INTRODUCTION

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OUR RECENT STUDIES (1, 10) in baboons (Papio hamadryas sensu lato) found that prolonged intracerebroventricular (ICV) infusion of ANG II increased the daily voluntary intake of water and 300 mM NaCl solution. Concurrent ICV infusions of ANG II receptor antagonists inhibited those responses and also inhibited the increased water intake caused by water restriction and the increased 300 mM NaCl intake caused by furosemide administration. These findings, consistent with results in some other nonprimate mammals, led to the proposal that brain ANG II was involved in both thirst and Na appetite in baboons (1, 10).

In peripheral ANG systems, ANG II is the main effector peptide in the systemic circulation, although exogenous ANG III can be as potent as ANG II in, for example, stimulating aldosterone secretion (2) or inhibiting renin release (3) in sheep. In the rat brain, ANG III was found to be equipotent with ANG II as a pressor agent or dipsogen (4, 7, 13-15), and then Harding et al. (6) observed that 93% of ANG released in the paraventricular nucleus was ANG III. ICV ANG III was not as potent as ANG II in stimulating water intake in sheep (11).

The importance of locally produced ANG III in brain regulatory systems concerned with arterial blood pressure, water and salt intake, and release of pituitary hormones (12) has been highlighted by the findings of new experiments using ICV infusions of specific aminopeptidase A and N inhibitors (8, 9, 16). The authors concluded that the conversion of ANG II to ANG III is actually required for ANG II to regulate brain pathways concerned with vasopressin release and arterial blood pressure.

Accordingly, it seemed important to determine whether central ANG III was equipotent with ANG II in stimulating water and salt intake in a primate, using ICV infusions of these peptides.

	METHODS

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Animals and maintenance. Six adult male baboons weighing 25-35 kg were studied in individual metabolism cages fitted with stainless steel urine collection pans. They were fed 500 g/day of pelleted food (prepared at Southwest Foundation from Purina Monkey Chow 25-5045-6) containing ~20 mmol Na/kg and ~190 mmol K/kg. Water and 300 mM NaCl were continuously available from containers mounted on the roof of the cage and connected to pressure-sensitive drinking valves. Twenty-four-hour intakes of food, water, and NaCl solution and 24-h urine volumes were measured daily at 1200-1300. Experiments began when daily intakes of food and fluids were constant.

Surgical procedures. The procedures involved in 1) implanting a lateral cerebral ventricular cannula and 2) connecting and changing Alzet osmotic pumps (Alza, Palo Alto, CA) subcutaneously for intraventricular infusions have been described (1). Baboons were fasted from 1600 before pump implants and had access to food for only 3-4 h on the day of surgery. On that day, they were anesthetized or in recovery throughout the morning. For those reasons, the intakes of water, NaCl solution, and food were often reduced in that 24-h period.

Agents used. The vehicle for infusions was sterile 0.9% saline. Solutions were prepared in sterile syringes and Millipore filtered into osmotic pumps. 2-ML-2 or 2-ML-4 pumps were used during baseline periods, and 2-ML-1 (10 µl/h) pumps were used for 7-day infusions of agents.

Experimental procedures. After a baseline of 6 days, the pump containing 0.9% saline was changed at ~0900 to a pump containing ANG III (4.6 µg/10 µl). After 7 days of observations, this pump was changed at ~0900 to a pump containing saline. Observations were continued for 7 recovery days. After a rest period of at least 1 mo, the experiment was repeated with ICV infusion of ANG II (5.0 µg/10 µl).

Statistical analysis. Data are presented as means ± SE. A two-way repeated-measures ANOVA (2-factor repetition) was used to test for significant differences between the effects of ANG III and ANG II. A one-way repeated-measures ANOVA was used to detect significant time effects within each infusion treatment with post hoc least significant difference tests to establish significant changes from the baseline condition.

	RESULTS

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Daily intakes of fluids during baseline and ANG infusion periods varied considerably between baboons (Fig. 1). For example, in the ANG III experiments, the baboon with the lowest NaCl intake in the baseline period consumed 5.0 ± 1.2 mmol/day (mean of the baboon's 6 baseline day values), whereas the highest consumption was 230 ± 24 mmol/day. ANG III infusion increased daily NaCl intake in these two baboons to maxima of 57 and 818 mmol, respectively. One baboon had little or no increase in daily fluid intakes with ANG III infusion, whereas the other five baboons increased NaCl intake 3- to 15-fold and almost doubled water intake. Similar variability was observed in the ANG II experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Effects of 7 days of intracerebroventricular infusion of ANG II (circles) at 5.0 µg/h or ANG III (diamonds) at 4.6 µg/h on daily intakes of 300 mM NaCl (top) and water (bottom). Daily Na balance (Na intake in NaCl solution and food minus Na loss in urine) is also shown (middle). Values are means ± SE (n = 6). * P < 0.05 and *** P < 0.001, comparing combined ANG II/ANG III (difference between them not significant) with baseline.

There were no significant differences (2-factor ANOVA, repeated measures) between the effects of ANG III and ANG II (Fig. 1) on daily NaCl intake, Na balance (Na intake from NaCl solution and food minus Na loss in urine), water intake, or food intake (not shown in Fig. 1). For the combined ANG III and ANG II data, 1) NaCl intake (Fig. 1, top) was significantly increased compared with baseline on all infusion days except day 1, 2) water intake (Fig. 1, bottom) was significantly increased on infusion days 3-6, and 3) Na balance was not significantly changed (Fig. 1, middle).

When the ANG III and ANG II data were tested separately (1-factor ANOVA, repeated measures), both of the peptides significantly increased NaCl intake on infusion days 3-7 compared with baseline. Both peptides significantly increased water intake on infusion day 3. The increase continued for 4 days with ANG III and for 3 days with ANG II infusion.

The mean daily Na balances were all small positive values throughout the baseline period (consistent with the unmeasured daily loss of Na in feces) except on baseline day 6, when pre- and postsurgical procedures reduced NaCl and water intakes (Fig. 1). There was no evidence that the baboons went into negative Na balance before the increases in NaCl intake caused by peptide infusion. Mean daily Na balances varied more widely during the infusion period than in the baseline period. Baseline values were almost restored by the end of the recovery period.

Three of six baboons reduced daily food intake during the ANG II infusion; two of those baboons had also eaten less during the ANG III infusion. Those three baboons were the first to increase daily NaCl intake in response to ANG infusion, and their intakes rose to the higher levels.

	DISCUSSION

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If ANG III were the main effector peptide of the brain ANG system, then it ought to be as potent as ANG II in mimicking the central actions of this peptide system. The close similarity of the actions of ANG III and ANG II on water and NaCl intake in these baboons suggests that the role of ANG III in stimulating these ingestive behaviors could be analogous with the major role proposed for ANG III in regulating pressor and vasopressin release systems in the rat brain (8, 9, 16).

The comparison of ANG II and ANG III in the present experiments was limited to a single equimolar dose level. The 5-µg/h level of ANG II was shown to be consistently effective in stimulating NaCl and water intake in earlier experiments (1, 10), and the same responses were observed here. Evidence presented here, and earlier with respect to ANG II (1, 10), indicates that the increases of NaCl intake beginning on day 1 or day 2 of peptide infusion were not secondary to urinary loss of Na caused by possible central and cardiovascular actions of exogenous angiotensins.

Conclusions based on a single large equimolar dose level of the peptides must be treated with some caution because studies with large doses in rats and other species tend to favor the argument that ANG II is a more potent dipsogen than ANG III (for review, see Ref. 5). There is also uncertainty arising from the unknown relative stability of these peptides in cerebrospinal fluid and brain tissue in baboons.

The high NaCl and water intakes reached by days 3-4 of the two ANG infusions were not sustained through the remainder of the infusion period. Late declines were observed in most baboons, but the greatest impact on mean intake values was due to the large declines that occurred in the baboons with the largest increases in water and NaCl intakes. For those baboons, the declines may be explained by feedback from cardiovascular and gastrointestinal systems, including malaise. Such reactions may also explain the transient reduction in daily food intake in some baboons.

In summary, the equipotency of ICV infusions of ANG III and ANG II in evoking water and salt ingestions in baboons satisfies one of the prerequisites for establishing a role for brain ANG III in the regulation of these behaviors. However, proof of that role, up to the level of proving that the conversion of ANG II to ANG III is a requirement for this function of the brain ANG system, must wait on the availability of specific aminopeptidase inhibitors.

Perspectives