increased daily 3 % NaCl intake, but large variability was observed in the response.

Transcription

1 J. Physiol. (1987), 393, pp With 7 text-figures Printed in Great Britain SALT APPETITE IN THE PIGEON IN RESPONSE TO PHARMACOLOGICAL TREATMENTS BY A. N. EPSTEIN AND M. MASSI* From the Department of Biology and Mahoney Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. and * Institute of Pharmacology, University of Camerino, Camerino, Italy (Received 9 December 1986) SUMMARY 1. In response to furosemide-induced sodium depletion pigeons showed a robust salt appetite. Following the 1st depletion they started to ingest 3% NaCl after a latency of s and in 24 h they took ml of this solution (vs. a daily mean intake of 1-2 ml prior to the depletion). 2. The appetite was selective as shown by the fact that when, after depletion, 0 34 M-CaCl2 was offered (which is equiosmotic to 3 % NaCl) pigeons took just a trivial amount of it. 3. Analysis of sodium losses following the natriuretic treatment revealed that pigeons respond to sodium depletion with an excessive overconsumption of NaCl solution. In the 2 h after access to salt they took about 3 times the amount of sodium lost. 4. Repeated sodium depletions sharply reduced the latency to the ingestion of salt and produced larger intakes. However, the overall amount of salt taken in 24 h after the later depletions was very similar and statistically indistinguishable from that taken following the 1st depletion. 5. Subchronic deoxycorticosterone acetate treatment (2 mg pigeon-' day-' I.M.) increased daily 3 % NaCl intake, but large variability was observed in the response. 4 mg pigeon-' day-' evoked a reliable 3% NaCl intake which was particularly marked from the 5th day of the treatment. 6. Pulse intracerebroventricular (I.c.v.) injection of purified hog renin evoked water intake within about 1 min of injection, followed (about 6 h later) by increased salt intake. In the 24 h after renin injection pigeons took ml of 3% NaCl. On the 2nd day following injection salt intake was still higher than in controls. 7. In conclusion, our results show that pigeons respond to sodium depletion with a robust salt appetite. Moreover, salt appetite can be evoked by deoxycorticosterone acetate as well as by renin. These findings suggest that in the pigeon salt appetite may be an endocrine-induced behaviour controlled by mineralocorticoids and by the renin-angiotensin system.

2 556 A. N. EPSTEIN AND M. MASSI INTRODUCTION Mammals maintain their sodium content relatively constant and to do so they must restore their losses of sodium by salt intake. Several species display an innate appetite for salt when they are sodium depleted. This has been shown in herbivores like the rabbit and the sheep (see for review, Denton, 1982), and in the carnivorous dog (Fitzsimons & Moore-Gillon, 1980; Ramsay & Reid, 1979), as well as in the omnivorous rat (see for review, Denton, 1982; Fregly & Rowland, 1986) and monkey (Schulkin, Leibman, Ehrman, Norton & Ternes, 1984). There is also both clinical and laboratory evidence demonstrating that humans show an appetite for salt when sodium deficient (McCance, 1936; Wilkins & Richter, 1940). Although many animal species may express the appetite, the physiological mechanisms which generate it may be different from one species to another. Most of the work on this problem has been done in rats and in sheep. In sheep, the sodium concentration in the cerebrospinal fluid and therefore in the adjacent brain appears to dictate the expression of the appetite (Weisinger, Considine, Denton, Leksell, McKinley, Mouw, Muller & Tarjan, 1982). On the other hand, in the rat the cerebrospinal fluid sodium concentration does not influence the arousal and the expression of the appetite (Osborne, Weisinger & Denton, 1983; Epstein, Zhang, Schultz, Rosenberg, Kupsha & Stellar, 1984). Instead, salt appetite appears to be an endocrine-induced behaviour, which can be aroused by both angiotensin and aldosterone treatment even in the sodium-replete animal (Rice & Richter, 1943; Fregly & Waters, 1966; Avrith & Fitzsimons, 1980; Bryant, Epstein, Fitzsimons & Fluharty, 1980; Epstein, 1982; Fluharty & Epstein, 1983). These differences between rats and sheep leave us ignorant of the general mechanism for salt appetite that may have evolved among complex animals. This issue can only be understood by studying the problem in a broader range of animal species. We have therefore extended the investigation of salt appetite and its physiological mechanisms to the pigeon in order to study it in another order of animals. The pigeon is a common experimental animal that habituates easily to laboratory confinement. It is granivorous and its diet, in the wild, is therefore not high in sodium. Moreover, it is very sensitive to the behavioural effects of angiotensin II (Evered & Fitzsimons, 1981), and it depends on mineralocorticoids for its sodium homeostasis (Sandon, Farekas & Robinson, 1976). For these reasons we thought it interesting to investigate in this animal species (a) whether it expresses an appetite for salt when sodium depleted and (b) whether the appetite can be evoked by the same hormones that elicit it in the rat. METHODS Animals The subjects in all experiments were male White Carneoux pigeons (Columba livia) (Palmettos Pigeon Plant, Sumter, SC, U.S.A.) weighing between 400 and 600 g. They were housed in individual cages in a temperature-controlled room ( C) on natural lighting. They had continuous access to tap water, 3 % NaCl solution, mineral grit (Gran-I-grit, North Carolina Granite Corp., Mt Airy, NC, U.S.A.) and grain food (Pro-Il Breeder Pigeon Grains, No. HP 5431, Purina, Richmond, IN, U.S.A.) except when noted.

3 SALT APPETITE IN THE PIGEON 557 Sub8tances The following substances were employed: (1) furosemide (Lasix, American Hoechst Corporation, Somerville, NJ, U.S.A.); (2) deoxycorticosterone acetate (Sigma Chemical Co., St Louis, MO, U.S.A.); and (3) renin, which was generously supplied by Professor Detlev Ganten of the German Institute for High Blood Pressure Research. It was their preparation E III which is purified from hog kidneys by affinity chromatography as described by Ganten, Speck, Meyer, Loos, Schelling, Rettig & Unger (1980). Surgery Under ketamine-acepromazine anaesthesia ( p1l/100 g body weight of a solution containing ketamine, mg/ml, and acepromazine, 1.37 mg/ml)- a stainless-steel guide cannula (o.d. = 600him) was stereotaxically implanted 1 mm above the third ventricle according to the technique described by Evered & Fitzsimons (1977) and employing the stereotaxic co-ordinates of the Karten & Hodos atlas (1968). The animals were allowed at least 1 week to recover from surgery before being tested. Elicitation of 8alt appetite Sodium depletion. Sodium depletion was produced by an adaptation of the method developed by Wolf (1982), which combines pharmacological natriuresis with removal of sodium from the experimental environment. Natriuresis was produced by intramuscular (I.M.) injection of furosemide (two injections of 5 mg 0.5 ml-' pigeon-', separated by 2 h). Mineral grit as well as 3% NaCl solution were removed from the pigeon's cage 20 h before the first furosemide injection. Since pigeons can store ingested stuffs in their crop for several hours, this interval of 20 h was adopted to assure that at the moment of the natriuretic treatment, sodium would not have been available from the crop. At the time of the first furosemide injection the cages were carefully washed to remove adherent salt. Also food cups were washed and fresh food (Pro-II Breeder Pigeon Grains, No. 5431, Purina, Richmond, IN, U.S.A.) was offered. It was possible to use the standard food during the depletion period because the sodium content of these air-washed grains proved to be extremely low (about 0-03 %, w/w). Tap water was always available h after the first furosemide injection 3% NaCl was returned to the animals and their consumption of it and of water was recorded in a 2 h test, as well as at 24 h. The latency to drink the salt solution was also measured. The same animals received four additional depletions at intervals of about a week. Daily intakes of 3 % NaCl and of water were measured between depletions. Deoxycortico8terone acetate (DOCA). In another group of pigeons, sodium appetite was aroused by DOCA treatment. They received a daily i.m. injection of 2 or 4 mg/pigeon of DOCA in sesame oil (1 ml) for 7 days. Animals had free access to tap water, 3% NaCl and food throughout the experiment, but they were not offered mineral grit. Daily intakes of water and 3% NaCl were measured, before, during and after the DOCA treatment and were compared for statistical analysis to the daily intakes of the day prior to the beginning of the treatment. Renin. Renin (1 ng/pigeon), which activates brain angiotensin, was given in 1,e1 of isotonic saline by pulse intracerebroventricular (I.c.v.) injection through a stainless-steel injector (o.d. = 300,um) temporarily inserted into the chronic guide cannula and protruding 2 mm beyond its tip. 7 days after the first i.c.v. injection of renin, the animals received a 2nd renin injection to determine whether repeated treatments might affect the behavioural response. Control animals received a pulse i.c.v. injection of 1 u1 of isotonic saline. Injections were made between 1.30 and 2.00 pm. The intakes of water and of 3% NaCl were determined at intervals of 1, 2, 6, 18 and 24 h thereafter. Daily intakes of 3 % NaCl and water were measured up to 4 days after the pulse i.c.v. treatments. Animals employed for this experiment had already experienced sodium depletions. Selectivity of the appetite To address the problem of the selectivity of salt appetite in the pigeon, six animals that had already been depleted 4 times were depleted of sodium again (by combining furosemide with withdrawal of sodium for 24 h) and then offered a solution of 0 34 M-CaCl2 instead of the usual NaCl solution. The CaCl2 solution was equiosmotic to 3 % NaCl and had been available to the pigeons for 3 days before the depletion.

4 558 A. N. EPSTEIN AND M. MASSI Furosemide-induced sodium excretion The sodium loss elicited by the sodium-depletion treatment was determined by collecting urines and faeces in stainless-steel trays placed under the pigeons' cages at the moment of the first furosemide injection h later, just before access to 3 % NaCl, each tray was removed from the 30 C 0 0) Z" 20 E w 11 -/ 1#1.1 C a Z 10 c,,.1 I min 24h Time Fig. 1. Cumulative 3% NaCl intake of six pigeons in response to repeated sodium depletions. Values are means + s.e. of mean. Difference from 1st depletion intake: * P < 0 05; ** P < 0-01; where not indicated, the difference was not significant. O, 1st; *, 2nd; E, 3rd; *, 4th; and *, 5th depletions. cage, and its content was filtered. Distilled water was added to the tray to maximize collection of salts. The final filtered volume was brought up to 50 ml (or 100 ml if the urine volume was close to or larger than 50 ml) by adding distilled water. The concentration of sodium, as well as of potassium, in each sample was measured by flame photometer (Instrumentation Laboratories, Model 143). Statistical analysis All data are presented as means+ S.E. of mean. Statistical analysis was performed by means of the paired t test, except for the renin experiment when the unrelated t test was employed. Statistical significance was set at P < 005. RESULTS Depletion-induced salt appetite First sodium depletion. As shown in Fig. 1, in response to the first sodium depletion pigeons showed a robust salt appetite. Of the six animals tested, four took saline solution by pecking repeatedly at the drinking spout, while the others dipped their beak into it and drank vigorously. These rapid drinkers took their 30 min total of 3 % NaCl in just a few seconds and they drank the largest volumes of salt. Latencies to the ingestion of 3 % NaCl solution were rather long after the first

5 SALT APPETITE IN THE PIGEON 559 depletion, the mean of the six pigeons being s. The shortest latency observed was 100 s. As shown in Fig. 1, after the first 30 min the rate of salt intake declined but drinking continued for the whole 2 h test. A marked intake took place in the 22 h after the test, so that at 24 h after salt presentation the overall amount of 3 % NaCl > j st 2nd 3rd 4th 5th Depletion Fig. 2. Latency to the intake of 3% NaCl solution following five consecutive sodium depletions. Values are means+s.e. of mean of six data. Difference from 1st depletion latency: * P < 005; ** P < 001. ingested was ml (vs. a daily mean intake of 1-2 ml prior to the depletion). Water intake followed that of salt in all the animals tested, most of it being drunk after the end of the 2 h test. Repeated sodium depletions. As shown in Fig. 2 pigeons that were sodium depleted for a second time showed a mean latency to the ingestion of salt that was markedly shorter than after the 1st depletion ( vs. 373 ± 69 s; P < 0-01). Latency became even shorter following the 3rd depletion (59+7 s) and remained the same thereafter. Figure 1 shows that repeated depletions affected also the amount of salt ingested. In fact, the second, and subsequent depletions produced salt intakes that were higher than those observed after the first depletion at 30 and 60 min after salt was offered. At 120 min the difference from the 1st depletion intake was significant for the 3rd, 4th and 5th depletion. At 24 h after salt presentation the difference in salt intake between the 1st and subsequent depletions was significant between only the 1st and the 4th depletions. The intakes of salt induced by the 2nd, 3rd, 4th and 5th depletions were indistinguishable at all times of observation. Water intake always started after that of salt in all the animals tested. Unlike salt intake in the 2 h test no differences were observed in water intake between the 1st and the following depletions.

6 560 A. N. EPSTEIN AND M. MASSI Repeated depletion and sodium excretion. Another group of seven pigeons were depleted twice in order to replicate the findings obtained in the first group, and to evaluate whether the larger salt intake after the 1st depletion might be related to increased sodium excretion. Again, the latency to the ingestion of salt following the 30 4 Ar' 0.9~~~~ 20 / 31 E* E C~~~0 2 2 C~~~~~ El Cl) 0 1 St 2nd min 24 h Depletion Time Fig. 3. Sodium excretion and subsequent 3 % NaCl intake in a group of seven pigeons in response to the 1st and 2nd sodium depletion. Left panel: 24 h sodium excretion (from the 1st furosemide injection to access to salt). Right panel: cumulative 3 % NaCl intake after access to salt. In both panels, values are means + s.e. of mean. Difference between the two depletion data: * P < 0O05; ** P < 001; where not indicated, the difference was not significant. O, 1st; and 0, 2nd depletions. 2nd depletion was markedly faster than that following the 1st depletion (69+13 vs s; P < 0-01). Moreover, the intake of salt in the 2 h test was again higher after the 2nd depletion, the difference being significant at 30 and 60 min (Fig. 3). At 24 h, however, salt intake was almost exactly the same following the two depletions. Water intake following the 1st and 2nd depletion was indistinguishable. Analysis of the furosemide-induced sodium losses revealed that the losses during the two depletions were virtually identical: 3X37 + 0X28 sodium mequiv/pigeon was lost during the 1st 24 h depletion and mequiv/pigeon during the 2nd. Selectivity of the appetite. At the end of the depletion period pigeons offered the CaCl2 solution started to take it after a latency of s. However, after they tasted the solution offered, they abruptly stopped drinking. At 30 min after CaCl2 presentation the mean intake was as low as ml/ pigeon, and at 60 min it was ml/pigeon. After this 1 h test, the pigeons were offered 3 % NaCl and at 30 and 60 min they took respectively and ml/pigeon. Effect of repeated depletion on daily salt intake. In the group of six pigeons that received five consecutive depletions at intervals of 6-7 days, we measured daily salt intake on each of the days before and between each depletion. As shown in Fig. 4,

7 SALT APPETITE IN THE PIGEON the baseline pre-depletion intakes ranged between and ml. Usually the daily intakes were slightly higher at day 1 or 2 following the repletion day, but they rapidly declined back to baseline values. Even after the 5th depletion, the daily intakes measured at days after the last depletion were very much the same as those of the pre-depletion period a :_ 20 - E._ z 3 1st 2nd 3rd 4th 5th depletion Time (days) Fig. 4. Daily 3% NaCi intake before and after five subsequent sodium depletions. Each point is the mean + 5.E. of mean of six data. Values in the upper ordinate scale are 24 h intake since access to salt following a depletion treatment. DOCA-indced salt appetite In response to a daily 2 mg (I.M.) injection of DOCA, pigeons increased their daily intake of 3% NaCl solution slightly beginning with the 2nd day of treatment (Fig. 5). The intake remained stable for the following 2 days and then increased sharply from the 5th to the 7th day. However, a large variability was observed in the response. For instance, on the last day of the treatment two pigeons showed no response, their daily intake being 1 andjs ml; three animals took between 5t7 and 17*5 ml, and one showed the impressive intake of 30 ml of 3% NaCl. Owing to the large variability the mean intake of 10 ml on the 7th day was not statistically higher than that of the day preceding the beginning of the treatment (1I ml). Immediately after the DOCA treatment ended, daily salt intake came back to baseline values. Daily treatment with 4 mg/pigeon of DOCA proved to be much more effective. Again salt intake increased on the 2nd day of the treatment, then rose slowly in the 3rd and 4th days, while a marked further increase was observed on the 5th day. On the 7th day of treatment, the daily salt intake averaged *43 ml/pigeon (Fig. 5). In comparison to the intake of the day preceding the beginning of the treatment, significant differences in daily salt intake were detected from the 3rd to the 7th

8 562 A. N. EPSTEIN AND M. MASSI day of DOCA administration. After the treatment ceased, the daily salt intake remained above baseline for two more days. Water intake sharply increased during the pharmacological treatment mirroring the increase in salt intake. In response to 2 mg/day of DOCA a marked and abrupt 20 C 0, -X 10F '' E a) Pre-treatment I Treatment l I ll I Post-treatment L I Time (days) Fig. 5. Daily 3% NaCl intake before, during and after a 7 day treatment with s.c. injections of 2 (continuous line) or 4 mg pigeon-' day-' of DOCA. Each point is the mean+ S.E. of mean of six data. Difference from the intake of the day prior to the beginning of the treatment: * P < 0 05; ** P < 0-01; where not included the difference was not significant. increase in the intake was observed on the 5th day of treatment, then reached a plateau (at about 150 ml pigeon-' day-') for the following days of treatment and remained high for 1 day after it ceased. Again large variability was observed between animals and the difference from pre-treatment intake was never significant. In response to 4 mg/day a clear-cut increase was observed on the 4th day. Water intake reached a maximum on the 7th day ( ml), remained very high during the 2 days following the treatment and then came back to pre-treatment values. Renin-induced salt appetite The first behavioural response to the pulse i.c.v. injection of 1 ng of renin was a prompt and copious intake of water (Fig. 6). Latency to drink water following the first renin injection ranged between 25 and 210 s, the mean latency being s. 1 h after injection the mean intake of water was ml, which is about the volume of water that these animals would have ingested during an entire day in the absence of treatment. At 24 h after injection, the volume of water intake reached the impressive amount of ml/pigeon, that is about 50% of their body weight. On the 2nd day following the pulse i.c.v. renin injection, the daily water intake was down to ml and was not different from that of control pigeons ( ml/pigeon).

9 SALT APPETITE IN THE PIGEON When i.c.v. renin (1 ng/pigeon) was given again 1 week later to the same animals the dipsogenic response was strictly similar and indistinguishable from that observed following the first injection. In addition to increasing water intake, pulse i.c.v. renin produced also a sharp * 0 E * X C Pulse.c.v. * 1 ng renin / (or saline) * * 0 I h 2nd 3rd 4th day Time Fig. 6. Water intake following i.c.v. injection of saline or of renin, 1 ng/pigeon, which was given to the same animals twice with an interval of 7 days. Left panel: cumulative water intake in the first 24 h after renin injection. Right panel: daily water intake in the 2nd, 3rd and 4th day after renin injection. Values are means+ S.E. of mean of six data. Difference from controls: * P < 005, ** P < 0 01; where not indicated the difference was not significant. *, 1st renin; *, 2nd renin; and 0, saline. increase in the intake of 3% NaCl (Fig. 7). The natriorexigenic effect of renin was clearly delayed: following the 1st renin injection no consistent intake was observed in the first 2 h, and only some small ingestion of salt occurred in the 6 h period ( vs ml in control animals; P < 005). A clear-cut intake of salt was detected at 18 h ( vs ml in controls). At 24h the cumulative intake of 3 % NaCl was ml. On the 2nd day after injection, the intake of salt was still significantly higher than in controls ( vs ml). The natriorexigenic effect following the second renin injection was strictly similar to that observed after the first. DISCUSSION The results of the present study show that in response to a pharmacologically induced sodium depletion, pigeons express a robust salt appetite. Following their 1st depletion and after only a few minutes of latency, they ingest large volumes of a 3 % NaCl solution that they had previously avoided, and it was impressive how rapid the ingestion was in some of them.

10 564 A. N. EPSTEIN AND M. MASSI 18 0, W E C z 0 Pulse i.c.v. 1 ng renin (or saline) I, * h 2nd 3rd 4th day Time Fig % NaCl intake following pulse i.c.v. injection of saline or of renin (1 ng/pigeon) which was given to the same animals twice with an interval of 7 days. Left panel: cumulative salt intake in the first 24 h after renin administration. Right panel: daily salt intake in the 2nd, 3rd and 4th day after renin injection. Values are means+ S.E. of mean of six data. Difference from controls: * P < 0-05; ** P < 0 01; where not indicated the difference was not significant. E, 1st 2nd renin; and 0, saline. Taking into account the sodium loss during the day of the depletion, our results show that even after their first depletion and by the end of the first 30 min following NaCl presentation, the animals had ingested salt in excess to their losses. The seven pigeons used for this study lost about 3-5 mequiv during their first natriuretic treatment, and took 9 0 ml of 3 % NaCl or about 4-5 mequiv/pigeon at the end of the first 30 min of access to salt. After their second depletion, the excessive sodium ingestion at 30 min was even larger than after the first depletion. The further intake that took place in the remaining 90 min of the 2 h test, shows that pigeons respond to sodium depletion with a marked overconsumption. In this respect, they differ from sheep and rabbits whose salt appetites almost exactly replace their sodium losses (Denton, 1982). The excessive overconsumption in the pigeon is strictly reminiscent of the rat. Following the same furosemide treatment rats lose about 2-5 mequiv of sodium, and in a 2 h appetite test they ingest times that amount of sodium (Sakai, Nicolaidis & Epstein, 1986). Our pigeons lost about 3-5 mequiv and took about 20 ml of 3 % NaCl in the 2 h test after their 2nd depletion which is roughly 3 times their sodium losses. Evidence in the rat indicates that sodium-deficient rats are searching for salty tasting solutions rather than specifically for NaCl solutions, as shown by the fact that they ingest also non-sodium salts (Schulkin, 1986), even though in lower amount. The results obtained in pigeons suggest that the appetite is selective and not simply directed to the ingestion of any salty substance. In fact, when they were offered CaCl2 they consumed just a trivial amount of it, apparently just the amount necessary to taste it. Further studies are required to understand whether the appetite of sodium-depleted pigeons is completely specific for sodium salts.

11 SALT APPETITE IN THE PIGEON 565 Repeated depletions had two effects: (a) they sharply reduced the latency to the ingestion of salt and (b) they increased the rate of 3% NaCl intake in the 2 h test. The latency dropped from the 1st to the 3rd depletion; after the 3rd depletion, no further significant change was observed. In addition, the intake of 3% NaCl after the 2nd or subsequent depletions was larger than in response to the 1st depletion in the 2 h test and particularly in the first hour following salt presentation. This decreased latency to ingest NaCl and the increase in the rate of its ingestion between the 1st and 2nd depletion were not related to increased sodium loss which was practically the same in the two depletions. Thus, the findings obtained with repeated depletions are similar to those reported in rats by Epstein & Sakai (1986), who described a more rapid and copious ingestion of 3 % NaCl after the 2nd or subsequent sodium depletions. However, rats show an increased salt intake after the 2nd and subsequent depletions which is large (the intake is doubled in comparison to the 1st depletion), consistent and significant for the whole 2 h test, as well as at 24 h after NaCl presentation. In the pigeon, instead, a marked and reliable increase in 3% NaCl intake was detected only at 30 and 60 min, while at the end of the 2 h test the increase was smaller and only in some instances was the difference significant. On the other hand the 24 h intakes were always very much the same after the 1st, or subsequent depletions. These findings apparently indicate an acceleration of the behavioural response beginning with the 2nd depletion, rather than an overall increase in the intake of salt as is observed in the rat. A very interesting finding reported in rats (Epstein & Sakai, 1985; Frankmann, Dorsa, Sakai & Simpson, 1986) is that their daily salt intake rises after each depletion to reach (after three to five depletions) extraordinary levels. This intake, which occurs in the absence of need for salt, is persistent and is greater in females than in males. In the six male pigeons tested, however, even after five depletions no relevant change in daily salt intake was observed. In this regard it will be interesting to test whether female pigeons show increased avidity for salt after repeated depletions. In addition to the natriuretic treatment, the subchronic administration of high doses of DOCA also evoked salt appetite in the pigeon. On the 1st day of treatment, both with 2 and 4 mg pigeon-' day-' no increase in salt intake was detected; there was a slight increase in the following 3 days and then an abrupt increase occurred beginning with the 5th day of treatment. This temporal pattern of the effect is again strictly reminiscent of that observed in rats. Results obtained in them suggest that the natriorexigenic effect of mineralocorticoids is antagonized by the sodium retention they evoke and only after some days of treatment, when a renal escape with natriuresis takes place, can the natriorexigenic effect be fully expressed (Wolf, 1965). In birds, mineralocorticoids not only promote sodium renal conservation, but they also promote sodium reabsorption from the small intestine (Skadhauge, 1980) as well as from the cloaca (coprodeum and colon) where, owing to retrograde flow of the ureteral urine, a further reabsorption of urinary sodium can take place (Skadhauge, 1981). All these effects may produce a massive sodium retention that may antagonize the mechanisms that must be activated before the mineralocorticoid-induced natriorexia is expressed. A marked increase in sodium intake was also observed following pulse i.c.v. renin injection and activation of brain angiotensin. As in the rat, the first behavioural

12 566 A. N. EPSTEIN AND M. MASSI response to i.c.v. renin was a powerful dipsogenic response which started almost immediately after injection. Some intake of salt occurred between 2 and 6 h after injection, but most of the intake took place in the following morning. In this respect, the natriorexigenic effect induced by renin in the pigeon is much more delayed than in the rat in which a reliable sodium intake takes place at about1 h after i.c.v. renin. In the rat, sodium balance studies indicate that the stimulation of sodium intake induced by renin is not secondary to urinary losses and that treated animals remain in positive sodium balance for the 24 h after renin injection (Avrith & Fitzsimons, 1983). In the pigeon the long delay in the intake of salt raises the question whether this might be a direct effect of the action of angiotensin in the brain or whether it might be just secondary to natriuresis. High doses of angiotensinii, in fact, are known to produce a natriuretic effect in the rat (Severs, Daniels-Severs, Summy- Long & Radio, 1971). However, in the pigeon i.c.v. infusion of angiotensinii,1 ng/ min over 10 min, does not significantly affect sodium excretion (Fitzsimons, Massi & Thornton, 1982). Moreover, preliminary results in our laboratory indicate that sodium appetite induced by sodium depletion in the pigeon is markedly reduced by pharmacological blockade of the central renin-angiotensin system. These findings, taken together, suggest a role for the brain renin-angiotensin system in the elicitation of salt appetite, which is apparently not secondary to natriuresis. In conclusion, the results of the present study indicate that the pigeon is equipped by its evolution to express a robust and selective salt appetite in response to sodium depletion. Moreover, pharmacological doses of the same hormones that evoke the appetite in the rat also produce it in the pigeon. In birds, like in the rat, sodium depletion increases angiotensin and mineralocorticoid plasma levels (Skadhauge, 1981) and high endogenous levels of these hormones, even though much lower than those produced by our pharmacological treatments, may act in synergy to induce salt appetite (Epstein, 1982). These findings encourage the idea that the general vertebrate mechanism for salt appetite is an endocrine-induced behaviour controlled by the mineralocorticoid and renin-angiotensin system. We are grateful to Mrs Barbara Boggan for her skilled technical assistance and to Ms Keely Byford for typing the manuscript. We wish also to thank Dr Detlev Ganten of the German Institute for High Blood Pressure for his generous gift of purified renin. This research was supported by Research Fellowship of the Fogarty International Center (1 Massi and by NIH grant NS to Alan N. Epstein. REFERENCES F05 TW BI-5) to Maurizio AvRITH, D. B. & FITZSIMONS, J. T. (1980). Increased sodium appetite in the rat induced by intracranial administration of components of the renin-angiotensin system. Journal of Physiology 301, AvRiTH, D. B. & FITZSIMONS, J. T. (1983). Renin-induced sodium appetite: effects on sodium balance and mediation by angiotensin in the rat. Journal of Physiology 337, BRYANT, R. W., EPSTEIN, A. N., FITZSIMONS, J. T. & FLUHARTY, S. J. (1980). Arousal of a specific and persistent sodium appetite in the rat with continuous intracerebroventricular infusion of angiotension II. Journal of Physiology 301, DENTON, D. A. (1982). The Hunger for Salt. New York: Springer-Verlag. EPSTEIN, A. N. (1982). Mineralocorticoids and cerebral angiotensin may act together to produce sodium appetite. Peptides 3,

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