maximum value]. (C) varying concentrations of Ca2+. [Significant at *P<0.05 as compared to the value
without addition of Ca2+, significant at **P<0.05 as compared to the maximum value in the presence of Ca2+]; and (D) varying
concentration of calmodulin. [Significant at *P<0.05 as compared to the basal level of phosphorylation without CaM].
SARKAR et al.: NON-GENOMIC EFFECT OF L-T3
maximal incorporation of 32P was calculated as 7600
CPM/mg protein. To assure maximum Ca2+/CaMdependent protein phosphorylation, 2 µM CaM and
0.5 mM Ca2+ were used in subsequent experiments.
Inclusion of 0.5 mM Ca2+ enhanced 32P
incorporation by ~1.5-fold (P<0.05) over the basal
phosphorylation. Presence of both 0.5 mM Ca2+ and
2 µM CaM caused 2-fold higher (0.189 pmol/min/mg
protein) 32P incorporation compared to the basal
(control) level (0.097 pmol/min/mg protein P<0.05;
Fig. 2). The phosphorylation in the presence of both
0.5 mM Ca2+ and 2 µM CaM was further considered
as a control during evaluation of the L-T3-effect in
the L-T3 + Ca2+/CaM treatment group.
In vitro effect of L-T3 on total protein
phosphorylation: Interaction with calmodulin—
Levels of synaptosomal protein phosphorylation were
determined with and without standardized doses of
Ca2+ or Ca2+/CaM, and graded doses of L-T3 (10 to
1 µM) (Fig. 2). Addition of 0.5 mM Ca2+ (final
concentration) significantly (P<0.05) increased the
Fig. 2—Effect of L-T3 on total protein phosphorylation (32P
incorporation) in the presence of Ca2+ and CaM in adult rat
cerebrocortical synaptosomes. Net incorporation of 32P into
synaptosomal proteins as a function of L-T3 concentration in an in
vitro phosphorylation reaction assay mixture containing
synaptosomal protein (50 µg/mL) incubated for a fixed time
period of 1 min, with 2 µM CaM and 0.5 mM Ca2+ (▼), 0.5 mM
Ca2+ (∆) or no additions (●). [Values are mean ± SEM of 4
independent experiments and vertical bars indicate SEM. Values
are significant at * P<0.05 as compared to the basal level of
phosphorylation without L-T3; #P<0.05 as compared to the level
of phosphorylation with 0.5 mM Ca2+ and no L-T3; α P<0.05 as
compared to the level of phosphorylation with 2 µM CaM, 0.5
mM Ca2+ and no L-T3].
173
level of protein phosphorylation (0.148 pmole/min/mg
protein) by ~ 1.5-fold over the basal phosphorylation
(0.097 pmole/min/mg protein). Addition of 2 µM
CaM (final concentration) to the Ca2+-stimulated
treatment group additionally increased the level of
phosphorylation significantly (#P<0.05) by ~ 1.3-fold
(0.189 pmole/min/mg protein) and by ~2-fold over the
basal value.
In vitro addition of 10 and 100 nM doses of L-T3
alone did not alter significantly the basal
phosphorylation. However, the 1 µM dose of L-T3
significantly amplified the signal by ~1.3-fold
compared to the basal level (P<0.05).
Next, to determine whether Ca2+ enhances protein
phosphorylation in the presence of L-T3, increasing
doses of L-T3 (10 nM to 1 µM) were added in vitro
with a standardized concentration of Ca2+ (0.5 mM) in
the phosphorylation assay reaction. Although 0.5 mM
Ca2+ was able to significantly increase the basal
phosphorylation level, no further significant changes
were seen with additional 10 or 100 nM L-T3.
However, 1 µM concentration of L-T3 augmented
the signal significantly (P<0.05) by ~ 1.5-fold
(2167 pmols/min/mg protein) as compared to the
Ca2+-treated baseline (0.1475 pmols/min/mg protein),
and by ~ 2.2-fold over the basal phosphorylation (Fig. 2).
Thus it was demonstrated that brain physiological
concentrations of 10 nM LT3 and a 10-times higher
dose of L-T3 (100 nM) alone or with Ca2+ did not
significantly alter the levels of synaptosomal protein
phosphorylation in comparison with their respective
control values, i.e., with the basal control level of
phosphorylation and Ca2+-treated controls. This
indicated that these concentrations of L-T3 were not
strongly influenced by the addition of Ca2+. Only after
incubation with higher dose of L-T3 (1 µM), generally
considered to be a pharmacological dose, a significant
increase in phosphorylation was noted (Fig. 2).
In contrast, the effects of low physiological
concentrations of L-T3 were dramatically enhanced
when 2 µM CaM was added to the Ca2+ + L-T3treatment group (Fig. 2). In the presence of Ca2+ and
CaM, L-T3 (10 nM to 1 µM) induced a dosedependent increase in 32P incorporation into
synaptosomal protein, by 47 ± 8 , 74 ± 13 and 52 ± 11%
(P<0.05) (F = 6.77, P<0.0001) rapidly within 1 min
compared with the Ca2+/CaM-treated control
phosphorylation (0.189 pmols/min/mg protein;
Fig. 2).These results suggested a marked Ca2+/CaM
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INDIAN J EXP BIOL, MARCH 2011
dependence of the effect of L-T3 on synaptosomal
protein phosphorylation.
Further studies compared the effect of 1 nM L-T3
in the presence or absence of Ca2+, CaM, and EGTA
(1 mM) on phosphorylation of proteins in
synaptosomal lysates (Fig. 3). In the absence of L-T3,
more 32P was incorporated into the synaptosomal
proteins following the combined treatment with Ca2+
and CaM (143% of the control basal phosphorylation
without additions), although this effect was not
statistically significant. Addition of L-T3 to the
combined treatment with Ca2+ and CaM caused an
increase in phosphorylation (to 295% of basal
phosphorylation, P<0.05). Addition of 1 mM EGTA,
a Ca2+-chelating agent, to the Ca2+/CaM + 100 nM LT3 group considerably decreased (57 ± 8%) the
observed incorporation of 32P (P<0.05). The CaMdependent effect of L-T3 to stimulate phosphorylation
of synaptosomal proteins required the presence
of Ca2+ and was lost with chelation of divalent
cation (Fig. 3).
Fig.3— Effect of EGTA on Ca2+/CaM stimulation of protein
phosphorylation levels in synaptosomes from adult rat cerebral
cortex. Synaptosomal proteins were incubated in an in vitro
phosphorylation reaction assay mixture containing synaptosomal
protein (50 µg/mL) and incubated for 1 min in absence (control)
or presence of 2 µM CaM and 0.5 mM Ca2+ (Ca2+/CaM groups), 1
nM L-T3 (as T3 groups) or 1 mM EGTA (as Ca2+/CaM + T3 +
EGTA group). [Values are mean ± SEM of 4 independent
experiments and vertical bars indicate SEM. [Significant at*
P<0.05 as compared to the basal level (control) without the
additions of Ca2+/CaM, L-T3 or EGTA].
Discussion
Rapid and nongenomic effects of TH on
phosphorylation of neuronal proteins in adult nervous
system are still not fully understood. Phosphorylationdephosphorylation of proteins mediated by protein
kinases and protein phosphatases are major regulatory
events in cellular physiology and signal transduction
mechanisms19. Regulation of phosphorylation of
numerous Ca2+-dependent proteins in neuronal signal
transduction are well-documented and several of these
require the Ca2+-binding protein, CaM20.
The present study demonstrates that Ca2+/CaMdependent mechanisms synergistically enhance the
effect of L-T3 to increase the overall rate of
phosphorylation of adult rat cerebrocortical
synaptosomal proteins. The Ca2+/CaM-dependent
effects could be due to an activation of an unknown
CaM-dependent protein kinase(s), deactivation of
protein phosphatase(s) or a combination of effects. Of
particular note, the net Ca2+/CaM-mediated increase
in phosphorylation levels of synaptosomal proteins
observed in the current study is highly sensitive to LT3 stimulation.
Initially, optimal conditions were selected to
observe the maximum levels of 32P incorporation into
the synaptosomal phosphoproteins (Fig. 1). Linear
relationship of the phosphorylation with increasing
concentrations of synaptosomal proteins and time of
incubation was observed. As the time of incubation
was increased, the phosphorylation level reached a
plateau, suggesting a saturation of potential
phosphorylation sites, a balancing of kinase and
phosphatase activities or an exhaustion of the
exogenously added ATP. Optimization of basal level
of Ca2+/CaM-dependent protein phosphorylation was
observed at 0.5 mM Ca2+ and 2 µM CaM.
Effect of L-T3 alone (10 and 100 nM) in absence of
Ca2+ and CaM did not significantly increase the 32P
incorporation indicative of phosphorylation of
endogenous synaptosomal phosphoproteins, except at
the higher dose (1 µM), compared with control values.
While physiological levels of L-T3 in brain terminals
are difficult to measure, estimates of hormone in the
synapse have ranged from ~10 to 64 nM15,21. It
has been reported previously that during
6-n-propylthiouracil
(PTU)-induced
peripheral
hypothyroidism in adult rats, the synaptosomal level
of L-T3 rises endogenously up to ~126 nM15,21. L-T3
(1 µM) is well above this range, and would be
considered to have a more pharmacological type of
action on 32P incorporation to synaptosomal
SARKAR et al.: NON-GENOMIC EFFECT OF L-T3
phosphoproteins (Fig. 2). Similarly, Ca2+ alone did
not show alterations in 32P incorporation with 10 and
100 nM doses of L-T3, while 1 µM dose of hormone
significantly increased the phosphorylation compared
with the Ca2+-control value (Fig. 2). While euthyroid
or hypothyroid brain concentrations of L-T3 alone or
together with Ca2+ remain ineffective in the
modulation of synaptosomal protein phosphorylation,
the presence of Ca2+ can potentially enhance the
increment in phosphorylation induce by a higher,
pharmacologic dose of L-T3 (1 µM) in adult brain.
Treatment with agents regulating Ca2+ could be a
potential strategy for enhancing clinical treatment of
conditions, such as certain affective disorders, which
may be responsive to pharmacological doses of TH2.
Regulation of intracellular Ca2+ ion concentration is
of vital importance in several aspects of neuronal
signal transduction mechanisms. Entry of Ca2+ ions
into the cytoplasm increases intracellular Ca2+
concentration leading to greater binding to
recognition sites within the cell. One protein with
which Ca2+ interacts efficiently is CaM, a small Ca2+binding protein, that regulates Ca2+/CaM-dependent
protein kinase activities22. The present study showed
effects of physiological concentrations of L-T3 in
mediating a net increase in phosphorylation of
synaptosomal proteins were dependent on the
presence of both Ca2+ and CaM in the incubation.
Filter binding studies using phosphocellulose paper to
trap phosphorylated proteins after in vitro addition of
increasing concentrations of L-T3 (10 nM – 1 µM)
showed dose-dependent effect on Ca2+/CaMdependent increase in protein phosphorylation within
one min of L-T3 exposure in synaptosomes (Figs 2, 3)
with maximal effectiveness at the 100 nM
concentration. Effects on gene expression can
essentially be ruled out within this short period of
time, suggesting a rapid nongenomic action of L-T3.
The results further suggest that an important function
of Ca2+/CaM in adult brain is to enhance the effect of
L-T3. Furthermore, the effect of L-T3 on
synaptosomal phosphoproteins was blocked by 1 mM
dose of EGTA confirming its dependency upon the
presence of Ca2+ and thereby upon CaM.
In an earlier in vitro study, a range of L-T3 doses
(0.1 to 100 nM), have been found to induce a rise in
intrasynaptosomal Ca2+ levels with an optimum at
100 nM of L-T3 (Ref. 11). In that study, the higher
concentration of L-T3 (1 µM) caused a slight
depression of intrasynaptosmal Ca2+ levels11. An
earlier report has shown that even picomolar
175
concentrations of L-T3 are able to significantly
increase intrasynaptosomal Ca2+ levels in vitro12.
Although the present studies used a lysate of
synaptosomal membranes and therefore, would not
detect changes in Ca2+ accumulation, the synergistic
effect of L-T3 and Ca/CaM on protein
phosphorylation would likely be further amplified by
the effect of L-T3 to increase Ca levels intracellularly
in the physiological situation.
Several phosphoproteins are highly regulated by
protein kinase A (PKA) and protein kinase C (PKC)
in a Ca2+- and/or CaM-dependent way23. Often Ca2+
also works in conjunction with CaM or with
phosphoinositides/diacyl glycerol to induce additional
signal transduction pathways within the synaptic
network14. Regulation of intracellular Ca 2+, CaM and
subsequent protein phosphorylation are important for
brain and cognitive functions affected by various
psychiatric disorders24. Membrane depolarizationinduced Ca2+ influx activates extracellularly regulated
kinases/MAPK in a Ca2+/CaM-dependent way in
PC12 cells25. THs also promote MAPK-mediated
serine phosphorylation of the nuclear TH receptor β-1
isoform nongenomically in 293T cells26. Differential
regulation by Ca2+ and CaM of TH-induced
phosphorylation of specific electrophoreticallyseparated proteins in adult rat brain has also been
reported27. L-T3-induced protein phosphorylation
reactions are too early to be accounted for by genomic
actions. The present study establishes the critical role
of CaM in the presence of Ca2+ to the TH regulation
of protein phosphorylation in young adult rat brain
neurons. The results contribute to further understand
potential nongenomic effector mechanisms for TH in
adult brain and will be essential to the ongoing
analysis of proposed neurotransmitter-like or
neuromodulatory actions of the hormones in some
classes of neurons6,21,27-29.
Conclusion
The current investigation demonstrated a rapid
nongenomic effect of L-T3 on overall levels of
phosphorylation of synaptosomal proteins. In
particular, the present results showed a requirement
for Ca2+/CaM signaling to elicit the overall
stimulatory effect of brain physiological levels of TH
on neuronal protein phosphorylation. The findings
suggested a critical interaction between TH and
Ca2+/CaM signaling in the delicate management of
physiologic functions in adult mammalian central
nervous system.
INDIAN J EXP BIOL, MARCH 2011
176
Acknowledgement
Financial support for undertaking this work was
provided by grants IBN-0110961 (JVM, PKS), DBI0421079 (JVM, PKS) and IOS-0724962 (JVM) from
the National Science Foundation.
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References
1
2
3
4
5
6
7
8
9
10
11
12
13
Oetting A & Yen P M, New insights into thyroid hormone
action, Best Pract Res Clin Endocrinol Metab, 21 (2007) 193.
Samuels M H, Schuff K G, Carlson N E, Carello P &
Janowsky J S, Health status, mood, and cognition in
experimentally induced subclinical hypothyroidism, J Clin
Endocrinol Metab, 92 (2007) 2545.
Tang H Y, Lin H Y, Zhang S, Davis F B, & Davis P J,
Thyroid hormone causes mitogen-activated protein kinasedependent phosphorylation of the nuclear estrogen receptor,
Endocrinol, 145 (2004) 3265.
Davis P J, Leonard J L & Davis F B, Mechanisms of
nongenomic actions of thyroid hormone, Front
Neuroendocrinol, 29 (2008) 211.
Sarkar P K, Durga N D, Morris J J & Martin J V, In vitro
thyroid hormone rapidly modulates protein phosphorylation
in cerebrocortical synaptosomes from adult rat brain,
Neurosci, 137 (2006) 125.
Sarkar P K, In quest of thyroid hormone function in mature
mammalian brain, Indian J Exp Biol, 40 (2002) 865.
Bibbs J A & Nestler E J, Serine and Threonine
Phosphorylation, in Basic Neurochemistry: Molecular,
Cellular and Medical Aspects, edited by G J Siegel , R W
Alberts , B Brady , & D L Price , (Elsevier Inc,
Massachusetts) 2005, 391.
Lau F & Huganir R, Tyrosine Phosphorylation, in Basic
Neurochemistry: Molecular, Cellular and Medical Aspects,
edited by G J Siegel , R W Alberts , B Brady & D L Price ,
(Elsevier Inc, Massachusetts) 2005, 415.
Patwardhan P & Miller W T, Processive phosphorylation:
Mechanism and biological importance, Cell Signal, 19
(2007) 2218.
Mason G A, Walker C H & Prange A J Jr., Depolarizationdependent 45Ca uptake by synaptosomes of rat cerebral
cortex
is
enhanced
by
L-triiodothyronine,
Neuropsychopharmacol, 3 (1990) 291.
Chakrabarti N & Ray A K, Rise of intrasynaptosomal Ca2+
level and activation of nitric oxide synthase in adult rat
cerebral cortex pretreated with 3-5-3'-L- triiodothyronine,
Neuropsychopharmacol, 22 (2000) 36.
Sarkar P K & Ray A K, Calcium mobilization within
hypothyroid adult rat brain synaptosomes, Horm Metab Res,
35 (2003) 562.
Iqbal Z, Koenig H & Trout J H, Triiodothyronine (T3)
stimulates calcium influx and membrane trasport processes
16
17
18
19
20
21
22
23
24
25
26
27
28
29
in nerve cells and terminals of hypothyroid mouse cortex,
Fed Proc, 43 (2002) 735.
Segal J, Calcium is the first messenger for the action of
thyroid hormone at the level of the plasma membrane: first
evidence for an acute effect of thyroid hormone on calcium
uptake in the heart, Endocrinol, 126 (1990) 2693.
Sarkar P K & Ray A K, Synaptosomal T3 content in cerebral
cortex of adult rat in different thyroidal states,
Neuropsychopharmacol, 11 (1994) 151.
Sarkar P K & Ray A K, A simple biochemical approach to
differentiate synaptosomes and non-synaptic mitochondria
from rat brain, Methods Find Exp Clin Pharmacol, 14
(1992) 493.
Sarkar P K & Ray A K, Specific binding of L-triiodothyronine
modulates Na+-K+-ATPase activity in adult rat cerebrocortical
synaptosomes, NeuroReport, 9 (1998) 1149.
Vera J C, Measurement of microgram quantities of protein
by a generally applicable turbidimetric procedure, Anal
Biochem, 174 (1988) 187.
Walaas S I & Greengard P, Protein phosphorylation and
neuronal function, Pharmacol Rev, 43 (1991) 299.
Schulman H, The multifunctional Ca2+/calmodulin-dependent
protein kinases, Curr Opin Cell Biol, 5 (1993) 247.
Martin J V, Williams D B, Fitzgerald R M, Im H K &
vonVoigtlander P F, Thyroid hormonal modulation of the
binding and activity of the GABAA receptor complex of
brain, Neurosci, 73 (1996) 705.
Fujisawa H, Regulation of the activities of multifunctional
Ca2+/calmodulin-dependent protein kinases, J Biochem, 129
(2001) 193.
Xie Z & Xie J, The Na/K-ATPase-mediated signal
transduction as a target for new drug development, Front
Biosci, 10 (2005) 3100.
Monnet F P, Sigma-1 receptor as regulator of neuronal
intracellular Ca2+: clinical and therapeutic relevance, Biol
Cell, 97 (2005) 873.
Egea J, Espinet C & Comella J X, Calcium influx activates
extracellular-regulated kinase/mitogen-activated protein
kinase pathway through a calmodulin-sensitive mechanism in
PC12 cells, J Biol Chem, 274 (1999) 75.
Shih A, Lin H Y, Davis F B & Davis P J, Thyroid hormone
promotes serine phosphorylation of p53 by mitogen-activated
protein kinase, Biochemistry, 40 (2001) 2870.
Sarkar P K, L-triiodothyronine differentially and
nongenomically
regulates
synaptosomal
protein
phosphorylation in adult rat brain cerebral cortex: role of
calcium and calmodulin, Life Sci, 82 (2008) 920.
Mason G A, Walker C H & Prange A J, L-Triiodothyronine:
Is this peripheral hormone a central neurotransmitter?
Neuropsychopharmacol, 8 (1993) 253.
Dratman M B & Gordon J T, Thyroid hormones as
neurotransmitters, Thyroid, 6 (1996) 639.