Suppression of drinking but not feeding by central eledoisin and physalaemin in the rat

Appetite, 1986, 7,63-70

Suppression of Drinking But Not Feeding by Central Eledoisin and Physalaemin in the Rat M. MASSI, L. G. MICOSSI, G. DE CARO Institute of Pharmacology, University of Camerino

A. N. EPSTEI N Department of Biology, University of Pennsylvania

The tachykinins, eledoisin and physalaemin, given by intracerebroventricular (i.c. v.) injection have been shown to be potent antidipsogenic agents in rats. To evaluate their selectivity of action on rat ingestive behaviors, we compared their effects following i.c.v. injection on the intake of water, of milk containing 3·5 or 15% fat, and of solid food. The two tachykinins inhibited water intake induced by i.c.v. angiotensin II or by cellular dehydration, but did not reduce the intake of 15% fat milk or of solid food. The intake of 3'5% fat milk was inhibited only by the highest dose (1000 ng/rat) of eledoisin which also increased grooming and locomotion. The present findings suggest that in adult rats central eledoisin and physalaemin exert a selective suppressive effect on drinking behavior without affecting feeding.

INTRODUCTION

Eledoisin and physalaemin are two tachykinins of non-mammalian origin. They are similar to each other both chemically and biologically and both produce a rapid and profound hypotension in mammals (Erspamer, 1971; Erspamer & Anastasi, 1966). Recently physalaemin-like immunoreactivity has been detected in the gut and in the central nervous system of rats and guinea pigs (Lazarus, Linnoila, Hernandez & Di Augustine, 1980; Erspamer & Melchiorri, 1980), and a peptide similar to amphibian physalaemin has been found in human lung small-cell carcinoma (Lazarus, Di Augustine, Jahnke & Hernandez, 1983). Intracerebroventricular (i.c.v.) injection of eledoisin or physalaemin inhibits water intake induced by i.c.v. angiotensin II (Ang II), i.c.v. carbachol, water deprivation and cellular dehydration (De Caro, Micossi & Piccinin, 1977; De Caro, Massi, Micossi & Venturi, 1978) in the rat. The anterior hypothalamus and the medial preoptic area have proved to be very sensitive to the antidipsogenic effect of these peptides (De Caro, Massi, Perfumi & Venturi, 1983). Recent ontogenetic studies show, first, that eledoisin and physalaemin inhibit both water and milk intake in newborn rats three to five days This research was supported by NATO Grant RG 0502 82, and by NS 03469. We would like to thank Marino Cucculelli, Ettore Caraffil and Alfredo Fiorelli for their technical assistance. Current address for A. N. Epstein is 326 Leidy Laboratory, Department of Biology/G7, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. Reprint requests should be addressed to: Maurizio Massi, Institute of Pharmacology. University of Camerino, 62032 Camerino (MC), Italy. 0195--6663/86/010063 +08 $03'00/0

© 1986 Academic Press Inc. (London) Limited

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old, and, second, that while the inhibitory effect on water intake increases with age, that on milk intake decreases progressively and completely disappears at the age of 15 days (Cantalamessa, De Caro, Epstein & Perfumi, t 986). Similar studies comparing water and food intake after central administration of tachykinins have not yet been reported in adult animals. The present experiments were therefore designed to evaluate the selectivity of the effect of eledoisin and physalaemin on the ingestive behaviors of adult rats. In them we compare the effects of their i.c.v. injection on the intake of water, of milk containing 3·5 or 15% fat, and of solid food.

METHODS

Animals

The subjects in all experiments were male, albino, Wistar rats (Charles River, Caico, Italy) weighing between 250 and 300 g at the time intracranial cannulas were implanted. They were housed individually in plastic cages in a temperature controlled room (18-20°C) with 8 photoperiod of 12 h of light and 12 h of darkness (lights on at 0700 hrs). They had continuous access to tap water and to food in pellets (Mill, Morini, Reggio Emilia, Italy) except where noted. Substances

Both eledoisin and physalaemin were a gift of the Farmitalia Research Laboratories, Milano, Italy. lieS-angiotensin II was purchased from Peninsula Laboratories Inc., St. Louis, U.S.A. I.C. V. Cannula Implantation

Under equithesin anaesthesia (0·3 ml/lOOgBW, i.m.) an indwelling guide-cannula was sterotaxically implanted t mm above the left lateral cerebroventricle according to the technique described by Miselis and Epstein, 1975. The animals were allowed at least one week to recover from surgery before being tested. I.e. v. Injections

Injections into the lateral ventricle were made by means of a stainless steel injector temporarily inserted into the guide cannula and protruding 2 mm beyond the cannula tip. Eledoisin and physalaemin solutions in sterile isotonic sodium chloride were prepared just before the experiment began from stock solutions stored at - 25°C and were always administered in a volume of 1 JlI per rat. Control animals received an injection of 0'9% saline. At the beginning of the experiments the rats were divided into two groups. Each group was used over a period of about three months to test .the effect of the same tachykinin (eledoisin or physalaemin) on drinking in response to Ang II and to cellular dehydration, and on the intake of 3·5 and 15% fat milk. Later experiments concerning the effect of eledoisin and physalaemin on solid food intake were done in two additional groups of animals. Each animal was tested with saline (control) as well as with all the

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doses of the peptide employed in each group of experiments. The sequence of treatments for a single rat in each group of experiments was random. The animals were not tested more than twice a week. ImmedIately after injection, animals were returned to their home cage where their ingestive behaviors were studied. Moreover, attention was paid to evidence of other behavioral alterations (grooming, locomotion) induced by the peptides. However, behavioral effects, others that those on ingestive behaviors, were not quantified.

Water Intake

Drinking was elieted either by i.c.v. Ang II or by subcutaneous (s.c.) injection of hypertonic NaCl. I.c.v. injection of Ang II (lOOng/rat) in isotonic saline was given one minute after that of eledoisin or physalaemin. Immediately after the second i.c.v. injection, the animals were returned to their home cages where they had water and food available. Hypertonic NaCI (8'76 g/100 ml, 1 ml/lOO g BW) was given s.c. in the loose skin of the back. After s.c. injection the animal was returned to its cage from which water had been temporarily removed. Fifteen minutes after NaCl administration the animals received the i.c.v. injection of eledoisin or physalaemin or of simple saline (controls), and immediately afterwards they had access to water. Water intake was measured to the nearest 0·1 ml at 20 min intervals by means of graduated drinking tubes and expressed as ml/l00 g BW. Measurements were made for 1 h after i.c.v. Ang II, and for 2 h after s.c. hypertonic NaCl. Milk Intake

In some experiments animals were offered whole cow milk (Parmalat, Parma, Italy) containing 3·5% fat (and 88'5% water) or, in later experiments, a milk in which fat content was raised to 15% (79% water) by adding cow cream (Panna Chef, Parmalat, Parma, Italy). In both experiments milk was offered only I h per day between 0930 and 1030 hrs, while tap water and food pellets were freely available. During the first days of milk presentation intake gradually increased. Experiments began after 9 to 13 days of exposure to either kind of milk, when intake was stable. The peptides to be tested were injected i.c.v. immediately before milk presentation. Milk intake was measured at 20 min intervals for one hour by means of graduated drinking tubes and expressed as ml/lOO g BW. Under the experimental procedure used here in which milk was offered for only one hour per day, intolerance to milk lactose was not observed.

Solid Food Intake

The intake of food in pellets (13% water) was elicited by depriving the animals of food for 24 h while water was always available. After i.c.v. injection of eledoisin or physalaemin the animals had immediate access to solid food. Intake was measured at 20 min intervals for two hours by weighing the pellets to the nearest 0·01 g. Spillage was collected and its weight taken into account. These experiments began after a training period of about two weeks, during which the rats were deprived of food twice, at intervals of 5-6 days.

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Statistical Analysis

Analysis of variance was performed on all data. Statistically significant effects identified by the analysis of variance were further evaluated by post-hoc comparisons (Bonferroni's t-test) (Dunn & Clark, 1974). Statistical significance was set at p < 0·05.

RESULTS

Eledoisin

The effects of eledoisin on water, milk and solid food intakes are reported in Figure 1. Only data obtained in the first 20 min of observation are reported since significant differences in intake were not observed in the following 20 min periods. 120 80

eE 0

u

'0 #.

40

0 120 80

Cl>

...cco

.>tt.

c

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0 80 40

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FIGURE 1. Effects of eledoisin on the intake of water (induced by cell dehydration, open bars labeled CD; or i.c.v. angiotensin II, open bars labeled A II), milk (containing 3'S% fat, stippled bars labeled 3-S; or lS% fat, stippled bars labeled lS) and solid food (in pellets, solid bar). Values are means ± SEM. Difference from controls: **, p < 0'01; *, P < O·OS. Where not indicated, p>O·OS. Numbers as shown for each group.

Inhibition of water intake

Eledoisin proved to be extremely potent in inhibiting water intake induced by cellular dehydration (s.c. hypertonic NaCI). In fact, while control rats drank 2·25±0-4ml/100gBW, animals receiving a dose of eledoisin as low as lOng/rat took only 0·99 ±0'l5 ml/lOO g BW (44'0% of control intake p«O·Ol)). At the doses of 100 or 1000 ng/rat eledoisin almost completely inhibited drinking during the first 20 min of observation. Similar results were obtained when drinking was elicited by i.c.v. injection of Ang II. Controls drank 5·3±0·35ml/100g in 20 min following Ang II administr-

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ation, while animals treated with 10 ng of eledoisin drank 4·13 ± 0·2 mil 100 g (77,9% of control intake p<0·05). Again, very potent drinking inhibitions were observed at the doses of 100 and 1000 ng/rat. Effects of food intake

On the other hand, the only effects of eledoisin on food intake were an inhibition of 3,5% fat milk intake (p < 0,01) and an increased latency to eat solid food (568 ± 117 sec vs. 265 ± 51 sec in the controls,p <0'01). Both of these effects were produced only by the highest dose (lOOOng/rat) which also increased grooming and locomotion. When 15% fat milk was offered, control rats took 3·27 = ±0'32 ml/lOO g BW in 20 min. Eledoisin did not inhibit intake, even at the 1000 ng/ rat dose. In response to 24 h of food deprivation, control rats took 0·81 ± 0·08 gi l 00 g BW of solid food. Eledoisin at 10 and 100 ng/rat did not inhibit intake. At the highest dose employed (1000 ng/rat) the intake of treated rats (0·60 ±0'12 g/100 g BW) was slightly lower than that of control, but the difference was not statistically significant. The highest dose of eledoisin elicited intense grooming, and between episodes of grooming, an increase in locomotion. Grooming and locomotion were particularly evident in the first three to four min after injection and rapidly declined thereafter. On the other hand, lower doses of eledoisin, as well all of the doses of physalaemin employed, did not consistently have these effects. Physalaemin

The results obtained 20 min after physalaemin administration are reported in Figure 2. Again, no differences were observed during the following 20 min periods of observation except in the second 20 min period when physalaemin (1000 ng/rat) was tested on drinking induced by s.c. hypertonic NaCl, as reported below.

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80

0

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40 0 120

ro

C c

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80 40

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FIGURE 2. Effects of physalaemin on the intake of water (induced by cell dehydration or i.c.v. angiotensin II), milk (containing 3'5%, or 15% fat) and solid food (in pellets). Abbreviations and bars as in Figure 1. Values are means ±SEM. Difference from controls: **, pO·05. Numbers as shown for each group.

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Inhibition of water intake

Physalaemin proved to be less potent than eledoisin in inhibiting water intake both in response to Ang II and to cellular dehydration. In response to s.c. hypertonic NaCl, controls took 2·56 ± 0·59 ml/100 g BW and physalaemin treated rats showed a significantly lower intake only in response to 1000ng/rat (0·62±0·32ml/100gBW; p < 0·01). However, their intake in the second 20 min period of observation was significantly larger than that of controls (1·27 ± O· 36 vs. 0·03 ± 0·03 respectively; p
When the same animals were offered 3·5 or 15% fat milk, controls took respectively 4·71±0·44 and 3·55±0·17ml/100gBW during the first 20min of observation. Physalaemin did not inhibit the intake of either kind of milk even at the dose of 1000 ng/rat. Similar findings were obtained when the effect of physalaemin was tested on solid food intake. In fact, controls took 1·01 ± 0·12 g/100 BW during the first 20 min after food presentation, while in response to 100 and 1000 ng/rat the same animals ate (1·89±0·05ml/100gBW; p<0·01).

DISCUSSION

The present results confirm those of previous studies (De Caro et ai., 1977, 1978), showing that eledoisin and physalaemin are general antidipsogenic agents in adult rats. In fact, the two tachykinins at the same dose levels inhibited not only Ang II-induced drinking, but also drinking in response to cellular dehydration, a dipsogenic determinant which employs a neurological system qualitatively different from that for Ang II (Blass & Epstein, 1971; Mogenson & Kucharczyk, 1978; Kucharczyk, Assaf & Mogenson, 1976; Simpson et al., 1978). The higher potency of eledoisin and physalaemin in inhibiting drinking to cell dehydration in comparison to previous studies (De Caro et al., 1977, 1978) is most likely the result of the smaller osmotic challenge used here. It is interesting to note that the inhibitory effect of physalaemin on cell dehydrationinduced drinking lasted only for about 20 min. In fact, in the second 20 min period, the intake of rats treated with physalaemin (1000ng) was significantly larger than that of controls. On the other hand this was not observed after eledoisin injection, suggesting that its effect is longer lasting. A similar difference in the duration of the effects of the two tachykinins on Ang II-induced drinking was not observed, probably owing to the short-lasting dipsogenic action of Ang II (about 15 min). On the other hand, physalaemin while inhibiting water intake, did not reduce either milk or solid food intake, and eledoisin, which at 100 ng/rat almost completely inhibited drinking, did not reduce either milk or solid food intake. At the highest dose of eledoisin (1000 ng) 3·5% fat milk intake was inhibited and the latency to eat solid food was prolonged but, at the same dose, it did not reduce the amount of either 15% fat milk or solid food intake. This dose evoked grooming and locomotion that lasted three to four minutes, which may account for its slight effects on feeding.

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These findings clearly indicate that the inhibitory effects of physalaemin and eledoisin on drinking are specific for that ingestive behavior and are not due to malaise, illness or neurological arti.facts. The differences observed in the inhibitory effects of eledoisin and physalaemin on the intake of the different foods offered here are not explained by differences in their water contents. If water content were the principle factor, the failure of both tachykinins to inhibit milk intake would be unexplained, and if water content were the important difference the tachykinins should have had different effects on 15% fat milk (which is 79% water) and on solid food intake (which is about 13% water) while our results indicate that both peptides are ineffective in both instances. On the contrary, the qualitative differences in the effects of eledoisin and ph.ysalaemin on fluid intakes can be more easily explained on the basis of the nutrient content of the substances offered for ingestion. Milk, even at the lowest fat content used, provides not only water but also nutrients, and in this respect it can be reasonably considered to be a food for the rat. Several studies of rat ingestive behavior support the idea that milk is treated by rats as a food, and indicate that the ability to distinguish milk from water develops in the first days of life. Bruno, Hall and Grill (1980), for instance, reported that dehydration inhibits the consumption of milk in hungry rats at 20 days of age, and Bruno (1981) showed that 15-day-old pre-weaning rats offered either milk or water from a distant source responded to dehydration by increasing their water intake, while reducing their intake of milk. Moreover, Ellis, Axt and Epstein (1984) showed that intracranial injections of Ang II arouse both water and milk intake until the eighth day after birth when its effect becomes abruptly selective for water. All of these observations suggest the existence of two distinct intake control systems, one for water and the other for milk. The present experiments show that the effects on the brain of eledoisin and physalaemin are highly selective for the system controlling water intake. The inhibition of 3'5% fat milk by eledoisin (1000 ng/rat) may be due to the fact that rat milk is more concentrated than cow milk (Luckey, Maude & Pleasants, 1954; Altman & Dittmer, 1961) so that its intake might involve both intake control systems. On the other hand, 15% fat milk is more food-like as, of course, is solid food itself, and the intake of both was not inhibited by eledoisin. It is important to point out that the inhibition of 3·5 but not of 15% fat milk by eledoisin (1000ng/rat) cannot be due to a higher palatability ofthe second fluid, since the control intake of 15% fat milk was never larger than that of 3'5% fat milk. The results of the present study in adult rats confirm and extend those obtained in neonates. As stated in the introduction, physalaemin and eledoisin have already been shown to be specific antidipsogenic agents in pups older than five days. The findings of the present study show that they continue to exert a highly specific effect on water intake in the adult rat without affecting feeding behaviors. REFERENCES

Altman. P. L., & Ditmmer, D. S. Blood and other body fluids. In Biological Handbook Series, Federation of American Societies of Experimental Biology, Bethesda, MD, 1961. Blass, E. M., & Epstein, A. N. A lateral preoptic osmosensitive zone for thirst in the rat. Journal of Comparative Physiology and Psychology, 1971, 76, 378-304. Bruno, J. P. Development of drinking behavior in preweanling rats. Journal of Comparative Physiology and Psychology, 1981,95, 1016-1027.

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Bruno, J. P., Hall, W. G., & Grill, H. J. Dehydration-induced anorexia. Neuroscience Abstracts, 1980, 6, 517. Cantalamessa, F., De Caro, G., Epstein, A. N., & Perfumi, M. Effects of the tachykinins eledoisin and physalaemin on drinking behavior of baby rats. In G. de Caro, A. N. Epstein and M. Massi (Eds.), The physiology of thirst and sodium appetite. New York: Plenum, 1986. De Caro, G., Micossi, L. G., & Piccinin, G. L. Antidipsogenic effect of intraventricular administration of eledoisin to rats. Pharmacological Research Communications, 1977,9,489500. De Caro, G., Massi, M., Micossi, L. G., & Venturi, F. Physalaemin: A new potent antidipsogen in the rat. Neuropharmacology, 1978, 17, 925-928. De Caro, G., Massi, M., Perfumi, M., & Venturi, F. Sensitivity to different nuclei ofrat brain to the antidipsogenic effect of tachykinins. Appetite, 1983,4, 198. Dunn, D. J., & Clark, A. Applied statistics: Analysis of variance and regression. New Yark: J. Wiley, 1974. Ellis, S., Axt, K., & Epstein, A. N. The arousal of ingestive behavior by chemical injection into the brain of the suckling rat. Journal of Neuroscience, 1984,4,945-955. Erspamer, V. Biogenic amines and active polypeptides ofthe amphibian skin. Annual Reviews of Pharmacology, 1971,11,327-350. Erspamer, V., & Anastasi, A. Polypeptides active on plain muscle in the amphibian skin. In E. G. Erdos, N. Beck, F. Sicuteri & A. F. Wilde (Eds.), Hypotensive polypeptides. Pp. 66-75. New York: Springer, 1966. Erspamer, V., & Melchiorri, P. Active polypeptides: from amphibian skin to gastrointestinal tract and brains of mammals. Trends in Pharmacological Science, 1980, 1, 392-395. Kucharczyk, J., Assaf, S. Y, & Mogenson, G. J. Differential effects of brain lesions on thirst induced by the administration of angiotensin II to the preoptic region, subfornical organ and anterior third ventricle. Brain Research, 1976, 108, 327-337. Lazarus, L. H., Linnoila, R I., Hernandez, 0., & DiAugustine, R P. A neuropeptide in mammalian tissues with physalaemin-like immuno-reactivity. Nature, 1980,287,555-558. Lazarus, L. H., DiAugustine, R. P., Jahnke, G. D., & Hernandez, O. Physalaemin: An amphibian tachykinin in human lung small-cell carcinoma. Science, 1983,219, 79-81. Luckey, T. D., Maude, T. J., & Pleasants, J. The physical and chemical characteristics of rat's milk. Journal of Nutrition, 1954, 54, 345-353. Miselis, R. R, & Epstein, A. N. Feeding induced by intracerebroventricular 2-deoxy-n-glucose in the rat. American Journal of Physiology, 1975,229, 1438-1447. Mogenson, G. 1., & Kucharczyk, J. Central neural pathways for angiotensin-induced thirst. Federation Proceedings, 1978, 37, 2683-2688. Simpson, J. B., Epstein, A. N., & Camardo, 1. S. Localization of receptors for the dipsogenic action of angiotensin II in the subfornical organ of rat. Journal of Comparative and Physiological Psychology, 1978, 92, 581-608.

Received 15 March 1985, revision 15 June 1985