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Effects of angiotensin and vasopressin V1 receptors on water and sodium intake induced by injection of vasopressin into lateral septal area
Regulatory Peptides 118 (2004) 159 – 164 www.elsevier.com/locate/regpep
Effects of angiotensin and vasopressin V1 receptors on water and sodium intake induced by injection of vasopressin into lateral septal area E.G.O. Mima a, R.R. Andrade b, L.A.A. Camargo b,*, W.A. Saad b a
b
Department of Dental Materials and Prosthodontics, School of Dentistry, Paulista State University, UNESP, Araraquara, SP, Brazil Department of Physiology and Pathology, School of Dentistry, Paulista State University, UNESP, Rua Humaita´, 1680, Araraquara, SP 14801-903, Brazil Received 6 August 2003; accepted 23 December 2003
Abstract The specific arginine8-vasopressin (AVP) V1 receptors antagonist (AAVP) was injected (20, 40 and 80 nmol) into the lateral septal area (LSA) to determine the effects of selective septal V1 receptor on water and 3% sodium intake in rats. Was also observed the effects of losartan and CGP42112A (select ligands of the AT1 and AT2 ANG II receptors, respectively) injected into LSA prior AVP on the same appetites. Twenty-four hours before the experiments, the rats were deprived of water. The volume of drug solution injected was 0.5 Al. Water and sodium intake were measured at 0.25, 0.5, 1.0 and 2.0 h. Injection of AVP reduced the water and sodium ingestion vs. control (0.15 M saline). Pre-treatment with AAVP (40, 80 and 160 nmol) did not alter the decrease in the water ingestion induced by AVP, whereas AAVP abolished the action of AVP-induced sodium intake. Losartan (40, 80 and 160 nmol) did not alter the effect of AVP on water and sodium intake, whereas CGP42112A (20, 40 and 60 nmol) at the first 30 min increased water ingestion. Losartan and CGP42112A together increased the actions of AVP, showing more pronounced effects than when the two antagonists were injected alone. The results showed that AVP inhibited the appetites and these effects were increased by the AAVP. The involvement of angiotensinergic receptors in the effects of AVP is also suggested. D 2004 Elsevier B.V. All rights reserved. Keywords: AVP; AAVP; AT1 receptor; AT2 receptor; Water intake; Sodium intake
1. Introduction The neuronal peptide arginine8-vasopressin (AVP) has an important role in the regulation of the hydrolic balance. Many papers show the presence of vasopressinergic innervation at the central nervous system (CNS) [1– 4], including septal area (SA) [5 –7]. The pharmacologic properties of the AVP receptors at the SA are similar to V1 peripheric receptors [8– 10]. The administration of d(CH2)5Tyr(Me)AVP (AAVP) at CNS has shown a stronger antagonist on the AVP V1 receptors [4,11,12]. The systemic or central administration of angiotensin II (ANG II) increases the secretion of AVP [13,14]. In regard to water intake, sodium appetite and secretion of AVP, in
* Corresponding author. Tel./fax: +55-16-201-6488. E-mail address: [email protected] (L.A.A. Camargo). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2003.12.006
response to intracerebroventricular (i.c.v.) administration of ANG II, many studies have demonstrated that the administration of losartan, ANG II AT1 receptor antagonist, blocks this answers in rats [15,16]. Studies with central administration of AT2 antagonists (PD123319, PD123177 and CGP42112A) showed many answers in regard to water and sodium intake. Some investigators observed inhibitory influences after administration AT2 blockers i.c.v. on water and sodium intake and AVP release in rats [16 – 19]. The reason for different effects of some AT2 antagonists in rats still has not been explained. However, it has been suggested that the inhibitory effects of high doses of these antagonists applied i.c.v. can be due to an action at the central AT1 receptors or an ‘‘in vivo’’ conversion in AT1 antagonists [18]. It has also been suggested that other subtypes of AT2 receptors, sensitive to an antagonist, can exist in the brain [20]. At the present investigation, we studied the effect of AVP V1 receptors antagonist, as well as AT1 an AT2 receptors
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of AVP into the same area. Water and sodium intake was studied in different experiments and in several groups of animals, after antagonists and AVP injection into LSA, as shown below:
Fig. 1. Photomicrograph of a hematoxylin-stained section of the rat brain showing the site of injection into the LSA (arrow).
antagonists found in the lateral septal area (LSA), on thirst and sodium appetite by administration of AVP at this same area.
U Control (0.15 M NaCl), U AVP (20, 40 and 80 nmol), U d(CH2)5Tyr(Me)AVP (40, 80 and 160 nmol) + AVP (40 nmol), U Losartan (40, 80 and 160 nmol) + AVP (40 nmol), U CGP42112A (20, 40, 60 and 80 nmol) + AVP (40 nmol), U Losartan (40, 80 and 160 nmol) + CGP42112A (20, 40 and 60 nmol) + AVP (40 nmol). Water and sodium intake was recorded over a 2-h period (one sample each at 0.25, 0.5, 1 and 2 h), using a burette with 0.1-ml divisions fitted with a metal spout for liquid ingestion.
2. Materials and methods 2.1. Intracranial cannulation Male Holtzman rats weighing 250– 300 g were used in all experiments. The rats were individually housed in a temperature-controlled room (23 F 2 jC) with a 12:12h light/dark cycle. Food and water were available ad libitum except as noted. Stainless steel cannulae (0.6 mm outer diameter, 0.33 mm inner diameter) were implanted into the ASL of each animal under 2,2,2-tribromoethanol anesthesia according to standard stereotaxic techniques based on the following coordinates: 0.8 mm rostral to the bregma, 0.8 mm lateral to the sagittal line and 4.2 mm below the duramater. Cannulae were attached to the skull using acrylic cement and small screws. Insertion of a close-fitting stylet kept the lumen free from debris and clots. After brain surgery, the animals received a dose of 50 000 U of penicillin and were returned to individual metabolic cages, with free access to granular ration, tap water and 3% NaCl solution for 1 week until the day of experiment. A dental needle was connected by PE tubing to a Hamilton-type syringe (10 Al) kept outside the cage. All drugs were injected in 0.5 Al volumes over 10 –15 s. 2.2. Drugs AVP (Sigma), losartan (DuPont Merck), CGP42112A (RBI) and d(CH2)5Tyr(Me)AVP (Bachem). 2.3. Procedures Food, water and 3% NaCl solution were removed 24 h before the intracranial injection for sodium intake register. For water intake register, only liquids were removed. Antagonists were injected into LSA 15 min before injection
Fig. 2. The water intake responses of rats injected with AVP into the LSA (A) and rats pre-treated with AAVP (B). Results are reported as mean F S.E.M. N = number of animals. *P < 0.05 compared with saline injection.
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2.4. Brain histology At the end of the experiments, the rats were anesthetized with ether and perfused though the heart with 10% formalin. The brains were removed, stored in 10% formalin for 1 week, frozen and cut. Coronal section (20 Am) was stained with hematoxylin and eosin for analysis by light microscopy to confirm the position of injection. Only the results obtained from rats with typical injection into the LSA were used (Fig. 1). 2.5. Statistics Data are reported as means F S.E.M. Analysis of variance and Newmans– Keuls test were used to test statistical significance. Differences were considered significant at P < 0.005.
Fig. 3. The water intake responses of rats injected with antagonists losartan (A), CGP42112A (B) and both (C) previous to AVP. Results are reported as mean F S.E.M. N = number of animals. *P < 0.05 compared with saline injection. +P < 0.05 compared with AVP 40 nmol.
Fig. 4. The 3% NaCl intake responses of rats injected with AVP into the LSA (A) and rats pre-treated with AAVP (B). Results are reported as mean F S.E.M. N = number of animals. *P < 0.05 compared with saline injection. +P < 0.05 compared with AVP 40 nmol.
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3. Results The water intake responses of rats injected with AVP into the LSA and rats pre-treated with AAVP are summarized in Fig. 2. Rats submitted to AVP injection showed a significant reduction in water intake compared with animals injected with 0.15 M NaCl (control). AAVP injected into LSA potentates the decrease in the water intake induced by the central injection of AVP when compared with control group. Rats injected with antagonists losartan, CGP42112A and both previous to AVP are shown in Fig. 3. Losartan and CGP42112A alone diminish water intake induced by AVP compared with control. Both, losartan and CGP42112A together, increased AVP effect, reducing significantly lesser than one caused by AVP after 2 h. Three percent of NaCl intake following the injection of AVP into LSA of controls and rats injected with AAVP are shown in Fig. 4. Rats injected with AVP into LSA showed a significant reduction in sodium intake at 0.25 and 0.5 h compared with control. AAVP inhibited sodium ingestion induced by AVP compared with control. At 2 h, AAVP showed a sodium intake smaller than one caused by AVP. Rats pre-treated with antagonists losartan, CGP42112A and both previous to AVP are shown in Fig. 5. Losartan reduced significantly sodium intake induced by AVP at initials periods compared with control. CGP42112A did not show significant differences in sodium intake induced by AVP compared with control. Both, losartan and CGP42112A together, increased AVP effect on diminution sodium intake induced by AVP compared with control. This diminution were significantly lesser than one caused by AVP at 2 h.
4. Discussion The results of the present study show a dose-dependent reduction on water and sodium intake induced by AVP injected into LSA in water-deprived rats. Previous studies show contradictory results about the effect of AVP injected centrally [21]. Water privation causes loss of water in extra and intracellular components. Consequently, the solute concentration of extra- and intracellular fluids increase, and both compartments volume diminish themselves. These events stimulate AVP and angiotensin secretion to increase corporal water renal conservation, with the aim to re-establish water deficit [22]. The precise mechanism of AVP action has not been defined precisely yet. Thus, AVP can directly stimulate some neurons that intervene on thirst. Therefore, AVP can be found provoking
Fig. 5. The 3% NaCl intake responses of rats injected with antagonists losartan (A), CGP42112A (B) and both (C) previous to AVP. Results are reported as mean F S.E.M. N = number of animals. *P < 0.05 compared with saline injection. +P < 0.05 compared with AVP 40 nmol.
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changes in neuronal membrane properties modulating the neurons excitability [23]. Other pathway would be that AVP can change cellular permeability of other brain membranes, facilitating water movement through osmotic gradient. It was shown that water permeability in brain increases after AVP administration into the lateral ventriculus, which surrounds the LSA [24]. Another evidence is that AVP influences the central catecholamines metabolism, as well as angiotensin, on specific brain regions including limbic system and hypothalamus [25]. It is known that the noradrenergic system exercises an essential role at water and salt ingestion control [26]. The present and previous results indicate that AVP effect on thirst depends on its concentration: AVP high doses inhibit water ingestion [21]. A part of this inhibitory effect probably is caused by a vasoconstrictor action of AVP and an increase of inhibitory impulses of cardiovascular receptors [27]. Biological bases of salt appetite have not been understood completely yet. Many papers suggest that NaCl consume cannot be due to excitatory stimulus associated with a sodium deficiency, but due to a complex interaction between excitatory and inhibitory stimulus [28,29]. Reflex and behavior answers are necessary to correct the balance and maintenance of corporal fluid homeostasis. Reflex mechanisms use the autonomic nervous system and endocrine answers (as angiotensin and AVP) to change the water and sodium loss on dehydrating situations. Behavior answers include water and sodium ingestion from food and liquids [30]. The present data show that AVP administration into LSA of dehydrating rats diminishes sodium ingestion. Specially, the LSA of rats contains a dense and dimorphic vasopressinergic innervation [31]. AVP administration into ventral region of third ventriculus at preoptic area also shows a reduction on sodium ingestion [32]. Another aim of this paper was to determine the relative contribution of LSA AT1 and AT2 receptors on water and sodium ingestion induced by AVP. The results show that a reduction in thirst and sodium ingestion provoked by AVP injection into LSA of rats was not changed by previous application of losartan, and CGP42112A blockage induced an increase of this behaviors as soon as the dose was applied and time period observed. These facts suggest that AVP action into LSA on water and sodium intake is depending on two subtypes of angiotensin receptors, with predominance of AT1 receptors. It is known that ANG II administration into several central areas increases water ingestion and sodium appetite, diminishing the arterial baroreceptor reflex sensibility and increasing the mean arterial pressure. It is also known that ANG II release is linked to AVP synthesis and the release on several central areas with regard to hydric and mineral balance. It should be noted that, specifically, ASL has high density of AT2 receptors, besides having AT1 receptors. It was shown that central AT1 receptors blockage reduces pressure answer of ANG II at 70% and, although some
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authors suggest the AT2 receptors role on AVP release, other studies suggest that AVP secretion is due to AT1 receptors (see Ref. [33] for review). The results of this study also show that losartan and CGP42112A administered together reduces water and sodium intake induced by AVP higher than the administration of blockers AT1 and AT2 alone into LSA. Although autoradiographic researches have shown a predominant distribution of AT1 receptors in para-ventricular nuclei (PVN) [34,35], there are studies that show that AT2 receptors antagonists attenuate AVP release by ANG II [36] and neuronal excitation in PVN [37]. Previous results [38] demonstrate that about 25% of ANG II receptors from hypothalamus do not bind to losartan, and this receptor bind to CGP42112A and [Sar1, Ile8]ANG II. Thus, the present results suggest that AVP action on water and sodium intake depend on both subtypes receptor activation. Several studies suggest that a small number of sensitive sites binding to losartan are revealed when AT2 receptors are activated, or CGP42112A act increasing losartan affinity to AT1 receptors [39,40]. An alternative mechanism can be responsible for the big deficit of water and sodium induced at this study; it can be explained by papers that identify a G protein that is bound to AT1 receptors angiotensin [23]. This new receptor is called AT1B, and it seems to have a bigger affinity to PD123319 and lower affinity to losartan [41]. The present results also show that AVP antagonist type V1 d(CH2)5Tyr(Me)AVP injected into LSA of rats shows a tendency to reduction of water and sodium intake induced by AVP, mainly with previous application of high doses AAVP. Many biochemistry, autoradiographic and electrophysiologic data show the existence of V1 receptors in CNS [8,42 –45]. It was demonstrated that endogenous AVP was blocked by AAVP administration into lateral central ventriculus that surrounds lateral septum [4]. In conclusion, the present data show that AVP, at administered dose at this work, reduces water and sodium intake into LSA. The present data also show that angiotensin AT1 and AT2 receptors and vasopressinergic V1 receptors increase these effects. The present data also show that angiotensin AT1 and AT2 receptors activated simultaneously reduce in higher proportion water and sodium ingestion induced by AVP into LSA, when they are blocked by losartan and CGP42112A.
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Effects of angiotensin and vasopressin V1 receptors on water and sodium intake induced by injection of vasopressin into lateral septal area E.G.O. Mima a, R.R. Andrade b, L.A.A. Camargo b,*, W.A. Saad b a
b
Department of Dental Materials and Prosthodontics, School of Dentistry, Paulista State University, UNESP, Araraquara, SP, Brazil Department of Physiology and Pathology, School of Dentistry, Paulista State University, UNESP, Rua Humaita´, 1680, Araraquara, SP 14801-903, Brazil Received 6 August 2003; accepted 23 December 2003
Abstract The specific arginine8-vasopressin (AVP) V1 receptors antagonist (AAVP) was injected (20, 40 and 80 nmol) into the lateral septal area (LSA) to determine the effects of selective septal V1 receptor on water and 3% sodium intake in rats. Was also observed the effects of losartan and CGP42112A (select ligands of the AT1 and AT2 ANG II receptors, respectively) injected into LSA prior AVP on the same appetites. Twenty-four hours before the experiments, the rats were deprived of water. The volume of drug solution injected was 0.5 Al. Water and sodium intake were measured at 0.25, 0.5, 1.0 and 2.0 h. Injection of AVP reduced the water and sodium ingestion vs. control (0.15 M saline). Pre-treatment with AAVP (40, 80 and 160 nmol) did not alter the decrease in the water ingestion induced by AVP, whereas AAVP abolished the action of AVP-induced sodium intake. Losartan (40, 80 and 160 nmol) did not alter the effect of AVP on water and sodium intake, whereas CGP42112A (20, 40 and 60 nmol) at the first 30 min increased water ingestion. Losartan and CGP42112A together increased the actions of AVP, showing more pronounced effects than when the two antagonists were injected alone. The results showed that AVP inhibited the appetites and these effects were increased by the AAVP. The involvement of angiotensinergic receptors in the effects of AVP is also suggested. D 2004 Elsevier B.V. All rights reserved. Keywords: AVP; AAVP; AT1 receptor; AT2 receptor; Water intake; Sodium intake
1. Introduction The neuronal peptide arginine8-vasopressin (AVP) has an important role in the regulation of the hydrolic balance. Many papers show the presence of vasopressinergic innervation at the central nervous system (CNS) [1– 4], including septal area (SA) [5 –7]. The pharmacologic properties of the AVP receptors at the SA are similar to V1 peripheric receptors [8– 10]. The administration of d(CH2)5Tyr(Me)AVP (AAVP) at CNS has shown a stronger antagonist on the AVP V1 receptors [4,11,12]. The systemic or central administration of angiotensin II (ANG II) increases the secretion of AVP [13,14]. In regard to water intake, sodium appetite and secretion of AVP, in
* Corresponding author. Tel./fax: +55-16-201-6488. E-mail address: [email protected] (L.A.A. Camargo). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2003.12.006
response to intracerebroventricular (i.c.v.) administration of ANG II, many studies have demonstrated that the administration of losartan, ANG II AT1 receptor antagonist, blocks this answers in rats [15,16]. Studies with central administration of AT2 antagonists (PD123319, PD123177 and CGP42112A) showed many answers in regard to water and sodium intake. Some investigators observed inhibitory influences after administration AT2 blockers i.c.v. on water and sodium intake and AVP release in rats [16 – 19]. The reason for different effects of some AT2 antagonists in rats still has not been explained. However, it has been suggested that the inhibitory effects of high doses of these antagonists applied i.c.v. can be due to an action at the central AT1 receptors or an ‘‘in vivo’’ conversion in AT1 antagonists [18]. It has also been suggested that other subtypes of AT2 receptors, sensitive to an antagonist, can exist in the brain [20]. At the present investigation, we studied the effect of AVP V1 receptors antagonist, as well as AT1 an AT2 receptors
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of AVP into the same area. Water and sodium intake was studied in different experiments and in several groups of animals, after antagonists and AVP injection into LSA, as shown below:
Fig. 1. Photomicrograph of a hematoxylin-stained section of the rat brain showing the site of injection into the LSA (arrow).
antagonists found in the lateral septal area (LSA), on thirst and sodium appetite by administration of AVP at this same area.
U Control (0.15 M NaCl), U AVP (20, 40 and 80 nmol), U d(CH2)5Tyr(Me)AVP (40, 80 and 160 nmol) + AVP (40 nmol), U Losartan (40, 80 and 160 nmol) + AVP (40 nmol), U CGP42112A (20, 40, 60 and 80 nmol) + AVP (40 nmol), U Losartan (40, 80 and 160 nmol) + CGP42112A (20, 40 and 60 nmol) + AVP (40 nmol). Water and sodium intake was recorded over a 2-h period (one sample each at 0.25, 0.5, 1 and 2 h), using a burette with 0.1-ml divisions fitted with a metal spout for liquid ingestion.
2. Materials and methods 2.1. Intracranial cannulation Male Holtzman rats weighing 250– 300 g were used in all experiments. The rats were individually housed in a temperature-controlled room (23 F 2 jC) with a 12:12h light/dark cycle. Food and water were available ad libitum except as noted. Stainless steel cannulae (0.6 mm outer diameter, 0.33 mm inner diameter) were implanted into the ASL of each animal under 2,2,2-tribromoethanol anesthesia according to standard stereotaxic techniques based on the following coordinates: 0.8 mm rostral to the bregma, 0.8 mm lateral to the sagittal line and 4.2 mm below the duramater. Cannulae were attached to the skull using acrylic cement and small screws. Insertion of a close-fitting stylet kept the lumen free from debris and clots. After brain surgery, the animals received a dose of 50 000 U of penicillin and were returned to individual metabolic cages, with free access to granular ration, tap water and 3% NaCl solution for 1 week until the day of experiment. A dental needle was connected by PE tubing to a Hamilton-type syringe (10 Al) kept outside the cage. All drugs were injected in 0.5 Al volumes over 10 –15 s. 2.2. Drugs AVP (Sigma), losartan (DuPont Merck), CGP42112A (RBI) and d(CH2)5Tyr(Me)AVP (Bachem). 2.3. Procedures Food, water and 3% NaCl solution were removed 24 h before the intracranial injection for sodium intake register. For water intake register, only liquids were removed. Antagonists were injected into LSA 15 min before injection
Fig. 2. The water intake responses of rats injected with AVP into the LSA (A) and rats pre-treated with AAVP (B). Results are reported as mean F S.E.M. N = number of animals. *P < 0.05 compared with saline injection.
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2.4. Brain histology At the end of the experiments, the rats were anesthetized with ether and perfused though the heart with 10% formalin. The brains were removed, stored in 10% formalin for 1 week, frozen and cut. Coronal section (20 Am) was stained with hematoxylin and eosin for analysis by light microscopy to confirm the position of injection. Only the results obtained from rats with typical injection into the LSA were used (Fig. 1). 2.5. Statistics Data are reported as means F S.E.M. Analysis of variance and Newmans– Keuls test were used to test statistical significance. Differences were considered significant at P < 0.005.
Fig. 3. The water intake responses of rats injected with antagonists losartan (A), CGP42112A (B) and both (C) previous to AVP. Results are reported as mean F S.E.M. N = number of animals. *P < 0.05 compared with saline injection. +P < 0.05 compared with AVP 40 nmol.
Fig. 4. The 3% NaCl intake responses of rats injected with AVP into the LSA (A) and rats pre-treated with AAVP (B). Results are reported as mean F S.E.M. N = number of animals. *P < 0.05 compared with saline injection. +P < 0.05 compared with AVP 40 nmol.
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3. Results The water intake responses of rats injected with AVP into the LSA and rats pre-treated with AAVP are summarized in Fig. 2. Rats submitted to AVP injection showed a significant reduction in water intake compared with animals injected with 0.15 M NaCl (control). AAVP injected into LSA potentates the decrease in the water intake induced by the central injection of AVP when compared with control group. Rats injected with antagonists losartan, CGP42112A and both previous to AVP are shown in Fig. 3. Losartan and CGP42112A alone diminish water intake induced by AVP compared with control. Both, losartan and CGP42112A together, increased AVP effect, reducing significantly lesser than one caused by AVP after 2 h. Three percent of NaCl intake following the injection of AVP into LSA of controls and rats injected with AAVP are shown in Fig. 4. Rats injected with AVP into LSA showed a significant reduction in sodium intake at 0.25 and 0.5 h compared with control. AAVP inhibited sodium ingestion induced by AVP compared with control. At 2 h, AAVP showed a sodium intake smaller than one caused by AVP. Rats pre-treated with antagonists losartan, CGP42112A and both previous to AVP are shown in Fig. 5. Losartan reduced significantly sodium intake induced by AVP at initials periods compared with control. CGP42112A did not show significant differences in sodium intake induced by AVP compared with control. Both, losartan and CGP42112A together, increased AVP effect on diminution sodium intake induced by AVP compared with control. This diminution were significantly lesser than one caused by AVP at 2 h.
4. Discussion The results of the present study show a dose-dependent reduction on water and sodium intake induced by AVP injected into LSA in water-deprived rats. Previous studies show contradictory results about the effect of AVP injected centrally [21]. Water privation causes loss of water in extra and intracellular components. Consequently, the solute concentration of extra- and intracellular fluids increase, and both compartments volume diminish themselves. These events stimulate AVP and angiotensin secretion to increase corporal water renal conservation, with the aim to re-establish water deficit [22]. The precise mechanism of AVP action has not been defined precisely yet. Thus, AVP can directly stimulate some neurons that intervene on thirst. Therefore, AVP can be found provoking
Fig. 5. The 3% NaCl intake responses of rats injected with antagonists losartan (A), CGP42112A (B) and both (C) previous to AVP. Results are reported as mean F S.E.M. N = number of animals. *P < 0.05 compared with saline injection. +P < 0.05 compared with AVP 40 nmol.
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changes in neuronal membrane properties modulating the neurons excitability [23]. Other pathway would be that AVP can change cellular permeability of other brain membranes, facilitating water movement through osmotic gradient. It was shown that water permeability in brain increases after AVP administration into the lateral ventriculus, which surrounds the LSA [24]. Another evidence is that AVP influences the central catecholamines metabolism, as well as angiotensin, on specific brain regions including limbic system and hypothalamus [25]. It is known that the noradrenergic system exercises an essential role at water and salt ingestion control [26]. The present and previous results indicate that AVP effect on thirst depends on its concentration: AVP high doses inhibit water ingestion [21]. A part of this inhibitory effect probably is caused by a vasoconstrictor action of AVP and an increase of inhibitory impulses of cardiovascular receptors [27]. Biological bases of salt appetite have not been understood completely yet. Many papers suggest that NaCl consume cannot be due to excitatory stimulus associated with a sodium deficiency, but due to a complex interaction between excitatory and inhibitory stimulus [28,29]. Reflex and behavior answers are necessary to correct the balance and maintenance of corporal fluid homeostasis. Reflex mechanisms use the autonomic nervous system and endocrine answers (as angiotensin and AVP) to change the water and sodium loss on dehydrating situations. Behavior answers include water and sodium ingestion from food and liquids [30]. The present data show that AVP administration into LSA of dehydrating rats diminishes sodium ingestion. Specially, the LSA of rats contains a dense and dimorphic vasopressinergic innervation [31]. AVP administration into ventral region of third ventriculus at preoptic area also shows a reduction on sodium ingestion [32]. Another aim of this paper was to determine the relative contribution of LSA AT1 and AT2 receptors on water and sodium ingestion induced by AVP. The results show that a reduction in thirst and sodium ingestion provoked by AVP injection into LSA of rats was not changed by previous application of losartan, and CGP42112A blockage induced an increase of this behaviors as soon as the dose was applied and time period observed. These facts suggest that AVP action into LSA on water and sodium intake is depending on two subtypes of angiotensin receptors, with predominance of AT1 receptors. It is known that ANG II administration into several central areas increases water ingestion and sodium appetite, diminishing the arterial baroreceptor reflex sensibility and increasing the mean arterial pressure. It is also known that ANG II release is linked to AVP synthesis and the release on several central areas with regard to hydric and mineral balance. It should be noted that, specifically, ASL has high density of AT2 receptors, besides having AT1 receptors. It was shown that central AT1 receptors blockage reduces pressure answer of ANG II at 70% and, although some
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authors suggest the AT2 receptors role on AVP release, other studies suggest that AVP secretion is due to AT1 receptors (see Ref. [33] for review). The results of this study also show that losartan and CGP42112A administered together reduces water and sodium intake induced by AVP higher than the administration of blockers AT1 and AT2 alone into LSA. Although autoradiographic researches have shown a predominant distribution of AT1 receptors in para-ventricular nuclei (PVN) [34,35], there are studies that show that AT2 receptors antagonists attenuate AVP release by ANG II [36] and neuronal excitation in PVN [37]. Previous results [38] demonstrate that about 25% of ANG II receptors from hypothalamus do not bind to losartan, and this receptor bind to CGP42112A and [Sar1, Ile8]ANG II. Thus, the present results suggest that AVP action on water and sodium intake depend on both subtypes receptor activation. Several studies suggest that a small number of sensitive sites binding to losartan are revealed when AT2 receptors are activated, or CGP42112A act increasing losartan affinity to AT1 receptors [39,40]. An alternative mechanism can be responsible for the big deficit of water and sodium induced at this study; it can be explained by papers that identify a G protein that is bound to AT1 receptors angiotensin [23]. This new receptor is called AT1B, and it seems to have a bigger affinity to PD123319 and lower affinity to losartan [41]. The present results also show that AVP antagonist type V1 d(CH2)5Tyr(Me)AVP injected into LSA of rats shows a tendency to reduction of water and sodium intake induced by AVP, mainly with previous application of high doses AAVP. Many biochemistry, autoradiographic and electrophysiologic data show the existence of V1 receptors in CNS [8,42 –45]. It was demonstrated that endogenous AVP was blocked by AAVP administration into lateral central ventriculus that surrounds lateral septum [4]. In conclusion, the present data show that AVP, at administered dose at this work, reduces water and sodium intake into LSA. The present data also show that angiotensin AT1 and AT2 receptors and vasopressinergic V1 receptors increase these effects. The present data also show that angiotensin AT1 and AT2 receptors activated simultaneously reduce in higher proportion water and sodium ingestion induced by AVP into LSA, when they are blocked by losartan and CGP42112A.
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