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Am J Physiol Regul Integr Comp Physiol 282: R10-R18, 2002;
0363-6119/02 $5.00
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Vol. 282, Issue 1, R10-R18, January 2002

Ingestive responses to administration of stress hormones in baboons

R. E. Shade1, J. R. Blair-West1,2, K. D. Carey1,3, L. J. Madden1, R. S. Weisinger4, J. E. Rivier5, W. W. Vale5, and D. A. Denton1,4

1 Department of Physiology and Medicine and 3 Southwest Regional Primate Research Center, Southwest Foundation for Biomedical Research, San Antonio, Texas 78245-0549; 2 Department of Physiology and 4 Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Melbourne, Victoria 3010, Australia; and 5 The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental stress and the administration of the stress hormone ACTH have been reported to stimulate sodium appetite in many nonprimate species. Experiments were conducted to determine whether prolonged intracerebroventricular infusions of the neuropeptides corticotropin-releasing factor (CRF) and urocortin (Ucn), or systemic administration of ACTH, affected ingestive behaviors in a nonhuman primate, the baboon. Intracerebroventricular infusions of CRF or Ucn significantly decreased daily food intake. The decrease with Ucn continued into the postinfusion period. These infusions did not alter daily water intake. Daily voluntary intake of 300 mM NaCl solution was not increased, and there was evidence of reductions on days 2-4 of the infusions. Intramuscular injections of porcine ACTH or synthetic ACTH (Synacthen) for 5 days did not affect daily NaCl intake, although the doses were sufficient to increase cortisol secretion and arterial blood pressure. Sodium depletion by 3 days of furosemide injections did induce a characteristic sodium appetite in the same baboons. These results demonstrate the anorexigenic action of CRF and Ucn in this primate. Also, CRF, Ucn, and ACTH did not stimulate sodium appetite at the doses used.

food intake; sodium appetite; corticotropin-releasing factor; urocortin; adrenocorticotropic hormone; sodium deficiency


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PREVIOUS STUDIES IN BABOONS from this laboratory have shown that sodium depletion induces a salt appetite (11) and that brain angiotensin is involved in the ingestive behaviors of thirst and sodium appetite (3, 31). We have now investigated the peptides implicated in stress, corticotropin-releasing factor (CRF), urocortin (Ucn), and ACTH. There is an extensive literature on the effects of stress, and in particular CRF and ACTH, on the ingestion of food, water, and salt in various mammalian species, but there are very few reports concerning ingestive and other behavioral responses in primates (13, 15).

CRF is a primary hormone involved in the initiation of the behavioral and physiological responses to stress (32, 37). The major actions of CRF (for reviews, see Refs. 20, 25, 32) are anorexia, release of ACTH, activation of the sympathetic nervous system, increased locomotor activity, thermogenesis, and modulation of the immune response. The effect of CRF on food intake was unaffected by hypophysectomy (14, 21), and the reversal by adrenalectomy was attributed to the removal of medullary catecholamine (14, 26). The actions of CRF are mediated by CRF-R1 and CRF-R2alpha receptors in the brain and CRF-R2beta receptors in the brain and peripheral tissues (16, 19, 24, 25). The recently discovered Ucn (36), a 41-amino acid peptide, has CRF receptor-binding characteristics and a potency that suggest that Ucn may mediate some of the actions previously attributed to CRF (23, 33).

CRF and Ucn have been identified in brain areas known to be involved in the control of fluid and electrolyte metabolism, e.g., supraoptic and paraventricular nuclei and the organum vasculosum of the lamina terminalis (6, 17, 38). Experiments in rabbits (35, 36) and in mice (10) indicated that centrally infused CRF stimulated NaCl intake in those species. Those results are consistent with other evidence that experimental stresses increased NaCl intake in several species (8-10, 18).

When the present experiments began, it was expected that CRF and, in its turn, ACTH would stimulate sodium appetite in the baboon. The systemic administration of ACTH has been shown to increase arterial blood pressure in several species (1, 28, 43) associated with specific stimulation of sodium chloride ingestion in rabbits (2), sheep (41), rats (42), and mice (4, 10). This effect on sodium appetite was reproduced in rabbits (2), sheep (41), and mice (4) by administration of a mixture of corticosteroids. A major aim of the present study was, therefore, to determine whether ACTH also stimulates sodium intake in baboons.

The results of recent experiments in sheep (40), however, are at variance with the proposal that the stress cascade of CRF-ACTH-corticosteroid secretion increases NaCl intake. The central administration of CRF or Ucn or ACTH, in sheep, all decreased food intake, but each of them also decreased need-free NaCl intake. Systemic infusion of ACTH did increase NaCl intake, as expected, but systemic infusion of CRF or Ucn decreased NaCl intake, and the concurrent infusion of Ucn with ACTH blocked the salt appetite response to ACTH. Thus it appeared that the hormones associated with stress might have both excitatory and inhibitory effects on NaCl intake.

Against the background of these conflicting findings, experiments were conducted in baboons to determine whether CRF, Ucn, or ACTH could effect changes in food, water, or salt intake in a primate species. Because CRF and Ucn are neuropeptides with major sites of action within the hypothalamus, the effects of these two peptides were tested in baboons by chronic intracerebroventricular infusions (3). The physiological effects of ACTH are achieved through its release into the systemic circulation, and, therefore, the effects of ACTH administration were tested by a course of intramuscular injections. Experiments with administration of furosemide were included to establish that the stimulus of Na depletion evoked a sodium appetite in the baboons used for ACTH experiments.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Maintenance

Twelve adult male baboons weighing 25-35 kg were studied in individual metabolism cages fitted with stainless steel urine collection pans. All animals were habituated to the cages for 6-8 wk before starting experimental observations. The daily food ration consisted of 500 g of pelleted food (Purina Monkey Chow 25-5045-6 from Purina Mills was the base for this diet) formulated by the Southwest Foundation for Biomedical Research experimental diet facility to contain 20 mmol Na/kg and ~190 mmol K/kg. The sodium content of ingested food was included in all sodium balance calculations. On this diet, the baboons maintained body weight and good health. Most of the ingested sodium appeared in the daily urine loss.

Water and 300 mM NaCl (when it was presented for experiments) were available ad libitum from containers connected to valves attached to the cage sides and activated by the baboon's tongue (Lixit). Twenty-four-hour intakes of food, water, and 300 mmol NaCl as well as 24-h urine volume were measured daily at 1200-1300. Infusion experiments were started when daily intakes of food and fluids were constant.

All procedures and protocols were approved by the Southwest Foundation Institutional Animal Care and Use Committee.

Surgical Preparation

Six baboons were prepared for intracerebroventricular infusion by placing a 22-gauge stainless steel cannula in the left lateral brain ventricle as described previously (3). This preparation permitted infusion of CRF, Ucn, or 0.9% NaCl into cerebrospinal fluid from osmotic pumps (Alzet, Alza, Palo Alto, CA) placed subcutaneously in the midscapular region of the baboon's back. The surgical procedures (under 1-2% isoflurane anesthesia) used for replacing osmotic pumps for intracerebroventricular infusions of test agents during experimental periods or 0.9% NaCl during control and recovery periods have been described (3).

Test Agents

The human forms of CRF and Ucn were synthesized in the Clayton Foundation Laboratories for Peptide Biology at the Salk Institute. For intracerebroventricular infusion, the peptides were dissolved in sterile water using gentle, magnetic stirring and slight warming until the solution was complete (up to 1 h for Ucn). Solutions were then passed through sterile 0.22-µm filters using aseptic techniques and then placed into 2ML1 (1 wk) osmotic pumps.

CRF and Ucn solutions were prepared at a concentration that delivered 5 µg/h from osmotic pumps with a nominal infusion rate of 10 µl/h. This dose for intracerebroventricular infusion of CRF was chosen after consideration of doses used in other species (7, 12, 29, 36) and after conducting preliminary studies in two baboons with intracerebroventricular infusions of 1 and 10 µg/h for 7 days. The lower dose had no effects on food, water, or salt intake; the higher dose caused large reductions in each of those intakes in only one of the baboons. This variability in response between the baboons used for the preliminary experiments suggests that the 5-µg/h dose of CRF used for the main study was probably at the lower end of the dose-response range for baboons. The amounts of CRF and Ucn required for 7 days of infusion precluded performing more dose-response studies. The 5-µg/h dose of Ucn was chosen so that it would be almost equimolar with the dose of CRF, noting that Ucn was more potent than CRF in effects on hemodynamic parameters and stimulation of ACTH and cortisol release (23).

Two preparations of ACTH were used for this study. One set of experiments with porcine ACTH (ACTHAR, corticotropin for injection, Armour Pharmaceutical, Blue Bell, PA) used intramuscular injections of 40 U.S.P. units, 2 times/day = 80 U/day for 5 days in six baboons. The experiments were repeated in five of six baboons using a long-acting form of synthetic ACTH (Synacthen Depot, CIBA) given intramuscularly at a dose of 25 IU, 2 times/day = 50 IU/day for 5 days. These doses of synthetic ACTH were similar to doses used in sheep (28, 41) to produce increases in blood pressure and salt ingestion. Similar doses of this synthetic preparation of ACTH in humans have also produced increases in blood pressure and metabolic effects (5, 43).

Experimental Protocols

1. Intracerebroventricular infusions of CRF and Ucn. Baseline observations were collected for 4 days with intracerebroventricular infusion of physiological saline (0.9% NaCl). The osmotic pump was changed at ~0900 to a pump containing either CRF or Ucn at a concentration that delivered 5 µg/h. After 7 days of peptide infusion, the osmotic pump was replaced with another pump containing 0.9% NaCl, and recovery observations were collected for 7 days.

The delivery of CRF or Ucn by intracerebroventricular infusion was verified by intracerebroventricular injection of 5 µg of ANG II (human, Bachem, Torrance, CA) into the infusion cannula before attaching the replacement pump. This procedure and its purpose have been described (3). Under 1-2% isoflurane anesthesia, the pump containing peptide was detached and the infusion system was flushed with 2 ml of 0.9% NaCl. Then 5 µg of ANG II were injected into the catheter and flushed with 2 ml of 0.9% NaCl. This injection increased arterial blood pressure 10-20 mmHg for 5-15 min with a small increase in heart rate. Previous experience with this bolus injection of ANG II indicated that a large intake of 300 mmol NaCl solution would occur on the postoperative day. This intake was not preceded by a natriuresis, and intracerebroventricular infusion of ANG II stimulated NaCl intake in baboons before the increased intake caused an increase in urinary Na excretion (3). To avoid this response to the ANG II test of patency, NaCl solution was withheld through the first day of recovery in the CRF and Ucn experiments.

The preparation of baboons for surgery to change osmotic pumps included removing the food bin from the animal's cage at ~1600 on the preceding day. Thus the baboons had only 3-4 h of access to food on that day, and sometimes there was food in the bin when it was removed.

Body weight was measured, and a blood sample for plasma chemistries was collected when the baboons were sedated at the time of the surgery for changing osmotic pumps.

2. ACTH administration. After 5 days of baseline observations, ACTH preparations were administered as intramuscular injections given twice per day (at 0800 and 1800) for 5 days. Baboons were immobilized during baseline and treatment periods by moving the back of the cage forward with a crank mechanism. This procedure typically required no more than 1 min to achieve an intramuscular injection into the thigh. Recovery observations were then made over a period of 5 days.

The six baboons used for the ACTH administration experiments were tested for the ability to increase NaCl intake in response to a sodium-depletion protocol described previously (11). This experience was their first exposure to NaCl solution. Briefly, daily water, 300 mM NaCl, and food intakes were measured for a baseline period of 5 days. This was followed by 3 days of diuretic treatment in which furosemide (Lasix, Hoechst) was given at 1 mg/kg im, 2 times/day. Recovery observations followed for a period of 7 days. Daily urinary sodium excretion and total sodium intake were used to calculate daily sodium balance to compare the amount of sodium depletion produced by furosemide treatment and the amount of the increase in sodium intake. Fecal sodium excretion was not measured. ACTH infusion experiments were conducted weeks to months after this test.

The physiological activity of Synacthen was verified in separate experiments using baboons placed on a tether system that permits blood sampling and measurements of blood pressure and heart rate of conscious, unrestrained baboons while being maintained in their home cage (30). Arterial blood pressure and heart rate were monitored in one baboon for 7 control days, 5 days of intramuscular injection of Synacthen at 25 IU 2 times/day at 0800 and 1800, and for 7 recovery days. In another baboon, blood samples were collected at 0800, 1200, and 1600 for 3 days. Synacthen was injected intramuscularly at 25 IU at 0800 and 1600 on day 2. Plasma cortisol was measured in the samples by radioimmunoassay. Blood samples were also obtained from three baboons, under ketamine anesthesia, at 0800 on the fifth baseline day and on the fifth injection day of the Synacthen experiment. Plasma cortisol was also assayed in these samples.

Analytic Procedures

Urine sodium concentration was measured by flame photometry (Corning model 450). Plasma cortisol concentration was measured by radioimmunoassay (Coat-a-Count, Diagnostic Products, Los Angeles, CA). Hematocrit and plasma creatinine, total protein, glucose, sodium, and potassium were measured in blood samples by standard methods.

Statistical Analysis

Data are presented as means ± SE. During baseline periods, the baboons typically consumed the entire daily 500-g ration and there were variable degrees of decrease in food intake during the intracerebroventricular infusions of CRF and Ucn. Thus the distribution of food intake data in baseline and infusion periods did not meet the assumptions required to conduct parametric and some nonparametric analyses of these data. For this reason, a Fisher Exact Test was used to compare daily food intake during the baseline period with intake during the infusion or recovery period by categorizing each day's food intake as <= 500 g. A Friedman repeated-measures analysis of variance and a subsequent Student-Newman-Keuls test for ranked data (Sigmastat) were used to compare the baseline intake of water (or NaCl) with the daily intake of water (or NaCl) during or after infusion of CRF or Ucn or treatment with ACTH. The mean of the values obtained on the 3-5 days before each infusion or treatment for each animal was used in determining the baseline value. The same statistical tests were applied to the results of daily NaCl intake in the furosemide treatment experiment. A two-tailed probability of <0.05 was considered significant. Effects of treatments on body weight were assessed by paired t-test.

Note that one baboon was given three doses of CRF, 1 and 10 µg/h in preliminary experiments and 5 µg/h in the main experiment, and 5 µg/h of Ucn. In all of these experiments, this baboon ate all of the 500 g of food offered daily during the four infusion periods (except for 1 postoperative day). It also responded normally to the angiotensin test at pump change, indicating patency of the infusion cannula. This unique set of results suggests that all of the doses of CRF and Ucn were beneath the threshold in this baboon. Note also that this same baboon had average daily NaCl intakes of 2.3 ± 0.4 and 2.8 ± 0.6 mmol/day during baseline periods. This amounts to a volume of 7-8 ml/day, which is similar to the small volumes that were lost daily in disconnecting and reconnecting the delivery system to weigh the water and NaCl solution bottles. Therefore, this baboon could not contribute to the results of experiments in which NaCl intake was reduced, and it diminished the statistical significance of changes for the other five baboons with respect to reductions in both NaCl intake and food intake. The baboon was, however, included in the analysis of grouped data.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of CRF Infusion

The results for the six CRF experiments are shown in Fig. 1. Mean daily food intake (Fig. 1A) was 500 g during the baseline period, except for day 4 when one baboon ate only 380 g. This single variation from 500 g was attributed to the routine removal of the food bin at 1600 on the day preceding surgery (as described in METHODS). Food intake fell to 282 ± 57 g on the first day of CRF infusion and 289 ± 78 g on the second day. The mean intake then increased gradually to day 7 of infusion, except for the same baboon who ate less food on day 7 than he did on day 6, probably because, again, the food bin was removed at 1600 on day 7. The food intake during CRF infusion was significantly less than the intake during the baseline period. Daily food intake was similar to baseline during the recovery period.


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  		Fig. 1.   Effect of intracerebroventricular (ICV) infusion of corticotropin-releasing factor (CRF) for 7 days at 5 µg/h (n = 6 baboons) on daily intakes of food (A), water (B), and 300 mM NaCl solution (C). Values are means ± SE. *P < 0.05 compared with the baseline period (food intake) and baseline values (water and NaCl intakes).

The mean body weight of the baboons was 31.4 ± 1.3 kg on the day CRF infusion started. Mean body weight was 30.9 ± 1.3 kg [not significant (NS)] when the pump was replaced 7 days later.

Mean daily water intake during CRF infusion (Fig. 1B) was not significantly different from that during the baseline period. Two baboons did have reduced water intakes 1 or 2 days early in the CRF infusion period. This was not observed in the other four baboons. Thus, there was no regular relation between the daily water intake and the changes of daily food intake during CRF infusion.

Mean daily hypertonic NaCl intake (Fig. 1C) on the 4 baseline days varied from 132 ± 61 (day 1) to 111 ± 53 (day 4) mmol/day. This variability was mainly due to interanimal and not to intra-animal variation. Individual baboons drank 2 ± 1, 12 ± 3, 65 ± 6, 85 ± 15, 235 ± 23, and 326 ± 6 mmol/day; some baboons consistently showing little or no interest in the NaCl solution and others consistently having an avid appetite. On these grounds, the NaCl intake was confirmed as need-free (see METHODS).

Five baboons drank considerably less than usual NaCl solution on 2 days during days 2-4 of CRF infusion. One baboon whose baseline NaCl intake was 65 ± 6 mmol/day drank 1.2 mmol on day 2. Three other baboons drank <1.0 mmol of NaCl on day 3, and three baboons drank 2-6 mmol of NaCl on day 4. The mean intake on day 3 was 30 ± 17 mmol (P < 0.05), which was 20-25% of the mean baseline values. After day 4, mean NaCl intake increased to the levels and to the variability of the baseline period. The intake of 0 mmol on day 1 of recovery was due to withholding the NaCl supply for 1 day postoperatively (see METHODS). The low intakes on day 3 of recovery followed the very high intake that occurred when NaCl was returned to the baboons on recovery day 2. NaCl intake was similar to baseline values during recovery days 4-7.

Total sodium intake was less than urinary sodium output in all six baboons on either day 2 (n = 4) or day 3 (n = 2) of the CRF infusion and on recovery day 1 (n = 6) when NaCl was not available (data not shown). At all other times, this balance was positive.

Seven days of CRF infusion had no effect on hematocrit or plasma concentrations of creatinine, total protein, glucose, sodium, and potassium.

Effects of Ucn Infusion

The effects of Ucn infusion in all six baboons are shown in Fig. 2. Mean food intake (Fig. 2A) was 500 g/day during the 4-day baseline period, except for one baboon who had not eaten all of his food when it was removed at 1600 on the day before surgery. Food intake fell to 348 ± 68 g on the first day of Ucn infusion and remained low into the end of the recovery period. Food intake, relative to baseline, was significantly decreased during both the infusion and the recovery periods.


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  		Fig. 2.   Effect of ICV infusion of urocortin for 7 days at 5 µg/h (n = 6 baboons) on daily intakes of food (A), water (B), and 300 mM NaCl solution (C). Values are means ± SE. *P < 0.05 compared with the baseline period (food intake) and baseline values (water and NaCl intakes).

The mean body weight of the baboons was 32.2 ± 1.4 kg on the day the pump with Ucn was introduced. The weight was 32.0 ± 1.2 kg (NS) when this pump was replaced 7 days later.

Mean daily water intake (Fig. 2B) appeared to be slightly less than baseline values, beginning at the start of the Ucn infusion and continuing through the Ucn infusion period. Despite this trend, the changes were not significant.

Daily intake of NaCl solution (Fig. 2C) averaged 106 ± 48 mmol/day during the baseline period. Again, baseline daily NaCl intakes varied widely between baboons, but were consistent for individuals, and were similar to their values for the CRF experiments; they were 3 ± 1, 22 ± 2, 33 ± 2, 83 ± 13, 269 ± 28, and 226 ± 37 mmol/day in the same animal sequence. Mean NaCl intake was 56 ± 23 mmol on day 2 and 59 ± 27 mmol on day 3 of Ucn infusion. Subsequently, all mean daily NaCl intakes were equal to or greater than baseline values, except for recovery day 1 when NaCl was not available. The high intakes on day 2 of recovery were probably related to that withholding, as occurred in the CRF experiment.

Total sodium intake was less than urinary sodium output (data not shown) in all six baboons on either day 2 (n = 3) or day 3 (n = 3) of the Ucn infusion (2 baboons were still in negative balance on day 4) and on recovery day 1 (n = 6) when NaCl solution was not available. At all other times, this balance was positive.

Seven days of Ucn infusion had no effect on hematocrit or plasma concentrations of creatinine, total protein, glucose, sodium, and potassium.

Effects of ACTH Injection

Preliminary sodium-depletion experiment. The ability of sodium depletion by furosemide injection to stimulate a 300-mM NaCl intake in the six baboons that were to be given ACTH is shown in Fig. 3. The mean daily NaCl intake was 5.4 ± 2.8 mmol/day for the 5 days of baseline observations. Three days of furosemide injections (1 mg/kg, 2 times/day) increased NaCl intake to 28 ± 12 mmol on the third day of furosemide treatment. There was a cumulative average net urinary loss of 39 mmol of sodium for the 3 days of furosemide treatment despite the increase in NaCl intake. Stimulation of NaCl intake continued during the 3 days after furosemide with peak intake occurring on recovery day 2 (91 ± 32 mmol). Hypertonic NaCl intake then returned to the values observed during the baseline period. The NaCl intakes for the groupings, baseline days 3-5, furosemide days 1-3, recovery days 1-3, and recovery days 4-6 differed significantly (P < 0.05).


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  		Fig. 3.   Effect of intramuscular injection of furosemide for 3 days at 1 mg/kg, 2 times/day (n = 6 baboons), on daily intake of 300 mM NaCl solution (A) and daily urinary sodium excretion (B). Values are means ± SE.

Effect of Porcine ACTH on NaCl Intake

The effect of porcine ACTH administration (80 U.S.P./day, n = 6) on hypertonic NaCl intake and sodium balance is shown in Fig. 4, left. Daily hypertonic NaCl intake averaged 8.7 ± 2.7 mmol during the baseline period. There was no significant effect of ACTH administration for 5 days on NaCl intake (average = 10.0 ± 3.3 mmol/day). There was a small increase in NaCl intake during the recovery period after the ACTH injections were stopped (5-day average = 13.7 ± 3.7 mmol/day, P < 0.05 compared with baseline and ACTH periods).


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  		Fig. 4.   Effect of intramuscular injection of 2 ACTH preparations for 5 days on daily intake of 300 mM NaCl solution (top) and daily sodium balance (bottom). Left: porcine ACTH at 40 U.S.P. units 2 times/day = 80 U/day; n = 6 baboons used for the furosemide study (Fig. 3). Right: synthetic ACTH at 25 IU 2 times/day = 50 IU/day; n = 5 of the same baboons. Values are means ± SE.

There was no significant effect of ACTH on daily total sodium intake or urinary sodium excretion (data not shown). A small positive daily sodium balance of 4.0 ± 4.8 mmol/day was observed during the baseline period (Fig. 4, bottom left). There was no significant effect on sodium balance during ACTH administration or during the recovery period. Porcine ACTH also had no significant effect on food or water intake (data not shown).

Effect of Synthetic ACTH on NaCl Intake

The effects of administration of a long-acting synthetic form of ACTH (50 IU/day) in five of the baboons studied above are shown in Fig. 4, right. Hypertonic NaCl intake averaged 9.7 ± 4.2 mmol/day during baseline observations, 4.9 ± 2.3 mmol/day during Synacthen administration, and 12.0 ± 6.2 mmol/day during the recovery period (Fig. 4, top right) (differences NS).

There were also no significant effects of Synacthen administration on daily total sodium intake and urinary sodium excretion (data not shown), on daily sodium balance (Fig. 4, bottom right), or on food and water intakes (data not shown).

Physiological Activity of Synthetic ACTH

In a tethered baboon, the mean 24-h average blood pressure varied between 81 and 86 mmHg (average 83.4 ± 1.8 mmHg) during the 7 baseline days. Synacthen administration (50 IU im) was associated with a gradual increase in blood pressure over the 5 days of treatment so that blood pressure was increased by 12-14 mmHg for days 3-5 of ACTH administration. Blood pressure returned to baseline on the first recovery day. The mean 24-h heart rate ranged from 59 to 64 beats/min during baseline (average 61 ± 3 beats/min), fell to 54-59 beats/min during ACTH treatment, and increased to 63-72 beats/min during the recovery period.

Plasma cortisol concentrations at 0800 in three of the baboons used for the Synacthen experiment (Fig. 4) were 23, 24, and 35 µg/dl on baseline day 5 and 67, 68, and 77 µg/dl on day 5 of the ACTH treatment. The effect of 25 IU 2 times/day of Synacthen on diurnal plasma cortisol concentration in a single-tethered baboon is shown in Fig. 5. Plasma cortisol concentration increased five- to sixfold at 1200 and 1600 on the day of administration, and it increased threefold at 0800 the next day. Plasma cortisol was still increased twofold at 1200 on the day after treatment, indicating the prolonged activity of the preparation.


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  		Fig. 5.   Effect of intramuscular injection of 25 IU synthetic ACTH at 0800 and 1600 on plasma cortisol concentration in 1 baboon.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of these experiments in a nonhuman primate were that the neuropeptides CRF and Ucn reduced daily food intake during the course of prolonged intracerebroventricular infusion and that these infusions or prolonged systemic administration of ACTH did not increase NaCl intake. The most unexpected finding was that ACTH was without effect on NaCl intake despite evidence that the doses were sufficient to increase plasma cortisol concentration and arterial blood pressure in baboons. Evidence is also presented that the infusions of CRF and Ucn reduced NaCl intake on day 2 or day 3 of infusion, associated with a brief period of negative Na balance. This latter result is different from earlier findings that intracerebroventricular infusion of ovine CRF increased NaCl intake in rabbits (35, 36) and in mice (10). The result is, however, consistent with more recent findings that human CRF and rat Ucn, administered intracerebroventricularly or intravenously, inhibited need-free NaCl intake in sheep (40).

Although the reduction of NaCl intake during CRF or Ucn infusions was not sustained, the reduction of food intake was sustained during these infusions and appeared to continue into the recovery period in the Ucn experiments. There are few studies of prolonged intracerebroventricular infusion of CRF or Ucn in other species to compare with these results. But, in 4 days of intracerebroventricular infusions of CRF or Ucn in sheep at 5 µg/h, the same dose as used in the present experiments, food intake was significantly reduced throughout the infusion, and the reduction continued into the recovery period, particularly with the Ucn infusion (40). Also, in rhesus monkeys, repeated daily intracerebroventricular injections of CRF decreased home cage food intake, body weight, and responding for food (13). More pronounced effects of Ucn on food intake, compared with CRF, may be related to the observation that the mediating CRF-R2 receptors have 6-40 times greater affinity for Ucn than for CRF (33, 38).

The result that ACTH administration did not evoke a Na appetite in baboons was unexpected. Studies in nonprimate species have shown that experimental stress can increase NaCl intake in several species (8-10, 18), that intracerebroventricular infusion of CRF stimulated NaCl intake in rabbits (35, 36) and in mice (10), and that systemic administration of ACTH increased NaCl intake in several species (2, 4, 10, 41, 42). The results of the present study do not support a CRF-ACTH-corticosteroid pathway of stimulation of Na appetite by stress in this nonhuman primate. In addition, intracerebroventricular administration of CRF or Ucn, at doses that significantly decreased food intake, did not stimulate Na appetite in baboons.

Further interpretation of these data is made difficult by the relatively small number of baboons available for study, compounded by the wide variation in daily NaCl intake. This variability and the tendency for repeated exposures to NaCl solution to enhance its intake were noted previously (3, 31). Wide variations of hedonic intake of NaCl are not uncommon in other primates, notably humans (9). The consistently high intake of NaCl solution by some baboons is not evidence that they were sodium deficient on the diet alone. All baboons maintained body weight and good health before and between experiments when they had no access to the NaCl solution. Most of the daily dietary sodium appeared in the daily urine loss. Finally, when experimentally naive baboons had their first access to NaCl solution, intakes were consistently low (e.g., baselines of ACTH and furosemide experiments). When experienced baboons had access to NaCl, the intakes varied from 2 ± 1 to 326 ± 6 mmol/day, intakes being characteristic of each baboon and independent of any dietary factor as the diet was common to all baboons. An earlier publication (3) noted that "with time and the number of experiments, the baseline mean intake of 300 mM NaCl had increased to ~100 mmol/day compared with ~10 mmol/day in Fig. 1" (the first experiment). Very similar mean baseline values for groups of naive and experienced baboons were observed in the present experiments.

The failure of ACTH to stimulate Na appetite in baboons provides a ready, although not necessarily complete, explanation for the failure of CRF and Ucn to increase Na intake as has been reported in other species (10, 35, 36). This finding, however, does not seem to be related to the observed transient reduction of NaCl intake by CRF and Ucn. That observation seemed anomalous until the recent discovery of similar inhibitory actions of CRF and Ucn on daily NaCl intake in sheep (40). Intriguing features of those experiments were that intracerebroventricular infusions of CRF or Ucn inhibited need-free NaCl intake, that peripheral infusion of Ucn blocked the NaCl intake caused by ACTH administration, but that intracerebroventricular infusion of Ucn did not block the NaCl intake of Na-depleted sheep. The brain mechanism regulating the Na appetite response to Na deficiency could not be overridden by this neuropeptide.

Little is known about the pathway concerned with hedonic or need-free Na intake, whereas there is information on Na appetites that arise from stimulation by corticosteroids or stimulation by Na depletion. In rats, the amygdala and bed nucleus of the stria terminalis may be the sites of Na appetite stimulation by corticosteroids (27), whereas angiotensin may stimulate Na appetite in the anterior third ventricle region in baboons (3) as in rats (26) and other species (9). How CRF or Ucn modulates Na intake in need-free situations is not clear, but evidence from sheep studies (40) indicates that Ucn blocked the central action of corticosteroids in causing Na appetite but did not block the central action of Na depletion in causing Na appetite. The effect of Ucn on Na depletion-induced appetite in baboons is unknown.

In the present experiment, treatment with ACTH did not cause a decrease in food intake. Thus, in baboons, as in rats (14, 21) and in mice (4, 10), the decrease in food intake caused by CRF administration appears to be independent of the release of ACTH and the secretion of glucocorticoid hormones. Another possibility is that the decrease in food intake caused by CRF or UCN is due to the release of oxytocin. Evidence has shown that the decrease in food intake caused by intracerebroventricular administration of CRF in rats is blocked by the prior administration of an oxytocin antagonist (22). Interestingly, oxytocin appears to be one of the main inhibitory factors that limit the expression of sodium appetite (34, 39). Thus, an influence of CRF or Ucn on the release of oxytocin, as in rats, could explain the decrease in food intake and the failure to stimulate Na intake.

In summary, these experiments in baboons have shown that both CRF and Ucn are anorexigenic, and CRF also transiently reduced need-free NaCl intake. This effect of these neuropeptides on food intake is not novel, but it extends the phenomenon into primate species. The effects on NaCl intake were unexpected because, at the time the experiments were carried out, there was supportive evidence that experimental stress and administration of CRF stimulated Na appetite in several species. The finding that systemic administration of ACTH did not alter Na intake in baboons raises the question of whether this is the case in other nonhuman primates as well as humans.

Perspectives

Data have been reported in many species that support the hypothesis that several components of the hormonal cascade stimulated by stress contribute to an increase in salt intake behavior. This effect has been interpreted as an appropriate response for adaptation to environmental conditions where increasing total body sodium would potentially increase survival. The present study shows that the factors associated with stimulation of the hypothalamic-pituitary-adrenal (HPA) axis did not promote salt intake in baboons. This pathway may be of little significance in stressed primates compared with a role in other mammals. However, the HPA axis is only one of several hormonal systems stimulated by stress, and there is little information available in regard to whether stress has any effect on salt intake behavior in primates. For example, several studies have shown that stress stimulates the renal renin-angiotensin system, and we have shown that the central infusion of angiotensin produces large increases in sodium appetite in the baboon. Clearly, additional studies are required to determine whether stress influences sodium appetite in primates.


    ACKNOWLEDGEMENTS

The assistance provided by R. Ison, D. Weaver, and E. Jackson is greatly appreciated.


    FOOTNOTES

This study was supported by grants from the Robert J. Kleberg, Jr., and Helen C. Kleberg Foundation; the G. Harold and Leila Y. Mathers Charitable Foundation; National Institutes of Health Grant P51-RR-13986 to the Southwest Regional Primate Research Center; and the National Health and Medical Research Council of Australia.

Address for reprint requests and other correspondence: R. E. Shade, Southwest Foundation for Biomedical Research, P.O. Box 760549, San Antonio, TX 78245-0549 (E-mail: bshade{at}sfbr.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 25 July 2000; accepted in final form 28 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Regul Integr Comp Physiol 282(1):R10-R18
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