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Behavioral, neuroendocrine and thermoregulatory actions of apelin-13
Neuroscience 129 (2004) 811– 816
BEHAVIORAL, NEUROENDOCRINE AND THERMOREGULATORY ACTIONS OF APELIN-13 M. JÁSZBERÉNYI, E. BUJDOSÓ AND G. TELEGDY*
(Tatemoto et al., 1998). It is derived from a 77-amino-acid precursor, preproapelin, which is processed to several molecular forms in different tissues. Synthetic C-terminal fragments of preproapelin consisting of 13–19 amino acids were found to exhibit significantly higher activity at the receptor than that of apelin-36 (Tatemoto et al., 1998; Kawamata et al., 2001). The expression of the rat apelin receptor has been detected throughout the CNS, and especially in the limbic structures, the hypothalamus and the pituitary. In the hypothalamus, the most intense expression is in the supraoptic and paraventricular nuclei (De Mota et al., 2000; O’Carroll et al., 2000), which suggests a prominent role of apelin in the regulation of homeostatic, behavioral and endocrine processes. Some physiological effects of apelin have already been described: the administration of apelin-13 increased the water intake (Lee et al., 2000; Taheri et al., 2002) and it was demonstrated to have an impact on the blood pressure (Lee et al., 2000; Seyedabadi et al., 2002) and vasopressin release (Reaux et al., 2001; Taheri et al., 2002). The role of pyroglutamylated apelin-13 in the control of appetite and pituitary hormone release has also been revealed (Taheri et al., 2002). The hypothalamus, the major site of apelin-positive nerve fibers (Reaux et al., 2002), plays a central role in the control of neuroendocrine processes and homeostatic responses. Accordingly, in the present study, the effects of apelin on the hypothalamo–pituitary–adrenal (HPA) response and thermoregulation were examined. Since the limbic structures, which regulate behavior, also display a high expression of the apelin receptor (De Mota et al., 2000), its action on spontaneous locomotion and exploratory behavior was also investigated. Since the corticotropin-releasing hormone (CRH) is one of the most potent regulators of stress-related behavior (Menzaghi et al., 1994) and HPA activation (Vale et al., 1981), animals were pretreated with the CRH antagonist ␣-helical CRH9 – 41 in order to shed light on the involvement of CRH transmission in the behavioral and hormonal responses elicited by apelin. Strijbos et al. (1992) revealed the effects of CRH on thermogenesis and body temperature; therefore, we investigated the role of CRH in the apelin-induced hyperthermic response, too. Recent data provided evidence of the role of nitric oxide (NO) in the mediation of the action of apelin on blood pressure (Tatemoto et al., 2001). With the aim of investigating the regulatory role of NO in the neuroendocrine, behavioral and thermoregulatory responses evoked by apelin, therefore
Department of Pathophysiology, University of Szeged, Neurohumoral Research Group, Hungarian Academy of Sciences, Semmelweis u. 1, PO Box 427, H-6701, Szeged, Hungary
Abstract—As the distribution of apelinergic neurons in the brain suggests an important role of apelin-13 in the regulation of neuroendocrine processes, in the present experiments the effects of this recently identified neuropeptide on the open-field activity, the hypothalamo–pituitary–adrenal (HPA) system and the body temperature were investigated. I.c.v. administration of apelin-13 (1–10 g) to rats caused significant increases in square crossing, rearing, plasma corticosterone release and core temperature, whereas it did not influence the spontaneous motor activity during telemetric observation. To determine the mediation of the actions of apelin, a corticotropin-releasing hormone (CRH) antagonist, the nonselective dopamine antagonist haloperidol, the selective dopamine D1 receptor antagonist SCH-23390 and the nitric oxide synthase inhibitor N-nitro-L-arginine-methyl ester (L-NAME) were administered to the rats. The apelinevoked HPA activation was diminished by preadministration of the CRH antagonist, while the dopamine antagonist and L-NAME attenuated only the square crossing and rearing induced by apelin-13. To characterize the transmission of the thermoregulatory action of apelin, animals were pretreated either with L-NAME, the CRH antagonist or with the cyclooxygenase inhibitor noraminophenazone. L-NAME and the CRH antagonist did not cause significant inhibition of the apelinevoked increase in core temperature, while the cyclooxygenase inhibitor, applied 30 min before peptide treatment, did not prove effective in preventing the apelin-evoked thermoregulatory response, whereas when it was administered 2 h after the peptide treatment, it transiently and significantly reduced the hyperthermic response. The present data suggest that apelin-13 plays an important role in the regulation of behavioral, endocrine and homeostatic responses in the CNS, and dopamine, nitric oxide and prostaglandins seem to take part in the mediation of its effects. Since the corticosterone response could be blocked by the CRH antagonist, it is likely to be mediated through the activation of the CRH neurons. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: open-field behavior, HPA axis, telemetry.
The recently discovered peptide apelin has been demonstrated to be the endogenous ligand for an orphan G protein-coupled receptor, APJ, identified in a human gene by O’Dowd et al. (1993). The putative endogenous ligand apelin-36 was first isolated from bovine stomach extracts *Corresponding author. Tel: ⫹36-6254-5797; fax: ⫹36-6254-5710. E-mail address: [email protected] (G. Telegdy). Abbreviations: CRH, corticotropin-releasing hormone; HPA, hypothalamo-pituitary-adrenal; NAP, noraminophenazone; NO, nitric oxide; L-NAME, N-nitro-L-arginine-methyl ester; NOS, nitric oxide synthase.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.08.007
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Fig. 1. Apelin-13 evoked response in square crossing and rearing. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of animals used. * P⬍0.005 vs. control.
the effects of the NO synthase (NOS) inhibitor N-nitro-Larginine-methyl ester (L-NAME) were tested, too. Previous studies suggested that dopaminergic mediation might be involved in hyperlocomotion (Matsuzaki et al., 2002) and in the HPA response (Jezova et al., 1985). Hence, in a further set of experiments, to characterize dopaminergic mediation involved in the open-field behavior and HPA activation induced by apelin, animals were pretreated either with the nonselective dopamine antagonist haloperidol or with the D1 receptor-selective SCH 23390. Several publications have demonstrated that the prostaglandins play an indispensable role in the mediation of hyperthermic processes (Matsumura et al., 1990). Accordingly, by using the cyclooxygenase inhibitor noraminophenazone (NAP), we attempted to clarify the mediation of the thermoregulatory actions of apelin.
EXPERIMENTAL PROCEDURES Animals The animals were kept and handled during the experiments in accordance with the Council Directive of the European Economic Community regarding the protection of animals for experimental and other scientific purposes (86/609/EEC). Further, all efforts
Fig. 2. Effects of haloperidol (HAL) on apelin-induced response in square crossing and rearings. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of animals used. * P⬍0.05 vs. control.
Fig. 3. Effects of selective D1 receptor antagonist SCH-23390 on apelin-induced response in square crossing and rearings. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of animals used. * P⬍0.005 vs. control.
were made to minimize the number of animals used and their suffering. Male Wistar rats weighing 150 –250 g upon arrival were used. The rats were kept in their home cages at a constant room temperature on a standard illumination schedule with 12-h light/ dark period (lights on from 6.00 a.m.). Commercial food and tap water were available ad libitum. The rats were allowed a minimum of 1 week to acclimatize before surgery. To minimize the effects of nonspecific stress, the rats were handled daily.
Surgery For i.c.v. peptide administration, the rats were implanted with a stainless steel 20 G 1.5 Luer cannula (10 mm long) aimed at the right lateral cerebral ventricle under Nembutal (35 mg/kg, i.p.) anesthesia. The stereotaxic coordinates were 0.2 mm posterior; 1.7 mm lateral to the bregma; 3.7 mm deep from the dural surface, according to the atlas of Pellegrino et al. (1979). Cannulas were secured to the skull with dental cement and acrylate. The rats were used after a recovery period of at least 5 days. In the case of i.c.v. administration, Methylene Blue was injected into each decapitated head and the brains were dissected to verify the correct positioning and the permeability of the cannulas. Only data from rats in which the placement was correct were considered for the statistical evaluation. For implantation of an internal radio transmitter (E-Mitter), the rats were anesthetized with Nembutal (35 mg/kg, i.p.). The abdomen was opened by making a 2 cm midline incision along the linea alba. The E-Mitter was placed in the abdominal cavity along the sagittal plane in front of the caudal arteries and veins, but dorsal to the digestive organs. The abdom-
Fig. 4. Effects of i.c.v. administration of L-NAME on apelin-induced response in square crossing and plasma corticosterone. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of mice used. * P⬍0.05 vs. control.
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Table 1. Effects of i.c.v. administration of CRH antagonist ␣-helical CRH9 – 41 on apelin-induced behavioural responses (behavioural activity was characterized by the total number of explored squares or rearings/5 min test sessions)a
Locomotion Rearing
Control (8)
CRH antagonist (1 g) (8)
CRH antagonist⫹apelin (9)
Apelin (1 g) (10)
65.38⫾9.27 8.63⫾0.98
77.50⫾3.70 10.00⫾0.82
103.78⫾6.19* 14.33⫾1.32*
113.13⫾9.23* 17.00⫾2.14*
a Numbers in brackets are numbers of animals used. * P⬍0.05 vs. control.
inal opening was than closed with absorbable suture material, using a continuous interlocking running stitch, while the skin was closed with stainless steel suture material, using interrupted mattress stitches.
Treatment Protocol 1. Different doses of apelin-13 (Bachem, Bubendorf, Switzerland) dissolved in saline, or saline alone (control animals), in a volume of 2 l, were injected i.c.v. into conscious rats with a Hamilton microsyringe over 30 s, immobilization of the rats being avoided during handling. Protocol 2. For this experimental setting, animals were subjected to combined treatment with ␣-helical CRH9 – 41 (Bachem), the nonselective dopamine receptor blocker haloperidol (Richter, Budapest, Hungary), the dopamine D1 receptor-selective antagonist SCH 23390 (Tocris, Bristol, UK), the NOS inhibitor L-NAME (Sigma, Budapest, Hungary), the pyrazolone derivative NAP (Algopyrin; Chinoin, Budapest, Hungary) and apelin-13. L-NAME, ␣-helical CRH9 – 41 and SCH 23390 dissolved in 0.9% saline were injected i.c.v. Haloperidol (dissolved in 0.9% saline) was administered i.p. while NAP was injected i.m. in a volume of 0.5 ml. The concentrations of the antagonists or inhibitors were the concentrations that had proved most effective in previous experiments (Telegdy, 1984; Pataki et al., 1999; Bujdoso et al., 2001, 2003; Yamauchi et al., 2003), but which per se did not affect the behavioral paradigms. Thirty minutes after the pretreatment, the animals were treated i.c.v. with the dose of apelin-13 that had proved most effective in protocol 1. The control animals received saline alone. Thirty minutes after peptide administration, the animals were subjected to behavioral tests, or the plasma corticosterone level was measured, and the core temperature was recorded continuously. In a further set of experiments, animals were treated with NAP 2 h after the apelin injection.
Open-field test Thirty minutes after the apelin or vehicle administration, the behavioral paradigms were monitored over 5-min test sessions. The rats were removed from their home cages and placed in the center of an open-field box. The locomotor activity was analyzed by counting the number of passages from one to another of 36 equally sized (10⫻10 cm each) squares. The number of rearings was also recorded. The grooming frequency was characterized by the numbers of face washings, forepaw lickings or head strokings.
Determination of plasma corticosterone Thirty minutes after apelin treatment, the rats were killed by decapitation and approximately 3 ml blood was collected in heparinized tubes for corticosterone assay. The plasma corticosterone level was determined by fluorescence assay (Purves and Sirett, 1965).
Telemetry Apelin-13 was administered i.c.v. to the rats (9:00 a.m.) and the spontaneous motor activity and core temperature were recorded
continuously. The system uses E-Mitter, an implanted radiotelemetry device (Mini Mitter, Bend, OR, USA), to determine the animal temperature and motor activity data. The E-Mitter obtains power from a radiofrequency field produced by an energizer/receiver placed below the cage of the animals. The counts were recorded continuously and the output from the receivers was managed by VitalView, a Windows-based data acquisition system.
Statistical analysis Values are presented as means⫾S.E.M. Statistical analysis of the results was performed by analysis of variance, and differences between groups were examined by Tukey’s post hoc comparison test (Statistica 5.0). In the case of the telemetric observations the groups were compared at each point of time. A probability level of 0.05 was accepted as indicating a statistically significant difference.
RESULTS Effects of apelin-13 on open-field behavior and spontaneous locomotion The treatment with apelin-13 enhanced the numbers of both rearings and square crossings during the open-field exploration test (Fig. 1). The dose of 1 g increased the number of squares explored (F(3, 36)⫽7.24; P⬍0.005 vs. control), but further elevation of the dose (5 g) did not result in an additional increase. Apelin-13 also facilitated the rearing activity (Fig. 1): the most effective dose was 1 g (F(3, 36)⫽6.99; P⬍0.001 vs. control), and a higher dose of apelin-13 (5 g) proved less effective. As concerns grooming, apelin-13 did not evoke a significant response and neither of the applied doses (1 and 10 g) of the peptide altered the spontaneous motor activity during telemetric observation (data not shown). Effects of CRH antagonist, dopamine antagonist and L-NAME on behavioral responses evoked by apelin-13 Haloperidol pretreatment inhibited both the square crossing and the rearing response brought about by apelin (F(3, 39)⫽4.08; P⬍0.05 vs. control for square crossing and F(3, 39)⫽8.77; P⬍0.005 vs. control for rearing; Fig. 2). The selective D1 antagonist SCH 23390 completely abolished the behavioral responses induced by apelin-13 (F(3, 37)⫽6.30; P⬍0.005 vs. control for square crossing and F(3, 37)⫽10.59; P⬍0.0005 vs. control for rearing; Fig. 3). L-NAME also mitigated the apelin-evoked increases (F(3, 49)⫽9.68; P⬍0.005 vs. control for square crossing and F(3, 49)⫽5.30; P⬍0.05 vs. control for rearing) in square crossing and rearing (Fig. 4); however, the CRH antagonist ␣-helical CRH9 – 41 failed to modify the apelin-induced locomotor and rearing activity (Table 1).
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Fig. 5. Effects of apelin-13 on HPA response. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of animals used. * P⬍0.05 vs. control.
Fig. 6. Effects of i.c.v. administration of CRH antagonist ␣-helical CRH9 – 41 (CRH a.) on HPA response evoked by apelin-13. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of rats used. * P⬍0.05 vs. control.
DISCUSSION
Effects of apelin-13 on HPA system Apelin-13 brought about a prominent activation of the HPA system (Fig. 5). In a dose of 10 g, apelin-13 elevated the plasma concentration of corticosterone by 153% as compared with the control (F(3, 22)⫽5.48; P⬍0.05 vs. control). Effects of CRH antagonist, haloperidol and L-NAME on HPA activation elicited by apelin-13 The CRH antagonist ␣-helical CRH9 – 41 produced inhibition of the apelin-evoked increase (F(3, 20)⫽3.80; P⬍0.05 vs. control) in corticosterone secretion (Fig. 6). On the other hand, the dopamine antagonist haloperidol and LNAME turned out to be ineffective on the HPA response elicited by apelin-13 (Table 2 and Table 3), and the antagonists did not cause further increase in the apelin-induced corticosterone level. Effects of CRH antagonist, L-NAME and NAP on hyperthermic effect of apelin-13 Apelin in a dose of 10 g increased the core temperature (F(4, 47)⫽4.74; P⬍0.005 vs. control; Fig. 7). Pretreatment with CRH antagonist and L-NAME did not modify the apelin-evoked hyperthermic effect. The cyclooxygenase inhibitor NAP, applied 30 min before peptide treatment, did not prove effective in reducing the apelin-evoked thermoregulatory response (data not shown). However, the administration of NAP 2 h after the i.c.v. apelin injection, significantly and transiently reduced the apelin-induced core temperature (P⬍0.05 vs. apelin).
The present experiments clearly demonstrate that apelin-13 leads to a marked activation of locomotion and rearing in a dose-dependent manner and the dose-response curves exhibit an inverted U-shape. Lee et al. (2000) demonstrated sequence and distribution similarities between apelin and angiotensin II, and our results revealed that the locomotor-activating effect of apelin resembles the action of angiotensin II (Braszko et al., 1988). Several studies have demonstrated that CRH acts as a mediator of behavioral processes (Bujdoso et al., 2001; Menzaghi et al., 1994), but in our experiment, CRH antagonist pretreatment failed to decrease the locomotor hyperactivity induced by apelin. Further experiments were therefore designed to shed light on the mediation of the behavioral responses brought about by apelin. Haloperidol pretreatment reduced the numbers of both the square crossing and of rearing, suggesting that the nigrostriatal system or mesolimbic structures such as the nucleus accumbens mediate the behavioral actions of the apelinergic neurons (Gysling and Wang, 1983; Johnson and North, 1992). In our experiments, SCH 23390, a selective dopamine receptor blocker, completely reduced the behavioral action of apelin-13, indicating an indispensable role of D1 receptors in these processes. Our findings are in harmony with those of previous behavioral studies (Hoffman and Beninger, 1985; Meyer et al., 1993), showing that D1 receptors play a noteworthy role in the control of locomotor activity and rearing. The results of the antagonist studies led to a critical role being ascribed to dopamine in the apelinevoked behavioral processes. We found that L-NAME pretreatment caused a moderate inhibition of the behavioral
Table 2. Effects of haloperidol on apelin-induced corticosterone level (corticosterone concentration is given in g/100 ml)a
Corticosterone level mean⫾S.E.M
Control (6)
Haloperidol (10 g) (6)
Haloperidol⫹apelin (6)
Apelin (10 g) (8)
11.08⫾0.70
12.53⫾2.57
18.72⫾1.82*
19.16⫾1.66*
a Numbers in brackets are numbers of animals used. * P⬍0.05 vs. control.
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Table 3. Effects of i.c.v. administration of L-NAME on apelin-induced corticosterone level (corticosterone concentration is given in g/100 ml)a
Corticosterone level mean⫾S.E.M
(20 g) (7)
Control (7)
L-NAME
9.84⫾0.77
13.09⫾1.87
L-NAME⫹apelin
(7) 22.99⫾3.04*
Apelin (10 g) (5) 18.40⫾0.76*
a Numbers in brackets are numbers of animals used. * P⬍0.05 vs. control.
responses elicited by apelin-13. Our data reinforce the hypothesis of the NO-dependent action of apelin (Tatemoto et al., 2001). On the other hand, apelin-13 did not influence the spontaneous locomotion during telemetric observations. The discrepancy observed between its effects on exploratory behavior and spontaneous locomotion corroborates the hypothesis that the motor responses are regulated by different mechanisms (Diamant and de Wied, 1991). The present data regarding apelin-evoked HPA activation lend support to the earlier conclusion (Taheri et al., 2002) that apelin has a marked impact on neuroendocrinological processes. The CRH antagonist diminished the corticosterone secretion evoked by apelin, but the role of other mediators (e.g. vasopressin) (Taheri et al., 2002) in the activation of the HPA axis cannot be excluded. In our experiments, the HPA activation evoked by apelin was not inhibited by haloperidol, which supports the view that the actions of apelin on the HPA system do not involve dopaminergic transmission (Amar et al., 1982; Bujdoso et al., 2003). The differences between the effective apelin dose on behavior (1 g) and plasma corticosterone (10 g), reflect the situation that distinct neural pathways may be involved in the regulation of exploratory behavior and HPA activation. Our data also demonstrate that apelin-13 increased the core temperature, which might indicate a physiological
role of the dense apelin-positive projections described in the preoptic region of the hypothalamus (Reaux et al., 2002). These findings also help to harmonize the behavioral observations, since, under resting conditions, the heat-conserving processes (curling-up, etc.) may interfere with a statistically significant locomotor response. There is evidence of several distinct pathways for the induction of fever, and it seems that CRH (Strijbos et al., 1992) acts to mediate cytokine-induced (Busbridge et al., 1989) fever. The cytokines increase the body temperature at least partially via the prostaglandins (Feldberg and Saxena, 1975). Further, the synthesis of NO has been regarded as an important step in thermoregulation (Monroy et al., 2001). In our experiment he NOS inhibitor L-NAME and the CRH antagonist did not cause significant inhibition in the apelinevoked increase in core temperature. The cyclooxygenase inhibitor significantly decreased the apelin-induced elevation of the core temperature, but this effect did not prove permanent. Our results reflect the fact that apelin increase the core temperature, probably in part, through the production of prostaglandins. In conclusion, our results, taken together with the histological data, indicate that apelin may play a significant role in the regulation of the behavioral processes, endocrine secretion and the autonomic nervous system. In the behavioral responses evoked by apelin, the action of the dopaminergic neurons of the subcortical motor system and
Fig. 7. Effects of L-NAME, the CRH antagonist ␣-helical CRH9 – 41 (CRH a.) and NAP on the hyperthermic effect of apelin-13. Data are expressed as mean⫾S.E.M. Numbers in brackets are numbers of identical experiments. * P⬍0.05 for apelin-13 vs. control; # P⬍0.05 for NAP⫹apelin-13 vs. control.
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NO transmission might be involved. Apelin appears to activate the paraventricular CRH neurons, but dopaminergic transmission does not seem to play a significant role in its hypothalamic action. Further, our findings suggest that prostaglandins may relay the hyperthermic effect of apelin-13. Acknowledgments—This work was supported by grants from OTKA (T-037224), ETT (123-04), MTA-AKP (2000-114 3,2) and NKFP 1/027/2001.
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(Accepted 4 August 2004)
BEHAVIORAL, NEUROENDOCRINE AND THERMOREGULATORY ACTIONS OF APELIN-13 M. JÁSZBERÉNYI, E. BUJDOSÓ AND G. TELEGDY*
(Tatemoto et al., 1998). It is derived from a 77-amino-acid precursor, preproapelin, which is processed to several molecular forms in different tissues. Synthetic C-terminal fragments of preproapelin consisting of 13–19 amino acids were found to exhibit significantly higher activity at the receptor than that of apelin-36 (Tatemoto et al., 1998; Kawamata et al., 2001). The expression of the rat apelin receptor has been detected throughout the CNS, and especially in the limbic structures, the hypothalamus and the pituitary. In the hypothalamus, the most intense expression is in the supraoptic and paraventricular nuclei (De Mota et al., 2000; O’Carroll et al., 2000), which suggests a prominent role of apelin in the regulation of homeostatic, behavioral and endocrine processes. Some physiological effects of apelin have already been described: the administration of apelin-13 increased the water intake (Lee et al., 2000; Taheri et al., 2002) and it was demonstrated to have an impact on the blood pressure (Lee et al., 2000; Seyedabadi et al., 2002) and vasopressin release (Reaux et al., 2001; Taheri et al., 2002). The role of pyroglutamylated apelin-13 in the control of appetite and pituitary hormone release has also been revealed (Taheri et al., 2002). The hypothalamus, the major site of apelin-positive nerve fibers (Reaux et al., 2002), plays a central role in the control of neuroendocrine processes and homeostatic responses. Accordingly, in the present study, the effects of apelin on the hypothalamo–pituitary–adrenal (HPA) response and thermoregulation were examined. Since the limbic structures, which regulate behavior, also display a high expression of the apelin receptor (De Mota et al., 2000), its action on spontaneous locomotion and exploratory behavior was also investigated. Since the corticotropin-releasing hormone (CRH) is one of the most potent regulators of stress-related behavior (Menzaghi et al., 1994) and HPA activation (Vale et al., 1981), animals were pretreated with the CRH antagonist ␣-helical CRH9 – 41 in order to shed light on the involvement of CRH transmission in the behavioral and hormonal responses elicited by apelin. Strijbos et al. (1992) revealed the effects of CRH on thermogenesis and body temperature; therefore, we investigated the role of CRH in the apelin-induced hyperthermic response, too. Recent data provided evidence of the role of nitric oxide (NO) in the mediation of the action of apelin on blood pressure (Tatemoto et al., 2001). With the aim of investigating the regulatory role of NO in the neuroendocrine, behavioral and thermoregulatory responses evoked by apelin, therefore
Department of Pathophysiology, University of Szeged, Neurohumoral Research Group, Hungarian Academy of Sciences, Semmelweis u. 1, PO Box 427, H-6701, Szeged, Hungary
Abstract—As the distribution of apelinergic neurons in the brain suggests an important role of apelin-13 in the regulation of neuroendocrine processes, in the present experiments the effects of this recently identified neuropeptide on the open-field activity, the hypothalamo–pituitary–adrenal (HPA) system and the body temperature were investigated. I.c.v. administration of apelin-13 (1–10 g) to rats caused significant increases in square crossing, rearing, plasma corticosterone release and core temperature, whereas it did not influence the spontaneous motor activity during telemetric observation. To determine the mediation of the actions of apelin, a corticotropin-releasing hormone (CRH) antagonist, the nonselective dopamine antagonist haloperidol, the selective dopamine D1 receptor antagonist SCH-23390 and the nitric oxide synthase inhibitor N-nitro-L-arginine-methyl ester (L-NAME) were administered to the rats. The apelinevoked HPA activation was diminished by preadministration of the CRH antagonist, while the dopamine antagonist and L-NAME attenuated only the square crossing and rearing induced by apelin-13. To characterize the transmission of the thermoregulatory action of apelin, animals were pretreated either with L-NAME, the CRH antagonist or with the cyclooxygenase inhibitor noraminophenazone. L-NAME and the CRH antagonist did not cause significant inhibition of the apelinevoked increase in core temperature, while the cyclooxygenase inhibitor, applied 30 min before peptide treatment, did not prove effective in preventing the apelin-evoked thermoregulatory response, whereas when it was administered 2 h after the peptide treatment, it transiently and significantly reduced the hyperthermic response. The present data suggest that apelin-13 plays an important role in the regulation of behavioral, endocrine and homeostatic responses in the CNS, and dopamine, nitric oxide and prostaglandins seem to take part in the mediation of its effects. Since the corticosterone response could be blocked by the CRH antagonist, it is likely to be mediated through the activation of the CRH neurons. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: open-field behavior, HPA axis, telemetry.
The recently discovered peptide apelin has been demonstrated to be the endogenous ligand for an orphan G protein-coupled receptor, APJ, identified in a human gene by O’Dowd et al. (1993). The putative endogenous ligand apelin-36 was first isolated from bovine stomach extracts *Corresponding author. Tel: ⫹36-6254-5797; fax: ⫹36-6254-5710. E-mail address: [email protected] (G. Telegdy). Abbreviations: CRH, corticotropin-releasing hormone; HPA, hypothalamo-pituitary-adrenal; NAP, noraminophenazone; NO, nitric oxide; L-NAME, N-nitro-L-arginine-methyl ester; NOS, nitric oxide synthase.
0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.08.007
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Fig. 1. Apelin-13 evoked response in square crossing and rearing. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of animals used. * P⬍0.005 vs. control.
the effects of the NO synthase (NOS) inhibitor N-nitro-Larginine-methyl ester (L-NAME) were tested, too. Previous studies suggested that dopaminergic mediation might be involved in hyperlocomotion (Matsuzaki et al., 2002) and in the HPA response (Jezova et al., 1985). Hence, in a further set of experiments, to characterize dopaminergic mediation involved in the open-field behavior and HPA activation induced by apelin, animals were pretreated either with the nonselective dopamine antagonist haloperidol or with the D1 receptor-selective SCH 23390. Several publications have demonstrated that the prostaglandins play an indispensable role in the mediation of hyperthermic processes (Matsumura et al., 1990). Accordingly, by using the cyclooxygenase inhibitor noraminophenazone (NAP), we attempted to clarify the mediation of the thermoregulatory actions of apelin.
EXPERIMENTAL PROCEDURES Animals The animals were kept and handled during the experiments in accordance with the Council Directive of the European Economic Community regarding the protection of animals for experimental and other scientific purposes (86/609/EEC). Further, all efforts
Fig. 2. Effects of haloperidol (HAL) on apelin-induced response in square crossing and rearings. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of animals used. * P⬍0.05 vs. control.
Fig. 3. Effects of selective D1 receptor antagonist SCH-23390 on apelin-induced response in square crossing and rearings. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of animals used. * P⬍0.005 vs. control.
were made to minimize the number of animals used and their suffering. Male Wistar rats weighing 150 –250 g upon arrival were used. The rats were kept in their home cages at a constant room temperature on a standard illumination schedule with 12-h light/ dark period (lights on from 6.00 a.m.). Commercial food and tap water were available ad libitum. The rats were allowed a minimum of 1 week to acclimatize before surgery. To minimize the effects of nonspecific stress, the rats were handled daily.
Surgery For i.c.v. peptide administration, the rats were implanted with a stainless steel 20 G 1.5 Luer cannula (10 mm long) aimed at the right lateral cerebral ventricle under Nembutal (35 mg/kg, i.p.) anesthesia. The stereotaxic coordinates were 0.2 mm posterior; 1.7 mm lateral to the bregma; 3.7 mm deep from the dural surface, according to the atlas of Pellegrino et al. (1979). Cannulas were secured to the skull with dental cement and acrylate. The rats were used after a recovery period of at least 5 days. In the case of i.c.v. administration, Methylene Blue was injected into each decapitated head and the brains were dissected to verify the correct positioning and the permeability of the cannulas. Only data from rats in which the placement was correct were considered for the statistical evaluation. For implantation of an internal radio transmitter (E-Mitter), the rats were anesthetized with Nembutal (35 mg/kg, i.p.). The abdomen was opened by making a 2 cm midline incision along the linea alba. The E-Mitter was placed in the abdominal cavity along the sagittal plane in front of the caudal arteries and veins, but dorsal to the digestive organs. The abdom-
Fig. 4. Effects of i.c.v. administration of L-NAME on apelin-induced response in square crossing and plasma corticosterone. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of mice used. * P⬍0.05 vs. control.
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Table 1. Effects of i.c.v. administration of CRH antagonist ␣-helical CRH9 – 41 on apelin-induced behavioural responses (behavioural activity was characterized by the total number of explored squares or rearings/5 min test sessions)a
Locomotion Rearing
Control (8)
CRH antagonist (1 g) (8)
CRH antagonist⫹apelin (9)
Apelin (1 g) (10)
65.38⫾9.27 8.63⫾0.98
77.50⫾3.70 10.00⫾0.82
103.78⫾6.19* 14.33⫾1.32*
113.13⫾9.23* 17.00⫾2.14*
a Numbers in brackets are numbers of animals used. * P⬍0.05 vs. control.
inal opening was than closed with absorbable suture material, using a continuous interlocking running stitch, while the skin was closed with stainless steel suture material, using interrupted mattress stitches.
Treatment Protocol 1. Different doses of apelin-13 (Bachem, Bubendorf, Switzerland) dissolved in saline, or saline alone (control animals), in a volume of 2 l, were injected i.c.v. into conscious rats with a Hamilton microsyringe over 30 s, immobilization of the rats being avoided during handling. Protocol 2. For this experimental setting, animals were subjected to combined treatment with ␣-helical CRH9 – 41 (Bachem), the nonselective dopamine receptor blocker haloperidol (Richter, Budapest, Hungary), the dopamine D1 receptor-selective antagonist SCH 23390 (Tocris, Bristol, UK), the NOS inhibitor L-NAME (Sigma, Budapest, Hungary), the pyrazolone derivative NAP (Algopyrin; Chinoin, Budapest, Hungary) and apelin-13. L-NAME, ␣-helical CRH9 – 41 and SCH 23390 dissolved in 0.9% saline were injected i.c.v. Haloperidol (dissolved in 0.9% saline) was administered i.p. while NAP was injected i.m. in a volume of 0.5 ml. The concentrations of the antagonists or inhibitors were the concentrations that had proved most effective in previous experiments (Telegdy, 1984; Pataki et al., 1999; Bujdoso et al., 2001, 2003; Yamauchi et al., 2003), but which per se did not affect the behavioral paradigms. Thirty minutes after the pretreatment, the animals were treated i.c.v. with the dose of apelin-13 that had proved most effective in protocol 1. The control animals received saline alone. Thirty minutes after peptide administration, the animals were subjected to behavioral tests, or the plasma corticosterone level was measured, and the core temperature was recorded continuously. In a further set of experiments, animals were treated with NAP 2 h after the apelin injection.
Open-field test Thirty minutes after the apelin or vehicle administration, the behavioral paradigms were monitored over 5-min test sessions. The rats were removed from their home cages and placed in the center of an open-field box. The locomotor activity was analyzed by counting the number of passages from one to another of 36 equally sized (10⫻10 cm each) squares. The number of rearings was also recorded. The grooming frequency was characterized by the numbers of face washings, forepaw lickings or head strokings.
Determination of plasma corticosterone Thirty minutes after apelin treatment, the rats were killed by decapitation and approximately 3 ml blood was collected in heparinized tubes for corticosterone assay. The plasma corticosterone level was determined by fluorescence assay (Purves and Sirett, 1965).
Telemetry Apelin-13 was administered i.c.v. to the rats (9:00 a.m.) and the spontaneous motor activity and core temperature were recorded
continuously. The system uses E-Mitter, an implanted radiotelemetry device (Mini Mitter, Bend, OR, USA), to determine the animal temperature and motor activity data. The E-Mitter obtains power from a radiofrequency field produced by an energizer/receiver placed below the cage of the animals. The counts were recorded continuously and the output from the receivers was managed by VitalView, a Windows-based data acquisition system.
Statistical analysis Values are presented as means⫾S.E.M. Statistical analysis of the results was performed by analysis of variance, and differences between groups were examined by Tukey’s post hoc comparison test (Statistica 5.0). In the case of the telemetric observations the groups were compared at each point of time. A probability level of 0.05 was accepted as indicating a statistically significant difference.
RESULTS Effects of apelin-13 on open-field behavior and spontaneous locomotion The treatment with apelin-13 enhanced the numbers of both rearings and square crossings during the open-field exploration test (Fig. 1). The dose of 1 g increased the number of squares explored (F(3, 36)⫽7.24; P⬍0.005 vs. control), but further elevation of the dose (5 g) did not result in an additional increase. Apelin-13 also facilitated the rearing activity (Fig. 1): the most effective dose was 1 g (F(3, 36)⫽6.99; P⬍0.001 vs. control), and a higher dose of apelin-13 (5 g) proved less effective. As concerns grooming, apelin-13 did not evoke a significant response and neither of the applied doses (1 and 10 g) of the peptide altered the spontaneous motor activity during telemetric observation (data not shown). Effects of CRH antagonist, dopamine antagonist and L-NAME on behavioral responses evoked by apelin-13 Haloperidol pretreatment inhibited both the square crossing and the rearing response brought about by apelin (F(3, 39)⫽4.08; P⬍0.05 vs. control for square crossing and F(3, 39)⫽8.77; P⬍0.005 vs. control for rearing; Fig. 2). The selective D1 antagonist SCH 23390 completely abolished the behavioral responses induced by apelin-13 (F(3, 37)⫽6.30; P⬍0.005 vs. control for square crossing and F(3, 37)⫽10.59; P⬍0.0005 vs. control for rearing; Fig. 3). L-NAME also mitigated the apelin-evoked increases (F(3, 49)⫽9.68; P⬍0.005 vs. control for square crossing and F(3, 49)⫽5.30; P⬍0.05 vs. control for rearing) in square crossing and rearing (Fig. 4); however, the CRH antagonist ␣-helical CRH9 – 41 failed to modify the apelin-induced locomotor and rearing activity (Table 1).
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Fig. 5. Effects of apelin-13 on HPA response. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of animals used. * P⬍0.05 vs. control.
Fig. 6. Effects of i.c.v. administration of CRH antagonist ␣-helical CRH9 – 41 (CRH a.) on HPA response evoked by apelin-13. Data are expressed as mean⫾S.E.M. Figures within bars are numbers of rats used. * P⬍0.05 vs. control.
DISCUSSION
Effects of apelin-13 on HPA system Apelin-13 brought about a prominent activation of the HPA system (Fig. 5). In a dose of 10 g, apelin-13 elevated the plasma concentration of corticosterone by 153% as compared with the control (F(3, 22)⫽5.48; P⬍0.05 vs. control). Effects of CRH antagonist, haloperidol and L-NAME on HPA activation elicited by apelin-13 The CRH antagonist ␣-helical CRH9 – 41 produced inhibition of the apelin-evoked increase (F(3, 20)⫽3.80; P⬍0.05 vs. control) in corticosterone secretion (Fig. 6). On the other hand, the dopamine antagonist haloperidol and LNAME turned out to be ineffective on the HPA response elicited by apelin-13 (Table 2 and Table 3), and the antagonists did not cause further increase in the apelin-induced corticosterone level. Effects of CRH antagonist, L-NAME and NAP on hyperthermic effect of apelin-13 Apelin in a dose of 10 g increased the core temperature (F(4, 47)⫽4.74; P⬍0.005 vs. control; Fig. 7). Pretreatment with CRH antagonist and L-NAME did not modify the apelin-evoked hyperthermic effect. The cyclooxygenase inhibitor NAP, applied 30 min before peptide treatment, did not prove effective in reducing the apelin-evoked thermoregulatory response (data not shown). However, the administration of NAP 2 h after the i.c.v. apelin injection, significantly and transiently reduced the apelin-induced core temperature (P⬍0.05 vs. apelin).
The present experiments clearly demonstrate that apelin-13 leads to a marked activation of locomotion and rearing in a dose-dependent manner and the dose-response curves exhibit an inverted U-shape. Lee et al. (2000) demonstrated sequence and distribution similarities between apelin and angiotensin II, and our results revealed that the locomotor-activating effect of apelin resembles the action of angiotensin II (Braszko et al., 1988). Several studies have demonstrated that CRH acts as a mediator of behavioral processes (Bujdoso et al., 2001; Menzaghi et al., 1994), but in our experiment, CRH antagonist pretreatment failed to decrease the locomotor hyperactivity induced by apelin. Further experiments were therefore designed to shed light on the mediation of the behavioral responses brought about by apelin. Haloperidol pretreatment reduced the numbers of both the square crossing and of rearing, suggesting that the nigrostriatal system or mesolimbic structures such as the nucleus accumbens mediate the behavioral actions of the apelinergic neurons (Gysling and Wang, 1983; Johnson and North, 1992). In our experiments, SCH 23390, a selective dopamine receptor blocker, completely reduced the behavioral action of apelin-13, indicating an indispensable role of D1 receptors in these processes. Our findings are in harmony with those of previous behavioral studies (Hoffman and Beninger, 1985; Meyer et al., 1993), showing that D1 receptors play a noteworthy role in the control of locomotor activity and rearing. The results of the antagonist studies led to a critical role being ascribed to dopamine in the apelinevoked behavioral processes. We found that L-NAME pretreatment caused a moderate inhibition of the behavioral
Table 2. Effects of haloperidol on apelin-induced corticosterone level (corticosterone concentration is given in g/100 ml)a
Corticosterone level mean⫾S.E.M
Control (6)
Haloperidol (10 g) (6)
Haloperidol⫹apelin (6)
Apelin (10 g) (8)
11.08⫾0.70
12.53⫾2.57
18.72⫾1.82*
19.16⫾1.66*
a Numbers in brackets are numbers of animals used. * P⬍0.05 vs. control.
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Table 3. Effects of i.c.v. administration of L-NAME on apelin-induced corticosterone level (corticosterone concentration is given in g/100 ml)a
Corticosterone level mean⫾S.E.M
(20 g) (7)
Control (7)
L-NAME
9.84⫾0.77
13.09⫾1.87
L-NAME⫹apelin
(7) 22.99⫾3.04*
Apelin (10 g) (5) 18.40⫾0.76*
a Numbers in brackets are numbers of animals used. * P⬍0.05 vs. control.
responses elicited by apelin-13. Our data reinforce the hypothesis of the NO-dependent action of apelin (Tatemoto et al., 2001). On the other hand, apelin-13 did not influence the spontaneous locomotion during telemetric observations. The discrepancy observed between its effects on exploratory behavior and spontaneous locomotion corroborates the hypothesis that the motor responses are regulated by different mechanisms (Diamant and de Wied, 1991). The present data regarding apelin-evoked HPA activation lend support to the earlier conclusion (Taheri et al., 2002) that apelin has a marked impact on neuroendocrinological processes. The CRH antagonist diminished the corticosterone secretion evoked by apelin, but the role of other mediators (e.g. vasopressin) (Taheri et al., 2002) in the activation of the HPA axis cannot be excluded. In our experiments, the HPA activation evoked by apelin was not inhibited by haloperidol, which supports the view that the actions of apelin on the HPA system do not involve dopaminergic transmission (Amar et al., 1982; Bujdoso et al., 2003). The differences between the effective apelin dose on behavior (1 g) and plasma corticosterone (10 g), reflect the situation that distinct neural pathways may be involved in the regulation of exploratory behavior and HPA activation. Our data also demonstrate that apelin-13 increased the core temperature, which might indicate a physiological
role of the dense apelin-positive projections described in the preoptic region of the hypothalamus (Reaux et al., 2002). These findings also help to harmonize the behavioral observations, since, under resting conditions, the heat-conserving processes (curling-up, etc.) may interfere with a statistically significant locomotor response. There is evidence of several distinct pathways for the induction of fever, and it seems that CRH (Strijbos et al., 1992) acts to mediate cytokine-induced (Busbridge et al., 1989) fever. The cytokines increase the body temperature at least partially via the prostaglandins (Feldberg and Saxena, 1975). Further, the synthesis of NO has been regarded as an important step in thermoregulation (Monroy et al., 2001). In our experiment he NOS inhibitor L-NAME and the CRH antagonist did not cause significant inhibition in the apelinevoked increase in core temperature. The cyclooxygenase inhibitor significantly decreased the apelin-induced elevation of the core temperature, but this effect did not prove permanent. Our results reflect the fact that apelin increase the core temperature, probably in part, through the production of prostaglandins. In conclusion, our results, taken together with the histological data, indicate that apelin may play a significant role in the regulation of the behavioral processes, endocrine secretion and the autonomic nervous system. In the behavioral responses evoked by apelin, the action of the dopaminergic neurons of the subcortical motor system and
Fig. 7. Effects of L-NAME, the CRH antagonist ␣-helical CRH9 – 41 (CRH a.) and NAP on the hyperthermic effect of apelin-13. Data are expressed as mean⫾S.E.M. Numbers in brackets are numbers of identical experiments. * P⬍0.05 for apelin-13 vs. control; # P⬍0.05 for NAP⫹apelin-13 vs. control.
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NO transmission might be involved. Apelin appears to activate the paraventricular CRH neurons, but dopaminergic transmission does not seem to play a significant role in its hypothalamic action. Further, our findings suggest that prostaglandins may relay the hyperthermic effect of apelin-13. Acknowledgments—This work was supported by grants from OTKA (T-037224), ETT (123-04), MTA-AKP (2000-114 3,2) and NKFP 1/027/2001.
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(Accepted 4 August 2004)