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Department of Pharmacology, University of Tartu (A.K., R.P., L.R.), Ulikooli 18, 50090 Tartu, Estonia; the Department of Medicinal Chemistry (F.M., I.M.) and the Laboratory of Pharmacology (R.M.), Institute of Organic Synthesis, Aizkraukles 21, LV-1006, Riga, Latvia; and the Department of Pharmaceutical Pharmacology, Uppsala University (F.M., R.M., I.M., J.E.S.W., H.B.S.), 751 24 Uppsala, Sweden
Address all correspondence and requests for reprints to: Dr. Helgi B. Schiöth, Department of Pharmaceutical Pharmacology, Biomedical Center, Box 591, 751 24 Uppsala, Sweden. E-mail: helgis{at}bmc.uu.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-MSH-(3–11))
has a 29-membered atom ring structure that includes an Arg in position
5. HS024 was found to antagonize an MSH-induced cAMP response in
cells expressing the human MC1, MC3, MC4, and MC5 receptor DNAs. HS024
also caused a dose-dependent increase in food intake, with a maximum
response (4-fold increase) at a 1-nmol dose injected
intracerebroventricularly in free feeding rats. We also tested SHU9119,
a previously described nonselective MC receptor antagonist, and found
HS024 and SHU9119 to have similar potencies for increasing food intake,
although SHU9119 appeared to induce more serious side-effects. HS024
increased the food intake of free feeding rats to levels comparable to
those in food-deprived rats, indicating that blockade of the MC4
receptor is a highly effective way to increase feeding. Moreover, we
tested the effects of intracerebroventricular injections of HS024 in
elevated plus-maze and open-field experiments on rats. In these tests,
HS024 did not appear to affect emotionality or locomotor activity,
suggesting that the MC4 receptor does not mediate the anxiogenic-like
and locomotor effects related to the melanocortic peptides. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MSH with high
affinity. The MC1 receptor plays an important role in skin and fur
pigmentation in a variety of vertebrates (6). The MC2 receptor
(i.e. the ACTH receptor) has a well defined function in the
regulation of steroid production in the adrenal gland. The MC3 receptor
is found in the brain, placenta, and gut tissues (3, 7). The MC4 is
found only in the central nervous system, where it is expressed in
several different sites, including the cortex, thalamus, hypothalamus,
brain stem, and spinal cord (4, 8). The MC4 and MC5 receptors have been
studied in knock-out mice, and the MC4 receptor was shown to be
involved in weight homeostasis (9), whereas the MC5 receptor was found
to have a role in exocrine gland function (10).
The natural melanocortic peptides (MSH, ßMSH, 
MSH, and ACTH)
each have a specific affinity profile for the MC receptor subtypes but
are not selective for the different subtypes, with the exception that
MSH is selective for the MC1 receptor and ACTH is selective for the
MC2 receptor (11, 12, 13). The MC2 receptor is distinguishable from the
other MC receptors in that it does not bind the MSH peptides. The lack
of selective compounds has hampered clarification of the physiological
roles of the MC3, MC4, and MC5 receptors. Major progress was made with
the development of the synthetic MSH analogs, SHU9119 (14), a
nonselective MC3 and MC4 receptor antagonist, and HS014 (15), a
selective MC4 receptor antagonist. SHU9119 and HS014 are antagonists
for the MC3 and MC4 receptors but agonists for the MC1 and MC5
receptors. SHU9119 has been used in some important initial studies for
elucidation of the roles of the neural MC3 and MC4 receptors (16, 17, 18),
and the new analog HS014 was shown to increase the food intake of
freely feeding rats (19, 20).
In this study, we synthesized several new cyclic MSH analogs and discovered a novel substance, HS024, that is a selective MC4 antagonist with a 10-fold higher affinity for the MC4 receptor than HS014. Moreover, we tested the ability of HS024 to influence food intake in rats and compared it with those of SHU9119 and HS014. In addition to food consumption, we measured spillage behavior, which may be a useful index of toxicity-related changes in food intake (21). Orexigens, such as neuropeptide Y (NPY) and benzodiazepines, have previously shown anxiolytic-like and sedative effects. An increase in food intake may also be due to arousal or changes in general activity. Melanocortic peptides have been shown to affect some anxiety-related measures (22, 23, 24, 25). Accordingly, it may be anticipated that melanocortin receptor antagonists might also affect behavioral measures modified by fear. Therefore, we performed elevated plus-maze and open-field experiments on rats after intracerebroventricular (icv) injection of HS024 to assess potential anxiolytic/anxiogenic activity and locomotor effects of MC4 receptor blockade.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MSH
(NDP-MSH) (26) was radioiodinated by the chloramine-T method and
purified by HPLC. NDP-MSH, SHU9119, and all amino acid derivatives were
purchased from Neosystem (Strasbourg, France).
Peptide synthesis
The novel peptides tested in this study were synthesized in our
laboratory using the solid phase approach and then purified by HPLC.
The correct mol wt of the peptides were confirmed by mass spectrometry.
The peptide sequences were assembled using the Pioneer PerSeptive
Biosystems peptide synthesis system. F-moc
(9-fluorenylmethoxycarbonyl)-amino acid derivatives were used in
coupling steps. When pentafluorophenyl esters were used, the synthesis
cycle was as follows: 1) the F-moc group was removed by 20% piperidine
in N,N-dimethylformamide (DMF; 5 min); 2) to form
a new peptide, bound side-chain-protected F-moc-amino acid
pentafluorophenyl ester (4 eq.) and
1-hydroxy-7-azabenzotriazole (4 eq.) were dissolved in DMF
and circulated through the column for 30–60 min; and 3) to cap
residual amino groups, the support was treated with 0.3 M
Ac2O (acetic anhydride) in DMF for 5 min. If free acids
were used, then in step 2, side-chain-protected F-moc-amino acid (4
eq.),
O-[7-azabenzotriazol-1-yl]1,1,3,3-tetramethyluronium
hexafluorophosphate (4 eq.), and
N,N-diisopropylethylamine (4 eq.) were
applied. For deprotection, a reagent mixture (trifluoroacetic
acid-phenol-anisole-1,2-ethanedithiol-water, 82:2:2:2:2; 2.5 h)
was used. The raw peptides formed were purified by HPLC (10 x
250-mm column, Vydac RP C18, 90A, 201HS1010; eluent,
20–35% MeCN (acetonitrile) in water and 0.1% trifluoroacetic acid;
detection at 240 nm).
Expression of receptor clones
The human MC1 and human MC5 receptors (1, 5) were cloned into
the expression vector pRc/CMV (Invitrogen, San Diego, CA).
The human MC3 and human MC4 receptors, cloned into the expression
vector pCMV/neo, were gifts from Dr. Ira Gantz (3, 4). For receptor
expression, COS-1 (CV-1 origin, simian virus 40) cells were grown in
DMEM with 10% FCS. Eighty percent confluent cultures were transfected
with the DNA mixed with liposomes in serum-free medium (for details,
see Ref. 12). After transfection, the serum-free medium was replaced by
serum-containing medium, and the cells were cultivated for about
48 h. Cells were then scraped off, centrifuged, and used for
radioligand binding.
Binding studies
The transfected cells were washed with binding buffer (11) and
distributed into 96-well nonculture-coated plates that were
centrifuged, and the binding buffer was removed. The cells were then
immediately incubated in the well plates for 2 h at 37 C with 0.05
ml binding buffer in each well containing a constant concentration of
[125I]NDP-MSH and appropriate concentrations of competing
unlabeled ligand. After incubation, the cells were washed with 0.2 ml
ice-cold binding buffer and detached from the plates with 0.2 ml 0.1
N NaOH. Radioactivity was counted (Wizard automatic
-counter, Wallac, Turku, Finland), and data were analyzed
with a software package suitable for radioligand binding data analysis
(Wan System AB, Umea, Sweden). Data were analyzed by fitting to
formulas derived from the law of mass action by the method generally
referred to as computer modeling. The Kd values for
[125I]NDP-MSH for the MC receptors were taken from
previous studies (11, 12). The binding assays were performed in
duplicate wells and repeated three times. Untransfected COS-1 cells did
not show any specific binding for [125I]NDP.
cAMP assay
The transfected cells, cultivated in the same medium as the COS
cells (see above), were harvested and incubated for 30 min at 37 C with
0.05 ml serum-free DMEM in each tube containing 0.5 mM
isobutylmethylxanthine and appropriate concentrations of MSH or
HS024. After incubation with the indicated drugs, cAMP was extracted
with perchloric acid at a final concentration of 0.4 M.
After centrifugation, the protein-free supernatants were neutralized
with 5 M KOH-1 M Tris
(Tris-(hydroxymethyl)aminomethane). Neutralized cAMP extract (0.05
ml) or a cAMP standard (dissolved in distilled water) was added to a
96-well microtiter plate. The cAMP content was then estimated
essentially according to the method described by Nordstedt and Fredholm
(27) by adding to each well [3H]cAMP (0.14 pmol;
11,000 cpm; SA, 54 Ci/mmol; Amersham, Arlington
Heights, IL) and bovine adrenal binding protein and incubating at 4 C
for 150 min. Standards containing nonlabeled cAMP were also assayed
concomitantly with the samples. The incubates were thereafter harvested
by filtration on Whatman GF/B filters (Clifton, NJ) using
a semiautomatic Brandel cell harvester (Bethesda, MD). Each filter was
rinsed with 3 ml 50 mM Tris-HCl, pH 7.4. The filters were
punched out, put into scintillation vials with scintillation fluid, and
counted. The cAMP assays were performed in duplicate wells and repeated
three times.
Animals and surgery
Adult male Wistar rats (National Laboratory Animal Center,
Kuopio, Finland), weighing 330–380 g at the time of surgery, were
housed individually in hanging wire mesh (45 x 37 x 19
cm) or polypropylene cages (rats used in elevated plus-maze and
open-field tests) with free access to food and water at controlled
temperature (20 ± 1 C) and light (12-h light, 12-h dark cycle;
lights on at 0800 h). The rats had free access to food pellets
(R35 or R70, Lactamin, Stockholm, Sweden) and tap water. Rats
were anesthetized with chloral hydrate (350 mg/kg·10 ml, ip) and
secured in a stereotaxic instrument where a 11-mm-long 23-gauge
stainless steel cannula was lowered to within 1.5 mm of the ventricle
and anchored to the skull with two screws and dental acrylic. With the
tooth bar 3.0 mm above interaural zero, implantation coordinates were
0.7 posterior to bregma, 1.4 mm lateral, and 3.2 mm below the skull at
the point of entry. After surgery, the cannula was closed with a
stylet. Rats were handled and weighed during the recovery period (7
days) to minimize nonspecific stress. Substances were dissolved in
saline and administered by a 31-gauge stainless steel injector (Plastic
One, Inc., Roanoke, VA) projecting 1.5 mm below the tip of the guide
cannulas. The injector was connected to the gas-tight 50-µl Hamilton
syringe (Reno, NV) by polyethylene tubing (id, 0.58 mm; od, 1.27 mm).
Drugs were infused by infusion pump (World Precision Instruments,
Sarasota, FL) at a speed of 10 µl/min. The movement of an air bubble
inside the polyethylene tubing confirmed the drug flow. The needle was
left in place for 15 sec, then the cannula was closed with a stylet,
and rats were returned to the home cage. All injections were carried
out between 1200–1600 h every third day and were given in randomized
order in a such way that none of the rats received the same dose of
peptide twice.
Feeding experiments
On the day of the experiment, the food was removed from wire
baskets, and the rats were injected icv with SHU9119 (0.3, 1.0, 3.0,
and 6 nmol) and HS024 (0.1, 0.3, and 1.0 nmol) and returned to the home
cage. Seven preweighted pellets (20 g) were presented on clean
plastic petri dishes. Food intake was measured to the nearest 0.01
g 1, 2, 3, and 4 h after the icv injection by weighing remaining
pellets and spillage using a Mettler (Stockholm, Sweden) PB3002
balance.
Elevated plus-maze and open-field
Rats were tested in an elevated plus-maze test 20 min after icv
injection of HS024 (0.02 and 0.1 nmol) or vehicle. Tests were conducted
in quiet, separate rooms. The elevated plus-maze was made of
polypropylene (walls pale yellow, floor black) and consisted of 2 open
arms measuring 50 x 10 cm, and 2 closed arms that were of the
same size but had 40-cm high end and side walls. The arms were
connected by a central area measuring 10 x 10 cm, and the maze
was 65 cm above the floor. Behavior was observed by a trained observer
blind to treatment conditions. Before testing, rats were placed in a
novel environment (clean empty polypropylene cage) for 5 min; this has
been shown to increase the sensitivity of the elevated plus-maze test
(28). Rats were then placed in the central area of the maze facing 1 of
the open arms. The number of entries into open and closed arms, the
time spent on open arms, and line crossings on open part were recorded
over a 4-min testing period. An arm entry was defined as all 4 paws
into an arm. Immediately after the plus-maze testing, the rats were
tested on an open field. The open field was a wooden arena (100 x
100 cm with 40-cm-high side walls) painted dark gray. Black lines
divided the arenas into 16 equal squares. The number of squares visited
(with all 4 feet on 1 square) and the number of rearing was registered
in a 4-min test. The elevated plus-maze and open-field were cleaned
with a dampened cloth and dried with paper towels between sessions.
Verification of injection sites
Upon completion of the study, the rats were overdosed with
chloral hydrate (600 mg/kg), and fast green dye was infused to mark the
injection site. The brains were removed, and the distribution of the
dye was examined. Only the animals with uniform distribution of the dye
in the ventricles were included in the data analysis.
Statistical evaluation
All results are expressed as the mean ± SEM.
The cumulative food intake data were analyzed by one-way ANOVA for
repeated measures; elevated plus-maze test and open-field data were
evaluated by factorial ANOVA. Where appropriate, a post-hoc
analysis were carried out using the least significant difference (LSD)
test.
Animal ethics
Experimental procedures were approved by the ethics committee of
animal experiments at the University of Tartu and were carried out in
accordance with guidelines of the European Community.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MSH, NDP-MSH,
MTII, SHU9119, and HS014 in Fig. 1
. The
human DNAs for the MC1, MC3, MC4, and MC5 receptors were transiently
and independently expressed in COS-1 cells for competitive receptor
binding using [125I]NDP-MSH as radioligand. The
expression levels of the different receptor subtypes were similar (data
not shown). The Ki values for the different peptides
resulting from calculations of the competition curves of binding with
[125I]NDP-MSH are summarized in Table 1. For comparison, we also included in
Table 1 the Ki values of MSH, NDP-MSH, MTII, SHU9119,
and HS014, values that we recently reported (15, 29) from tests using
the same methodology as that used in the present study.
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However, when we combined the chemical properties of HS007
(Arg5) and HS010 (Nle4) in HS024, we found a
substance that is highly potent and selective for the MC4 receptor.
HS024 has an 11-fold higher affinity for the MC4 receptor compared with
HS014 and also a slightly higher affinity for the MC4 receptor compared
with SHU9119. HS024 has similar selectivity as HS014 for the MC4
receptor relative to the MC3 receptor. The primary structure of HS024
is shown in Fig. 2. The competition
curves for HS024 at the different MC receptor subtypes are shown in
Fig. 3.
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MSH-(4–11)
analog (HS964) generated HS014, which led to an increase in both the
affinity and the selectivity for the MC4 receptor (15). The addition of
(Pro12-Pro13-Lys14-Asp15)
to HS024 resulted in HS032, which barely affected the affinity for the
MC4 receptor and lowered the selectivity. The addition of two residues
(Tyr1-Pro2) from the N-terminal of ßMSH to
HS024, resulting in HS031, gave lower affinity for all of the MC
receptors. HS031 was also virtually nonselective.
In addition to these "tailed" substances, we made single amino acid
substitutions in HS024 at positions 4, 5, 6, and 9. When the basic
hydrophilic His6 was replaced by the acidic hydrophilic
Glu6 (in HS052), affinity was lost for all of the MC
receptors, in particular for the MC4 receptor. HS052 is practically
nonselective. Substitution of the basic hydrophilic Arg5
with the structurally related residue Lys5 (HS053) resulted
in slightly lower affinity for the MC1 and MC3 receptors (<2-fold),
but brought about a considerable loss in affinity for the MC4 and MC5
receptors (16- and 88-fold, respectively). Replacement of the nonpolar
hydrophobic residue Nle4 by the polar and neutral
Asn4 (HS055) slightly improved binding to the MC3 receptor
and slightly lowered the affinity for the MC4 and the MC5 receptors.
However, this change in the structure of the peptide resulted in a more
than 28-fold increase in the affinity for the MC1 receptor. Tic
(1,2,3,4-L-tetrahydroisoquinoline-3-carboxylic acid) is a
nonpolar hydrophobic amino acid similar to Trp. Tic, however, has a
more rigid conformation than Trp. When Trp was exchanged for Tic in
position 9 (HS024
HS057), the affinity was reduced for all of the MC
receptors, especially MC3 (190-fold), MC4 (3400-fold), and MC5
(370-fold) receptors. However, the affinity of HS057 for the MC1
receptor was only 2-fold lower than that of HS024.
We selected HS024 for further investigations and tested the cAMP
responses to MSH and HS024 in COS-1 cells expressing the human MC1,
MC3, MC4, and MC5 receptors (see Fig 4).
As shown in Fig. 4, MSH stimulated accumulation of cAMP at all
receptor types. COS-1 cells that had not been transfected with any of
the MC receptors did not respond to MSH (data not shown). HS024, in
concentrations up to 100 µM, did not affect the cAMP
levels of any of the MC receptor-expressing cells. Instead, 0.1
µM HS024 completely blocked the cAMP increase induced by
MSH for all four MC receptors (Fig. 4).
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together with our earlier results for
HS014 using the same methodological approach (19). The injection of
either HS024 or SHU9119 induced behavioral activation in the rat,
followed by increased ingestion of its regular food. As shown in Fig. 5, HS024 produced strong dose-dependent stimulation of feeding. The
drug, time, and interaction effects were, respectively:
F(3,29) = 15.62, P < 0.0001;
F(3,87) = 194.77, P < 0.0001; and
F(9,87) = 6.21, P < 0.0001. Food intakes
differed at 1, 2, 3, and 4 h (F(3,29) = 3.76, 14.25,
13.13, and 15.76, respectively; P < 0.05). Individual
comparisons with LSD tests indicated that at 1 h, only 1 nmol
HS024 increased food intake, whereas at 2, 3, and 4 h, all doses
of HS024 increased food intake (Fig. 5).
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. The drug, time, and interaction effects as determined by repeated
measures ANOVA were, respectively: F(4,25) = 5.89,
P < 0.005; F(3,75) = 53.67,
P < 0.001; and F(12,75) = 4.13,
P < 0.001. Factorial ANOVA indicated that food intake
was different at 1, 2, 3, and 4 h (F(4,25) = 3.29,
3.44, 6.27, and 7.02, respectively; P < 0.005).
Individual post-hoc comparisons indicated that 1 nmol
SHU9119 increased food intake at all points of measurement and that a
0.3 nmol dose of SHU9119 increased food intake at 3 and 4 h
(P < 0.05). The two highest doses of SHU9119 tested (3
and 6 nmol) did not affect food intake significantly at any point of
measurement.
As shown in Fig. 5, the effects of HS024 and SHU9119 on food intake
were similar in magnitude. The dose-response curve for SHU9119 was
bell-shaped; maximal stimulation of feeding occurred at 1 nmol SHU9119.
HS024 also stimulated food intake, with a maximal increase in feeding
at 1 nmol. We did not test higher doses of HS024, as some rats treated
with 0.3 (1 of 9) and 1.0 nmol (1 of 10) of HS024 developed
exopthalamus and episodes of rotation along the long axis of the body
(barrel-rolling). This repetitive motor disturbance also developed in
SHU9119-treated rats [3 nmol (1 of 7) and 6 nmol (3 of 5; 2 of these 3
rats died]. The effects of HS014 are also shown in Fig. 5 for
comparison (data taken from Ref. 19). All rats treated with HS014 at 10
nmol (n = 4) developed fear-like reactions and thereafter became
sedated for approximately 20 min. However, the rats treated with HS014
did not develop barrel-rolling.
Spillage behavior during the feeding experiments was increased by all
three drugs (Fig. 6). Spillage was
significantly increased by HS024 (0.33 and 1.0 nmol) and SHU9119 (0.33,
1.0, and 3.0 nmol) as well as by HS014 (1.0 and 3.3 nmol) [previously
unpublished data from our earlier experiments (19)]. When the data
were expressed as the percentage of spillage from cumulative food
intake after the fourth hour (% spillage/CFI), we found that SHU9119
had a significant effect on this parameter (F(4.22) = 2.89,
P < 0.05). Individual comparisons with LSD tests
indicated that 1 and 3 nmol SHU9119 increased the % spillage/CFI.
Notably, the 3.0-nM dose of SHU9119 increased spillage, but
this dose did not increase cumulative food intake. HS024 did not
significantly affect % spillage/CFI. Only one animal of four displayed
increased spillage under the maximum HS014 dosage (10 nmol; data not
shown).
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. The open-field test was carried
out immediately after the elevated plus-maze test, and no differences
in horizontal and vertical activity were detected (Table 2). The number
of fecal boli deposited was increased at 0.02 nmol HS024 in the
elevated plus-maze test. In the open-field test, where the defecation
score has been shown to correlate with emotionality (32), the number of
fecal boli was unaffected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MSH analogs (33, 34) were found to be long acting, enzymatically stable, and highly
potent in frog skin-tanning assays and other pigment dispersion assays
using amphibians (for review, see Ref. 35). These assay systems relate
to the amphibian MC1 receptors, which are shown to behave somewhat
differently from the mammalian MC1 receptors. Further development of
the 23-atom membered ring structure of cyclic
[Cys4,Cys10]MSH resulted in another
23-atom membered group of cyclic analogs possessing another distinct
ring closure, namely the so-called lactam analogs, which include MTII
(36). MTII is selective for the MC1 receptor, and it was suggested that
23 membered rings are optimal for activity of MSH analogs in relation
to the MC1 receptor. Substitution of Phe7 for
ß-(2-naphthyl)-D-alanine in MTII resulted in SHU9119, the
first antagonist for the MC3 and MC4 receptors (14). In a joint study
by ourselves and Dr. Hruby, we found that SHU9119 is virtually
nonselective for the MC1, MC3, MC4, and MC5 receptors (29), in line
with earlier data (14). We recently found a novel compound, HS014,
which has a 26-membered ring structure with a disulfide ring closure
(15). HS014 has an approximately 20-fold higher affinity for the MC4
receptor than for the MC3 receptor. In the same study we observed that
a compound with a 29-membered ring structure had preference for the MC3
receptor. Another compound with an Arg residue in position 5 also
seemed to have a preference for the MC3 receptor. In our present study
we combined these chemical properties and synthesized HS024. HS024,
however, somewhat surprisingly turned out to be superpotent for the MC4
receptor (>3000-fold higher affinity than the natural hormone MSH)
and showed similar as HS014 selectivity for the MC4 receptor above the
MC3 receptor. Addition of a C-terminal tail to HS024, which had proven
to be successful in the development of HS014, only slightly enhanced
the affinity and reduced selective binding for the MC4 receptor.
Addition of an N-terminal tail to HS024 did not enhance the affinity
and eradicated the MC4 receptor selectivity. However, this was not very
surprising, as we recently showed that the N-terminal part of the
linear NDP-MSH is not important for the binding of the MC4 receptor,
whereas it is important for the MC3 and MC5 receptors (37). Replacement
of specific residues within the ring structure of HS024 did not improve
the selectivity or the affinity for the MC4 receptor. Thus, we selected
HS024 for further analysis. In the cAMP assay, HS024 was shown to be an antagonist for the MC3 and MC4 receptors, similar to what has been reported for SHU9119 and HS014. However, in contrast to these latter peptides, HS024 was also an antagonist for the MC1 and MC5 receptors. There are no reports of potent antagonists for the MC1 and MC5 receptors apart from the findings of Jayawickreme et al. (38), who showed 153N-6 to be an antagonist of pigmentation in dermal melanophores of amphibians. However, 153N-6 appears not to be an antagonist for the human MC1 receptor (39) (Schiöth, H. B., et al., unpublished results).
In the present study, both HS024 and SHU9119 increased food intake in free feeding rats. Our data for SHU9119 are in general agreement with those from studies performed by Seeley et al. (18) that reported an increase in food intake after icv administration of 1 nmol SHU9119. In the same study, 0.5 nmol SHU9119 did not increase food intake, but this dose antagonized the suppressive effects of leptin on food intake (18). In the present study, 0.3 nmol SHU9119 increased food intake; the effect was statistically significant 3 h after injection. Interestingly, SHU9119 (6 nmol) did not affect daytime food intake in mice (17). We confirm that at this dose, SHU9119 does not affect feeding in rats, whereas lower doses significantly stimulate feeding. The failure of higher doses of MC receptor antagonists to modify food intake may be related to sedation and toxicity. It has been shown that the first MC4-selective antagonist HS014 increased food intake in free feeding rats (19, 20). The potency of HS024 at the MC4 receptor, as determined in binding assays, is very similar to that of SHU9119, whereas HS014 is about 10 times less potent. This order of potency was also observed in the feeding assays. One nanomole of SHU9119 or HS024 caused maximal stimulation of feeding, whereas higher doses caused side-effects. The maximal effect of HS014 was also observed at 1 nmol.
Our present data support the hypothesis that MC4 receptors exert a tonic inhibitory influence on the ingestion of nutrients, as indicated in previous reports (9, 17, 19, 20). It is interesting to note that HS024 and SHU9119 were able to induce food intake in free feeding rats to levels comparable to those in rats deprived of food for 24 h (40). The rats treated with 1 nmol SHU9119 and HS024 had consumed about 6–8 g food by the fourth hour of testing. This effect is similar to that of icv injections of NPY (40, 41) and recently discovered orexins/hypocretins (42, 43), which both appear to be the most potent natural stimulants of feeding yet described. The data indicate that the sole blockage of the MC4 receptor is a highly effective way to induce feeding.
Spillage was increased dose dependently by all drugs tested. In general, the spillage increased concomitantly with increases in food intake. Interestingly, however, 3 nmol SHU9119 increased the spillage but not the food intake. This finding might indicate that there is a conflict between the motivation and the ability to eat after the administration of SHU9119. It can be speculated that these effects are related to actions on MC5 receptors and reduction in saliva production, as MC5 knock-out animals have been shown to have defective function of exocrine glands (10). The difference between SHU9119 and the other substances may be related to the lack of selectivity of SHU9119 for the MC4 receptor. An increase in spillage could also be taken as a general index of toxicity (21).
Ligands activating MC receptor subtypes (ACTH and MSH) have been
shown to inhibit punished responding in the Vogel conflict test (25),
and microinjection of MSH into the medial preoptic area has been
shown to increase anxiety and aggressive behavior (22). These data
indicate that MC receptors may be involved in the regulation of
emotional behavior. We, therefore, performed elevated plus-maze tests
that can be used to screen for compounds with
anxiolytic/anxiogenic-like activity. We selected the dose of HS024 that
produced half-maximal stimulation of feeding (0.1 nmol) and evaluated
the effects of HS024 on anxiety-related measures using the elevated
plus-maze test. HS024 (0.02 and 0.1 nmol) did not affect elevated
plus-maze exploration. Thus, it can be concluded that HS024 does not
affect anxiety-related behavior at the doses tested. This is not
unexpected, as upon exposure to a novel environment, several other
neurotransmitters are released in addition to POMC-related peptides.
Numerous studies suggest that CRF may play a central role in the stress
response (reviewed in Ref. 44). A nonselective antagonist of CRF
receptors, hCRF-(9–41) has anxiolytic-like effects in the elevated
plus-maze test (45). hCRF-(9–41) has also been shown to block the
anxiogenic-like effects of the pharmacological manipulations of other
neurotransmitter systems, including cholecystokinin-ergic (46) and
NPY-ergic systems (30). These findings indicate that CRF is an
important mediator of the fear reaction, and therefore, the sole
blockage of the MC4 receptors may be insufficient to modify behavior in
the elevated plus-maze paradigm.
Several compounds that influence food intake also modulate locomotor activity. The doses of NPY and benzodiazepines that are sedative and suppress open-field activity increase food intake (47), whereas the drugs that decrease food consumption may affect locomotion or produce anxiogenic-like effects. Open-field behavior was not affected by HS024. These findings suggest that MC4 receptors are not directly involved in locomotion and emotional behavior and that the increase in food intake seen after icv injection of HS024 differs in this regard from ingestive responses evoked by NPY or benzodiazepines.
High doses of HS024 and SHU9119 produced a repetitive motor disturbance called barrel-rolling. These effects seemed to be most severe for SHU9119, culminating in the death of two of five rats treated with the highest dose, whereas no lethality was observed for the other substances. The reasons for this are unknown. One can speculate that the motor disturbances are related to direct modulation of calcium metabolism, as agouti peptide, which acts as an antagonist at melanocortin receptor subtypes, has been shown to elevate the intracellular free Ca2+ concentration (48), and Ca2+ antagonists are effective in animal models of epilepsy (49).
In conclusion, we have developed a very potent selective MC4 antagonist that may be a valuable tool for evaluation of the role of MC4 receptors in physiological processes, including feeding behavior, and this substance may be used as a prototype for agents for treatment of eating disorders associated with anorexia. Moreover, our data indicate that selective MC4 receptor blockage does not affect locomotion or emotional behavior.
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Received May 11, 1998.
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), MC3 (•), MC4 (
), or MC5
(
) receptor clones, obtained by using a fixed concentration of
[125I]NDP-MSH and varying concentrations of the
nonlabeled competing peptide. All experiments were performed in
duplicate and repeated three times.
