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Interleukin-1 inhibits drinking behaviour through prostaglandins, but not by nitric oxide formation
Life .%ences.,Vol. 60, No. 7, pp. 457464,1597 cqyfight 0 1997 l2kvier Scicm Inc. Printed in the USA. All tights reserved oD24-3255p $17.00 t .oo ELSEVIER
PI1 soo24-3205(96)00675-3
INTERLEUKIN-1
Gioacchino
INHIBITS DRINKING BEHAVIOUR THROUGH PROSTAGLANDINS, BUT NOT BY NITRIC OXIDE FORMATION
Calapai, *Luca Parente, Felice Nava, Gabriella Facciola, Maria Concetta Marciano and Achille P. Caputi
Institute of Pharmacology, School of Medicine, University of Messina and *Institute of Pharmacology and Pharmacognosy, School of Pharmacy, University of Palermo, Italy
(Received in final form November 27, 19%)
Summary Interleukin-1 p (IL-l B) causes inhibition of drinking behaviour. Moreover it induces formation of prostaglandins (PGs) and nitric oxide (NO). Both PGs and NO are able to inhibit drinking stimulated by water deprivation or by intracerebroventricular (i.c.v.) administration of angiotensin II. In this study, we studied in the preoptic area (POA) the possible role of PGs and NO in the antidipsogenic action induced by IL1P. IL-ll3 was injected in the lateral cerebral ventricle (icv.) (2.5, 10, 20, and 40 t&at) or into POA (0.625, 1.25, 2.5, and 10 rig/rat). Larginine (12.5, 25, 50, and 100 @rat), the precursor of NO, or No-nitroL-arginine methyl ester (L-NAME) (25, 50, and 100 @rat), an inhibitor of nitric oxide synthase (NOS), were injected only into POA. Drinking behaviour was induced by water deprivation (24 h). IL-ll3 injected either i.c.v. or mto POA caused dose dependent inhibition of drinking. In the POA a treatment with acetylsalicylic acid (ASA) (33, 66, and 135 &at), but not with L-NAME, antagonized the inhibition of drinking behaviour induced by the highest doses of IL-l p in the POA. In the POA, a treatment with ASA or L-NAME antagonized the inhibition of drinking behaviour caused by injection of the highest doses of L-arginine. Our data suggest that the central inhibition of drinking behaviour of IL-10 is mediated through the formation of PGs, but not NO, in the POA. Key Words: water intake, interleukin-la, cyt~kine.s, preoptic area
______~__~_~________~~~~_~_~~~~~~~~~~______~_~~~~~_________~~~~~__________~~~___________ Correspondence: Ginacchino Calapai, M.D.. Institute of Pharmacology, School of Medicine, University of Mcssina, Piazza XX Settembre 4, 98122 Messina, Italy, Phone: ~39-90-712533, FAX:+39-90-66 1029
458
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Interleukin-1 (IL-l) is a cytokine released by monocytes/macrophages and in central nervous system (CNS) by glial cells (1,2). IL-l exists in two forms: IL-la, and IL-1S (2). IL-ll3 is the major secreted form, and is evident in a high concentration in plasma, tissue fluids, and brain (3). It has been suggested that IL-ll3 can mediate different neuroendocrine functions (3-5). IL-1 p administration induces fever, sleep, hypotension and anorexia (2, 3,6-8). IL-lp given centrally or peripherally elicits also an inhibitory effect on drinking behaviour through an action on the central sites controlling water intake (7). This effect is not related on the anorexia induced by the cytokine (7). However, the mechanisms underlying its antidipsogenic effect are not clear. In previous studies we have observed that intravenous (i.v.) or intraperitoneal (i.p.) administration of lipopolysaccharide of Escherichia coli (LPS) inhibits drinking behaviour in an overlapping way to the antidipsogenic effect produced by IL- l/3 and other molecules (9- 12). Our recent studies suggest that IL-1 is not directly involved in thirst induced by LPS but indirectly trought the mediation of other substances (12). The preoptic area (POA) is one of the most sensitive cerebral nucleus among the sites involved in drinking behaviour (13). Since various prostaglandins (PGs) including PGIr injected in the POA are antidipsogenic (14) and nitric oxide (NO) formation in POA is responsible for the reduction of water intake stimulated by either water deprivation or angiotensin II (15), we have investigated the role of PGs and NO in antidipsogenic effects induced by IL-10 in 24 h water deprived rats. Materials and Methods Animals: Adult male Sprague-Dawley rats weighing 280-320 g were used. The animals were housed at a constant temperature of 22 5 2” C under a 12/12 h light-dark cycle (lights on at 6:00 a.m.), with free access to Purina rat chow pellets and tap water, unless otherwise stated. Treatments: In a first group of 24 h water deprived rats IL-1S was injected in the lateral cerebral ventricle (i.c.v.) (2.5, 10, 20, and 40 ng/rat) or into the POA (0.625, 1.25, 2.5, and 10 @rat), while naive water deprived animals received saline solution (3 pi/rat i.c.v. or 1 ul/rat in the POA). In a second group of 24 h water deprived rats L-arginine (12.5, 25, 50, and 100 @rat) was injected into the POA, while control animals received saline solution (1 ul/rat). In a last group of 24 h water deprived animals acetyl salycilic acid (ASA) (33, 66, and 135 l&rat) or No-nitro-L-arginine methyl ester (L-NAME) (25, 50, and 100 rig/rat), was given into POA 30 min before injection of IL- 1S or L-arginine. Each group of animals was composed of six rats. Intracerebroventricular iniections: Stainless steel guide cammlae (o.d. = 0.66 mm) were inserted seven days before the experiments. The rats were anesthetized with chloral hydrate (400 mgkg, i.p.) and placed in a stereotaxic instrument (Stellar, Stoelting). For i.c.v. injections, cannulae were implanted i.c.v. (stereotaxic coordinates: AP = 1 mm behind the bregma, L = 2.5 mm from the midsagittal suture and V = 2 mm from the dura). For injections into POA cannulae were implanted, aimed 2 mm above the left medial POA, (stereotaxic coordinates: AP = 7.9 mm anterior to the interaural line, L = 0.7 mm from the midsagittal suture, V = 3.4 mm up from the interaural line) (16). The incisor bar of the stereotaxic instrument was elevated 5 mm above the interaural line (16). Injections into the lateral cerebral ventricle (3 ~1) or into the POA (1 ~1) were made by a 30 gauge injector temporarily inserted into the guide-cannula and protruding 2 mm beyond the cannula tip. Injections were carried out over a period of l-2 min (15).
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Water intake evaluation: Drinking was elicited by 24 h water deprivation. The rats had free access to food during this period. Water intake following deprivation was monitored for a 60 min period and expressed as ml/ rat. Water intake following deprivation was monitored 60 min after IL-18 administration and 30 min after L-arginine injection for a 60 min period and expressed as ml/rat. In order to avoid appearance of tolerance to IL-l 8, each animal was treated only once with this substance and not used for other experiments. Drugs: Interleukin-18 (IL-18) was a gift from Istituto Ricerche Immunobiologiche Siena (I.R.I.S.). Acetylsalicylic acid (ASA) as the soluble lysine salt was purchased from MaggioniWinthrop while L-arginine and No-nitro-L-arginine methyl ester (L-NAME) were obtained from Sigma Chemical Company. IL-18 was dissolved in phosphate buffered saline solution (PBS), ASA in alkaline solution, L-arginine, and L-NAME in 0.9% NaCl solution. Statistical Analysis: Data are expressed as the means + SD. Statistical analysis of data was performed using one way analysis of variance (ANOVA) and Duncan’s test was used to compare group means. Statistical significance was set at P < 0.01. Results I.c.v. injections of IL-18 induced a significant and dose-dependent reduction of water intake. This effect was evident starting from dose of 10 @rat (Fig. 1). A similar effect was observed following IL-18 administration in different doses into POA. In particular an evident and significant water intake inhibition was present starting with the dose of 2.5 &rat (Fig. 2). The same dose did not produce inhibition of drinking when injected i.c.v. in water deprived rats (Fig. 1).
*
1
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FIG. 1 Water intake, induced by 24 h water deprivation, after IL-18 administration in the left lateral ventricle (i.c.v.). IL-1 8 doses are expressed as ng/rat. Water was presented to the animals 60 min after IL18 treatment for a 60 min period. Each column represents the mean + S.D. of six animals. *P < 0.01 vs control.
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FIG. 2 Water intake, induced by 24 h water deprivation, after IL-IS administration in the POA. IL-ll3 doses are expressed as ngkat. Water was presented to the animals 60 min after IL-ll3 treatment for a 60 min period. Each column represents the mean + SD. of six animals. *P < 0.01 vs control. L-arginine (12.5, 25,50, and 100 rig/rat) injected into POA inhibited, in evident manner and significantly, water intake starting by the dose of 25 @rat (Fig. 3).
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4L A FIG. 3 i Water intake, induced by 24 h water deprivation, after L-arginirk administration into POA. L-arginine doses are expressed as ngkat. Water was presented to the animals 30 min after L-arginine treatment for a 60 min period. Each column represents the mean + S.D. of six animals. *P < 0.01 vs control.
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A pretreatment with ASA (33, 66, and 135 &at) into the POA, 30 min before IL-1S treatment, was able to antagonize antidipsogenic effects induced by injection of IL-1 g at dose of 10 @rat (Fig. 4). On the other hand a pretratment with L-NAMJZ(25, 50, and 100 ng/rat) into the POA, 30 min before IL-ll3 administration, was unable to influence thirst induced by IL-l S administration in POA at dose of 10 &rat (Fig. 4). 14
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Y 4 i FIG. 4 Water intake induced by 24 h water deprivation, after IL-ll3 (at the highest dose used) + ASA or IL-lp (at the highest dose used) + LNAME administration into POA. ASA doses are expressed as I.&at, while IL-1 p or L-NAME doses as @at. Water was presented to the animals 60 min after IL-1 /3 treatment for a 60 min period. Each column represents the mean + S.D. of six animals. *P < 0.01 vs control. Injection into POA of ASA (33, 66, and 135 @rat) or L-NAME (25, 50, and 100 n&at), 30 min before L-arginine, was able to abolish, significantly and in a dose-dependent manner, thirst induced by L-arginine (Fig. 5).
PGS and ILlp Drink& Inb.i.ition
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12 10 8 8 4 2
FIG. 5 Water intake induced by 24 h water deprivation, after Garginine (at the highest dose used) + ASA or L-arginine (at the highest dose used) + L-NAME administration into POA. ASA doses are expressed as @rat, while IL-IS or L-NAME doses as r&at. Water was presented to the animals 30 min after L-arginine treatment for a 60 min period. Each column represents the mean t S.D. of six animals. *P < 0.0 1 vs control.
It is now evident that IL-IS, like other cytokines, acts on different target cells and modulates many biological functions (1-4, 17). The role that this cytokine plays in the communication between the central nervous system (CNS) and the periphery is one of the most interesting features of its biology (3-5). IL-Ip is produced in the brain by astrocytes and is able to alter neuroendocrine activity, slow-wave sleep and body temperature (3,6-S, 17). It has been shown that IL-ll3 can induce its effects acting directly on specific target tissues or, indirectly, as an effector molecule (12). This second way may explain, for example, the hypotension caused by IL-l l3 through the formation of substances like PGs and NO ( 18). It is well known that IL-1 p can influence NO release, it increases the expression of inducible NOS in several rodent cell types and causes nitrite and nitrate formation (19, 20). A reduction of water intake, like other changes (temperature, sleep, feeding) occurring during infections, can be considered a result of toxic agents action (21) or various substances and cytokines including IL-1 l3 (12). In particular various works have shown that peripheral administration of endotoxin (9, 11, 12) or central injection of tumor necrosis factor a (TNF a) (11,22,23), both releasing IL- 1, produce similar inhibitory effects on drinking behaviour. More recently, we also demonstrated that either LPS or TNFa cause inhibition of drinking behaviour through the release of NO in the POA (I 1). POA is one of the most important cerebral regions involved in the mechanisms regulating drinking behaviour (13) and fever (3).
Vol. 60,No. 7, 1997
PGsandILj3DrinkiqIllhibson
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Our data show that high doses of IL-l l3 injected in the POA inhibit water intake induced by 24 h water deprivation. Furthermore, doses of 2.5 and 5 r&at, able to inhibit drinking behaviour when injected into POA, are not effective when administered in the lateral cerebral ventricle. Injection of IL-l B at the dose of 10 @rat in the lateral cerebral ventricle causes a slight but significant reduction of water intake, but the administration of the same dose into POA induces a more potent inhibition of drinking behaviour, thus indicating that POA is a specific site for control of drinking behaviour. The present results confirm previous studies showing that antidipsogenic effect can be produced by L-arginine injection, the precursor of NO, in the POA and that this action is specific because it can be reverted by L-NAME, an inhibitor of NO formation (11). Since our precedent experiments have demonstrated that IL-lp is not a direct mediator implicated in inhibition of thirst induced by LPS (12) we have investigated the possibility that the Larginine/NO pathway and/or PGs could be activated by IL-ll3 in order to inhibit drinking behaviour. Our data show that L-NAME, an inhibitor of NOS, given into POA, is not able to antagonize IL-lb inhibition of water intake, while ASA into POA is very effective. The precursor of NO, L-arginine, when injected into POA, inhibits drinking and this effect is abolished either by L-NAME or ASA. Therefore, it is possible to argue that IL-lb needs arachidonic acid metabolites to inhibit drinking, while NO formation is not necessary. This result is in accord with precedent studies showing that IL-lb is able to increase PGEr secretion in astrocyte culture in the rat (18) and that PGE, is a potent antidipsogenic compound (24). On the contrary, the antidipsogenic effect of L-arginine requires an increased synthesis of NO, since it is abolished by L-NAME. Moreover, the antidipsogenic effect of L-arginine requires arachidonic acid metabolites because it is blocked by ASA. So, at the moment, we can hypothesize that in the POA an increase of NO formation leads to an increased metabolism of arachidonic acid. The above findings induce to believe that effects of IL-lp on drinking behaviour in 24 h water deprived rats are not mediated directly by NO formation but by PG levels. These results are in accord to various studies demonstrating that many of IL-lp central effects are dependent on the synthesis .of eicosanoids, since they are attenuated by cyclooxygenase inhibitors (2, 3, 25, 26). We believe that PGs might be involved not only in fever and in other central effects but also in reduction of water intake observed after IL-ll3 administration. This is also evident from our data demonstrating that POA, a crucial site for control of fever and water intake, is very sensible to eicosanoids levels. By contrast, various central effects of IL-lb such as sleep, drastic changes in behaviour like depression or lack of interest for social activities are independent of PGs levels (3,27). Our precedent experiments demonstrating that human interleukin-1 receptor antagonist (IL-lra) is able to control LPS fever (3), but does not reverse the antidipsogenic effects of LPS treatment (12), suggest that IL-l induced fever is not a true impediment to drinking behaviour (12). We think that inhibition of water intake induced IL-lb and NO, is due, directely or indirectely, through PGs levels in brain (2, 28,29). Acknowledgements We would like to express our appreciation to Mr. Fabio Giuffre for his skilful technical assistance. This work was supported by grants from MURST (40% and 60%). This work was carried out in accordance with the internationally accepted principles concerning the care and the use of laboratory animals.
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References I. 2. 3. 4. 5.
CA. DINARELLO. FASEB J. 2.108-I 15 (1988). CA. DINARELLO. Blood n 1627-1652 (1991). N.J. ROTHWELL. TiPS 12 430-436 (1991). E.T. CUNNINGHAM, Jr. and E.B. DE SOUZA. Immunology Today 14 171- 176 ( 1993). R. BERNARDINI, A.E. CALOGERO, G. MAUCERI and G.P. CHROUSOS. Life Sci. 47 1601-1607 (1990). 6. M.R. OPP and J.M. KRUEGER. Am. J. Physiol. 29 R453-457 (1991). 7. C. MASOTTO, G. CASPANI, M.G. DE SIMONI, M. MENGOZZI, M. SCATTURM, M. SIROM, A. CORENZI and P. GHEZZI. Brain Res. Bull. 28 161-165 (1992). 8. C.R. PLATA-SALAMAN and J.M.H. FFRENCH-MULLEN. Physiol. Behav. a 1277-1279 ( 1992). 9. A. FOCA’, P. NICOLETTA, G. MATERA, P. MASTROENl and A.P. CAPUTI. Circ. Shock 1_1 34 I-350 (1983). 10. G. CALAPAI, G. MAZZAGLIA, M.C. MARCIANO, F. SQUADRITO, D. ALTAVILLA, B.ZINGARELLI, M. PROSDOCIMI and A.P. CAPUTI. Life Sci. j_l204 l-2048 (1992). 11. G. CALAPAI, G. MAZZAGLIA, M. CILIA, B. ZINGARELLI, F. SQUADRITO and A.P. CAPUTI. Br. J. Pharmacol. 111 1328-1332 (1994). 12. F.NAVA, G. CALAPAI, A. DE SARRO and A. P. CAPUTI. Eur. J. Pharmacol. 309 223227 (1996). 13. A.N. EPSTEIN, J.T. FITZSIMONS and B.J. ROLLS. J. Physiol., 210 457-474 ( 1970). 14. A. FOCA’, G. MATERA, P. MASTROENI and A.P. CAPUTI. Circ. Shock u 137-145 (1985). 15. G. CALAPAI, F. SQUADRITO, D. ALTAVILLA, B. ZINGARELLI, G.M. CAMPO, M. CILIA and A.P. CAPUTI. Neuropharmacology 31 761-764 (1992). 16. L.J. PELLEGRINO, A.S. PELLEGRINO and A.J. CUSHMAN. A stereotuxic atlus ofthe rut bruin, Plenum Press, New York, (1975). 17. M. MARTIN and K. RESCH. TiPS2 171-177 (1988). 18. GKATSUURA, P.E. GOTSCHALL, R.R. DAHL and A. ARIMURA. Endocrinology 124 3125-3127(1989). 19. C. F. NATHAN and D.J. STUEHR. J. Natl. Cancer Inst. 82 726-728 (1990). 20. A. K. NUSSLER, M. DI SILVIO, T.R. BILLIAR, R.A. HOFFMAN, D.A. GELLER, R. SELBY, J. MADARIAGA and R.L. SIMMONS. J. Exp. Med. 176 261-264 (1992). 21. S. KENT, R. BLUTHE’, K.W. KELLEY and R. DANTZER. TiPS 13 24-28 (1992). 22. G. CALAPAI, F. SQUADRITO, D. ALTAVILLA, B. ZINGARELLI, G.M. CAMPQ, A. SAITTA, M. IOCULANO, G. URNA, A. SARDELLA and A.P. CAPUTI. Neurosci. Res. Commun. 9 53-61 (1991). 23. C.R. PLATA-SALAMAN, Y. OOMURA and Y. KAI. Brain Res. 448 106-I 14 (1988). 24. N.J. KENNEY and A. EPSTEIN. J. Comp. Physiol. Phychol. 92 204-219 (1978). 25. A. UEHARA, Y. ISHIKAWA, T. OKUMURA, K. OKAMURA, C. SEKIYA, Y. TAKASUGI and M. NAMIKI. Eur. J. Pharmacol. 170 257-260 (1989). 26. A. UEHARA, T. OKUMURA, C. SEKIYA, K. OKAMURA, Y. TAKASUGI and M. NAMIKI. Biophys. Res. Commun. 162 1578-1584 (1989). 27. K. NAITO, H. OSAMA, R. VENO, 0. HAYAISHI, K. HONDA and S. INOVE’. Brain Res. 453 329-336 (1988). 28. M.J. KLUGER. Phys. Rev. 71 1627-1652 (1991). 29. J. GARTHWHITE. TINS 14 60-67 (199 1).
PI1 soo24-3205(96)00675-3
INTERLEUKIN-1
Gioacchino
INHIBITS DRINKING BEHAVIOUR THROUGH PROSTAGLANDINS, BUT NOT BY NITRIC OXIDE FORMATION
Calapai, *Luca Parente, Felice Nava, Gabriella Facciola, Maria Concetta Marciano and Achille P. Caputi
Institute of Pharmacology, School of Medicine, University of Messina and *Institute of Pharmacology and Pharmacognosy, School of Pharmacy, University of Palermo, Italy
(Received in final form November 27, 19%)
Summary Interleukin-1 p (IL-l B) causes inhibition of drinking behaviour. Moreover it induces formation of prostaglandins (PGs) and nitric oxide (NO). Both PGs and NO are able to inhibit drinking stimulated by water deprivation or by intracerebroventricular (i.c.v.) administration of angiotensin II. In this study, we studied in the preoptic area (POA) the possible role of PGs and NO in the antidipsogenic action induced by IL1P. IL-ll3 was injected in the lateral cerebral ventricle (icv.) (2.5, 10, 20, and 40 t&at) or into POA (0.625, 1.25, 2.5, and 10 rig/rat). Larginine (12.5, 25, 50, and 100 @rat), the precursor of NO, or No-nitroL-arginine methyl ester (L-NAME) (25, 50, and 100 @rat), an inhibitor of nitric oxide synthase (NOS), were injected only into POA. Drinking behaviour was induced by water deprivation (24 h). IL-ll3 injected either i.c.v. or mto POA caused dose dependent inhibition of drinking. In the POA a treatment with acetylsalicylic acid (ASA) (33, 66, and 135 &at), but not with L-NAME, antagonized the inhibition of drinking behaviour induced by the highest doses of IL-l p in the POA. In the POA, a treatment with ASA or L-NAME antagonized the inhibition of drinking behaviour caused by injection of the highest doses of L-arginine. Our data suggest that the central inhibition of drinking behaviour of IL-10 is mediated through the formation of PGs, but not NO, in the POA. Key Words: water intake, interleukin-la, cyt~kine.s, preoptic area
______~__~_~________~~~~_~_~~~~~~~~~~______~_~~~~~_________~~~~~__________~~~___________ Correspondence: Ginacchino Calapai, M.D.. Institute of Pharmacology, School of Medicine, University of Mcssina, Piazza XX Settembre 4, 98122 Messina, Italy, Phone: ~39-90-712533, FAX:+39-90-66 1029
458
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Vol. 60,No. 7, 1997
Interleukin-1 (IL-l) is a cytokine released by monocytes/macrophages and in central nervous system (CNS) by glial cells (1,2). IL-l exists in two forms: IL-la, and IL-1S (2). IL-ll3 is the major secreted form, and is evident in a high concentration in plasma, tissue fluids, and brain (3). It has been suggested that IL-ll3 can mediate different neuroendocrine functions (3-5). IL-1 p administration induces fever, sleep, hypotension and anorexia (2, 3,6-8). IL-lp given centrally or peripherally elicits also an inhibitory effect on drinking behaviour through an action on the central sites controlling water intake (7). This effect is not related on the anorexia induced by the cytokine (7). However, the mechanisms underlying its antidipsogenic effect are not clear. In previous studies we have observed that intravenous (i.v.) or intraperitoneal (i.p.) administration of lipopolysaccharide of Escherichia coli (LPS) inhibits drinking behaviour in an overlapping way to the antidipsogenic effect produced by IL- l/3 and other molecules (9- 12). Our recent studies suggest that IL-1 is not directly involved in thirst induced by LPS but indirectly trought the mediation of other substances (12). The preoptic area (POA) is one of the most sensitive cerebral nucleus among the sites involved in drinking behaviour (13). Since various prostaglandins (PGs) including PGIr injected in the POA are antidipsogenic (14) and nitric oxide (NO) formation in POA is responsible for the reduction of water intake stimulated by either water deprivation or angiotensin II (15), we have investigated the role of PGs and NO in antidipsogenic effects induced by IL-10 in 24 h water deprived rats. Materials and Methods Animals: Adult male Sprague-Dawley rats weighing 280-320 g were used. The animals were housed at a constant temperature of 22 5 2” C under a 12/12 h light-dark cycle (lights on at 6:00 a.m.), with free access to Purina rat chow pellets and tap water, unless otherwise stated. Treatments: In a first group of 24 h water deprived rats IL-1S was injected in the lateral cerebral ventricle (i.c.v.) (2.5, 10, 20, and 40 ng/rat) or into the POA (0.625, 1.25, 2.5, and 10 @rat), while naive water deprived animals received saline solution (3 pi/rat i.c.v. or 1 ul/rat in the POA). In a second group of 24 h water deprived rats L-arginine (12.5, 25, 50, and 100 @rat) was injected into the POA, while control animals received saline solution (1 ul/rat). In a last group of 24 h water deprived animals acetyl salycilic acid (ASA) (33, 66, and 135 l&rat) or No-nitro-L-arginine methyl ester (L-NAME) (25, 50, and 100 rig/rat), was given into POA 30 min before injection of IL- 1S or L-arginine. Each group of animals was composed of six rats. Intracerebroventricular iniections: Stainless steel guide cammlae (o.d. = 0.66 mm) were inserted seven days before the experiments. The rats were anesthetized with chloral hydrate (400 mgkg, i.p.) and placed in a stereotaxic instrument (Stellar, Stoelting). For i.c.v. injections, cannulae were implanted i.c.v. (stereotaxic coordinates: AP = 1 mm behind the bregma, L = 2.5 mm from the midsagittal suture and V = 2 mm from the dura). For injections into POA cannulae were implanted, aimed 2 mm above the left medial POA, (stereotaxic coordinates: AP = 7.9 mm anterior to the interaural line, L = 0.7 mm from the midsagittal suture, V = 3.4 mm up from the interaural line) (16). The incisor bar of the stereotaxic instrument was elevated 5 mm above the interaural line (16). Injections into the lateral cerebral ventricle (3 ~1) or into the POA (1 ~1) were made by a 30 gauge injector temporarily inserted into the guide-cannula and protruding 2 mm beyond the cannula tip. Injections were carried out over a period of l-2 min (15).
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Water intake evaluation: Drinking was elicited by 24 h water deprivation. The rats had free access to food during this period. Water intake following deprivation was monitored for a 60 min period and expressed as ml/ rat. Water intake following deprivation was monitored 60 min after IL-18 administration and 30 min after L-arginine injection for a 60 min period and expressed as ml/rat. In order to avoid appearance of tolerance to IL-l 8, each animal was treated only once with this substance and not used for other experiments. Drugs: Interleukin-18 (IL-18) was a gift from Istituto Ricerche Immunobiologiche Siena (I.R.I.S.). Acetylsalicylic acid (ASA) as the soluble lysine salt was purchased from MaggioniWinthrop while L-arginine and No-nitro-L-arginine methyl ester (L-NAME) were obtained from Sigma Chemical Company. IL-18 was dissolved in phosphate buffered saline solution (PBS), ASA in alkaline solution, L-arginine, and L-NAME in 0.9% NaCl solution. Statistical Analysis: Data are expressed as the means + SD. Statistical analysis of data was performed using one way analysis of variance (ANOVA) and Duncan’s test was used to compare group means. Statistical significance was set at P < 0.01. Results I.c.v. injections of IL-18 induced a significant and dose-dependent reduction of water intake. This effect was evident starting from dose of 10 @rat (Fig. 1). A similar effect was observed following IL-18 administration in different doses into POA. In particular an evident and significant water intake inhibition was present starting with the dose of 2.5 &rat (Fig. 2). The same dose did not produce inhibition of drinking when injected i.c.v. in water deprived rats (Fig. 1).
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FIG. 1 Water intake, induced by 24 h water deprivation, after IL-18 administration in the left lateral ventricle (i.c.v.). IL-1 8 doses are expressed as ng/rat. Water was presented to the animals 60 min after IL18 treatment for a 60 min period. Each column represents the mean + S.D. of six animals. *P < 0.01 vs control.
pGsandIL~DrhkiugIchii
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FIG. 2 Water intake, induced by 24 h water deprivation, after IL-IS administration in the POA. IL-ll3 doses are expressed as ngkat. Water was presented to the animals 60 min after IL-ll3 treatment for a 60 min period. Each column represents the mean + SD. of six animals. *P < 0.01 vs control. L-arginine (12.5, 25,50, and 100 rig/rat) injected into POA inhibited, in evident manner and significantly, water intake starting by the dose of 25 @rat (Fig. 3).
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4L A FIG. 3 i Water intake, induced by 24 h water deprivation, after L-arginirk administration into POA. L-arginine doses are expressed as ngkat. Water was presented to the animals 30 min after L-arginine treatment for a 60 min period. Each column represents the mean + S.D. of six animals. *P < 0.01 vs control.
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A pretreatment with ASA (33, 66, and 135 &at) into the POA, 30 min before IL-1S treatment, was able to antagonize antidipsogenic effects induced by injection of IL-1 g at dose of 10 @rat (Fig. 4). On the other hand a pretratment with L-NAMJZ(25, 50, and 100 ng/rat) into the POA, 30 min before IL-ll3 administration, was unable to influence thirst induced by IL-l S administration in POA at dose of 10 &rat (Fig. 4). 14
T
12
0
Y 4 i FIG. 4 Water intake induced by 24 h water deprivation, after IL-ll3 (at the highest dose used) + ASA or IL-lp (at the highest dose used) + LNAME administration into POA. ASA doses are expressed as I.&at, while IL-1 p or L-NAME doses as @at. Water was presented to the animals 60 min after IL-1 /3 treatment for a 60 min period. Each column represents the mean + S.D. of six animals. *P < 0.01 vs control. Injection into POA of ASA (33, 66, and 135 @rat) or L-NAME (25, 50, and 100 n&at), 30 min before L-arginine, was able to abolish, significantly and in a dose-dependent manner, thirst induced by L-arginine (Fig. 5).
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FIG. 5 Water intake induced by 24 h water deprivation, after Garginine (at the highest dose used) + ASA or L-arginine (at the highest dose used) + L-NAME administration into POA. ASA doses are expressed as @rat, while IL-IS or L-NAME doses as r&at. Water was presented to the animals 30 min after L-arginine treatment for a 60 min period. Each column represents the mean t S.D. of six animals. *P < 0.0 1 vs control.
It is now evident that IL-IS, like other cytokines, acts on different target cells and modulates many biological functions (1-4, 17). The role that this cytokine plays in the communication between the central nervous system (CNS) and the periphery is one of the most interesting features of its biology (3-5). IL-Ip is produced in the brain by astrocytes and is able to alter neuroendocrine activity, slow-wave sleep and body temperature (3,6-S, 17). It has been shown that IL-ll3 can induce its effects acting directly on specific target tissues or, indirectly, as an effector molecule (12). This second way may explain, for example, the hypotension caused by IL-l l3 through the formation of substances like PGs and NO ( 18). It is well known that IL-1 p can influence NO release, it increases the expression of inducible NOS in several rodent cell types and causes nitrite and nitrate formation (19, 20). A reduction of water intake, like other changes (temperature, sleep, feeding) occurring during infections, can be considered a result of toxic agents action (21) or various substances and cytokines including IL-1 l3 (12). In particular various works have shown that peripheral administration of endotoxin (9, 11, 12) or central injection of tumor necrosis factor a (TNF a) (11,22,23), both releasing IL- 1, produce similar inhibitory effects on drinking behaviour. More recently, we also demonstrated that either LPS or TNFa cause inhibition of drinking behaviour through the release of NO in the POA (I 1). POA is one of the most important cerebral regions involved in the mechanisms regulating drinking behaviour (13) and fever (3).
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Our data show that high doses of IL-l l3 injected in the POA inhibit water intake induced by 24 h water deprivation. Furthermore, doses of 2.5 and 5 r&at, able to inhibit drinking behaviour when injected into POA, are not effective when administered in the lateral cerebral ventricle. Injection of IL-l B at the dose of 10 @rat in the lateral cerebral ventricle causes a slight but significant reduction of water intake, but the administration of the same dose into POA induces a more potent inhibition of drinking behaviour, thus indicating that POA is a specific site for control of drinking behaviour. The present results confirm previous studies showing that antidipsogenic effect can be produced by L-arginine injection, the precursor of NO, in the POA and that this action is specific because it can be reverted by L-NAME, an inhibitor of NO formation (11). Since our precedent experiments have demonstrated that IL-lp is not a direct mediator implicated in inhibition of thirst induced by LPS (12) we have investigated the possibility that the Larginine/NO pathway and/or PGs could be activated by IL-ll3 in order to inhibit drinking behaviour. Our data show that L-NAME, an inhibitor of NOS, given into POA, is not able to antagonize IL-lb inhibition of water intake, while ASA into POA is very effective. The precursor of NO, L-arginine, when injected into POA, inhibits drinking and this effect is abolished either by L-NAME or ASA. Therefore, it is possible to argue that IL-lb needs arachidonic acid metabolites to inhibit drinking, while NO formation is not necessary. This result is in accord with precedent studies showing that IL-lb is able to increase PGEr secretion in astrocyte culture in the rat (18) and that PGE, is a potent antidipsogenic compound (24). On the contrary, the antidipsogenic effect of L-arginine requires an increased synthesis of NO, since it is abolished by L-NAME. Moreover, the antidipsogenic effect of L-arginine requires arachidonic acid metabolites because it is blocked by ASA. So, at the moment, we can hypothesize that in the POA an increase of NO formation leads to an increased metabolism of arachidonic acid. The above findings induce to believe that effects of IL-lp on drinking behaviour in 24 h water deprived rats are not mediated directly by NO formation but by PG levels. These results are in accord to various studies demonstrating that many of IL-lp central effects are dependent on the synthesis .of eicosanoids, since they are attenuated by cyclooxygenase inhibitors (2, 3, 25, 26). We believe that PGs might be involved not only in fever and in other central effects but also in reduction of water intake observed after IL-ll3 administration. This is also evident from our data demonstrating that POA, a crucial site for control of fever and water intake, is very sensible to eicosanoids levels. By contrast, various central effects of IL-lb such as sleep, drastic changes in behaviour like depression or lack of interest for social activities are independent of PGs levels (3,27). Our precedent experiments demonstrating that human interleukin-1 receptor antagonist (IL-lra) is able to control LPS fever (3), but does not reverse the antidipsogenic effects of LPS treatment (12), suggest that IL-l induced fever is not a true impediment to drinking behaviour (12). We think that inhibition of water intake induced IL-lb and NO, is due, directely or indirectely, through PGs levels in brain (2, 28,29). Acknowledgements We would like to express our appreciation to Mr. Fabio Giuffre for his skilful technical assistance. This work was supported by grants from MURST (40% and 60%). This work was carried out in accordance with the internationally accepted principles concerning the care and the use of laboratory animals.
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References I. 2. 3. 4. 5.
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