(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 [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.
A
tetrapeptide corresponding to a novel amino acid sequence
Leu-Ser-Ala-Leu (LSAL) was identified. It reduces the binding of
[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).
Two other peptides,
Leu-Ser (LS) and Ala-Leu (AL), were also purified. They hardly affected
[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.
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 ()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) .
Furthermore, it
was also shown that antidepressant drugs could act on 5-HT 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) .
Among 5-HT 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) .
According to these results, the hypothesis of the existence of an
endogenous factor acting at 5-HT 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.
Herein, we report the isolation and
characterization of a cerebral compound, which specifically interacts
with 5-HT binding sites.
The TSK HW 40S column was
obtained from Merck, and the Sephadex G 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 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 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).
The uptake of 5-HT, dopamine, noradrenaline,
-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.
The retention time of
[H]LSAL under the same experimental conditions
was determined by a control injection.
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 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
CH
COONH
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
(
-
).
Figure 2:
Reverse phase chromatography of rat
F 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
(
-
).
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, -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.
P fraction represented the main activity inducing
83 ± 13% inhibition of the 5-HT 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).
Figure 3:
Purification of rat P fraction. P fraction
(rat) was purified by four successive chromatographic steps: Sephadex
G (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).
The biological activities observed for rat
P 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).
Figure 4:
Pattern of activity of the bovine F 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.
Figure 5:
Purification of bovine P
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).
Amino acid analysis of bovine P 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
.
Figure 7:
Stability of LSAL. 3 µCi of
[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).
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 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.
The inhibitory effect of LSAL on
5-HT 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).
Figure 9:
Interaction of synthetic P (LSAL), P (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.
The results reported herein describe the isolation and
purification of a cerebral factor able to specifically interact with
the 5-HT receptor.
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 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.
The
initial size exclusion chromatography led to the separation of the
F 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).
The further
C 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.
The major activity retained on the column was the P fraction (t = 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.
The P fraction was purified by
gel permeation (Sephadex G) 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.
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 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.
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 8.10M, 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.
The activities of rat P 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.
LS and AL were poorly active compared with
LSAL (100,000 and 10,000 times less efficient, respectively).
Accordingly, P 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.
LSAL exhibited similar properties of binding
inhibitions in rat brain and in bovine brain cortical membranes,
indicating that it also interacted with 5-HT 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
.
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 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.
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 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.