Brain Research Bulletin, Vol. 26, pp. 155-160. 0 Pergamon Press plc, 1991. hinted in the U.S.A.
0361-9230191 $3.00 + .OO
Tachykinin Receptor Subtypes Involved in the Central Effects of Tachykinins on Water and Salt Intake’ M. MASSI,* C. POLIDORI, Institute of Pharmacology,
M. PERFUMI,
University of Camerino,
Received
L. GENTILI
AND G.
Via Scalzino 5, 62032
DE
CAR0
Camerino
(MC), Italy
5 July 1990
MASSI, M., C. POLIDORI, M. PERFUMI, L. GENTILI AND G. DE CARO. Tachykinin receptor subtypes involved in the cenfral efjcecrsof tachykinins on water and salt intake. BRAIN RES BULL 26(l) 155-160, 1991.--The present study was aimed at investigating which tachykinin receptor subtypes mediate the inhibitory effects of tachykinins a) on salt intake induced by sodium depletion, b) on water intake induced by subcutaneous hypertonic NaCl administration and c) on water intake induced by central angiotensin II injection. The study was carried out by evaluating the potency of action, following intracerebroventricular injection, of several peptides, including both naturally occurring tachykinins and synthetic peptides selective for a given receptor subtype. The results obtained show different rank orders of potency of the agonists in the different behavioral tests, thus suggesting that different receptor subtypes are involved in the effects of tachykinins on water and salt intake. NK-3 receptors appear to be involved in the inhibitory effect of tachykinins on depletion-induced salt appetite. NK-2 receptors apparently mediate the inhibitory effect of tachykinins on drinking induced by hyperosmotic NaCl administration, while NK-1 receptors are probably involved in the inhibition of angiotensin II-induced drinking. Tachykinins
Tachykinin
receptor subtypes
Salt appetite
Water intake
TACHYKININS (TKs) are biologically active peptides sharing the common carhoxyterminal sequence PHE-X-GLY-LEU-MET* NH, (9). Four TKs have been so far identified in the gastrointestinal tract and in the central nervous system of mammals: substance P (SP), neurokinin B (NKB), neurokinin A (NKA) and its extended form neuropeptide K (NPK) (1, 10, 22, 23). According to the occurrence of multiple TKs, at least 3 different TK receptor subtypes have been proposed: the NK-1, the NK-2 and the NK-3 for which SP, NKA and NKB, respectively, are considered to be the endogenous ligand (12-14, 20, 21). In the absence of potent and selective TK antagonists the rank order of potency of agonists has been used for receptor classification both in in vitro and in receptor binding assays. Previous studies have shown that the intracerebroventricular (ICV) injection of the naturally occurring TKs eledoisin (ELE), physalaemin (PHYS) and SP exert potent effects of central origin on the regulation of body fluids in the rat. In fact, they potently inhibit water and salt intake induced by a variety of dipsogenic and natriorexigenic determinants (3-6); moreover, ELE and PHYS,
but not SP, produce antidiuresis in water replete rats, which appears to be mediated by vasopressin release (2,19). The abovementioned effects have been so far studied with naturally occurring TKs which are rather unselective for the different TK receptor subtypes. This lack of selectivity makes it impossible to understand which TK receptor is involved in the different behavioral effects of TKs and to understand the functional relationship between these effects. The present study was therefore designed to investigate the subtypes of TK receptor involved in 3 behavioral effects of TKs: 1) the inhibition of sodium depletion-induced salt appetite, 2) the suppression of water intake induced by subcutaneous (SC) hypertonic NaCl load and 3) the inhibition of water intake induced by ICV angiotensin II (Ang II). The study was carried out employing: a) two highly selective NK-3 agonists, [MePhe’]NKB (7,8) and the senktide analogue ([AspS.6,MePhe8]SP5-1 1) (NH,SENK) (ll), b) the NK-1 selective agonist SP methyl ester (SPOMe) (8),
‘This article was presented at the Xth International Congress on the Physiology of Food and Fluid Intake held in Paris, France, July 4-8, 1989. Other selected articles from this meeting have been published in Physiology & Behavior, Volume 48, Number 6, 1990, Volume 49, Number 1, 1991 and Brain Research Bulletin, Volume 25, Number 6, 1990. *Requests for reprints should be addressed to Dr. Maurizio Massi, Istituto di Farmacologia, Universita’ di &merino, Via Scalzino 5, 62032 Camerino (MC), Italy.
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c) two SP derivatives, [Sar9]SP (8) and [MePhe’,Sar’]SP, endowed with NK-1 and NK-3, but devoid of NK-2, activity; d) in addition we tested also several naturally occurring TKs, both mammalian and nonmammalian. Although being rather unselective, they show different agonist receptor profiles, which were considered helpful to our purposes. METHOD
Animals Male albino Wistar rats (Charles River, Calco, Como, Italy), averaging 300-325 g at the moment of the intracranial surgery, were employed. They were housed individually in a temperaturecontrolled room on a 12: 12 light-dark cycle. Food pellets (MILL, Morini, Reggio Emilia, Italy) and tap water were available ad lib. Rats used for the experiments concerning salt intake were offered access to 3% NaCl, as described below in detail. Drugs The following drugs were employed: 1) SP, 2) NKA, 3) ELE, 4) PHYS, 5) kassinin (KASS), 6) SPOMe, 7) [Sar’]SP and 8) [MePhe’,Sar’]SP, which were purchased from Peninsula Labs Europe, Merseyside, U.K. We also employed 9) [MePhe’]NKB 1) which were a generous gift of the and ([As~‘,~ ,MePhe*]SPS--1 Menarini Pharmaceuticals, Florence, Italy. Intracranial Surgery All the animals employed were anaesthetized (Equithesin, 3 ml/kg b.wt.; intraperitoneally) and fitted by stereotaxic surgery with a stainless steel cannula (o.d. 0.6 mm), aimed at the anteroventral third ventricle for rats employed in salt intake experiments and at the lateral ventricle for water intake experiments. The guide cannula was attached to the skull by jewelry screws and dental acrylic cement. The rats were allowed 1 week to recover from surgery before testing began. During the postoperative period the animals were handled and mock-injected to adapt them to the testing procedure. Intracranial Injections All the peptides tested were dissolved in sterile isotonic saline (ISO) and were administered through a stainless steel injector (o.d. 0.3 mm) temporarily inserted into the guide cannula and protruding into the ventricle. All the drugs were given in a volume of 1 (*l. Experimental Procedure Experiment I: Effect of ICV injection of TKs on sodium depletion-induced salt appetite. Salt appetite was elicited by an adaptation of the method of Wolf (24), in which depletion is produced by combining pharmacological natriuresis with a sodium-deficient diet. Natriuresis was produced by SC injection of furosemide (Lasix; 2 injections of 5 mg/rat, separated by 2 h). At the time of the first injection, the pellets were replaced by a sodium-deficient powdered diet (ICN Nutritional Biochemicals, Cleveland, OH USA, No. 902903) offered in a glass cup, 3% NaCl was removed from the cages and the cages were washed to remove adherent salt. The animals were not deprived of water. Twenty-two to 24 h later they received a pulse ICV injection of the TK peptide tested or of simple IS0 (controls) and immediately afterwards 3% NaCl was returned to them. Consumption of 3% NaCl and water, as well as latency to drinking each solution, was recorded at 15, 30, 60 and 120 min. Each animal received different treat-
ments at intervals of 7 days. Testing began with the 3rd depletion, when salt intake in response to sodium depletion had reached a plateau. The experiment was carried out according to a withinsubject design to minimize the individual variability in salt intake. Each rat received both IS0 and all the doses of each peptide. Different groups of rats were used for different TKs. Experiment 2: Effect of ICV injection of TKs on drinking induced by SC hypertonic NaCl. Hypertonic NaCl(l.5 M; 1 ml/l00 g b.wt.) was given subcutaneously in the loose skin of the back. After the SC injection rats were returned to their cage from which water had been temporarily removed. Fifteen min after the NaCl administration animals received the ICV injection of the TK tested or of simple IS0 (controls) and immediately afterwards they had access to water. The intake of water was measured at intervals of 15 min for a period of 2 h after access to it. The experiment was carried out according to a randomized block design. Experiment 3: Eflect of ICV injection of TKs on Ang Il-induced drinking. Ang II (100 ng/rat) was given by ICV injection 1 min after that of IS0 or of the TK tested. Immediately after the second ICV injection the animals had free access to water and food. Water intake was measured at intervals of 15 min for 1 h after Ang II administration. The experiments were carried out according to a randomized block design. Statistical analysis. Data are presented as averages (means ? SEM). Statistical analysis of data for each peptide was performed by multifactorial analysis of variance (ANOVA). Planned pairwise comparisons were carried out by means of t-tests, when the ANOVA revealed a significant drug effect. Statistical significance was set at pcO.05. RESULTS
Experiment 1: Effect of ICV Injection of TKs on Sodium Depletion-Induced Salt Appetite The results of this experiment are summarized in Fig. 1. a) The selective NK-3 agonists [MePhe’]NKB and NH,SENK proved to be very potent inhibitors of salt intake. [MePhe’]NKB inhibited the 15-min salt intake even at the dose of 31.2 ng (24.5 picomoles)/rat, the intake of treated rats (4.01 kO.6) being statistically lower than that of controls (6.08kO.46, p
0361-9230191 $3.00 + .OO
Tachykinin Receptor Subtypes Involved in the Central Effects of Tachykinins on Water and Salt Intake’ M. MASSI,* C. POLIDORI, Institute of Pharmacology,
M. PERFUMI,
University of Camerino,
Received
L. GENTILI
AND G.
Via Scalzino 5, 62032
DE
CAR0
Camerino
(MC), Italy
5 July 1990
MASSI, M., C. POLIDORI, M. PERFUMI, L. GENTILI AND G. DE CARO. Tachykinin receptor subtypes involved in the cenfral efjcecrsof tachykinins on water and salt intake. BRAIN RES BULL 26(l) 155-160, 1991.--The present study was aimed at investigating which tachykinin receptor subtypes mediate the inhibitory effects of tachykinins a) on salt intake induced by sodium depletion, b) on water intake induced by subcutaneous hypertonic NaCl administration and c) on water intake induced by central angiotensin II injection. The study was carried out by evaluating the potency of action, following intracerebroventricular injection, of several peptides, including both naturally occurring tachykinins and synthetic peptides selective for a given receptor subtype. The results obtained show different rank orders of potency of the agonists in the different behavioral tests, thus suggesting that different receptor subtypes are involved in the effects of tachykinins on water and salt intake. NK-3 receptors appear to be involved in the inhibitory effect of tachykinins on depletion-induced salt appetite. NK-2 receptors apparently mediate the inhibitory effect of tachykinins on drinking induced by hyperosmotic NaCl administration, while NK-1 receptors are probably involved in the inhibition of angiotensin II-induced drinking. Tachykinins
Tachykinin
receptor subtypes
Salt appetite
Water intake
TACHYKININS (TKs) are biologically active peptides sharing the common carhoxyterminal sequence PHE-X-GLY-LEU-MET* NH, (9). Four TKs have been so far identified in the gastrointestinal tract and in the central nervous system of mammals: substance P (SP), neurokinin B (NKB), neurokinin A (NKA) and its extended form neuropeptide K (NPK) (1, 10, 22, 23). According to the occurrence of multiple TKs, at least 3 different TK receptor subtypes have been proposed: the NK-1, the NK-2 and the NK-3 for which SP, NKA and NKB, respectively, are considered to be the endogenous ligand (12-14, 20, 21). In the absence of potent and selective TK antagonists the rank order of potency of agonists has been used for receptor classification both in in vitro and in receptor binding assays. Previous studies have shown that the intracerebroventricular (ICV) injection of the naturally occurring TKs eledoisin (ELE), physalaemin (PHYS) and SP exert potent effects of central origin on the regulation of body fluids in the rat. In fact, they potently inhibit water and salt intake induced by a variety of dipsogenic and natriorexigenic determinants (3-6); moreover, ELE and PHYS,
but not SP, produce antidiuresis in water replete rats, which appears to be mediated by vasopressin release (2,19). The abovementioned effects have been so far studied with naturally occurring TKs which are rather unselective for the different TK receptor subtypes. This lack of selectivity makes it impossible to understand which TK receptor is involved in the different behavioral effects of TKs and to understand the functional relationship between these effects. The present study was therefore designed to investigate the subtypes of TK receptor involved in 3 behavioral effects of TKs: 1) the inhibition of sodium depletion-induced salt appetite, 2) the suppression of water intake induced by subcutaneous (SC) hypertonic NaCl load and 3) the inhibition of water intake induced by ICV angiotensin II (Ang II). The study was carried out employing: a) two highly selective NK-3 agonists, [MePhe’]NKB (7,8) and the senktide analogue ([AspS.6,MePhe8]SP5-1 1) (NH,SENK) (ll), b) the NK-1 selective agonist SP methyl ester (SPOMe) (8),
‘This article was presented at the Xth International Congress on the Physiology of Food and Fluid Intake held in Paris, France, July 4-8, 1989. Other selected articles from this meeting have been published in Physiology & Behavior, Volume 48, Number 6, 1990, Volume 49, Number 1, 1991 and Brain Research Bulletin, Volume 25, Number 6, 1990. *Requests for reprints should be addressed to Dr. Maurizio Massi, Istituto di Farmacologia, Universita’ di &merino, Via Scalzino 5, 62032 Camerino (MC), Italy.
155
MASS1 ET AL.
156
c) two SP derivatives, [Sar9]SP (8) and [MePhe’,Sar’]SP, endowed with NK-1 and NK-3, but devoid of NK-2, activity; d) in addition we tested also several naturally occurring TKs, both mammalian and nonmammalian. Although being rather unselective, they show different agonist receptor profiles, which were considered helpful to our purposes. METHOD
Animals Male albino Wistar rats (Charles River, Calco, Como, Italy), averaging 300-325 g at the moment of the intracranial surgery, were employed. They were housed individually in a temperaturecontrolled room on a 12: 12 light-dark cycle. Food pellets (MILL, Morini, Reggio Emilia, Italy) and tap water were available ad lib. Rats used for the experiments concerning salt intake were offered access to 3% NaCl, as described below in detail. Drugs The following drugs were employed: 1) SP, 2) NKA, 3) ELE, 4) PHYS, 5) kassinin (KASS), 6) SPOMe, 7) [Sar’]SP and 8) [MePhe’,Sar’]SP, which were purchased from Peninsula Labs Europe, Merseyside, U.K. We also employed 9) [MePhe’]NKB 1) which were a generous gift of the and ([As~‘,~ ,MePhe*]SPS--1 Menarini Pharmaceuticals, Florence, Italy. Intracranial Surgery All the animals employed were anaesthetized (Equithesin, 3 ml/kg b.wt.; intraperitoneally) and fitted by stereotaxic surgery with a stainless steel cannula (o.d. 0.6 mm), aimed at the anteroventral third ventricle for rats employed in salt intake experiments and at the lateral ventricle for water intake experiments. The guide cannula was attached to the skull by jewelry screws and dental acrylic cement. The rats were allowed 1 week to recover from surgery before testing began. During the postoperative period the animals were handled and mock-injected to adapt them to the testing procedure. Intracranial Injections All the peptides tested were dissolved in sterile isotonic saline (ISO) and were administered through a stainless steel injector (o.d. 0.3 mm) temporarily inserted into the guide cannula and protruding into the ventricle. All the drugs were given in a volume of 1 (*l. Experimental Procedure Experiment I: Effect of ICV injection of TKs on sodium depletion-induced salt appetite. Salt appetite was elicited by an adaptation of the method of Wolf (24), in which depletion is produced by combining pharmacological natriuresis with a sodium-deficient diet. Natriuresis was produced by SC injection of furosemide (Lasix; 2 injections of 5 mg/rat, separated by 2 h). At the time of the first injection, the pellets were replaced by a sodium-deficient powdered diet (ICN Nutritional Biochemicals, Cleveland, OH USA, No. 902903) offered in a glass cup, 3% NaCl was removed from the cages and the cages were washed to remove adherent salt. The animals were not deprived of water. Twenty-two to 24 h later they received a pulse ICV injection of the TK peptide tested or of simple IS0 (controls) and immediately afterwards 3% NaCl was returned to them. Consumption of 3% NaCl and water, as well as latency to drinking each solution, was recorded at 15, 30, 60 and 120 min. Each animal received different treat-
ments at intervals of 7 days. Testing began with the 3rd depletion, when salt intake in response to sodium depletion had reached a plateau. The experiment was carried out according to a withinsubject design to minimize the individual variability in salt intake. Each rat received both IS0 and all the doses of each peptide. Different groups of rats were used for different TKs. Experiment 2: Effect of ICV injection of TKs on drinking induced by SC hypertonic NaCl. Hypertonic NaCl(l.5 M; 1 ml/l00 g b.wt.) was given subcutaneously in the loose skin of the back. After the SC injection rats were returned to their cage from which water had been temporarily removed. Fifteen min after the NaCl administration animals received the ICV injection of the TK tested or of simple IS0 (controls) and immediately afterwards they had access to water. The intake of water was measured at intervals of 15 min for a period of 2 h after access to it. The experiment was carried out according to a randomized block design. Experiment 3: Eflect of ICV injection of TKs on Ang Il-induced drinking. Ang II (100 ng/rat) was given by ICV injection 1 min after that of IS0 or of the TK tested. Immediately after the second ICV injection the animals had free access to water and food. Water intake was measured at intervals of 15 min for 1 h after Ang II administration. The experiments were carried out according to a randomized block design. Statistical analysis. Data are presented as averages (means ? SEM). Statistical analysis of data for each peptide was performed by multifactorial analysis of variance (ANOVA). Planned pairwise comparisons were carried out by means of t-tests, when the ANOVA revealed a significant drug effect. Statistical significance was set at pcO.05. RESULTS
Experiment 1: Effect of ICV Injection of TKs on Sodium Depletion-Induced Salt Appetite The results of this experiment are summarized in Fig. 1. a) The selective NK-3 agonists [MePhe’]NKB and NH,SENK proved to be very potent inhibitors of salt intake. [MePhe’]NKB inhibited the 15-min salt intake even at the dose of 31.2 ng (24.5 picomoles)/rat, the intake of treated rats (4.01 kO.6) being statistically lower than that of controls (6.08kO.46, p
