| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Division of Endocrinology, Diabetes, Metabolism and Molecular Medicine, Departments of 1 Pharmacology and Experimental Therapeutics and 3 Medicine, Tupper Research Institute, Tufts University School of Medicine and Tufts-New England Medical Center, Boston, Massachusetts 02111; and 2 Department of Neuroscience, Uppsala University, 751 24 Uppsala, Sweden
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Activation of central melanocortin receptors (MCR) inhibits fever, but the identity of the MCR subtype(s) mediating this antipyretic effect is unknown. To determine whether selective central melanocortin receptor-4 (MC4R) activation produces antipyretic effects, the MC4R selective agonist MRLOB-0001 (CO-His-D-Phe-Arg-Trp-Dab-NH2) was administered intracerebroventricularly to rats treated with Escherichia coli lipopolysaccharide (LPS, 30 µg/kg ip). Treatment with MRLOB-0001 (150 ng icv) did not lower core body temperature (Tc) in afebrile rats but did suppress LPS-induced increases in Tc and associated decreases in tail skin temperature (Tsk), an indicator of vasomotor thermoeffector function. In contrast, systemic treatment with MRLOB-0001 (150 ng iv) did not produce similar antipyretic effects. Coadministration of the selective MC4R antagonist HS014 (1 µg icv) blocked the antipyretic effects of MRLOB-0001. HS014 alone (1 µg icv) had no significant effect on LPS-induced increases in Tc or decreases in Tsk and in afebrile rats had no significant effects on Tc or Tsk. We conclude that pharmacological activation of central MC4R suppresses febrile increases in Tc and that inhibition of heat conservation pathways may contribute to this effect. These findings suggest that the central MC4R may mediate the long-recognized antipyretic effects of centrally administered melanocortins.
antipyretic; thermoregulation; thermoeffector; peripheral vasoconstriction; HS014
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
FEVER IS A HALLMARK of the acute phase response to infection that is orchestrated by the central nervous system (CNS) and is believed to have adaptive value in optimizing host immune responses against microbial invasion (17). However, because excessive fever can prove dangerous to the host, it is believed that counterregulatory systems have evolved to modulate the duration and magnitude of the febrile response (20, 29). Among these systems, the central melanocortinergic system is a prominent pharmacological target capable of suppressing fever.
Activation of central melanocortin receptors (MCRs) suppresses fever.
For example, the endogenous melanocortin 
-melanocyte stimulating
hormone (-MSH), a 13-amino acid peptide derived from proopiomelanocortin, has well-documented antipyretic activity. Administration of exogenous -MSH, which acts as a nonselective MCR
agonist, suppresses experimental fevers induced by immune stimuli,
including the bacterial endotoxin lipopolysaccharide (LPS; see Refs.
6 and 29). It has been proposed that the MCR-3 (MC3R)
and/or MCR-4 (MC4R), both of which are CNS-associated MCR subtypes,
mediate these antipyretic effects, because intracerebroventricular administration of SHU-9119, a nonselective synthetic MC3R/MC4R antagonist, blocked suppression by -MSH of LPS-induced fever in rats
(12, 13). However, the specific roles of MC3R and MC4R in
mediating these effects are unknown.
It is of fundamental interest to determine the pharmacological mechanisms involved in mediating the antipyretic actions of melanocortins. The MC4R, which has been the focus of recent interest as a key central regulator of energy homeostasis (4), is expressed widely among thermoregulatory and other autonomic centers in the CNS (21, 30, 31). Its role in regulation of fever remains unknown, and it is unknown whether selective MC4R activation is sufficient to produce antipyretic effects. The recent development of novel synthetic ligands having improved selectivity for MC4R has enhanced the prospects for investigating the specific role of this receptor in regulation of various CNS functions, including fever.
The aim of this study was to test the hypothesis that pharmacological activation of central MC4R signaling suppresses febrile responses. The effect of central infusion of a novel selective MC4R agonist, MRLOB-0001 (CO-His-D-Phe-Arg-Trp-Dab-NH2) (Dab: 2,4-di-amino-butyric acid; see Ref. 3), on core body temperature (Tc) in LPS-challenged febrile rats was determined in the presence and absence of coadministration of the selective MC4R antagonist, HS014 (16). The potential contribution of general motor activity and heat-conserving thermoeffectors to these responses was assessed by measuring gross motor activity and changes in local skin temperature of the tail, the major thermoeffector organ controlling radiant heat loss in rats, reflecting alterations in peripheral vasomotor tone (11). The results indicate that selective central MC4R activation by MRLOB-0001 suppresses fever and that this suppression is associated with inhibition of fever-associated increases in peripheral vasomotor tone.
| |
MATERIAL AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Animals and Surgical Procedures
Adult male Sprague-Dawley rats (Taconic, Germantown, NY) initially weighing 250-300 g were used. Rats were initially housed three per cage in a room with temperature maintained at 21 ± 1°C and a 14-h light cycle (lights on 0600-2000). Standard rodent chow (Harlan, Madison, WI) and tap water were provided ad libitum throughout the experiment. All procedures were approved by the Institutional Animal Care and Use Committee of Tufts University Medical School and New England Medical Center, and these studies comply with the guiding principles for research of the American Physiological Society (2).Before experiments (7-10 days), each rat was anesthetized with pentobarbital sodium (45 mg/kg ip) and implanted intraperitoneally with a radiotelemetry transmitter (E-Mitter 4000 System; Mini Mitter, Bend, OR) for monitoring Tc and gross motor activity. A 22-gauge stainless steel guide cannula (Plastics One, Roanoke, VA) was permanently implanted in the right lateral ventricle for intracerebroventricular injection, as described earlier (12). Coordinates for right lateral ventricular cannulation, determined using a rat brain atlas (22), were as follows (incisor bar set 3.3 mm below the horizontal plane): 0.8 mm posterior to bregma, 1.4 mm lateral, and 3.4 mm ventral to the skull surface. After surgery, the rats were housed individually in cages in a separate room maintained at 28 ± 1°C, within the thermoneutral range for rats (11), and with a 12:12-h light-dark cycle (lights on 0600-1800). Examination of the diurnal Tc and motor activity profiles for the day preceding experiments confirmed that the rats were entrained to the 12:12-h light-dark cycle (data not shown). Correct placement of intracerebroventricular cannulas was verified at the end of the experiment by injection of 10 µl of 0.1% trypan blue followed by postmortem brain dissection. Data obtained from rats with misplaced cannulas were excluded from analysis. For studies involving intravenous injections, 7 days before experiments, rats were anesthetized using ketamine (40 mg/kg)-xylazine (8 mg/kg) and implanted with permanent intravenous catheters in the right jugular vein followed immediately by radiotelemetry transmitter implantation, as described above. Catheters were constructed of an 8.2-cm length of Micro-Renathane tubing (0.04 in. OD × 0.025 in. ID; Braintree Scientific, Braintree, MA) fused via a short length of 22-gauge tubing to a 2.5-cm length of medical-grade silicone tubing (Silastic, 0.047 in. OD × 0.025 in. ID; Dow-Corning, Midland, MI). The silicone tubing was inserted to the level of the right atrium, and the catheter was anchored in place with ligatures. The Micro-Renathane end was fitted with a subcutaneous lug constructed of a short length of intravenous set tubing affixed with silicone cement, and the proximal end was exteriorized at the nape of the neck, secured in place with wound clips, and closed with a 22-gauge stainless steel obdurator. Catheters were flushed with 0.4 ml heparinized saline daily to maintain patency. Heparinized saline was prepared by rinsing a 1-ml tuberculin syringe with heparin sodium (10,000 U/ml; Pharmacia & Upjohn, Kalamazoo, MI) and then mixing the residual solution in the syringe with 1 ml pyrogen-free 0.9% NaCl.
Animal Handling and Intracerebroventricular Injections
Studies were performed on conscious, unrestrained rats. Each rat was subjected to only one experiment. To minimize the potential influence of stress on results, each rat was conditioned daily for five consecutive days before experiments to a regimen that included gentle handling and a simulated intracerebroventricular injection, performed by removing the dummy cannula and connecting the injection device to the intracerebroventricular guide cannula, or flushing of intravenous catheters. Intracerebroventricular injections were administered via an injection cannula designed to protrude 1 mm from the guide cannula and connected via PE-50 tubing to a 1-ml Hamilton syringe driven by a microinfusion pump (Bee, MF-9090; Bioanalytical Systems, West Lafayette, IN). Injections were delivered at a speed of 2.5 µl/min over a 2-min time period for a final intracerebroventricular injectate volume of 5 µl. Internal cannulas were left in place for 1 min after injection to prevent reflux of injectate.Tc, Motor Activity, and Tail Skin Temperature Measurements
The radiotelemetry signals emitted by each implanted transmitter were monitored continuously via a receiver (Mini Mitter) placed under each cage. These signals were transmitted to a personal computer (Dell, Round Rock, TX), integrated over 5-min intervals, and converted to Tc values according to the frequency-temperature calibration curves using the Vitalview software package (Mini Mitter). General motor activity was also measured using the Mini Mitter system. In this system, activity is detected as changes in signal strength arising from changes in position or orientation of the transmitter and are recorded as motor activity counts.To monitor vasomotor thermoeffector responses noninvasively in the conscious, unrestrained rats, tail skin temperatures (Tsk) were determined using an infrared pyrometry device (Omega Engineering, Stamford, CT). Permanent black ink marks were placed bilaterally on the skin overlying the lateral tail vein ~1 cm from the base of the tail. For determination of Tsk, the infrared pyrometer was held in a horizontal position, perpendicular to the tail skin surface, and aimed at the marked skin surface from a distance of ~8 mm. Bilateral Tsk readings were recorded and averaged at the indicated times.
Experimental Protocol
Before experiments (1 day), rats were weighed and arbitrarily assigned to body weight-matched experimental groups. On the day of the experiment, each rat received an intraperitoneal injection of either 200 µl vehicle (pyrogen-free 0.9% NaCl) or LPS (30 µg/kg in 200 µl vehicle) at a time (designated time 0) between 0900 and 1000. Thirty minutes after the intraperitoneal injection (time 0.5 h), rats received intracerebroventricular injections with either artificial cerebrospinal fluid [aCSF (in mM): 138 NaCl, 3.37 KCl, 1.5 CaCl2, 1.15 MgCl2, 1.45 Na2HPO4, and 4.85 NaH2PO4, pH 7.4], varying doses of MRLOB-0001 [50, 150 (180 pmol), or 500 ng], and/or 1 µg (600 pmol) HS014 diluted in aCSF. In one experiment, rats received similar intraperitoneal treatments with LPS or 0.9% NaCl, followed 30 min later by intravenous injections of MRLOB-0001 (150 ng in 0.15 ml of 0.9% NaCl) via intrajugular catheters. Tc and motor activity were recorded at 5-min intervals starting at 0900 the day before the experiment and ending at 0900 the day after the experiment. Just before intraperitoneal injection, baseline Tsk was determined; thereafter, Tsk was determined hourly starting at time 1.5 h and ending at time 6.5 h.Drugs
Stock solutions of LPS derived from Escherichia coli endotoxin (0111:B4; List Laboratories, Campbell, CA) were prepared by dissolving in vehicle at a concentration of 5 mg/ml and storing in aliquots at 4°C. On the experiment day, aliquots were warmed for 1 h at 37°C, briefly sonicated, and diluted further with vehicle to the respective injectate concentrations. MRLOB-0001 [selectivity for MC4R in radioligand binding and cAMP accumulation bioassays, respectively: MC3R, 46- and 55-fold; and MC5R, 210- and >1,000-fold (3); kindly provided by M. Bednarek and L. H. T. van der Ploeg] was diluted with sterile water to a concentration of 1 µg/µl and stored in aliquots at
70°C. At the conclusion of
these studies, light chromatography-mass spectrometry analysis of the
MRLOB-0001 peptide was performed to confirm its integrity and purity,
and no appreciable peptide degradation was observed (D. Weinberg, personal communication). HS014 (synthesized by Neosystem, Strasbourg, France) was diluted with sterile water to a concentration of 2 µg/µl and stored in aliquots at 70°C. On experiment day,
aliquots of the peptide stock solutions were thawed and diluted with
aCSF for intracerebroventricular injections, or with 0.9% NaCl for intravenous injections, to the respective injectate concentrations.
Data Handling and Statistics
All times and intervals are expressed with respect to time of LPS or vehicle intraperitoneal injections (time 0). Average Tc values for 30-min periods were computed from Tc recorded at 5-min intervals. For each rat, the change in Tc (
Tc) was calculated by subtracting from
each recorded Tc value the baseline temperature, defined as
the mean Tc during the 1-h period immediately preceding the
injection. Values for Tc were calculated for the period
beginning at the intraperitoneal injection (time 0) and
ending at lights-off of the dark cycle (time 8 h at 1800).
With the use of the trapezoidal method, areas under the
Tc vs. time curves (AUC Tc) were
calculated from the resulting Tc values for each of two
time periods: 1) the period beginning at the time of
intracerebroventricular or intravenous injection, 0.5 h, to
time 3.5 h (designated "phase 1"), during
which 30 µg/kg LPS consistently induced a 0.5-1°C peak in
Tc, in line with previous studies of LPS-induced fever (12, 13), and corresponding to the conventionally defined first phase of fever (reviewed in Ref. 23); and
2) the interval 4-8 h (phase 2), during
which a second peak in Tc was consistently observed,
corresponding to the conventionally defined second phase of fever
(23).
Change in Tsk (Tsk) was calculated by
subtracting the baseline Tsk from each subsequent
Tsk value. The AUC for Tsk (AUC Tsk) were calculated by the trapezoidal method for both
phase 1 (0.5-3.5 h) and phase 2. For
Tsk measurements, phase 2 refers to the 4.0- to 6.5-h sampling period.
Average gross motor activity values for 30-min periods were computed from motor activity counts recorded at 5-min intervals. Total gross motor activity was calculated by summation of average motor activity values over phases 1 and 2. Data are expressed as treatment group means ± SE except where otherwise indicated. Significant differences in AUC Tc, AUC Tsk, and total motor activity between treatment groups were determined by one-way ANOVA using SigmaStat software (SPSS Science, Chicago, IL) followed by post hoc tests using the Bonferroni correction for multiple comparisons. Differences showing a P value <0.05 after Bonferroni correction were deemed significant.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Effects of MC4R Agonist on LPS-Induced Changes in Tc, Tail Skin Vasomotor Response, and Motor Activity
Tc.
A small rise in Tc (<0.5°C), peaking at time
1-1.5 h, was observed in all groups (Fig.
1A) because of the minor
stress of handling, injections, and associated increase in motor
activity (Fig. 1E). After this, LPS (30 µg/kg ip)-treated
rats exhibited a biphasic rise in Tc, with the first peak
occurring during phase 1 (time 0-3.5 h) and
the second peak occurring during phase 2 (time
4-8 h). During both phases, the AUC Tc for
LPS-treated rats was significantly higher than that of vehicle-injected
rats (Fig. 1B).
|
150 ng icv were
maximally effective. To determine whether the antipyretic effects of
centrally administered MRLOB-0001 were mediated within the CNS, we
tested the effect of systemic injection of an antipyretic dose of
MRLOB-0001 (150 ng). In contrast with the marked antipyretic effect of
intracerebroventricular injection of MRLOB-0001, LPS-induced
Tc profiles were virtually identical in rats receiving
intravenous MRLOB-0001 and in those receiving 0.9% NaCl for at least
5 h (data not shown), and there were no significant treatment
effects on Tc AUC during either phase 1 (LPS/NaCl, 2.6 ± 0.6, n = 5; LPS/MRLOB-0001,
2.9 ± 0.6°C · h, n = 5)
or phase 2 (LPS/NaCl, 7.1 ± 1.0, n = 5; LPS/MRLOB-0001, 5.9 ± 1.3°C · h,
n = 5).
Tail skin vasomotor response. LPS (30 µg/kg ip)-treated rats exhibited a marked, significant reduction in Tsk values vs. those of vehicle-injected controls that persisted during phases 1 and 2, indicative of a heat-conserving vasomotor response activated during LPS-induced fever (Fig. 1, C and D). Intracerebroventricular treatment with MRLOB-0001 (150 ng) prevented the LPS-induced drop in Tsk during both phases 1 and 2 (Fig. 1, C and D).
Motor activity. During phase 1, all groups exhibited a brief increase in gross motor activity that lasted ~1.5 h after intraperitoneal and intracerebroventricular injections (Fig. 1E). There were no significant differences in motor activity among groups during phase 1 (Fig. 1F). During phase 2, LPS-treated rats receiving only aCSF intracerebroventricularly exhibited significant decreases in total motor activity compared with rats receiving intraperitoneal vehicle/intracerebroventricular aCSF (Fig. 1, E and F). Treatment with MRLOB-0001 (150 ng icv) had no significant effect on the LPS-induced decrease in total motor activity during phase 2 (Fig. 1F). These results indicate that the LPS- and MRLOB-0001-induced changes in Tc could not be attributed to corresponding changes in gross motor activity.
Effect of Central MC4R Blockade by Intracerebroventricular HS014 on Antipyretic Actions of MC4R Agonist
Tc.
To determine whether the antipyretic action of MRLOB-0001 was mediated
specifically via the MC4R, we tested the ability of a selective MC4R
antagonist to inhibit the effect. Coadministration of the MC4R
antagonist HS014 (1 µg, 600 pmol icv) with the lower effective dose
of MRLOB-0001 determined above (150 ng icv) prevented the suppression
by MRLOB-0001 of the LPS-induced increase in Tc during
phase 1. During phase 2, Tc responses
of LPS/(MRLOB-0001 + HS014)-treated rats were not significantly
different from those observed in either LPS/aCSF- or LPS/MRLOB-0001
treated rats (Fig. 1, A and B). In the absence of
coadministered MRLOB-0001, HS014 had no significant effect on
Tc responses in either LPS-treated or NaCl-treated rats
(Fig 2, A and B).
|
Skin vasomotor response and motor activity.
Coadministration of HS014 prevented the suppression by
MRLOB-0001 of the LPS-induced decrease in Tsk during
phase 1 of fever (Fig. 1, C and D).
During phase 2, the integrated changes in tail skin
temperature (AUC Tsk) seen in LPS/(MRLOB-0001 + HS014)-treated rats were not significantly different from those
observed in either LPS/aCSF- or LPS/MRLOB-0001-treated rats (Fig.
1D). Nevertheless, the time course of Tsk
indicated that the blockade of MRLOB-0001 effects on Tsk
responses by HS014 persisted at least through 4.5 h (Fig.
1C). In the absence of coadministered MRLOB-0001, HS014 had
no significant effect on Tsk responses in either
LPS-treated or NaCl-treated rats (Fig. 2, C and
D).
Effect of Selective MC4R Agonist in Afebrile Rats
Tc.
Administration of MRLOB-0001 (150 ng icv) in rats treated
intraperitoneally with vehicle caused a rise in Tc that was
significantly greater than that seen in intraperitoneal
NaCl/intracerebroventricular aCSF-treated controls during the period
0.5-3.5 h (corresponding to phase 1 in LPS-treated
rats; Fig. 3, A and
B). No differences in
Tc profiles were seen among these two groups during the 4- to 8-h period (corresponding to phase 2; Fig. 3,
A and B).
|
Skin vasomotor response and motor activity. There were no significant differences in Tsk profiles between NaCl/MRLOB-0001- and NaCl/aCSF-treated rats during either the 0.5- to 3.5-h or 4- to 8-h periods (Fig. 3, C and D), although there was a trend toward increased motor activity (P = 0.09) in the NaCl/MRLOB-0001 rats during the 0.5- to 3.5-h period (corresponding to phase 1; Fig. 3, E and F). During this period, all NaCl/MRLOB-0001-treated rats, but not NaCl/aCSF-treated rats, exhibited a marked increase in grooming behavior. Grooming, a classic behavioral response to intracerebroventricular administration of melanocortins (8), commenced within 3-5 min of MRLOB-0001 administration and lasted ~2 h. Because it was not a formal objective of this study to assess grooming responses, the induction of this marked and reproducible behavioral response was noted qualitatively but was not quantified. None of the rats in the LPS-treated groups exhibited this response.
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
These findings support the hypothesis that central activation of
the MC4R suppresses the febrile response. Intracerebroventricular administration of the selective MC4R agonist MRLOB-0001 suppressed LPS-induced increases in Tc but did not lower
Tc in afebrile rats. LPS administration caused a decrease
in Tsk, reflecting reduced blood flow to the skin via the
lateral tail vein, a well-established heat-conserving thermoeffector
response associated with the elevated body temperature set point during
fever (11). Treatment with MRLOB-0001 prevented this
LPS-induced peripheral vasoconstriction. In contrast, in afebrile rats
MRLOB-0001 treatment did not lower Tc or raise
Tsk, indicating that its suppression of LPS actions reflected an antipyretic, rather than a cryogenic effect, consistent with the well-documented antipyretic effects of centrally administered exogenous melanocortins (29). Furthermore, MC4R blockade
by coadministered HS014 completely blocked the antipyretic effects of
MRLOB-0001 during the first phase of fever. In addition, a dose of
MRLOB-0001 that was antipyretic when given intracerebroventricularly failed to inhibit fever when injected systemically, indicating that its
antipyretic action was mediated by activation of MC4R within the CNS.
Therefore, the antipyretic effect of the selective MC4R agonist and its
blockade by a selective MC4R antagonist strongly suggest that the
antipyretic effect of MRLOB-0001 is mediated by MC4R within the CNS.
Previous studies showed that centrally administered -MSH exerts an
antipyretic effect in rats that is qualitatively similar to the
presently observed effect of MRLOB-0001, and which is blocked by
coadministered SHU-9119, an MCR antagonist having equivalent potencies
at the MC3R and MC4R (12). Therefore, considered together
with the previous findings, the present findings further suggest a
probable role of MC4R in mediating the long-recognized antipyretic
actions of centrally administered -MSH, a nonselective MC4R agonist.
To determine whether the antipyretic effects of MRLOB-0001 were
mediated specifically via the MC4R, we used the MC4R-selective antagonist HS014. Central administration of HS014 had no significant effects on Tc in either febrile or afebrile rats,
indicating that it is not intrinsically hyperthermic. The
intracerebroventricular dose of HS014 used was predicted to be adequate
to block the action of coadministered MRLOB-0001 at the ~3:1 molar
dose ratio used, based on the in vitro potencies reported in binding
and biological assays (3, 25). For example, centrally
injected (icv) HS014 blocked the anorexic and several other behavioral
effects of coadministered -MSH when used at approximate molar dose
ratios of (2-3):1 (32). Furthermore, HS014 is
particularly selective for the MC4R of the rat, for which it has been
shown to have 85-100 times greater binding affinity than for the
MC3R (19, 25). Together, these lines of evidence strongly
support the conclusion that the blockade of the antipyretic effect of
MRLOB-0001 by HS014 is caused by its selective antagonism at MC4R.
Previous studies indicated that endogenous central melanocortins exert
antipyretic effects during experimental fever (12, 14,
27), probably by acting at MC3R or MC4R since central MCR
blockade using the MC3R/MC4R antagonist SHU-9119 (12, 14) exacerbated LPS-induced fevers in rats. The present finding that the
selective MC4R antagonist HS014 failed to exacerbate LPS-induced fever
suggests that endogenous melanocortins may not exert these physiological antipyretic effects by acting at the MC4R, but it does
not rule out a physiological antipyretic role of the MC4R. The dose of
HS014 used was found to be sufficient to block the antipyretic effects
of coadministered MRLOB-0001 at its site(s) of action. However, it is
conceivable that the antipyretic actions of endogenous neuronal
melanocortins are exerted at multiple CNS sites, in which case
intraparenchymal concentrations of HS014 in certain critical regions
could potentially have been insufficient to antagonize such actions,
e.g., at CNS sites more distant from the ventricles. Alternatively, it
is possible that the antipyretic actions of endogenous melanocortins
are mediated at least in part via the MC3R, at which HS014 is a less
potent antagonist than SHU-9119 (26), or that an
antipyretic influence of the MC4R is exerted in a time frame that
exceeds that of the biological activity of HS014 in the present study.
A recent study found that febrile responses to intracerebroventricular
injection of interleukin-1
were unaffected by a coinjected MC3R/MC4R
antagonist. This may indicate that MC3R and MC4R do not modulate the
thermoregulatory response to intracerebral interleukin-1, but
whether that model system is directly relevant to fever induced by
systemic microbial pyrogens such as LPS is not clear, and it is
uncertain whether intraparenchymal antagonist concentrations would have
been adequate to block antipyretic actions of endogenous melanocortins
at their target sites (18). Further studies will be
required to elucidate the nature and physiological significance of the
regulation of fever by the central melanocortin system.
The mechanisms involved and CNS sites at which MC4R activation produces
antipyretic effects are unknown. The effects are almost certainly
centrally mediated, because the effective systemic dose of
melanocortins required for antipyretic effects is ~100-fold greater
than that required by intracerebroventricular administration, and the
antipyretic effect of centrally injected -MSH was blocked by
coadministered MC3R/MC4R antagonist at a dose that was ineffective when
administered intravenously (12, 13). Furthermore, MC4R is
believed to be predominantly or exclusively expressed in the CNS, where
it is distributed in numerous autonomic centers, including hypothalamic
and preoptic regions containing circuitry believed to be involved in
establishing body temperature set point, integration of afferent
temperature sensory inputs, and descending control of thermogenesis
(5, 30, 31). These areas also receive substantial
innervation by endogenous proopiomelanocortin-producing neurons
(9, 30). Therefore, it is likely that activation of MC4R
expressed in thermoregulatory neurons either inhibits the elevation of
body temperature set point, or acts more distally and in a
physiological state-dependent manner to inhibit descending thermoeffector pathways. These possibilities remain to be tested.
In terms of effector systems, the present studies demonstrate that
selective MC4R activation results in the inhibition of at least one
thermoeffector system involved in the febrile response, the peripheral
vasomotor response. This is consistent with a previous study in rabbits
(10) that demonstrated antipyretic effects of -MSH and
ACTH, both of which are nonselective MCR agonists. Based on
thermodynamic considerations alone, this effect, which inhibits
retention of body heat, must contribute to the suppression of
Tc elevation by MC4R activation. Whether this effect alone is sufficient to account for the net suppression of febrile
Tc elevation by centrally administered melanocortins, or
whether additional thermoeffector systems are involved, remains to be determined.
In this connection, the present study provides no direct evidence that
MC4R activation during the febrile state modulates general motor
activity, one potential source of thermogenesis, since no significant
effects of MRLOB-0001 or HS014 treatments on LPS-induced suppression of
motor activity were observed. This contrasts somewhat with the moderate
exacerbation of the LPS-induced suppression of motor activity we
observed in response to intracerebroventricular -MSH treatment in a
related study (14). This difference could potentially be
attributable to activation by the nonselective agonist -MSH of MCR
subtypes other than MC4R, which influence locomotor activity during
fever. Alternatively, these differences in melanocortin influences on
motor activity may be attributable to other major differences in design
in the two studies, including the facts that the rats were fasted and
received a higher LPS dose in our earlier study (14). One
additional consideration in the present study is that all rats
exhibited a robust increase in motor activity during the first 1.5 h after intraperitoneal injections, which may have obscured subtle
treatment-associated changes in motor activity during that period. The
question of whether the antipyretic action of MC4R agonists could
potentially involve alterations in thermogenesis via altered motor
activity merits further study.
The suppressive effects of MRLOB-0001 on LPS-induced Tc
elevation and peripheral vasomotor responses persisted during both phases of fever, whereas the blockade of antipyretic effects by coadministration of HS014 subsided partially during the second phase
(4-8 h) of fever. The relatively greater persistence of MRLOB-0001
effects compared with those of HS014 may reflect pharmacokinetic differences, such as greater in vivo stability or decreased receptor dissociation rates of MRLOB-0001 compared with HS014, but available data are insufficient to address these possibilities directly. Nevertheless, the results provide additional evidence of the in vivo
potency of MRLOB-0001, as indicated by its brisk induction of grooming
activity in afebrile rats. Induction of grooming behavior is a classic
response to centrally administered melanocortins, including -MSH,
and is thought to be mediated by MC4R within the CNS (1).
By comparison, induction of grooming behavior by -MSH requires at
least a 10-fold higher molar dose than the presently effective dose of
MRLOB-0001 (8). In addition, treatment with MRLOB-0001
failed to elicit a grooming response in LPS-treated rats, indicating
that the induction of grooming behavior by MC4R activation, like its
effects on body temperature, is highly dependent on physiological state.
An alternative interpretation of the MRLOB-0001-induced grooming response, seen exclusively in afebrile controls, concerns its thermoregulatory implications. The induction of grooming behavior in afebrile rats after intracerebroventricular MRLOB-0001 administration was accompanied by a coincident increase in Tc. Grooming is thought to be an adaptive response employed by rodents to lower body temperature during hyperthermic states, i.e., when Tc exceeds body temperature set point, and is characterized by use of the tongue and paws to spread saliva over the body starting at the head, progressing to the torso, and ending with the limbs (11). Because the presently observed MRLOB-0001-induced grooming response followed this pattern, it is possible that activation of central MC4R by MRLOB-0001 induces a neurally mediated hyperthermic effect in afebrile rats, leading in turn to induction of grooming behavior, a secondary homeostatic response to the hyperthermia. This is consistent with reports that pharmacological activation of central MC4R and/or MC3R stimulates oxygen consumption and thermogenic activity in brown fat in normal rats and that MC4R-deficient mice exhibit decreased melanocortin- and leptin-induced thermogenesis (7, 24, 28). In contrast, LPS-treated rats receiving MRLOB-0001 treatment exhibited little or no rise in Tc during phase 1, and the MRLOB-0001-induced grooming response was absent in those animals. Because the results of MRLOB-0001 treatment were qualitatively opposite in febrile and afebrile rats (Tc lowering vs. Tc elevating, respectively), these findings underscore the exquisite specificity of the antipyretic effect of centrally administered MRLOB-0001 and further indicate that the effects of MC4R activation on Tc are highly dependent on physiological state.
In summary, these results indicate that central MC4R activation suppresses fever and that prevention of fever-associated peripheral vasoconstriction contributes to the antipyretic response. This study also demonstrates that the thermoregulatory effects of MC4R activation are markedly dependent on physiological state, suppressing the elevation of Tc associated with an inflammatory state while elevating Tc in afebrile rats. Determination of the neural mechanisms involved in these effects, and the potential role of the MC4R in mediating other recognized melanocortin-induced suppressive effects on neuroinflammatory processes in the CNS (15), will be of substantial interest.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Lex H. T. Van der Ploeg, Maria Bednarek, and David Weinberg for generously providing MRLOB-0001 and analytical data, Jerold Harmatz for statistical consultations, Dr. Joseph Cannon for helpful comments and a critical review of the manuscript, and Allison Ohrt and Latrice Goosby for technical assistance.
This work was supported by National Institute of Mental Health Grant MH-44694 (J. B. Tatro), the Swedish Research Council (VR, medicine; H. B. Schiöth), and Melacure Therapeutics AB, Uppsala, Sweden (H. B. Schiöth).
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: J. B. Tatro, Div. of Endocrinology, Diabetes, Metabolism, and Molecular Medicine, Box 268, Tufts-New England Medical Center, 750 Washington St., Boston, MA 02111 (E-mail: jtatro{at}tufts-nemc.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpregu.00581.2002
Received 18 September 2002; accepted in final form 14 February 2003.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Adan, RAH
Effects of melanocortins in the nervous system.
In: The Melanocortin Receptors, edited by Cone RD.. Totowa, NJ: Humana, 2000, p. 109-141.
2.
American Physiological Society.
Guiding principles for research involving animals and human beings.
Am J Physiol Regul Integr Comp Physiol
283:
R281-R283,
2002
3.
Bednarek, M,
MacNeil T,
Tang R,
Kalyani L,
Van der Ploeg L,
and
Weinberg DL.
Potent and selective peptide agonists of -melanotropin action at human melanocortin receptor 4: their synthesis and biological evaluation in vitro.
Biochem Biophys Res Commun
286:
641-645,
2001[Web of Science][Medline].
4.
Benoit, S,
Schwartz MW,
Baskin D,
Woods S,
and
Seeley R.
CNS melanocortin system involvement in the regulation of food intake.
Horm Behav
37:
299-305,
2000[Medline].
5.
Boulant, JA.
Role of the preoptic-anterior hypothalamus in thermoregulation and fever.
Clin Infect Dis
31:
S157-S161,
2000.
6.
Catania, A,
and
Lipton JM.
-Melanocyte stimulating hormone in the modulation of host reactions.
Endocr Rev
14:
564-576,
1993
7.
Chen, AS,
Metzger JM,
Trumbauer ME,
Guan X-M,
Yu H,
Frazier EG,
Marsh DJ,
Forrest MJ,
Gopal-Truder S,
Fisher J,
Camacho R,
Strack AM,
Mellin TN,
MacIntyre DE,
Chen HY,
and
Van der Ploeg LHT
Role of the melanocortin-4 receptor in metabolic rate and food intake in mice.
Transgenic Res
9:
145-154,
2000[Web of Science][Medline].
8.
De Wied, D.
Behavioral pharmacology of neuropeptides related to melanocortins and the neurohypophyseal hormones.
Eur J Pharmacol
375:
1-11,
1999[Web of Science][Medline].
9.
Elias, CF,
Saper CB,
Maratos-Flier E,
Tritos NA,
Lee C,
Kelly J,
Tatro JB,
Hoffman GE,
Ollmann MM,
Barsh GS,
Sakurai T,
Yanagisawa M,
and
Elmquist JK.
Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area.
J Comp Neurol
402:
442-459,
1998[Web of Science][Medline].
10.
Glyn, JR,
and
Lipton JM.
Hypothermic and antipyretic effects of centrally administered ACTH (1-24) and -melanotropin.
Peptides
2:
177-187,
1981[Web of Science][Medline].
11.
Gordon, CJ.
Temperature Regulation in Laboratory Rodents. New York, NY: Cambridge Univ. Press, 1993.
12.
Huang, Q-H,
Entwistle ML,
Alvaro JD,
Duman RS,
Hruby VJ,
and
Tatro JB.
Antipyretic role of endogenous melanocortins mediated by central melanocortin receptors during endotoxin-induced fever.
J Neurosci
17:
3343-3351,
1997
13.
Huang, Q-H,
Hruby VJ,
and
Tatro JB.
Systemic -MSH suppresses LPS fever via central melanocortin receptors independently of its suppression of corticosterone and IL-6 release.
Am J Physiol Regul Integr Comp Physiol
275:
R524-R530,
1998
14.
Huang, QH,
Hruby VJ,
and
Tatro JB.
Role of central melanocortins in endotoxin-induced anorexia.
Am J Physiol Regul Integr Comp Physiol
276:
R864-R871,
1999
15.
Ichiyama, T,
Sato S,
Okada K,
Catania A,
and
Lipton JM.
The neuroimmunomodulatory peptide -MSH.
Ann NY Acad Sci
917:
211-216,
2000.
16.
Kask, A,
Rago L,
Mutilis F,
Pahkla R,
Wikberg J,
and
Schiöth H.
Selective antagonist for the melanocortin 4 receptor (HS014) increases food intake in free-feeding rats.
Biochem Biophys Res Commun
245:
90-93,
1998[Web of Science][Medline].
17.
Kluger, MJ,
Kozak W,
Conn C,
Leon LR,
and
Soszynski D.
The adaptive value of fever.
In: Basic Mechanisms and Management, edited by Mackowiak PA.. Philadelphia, PA: Lippincott-Raven, 1997, p. 255-266.
18.
Lawrence, CB,
and
Rothwell NJ.
Anorexic but not pyrogenic actions of interleukin-1 are modulated by central melanocortin-3/4 receptors in the rat.
J Neuroendocrinol
13:
490-495,
2001[Web of Science][Medline].
19.
Lindblom, J,
Schioth HB,
Larsson A,
Wikberg JES,
and
Bergstrom L.
Autoradiographic discrimination of melanocortin receptors indicates that the MC3 subtype dominates in the medial rat brain.
Brain Res
810:
161-171,
1998[Web of Science][Medline].
20.
Mackowiak, PA,
and
Boulant JA.
Fever's upper limit.
In: Fever: Basic Mechanisms and Management, edited by Mackowiak PA.. New York, NY: Lipincott-Raven, 1997, p. 147-163.
21.
Mountjoy, KG,
Mortrud MT,
Low MJ,
Simerly RB,
and
Cone RD.
Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain.
Mol Endocrinol
8:
1298-1308,
1994
22.
Paxinos, G,
and
Watson C.
The Rat Brain in Stereotaxic Coordinates. New York, NY: Academic, 1986.
23.
Romanovsky, A,
Kulchitsky V,
Akulich N,
Koulchitsky S,
Simons c,
Sessler D,
and
Gourine V.
The two phases of biphasic fever-two different strategies for fighting infection?
Ann NY Acad Sci
813:
485-490,
1997[Medline].
24.
Satoh, N,
Ogawa Y,
Katsuura G,
Numata Y,
Masuzaki H,
Yoshimasa Y,
and
Nakao K.
Satiety effect and sympathetic activation of leptin are mediated by hypothalamic melanocortin system.
Neurosci Lett
249:
107-110,
1998[Web of Science][Medline].
25.
Schiöth, HB,
Bouifouri A,
Rudzish R,
Muceniece R,
Watanobe H,
Wikberg JES,
and
Larhammer D.
Pharmacological comparison of rat and human melanocortin 3 and 4 receptors.
Regul Pept
106:
7-12,
2002[Web of Science][Medline].
26.
Schiöth, HB,
Muceniece R,
Mutilis F,
Bouifouri AA,
Mutile I,
and
Wikberg JES
Further pharmacological characterization of the selective melanocortin 4 receptor antagonist HS014: comparison with SHU9119.
Neuropeptides
33:
191-196,
1999[Web of Science][Medline].
27.
Shih, ST,
Khorram O,
Lipton JM,
and
McCann SM.
Central administration of -MSH antiserum augments fever in the rabbit.
Am J Physiol Regul Integr Comp Physiol
250:
R803-R806,
1986
28.
Ste. Marie, L,
Miura GI,
Marsh DJ,
Yagaloff K,
and
Palmiter RD.
A metabolic defect promotes obesity in mice lacking melanocortin-4 receptors.
Proc Natl Acad Sci USA
97:
12339-12344,
2000
29.
Tatro, JB.
Endogenous antipyretics.
Clin Infect Dis
31:
S190-S201,
2000.
30.
Tatro, JB.
Melanocortin receptor expression, and function in the nervous system.
In: The Melanocortin Receptors, edited by Cone RD.. Totowa, NJ: Humana, 2000, p. 173-207.
31.
Tatro, JB,
Entwistle ML,
Fairchild-Huntress V,
and
Huszar D.
MC4-R is the principal melanocortin receptor subtype of the preoptic area and lateral hypothalamus (Abstract).
Soc Neurosci Abstr
27 (30):
2,
2001.
32.
Vergoni, A,
Schiöth HS,
and
Bertolini A.
Melanocortins and feeding behavior.
Biomed Pharmacother
54:
129-134,
2000[Medline].
This article has been cited by other articles:
|
|
 |
|
  A. A. Romanovsky Anorexia: the toll for lipopolysaccharide recognition Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R274 - R275. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
