Divergent Behavioral Roles of Angiotensin Receptor Intracellular Signaling Cascades
http://www.100md.com
《内分泌学杂志》
Departments of Animal Biology (D.D., D.K.Y., S.J.F.), Psychology (L.F.F., S.J.F.)
Pharmacology (S.J.F.) and the Institute of Neurological Sciences (S.J.F.), University of Pennsylvania, Philadelphia, Pennsylvania 19104
Abstract
Central injections of angiotensin II (AngII) increase both water and NaCl intake. These effects of AngII occur largely through stimulation of the AngII type 1 (AT1) receptor. Stimulation of the AT1 receptor leads to a number of intracellular events, including phospholipase C (PLC) activation and the subsequent formation of diacylglycerol and inositol trisphosphate (IP3), which then activate protein kinase C (PKC) and increase intracellular calcium, respectively. In addition, AT1 receptor stimulation leads to the activation of MAPK family members. Recent experiments using mutated AT1 receptor constructs or the AngII analog Sar1,Ile4,Ile8-AngII (SII) revealed that MAPK activation can occur independent of PLC/PKC/IP3 activation. The present experiments used in vitro and in vivo approaches to clarify the cellular and behavioral responses to SII. Specifically, SII mimicked AngII stimulation of MAPK in AT1 receptor-transfected COS-1 cells and rat brain but blocked the effects of AngII in two distinct settings: in vitro stimulation of IP3 and in vivo increases in water intake. Moreover, SII increased intake of 1.5% NaCl, despite the SII blockade of IP3 formation and water intake. Examination of brain tissue showed increases in Fos expression in several AngII-sensitive brain areas after injection of AngII, but not SII. The lack of SII-induced IP3 production, water intake, and Fos expression strongly suggest that the PLC/PKC/IP3 pathway is required for water intake, but not NaCl consumption stimulated by AngII. Collectively, these results support the hypothesis that divergent intracellular signals from a single receptor type can give rise to separable behavioral phenomena.
Introduction
DETERMINING THE RELEVANCE of intracellular signaling pathways has been a critical step toward understanding the mechanisms involved in the neural control of behavior. The body of work by Kandel and colleagues, for example, provided a wealth of information about the role of cAMP signaling in the regulation of learning and memory (for review see Ref.1). Progress of this magnitude has been arguably absent from investigations of other behaviors, although initial steps have been taken for many, including reproductive behavior (2, 3), circadian rhythms (4, 5), and the role of the octapeptide hormone angiotensin II (AngII) (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-COO–) in body fluid homeostasis (6, 7).
AngII is a critical component for the maintenance of body fluid and cardiovascular homeostasis. Circulating AngII increases blood pressure and acts on the brain to stimulate water and NaCl intake, both of which are critical for the restoration of body fluids in conditions of hypovolemia. At the behavioral level, AngII induction of water and salt intake have been well studied (for review see Refs.6 and 7). The water intake that follows central injections of AngII is so reproducible that such injections are commonly used to verify accurate intracerebroventricular cannula placement. Unlike the AngII-induced increases in water intake that occur rapidly and tend to plateau by 30 min, increases in salt intake stimulated by AngII tend to be more delayed (for review see Refs.8 and 9) but can be demonstrated in a short time frame when more dilute concentrations of NaCl are offered (e.g. Refs.10 and11) or when oxytocin receptor antagonists are provided (for review see Ref.12).
The increases in water and sodium intake resulting from AngII are initiated largely through stimulation of the angiotensin type 1 (AT1) receptor in forebrain circumventricular organs, including the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO) (13). Like other seven-membrane-spanning receptor family members, agonist binding at the AT1 receptor initiates a host of intracellular events (for review see Ref.14). For example, this receptor is well known to couple to the G protein Gq, which stimulates phospholipase C (PLC) upon agonist binding to generate the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG and IP3 then activate protein kinase C (PKC) and liberate intracellular stores of calcium, respectively. In addition to this more traditionally ascribed signaling pathway, there is growing evidence that AT1 receptor activation also stimulates intracellular events previously attributed primarily to growth factor receptors. The discovery that AT1 receptors can mediate these alternate signaling pathways, including regulation of tyrosine kinase and tyrosine phosphatase activity as well as increased expression of several early immediate genes including c-fos, c-jun, and c-myc (15, 16) provides cellular mechanisms whereby AngII can exert persistent physiological and behavioral changes.
Among the specific, alternate signaling pathways engaged by the AT1 receptor is the phosphorylation (activation) of MAPK family members, including p44/42MAPK, also known as ERK1/ERK2 (17). Although AngII-induced p44/42MAPK activation had been shown to require activation of PKC (18), recent work using AT1 receptor point mutants has revealed that these intracellular events can occur separately (19). As such, it is becoming increasingly clear that in addition to stimulating MAPK through PKC, the AT1 receptor can also stimulate MAPK through alternate means.
In addition to studies using AT1 receptor mutants, strategies to understand the signaling properties of AngII receptors have relied on analogs of AngII. One such analog is a double isoleucine substituted form, Sar1,Ile4,Ile8-AngII (SII) (20). Previous studies used this peptide analog exclusively in vitro as part of an effort to demonstrate that the Tyr4 and Phe8 residues of AngII are important pharmacophores, each of which is critical for the bioactivity of this peptide (20). Not surprisingly, these early experiments focused primarily on the ability of AT1 to activate the PLC/PKC/IP3 pathway. More recent studies, however, demonstrated that when MAPK rather than IP3 was measured, SII acted as an agonist at the AT1 receptor independent of G protein activation (21, 22, 23). Thus, there is growing evidence that the traditionally ascribed G protein-dependent pathway and the more recently discovered MAPK stimulation can occur independently.
Many studies have approached the mechanisms underlying AngII-induced water and NaCl consumption from an anatomical perspective, focusing on the circuitry that links AngII-responsive structures such as forebrain circumventricular organs with other brain areas (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Because there is growing evidence that the AT1 receptor employs multiple signaling pathways that can operate independently, it is possible that the divergent signaling pathways used by the AT1 receptor have different roles in the array of behaviors induced by AngII. Thus, it is critical not only to study the circuitry involved but also to examine the potential relevance of different intracellular events to specific behavioral responses stimulated by AngII. To this end, the present report describes experiments using in vitro and in vivo experiments to examine this potential divergence.
Materials and Methods
Materials
AngII was purchased from Bachem (King of Prussia, PA). SII was synthesized and obtained from Bachem. Tissue culture medium and supplements, including LipofectAMINE reagent were obtained from Invitrogen/Life Technologies (Gaithersburg, MD). [3H]Myoinositol was obtained from American Radiolabeled Chemicals (St. Louis, MO) and [125I]Sar1,Ile8-AngII was obtained from the University of Mississippi Peptide Center (directed by Dr. Robert Speth). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
Cell culture experiments
COS-1 cells were used for all of the in vitro experiments described here. Cells were grown in polystyrene tissue culture flasks in medium consisting of DMEM supplemented with 10% fetal bovine serum, L-glutamine, and penicillin-streptomycin in a humidified atmosphere of 5% CO2 and 95% O2 at 37 C. AT1 receptor was later introduced into the cells by transfection with LipofectAMINE for 5 h, after which the transfection medium was removed and replaced with normal growth medium. Transfected cells were used for ligand binding experiments, IP3 assays, and MAPK assays as described below.
Ligand binding experiments
Two days after transfection, the cells were rinsed with ice-cold Tris-buffered saline (20 mM Tris-HCl, pH 7.4, and 150 mM NaCl) and then harvested by scraping into 20 mM Tris-HCl (pH 7.4). After polytron homogenization and centrifugation, the membrane pellets were resuspended in assay buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 0.3 TIU/ml aprotinin, and 100 μg/ml 1,10-phenanthroline], and protein content was determined by bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL). The binding assays were initiated by addition of the desired amount of 5 μg membrane protein to the assay mixture containing various concentrations of [125I]Sar1,Ile8-AngII and unlabeled competitors (AngII and SII). Nonspecific binding was defined as the amount of radioligand binding remaining in the presence of 1 μM Sar1,Ile8 -AngII. The binding assays proceeded for 60 min at room temperature and were terminated by rapid dilution with 5 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1.5 mM CaCl2 and vacuum filtration on glass-fiber filters presoaked with 0.3% polyethylenimine, using a Brandell harvester (Brandell, Gaithersburg, MD). Radioligand binding was quantified by -counting of the filters. Data were analyzed and fit to a single-site model.
IP3 assays
IP3 formation was measured as described previously (19). Briefly, AT1-transfected cells were preloaded with [3H]myoinositol (4.5 μCi/ml DMEM) for 18 h before being treated with vehicle (DMEM), AngII, SII, or both AngII and SII for 30 sec. Cells then were washed, lysed, and processed for IP3 quantification by stepwise elution from anion-exchange resin columns. IP3 was quantified by liquid scintillation counting of the IP3-containing fraction. Protein quantification was performed by BCA assay.
In vitro MAPK assays
Activated p44/42MAPK was measured as described previously (35). Briefly, AT1-transfected cells were treated with vehicle (DMEM), AngII, or SII for 5 min before being washed, lysed by Dounce homogenization in cold lysis buffer [25 mM Tris-HCl (pH 8.0), 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM sodium molybdate, 1 mM phenylarsine oxide, 10 μg/ml pepstatin, 2 μg/ml aprotinin, and 10 μg/ml leupeptin], and processed for MAPK immunoblotting (described below) and protein quantification by BCA assay.
MAPK immunoblotting
Western blotting for activated (phosphorylated) MAPK was performed as described previously (35). Briefly, 20–30 μg protein was run on a 10% SDS-PAGE gel and subsequently transferred to a nitrocellulose membrane. Membranes were probed for activated MAPK by incubation with a monoclonal antibody directed against phospho-p44/42MAPK (1:1000) (Cell Signaling Technology, Beverly, MA). The membranes then were washed and incubated with peroxidase-conjugated goat antimouse IgG (1:1000 or 1:2000) (Jackson ImmunoResearch Laboratories, West Grove, PA) before immunoreactivity was detected using chemiluminescence reactions according to the manufacturer’s instructions (Western Lightning kit; PerkinElmer Life Sciences, Boston, MA).
Animals and surgery
Adult, male Sprague-Dawley rats were used for all of the experiments described. The number of animals within each treatment group for each experiment performed is indicated below. Animals were maintained in a temperature-controlled room (22 C) with a 12-h light, 12-h dark cycle and food and water available ad libitum. The handling and care of experimental animals conformed to the regulations provided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
A subset of animals in these experiments were implanted with chronic indwelling cannulas aimed at the third cerebral ventricle (coordinates were on the midline, 2.0 mm posterior to Bregma, and 7.5 mm ventral to dura). Animals were allowed to recover for at least 1 wk before the onset of the experiments. Proper cannula placement was verified by injection of 210 μg 5-thio-D-glucose in 2 μl saline with a subsequent increase in plasma glucose being indicative of proper placement in the ventricle (36). At least 48 h elapsed before onset of the experiments described below. All experiments described here were performed during the lights-on portion of the cycle.
Water intake
Two separate experiments were used to measure water intake. First, using a counterbalanced, repeated-measures design, animals (n = 4) received third ventricle injections of vehicle [1 μl Tris-buffered saline (TBS)], 10 ng AngII, or 1 μg SII with at least 48 h between injections. Water intake was measured 5, 10, 15, and 30 min after the injections. Second, separate groups of animals (n = 3 per group) were injected with either vehicle (1 μl TBS), 10 ng AngII, 10 μg SII, or both AngII and SII. Water intake again was measured 5, 10, 15, and 30 min after the injection.
NaCl intake
NaCl intake was measured using a two-bottle test in which animals had access to water and 1.5% NaCl. Animals (n = 4 per group) were injected with vehicle (1 μl TBS), 10 ng AngII, or 1 μg SII into the third ventricle, and intake was recorded 15 min, 3 h, and 7 h after the injection.
Fos immunohistochemistry
Each animal (n = 3 per group) received a third ventricle injection of either vehicle (1 μl TBS), 10 ng AngII, or 1 μg SII 1 h before being killed by transcardial perfusion with saline followed by phosphate-buffered 4% paraformaldehyde under anesthesia with ketamine and xylazine. Food and water were not available to the animals after the initial injection. Brains were removed from the crania and postfixed overnight before being transferred to 20% sucrose in 0.1 M phosphate buffer. Blocks of brain containing the anterior hypothalamus were cut into three sets of 40-μm coronal sections on a freezing microtome, and one set was processed immunohistochemically for Fos expression as described previously (37). Briefly, the sections were washed, soaked in TBS containing 0.3% H2O2 before being washed and further incubated overnight at room temperature in an antibody directed against the early immediate gene Fos (rabbit anti-c-Fos; 1:5000) (Santa Cruz Biotechnology, Santa Cruz, CA). Sections then were washed and further incubated in biotinylated donkey antirabbit IgG (1:1000) (Jackson ImmunoResearch) followed by another wash and incubation with an avidin-biotin-peroxidase complex (1:333) (Elite Kit; Vector Laboratories, Burlingame, CA). After being washed with TBS and then with 50 mM Tris, immunoreactivity was visualized by reacting the sections with 3,3'-diaminobenzidine (0.2 mg/ml) and 0.025% H2O2 in 50 mM Tris for 15 min. The reaction was stopped with TBS washes, after which the sections were floated onto gelatinized slides, dehydrated with increasing concentrations of alcohol followed by Hemo-De (Fisher Scientific, Pittsburgh, PA), and coverslipped with Permount (Fisher).
In vivo MAPK assays
An additional second set of animals were included in these studies. These animals received acute injections of vehicle (n = 5), 10 ng AngII (n = 5), or 1 μg SII (n = 6). Specifically, a 2-μl Hamilton syringe was loaded with 0.6 μl India Ink (diluted 1:1 in deionized water) followed by 1 μl vehicle (TBS), AngII, or SII. Animals were anesthetized and fixed in a stereotaxic apparatus, and the syringe was lowered into the lateral cerebral ventricle (coordinates were 1.4 mm lateral to midline, 0.9 mm posterior to Bregma, and 3.4 mm ventral to dura). The injections were made in two stages, the first microliter, containing the vehicle or drug, was injected followed by a 9-min delay before the final 0.6 μl was injected and the animal decapitated 1 min later for a total vehicle/drug exposure of 10 min. The brains were rapidly dissected from the crania, halved at the midsagittal plane, and frozen in heptane on dry ice. All animals included had ink in the cerebral ventricular system that was easily visualized after the sagittal cut was made.
A small block of tissue containing the anterior wall of the third ventricle, designed to include the forebrain circumventricular structures, was dissected from the midline of each brain. Tissue blocks were Dounce homogenized in 100 μl ice-cold lysis buffer. After centrifugation, the supernatant was assayed for protein by the BCA method and for activated MAPK by Western blotting as described above.
Data analysis
Radioligand binding data were processed using Prism (version 4.0; GraphPad Software, Inc., San Diego, CA) to calculate Bmax and Kd values for [125I]Sar1,Ile8-AngII and Ki values for AngII and SII. Films from MAPK immunoblots were digitized and OD of each band determined using Scion Image (version 4.0.2; Scion Corp., Frederick, MD). Statistical comparisons were conducted using Sigma Stat (version 2.03; SPSS Inc., Chicago, IL). Details of the tests used are provided for each experiment below. Fos immunoreactivity was quantified by counting labeled nuclei on printed digital micrographs that had been coded such that the treatment condition was unknown. The OVLT and immediately surrounding anterior portion of the median preoptic nucleus (MEPO) were identified with the aid of an atlas of rat brain sections (38). The data were expressed as the mean (±SEM) number of cells per section. A qualitative analysis of Fos immunoreactivity in other brain areas was also conducted. For this analysis, the relative amount of Fos immunoreactivity was rated on a 10-point scale in the posterior portion of the MEPO (the portion of the nucleus separated into dorsal and ventral components by the anterior commissure), the paraventricular hypothalamic nucleus (PVN), the SFO, and the supraoptic nucleus (SON).
Results
In vitro experiments
The overall goal of the in vitro experiments described here was to confirm and extend previous reports showing that the AngII analog SII binds the AT1 receptor, fails to activate the pathway including IP3 formation, but does stimulate MAPK activation (20, 21, 22, 23). We sought to extend these findings in vitro by testing the ability of SII to antagonize the IP3 production stimulated by AngII. The initial step toward these goals was to compare the binding affinities and the concomitant activated signaling pathways of AngII and its analog SII on the AT1 receptor. Using membrane preparations from AT1-transfected COS-1 cells, the receptor exhibited its typical high affinity for the radiolabeled antagonist [125I]Sar1,Ile8-AngII in saturation isotherms (Kd = 0.56 ± 0.09 nM; Bmax = 3.76 ± 1.15 pmol/mg protein; n = 3). Competition binding analysis then was used to determine the relative affinities of AngII and SII for the AT1 receptor. Both AngII and SII bound the AT1 receptor with high affinity, although the affinity for AngII was nearly 50-fold greater than for SII (Ki values: AngII, 18.1 ± 0.64 nM; SII, 972.5 ± 75.1 nM) (Fig. 1). Moreover, the competition curves demonstrate that AngII and SII compete for the same binding pocket.
Using our AT1-transfected COS-1 cell system, we investigated the relative abilities of AngII and SII to activate two distinct signaling pathways: stimulation of the PLC/PKC/IP3 pathway and activation of p44/42MAPK. Treatment of the transfected cells with 250 nM AngII led to reliable increases in both IP3 formation (Fig. 2A) (ANOVA, F2,6 = 13.5; P = 0.006, Student-Newman-Keuls post hoc comparisons) and in activation (i.e. phosphorylation) of MAPK (Fig. 3) (ANOVA, F2,8 = 32.7; P < 0.001, Student-Newman-Keuls post hoc comparisons). Cells treated with 30 μM SII also exhibited reliable increases in activated MAPK (Fig. 3) (ANOVA, F2,8 = 32.7; P < 0.001, Student-Newman-Keuls post hoc comparisons), despite the failure of this concentration of SII to stimulate IP3 formation (Fig. 2A) (ANOVA, F2,6 = 13.5; P = 0.006, Student-Newman-Keuls post hoc comparisons). Moreover, exposure to SII blocked AngII-induced increase in IP3 formation (Fig. 2B) (two-way ANOVA, main effect of AngII, F1,8 = 25.9, P < 0.001; main effect of SII, F1,8 = 9.7, P = 0.014; interaction effect, F1,8 = 16.9, P = 0.003, Student-Newman-Keuls post hoc comparisons). As such, these in vitro experiments with SII confirm and extend previous findings (21, 22, 23) by confirming that SII binds AT1 receptors with high affinity, confirming that SII acts as an agonist with respect to AT1 receptor-mediated MAPK activation and showing for the first time that SII serves as an antagonist with respect to AT1 receptor-mediated IP3 formation.
In vivo experiments
The ability of SII to stimulate only a subset of the intracellular events activated by the AT1 receptor presented a unique opportunity to examine the behavioral relevance of these now separable pathways. As such, after confirming that SII stimulation of the AT1 receptor led to an increase in MAPK without concomitant IP3 release in vitro, we sought to test the behavioral effects of this compound in rats fitted with chronic indwelling cannulas aimed at the third cerebral ventricle. In an initial series of experiments, we used a repeated-measures design to test for differences in water intake between third ventricle injection of vehicle (1 μl TBS), AngII (10 ng), or SII (1 μg). The different doses of AngII and SII were intended to compensate for the lower binding affinity of SII compared with AngII and were chosen based primarily on the effective concentrations used in the in vitro experiments. During the course of the testing, water intake was negligible when animals were treated with vehicle or SII, but predictably elevated when AngII was injected (Fig. 4A) (two-way repeated-measures ANOVA, main effect of drug, F2,18 = 22.5, P = 0.002; main effect of time, F3,18 = 49.1, P < 0.001; interaction, F6,18 = 16.7, P < 0.001, Student-Newman-Keuls post hoc comparisons). In a second experiment, we used a single-trial design in which animals were injected with vehicle, 10 ng AngII, 10 μg SII, or the combination of AngII and SII. Despite the elevated dose of SII used in this set of experiments, the behavioral response to SII alone was identical to before, with indistinguishable amounts of water consumed by vehicle- or SII-treated groups after 30 min. Moreover, like the antagonism of the IP3 response observed in vitro, when SII was given in combination with AngII, SII blocked nearly all of the AngII-induced water intake (two-way repeated-measures ANOVA, main effect of drug, F3,30 = 15.1, P < 0.001; main effect of time, F3,30 = 42.1, P < 0.001; interaction, F9,18 = 24.4, P < 0.001, Student-Newman-Keuls post hoc comparisons). The cumulative water intake data from these animals after 30 min is shown in Fig. 4B.
After determining that SII functioned as an antagonist with respect to both IP3 formation in vitro and water intake in vivo, we performed a set of experiments to evaluate whether NaCl intake could be increased by SII. To this end, we used a two-bottle test to measure water and 1.5% NaCl intake by animals treated with vehicle, 10 ng AngII, or 1 μg SII. Despite the finding that SII blocked AngII-induced IP3 in vitro and water intake in vivo, treatment with SII increased intake of 1.5% NaCl to levels nearly identical to those observed after AngII injection (Fig. 5A) (two-way repeated-measures ANOVA, main effect of treatment, F2,18 = 4.7, P = 0.045; main effect of time, F2,18 = 8.6, P = 0.002; interaction, F4,18 = 2.7, P = 0.06, Student-Newman-Keuls post hoc comparisons). Measures of water intake using this paradigm were elevated in animals treated with SII, but only at the longest time point measured and to a far lesser degree than observed after AngII treatment (Fig. 5B) (two-way repeated-measures ANOVA, main effect of treatment, F2,18 = 41.5, P < 0.001; main effect of time, F2,18 = 32.4, P < 0.001; interaction, F4,18 = 11.0, P < 0.001, Student-Newman-Keuls post hoc comparisons). As such, it is likely that this modest increase in water intake was the result of the osmotic requirements imposed by the increased NaCl intake rather than a direct effect of SII.
The difference in the behavioral responses to AngII and SII likely reflect differences in the intracellular events induced by these compounds. As a first step toward understanding these differences, we focused on the OVLT, a circumventricular brain structure that plays a critical role in the central response to AngII (13, 39, 40, 41, 42), and measured Fos expression, which has been shown to require increases in calcium in several systems (15, 43, 44, 45). As such, the absence of IP3 formation and the resultant increases in calcium after SII treatment would likely lead to measurable differences in Fos expression after AngII or SII treatment. As predicted, Fos immunoreactivity in the OVLT and immediately surrounding anterior MEPO, collectively referred to as the anteroventral wall of the third ventricle (AV3V) (46), was quantified and found to be greater in sections from animals treated with AngII compared with those treated with vehicle or SII (Fig. 6) (ANOVA, F2,8 = 7.38, P = 0.024, Student-Newman-Keuls post hoc comparisons). In contrast to the increases in Fos in the AV3V region after AngII treatment, injections of SII failed to increase Fos expression in this brain area. A qualitative analysis revealed a similar pattern of Fos expression in other brain areas in which AngII-induced Fos has been identified previously (47, 48, 49, 50). Specifically, as shown in Table 1, Fos expression was elevated in animals treated with AngII, but not SII, in the posterior MEPO (at the level of the nucleus where it is split into dorsal and ventral sections by the anterior commissure), PVN, SFO, and SON.
To further evaluate the intracellular events that occur after AngII or SII injection in vivo, we used acute injections of vehicle, AngII, or SII into the lateral ventricle of anesthetized rats. Extraction and examination of a small block of tissue containing the anterior wall of the third ventricle (Fig. 7A) revealed that, as demonstrated in vitro, treatment with either AngII or SII elevated levels of activated MAPK compared with that stimulated by vehicle (Fig. 7) (ANOVA, F2,15 = 4.22, P = 0.039, Student-Newman-Keuls post hoc comparisons). Unlike the data collected from in vitro experiments, however, levels of activated MAPK did not differ between the AngII or SII conditions when these drugs were applied in vivo.
Discussion
The regulation of behavioral state by AngII is a well-studied problem that has provided a wealth of information about the interface between peripheral hormones and central control of behavior. Although a good deal of attention has been paid to the intracellular signaling pathways under the control of the receptors for AngII, relatively little progress has been made toward understanding how these processes mechanistically affect behavior. The experiments described here confirm earlier findings that SII, an analog of AngII, failed to stimulate the traditionally ascribed Gq-mediated IP3 formation but led to increases in activated MAPK, whereas AngII was capable of activating both signaling pathways (21, 22, 23). Extending these findings, the present data provide the first demonstration that SII acts as an antagonist to AngII-induced IP3 formation in vitro and blocks AngII-stimulated water intake in vivo. These data are consistent with previous experiments demonstrating the attenuation of AngII-induced water intake by an inhibitor of PKC (51). As such, these data add to a growing literature indicating the requirement of the PLC/PKC/IP3 pathway for the water intake stimulated by AngII. More striking, however, was the finding that SII mimicked AngII in its ability to stimulate increases in NaCl intake, providing the first evidence that this intracellular pathway is critical for one, but not another, of the behaviors stimulated by AngII treatment. The present data suggest that the different intracellular signaling pathways employed by AngII map directly onto distinct behaviors, the PLC/PKC/IP3 pathway being critical for water but not NaCl intake, whereas activated MAPK may be more important for NaCl intake but is clearly insufficient to stimulate water intake.
When interpreting the present data, it is certainly noteworthy that SII-injected animals showed modest increases in water intake in the two-bottle paradigm used to measure NaCl ingestion; however, the increased water intake was not statistically significant until the longest time point, well after the observed increases in NaCl, and was far smaller than the AngII-induced water intake using the same paradigm. As such, it is very likely that such increases resulted from osmotic perturbations from the increased NaCl intake, rather than as a direct effect of SII. In fact, it would have been remarkable had the animals not compensated for the increased NaCl by ingesting more water.
The experiments showing increased levels of activated MAPK after rats were treated with either AngII or SII suggest that the findings from in vitro experiments can be extended to in vivo preparations. Even with these data and the fact that in vitro models are often excellent predictors of events that occur in vivo, without direct measures of IP3 formation in brain tissue, it is difficult to determine whether the profile of intracellular events stimulated by AngII in vitro is similar in vivo. The data from the experiments using Fos immunohistochemistry, however, provide indirect evidence that the activation profile of SII observed in vitro also occurs in brain. The increased Fos expression observed after AngII treatment in the brain areas examined is consistent with previous studies (47, 48, 49, 50). With respect to the lack of Fos in SII-treated animals, it is important to note that previous reports clearly demonstrate that stimulation of Fos expression depends on increases in intracellular calcium in a number of systems including those involving dopamine receptors (43), renal mesangial cells (44), lymphocytes (45), and AngII stimulation of adrenal cells (15). Moreover, experiments using cultured neuronal cells showed that activation of Fos-regulating kinase by AngII was blocked by PKC inhibition or by the calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (52). As such, the lack of Fos expression after SII treatment in vivo is entirely consistent with the inability of SII to stimulate the PLC/PKC/IP3 pathway in vitro, suggesting that the in vitro models used here are a faithful representation of events occurring in vivo. Moreover, the comparison of Fos expression after AngII and SII treatments further supports the hypothesis that the IP3 pathway is critical for the water intake stimulated by AngII.
The ability of SII to stimulate MAPK but not the IP3 pathway is temporally consistent with the subset of behaviors stimulated by SII. Specifically, IP3-mediated increases in intracellular calcium occur rapidly in comparison with the actions of MAPK. Thus, the finding that SII fails to stimulate rapid increases in water intake but does stimulate NaCl intake, which under physiological conditions is more delayed, fits well with the time course of the subset of the AngII effector pathways stimulated by SII.
The high affinity of SII for the AT1 receptor shown here and previously reported (20) strongly suggests that the responses observed here are specific to AngII receptor stimulation. Whether or not the present phenomenon relies on the AT1 or the type II (AT2) receptor remains an open question. Although the majority of evidence points to the AT1 receptor as the mediator of the behavioral effects of AngII (11, 53, 54, 55, 56), there is evidence, although limited, that AT2 receptor stimulation also plays a role (57). Given the hypothesis proposed here that the ingestion of NaCl stimulated by AngII depends on the activation of MAPK, it is, however, highly unlikely that the response to SII is AT2 mediated because the AT2 receptor does not appear to couple to MAPK (19). Indeed, in previous experiments using AT2-transfected COS-1 cells, agonist stimulation of this receptor subtype failed to activate p44/42MAPK (19). Nevertheless, determining which receptor subtypes are critical for the response could be addressed by future studies using receptor-subtype-specific antagonists in combination with SII administration.
In summary, the present report confirms previous findings that SII stimulates MAPK family members in vitro but fails to activate the intracellular pathway that includes IP3 formation. The present data notably extend these previous findings with the demonstration that SII blocked AngII-induced stimulation of IP3 formation in vitro and water intake in vivo. Furthermore, we describe the unexpected finding that central injections of SII increased NaCl intake. These data strongly suggest that the IP3 pathway is required for the water intake stimulated by AngII and offer the possibility that the activation of p44/42MAPK mediates AngII-induced NaCl intake but is insufficient for the stimulation of water intake. The present observations support the novel hypothesis that intracellular pathways stimulated by AT1 receptor activation can be separated based on behavioral relevance. These data highlight the importance of intracellular events in the regulation of behavioral state and provide novel information about the means through which single hormones can influence multiple behaviors.
Acknowledgments
We thank Drs. Harvey J. Grill and Joel M. Kaplan for help with the behavioral analysis portion of these experiments. Technical support was provided by Laiyi Luo and Aae Suzuki.
Footnotes
Funding for this work was provided by the following awards from the National Institutes of Health: DK064012 (to D.D.), HL058792 (to D.K.Y.), and DK052018 (to S.J.F.).
First Published Online August 25, 2005
1 D.D. and D.K.Y. contributed equally to this manuscript.
Abbreviations: AngII, Angiotensin II; AT1, AngII type 1; AT2, AngII type 2; AV3V, anteroventral wall of the third ventricle; BCA, bicinchoninic acid; DAG, diacylglycerol; IP3, inositol trisphosphate; MEPO, median preoptic nucleus; OVLT, organum vasculosum of the lamina terminalis; PKC, protein kinase C; PLC, phospholipase C; PVN, paraventricular hypothalamic nucleus; SII, Sar1,Ile4,Ile8-AngII; SFO, subfornical organ; SON, supraoptic nucleus; TBS, Tris-buffered saline.
Accepted for publication August 19, 2005.
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Pharmacology (S.J.F.) and the Institute of Neurological Sciences (S.J.F.), University of Pennsylvania, Philadelphia, Pennsylvania 19104
Abstract
Central injections of angiotensin II (AngII) increase both water and NaCl intake. These effects of AngII occur largely through stimulation of the AngII type 1 (AT1) receptor. Stimulation of the AT1 receptor leads to a number of intracellular events, including phospholipase C (PLC) activation and the subsequent formation of diacylglycerol and inositol trisphosphate (IP3), which then activate protein kinase C (PKC) and increase intracellular calcium, respectively. In addition, AT1 receptor stimulation leads to the activation of MAPK family members. Recent experiments using mutated AT1 receptor constructs or the AngII analog Sar1,Ile4,Ile8-AngII (SII) revealed that MAPK activation can occur independent of PLC/PKC/IP3 activation. The present experiments used in vitro and in vivo approaches to clarify the cellular and behavioral responses to SII. Specifically, SII mimicked AngII stimulation of MAPK in AT1 receptor-transfected COS-1 cells and rat brain but blocked the effects of AngII in two distinct settings: in vitro stimulation of IP3 and in vivo increases in water intake. Moreover, SII increased intake of 1.5% NaCl, despite the SII blockade of IP3 formation and water intake. Examination of brain tissue showed increases in Fos expression in several AngII-sensitive brain areas after injection of AngII, but not SII. The lack of SII-induced IP3 production, water intake, and Fos expression strongly suggest that the PLC/PKC/IP3 pathway is required for water intake, but not NaCl consumption stimulated by AngII. Collectively, these results support the hypothesis that divergent intracellular signals from a single receptor type can give rise to separable behavioral phenomena.
Introduction
DETERMINING THE RELEVANCE of intracellular signaling pathways has been a critical step toward understanding the mechanisms involved in the neural control of behavior. The body of work by Kandel and colleagues, for example, provided a wealth of information about the role of cAMP signaling in the regulation of learning and memory (for review see Ref.1). Progress of this magnitude has been arguably absent from investigations of other behaviors, although initial steps have been taken for many, including reproductive behavior (2, 3), circadian rhythms (4, 5), and the role of the octapeptide hormone angiotensin II (AngII) (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-COO–) in body fluid homeostasis (6, 7).
AngII is a critical component for the maintenance of body fluid and cardiovascular homeostasis. Circulating AngII increases blood pressure and acts on the brain to stimulate water and NaCl intake, both of which are critical for the restoration of body fluids in conditions of hypovolemia. At the behavioral level, AngII induction of water and salt intake have been well studied (for review see Refs.6 and 7). The water intake that follows central injections of AngII is so reproducible that such injections are commonly used to verify accurate intracerebroventricular cannula placement. Unlike the AngII-induced increases in water intake that occur rapidly and tend to plateau by 30 min, increases in salt intake stimulated by AngII tend to be more delayed (for review see Refs.8 and 9) but can be demonstrated in a short time frame when more dilute concentrations of NaCl are offered (e.g. Refs.10 and11) or when oxytocin receptor antagonists are provided (for review see Ref.12).
The increases in water and sodium intake resulting from AngII are initiated largely through stimulation of the angiotensin type 1 (AT1) receptor in forebrain circumventricular organs, including the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO) (13). Like other seven-membrane-spanning receptor family members, agonist binding at the AT1 receptor initiates a host of intracellular events (for review see Ref.14). For example, this receptor is well known to couple to the G protein Gq, which stimulates phospholipase C (PLC) upon agonist binding to generate the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG and IP3 then activate protein kinase C (PKC) and liberate intracellular stores of calcium, respectively. In addition to this more traditionally ascribed signaling pathway, there is growing evidence that AT1 receptor activation also stimulates intracellular events previously attributed primarily to growth factor receptors. The discovery that AT1 receptors can mediate these alternate signaling pathways, including regulation of tyrosine kinase and tyrosine phosphatase activity as well as increased expression of several early immediate genes including c-fos, c-jun, and c-myc (15, 16) provides cellular mechanisms whereby AngII can exert persistent physiological and behavioral changes.
Among the specific, alternate signaling pathways engaged by the AT1 receptor is the phosphorylation (activation) of MAPK family members, including p44/42MAPK, also known as ERK1/ERK2 (17). Although AngII-induced p44/42MAPK activation had been shown to require activation of PKC (18), recent work using AT1 receptor point mutants has revealed that these intracellular events can occur separately (19). As such, it is becoming increasingly clear that in addition to stimulating MAPK through PKC, the AT1 receptor can also stimulate MAPK through alternate means.
In addition to studies using AT1 receptor mutants, strategies to understand the signaling properties of AngII receptors have relied on analogs of AngII. One such analog is a double isoleucine substituted form, Sar1,Ile4,Ile8-AngII (SII) (20). Previous studies used this peptide analog exclusively in vitro as part of an effort to demonstrate that the Tyr4 and Phe8 residues of AngII are important pharmacophores, each of which is critical for the bioactivity of this peptide (20). Not surprisingly, these early experiments focused primarily on the ability of AT1 to activate the PLC/PKC/IP3 pathway. More recent studies, however, demonstrated that when MAPK rather than IP3 was measured, SII acted as an agonist at the AT1 receptor independent of G protein activation (21, 22, 23). Thus, there is growing evidence that the traditionally ascribed G protein-dependent pathway and the more recently discovered MAPK stimulation can occur independently.
Many studies have approached the mechanisms underlying AngII-induced water and NaCl consumption from an anatomical perspective, focusing on the circuitry that links AngII-responsive structures such as forebrain circumventricular organs with other brain areas (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Because there is growing evidence that the AT1 receptor employs multiple signaling pathways that can operate independently, it is possible that the divergent signaling pathways used by the AT1 receptor have different roles in the array of behaviors induced by AngII. Thus, it is critical not only to study the circuitry involved but also to examine the potential relevance of different intracellular events to specific behavioral responses stimulated by AngII. To this end, the present report describes experiments using in vitro and in vivo experiments to examine this potential divergence.
Materials and Methods
Materials
AngII was purchased from Bachem (King of Prussia, PA). SII was synthesized and obtained from Bachem. Tissue culture medium and supplements, including LipofectAMINE reagent were obtained from Invitrogen/Life Technologies (Gaithersburg, MD). [3H]Myoinositol was obtained from American Radiolabeled Chemicals (St. Louis, MO) and [125I]Sar1,Ile8-AngII was obtained from the University of Mississippi Peptide Center (directed by Dr. Robert Speth). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.
Cell culture experiments
COS-1 cells were used for all of the in vitro experiments described here. Cells were grown in polystyrene tissue culture flasks in medium consisting of DMEM supplemented with 10% fetal bovine serum, L-glutamine, and penicillin-streptomycin in a humidified atmosphere of 5% CO2 and 95% O2 at 37 C. AT1 receptor was later introduced into the cells by transfection with LipofectAMINE for 5 h, after which the transfection medium was removed and replaced with normal growth medium. Transfected cells were used for ligand binding experiments, IP3 assays, and MAPK assays as described below.
Ligand binding experiments
Two days after transfection, the cells were rinsed with ice-cold Tris-buffered saline (20 mM Tris-HCl, pH 7.4, and 150 mM NaCl) and then harvested by scraping into 20 mM Tris-HCl (pH 7.4). After polytron homogenization and centrifugation, the membrane pellets were resuspended in assay buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 0.3 TIU/ml aprotinin, and 100 μg/ml 1,10-phenanthroline], and protein content was determined by bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL). The binding assays were initiated by addition of the desired amount of 5 μg membrane protein to the assay mixture containing various concentrations of [125I]Sar1,Ile8-AngII and unlabeled competitors (AngII and SII). Nonspecific binding was defined as the amount of radioligand binding remaining in the presence of 1 μM Sar1,Ile8 -AngII. The binding assays proceeded for 60 min at room temperature and were terminated by rapid dilution with 5 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1.5 mM CaCl2 and vacuum filtration on glass-fiber filters presoaked with 0.3% polyethylenimine, using a Brandell harvester (Brandell, Gaithersburg, MD). Radioligand binding was quantified by -counting of the filters. Data were analyzed and fit to a single-site model.
IP3 assays
IP3 formation was measured as described previously (19). Briefly, AT1-transfected cells were preloaded with [3H]myoinositol (4.5 μCi/ml DMEM) for 18 h before being treated with vehicle (DMEM), AngII, SII, or both AngII and SII for 30 sec. Cells then were washed, lysed, and processed for IP3 quantification by stepwise elution from anion-exchange resin columns. IP3 was quantified by liquid scintillation counting of the IP3-containing fraction. Protein quantification was performed by BCA assay.
In vitro MAPK assays
Activated p44/42MAPK was measured as described previously (35). Briefly, AT1-transfected cells were treated with vehicle (DMEM), AngII, or SII for 5 min before being washed, lysed by Dounce homogenization in cold lysis buffer [25 mM Tris-HCl (pH 8.0), 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM sodium molybdate, 1 mM phenylarsine oxide, 10 μg/ml pepstatin, 2 μg/ml aprotinin, and 10 μg/ml leupeptin], and processed for MAPK immunoblotting (described below) and protein quantification by BCA assay.
MAPK immunoblotting
Western blotting for activated (phosphorylated) MAPK was performed as described previously (35). Briefly, 20–30 μg protein was run on a 10% SDS-PAGE gel and subsequently transferred to a nitrocellulose membrane. Membranes were probed for activated MAPK by incubation with a monoclonal antibody directed against phospho-p44/42MAPK (1:1000) (Cell Signaling Technology, Beverly, MA). The membranes then were washed and incubated with peroxidase-conjugated goat antimouse IgG (1:1000 or 1:2000) (Jackson ImmunoResearch Laboratories, West Grove, PA) before immunoreactivity was detected using chemiluminescence reactions according to the manufacturer’s instructions (Western Lightning kit; PerkinElmer Life Sciences, Boston, MA).
Animals and surgery
Adult, male Sprague-Dawley rats were used for all of the experiments described. The number of animals within each treatment group for each experiment performed is indicated below. Animals were maintained in a temperature-controlled room (22 C) with a 12-h light, 12-h dark cycle and food and water available ad libitum. The handling and care of experimental animals conformed to the regulations provided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
A subset of animals in these experiments were implanted with chronic indwelling cannulas aimed at the third cerebral ventricle (coordinates were on the midline, 2.0 mm posterior to Bregma, and 7.5 mm ventral to dura). Animals were allowed to recover for at least 1 wk before the onset of the experiments. Proper cannula placement was verified by injection of 210 μg 5-thio-D-glucose in 2 μl saline with a subsequent increase in plasma glucose being indicative of proper placement in the ventricle (36). At least 48 h elapsed before onset of the experiments described below. All experiments described here were performed during the lights-on portion of the cycle.
Water intake
Two separate experiments were used to measure water intake. First, using a counterbalanced, repeated-measures design, animals (n = 4) received third ventricle injections of vehicle [1 μl Tris-buffered saline (TBS)], 10 ng AngII, or 1 μg SII with at least 48 h between injections. Water intake was measured 5, 10, 15, and 30 min after the injections. Second, separate groups of animals (n = 3 per group) were injected with either vehicle (1 μl TBS), 10 ng AngII, 10 μg SII, or both AngII and SII. Water intake again was measured 5, 10, 15, and 30 min after the injection.
NaCl intake
NaCl intake was measured using a two-bottle test in which animals had access to water and 1.5% NaCl. Animals (n = 4 per group) were injected with vehicle (1 μl TBS), 10 ng AngII, or 1 μg SII into the third ventricle, and intake was recorded 15 min, 3 h, and 7 h after the injection.
Fos immunohistochemistry
Each animal (n = 3 per group) received a third ventricle injection of either vehicle (1 μl TBS), 10 ng AngII, or 1 μg SII 1 h before being killed by transcardial perfusion with saline followed by phosphate-buffered 4% paraformaldehyde under anesthesia with ketamine and xylazine. Food and water were not available to the animals after the initial injection. Brains were removed from the crania and postfixed overnight before being transferred to 20% sucrose in 0.1 M phosphate buffer. Blocks of brain containing the anterior hypothalamus were cut into three sets of 40-μm coronal sections on a freezing microtome, and one set was processed immunohistochemically for Fos expression as described previously (37). Briefly, the sections were washed, soaked in TBS containing 0.3% H2O2 before being washed and further incubated overnight at room temperature in an antibody directed against the early immediate gene Fos (rabbit anti-c-Fos; 1:5000) (Santa Cruz Biotechnology, Santa Cruz, CA). Sections then were washed and further incubated in biotinylated donkey antirabbit IgG (1:1000) (Jackson ImmunoResearch) followed by another wash and incubation with an avidin-biotin-peroxidase complex (1:333) (Elite Kit; Vector Laboratories, Burlingame, CA). After being washed with TBS and then with 50 mM Tris, immunoreactivity was visualized by reacting the sections with 3,3'-diaminobenzidine (0.2 mg/ml) and 0.025% H2O2 in 50 mM Tris for 15 min. The reaction was stopped with TBS washes, after which the sections were floated onto gelatinized slides, dehydrated with increasing concentrations of alcohol followed by Hemo-De (Fisher Scientific, Pittsburgh, PA), and coverslipped with Permount (Fisher).
In vivo MAPK assays
An additional second set of animals were included in these studies. These animals received acute injections of vehicle (n = 5), 10 ng AngII (n = 5), or 1 μg SII (n = 6). Specifically, a 2-μl Hamilton syringe was loaded with 0.6 μl India Ink (diluted 1:1 in deionized water) followed by 1 μl vehicle (TBS), AngII, or SII. Animals were anesthetized and fixed in a stereotaxic apparatus, and the syringe was lowered into the lateral cerebral ventricle (coordinates were 1.4 mm lateral to midline, 0.9 mm posterior to Bregma, and 3.4 mm ventral to dura). The injections were made in two stages, the first microliter, containing the vehicle or drug, was injected followed by a 9-min delay before the final 0.6 μl was injected and the animal decapitated 1 min later for a total vehicle/drug exposure of 10 min. The brains were rapidly dissected from the crania, halved at the midsagittal plane, and frozen in heptane on dry ice. All animals included had ink in the cerebral ventricular system that was easily visualized after the sagittal cut was made.
A small block of tissue containing the anterior wall of the third ventricle, designed to include the forebrain circumventricular structures, was dissected from the midline of each brain. Tissue blocks were Dounce homogenized in 100 μl ice-cold lysis buffer. After centrifugation, the supernatant was assayed for protein by the BCA method and for activated MAPK by Western blotting as described above.
Data analysis
Radioligand binding data were processed using Prism (version 4.0; GraphPad Software, Inc., San Diego, CA) to calculate Bmax and Kd values for [125I]Sar1,Ile8-AngII and Ki values for AngII and SII. Films from MAPK immunoblots were digitized and OD of each band determined using Scion Image (version 4.0.2; Scion Corp., Frederick, MD). Statistical comparisons were conducted using Sigma Stat (version 2.03; SPSS Inc., Chicago, IL). Details of the tests used are provided for each experiment below. Fos immunoreactivity was quantified by counting labeled nuclei on printed digital micrographs that had been coded such that the treatment condition was unknown. The OVLT and immediately surrounding anterior portion of the median preoptic nucleus (MEPO) were identified with the aid of an atlas of rat brain sections (38). The data were expressed as the mean (±SEM) number of cells per section. A qualitative analysis of Fos immunoreactivity in other brain areas was also conducted. For this analysis, the relative amount of Fos immunoreactivity was rated on a 10-point scale in the posterior portion of the MEPO (the portion of the nucleus separated into dorsal and ventral components by the anterior commissure), the paraventricular hypothalamic nucleus (PVN), the SFO, and the supraoptic nucleus (SON).
Results
In vitro experiments
The overall goal of the in vitro experiments described here was to confirm and extend previous reports showing that the AngII analog SII binds the AT1 receptor, fails to activate the pathway including IP3 formation, but does stimulate MAPK activation (20, 21, 22, 23). We sought to extend these findings in vitro by testing the ability of SII to antagonize the IP3 production stimulated by AngII. The initial step toward these goals was to compare the binding affinities and the concomitant activated signaling pathways of AngII and its analog SII on the AT1 receptor. Using membrane preparations from AT1-transfected COS-1 cells, the receptor exhibited its typical high affinity for the radiolabeled antagonist [125I]Sar1,Ile8-AngII in saturation isotherms (Kd = 0.56 ± 0.09 nM; Bmax = 3.76 ± 1.15 pmol/mg protein; n = 3). Competition binding analysis then was used to determine the relative affinities of AngII and SII for the AT1 receptor. Both AngII and SII bound the AT1 receptor with high affinity, although the affinity for AngII was nearly 50-fold greater than for SII (Ki values: AngII, 18.1 ± 0.64 nM; SII, 972.5 ± 75.1 nM) (Fig. 1). Moreover, the competition curves demonstrate that AngII and SII compete for the same binding pocket.
Using our AT1-transfected COS-1 cell system, we investigated the relative abilities of AngII and SII to activate two distinct signaling pathways: stimulation of the PLC/PKC/IP3 pathway and activation of p44/42MAPK. Treatment of the transfected cells with 250 nM AngII led to reliable increases in both IP3 formation (Fig. 2A) (ANOVA, F2,6 = 13.5; P = 0.006, Student-Newman-Keuls post hoc comparisons) and in activation (i.e. phosphorylation) of MAPK (Fig. 3) (ANOVA, F2,8 = 32.7; P < 0.001, Student-Newman-Keuls post hoc comparisons). Cells treated with 30 μM SII also exhibited reliable increases in activated MAPK (Fig. 3) (ANOVA, F2,8 = 32.7; P < 0.001, Student-Newman-Keuls post hoc comparisons), despite the failure of this concentration of SII to stimulate IP3 formation (Fig. 2A) (ANOVA, F2,6 = 13.5; P = 0.006, Student-Newman-Keuls post hoc comparisons). Moreover, exposure to SII blocked AngII-induced increase in IP3 formation (Fig. 2B) (two-way ANOVA, main effect of AngII, F1,8 = 25.9, P < 0.001; main effect of SII, F1,8 = 9.7, P = 0.014; interaction effect, F1,8 = 16.9, P = 0.003, Student-Newman-Keuls post hoc comparisons). As such, these in vitro experiments with SII confirm and extend previous findings (21, 22, 23) by confirming that SII binds AT1 receptors with high affinity, confirming that SII acts as an agonist with respect to AT1 receptor-mediated MAPK activation and showing for the first time that SII serves as an antagonist with respect to AT1 receptor-mediated IP3 formation.
In vivo experiments
The ability of SII to stimulate only a subset of the intracellular events activated by the AT1 receptor presented a unique opportunity to examine the behavioral relevance of these now separable pathways. As such, after confirming that SII stimulation of the AT1 receptor led to an increase in MAPK without concomitant IP3 release in vitro, we sought to test the behavioral effects of this compound in rats fitted with chronic indwelling cannulas aimed at the third cerebral ventricle. In an initial series of experiments, we used a repeated-measures design to test for differences in water intake between third ventricle injection of vehicle (1 μl TBS), AngII (10 ng), or SII (1 μg). The different doses of AngII and SII were intended to compensate for the lower binding affinity of SII compared with AngII and were chosen based primarily on the effective concentrations used in the in vitro experiments. During the course of the testing, water intake was negligible when animals were treated with vehicle or SII, but predictably elevated when AngII was injected (Fig. 4A) (two-way repeated-measures ANOVA, main effect of drug, F2,18 = 22.5, P = 0.002; main effect of time, F3,18 = 49.1, P < 0.001; interaction, F6,18 = 16.7, P < 0.001, Student-Newman-Keuls post hoc comparisons). In a second experiment, we used a single-trial design in which animals were injected with vehicle, 10 ng AngII, 10 μg SII, or the combination of AngII and SII. Despite the elevated dose of SII used in this set of experiments, the behavioral response to SII alone was identical to before, with indistinguishable amounts of water consumed by vehicle- or SII-treated groups after 30 min. Moreover, like the antagonism of the IP3 response observed in vitro, when SII was given in combination with AngII, SII blocked nearly all of the AngII-induced water intake (two-way repeated-measures ANOVA, main effect of drug, F3,30 = 15.1, P < 0.001; main effect of time, F3,30 = 42.1, P < 0.001; interaction, F9,18 = 24.4, P < 0.001, Student-Newman-Keuls post hoc comparisons). The cumulative water intake data from these animals after 30 min is shown in Fig. 4B.
After determining that SII functioned as an antagonist with respect to both IP3 formation in vitro and water intake in vivo, we performed a set of experiments to evaluate whether NaCl intake could be increased by SII. To this end, we used a two-bottle test to measure water and 1.5% NaCl intake by animals treated with vehicle, 10 ng AngII, or 1 μg SII. Despite the finding that SII blocked AngII-induced IP3 in vitro and water intake in vivo, treatment with SII increased intake of 1.5% NaCl to levels nearly identical to those observed after AngII injection (Fig. 5A) (two-way repeated-measures ANOVA, main effect of treatment, F2,18 = 4.7, P = 0.045; main effect of time, F2,18 = 8.6, P = 0.002; interaction, F4,18 = 2.7, P = 0.06, Student-Newman-Keuls post hoc comparisons). Measures of water intake using this paradigm were elevated in animals treated with SII, but only at the longest time point measured and to a far lesser degree than observed after AngII treatment (Fig. 5B) (two-way repeated-measures ANOVA, main effect of treatment, F2,18 = 41.5, P < 0.001; main effect of time, F2,18 = 32.4, P < 0.001; interaction, F4,18 = 11.0, P < 0.001, Student-Newman-Keuls post hoc comparisons). As such, it is likely that this modest increase in water intake was the result of the osmotic requirements imposed by the increased NaCl intake rather than a direct effect of SII.
The difference in the behavioral responses to AngII and SII likely reflect differences in the intracellular events induced by these compounds. As a first step toward understanding these differences, we focused on the OVLT, a circumventricular brain structure that plays a critical role in the central response to AngII (13, 39, 40, 41, 42), and measured Fos expression, which has been shown to require increases in calcium in several systems (15, 43, 44, 45). As such, the absence of IP3 formation and the resultant increases in calcium after SII treatment would likely lead to measurable differences in Fos expression after AngII or SII treatment. As predicted, Fos immunoreactivity in the OVLT and immediately surrounding anterior MEPO, collectively referred to as the anteroventral wall of the third ventricle (AV3V) (46), was quantified and found to be greater in sections from animals treated with AngII compared with those treated with vehicle or SII (Fig. 6) (ANOVA, F2,8 = 7.38, P = 0.024, Student-Newman-Keuls post hoc comparisons). In contrast to the increases in Fos in the AV3V region after AngII treatment, injections of SII failed to increase Fos expression in this brain area. A qualitative analysis revealed a similar pattern of Fos expression in other brain areas in which AngII-induced Fos has been identified previously (47, 48, 49, 50). Specifically, as shown in Table 1, Fos expression was elevated in animals treated with AngII, but not SII, in the posterior MEPO (at the level of the nucleus where it is split into dorsal and ventral sections by the anterior commissure), PVN, SFO, and SON.
To further evaluate the intracellular events that occur after AngII or SII injection in vivo, we used acute injections of vehicle, AngII, or SII into the lateral ventricle of anesthetized rats. Extraction and examination of a small block of tissue containing the anterior wall of the third ventricle (Fig. 7A) revealed that, as demonstrated in vitro, treatment with either AngII or SII elevated levels of activated MAPK compared with that stimulated by vehicle (Fig. 7) (ANOVA, F2,15 = 4.22, P = 0.039, Student-Newman-Keuls post hoc comparisons). Unlike the data collected from in vitro experiments, however, levels of activated MAPK did not differ between the AngII or SII conditions when these drugs were applied in vivo.
Discussion
The regulation of behavioral state by AngII is a well-studied problem that has provided a wealth of information about the interface between peripheral hormones and central control of behavior. Although a good deal of attention has been paid to the intracellular signaling pathways under the control of the receptors for AngII, relatively little progress has been made toward understanding how these processes mechanistically affect behavior. The experiments described here confirm earlier findings that SII, an analog of AngII, failed to stimulate the traditionally ascribed Gq-mediated IP3 formation but led to increases in activated MAPK, whereas AngII was capable of activating both signaling pathways (21, 22, 23). Extending these findings, the present data provide the first demonstration that SII acts as an antagonist to AngII-induced IP3 formation in vitro and blocks AngII-stimulated water intake in vivo. These data are consistent with previous experiments demonstrating the attenuation of AngII-induced water intake by an inhibitor of PKC (51). As such, these data add to a growing literature indicating the requirement of the PLC/PKC/IP3 pathway for the water intake stimulated by AngII. More striking, however, was the finding that SII mimicked AngII in its ability to stimulate increases in NaCl intake, providing the first evidence that this intracellular pathway is critical for one, but not another, of the behaviors stimulated by AngII treatment. The present data suggest that the different intracellular signaling pathways employed by AngII map directly onto distinct behaviors, the PLC/PKC/IP3 pathway being critical for water but not NaCl intake, whereas activated MAPK may be more important for NaCl intake but is clearly insufficient to stimulate water intake.
When interpreting the present data, it is certainly noteworthy that SII-injected animals showed modest increases in water intake in the two-bottle paradigm used to measure NaCl ingestion; however, the increased water intake was not statistically significant until the longest time point, well after the observed increases in NaCl, and was far smaller than the AngII-induced water intake using the same paradigm. As such, it is very likely that such increases resulted from osmotic perturbations from the increased NaCl intake, rather than as a direct effect of SII. In fact, it would have been remarkable had the animals not compensated for the increased NaCl by ingesting more water.
The experiments showing increased levels of activated MAPK after rats were treated with either AngII or SII suggest that the findings from in vitro experiments can be extended to in vivo preparations. Even with these data and the fact that in vitro models are often excellent predictors of events that occur in vivo, without direct measures of IP3 formation in brain tissue, it is difficult to determine whether the profile of intracellular events stimulated by AngII in vitro is similar in vivo. The data from the experiments using Fos immunohistochemistry, however, provide indirect evidence that the activation profile of SII observed in vitro also occurs in brain. The increased Fos expression observed after AngII treatment in the brain areas examined is consistent with previous studies (47, 48, 49, 50). With respect to the lack of Fos in SII-treated animals, it is important to note that previous reports clearly demonstrate that stimulation of Fos expression depends on increases in intracellular calcium in a number of systems including those involving dopamine receptors (43), renal mesangial cells (44), lymphocytes (45), and AngII stimulation of adrenal cells (15). Moreover, experiments using cultured neuronal cells showed that activation of Fos-regulating kinase by AngII was blocked by PKC inhibition or by the calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (52). As such, the lack of Fos expression after SII treatment in vivo is entirely consistent with the inability of SII to stimulate the PLC/PKC/IP3 pathway in vitro, suggesting that the in vitro models used here are a faithful representation of events occurring in vivo. Moreover, the comparison of Fos expression after AngII and SII treatments further supports the hypothesis that the IP3 pathway is critical for the water intake stimulated by AngII.
The ability of SII to stimulate MAPK but not the IP3 pathway is temporally consistent with the subset of behaviors stimulated by SII. Specifically, IP3-mediated increases in intracellular calcium occur rapidly in comparison with the actions of MAPK. Thus, the finding that SII fails to stimulate rapid increases in water intake but does stimulate NaCl intake, which under physiological conditions is more delayed, fits well with the time course of the subset of the AngII effector pathways stimulated by SII.
The high affinity of SII for the AT1 receptor shown here and previously reported (20) strongly suggests that the responses observed here are specific to AngII receptor stimulation. Whether or not the present phenomenon relies on the AT1 or the type II (AT2) receptor remains an open question. Although the majority of evidence points to the AT1 receptor as the mediator of the behavioral effects of AngII (11, 53, 54, 55, 56), there is evidence, although limited, that AT2 receptor stimulation also plays a role (57). Given the hypothesis proposed here that the ingestion of NaCl stimulated by AngII depends on the activation of MAPK, it is, however, highly unlikely that the response to SII is AT2 mediated because the AT2 receptor does not appear to couple to MAPK (19). Indeed, in previous experiments using AT2-transfected COS-1 cells, agonist stimulation of this receptor subtype failed to activate p44/42MAPK (19). Nevertheless, determining which receptor subtypes are critical for the response could be addressed by future studies using receptor-subtype-specific antagonists in combination with SII administration.
In summary, the present report confirms previous findings that SII stimulates MAPK family members in vitro but fails to activate the intracellular pathway that includes IP3 formation. The present data notably extend these previous findings with the demonstration that SII blocked AngII-induced stimulation of IP3 formation in vitro and water intake in vivo. Furthermore, we describe the unexpected finding that central injections of SII increased NaCl intake. These data strongly suggest that the IP3 pathway is required for the water intake stimulated by AngII and offer the possibility that the activation of p44/42MAPK mediates AngII-induced NaCl intake but is insufficient for the stimulation of water intake. The present observations support the novel hypothesis that intracellular pathways stimulated by AT1 receptor activation can be separated based on behavioral relevance. These data highlight the importance of intracellular events in the regulation of behavioral state and provide novel information about the means through which single hormones can influence multiple behaviors.
Acknowledgments
We thank Drs. Harvey J. Grill and Joel M. Kaplan for help with the behavioral analysis portion of these experiments. Technical support was provided by Laiyi Luo and Aae Suzuki.
Footnotes
Funding for this work was provided by the following awards from the National Institutes of Health: DK064012 (to D.D.), HL058792 (to D.K.Y.), and DK052018 (to S.J.F.).
First Published Online August 25, 2005
1 D.D. and D.K.Y. contributed equally to this manuscript.
Abbreviations: AngII, Angiotensin II; AT1, AngII type 1; AT2, AngII type 2; AV3V, anteroventral wall of the third ventricle; BCA, bicinchoninic acid; DAG, diacylglycerol; IP3, inositol trisphosphate; MEPO, median preoptic nucleus; OVLT, organum vasculosum of the lamina terminalis; PKC, protein kinase C; PLC, phospholipase C; PVN, paraventricular hypothalamic nucleus; SII, Sar1,Ile4,Ile8-AngII; SFO, subfornical organ; SON, supraoptic nucleus; TBS, Tris-buffered saline.
Accepted for publication August 19, 2005.
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