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(Received for publication, April 20,
1995; and in revised form, September 25, 1995) From the
The existence of endogenous compounds interacting with the
serotonergic system was previously postulated. In the present work, rat
brain tissues were extracted by acidic and organic procedures. The
resulting extract was tested for its capacity to interact with the
binding of [ A
tetrapeptide corresponding to a novel amino acid sequence
Leu-Ser-Ala-Leu (LSAL) was identified. It reduces the binding of
[ Two other peptides,
Leu-Ser (LS) and Ala-Leu (AL), were also purified. They hardly affected
[
The serotonergic system is thought to play an important role in
mental disorders and particularly in depression(1) . For a long
time, it had been proposed that this pathology was related to a deficit
in the serotonergic transmission(2, 3) . Accordingly,
antidepressant drugs essentially restore a normal level of 5-HT ( Furthermore, it
was also shown that antidepressant drugs could act on 5-HT Among 5-HT According to these results, the hypothesis of the existence of an
endogenous factor acting at 5-HT Herein, we report the isolation and
characterization of a cerebral compound, which specifically interacts
with 5-HT
The TSK HW 40S column was
obtained from Merck, and the Sephadex G
Rat brain cortices were dissected on ice
and rapidly homogenized for 30 s with an Ultraturrax apparatus (Ikka
Werk) in a 50 mM Tris-HCl buffer, pH 7.4, containing 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 5
IU/liter aprotinin. Homogenates were then incubated for 10 min at 37
°C to remove endogenous ligands, diluted in 30 volumes (v/w) of the
same medium, and centrifuged (17,500
The uptake of 5-HT, dopamine, noradrenaline,
The retention time of
[
Figure 1:
Size exclusion
chromatography of rat brain extract. Rat brain extract, prepared as
described under ``Experimental Procedures,'' was loaded on
the top of a TSK HW 40S column (700
Figure 2:
Reverse phase chromatography of rat
F
The NR
fraction was eluted in the void volume of the column. Further attempts
to purify it, on reverse or normal phase, on ion exchange, or on
hydrophobic columns, as well as on modified reverse phase columns, e.g. -NH P fraction represented the main activity inducing
83 ± 13% inhibition of the 5-HT
Figure 3:
Purification of rat P fraction. P fraction
(rat) was purified by four successive chromatographic steps: Sephadex
G
The biological activities observed for rat
P
Figure 4:
Pattern of activity of the bovine F
Figure 5:
Purification of bovine P
Amino acid analysis of bovine P
Figure 7:
Stability of LSAL. 3 µCi of
[
In brain, the hippocampal formation contained the
highest amount of inhibitory activity, followed by cortex; intermediate
levels were present in the striatum and cerebellum, whereas low levels
were detected in the brain stem. The relative quantities per gram of
original tissue were 14.28, 6.25, 2.22, 1.82, and 0.70 (arbitrary
units), respectively (Table 3). In the periphery, the inhibitory
activity was essentially found in kidney (2.5), heart (2.5), and lung
(1.4). Low activities were detected in stomach (0.5) and blood (0.14),
whereas it was undetectable in liver and spleen tissue (Table 3).
Figure 8:
Pharmacological specificity of the
synthetic LSAL. A, interaction of LSAL with 5-HT receptors.
Increasing concentrations of synthetic LSAL (10
The inhibitory effect of LSAL on
5-HT
Figure 9:
Interaction of synthetic P (LSAL), P
The results reported herein describe the isolation and
purification of a cerebral factor able to specifically interact with
the 5-HT The methodology developed to isolate
this factor is a classical acid and organic procedure followed by HPLC
chromatographic techniques. The initial step, including freezing and
lyophylization of the brain tissue, was introduced to decrease the
endogenous protease activity. The following step, which consisted of an
ultracentrifugation (120,000 The
initial size exclusion chromatography led to the separation of the
F The further
C The major activity retained on the column was the P fraction (t The P fraction was purified by
gel permeation (Sephadex G The identification of the
chemical structure of the P fraction, using amino acid analysis and
peptide sequencing, resulted in its characterization as the peptide
Leu-Ser-Ala-Leu. Complementary analysis using the NMR technique could
not detect the presence of any other compound, indicating that the
purification of the P fraction was conducted up to homogeneity. The
pharmacological specificity of the synthetic peptide was established
using dose-response curves. It was then demonstrated that only the
5-HT As expected, the
synthetic peptide exhibited a pharmacological profile very similar to
that of the P fraction tested at a dose corresponding to 1% of the
total purified fraction. Indeed, it was calculated that this dose
corresponded to a peptide concentration of 8.10 The activities of rat P LS and AL were poorly active compared with
LSAL (100,000 and 10,000 times less efficient, respectively).
Accordingly, P LSAL exhibited similar properties of binding
inhibitions in rat brain and in bovine brain cortical membranes,
indicating that it also interacted with 5-HT LSAL
is an original sequence not represented in any of the known peptides or
peptide precursors (Swissprot protein bank). Moreover, this peptide is
not homogeneously distributed within the brain but rather is present in
higher amounts in some brain areas (hippocampus, cortex) than in others
(brain stem). These brain areas have been shown to contain 5-HT Although it is too early to know the origin of
this compound and its potential pathophysiological implications, these
results demonstrate that LSAL is able to specifically interact with
5-HT
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 726-735
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
H]5-hydroxytryptamine
([H]5-HT) to 5-HT
receptors.
Compounds responsible for the observed inhibitory activities were
isolated and purified by high pressure liquid chromatography.H]5-HT to 5-HT receptors at low
concentration (IC = 10
M). This effect corresponds to a specific interaction at
5-HT
receptors since LSAL does not significantly affect
other neurotransmitter bindings. LSAL appears heterogeneously
distributed throughout the brain (hippocampus > cerebellum >
striatum > brain stem) and in peripheral tissues (kidney > lung
> stomach > blood > liver > spleen).
H]5-HT binding compared with LSAL. They
presumably represent degradation products of the functional peptide
LSAL. The fact that LSAL interacts specifically with 5-HT receptors that inhibit the release of neurotransmitters and
particularly that of 5-HT itself suggests that this peptide may be
involved in mechanisms controlling 5-HT neurotransmission and,
accordingly, may play an important role in pathophysiological functions
related to 5-HT activity.
)activity. Antidepressant drugs can be classified into
groups according to their primary mode of action, i.e. monoamine oxidase inhibitors, tricyclic antidepressants, and
selective serotonin reuptake inhibitors(4) . receptors(5, 6, 7) . The corresponding
mechanism of interaction was shown to be noncompetitive suggesting that
a site, distinct from that actually binding the amine, existed on these
receptors and specifically recognized these drugs and possibly
endogenous ligands(5) . receptors,
5-HT receptors, located on rat serotonergic neuron
terminals, play a crucial role in regulating the release of the
amine(8) . In non-rodents, 5-HT
receptors,
which are the species homolog of rodent 5-HT
, play the
same functional role(9, 10) . Experiments carried out
in rat in in vitro assays showed that several antidepressants
specifically interacted with 5-HT
receptor subtypes (11, 12, 13) modifying their sensitivity
after long term treatment (14, 15, 16, 17, 18) .
receptors was postulated.
Thus, we explored this hypothesis in examining the capacity of various
fractions, isolated from brain extracts, to interact with 5-HT binding sites. binding sites.
Materials
Male Wistar rats (150-200 g)
were obtained from IFFA CREDO (France). Bovine brains were collected at
the slaughterhouse. [H]8-OH-DPAT (3.7 TBq/mmol),
[H]ketanserin (2.22 TBq/mmol),
[H]BRL 43694 (1.85 TBq/mmol),
[H]DOB (0.37 TBq/mmol), and
[H]choline (3 TBq/mmol) were purchased from
DuPont NEN. [H]5-HT (3.26 TBq/mmol), I-cyanopindolol (81.4 TBq/mmol),
[
H]spiroperidol (0.55 TBq/mmol),
[H]quinuclidinyl benzylate (3.33 TBq/mmol),
[H]naloxone (1.48 TBq/mmol),
[H]mepyramine (0.74 TBq/mmol),
[H]prazosin (2.59 TBq/mmol),
[H]dihydroalprenolol (3.33 TBq/mmol),
[H]flunitrazepam (2.22 TBq/mmol),
[-
H]aminobutyric acid (3.33 TBq/mmol),
[H]dopamine (185 GBq/mmol), and
[H]noradrenaline (444 GBq/mmol) came from
Amersham Corp. [H]LSAL (4.14 TBq/mmol) was
synthesized by the Service des Molécules
Marquées, CEA-CEN. resin was from
Pharmacia Biotech Inc. The C
Ultrabase and Hypercarb
column were purchased from SFCC-Shandon. Synthetic peptides came from
Bachem for AL and LS and Neosystem for LSAL.Rat Brain Extracts
Assays were carried out using
brain tissue usually prepared from 60 rats (about 90 g). The tissues
were lyophilized and homogenized with an Ultraturrax apparatus (Ikka
Werk) in 10 volumes (w/v) of H
O containing 2 mM EDTA, 5 IU/liter aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at
17,500 
g for 40 min at 4 °C. The resulting
supernatant was kept at 4 °C, and the pellet was washed two
additional times. The three supernatants were then pooled, lyophilized,
and resuspended in 6 volumes (v/original weight) of 1 M acetic
acid. After a stirring period of 40 min at 4 °C, the mixture was
centrifuged (17,500 g for 40 min at 4 °C), and the
supernatant was lyophilized. An additional extraction was performed in
2 volumes (v/original weight) of 75% acetone. After centrifugation
(17,500 g for 20 min. at 4 °C), the resulting
supernatant was evaporated under vacuum at 40 °C in a rotavapor
apparatus (R110, Büchi). The dried extract was then
resuspended in 50 ml of HO and ultracentrifuged (120,000
g for 60 min at 25 °C). The upper lipid phase was
discarded, and the supernatant was lyophilized and stored at -70
°C until use.Localization of Biological Activity
At the
completion of each following chromatographic step, an aliquot of each
collected fraction (1%) was tested for its ability to displace the
binding of [H]5-HT to the 5-HT and
5-HT binding sites(20) . Rat brain cortical
membranes were incubated in a 50 mM Tris-HCl buffer, pH 7.4,
containing 0.1% ascorbic acid, 0.1% bovine serum albumin, 4 mM CaCl
, 1 µM pargyline, 5 nM
[H]5-HT with (5-HT) or without
(5-HT
) 0.1 µM 8-OH-DPAT, in the
presence/absence of the various fractions separated by the
chromatographic procedure. Incubation was carried out for 30 min at 25
°C in a total volume of 1 ml. Nonspecific binding was determined in
the presence of 10 µM of 5-HT. At the end of the
incubation period, free and bound radioactivities were separated by
filtration under vacuum on Whatman GF/B glass fiber filters. Each
tested tube was then washed twice with 5 ml of ice-cold incubation
buffer. The bound radioactivity retained on the filter was measured by
liquid scintillation counting (spectrometer Beta IV, Kontron). Active
fractions were then pooled and lyophilized before injection in the next
chromatographic column.Isolation of the Rat F
The
crude extract of rat brains was dissolved in 5 ml of 50 mM ammonium acetate buffer, pH 5, loaded onto a TSK HW 40S column
(700 Fraction 26 mm; M
separation range:
10,000-1,000) equilibrated in the same buffer, and eluted at a
flow rate of 2 ml/min. UV absorption was measured at 280 nm, and 60
fractions of 5 min each were collected.Separation of Rat P, P
Fractions 12-24 of the TSK HW 40S column
were pooled, lyophilized, and resuspended in 5 ml of 50 mM
ammonium acetate buffer, pH 5. Aliquots (1 ml) were injected into a
C, and P Fractions Ultrabase reverse phase column (250 10 mm),
equilibrated in the same buffer. The elution was performed at a flow
rate of 4 ml/min with a linear gradient of acetonitrile (0-12%)
followed by a 5-min step gradient (50% acetonitrile). The detection
wavelength was 240 nm. Thirty fractions of 1 min were collected. For
all the following chromatographic steps, absorbance was measured at 230
nm (except when indicated).Purification of Rat P Fraction
P fraction obtained
from the C Ultrabase chromatographic step was dissolved in
2 ml of a 10 mM ammonium acetate buffer, pH 5, and loaded onto
a Sephadex G column (450
16 mm; M separation range: 5,000-500). Elution was performed at a
flow rate of 0.3 ml/min with the same buffer. The chromatogram was
monitored at 280 nm, and 50 fractions of 20 min were collected. A
reverse phase HPLC column (C Ultrabase, 250 10 mm)
was then used. The equilibrating buffer consisted of a mixture of
ammonium acetate (50 mM, pH 5) and acetonitrile (85:15). The
elution was performed with a 15-min linear gradient of acetonitrile
(15-25%) followed by a 5-min step gradient at 50% acetonitrile at
a flow rate of 4 ml/min. 20 fractions of 1 min were then collected.
After lyophilization, the active fraction was injected into a carbon
column (Hypercarb, 100 3 mm) and eluted with a 50 mM
ammonium acetate buffer, pH 5. A 30-min linear gradient of acetonitrile
(0-30%) was used. The flow rate was 1 ml/min, and 40 fractions of
1 min were collected. Final purification step consisted of a reverse
phase chromatography using a C Ultrabase column (150
4 mm) under isocratic elution conditions (0.5% trifluoroacetic
acid, pH 2.5, and acetonitrile (83:17)) at a flow rate of 1 ml/min. The
active fraction was collected manually.Bovine Brain Extract
The same purification
procedure was carried out, i.e. acidic and organic
extractions, gel permeation on TSK HW 40S, and C Ultrabase
separations. Two bovine brains (about 600 g; 35.21 ± 0.62 g of
protein equivalent) were thus processed, which led to the recovery of
three fractions having precisely the same retention time as rat P,
P, and P fractions.Purification of Bovine P
P Fraction fraction was first loaded on the
Sephadex G column. The elution procedure was the same as
for rat P fraction. The active fractions were loaded onto a C
Ultrabase column (250 10 mm) equilibrated in a 50 mM ammonium acetate buffer, pH 5. Elution was run at 4 ml/min using a
linear gradient of acetonitrile (20 min, from 0 to 5%) followed by a
10-min step gradient at 30% acetonitrile. Thirty fractions of 1 min
were collected. This chromatographic step was repeated three times.
Active fractions were then subjected to a Hypercarb column (100
3 mm) in the same buffer conditions. A 60-min linear gradient of
acetonitrile was used (0-30%) at a flow rate of 1 ml/min. Sixty
fractions of 1 min were collected. Two further separations on the same
column, using isocratic elutions with a 50 mM ammonium acetate
buffer, pH 5, at a flow rate of 0.5 ml/min, were required to achieve
the purification of P fraction.Purification of Bovine P
The
first separation step was a gel filtration on a Sephadex G Fraction column. Chromatographic conditions were identical to those used
for rat P and bovine P
fractions. Then active fractions
were injected in a C Ultrabase column (250 10 mm).
The mobile phase was a 50 mM ammonium acetate buffer, pH 5.
Elution was carried out using a linear gradient of acetonitrile (40
min., from 0 to 10%) followed by a 10-min step gradient at 30%
acetonitrile. The flow rate was 4 ml/min, and 50 fractions of 1 min
were collected. A Hypercarb column (100 3 mm), equilibrated in
the same buffer, was then used. A 35-min gradient of acetonitrile
(0-20% in 30 min, 5 min at 30%) was carried out at 1 ml/min, and
40 fractions of 1 min were collected. Finally, active fraction was
reinjected in the same column, and an isocratic elution was performed
with a 50 mM ammonium acetate buffer, pH 5, at a flow rate of
0.5 ml/min.Amino Acid Analysis
Amino acid contents of bovine
P and P fractions were determined after a 24-h
hydrolysis in 6 N HCl at 120 °C. A model 6300 Beckman
apparatus was used. Amino acid separation was performed on a C reverse phase column after cadmium-ninhydride reaction.
Norleucine was used as an internal standard.NMR Analysis
The content of the various purified
fractions (P, P, P) and their chemical
structures (P, P) were examined in
DO and dimethylsulfoxide using a 500-MHz NMR spectrometer
(Varian).Protein Sequencing
The sequence of the purified
rat P fraction was determined using a 473 Applied Biosystems protein
sequencer using a microcartridge. Chemicals and methods were those
recommended by the manufacturer.Sequence Comparison
Purified peptide sequence was
compared with those published in the Swissprot protein bank (Genetic
Computer Group, Inc).Protein Measurement
The protein equivalents were
measured by the Lowry method(19) . The standard curve was
established with bovine serum albumin.Pharmacological Studies
Interaction of the
purified P fraction (1% of the total preparation) or synthetic LSAL (1
nM or increasing concentrations (10 to
10
M)) was examined either at various 5-HT
receptors or on other neurotransmitter receptors. Experiments were
carried out on rat (or bovine when indicated) brain cortical membranes
in a total volume of 1 ml.
g at 4 °C for
10 min). The pellet was resuspended in 30 volumes of the same buffer
and centrifuged as described above. The homogenate was then washed an
additional time, and the resulting pellet was resuspended in the
appropriate incubation buffer. Incubation buffers and the different
specific radiolabeled ligands are described in Table 1according
to the
literature(20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) .
Binding assays were performed after 30 min of incubation at 25 °C
with 500 µg of protein equivalents/incubate except for the one
using I-cyanopindolol, which was incubated for 60 min at
37 °C in the presence of 25 µg of proteins. At the end of the
incubation period, the tubes were cooled on ice for 10 min and filtered
under vacuum on Whatman GF/B glass fiber filters. Each filter was then
washed twice with 5 ml of ice-cold incubation buffer and dried. The
radioactivity retained on the filters was then measured either by
liquid scintillation counting as described previously (for tritiated
radioligands) or by
-counting (for iodinated radioligand)
(Spectrometer Crystal
multidetector radioimmune assay
system, Packard).
-aminobutyric acid, and choline were measured on rat cortical
synaptosomes prepared according to the method of Cotman and
Matthews(33) . Synaptosomes were incubated for 15 min at 37
°C in an oxygenated Krebs-Ringer buffer, pH 7.4 (118 mM NaCl, 4.7 mM KCl, 1.17 mM
KH
PO
, 1.22 mM CaCl, 1.25
mM MgSO, 25 mM NaHCO, and 10
mM glucose) in the presence of 20 nM of the different
neurotransmitters with or without aliquots of the purified P fraction
(1% of the total preparation) or increasing concentrations of the
synthetic LSAL (10 to 10
M). The final incubation volume was 250 µl. Passive uptake
was measured at 4 °C. Uptake reactions were stopped by the addition
of 2 ml of ice-cold incubation buffer (4 °C). Incubates were
rapidly filtered under vacuum on Whatman GF/B glass fiber filters. Each
filter was washed with 15 ml of cold incubation buffer (4 °C) and
dried. The radioactivity retained on the filters was then measured by
liquid scintillation counting.
Stability of LSAL
3 µCi of labeled LSAL were
added to the rat cerebral homogenate and extracted as described
previously. A blank of extraction was also carried out in the absence
of cerebral material. 10% aliquots of the radioactive extracts were
then analyzed on a C reverse phase column (C Ultrabase, 1504 mm) equilibrated with an ammonium acetate
buffer (50 mM, pH 5). Elution was run at 1 ml/min with an
isocratic step of 3 min followed by a linear gradient of acetonitrile
(0-30% in 30 min) and a 5-min step gradient at 50% acetonitrile.
Forty fractions of 1 min each were collected, and the radioactivity was
measured by liquid scintillation counting.H]LSAL under the same experimental conditions
was determined by a control injection.
Extractions
Brain extracts were prepared (from
about 90 g of tissue) using the usual extraction procedure, i.e. 1 M acetic acid and 75% acetone extractions and
ultracentrifugation. The crude homogenate contained an equivalent of
5.3 ± 0.2 g of proteins (mean ± S.E. of three independent
determinations). At the various extraction steps, the recovery of the
protein equivalent was 1.1 ± 0.1 g in the HO
supernatant, 0.54 ± 0.03 g in the acidic extract, 0.39 ±
0.01 g in the acetonic extract, and 0.049 ± 0.003 g in the final
ultracentrifugation supernatant. The resulting crude material obtained
is essentially deproteinized and delipidated.Isolation of the Rat F
Size
exclusion chromatography on a TSK HW 40S column was carried out. This
chromatographic step allowed to isolate two biological active fractions
able to interact with the binding of [ FractionH]5-HT to
5-HT sites (Fig. 1); the first fraction
(F
) eluted at 80 min and corresponded to a material having
an apparent molecular weight of 4,000 determined on the basis of
protein or peptide standard elutions. The second active fraction had an
elution time of 540 min and a low apparent molecular weight (less than
500). Using [H]5-HT as an internal standard, this
second fraction was identified as endogenous 5-HT.
26 mm; M separation range: 10,000-1,000). The elution was performed
at a flow rate of 2 ml/min with a 50 mM
CHCOONH buffer, pH 5.
Absorbance was observed at 280 nm. Aliquots corresponding to 1% of each
fraction were then tested for their abilities to displace
[
H]5-HT (5 nM) from its 5-HT binding sites on rat brain synaptosomal membranes
(
-). Each binding point is the mean ±
S.E. of three independent determinations. A coinjection of
[H]5-HT was realized under the same conditions as
internal standard
(-
).
Isolation of Rat P, P
The separation of the F, and P Fractions fractions on
a C reverse phase column led to the recovery of an active
fraction having a retention time of 21 min and called P fraction.
Additional active fractions were also obtained. Their retention times
were 3.30 min (NR), 9 min (P), and 12 min (P),
respectively. They were distinct from endogenous 5-HT as controlled by
using [H]5-HT (t
=17.45 min) (Fig. 2).
fraction. 2 ml of rat F fraction were
injected in a C Ultrabase column (250 10 mm). The
elution was performed as described under ``Experimental
Procedures.'' 1% aliquots of each fraction were tested for their
capacities to displace [H]5-HT (5 nM)
from its 5-HT binding sites on rat brain synaptosomal
membranes (
-). Each point is the mean ±
S.E. of three independent determinations. A coinjection of
[H]5-HT was realized under the same conditions as
internal standard
(-
).
, -OH, or -CN, did not lead to further
separation. Moreover, dialysis of this fraction (membrane cut-off:
1,000) led to the loss of the activity. This result suggests that NR
contains a high concentration of salts that interfere with the
biological test. binding (eight
independent determinations). The two other fractions, P
and
P, tested under the same experimental conditions, were less
efficient (60 ± 5% and 30 ± 8% inhibition of the
5-HT binding, respectively, eight independent extracts) (Fig. 2).
Purification of Rat P Fraction
The purification of
P fraction was carried out using size exclusion and reverse phase
chromatographies (Fig. 3). A first step of purification was
performed using a Sephadex G column, which possesses a
suitable separation field, and led to the recovery of an active
fraction having an elution time of 3.3 h. Successive reverse phase
chromatographies (C
Ultrabase, Hypercarb, and C Ultrabase) were required to obtain the final purification step
corresponding to an apparent homogeneous fraction. Each step of
separation led to the isolation of a single peak of activity. The use
of a carbone column (Hypercarb, interactions) allowed us to
separate P fraction from the majority of the material coeluted on a
classical C
reverse phase (C Ultrabase). At
these last steps of purification, the corresponding retention times for
P fraction were 5.30, 12.30, and 5.50 min for C Ultrabase,
Hypercarb, and C Ultrabase columns, respectively (Fig. 3, B, C, and D).
(A), C
Ultrabase (B),
Hypercarb column (C), C Ultrabase (D).
Chromatographic conditions were described under ``Experimental
Procedures.'' Biological activity (filled bars) was
determined in duplicates on 1% aliquots as described under
``Experimental Procedures.'' At each step of purification,
the respective retention times of the P fraction were 3.30 h (A), 5.30 min (B), 12.30 min (C) and 5.5 min (D). The detection wavelengths were 280 (A) and 230
nm (B, C, and D).
Identification of Rat P Fraction
NMR technique
demonstrated that the P fraction purified from rat brain extract was
homogeneous and only contained a peptide (not shown). It was
characterized by amino acid analysis and protein sequencing as LSAL.
The amounts of LSAL determined by this analytic process were 500 and
1000 pmol for the two independent batches sequenced. The molecular
weight of LSAL being 402.5, it represented a mean of 0.3 µg of the
peptide. Thus, the purification index was greater than
10
-fold (0.3 µg out of 60 rat brains corresponding to
90 g of wet weight). and P fractions were too low to identify them
in further purification steps. Therefore, this attempt was made using
bovine brains (600 g of initial material) that were extracted as
described above. The extract also contained three active fractions
corresponding to the same retention times as those already observed in
the rat brain extract (Fig. 4, solid line). When the
binding analysis was carried out on bovine brain cortical membranes
instead of rat brain membranes, the same pattern of activity was
observed (Fig. 4, dashed line). Moreover, the
inhibitory effects of the three fractions were not significantly
different from those measured on rat brain cortical membranes. Thus,
bovine P and P fractions were purified using
essentially the same purification procedure ( Fig. 5and 6).
fraction on reverse phase chromatography. 2 ml of bovine F fraction were injected in a C Ultrabase column (250
10 mm). The elution was performed as described under
``Experimental Procedures.'' 1% aliquots of each fraction
were tested for their capacities to displace
[H]5-HT (5 nM) from its 5-HT binding sites on rat (solid line) or bovine (dashed
line) brain cortical membranes. Each point is the mean ±
S.E. of three independent determinations.
fraction. Bovine P fraction was purified by five successive
chromatographic steps: Sephadex G (A),
C
Ultrabase (B), and Hypercarb column (C, D, and E). Chromatographic conditions
were described under ``Experimental Procedures.'' Biological
activity (filled bars) was determined in duplicates on 1%
aliquots as described before. For each purification step, the
respective retention times of the P fraction were 5.30 h (A), 8.30 min (B), 3 min (C), and 2.5 min (D and E). The detection wavelengths were 280 nm (A) and 230 nm (B, C, D, and E).
and P fractions showed that these compounds contained Leu, Ser, and
Ala, Leu, respectively. A ratio of 0.82 (Ser:Leu) and 1.06 (Ala:Leu)
between amino acids suggested that P and P fractions corresponded to dipeptidic structures. They were
identified as peptide LS for P fraction and peptide AL for
P fraction by two-dimensional NMR spectroscopic techniques.
Spin systems were identified via through-bond connectivities (TOCSY)
and sequential assignment was obtained via through-space connectivities
(ROESY) showing unambiguously that the two amino acids were linked (not
shown). Using norleucine as internal standard, amino acid analysis
showed that P and P fractions represented
relatively large amounts (200 µg for each of them) corresponding to
a purification index of 4 10
.Stability of LSAL
The stability of LSAL during the
different steps of the isolation procedure was tested using a labeled
LSAL. Under our experimental conditions, 83 and 81% of the added
radioactivities were recovered in the extraction control (blank
extract) and the cerebral extract, respectively. The analysis of the
content of these extracts on a reverse phase C column
showed that more than 80% of the recovered radioactivity corresponded
to native LSAL (t = 20.5 min) (85.4 and 83%
for the blank and cerebral extracts, respectively). Two other minor
peaks of radioactivity (t = 13 min and t = 26 min) were detected in both extracts.
They represented less than 10% of the total radioactivity. In the brain
extract, an additional peak (t = 5 min, 4%
of the total radioactivity) was observed (Fig. 7).
H]LSAL were added to the buffer of
homogenization (control of extraction or blank extract) or to the rat
cerebral homogenate (cerebral extract) and submitted to the extraction
process as described under ``Experimental Procedures.'' The
extracts were then analyzed on a C reverse phase column
(C Ultrabase) as described previously. 40 fractions of 1
min were collected, and their radioactivity was counted in a liquid
scintillation spectrometer after the addition of 4 ml of counting
scintillant liquid (BCS, Amersham).
Pharmacological Specificity of Rat P
Fraction
Aliquots of the purified P fraction (1% of the total
fraction) were used to examine its pharmacological properties. This
fraction specifically interacted with 5-HT receptors. More
precisely, it exhibited a specific interaction with the 5-HT receptor subtypes. Indeed, no significant inhibition was observed
on other serotonergic receptor bindings (5-HT
,
5-HT
, 5-HT
, 5-HT
) as well as
on other neurotransmitter receptor bindings (Table 2). Moreover,
at the same dose, the P fraction did not show any significant activity
either on the uptake of 5-HT or that of other neurotransmitters (or
their precursor), i.e. uptake of choline, -aminobutyric
acid, noradrenaline, and dopamine (Table 2).
Regional Distribution of the P Fraction in Rat Brain and
Peripheral Tissues
Tissues from 10 rats were extracted and
partially purified to the step corresponding to the separation of the
F fraction on C reverse phase chromatography
(as represented in Fig. 2). The resulting P fractions were then
tested for their abilities to inhibit 5-HT specific
binding. A dose-response curve was established for each P fraction, and
the corresponding ID
was used as a measurement of the
amount of LSAL in the fraction and expressed per gram of initial tissue (Table 3).
Synthetic Peptides
Binding assays measuring the
inhibitory effect of synthetic LSAL on the
[H]5-HT binding to 5-HT receptors
showed an apparent affinity corresponding to an IC
= 1.10
M with a maximal
effect reducing by 75-85% the specific binding. A very similar
effect was observed on the binding of
[
I]iodocyanopindolol to the same receptor
subtype (not shown). LSAL inhibits at higher concentrations the binding
of [
H]8-OH-DPAT to 5-HT receptors
(IC
= 1 µM) and is devoid of any
significant activity on other 5-HT
receptor subtypes
(5-HT, 5-HT
) as well as on other serotonergic
receptors (5-HT
, 5-HT
) or on other
neurotransmitter receptors (
and 
-adrenergic, D dopaminergic, H histaminergic, muscarinic, opiate,
and benzodiazepine), even at 1 or 10 µM (Fig. 8, A and B). Moreover, LSAL did not show any activity on
the uptake of different neurotransmitters or their precursors
(serotonin, noradrenaline, dopamine, choline, and -aminobutyric
acid) even at 10
M (Fig. 8C).
to
10
M) were incubated in the presence of
various specific radiolabeled ligands on rat brain cortical membranes.
Binding conditions were the same as for the purified P fraction (see Table 1). Each point is the mean ± S.E. of three
independent determinations. This experiment was repeated twice. B, Interaction of LSAL with other receptors. Increasing
concentrations of synthetic LSAL (10
to
10
M) were incubated in the presence of
various specific radiolabeled ligands on rat brain cortical membranes.
Binding conditions were the same as for the purified P fraction (see Table 1). Each point is the mean ± S.E. of three
independent determinations. This experiment was repeated twice. C, interaction of LSAL with the uptake of different
neurotransmitters (or precursors). 20 nM of the different
tritiated neurotransmitters (5-HT, dopamine, noradrenaline, choline,
and
-aminobutyric acid) were incubated in a Krebs-Ringer buffer
with rat brain synaptosomes (100 µg of proteins) with or without
increasing concentrations of synthetic LSAL (10
M to 10
M) for 10 min at 37
°C. Each point is the mean ± S.E. of three independent
determinations. This experiment was repeated three
times.
receptors was also compared with those of the
synthetic dipeptides (LS and AL). Indeed, LS and AL were, respectively,
100,000 and 10,000 times less efficient than the tetrapeptide (Fig. 9A). Finally, the synthetic peptides were also tested
on 5-HT
receptors using bovine brain cortical
membranes. Thus, LSAL inhibited the binding of
[
H]5-HT to 5-HT receptors with
an IC
= 7.10
M and a
maximal effect reducing by 75-85% the specific binding. The
synthetic dipeptides LS and AL were 500,000 and 100,000 times less
efficient than the tetrapeptide, respectively (Fig. 9B).
(LS), and P (AL) with 5-HT receptors. A, interaction with 5-HT
receptors. [
H]5-HT (5 nM) was
incubated for 30 min at 25 °C with increasing concentrations of
synthetic peptides (LSAL (-), LS
(
-), and AL (
-)) in
the presence of rat brain cortical membranes (100 µg of proteins)
in a 50 mM Tris-HCl buffer, pH 7.4, containing 4 mM CaCl, 0.1% ascorbic acid, 1 µM pargyline,
0.1 µM 8-OH-DPAT, and 0.1 µM mesulergine (V
= 200 µl). Nonspecific binding was
determined in the presence of 0.1 µM 5-CT. Specific
binding represented about 50% of the total binding and corresponded
typically to 1000 cpm. Each point is the mean ± S.E. of three
independent determinations. This experiment was repeated three times. B, interaction with 5-HT receptors.
[
H]5-HT (5 nM) was incubated for 30 min
at 25 °C with increasing concentrations of synthetic peptides (LSAL
(-), LS (-), and AL
(-)) in the presence of bovine brain
cortical membranes (100 µg of proteins) in a 50 mM Tris-HCl buffer, pH 7.4, containing 4 mM CaCl, 0.1% ascorbic acid, 1 µM pargyline,
0.1 µM 8-OH-DPAT (V = 200
µl). Nonspecific binding was determined in the presence of 0.1
µM 5-CT. Specific binding represented 50-60% of the
total binding and corresponded typically to 1500 cpm. Each point is the
mean ± S.E. of three independent determinations. This experiment
was repeated twice.
receptor.
g for 60 min at 25
°C) allowed us to separate and to discard endogenous lipids (upper
phase). Under these experimental conditions, the resulting aqueous
phase contained less than 1% of the original protein equivalent. fraction, which exhibited an inhibitory activity on the
binding of [H]5-HT to 5-HT sites. At
that early step of purification, the pharmacological profile of the
fraction already exhibited a clear selectivity for 5-HT receptors, since the fraction did not affect the binding of
specific radioligands to other neuroreceptors under study (not shown).
It was also demonstrated that F fraction did not correspond
to endogenous 5-HT, as the amine had a different elution time (t = 80 min for F; t = 540 min for 5-HT). reverse phase chromatography carried out to purify the
F fraction resulted in the separation of four peaks of
activity. One of them (NR) was eluted in the void volume of the column
and corresponded to a highly polar and dialyzable material (M < 1,000). This fraction was analyzed using
additional chromatographic systems, i.e. normal phase,
hydrophobic column, and ion exchange chromatography. In all of these
systems the fraction was retained on the column, suggesting that it
mainly consisted of salts. Previously, similar observations were
reported(34, 35, 36, 37, 38, 39) ,
which did not lead to the identification of any particular compound. = 21 min), which inhibited 83 ±
13% of the binding of [H]5-HT to 5-HT binding. The latter binding is the specific, high affinity
binding of the tritiated amine in the presence of nonradioactive
8-OH-DPAT, which specifically masks the 5-HT
receptors; 10
µM 5-CT is added to the medium to measure the nonspecific
binding. Under these conditions, the observed binding essentially
represents 5-HT
receptor subtypes in rat. Two other
fractions, P
and P, having shorter retention
times (t = 9 and 12 min, respectively),
also exhibited inhibitory activities.) and successive reverse phase
chromatographies using different matrices (C
Ultrabase and
Hypercarb columns) and various optimal mobile phases determined after
numerous trials. The pharmacological profile of the P fraction was
established by examining its effect on the specific binding of various
ligands as described under ``Experimental Procedures.'' These
assays were carried out in order to avoid the purification of a
fraction that would nonspecifically inhibit the binding of
[H]5-HT. Interestingly enough, at all
purification steps, the P fraction exhibited a clear serotonergic
specificity since, at the dose that maximally inhibited the
5-HT specific binding (1% of the total purified
fraction), it did not significantly interact with the binding of
[
H]mepyramine, [H]prazosin,
[H]dihydroalprenolol,
[H]spiroperidol,
[H]quinuclidinyl benzylate,
[H]naloxone, and
[H]flunitrazepam, which label histaminergic,
and adrenergic, dopaminergic, muscarinic, opiate, and
benzodiazepine receptors, respectively. Moreover, it did not affect the
binding of [H]ketanserin (antagonist) and
[H]DOB (agonist) to 5-HT receptors
and that of [H]BRL 43694 to 5-HT
receptors. The effect of the P fraction also appeared restricted to
5-HT receptors since the transport systems (uptake) of 5-HT
itself and that of other neurotransmitters (or their precursors) were
not affected (dopamine, noradrenaline, -aminobutyric acid,
choline). These results indicate that the P fraction is clearly
different from those previously reported, which efficiently inhibited
the uptake of biogenic
amines(34, 35, 36, 37, 38, 39) .
Furthermore, the purified P fraction specifically interacted with a
specific 5-HT
receptor subtype, as neither 5-HT (labeled by [
H]8-OH-DPAT) nor
5-HT or 5-HT
(labeled by
[
H]5-HT in the presence of 5-CT) were
significantly affected at a concentration that had a maximal inhibitory
effect on 5-HT receptors.
receptor subtype was inhibited in the nanomolar range
(IC
= 0.1 nM). At much higher
concentrations, LSAL also interacts with the 5-HT
receptors (IC
= 1 µM) and is
still devoid of any significant activity on the other receptors
examined. These results clearly demonstrate that LSAL specifically
interacts with 5-HT
receptor subtype.
M, namely 1% of 0.3 µg of LSAL (molecular weight
= 402.5) tested in a volume of 1 ml. Moreover, the synthetic
peptide had exactly the same retention time as the purified P fraction
on the C
Ultrabase reverse phase column (not shown). These
results strongly suggest that the active compound contained in the P
fraction corresponds to LSAL. and P fractions were too low to be traced accurately
through the following different chromatographic steps. The extract
prepared from bovine brains (600 g of initial weight) also contained
three fractions having the same retention times as P, P,
and P fractions obtained from rat brain extract. The
patterns of inhibitory activity on [H]5-HT
binding were very similar when measured on bovine brain membranes as
well as on rat brain membranes. Moreover, the activity of the bovine
brain extract closely resembled that of the rat brain extract. These
results tend to suggest that the three fractions observed in bovine
brain extract are identical to those present in rat. Nevertheless, in
bovine brain extract, the inhibitory activity was essentially localized
in the P and P fractions, which were identified
by amino acid and NMR analysis as AL and LS, respectively. Synthetic
peptides, namely LS, AL, and LSAL, coeluted with P,
P, and P fractions, respectively, whereas other peptides
such as SL, LA, and LASL have different retention times in the same
chromatographic system (not shown). These results further support the
hypothesis that P fraction in bovine brain corresponds to LSAL and that
P and P fractions in rat brain actually are LS
and AL, respectively. and P fractions were poorly
active compared with P fraction (using molecular weights of 211 and 213
for P (LS) and P (AL) fractions, respectively,
the 1% dose tested corresponded to an amount close to 0.3 µg/ml of
incubate and thus to a final concentration of 14 µM for
the two dipeptides). The fact that AL and LS are dipeptides
constitutive of LSAL suggests that these dipeptides may originate from
the degradation of LSAL. In favor of this hypothesis, it was shown that
the extraction procedure applied to the medium in the presence of
labeled LSAL and in the absence of tissue does not induce the
occurrence of the dipeptides. Moreover, the latter compounds were not
found in the extract of a brain homogenate in which
[H]LSAL was added prior to the the extraction
process. On the contrary, the major part of
[H]LSAL was found in the extract as the native
radioactive compound. This result indicates that the cleavage of the
tetrapeptide does not occur during the different steps of extraction
and purification. Thus, the presence of the dipeptides observed in the
brain extracts suggests that LSAL was degraded in LS and AL prior to
the extraction procedure. The fact that P and P fractions are relatively more important than P fraction in bovine
brain compared with rat brain extract supports the hypothesis that,
during the long post-mortem delay (2-3 h) before processing the
bovine brains, LSAL was cleaved in the two corresponding dipeptides.
This phenomenon occurred to a lesser extent in rat brain, which could
be processed more rapidly. These observations suggest that the cleavage
of LSAL in LS and AL may correspond to the inactivation process of this
endogenous peptide. receptor
subtype. It should be emphasized that 5-HT
receptors
are in non-rodent species the equivalent of the rodent 5-HT
receptor, as has been shown from their functional properties and
the close homology of the genes encoding for the corresponding receptor
proteins(40) . This observation suggests an important
functional role for this peptide since it has been conserved during the
evolution. Moreover, its activity is maintained despite the fact that
its functional target was modified under the pressure of the evolution
leading to different pharmacological properties of 5-HT
vis à vis 5-HT
.
receptors(41) ; however, on the basis of the herein
presented results, it is difficult to establish a direct relationship
between the distribution of LSAL and that of the 5-HT
receptors. Autoradiographic studies with the labeled peptide will
determine this point. LSAL is also present in peripheral tissues, i.e. in kidneys, which also contain a high density of
5-HT
receptors(42, 43) . The fact that
LSAL is not found in significant amounts in liver suggests that it is
not the result of the degradative procedure of a circulating protein.
These observations support the hypothesis that LSAL may be an
endogenous peptide.
receptors presumably via a particular binding site;
preliminary results indicate that the inhibition corresponds to a
noncompetitive interaction, which may suggest an allosteric mechanism.
Additional studies are necessary to test this hypothesis and to examine
the functional consequences of the existence of such a potential
modulator. Nevertheless, the existence of this mechanism of interaction
with the 5-HT
receptor, which controls the serotonergic
system activity, may lead to new directions of research in the
mechanisms involved in numerous pathophysiological functions
implicating the 5-HT system. Furthermore, the existence of a direct
interaction of an endogenous peptide with a G protein-coupled receptor
would result in new concepts in the mechanisms of regulation of the
central nervous system.
), retention time; LSAL, Leu-Ser-Ala-Leu; LS,
Leu-Ser; AL, Ala-Leu; HPLC, high pressure liquid chromatography; NR,
not retained.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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