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Effects of subtypes alpha- and beta-adrenoceptors of the lateral hypothalamus on the water and sodium intake induced by angiotensin II injected into the subfornical organ
Brain Research 881 (2000) 176–181 www.elsevier.com / locate / bres
Research report
Effects of subtypes alpha- and beta-adrenoceptors of the lateral hypothalamus on the water and sodium intake induced by angiotensin II injected into the subfornical organ a, ˜ Saad b , Luiz Antonio de Arruda Camargo *, Wilson Abrao a Gabriela Pavan de Arruda Camargo a
Department of Physiology, School of Dentistry, Paulista State University, UNESP, 1680 Humaita´ Street, Araraquara, Sao Paulo 14801 -903, Brazil b Department of Odontology, Taubate´ University, UNITAU, Taubabe´ , Sao Paulo, Brazil Accepted 8 August 2000
Abstract The present experiments were conducted to investigate the role of the a 1A -, a 1B , b 1 - and b 2 -adrenoceptors of the lateral hypothalamus (LH) on the water and salt intake responses elicited by subfornical organ (SFO) injection of angiotensin II (ANG II) in rats. 5-methylurapidil (an a 1A -adrenergic antagonist), cyclazosin (an a 1B -adrenergic antagonist) and ICI-118,551 (a b 2 -adrenergic antagonist) injected into the LH produced a dose-dependent reduction, whereas efaroxan (an a 2 -antagonist) increased the water intake induced by administration of ANG II into the SFO. These data show that injection of 5-methylurapidil into the LH prior to ANG II into the SFO increased the water and sodium intake induced by the injection of ANG II. The present data also show that atenolol (a b 1 -adrenergic antagonist), ICI-118,551, cyclazosin, or efaroxan injected into the LH reduced in a dose-dependent manner the water and sodium intake to angiotensinergic activation of SFO. Thus, the a 1 - and b-adrenoceptors of the LH are possibly involved with central mechanisms dependent on ANG II and SFO that control water and sodium intake. 2000 Elsevier Science B.V. All rights reserved. Theme: Neural bases of behaviors Topic: Ingestive behaviors Keywords: a-adrenergic antagonists; b-adrenergic antagonists; Water intake; Sodium intake; Subfornical organ; Lateral hypothalamus
1. Introduction It is well known that circulating angiotensin II (ANG II) has several physiological effects mediated through actions on the central nervous system (CNS) like water and sodium appetite [20,38]. Reductions of extracellular fluid induces an increase in water and sodium ingestion in part due to the actions of ANG II originated within the CNS [16,26] and are limited to stimulation of receptors that are situated in regions outside the blood–brain barrier, such as those in the circumventricular organs [12,33]. The subfornical organ (SFO) is a circumventricular structure that participates in the regulation of body fluid homeostasis *Corresponding author. Tel.: 155-16-201-6488; fax: 155-16-2224823. E-mail address: [email protected] (L.A.d.A. Camargo).
[25,37,43]. The lateral hypothalamus (LH) also is an area of CNS closely involved in the regulation of water and sodium [13,14,28]. It has been postulated that ANG II acts as a neuromodulator in the CNS because of its interactions with neurotransmitters, especially the catecholamines [9,31,34]. The extensive neural pathways from the SFO to the hypothalamus have been implicated in the control of body fluid intake regulation. Several lines of evidence indicate that fibers from the SFO converge on the nucleus medianus and also project to the supraoptic nucleus, paraventricular nucleus and throughout the lateral preoptic area–LH [36]. The central part of the SFO, which binds circulating ANG II [39], also contains an ANG II-immunoreactive terminal field that appears to arise from cells in the LH [24]. Considering the importance of the SFO and LH for the regulation of water and sodium in rats, the present
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L. A.d. A. Camargo et al. / Brain Research 881 (2000) 176 – 181
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experiments were designed to determine the participation of the a 1A , a 1B , b 1 , b 2 and a 2 -adrenoceptors of the LH in the thirst and sodium appetite induced by SFO application of ANG II.
2. Materials and methods
2.1. Animals Male Holtzman rats weighing 260–300 g at the beginning of the experiments were housed in individual metabolic cages. Standard Purina pellets (Na 1 content 5 nmols / 100 g), tap water and 3% NaCl solution were available ad libitum unless otherwise noted. Temperature in the animal colony was maintained at approximately 238C. The 12:12h light–dark cycle began with lights on at 7:00 A.M. All experiments began between 10:00 A.M. and 2:00 P.M.
2.2. Brain surgery After an acclimatization period of 7 days, the animals were maintained under tribromoethanol (Aldrich) (20 mg / 100 g b.wt., intraperitoneal [i.p.]) anesthesia throughout surgery. A stainless steel guide-cannula (1030.7 mm O.D.) was stereotaxically implanted into the brain with its opening protruding into the top of the SFO (coordinates: AP51.3 mm caudal to the bregma; V54.2 mm from the dura mater; L50.0 mm from the sagittal line). For LH cannulation the cannula (1430.7 mm O.D.) was positioned bilaterally as follows: AP51.6 mm posterior to the bregma; V57.5 mm below the dura mater; L51.5 mm from the sagittal midline. For cannula implantation the stereotaxic incisor bar was positioned 2.5 mm above the interaural line. The cannula was secured to the top of the skull with dental cement and fastened with two screws. The insertion of a close fitting stylet kept the lumen free of debris and clots. A prophylactic dose of penicillin (30.000 I.U.) was given intramuscularly (i.m.) presurgically.
Fig. 1. Photomicrograph of a hematoxylin-stained transverse section of the rat brain showing the site of injection into the SFO (arrow).
2.4. Drugs 5-methylurapidil, cyclazosin hydrochloride, efaroxan hydrochloride, (6)-atenolol, ICI-118,551 hydrochloride (RBI, Natick, MA, USA) and ANG II (Sigma Chemical Co., St. Louis, MO, USA).
2.5. Histology At the end of the experiments, the animals were anesthetized with ether and given a 2-ml injection of fast green dye via the intracranial cannula, followed by perfusion with saline and buffered formalin. The brains were removed, fixed in 10% formalin, frozen to 2258C and cut into 20–30-mm section. The presence or absence of dye in the ventricles was observed at this time. Only animals in which the presence of dye was noted to be restricted to the SFO and in which the injection was placed in the LH as described in the atlas of Paxinos [27] were used in this study. Figs. 1 and 2 present the site of the injection into the SFO and the LH, respectively.
2.3. Intracerebral injection techniques Single pulse intracranial injections were made after gently removing the animal from its cage, replacing the stylet with an injector that protruded 0.2 and 1.0 mm beyond the tip of the guide-cannula for SFO and LH, respectively, and was connected by PE-10 tubing to a 10-ml microsyringe, and injecting a total volume of 0.5 ml over a period of 30 s. Stylet and injector were always wiped with cotton soaked with 70% alcohol. After the injection, the injector was removed and replaced with the stylet, and the animal was returned to its cage for observation of its behavior.
Fig. 2. Photomicrograph of a hematoxylin-stained transverse section of the rat brain showing bilateral sites of injection into the LH (arrows).
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2.6. Statistical analysis The results are reported as mean6S.E.M. and were analyzed by one-way analysis of variance. Values were considered to be statistically significant when P,0.05. The Newman–Keuls post-hoc test was used to assess the difference between individual means.
2.7. Experimental protocol 2.7.1. Water or NaCl 3% intake Five days after brain surgery, different groups of animals for water or sodium intake were submitted to experimental sessions. Each animal was submitted to four or five experimental sessions at 3-day intervals. An aadrenergic antagonist (5-methylurapidil, cyclazosin or efaroxan) and the b-adrenergic antagonists (atenolol or ICI-118,551) were injected into the LH 20 min before injection of ANG II into the SFO. Recording of water or sodium intake started immediately after ANG II injection and continued for 4 h. Before the salt appetite test the next morning, the overnight water intake was measured, and graduated cylinders were replaced with glass burettes containing 3% NaCl. The burettes were calibrate to 0.1 ml and fitted with drinking spouts. The presence of sodium was announced to the drinking rats by sprinkling a few drops of the saline solution on their lips and whiskers.
Fig. 3. Effect of pretreatment with 5-methylurapidil, cyclazosin, and efaroxan into the LH on water intake induced by injection of ANG II into the SFO. Data are reported as mean6S.E.M. The number of animals is indicated at the top of each column. *P,0.05, compared with saline (control); 1 P,0.05, compared with ANG II.
3.2. Effects of 5 -methylurapidil, cyclazosin, efaroxan, atenolol, or ICI-118,551 into the LH on the sodium intake induced by ANG II injected into the SFO
3.1. Effects of 5 -methylurapidil, cyclazosin, efaroxan, atenolol, or ICI-118,551 into the LH on the water intake induced by ANG II injected into the SFO
Control rats (0.15 M NaCl into the SFO) consumed a mean amount of 1.360.4 ml of sodium over a period of 4 h. ANG II (10 pmol) injected into the SFO led to ingestion of 4.060.4 ml of sodium over the same period. Previous treatment with 5-methylurapidil (40 and 80 nmol) into the LH potentiated the effect on 3% NaCl intake induced by ANG II injected into the SFO [F(3.33)55.00, P,0.05]. Pretreatment with cyclazosin and efaroxan (40 and 80 nmol) into the LH reduced the sodium intake induced by ANG II [F(3.34)54.70, P,0.05 and F(3.35)57.30, P, 0.05, respectively] (Fig. 5). Previous treatment with atenolol and ICI-118,551 (40 and 80 nmol) into the LH
The water intake observed during a period of 4 h in the control experiment (0.15 M NaCl into the SFO) was 3.360.3 ml. Injection of ANG II (10 pmol) into the SFO produced an increase in water ingestion (12.961.6 ml / 2 h). Previous injection of 5-methylurapidil (20, 40, and 80 nmol) and cyclazosin (40 and 80 nmol) into the LH decreased the water intake induced by ANG II administration into the SFO [F (3,30)517.16, P,0.05 and F (3.35)517.45, P,0.05, respectively], whereas efaroxan (40 and 80 nmol) increased this effect [F(3.34)53.33, P,0.05] (Fig. 3). Previous treatment with ICI-118,551 (40 and 80 nmol) into the LH elicits a potentiation in water intake induced by ANG II injected into the SFO [F(3.33)5 23.96, P,0.05]. No changes in the dipsogenic effect of ANG II were observed after previous treatment with atenolol [F(3.33)51.03, P.0.05]. (Fig. 4). Injection of only 5-methylurapidil, cyclazosin, efaroxan, atenolol, or ICI-118,551 into the LH produced no alterations in water intake.
Fig. 4. Effect of pretreatment with atenolol and ICI-118,551 into the LH on water intake induced by injection of ANG II into the SFO. Data are reported as mean6S.E.M. The number of animals is indicated at the top of each column. *P,0.05, compared with saline (control); 1 P,0.05, compared with ANG II.
3. Results
L. A.d. A. Camargo et al. / Brain Research 881 (2000) 176 – 181
Fig. 5. Effect of pretreatment with 5-methylurapidil, cyclazosin, and efaroxan into the LH on sodium intake induced by injection of ANG II into the SFO. Data are reported as mean6S.E.M. The number of animals is indicated at the top of each column. *P,0.05, compared with saline (control); 1 P,0.05, compared with ANG II.
antagonized the sodium intake induced by ANG II administration into the SFO [F(3.33)511,73, P,0.05 and F(3.34)59.50, P,0.05, respectively] (Fig. 6). Injection of only 5-methylurapidil, cyclazosin, efaroxan, atenolol, or ICI-118,551 into the LH produced no alterations in sodium intake.
4. Discussion The present results show that the a 1A -adrenoceptor antagonist 5-methyl urapidil, the a 1B -adrenoceptor antagonist cyclazosin and the b 2 -adrenoceptor antagonist ICI118,551 injected into the LH reduces, whereas the a 2 adrenoceptor antagonist efaroxan increases the water intake induced by ANG II injected into the SFO.
Fig. 6. Effect of pretreatment with atenolol and ICI-118,551 into the LH on sodium intake induced by injection of ANG II into the SFO. Data are reported as mean6S.E.M. The number of animals is indicated at the top of each column. *P,0.05, compared with saline (control); 1 P,0.05, compared with ANG II.
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Several lines of evidence suggest that the adrenergic receptors in the LH are involved in the control of water intake [13,14,18,21,22]. A role of forebrain catecholaminergic systems in the mediation of ANG II-elicited drinking and blood pressure responses has also been suggested [4–6,32]. Interaction of ANG II with brain angiotensinergic receptors initiates a cascade of cellular events that results in stimulation of the NET system and catecholaminergic synthesis and turnover [1,30,35]. The central noradrenergic system has different effects on central ANG II-inducing drinking behavior. Water intake induced by central administration of ANG II is inhibited by a 1 -, a 2 - or b-antagonists depending on the site of injection. Prazosin and phentolamine, but not yohimbine, injected into the rostral hypothalamus reduced the water intake induced by intracerebroventricular (icv) ANG II [19]. The previous administration of prazosin and propranolol into the medial preoptic area (MPOA) inhibits the dipsogenic effect of ANG II injected into the same area [3]. Bilateral lesions of the LH reduced the dipsogenic effects of ANG II and noradrenaline injected into the median preoptic area (MnPO) [28]. Prazosin and yohimbine injected into the MnPO reduced the water intake induced by ANG II [29]. More recently it was demonstrated that the pretreatment with prazosin and propranolol into the LH enhanced, whereas yohimbine antagonized the water intake produced by ANG II injected into the SFO [7]. Taken together, these results show that the central injection of an a or b-adrenergic antagonist can disrupt the dipsogenic effect of ANG II in rats and suggest that the adrenergic pathways of LH can produce a dual effect on water intake. The release of noradrenaline may be an excitatory step along the ANG II-inducing thirst pathway and if we use an adrenergic antagonist we can block the dipsogenic response. In spite of this excitatory effect, the adrenergic pathways could also be involved in an inhibitory system for water intake. The present results show that the blockade of the a 1A -, a 1B -, or b 2 -adrenergic receptors into the LH increases, whereas the blockade of the a 2 adrenergic receptors decreases the water intake induced by ANG II. Recent data demonstrated that the action of noradrenaline on hypothalamus-brain stem areas of rats brain on angiotensinergic receptors (AT 1 receptors) [42] involves the a 1A -adrenergic receptors [40] and pharmacological findings indicate that activation of postsynaptic a 1 -adrenoceptors potentiates b-adrenoceptors [15]. The present data also show that 5-methylurapidil injected into the LH causes an increase, whereas cyclazosin, atenolol, ICI-118,551 and efaroxan causes a decrease in sodium intake induced by administration of ANG II into the SFO. Saline intake is also inhibited [11,40] or activated by noradrenaline. Thus, this double mediation of the control of water and sodium intake by noradrenaline is explained by the dual-role hypothesis which proposes that central noradrenaline participates in the inhibition and activation
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of both behaviors [10,14,41]. A relationship between noradrenaline and ANG II facilitation of sodium intake was first shown by Chiaraviglio and Taleisnik [8], who obtained 1% NaCl intake in normovolemic animals with noradrenaline injected into the third ventricle close to the columns of the fornix. Other authors reported the same kind of increase after noradrenaline was injected icv [2] or in the paraventricular nucleus [23]. However, it has been proposed [17] that the organum vasculosum laminae terminalis (OVLT) is the circumventricular organ site for a peripheral action of salt appetite because infusions of ANG II into the OVLT, but no SFO, elicit salt appetite and because lesions of the OVLT suppress salt appetite aroused either by a low dose of oral captopril or by sodium depletion. On the other hand, lesions of the SFO reduce salt appetite aroused by sodium depletion [38,43]. The injection procedure to localize brain areas receptive for the effects of ANG II on water and sodium intake has some limitations. A major problem is diffusion. Due to dose and volume utilized in the present work, it is possible that when ANG II was administered into the SFO, neurons in the MnPO as well as neurons in the adjacent periventricular parts of the SFO and the OVLT were activated as a result of the hormone being transported to these sites by cerebroventricular systems. The present results demonstrate that injection of 5methylurapidil, cyclazosin or ICI-118,551 into the LH reduces, whereas efaroxan increases the water intake induced by ANG II administration into the SFO. Previous treatment with 5-methylurapidil into the LH increases, whereas pretreatment with cyclazosin, atenolol, ICI118,551 or efaroxan reduces the salt ingestion produced by ANG II administration into the SFO. These findings show that the water and sodium intakes to ANG II injected into the SFO of rats are mediated by a 1A -, a 1B -, and a 2 as well as b 1 - and b 2 -adrenoceptor subtypes.
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The authors appreciate the technical assistance of Re´ Silas P. Barbosa, Silvia Foglia ´ ginaldo C. Queiroz, and Silvana A. D. Malavolta. They also thank Ana V. Oliveira for animal care. Research supported by CNPq and FAPESP.
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Research report
Effects of subtypes alpha- and beta-adrenoceptors of the lateral hypothalamus on the water and sodium intake induced by angiotensin II injected into the subfornical organ a, ˜ Saad b , Luiz Antonio de Arruda Camargo *, Wilson Abrao a Gabriela Pavan de Arruda Camargo a
Department of Physiology, School of Dentistry, Paulista State University, UNESP, 1680 Humaita´ Street, Araraquara, Sao Paulo 14801 -903, Brazil b Department of Odontology, Taubate´ University, UNITAU, Taubabe´ , Sao Paulo, Brazil Accepted 8 August 2000
Abstract The present experiments were conducted to investigate the role of the a 1A -, a 1B , b 1 - and b 2 -adrenoceptors of the lateral hypothalamus (LH) on the water and salt intake responses elicited by subfornical organ (SFO) injection of angiotensin II (ANG II) in rats. 5-methylurapidil (an a 1A -adrenergic antagonist), cyclazosin (an a 1B -adrenergic antagonist) and ICI-118,551 (a b 2 -adrenergic antagonist) injected into the LH produced a dose-dependent reduction, whereas efaroxan (an a 2 -antagonist) increased the water intake induced by administration of ANG II into the SFO. These data show that injection of 5-methylurapidil into the LH prior to ANG II into the SFO increased the water and sodium intake induced by the injection of ANG II. The present data also show that atenolol (a b 1 -adrenergic antagonist), ICI-118,551, cyclazosin, or efaroxan injected into the LH reduced in a dose-dependent manner the water and sodium intake to angiotensinergic activation of SFO. Thus, the a 1 - and b-adrenoceptors of the LH are possibly involved with central mechanisms dependent on ANG II and SFO that control water and sodium intake. 2000 Elsevier Science B.V. All rights reserved. Theme: Neural bases of behaviors Topic: Ingestive behaviors Keywords: a-adrenergic antagonists; b-adrenergic antagonists; Water intake; Sodium intake; Subfornical organ; Lateral hypothalamus
1. Introduction It is well known that circulating angiotensin II (ANG II) has several physiological effects mediated through actions on the central nervous system (CNS) like water and sodium appetite [20,38]. Reductions of extracellular fluid induces an increase in water and sodium ingestion in part due to the actions of ANG II originated within the CNS [16,26] and are limited to stimulation of receptors that are situated in regions outside the blood–brain barrier, such as those in the circumventricular organs [12,33]. The subfornical organ (SFO) is a circumventricular structure that participates in the regulation of body fluid homeostasis *Corresponding author. Tel.: 155-16-201-6488; fax: 155-16-2224823. E-mail address: [email protected] (L.A.d.A. Camargo).
[25,37,43]. The lateral hypothalamus (LH) also is an area of CNS closely involved in the regulation of water and sodium [13,14,28]. It has been postulated that ANG II acts as a neuromodulator in the CNS because of its interactions with neurotransmitters, especially the catecholamines [9,31,34]. The extensive neural pathways from the SFO to the hypothalamus have been implicated in the control of body fluid intake regulation. Several lines of evidence indicate that fibers from the SFO converge on the nucleus medianus and also project to the supraoptic nucleus, paraventricular nucleus and throughout the lateral preoptic area–LH [36]. The central part of the SFO, which binds circulating ANG II [39], also contains an ANG II-immunoreactive terminal field that appears to arise from cells in the LH [24]. Considering the importance of the SFO and LH for the regulation of water and sodium in rats, the present
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experiments were designed to determine the participation of the a 1A , a 1B , b 1 , b 2 and a 2 -adrenoceptors of the LH in the thirst and sodium appetite induced by SFO application of ANG II.
2. Materials and methods
2.1. Animals Male Holtzman rats weighing 260–300 g at the beginning of the experiments were housed in individual metabolic cages. Standard Purina pellets (Na 1 content 5 nmols / 100 g), tap water and 3% NaCl solution were available ad libitum unless otherwise noted. Temperature in the animal colony was maintained at approximately 238C. The 12:12h light–dark cycle began with lights on at 7:00 A.M. All experiments began between 10:00 A.M. and 2:00 P.M.
2.2. Brain surgery After an acclimatization period of 7 days, the animals were maintained under tribromoethanol (Aldrich) (20 mg / 100 g b.wt., intraperitoneal [i.p.]) anesthesia throughout surgery. A stainless steel guide-cannula (1030.7 mm O.D.) was stereotaxically implanted into the brain with its opening protruding into the top of the SFO (coordinates: AP51.3 mm caudal to the bregma; V54.2 mm from the dura mater; L50.0 mm from the sagittal line). For LH cannulation the cannula (1430.7 mm O.D.) was positioned bilaterally as follows: AP51.6 mm posterior to the bregma; V57.5 mm below the dura mater; L51.5 mm from the sagittal midline. For cannula implantation the stereotaxic incisor bar was positioned 2.5 mm above the interaural line. The cannula was secured to the top of the skull with dental cement and fastened with two screws. The insertion of a close fitting stylet kept the lumen free of debris and clots. A prophylactic dose of penicillin (30.000 I.U.) was given intramuscularly (i.m.) presurgically.
Fig. 1. Photomicrograph of a hematoxylin-stained transverse section of the rat brain showing the site of injection into the SFO (arrow).
2.4. Drugs 5-methylurapidil, cyclazosin hydrochloride, efaroxan hydrochloride, (6)-atenolol, ICI-118,551 hydrochloride (RBI, Natick, MA, USA) and ANG II (Sigma Chemical Co., St. Louis, MO, USA).
2.5. Histology At the end of the experiments, the animals were anesthetized with ether and given a 2-ml injection of fast green dye via the intracranial cannula, followed by perfusion with saline and buffered formalin. The brains were removed, fixed in 10% formalin, frozen to 2258C and cut into 20–30-mm section. The presence or absence of dye in the ventricles was observed at this time. Only animals in which the presence of dye was noted to be restricted to the SFO and in which the injection was placed in the LH as described in the atlas of Paxinos [27] were used in this study. Figs. 1 and 2 present the site of the injection into the SFO and the LH, respectively.
2.3. Intracerebral injection techniques Single pulse intracranial injections were made after gently removing the animal from its cage, replacing the stylet with an injector that protruded 0.2 and 1.0 mm beyond the tip of the guide-cannula for SFO and LH, respectively, and was connected by PE-10 tubing to a 10-ml microsyringe, and injecting a total volume of 0.5 ml over a period of 30 s. Stylet and injector were always wiped with cotton soaked with 70% alcohol. After the injection, the injector was removed and replaced with the stylet, and the animal was returned to its cage for observation of its behavior.
Fig. 2. Photomicrograph of a hematoxylin-stained transverse section of the rat brain showing bilateral sites of injection into the LH (arrows).
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2.6. Statistical analysis The results are reported as mean6S.E.M. and were analyzed by one-way analysis of variance. Values were considered to be statistically significant when P,0.05. The Newman–Keuls post-hoc test was used to assess the difference between individual means.
2.7. Experimental protocol 2.7.1. Water or NaCl 3% intake Five days after brain surgery, different groups of animals for water or sodium intake were submitted to experimental sessions. Each animal was submitted to four or five experimental sessions at 3-day intervals. An aadrenergic antagonist (5-methylurapidil, cyclazosin or efaroxan) and the b-adrenergic antagonists (atenolol or ICI-118,551) were injected into the LH 20 min before injection of ANG II into the SFO. Recording of water or sodium intake started immediately after ANG II injection and continued for 4 h. Before the salt appetite test the next morning, the overnight water intake was measured, and graduated cylinders were replaced with glass burettes containing 3% NaCl. The burettes were calibrate to 0.1 ml and fitted with drinking spouts. The presence of sodium was announced to the drinking rats by sprinkling a few drops of the saline solution on their lips and whiskers.
Fig. 3. Effect of pretreatment with 5-methylurapidil, cyclazosin, and efaroxan into the LH on water intake induced by injection of ANG II into the SFO. Data are reported as mean6S.E.M. The number of animals is indicated at the top of each column. *P,0.05, compared with saline (control); 1 P,0.05, compared with ANG II.
3.2. Effects of 5 -methylurapidil, cyclazosin, efaroxan, atenolol, or ICI-118,551 into the LH on the sodium intake induced by ANG II injected into the SFO
3.1. Effects of 5 -methylurapidil, cyclazosin, efaroxan, atenolol, or ICI-118,551 into the LH on the water intake induced by ANG II injected into the SFO
Control rats (0.15 M NaCl into the SFO) consumed a mean amount of 1.360.4 ml of sodium over a period of 4 h. ANG II (10 pmol) injected into the SFO led to ingestion of 4.060.4 ml of sodium over the same period. Previous treatment with 5-methylurapidil (40 and 80 nmol) into the LH potentiated the effect on 3% NaCl intake induced by ANG II injected into the SFO [F(3.33)55.00, P,0.05]. Pretreatment with cyclazosin and efaroxan (40 and 80 nmol) into the LH reduced the sodium intake induced by ANG II [F(3.34)54.70, P,0.05 and F(3.35)57.30, P, 0.05, respectively] (Fig. 5). Previous treatment with atenolol and ICI-118,551 (40 and 80 nmol) into the LH
The water intake observed during a period of 4 h in the control experiment (0.15 M NaCl into the SFO) was 3.360.3 ml. Injection of ANG II (10 pmol) into the SFO produced an increase in water ingestion (12.961.6 ml / 2 h). Previous injection of 5-methylurapidil (20, 40, and 80 nmol) and cyclazosin (40 and 80 nmol) into the LH decreased the water intake induced by ANG II administration into the SFO [F (3,30)517.16, P,0.05 and F (3.35)517.45, P,0.05, respectively], whereas efaroxan (40 and 80 nmol) increased this effect [F(3.34)53.33, P,0.05] (Fig. 3). Previous treatment with ICI-118,551 (40 and 80 nmol) into the LH elicits a potentiation in water intake induced by ANG II injected into the SFO [F(3.33)5 23.96, P,0.05]. No changes in the dipsogenic effect of ANG II were observed after previous treatment with atenolol [F(3.33)51.03, P.0.05]. (Fig. 4). Injection of only 5-methylurapidil, cyclazosin, efaroxan, atenolol, or ICI-118,551 into the LH produced no alterations in water intake.
Fig. 4. Effect of pretreatment with atenolol and ICI-118,551 into the LH on water intake induced by injection of ANG II into the SFO. Data are reported as mean6S.E.M. The number of animals is indicated at the top of each column. *P,0.05, compared with saline (control); 1 P,0.05, compared with ANG II.
3. Results
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Fig. 5. Effect of pretreatment with 5-methylurapidil, cyclazosin, and efaroxan into the LH on sodium intake induced by injection of ANG II into the SFO. Data are reported as mean6S.E.M. The number of animals is indicated at the top of each column. *P,0.05, compared with saline (control); 1 P,0.05, compared with ANG II.
antagonized the sodium intake induced by ANG II administration into the SFO [F(3.33)511,73, P,0.05 and F(3.34)59.50, P,0.05, respectively] (Fig. 6). Injection of only 5-methylurapidil, cyclazosin, efaroxan, atenolol, or ICI-118,551 into the LH produced no alterations in sodium intake.
4. Discussion The present results show that the a 1A -adrenoceptor antagonist 5-methyl urapidil, the a 1B -adrenoceptor antagonist cyclazosin and the b 2 -adrenoceptor antagonist ICI118,551 injected into the LH reduces, whereas the a 2 adrenoceptor antagonist efaroxan increases the water intake induced by ANG II injected into the SFO.
Fig. 6. Effect of pretreatment with atenolol and ICI-118,551 into the LH on sodium intake induced by injection of ANG II into the SFO. Data are reported as mean6S.E.M. The number of animals is indicated at the top of each column. *P,0.05, compared with saline (control); 1 P,0.05, compared with ANG II.
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Several lines of evidence suggest that the adrenergic receptors in the LH are involved in the control of water intake [13,14,18,21,22]. A role of forebrain catecholaminergic systems in the mediation of ANG II-elicited drinking and blood pressure responses has also been suggested [4–6,32]. Interaction of ANG II with brain angiotensinergic receptors initiates a cascade of cellular events that results in stimulation of the NET system and catecholaminergic synthesis and turnover [1,30,35]. The central noradrenergic system has different effects on central ANG II-inducing drinking behavior. Water intake induced by central administration of ANG II is inhibited by a 1 -, a 2 - or b-antagonists depending on the site of injection. Prazosin and phentolamine, but not yohimbine, injected into the rostral hypothalamus reduced the water intake induced by intracerebroventricular (icv) ANG II [19]. The previous administration of prazosin and propranolol into the medial preoptic area (MPOA) inhibits the dipsogenic effect of ANG II injected into the same area [3]. Bilateral lesions of the LH reduced the dipsogenic effects of ANG II and noradrenaline injected into the median preoptic area (MnPO) [28]. Prazosin and yohimbine injected into the MnPO reduced the water intake induced by ANG II [29]. More recently it was demonstrated that the pretreatment with prazosin and propranolol into the LH enhanced, whereas yohimbine antagonized the water intake produced by ANG II injected into the SFO [7]. Taken together, these results show that the central injection of an a or b-adrenergic antagonist can disrupt the dipsogenic effect of ANG II in rats and suggest that the adrenergic pathways of LH can produce a dual effect on water intake. The release of noradrenaline may be an excitatory step along the ANG II-inducing thirst pathway and if we use an adrenergic antagonist we can block the dipsogenic response. In spite of this excitatory effect, the adrenergic pathways could also be involved in an inhibitory system for water intake. The present results show that the blockade of the a 1A -, a 1B -, or b 2 -adrenergic receptors into the LH increases, whereas the blockade of the a 2 adrenergic receptors decreases the water intake induced by ANG II. Recent data demonstrated that the action of noradrenaline on hypothalamus-brain stem areas of rats brain on angiotensinergic receptors (AT 1 receptors) [42] involves the a 1A -adrenergic receptors [40] and pharmacological findings indicate that activation of postsynaptic a 1 -adrenoceptors potentiates b-adrenoceptors [15]. The present data also show that 5-methylurapidil injected into the LH causes an increase, whereas cyclazosin, atenolol, ICI-118,551 and efaroxan causes a decrease in sodium intake induced by administration of ANG II into the SFO. Saline intake is also inhibited [11,40] or activated by noradrenaline. Thus, this double mediation of the control of water and sodium intake by noradrenaline is explained by the dual-role hypothesis which proposes that central noradrenaline participates in the inhibition and activation
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of both behaviors [10,14,41]. A relationship between noradrenaline and ANG II facilitation of sodium intake was first shown by Chiaraviglio and Taleisnik [8], who obtained 1% NaCl intake in normovolemic animals with noradrenaline injected into the third ventricle close to the columns of the fornix. Other authors reported the same kind of increase after noradrenaline was injected icv [2] or in the paraventricular nucleus [23]. However, it has been proposed [17] that the organum vasculosum laminae terminalis (OVLT) is the circumventricular organ site for a peripheral action of salt appetite because infusions of ANG II into the OVLT, but no SFO, elicit salt appetite and because lesions of the OVLT suppress salt appetite aroused either by a low dose of oral captopril or by sodium depletion. On the other hand, lesions of the SFO reduce salt appetite aroused by sodium depletion [38,43]. The injection procedure to localize brain areas receptive for the effects of ANG II on water and sodium intake has some limitations. A major problem is diffusion. Due to dose and volume utilized in the present work, it is possible that when ANG II was administered into the SFO, neurons in the MnPO as well as neurons in the adjacent periventricular parts of the SFO and the OVLT were activated as a result of the hormone being transported to these sites by cerebroventricular systems. The present results demonstrate that injection of 5methylurapidil, cyclazosin or ICI-118,551 into the LH reduces, whereas efaroxan increases the water intake induced by ANG II administration into the SFO. Previous treatment with 5-methylurapidil into the LH increases, whereas pretreatment with cyclazosin, atenolol, ICI118,551 or efaroxan reduces the salt ingestion produced by ANG II administration into the SFO. These findings show that the water and sodium intakes to ANG II injected into the SFO of rats are mediated by a 1A -, a 1B -, and a 2 as well as b 1 - and b 2 -adrenoceptor subtypes.
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The authors appreciate the technical assistance of Re´ Silas P. Barbosa, Silvia Foglia ´ ginaldo C. Queiroz, and Silvana A. D. Malavolta. They also thank Ana V. Oliveira for animal care. Research supported by CNPq and FAPESP.
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