JOURNAL OF EXPERIMENTAL ZOOLOGY 286:343–349 (2000) Intra- and Extracellular Dehydration Has No Effect on Plasma Levels of Angiotensin II in an Amphibian LORETTA P. MAYER* AND CATHERINE R. PROPPER Northern Arizona University, Department of Biological Sciences, Flagstaff, Arizona 86001 ABSTRACT Previous studies have demonstrated that both dehydration (intra and extracellular) and treatment with angiotensin II (A-II) induce changes in thirst-related behavior in the spadefoot toad, Scaphiopus couchii. One of the steps in determining a causal relationship between a hormone and a behavior is to determine that there is association between an animal’s performance of the behavior and changes in endogenous hormonal concentrations. The hypothesis tested that plasma levels of the peptide hormone A-II would change as a result of dehydration known to induce water absorption response (WR) behavior in the spadefoot toad. Plasma samples were taken from toads dehydrated intracellularly by injection of hypertonic solutions of NaCl or sucrose at levels known to induce WR behavior. As an osmotic control, a group of animals was injected with urea, which has been demonstrated to not induce WR behavior. In order to determine the effects of extracellular dehydration on plasma, A-II levels in toads dehydrated by plasma volume depletion via cardiac puncture were compared to sham-punctured controls. None of the treatments in any experiment resulted in significant differences in plasma levels of angiotensin II among groups sampled at the time when WR behavior occurs. These results do not support the hypothesis that dehydration-induced thirst is stimulated by changes in plasma A-II concentrations at the onset of WR behavior. J. Exp. Zool. 286:343–349, 2000. © 2000 Wiley-Liss, Inc. Drinking is the primary behavioral response to dehydration for most vertebrates (Grossman, ’90). An understanding of the physiological mechanism that initiates drinking behavior is key to understanding how terrestrial vertebrates avoid debilitating dehydration. Fish, reptiles, birds, and mammals rehydrate through oral intake of fluids. However, in anuran amphibians, water is taken in across the ventral seat patch as an animal abducts its hind limbs and presses the pelvic region to a wet substrate (Stille, ’52; Bentley and Yorio, ’79). This behavior is termed water absorption response (WR) by Brekke et al. (’91) and can be induced by denying animals access to water (Brekke et al., ’91; Propper and Johnson, ’94). Although the behavior in amphibians is different from most other taxa, water-obtaining behavior is still elicited by dehydrating conditions. Primary drinking behavior is initiated by dehydration (Fitzsimons, ’79). Dehydration may occur as a result of intracellular loss of fluids or fluid depletion of the extracellular space. For example, when water is removed from cells (intracellular dehydration) or plasma volume is decreased (extracellular dehydration), oral drinking behavior is induced in dogs (Gilman, ’37; Bellows, ’39), rats (Adolph et al., ’54; Fitzsimons, ’61; Ramsay et al., © 2000 WILEY-LISS, INC. ’76; Janczewski et al., ’87; Salisbury and Rowland, ’90; Stricker ’91), mice (Rowland and Fregly, ’88), reptiles (Fitzsimons and Kaufman, ’76) and amphibians (Bentley and Yorio, ’78; Brekke et al., ’91; Taylor et al., ’99). Intracellular dehydration can be induced experimentally by administration of hypertonic solutions to an animal, thus increasing the osmotic gradient outside the cell resulting in intracellular dehydration. Intracellular dehydration induced by treatment with hypertonic saline solutions leads to drinking behavior. Although the specific behavioral response is different in anuran amphibians, again, treatment with hypertonic solutions initiates drinking (cutaneously). When water is removed from cells (intracellular dehydration), an increase in WR behavior is observed in the spadefoot toad Scaphiopus couchii (Taylor et al., ’99). In these studies, dehydration was induced by administration of solutions hypertonic to the animal, which increases the osmotic concentration in the extracellular space reGrant sponsor: National Science Foundation; Grant number: IBN 93-10352. *Correspondence to: Loretta P. Mayer, Northern Arizona University, Dept. of Biological Sciences, P.O. Box 5640, Flagstaff, AZ 86001. E-mail: firstname.lastname@example.org Received 1 December 1998; Accepted 20 July 1999 344 L.P. MAYER AND C.R. PROPPER sulting in an osmotic gradient that induces intracellular dehydration. Treatment with urea, which passes freely across the cell membrane and does not create an osmotic gradient, was ineffective in inducing any significant increase in WR behavior. Voluntary water intake has also been observed following extracellular dehydration resulting from a depletion of blood volume. In rats, a blood volume reduction of one-third induced an oral drinking response (Ramsay et al., ’76; Stricker, ’91). In iguanas, extracellular dehydration induced by injection of hyperoncotic polyethylene glycol caused a significant increase in drinking when compared to vehicle injected controls (Fitzsimons and Kaufman, ’76). These studies demonstrate that multiple forms of dehydration induce behavioral rehydration response across many taxa. Extracellular dehydration as a result of a reduction in blood volume has also been observed to be a stimulant of WR behavior in amphibians by Taylor et al. (’99). The specific signals that precipitate WR behavior are unclear. It has been suggested that the hormone angiotensin II (A-II) is an important dipsogen in other taxa (Epstein et al., ’70; Fitzsimons and Kaufman, ’76; Kobayashi et al., ’78; Gray and Erasmus, ’88; Wright et al., ’88; Volmert and Firman, ’91). Again, although the behavioral response is different, A-II also initiates WR behavior in amphibians (Hoff and Hillyard, ’91; Propper and Johnson, ’94; Propper et al., ’95; Hillyard et al., ’98). These studies demonstrated that administration of exogenous A-II resulted in a significant increase in WR behavior. The results of these studies suggest that plasma levels of AII may change with the hydration state of the animal, and that these changes may play a role in the induction of behavior. To determine if the role of plasma A-II is causal in the induction of WR behavior, it is necessary to establish that there is an association between the animal’s performance of the behavior and changes in endogenous plasma hormone concentrations. This study investigates whether intracellular and or extracellular dehydration induces changes in plasma levels of A-II in the spadefoot toad, Scaphiopus couchii. Prior to each experiment, the animals were weighed, then hydrated in dechlorinated tap water for 3 hr. Following this hydration period, the toads were again weighed, their bladders were emptied by cloacal cannulation with a small pipet tip, and they were weighed again before receiving treatment. Because toads can reabsorb water from the urinary bladder and this reabsorption is increased during dehydration, it is necessary to control for this condition by using animals with empty bladders (Ruibal, ’62). The vehicle for the intraperitoneal injections was amphibian Ringer’s (115 mM NaCl, 2.5 mM KHCO3, 1.0 mM CaCl2; pH 8.0) at 20.0 µl/g toad. After injection, the animals were placed in a dry plastic box (30 × 15 × 10 cm) for 30 min. Following the 30-min period, the toads were weighed and blood samples collected by cardiac puncture for hormone and osmolarity measurements. Toads were weighed again and returned to the 10-gallon tanks with 1 liter of dechlorinated tap water to rehydrate for 1 hr prior to being returned to their storage boxes. For experiment 1, toads (n = 8) were treated with (a) Ringer’s (b) Ringer’s + 0.5 M NaCl, and (c) Ringer’s +1.0 M NaCl. In experiment 2, toads (n = 7) were treated with (a) Ringer’s, (b) Ringer’s + 0.5 M sucrose, and (c) Ringer’s + 1.0 M sucrose. These doses have been demonstrated previously to induce WR behavior in this species (Taylor et al., ’99). In experiment 3, toads (n = 7) were treated with (a) Ringer’s and (b) Ringer’s + 1.7 M urea. All toads received all treatments in random order over the course of the experiment. Toads never received more than one treatment during any given week. MATERIALS AND METHODS Extracellular dehydration Scaphiopus couchii were collected from the Buenos Aires Wildlife Refuge, Pima County, Arizona, during the monsoon season (June–July) in 1996. The animals were transported to the laboratory in large containers (53 × 41 × 18 cm) that Toads (n = 10) were initially weighed and hydrated in dechlorinated tap water for 3 hr. Following this hydration period, the toads were bladder emptied by cloacal cannulation with a small pipet tip, and weighed again before receiv- contained 15 cm of loose peatmoss and water ad libitum. Approximately 10 toads (weight range, 14–30 g), were stored in each container. Once in the laboratory, the toads were transferred, one toad per container, to a plastic box (30 × 15 × 10 cm) containing 7 cm soil, and water was available ad libitum. The animals were fed crickets three times a week. The room was maintained at a temperature of 25°C and a 12 hour light:12 hour dark photoperiod. Intracellular dehydration DEHYDRATION AND PLASMA ANGIOTENSIN II ing treatment. A 0.5-cc syringe with a ½ inch, 25gauge heparinized needle was used for the heart puncture procedure. For experiment 4, the animals received (a) a sham puncture into the pericardial space with no blood removed or (b) a –± cardiac puncture through which 127.3 ± 10.4 (x SE) µl of blood was removed. After treatment, the animals were placed in a dry plastic box (30 × 15 × 10 cm) for 30 min. Toads were then weighed, sampled, weighed, and rehydrated as previously described. For each experiment, each individual received all treatments. There was a minimum of one week between each treatment regimen. Hormone measurements Hormone levels were assayed using a fluid phase radioimmunoassay (RIA). Samples were collected in peptidase inhibitor described by Ledwith et al. (’93). The RIA was based on the technique described by Hanssens et al. (’90). Briefly 125I-labeled A-II (Ile5) (specific activity = 2 × 106 µCi/ µMole), purchased from Amersham (Buckinghamshire, England), was suspended in 10 mM K2HPO4, 1 mM EDTA, 0.1% gelatin (P-gel buffer) buffer, pH 7.3, and incubated with rabbit A-II antiserum (Peninsula Laboratories, Inc., Belmont, CA) in Pgel buffer. Antiserum titer used was 1:100,000, and cross-reactivity with AIII was 100%, and results are reported as immunoreactive angiotensin (irANG) concentrations. Unbound label was separated from bound with a dextran/charcoal slurry. A Beckman LS5000TD gamma counter was used to detect the bound hormone-antibody complex. Serum concentrations of total angiotensin (AII and AIII) were calculated from premeasured standards (0.95–500 pg/100 µl) using four-parameter logistic analysis of the data using Assay-Zap software (Biosoft, Ferguson, MO). All samples within each experiment were run in the same assay. The limit of detection for the assay was 1.9 pg/100 µl with an intraassay cv of 11.29% (n = 10). A five-point serial dilution of S. couchii plasma was determined to be parallel to the standard curve. Osmolarity measurements were determined by vapor pressure osmometry using a Wescor 5500. During the collection of data, the osmometer was recalibrated every 20 samples with 100, 290, and 1000 mmole/kg standards to eliminate drift in the readings. Osmolarity measurements were taken from plasma samples following hormone measurement. Due to an insufficiency of plasma collected from some individuals, not all animals were measured. 345 Data analysis The differences in plasma concentrations of angiotensin and osmolarity measurements among treatments were analyzed using a one-way repeated measures ANOVA for experiments 1 and 2 and a paired t-test for experiments 3 and 4. The functional relationships between plasma hormone levels and osmolarity were analyzed by linear regression. RESULTS Intracellular dehydration Experiment 1 Sodium chloride treatment did not induce any significant changes in plasma levels of irANG (Fig. 1a; n = 8, P = 0.772). Osmolarity measurements among the three treatments were not significantly different (Table 1; n = 5, P = 0.652). There was no correlation between plasma irANG levels and osmolarity measurements in any of the NaCl treatment groups (Table 2; n = 5). Experiment 2 Sucrose treatment also did not induce any significant changes in plasma levels of irANG (Fig. 1b; n = 7, P = 0.068). Osmolarity measurements among the treatment groups were not significantly different (Table 1; n = 4, P = 0.215). There was no functional relationship between plasma irANG levels and osmolarity measurements in any of the sucrose treatment groups (Table 2; n = 4). Experiment 3 Treatment with urea had no significant effect on plasma levels of irANG (Fig. 1c; n = 7, P = 0.956). Osmolarity measurements among the treatment groups were not significantly different (Table 1; n = 6, P = 0.104). There was no functional relationship between plasma irANG levels and osmolarity measurements in any of the urea treated groups (Table 2; n = 6). Extracellular dehydration Experiment 4 Depleting blood volume by heart puncture did not cause any significant changes in plasma levels of irANG (Fig. 1d; n = 10, P = 0.181). Osmolarity measurements among the treatment groups were not significantly different (Table 1; n = 8, P = 0.407). There was no functional relationship between plasma irANG levels and osmolarity measurements in any of the treatment groups (Table 2; n = 8). 346 L.P. MAYER AND C.R. PROPPER Fig. 1. Plasma irANG concentrations following treatments with increasing doses of osmolytes or cardiac puncture in male S. couchii. (a) NaCl treatments; n = 8; P = 0.772; (b) sucrose treatments; n = 7; P = 0.068; (c) urea treatment; n = 7; P = 0.956; (d) blood volume reduction; n = 10; P = 0.181. Bars represent the mean for each treatment group ± SEM. No treatment resulted in changes in plasma irANG. DISCUSSION however, was not significantly different from the other two groups. The plasma osmolality for the second group was significantly elevated over animals in group 3, and there was no difference in plasma irANG between the three groups. Brain levels of irANG did differ among the groups in the periventricular nucleus. Animals displaying WR behavior had higher levels than those in the other two groups (Johnson, ’97). These data support the possibility that the role of A-II in amphibian thirst behavior may be a central one. Laboratory animals dehydrated by 5, 10, and 15% of their standard weight also did not have significantly different plasma levels of irANG compared with fully hydrated controls, even though animals dehydrated by 15% of their standard weight demonstrated significantly higher WR behavior from the other groups (Johnson, ’97). In these studies, the toads that were dehydrated by 15% had significantly higher plasma osmolalities when compared with controls. These data are similar to the results reported here in that dehy- Both intracellular and extracellular dehydration treatments known to induce WR behavior (Taylor et al., ’99) were ineffective in inducing any significant change in circulating plasma levels of irANG. Further, these treatments did not significantly elevate the osmolarity of circulating plasma. These results are consistent with other studies in amphibians. Plasma irANG measurements of spadefoot toads exhibiting WR behavior in field conditions were not significantly different from toads exhibiting breeding behavior or other activities (Johnson, ’97). In the field study of Johnson (’97), animals were collected during one of three behaviors: (1) displaying WR behavior on the first two nights of the monsoon rains, (2) active on the roadway not in WR behavior on nights 3 and 4, and (3) in a breeding pond on the fourth night. The animals in the first group were presumed to be displaying WR behavior to rehydrate following their lengthy estivation. The plasma osmolality for this group, DEHYDRATION AND PLASMA ANGIOTENSIN II TABLE 1. Effects of different dehydration treatments across all experiments on plasma osmolarity in S. couchii Treatment Mean osmolarity following treatment (±SE) mmol/kg n=5 0 M NaCl 0.5 M NaCl 1.0 M NaCl n=4 0 M sucrose 0.5 M sucrose 1.0 M sucrose n=6 0 M urea 1.7 M urea n=8 Sham puncture Cardiac puncture P value (repeated measures ANOVA) 274.6 (±5.64) 284.6 (±20.87) 291.0 (±12.46) 0.652 264.3 (±15.7) 314.0 (±27.7) 283.7 (±11.1) 0.215 263.8 (±13.2) 251.2 (±7.53) 0.104 283.4 (±14.45) 272.8 (±6.81) 0.407 drating conditions sufficient to induce WR behavior, and animals performing WR behavior in field conditions and the laboratory, do not have altered levels of plasma irANG when compared to hydrated individuals. Studies in rat are also consistent with these findings relative to plasma levels of A-II. Intravenous infusion of A-II (10–200 ng/kg/min) resulted in a drinking response that increased in a dosedependent manner to 100 ng/kg/min, then leveled off. The response to the highest dose was observed within eight min, and plasma levels of A-II for doses above 50 ng/kg/min were equivalent to plasma A-II levels after 48 hr of dehydration, however, there were no significant differences among A-II treated TABLE 2. Correlation of plasma levels of irANG and plasma osmolarity following each dehydration treatment in S. couchii Treatment n=5 0 M NaCl 0.5 M NaCl 1.0 M NaCl n=4 0 M sucrose 0.5 M sucrose 1.0 M sucrose n=6 0 M urea 1.7 M urea n=8 Sham puncture Cardiac puncture Correlation of plasma levels of irANG and osmolarity following treatment (r2) F value (linear regression) 0.653 0.343 0.002 5.65 1.57 0.005 0.179 0.652 0.258 0.218 1.87 0.348 0.387 0.325 2.52 0.237 0.481 0.385 5.51 3.75 347 groups at the onset of drinking behavior, which is achieved with the minimal (50 ng/kg/min) dose of A-II, indicating a threshold relationship may exist (Anke et al., ’88). These studies suggest that A-II plays a role in stimulating drinking behavior only when dehydration is severe. To investigate the potential dipsogenic role of A-II, studies have been conducted to compare methods of administration of exogenous A-II and their resultant effects on WR behavior. Induction of WR behavior by intracerebroventricular (icv) injection of A-II in the spadefoot toad was reported at 10 ng (Propper et al., ’95). This dose is approximately 1,000-fold less than the dose necessary to induce behavior via intraperitoneal (ip) injection in the same species (Propper and Johnson, ’94). These studies suggest that A-II may be functioning as a neurotransmitter in the induction of WR behavior in anurans, and that plasma A-II may not be a factor in generating a thirst response in this species. Recent studies on A-II induced gene expression addresses the potential role of A-II as a neurotransmitter with associated behavioral changes. Extracellular dehydration following hemorrhage in rats resulted in an increase in the production of Fos, a marker of cell activation and c-fos, an early-response gene, in the subfornical organ, organum vasculosum of the lamina terminalis, supraoptic and paraventricular nuclei (Badoer et al., ’93). Further studies showed that icv injections of A-II resulted in intense staining for c-fos in the median preoptic nucleus, organum vasculosum, subfornical organ, paraventricular nucleus, and supraoptic nucleus (Mahon et al., ’95). The expectation that this gene expression was a result of an increase in A-II was supported by the expression of c-fos following stimulation of the AT1 receptor in the midbrain and brainstem of rats that were treated with icv A-II (Lebrun et al., ’95). Mahon et al. (’95) subsequently observed similar increases in c-fos expression that correlated with an increase in drinking behavior. Further investigation of the potential mechanism of the A-II neurotransmission involved in drinking has shown mitogen-activated protein kinase (MAPK), an important signaling pathway for the stimulation of c-fos like genes, to be upregulated in rat neuronal cultures stimulated with A-II (Lu et al., ’96). These studies are the first to demonstrate this downstream signaling pathway in A-II-mediated neuromodulation in noradrenergic neurons. Studies of the kinetics of the A-II induced expression of cfos in vitro have shown that once c-fos expression 348 L.P. MAYER AND C.R. PROPPER is initiated, the process becomes independent of the hormone-receptor interaction (Garcia-Sainz et al., ’95), suggesting that plasma levels of A-II may spike to a threshold level necessary for induction of the signaling cascade and then recede to prestimulated levels. These studies, in total, support the hypothesis that the role of A-II in the initiation of a water response behavior as a result of dehydration occurs centrally. Therefore, changes in plasma levels of A-II would not necessarily be observed following, or even concurrent with, the performance of the behavior. This is consistent with observations in a natural field population of male S. couchii performing WR behavior at the onset of the monsoon season wherein levels of A-II in the periventricular nucleus of the hypothalamus were elevated over active or breeding toads. The irANG concentrations in the PVN of these animals did not correlate with plasma angiotensin concentrations (Johnson, ’97). Another possibility is that desert anurans are relatively insensitive with respect to an A-II response to moderate dehydration levels. Studies of birds have shown that plasma levels of A-II increase three-fold with a significant correlation in osmolality and drinking behavior following severe dehydration by water deprivation (Gray and Erasmus, ’88). Birds were water deprived for 3–5 days to induce a 5% reduction in standard weight. This amount of reduction in standard weight for this species is considerable and amounts to severe dehydration, whereas a reduction in standard weight of desert anurans of up to 45% can be tolerated. These studies suggest that dehydration induces an increase in plasma levels of A-II following severe dehydration and that this increase is positively correlated with osmolality that reflects the decrease in both intra- and extracellular water, and drinking that occurs following these levels of dehydration may be associated with plasma levels of A-II. This presents the possibility that A-II may play a role in dehydration-induced drinking when dehydration levels are severe. 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