513
Journal of Physiology (1991), 443, pp. 513-518 With 2 figures Printed in Great Britain
THE EFFECT OF ANALOGUES OF ANGIOTENSIN II ON DRINKING AND CARDIOVASCULAR RESPONSES TO CENTRAL ANGIOTENSIN II IN THE RAT
BY L. A. A. CAMARGO, L. A. DE LUCA JR, J. V. MENANI, A. RENZI, W. A. SAAD, WILLIAN A. SAAD* AND J. E. N. SILVEIRA From the Department of Physiology, School of Dentistry, UNESP, 14800, Araraquara, SP, Brazil, and the *Department of Surgery, School of Medicine, University of Sdo Paulo, SP, Brazil
(Received 6 July 1990) SUMMARY
1. Intracerebroventricular (i.c.v.) infusion (60 ng h-1) of Isoleu5-angiotensin II (Isoleu5-AngIl) and des-amine-angiotensin II (des-amine-AngII) in rats caused increased drinking behaviour and an increase in arterial blood pressure. 2. Des-amine-AnglI caused similar increases in heart rate and arterial blood pressure as AnglI. 3. Previous i.c.v. injection of the antagonists [Leu8]-AngII, des-amine-[Leu8]Angll and octanoyl-[Leu8]-AnglI prevented the increases in heart rate and blood pressure produced by i.c.v. infusion of AngII and caused partial reduction of the dipsogenic response.
4. The three antagonists had no effect on the increase in arterial blood pressure and heart rate caused by des-amine-AngII. The drinking response was reduced by previous injection of [Leu8]-Angll and des-amine-[Leu8]-Angll but not by octanoyl-
[Leu8]-AngII. 5. In conclusion, Isoleu5-AngII and des-amine-Angll increase drinking behaviour, arterial blood pressure and heart rate when infused into the cerebral ventricle of rats. The study with the antagonists showed that des-amine-AnglI probably binds more strongly to AnglI-receptors. INTRODUCTION
An abundance of evidence has shown that angiotensin II (AnglI) plays an important role in the forebrain circumventricular organs for the control of blood pressure and body fluid balance (Haywood, Ball, Lifschitz & Brennan, 1987; Bealer & Van Huysse, 1989; Thrasher, 1989; Unger, Horst, Bauer, Demmert, Rettig & Rohmeiss, 1989). Several studies on the renin-angiotensin system and its participation in different functions of the body, have indicated the action of angiotensin and its analogues in terms of structure-activity. Many AnglI analogues have been obtained by substituting one or more amino acids and radicals in the AnglI molecule (Fitzsimons, 1971). In relation to the myotropic and vasopressor MS 8633 17
PHY 443
514
L. A. A. CAMARGO AND OTHERS
activity of a number of antagonists of Angll analogues, it has been determined that the phenyl-group at position 8 has the information for the biological response, while the aromatic groups at positions 4 and 6 (tyrosine and histidine respectively), the guanide group at position 2 and the carboxyl C-ending are involved in the site of receptor binding. Also it has been determined that when the octapeptide Angll is deprived of its N-ending (N-terminal) this causes a progressive reduction in its central dipsogenic activity (Gildenberg, Ferrario & McCubbin, 1973; Ganten, Hutchinson, Schelling, Ganten & Fischer, 1976). The affinity of Angll and AngII analogues for converting enzymes is strongly infltienced by the charge of the NH2terminal amino acid residue. Inhibitory activity is enhanced by central or basic substituents and attenuated by acid NH2-terminal residues (Peach, 1977). In mammals, the structure of position 8 of the AngIl appears to be important in determining the affinity for angiotensin receptors, as well as acting as a part of the molecule which transcribes information to evoke intrinsic activity, presumably through the secondary mechanisms in the receptor (Nishimura, Norton & Bumpus, 1978). The present experiments were undertaken in order to test the effects of Isoleu5-AngII and its analogue des-amine-AngIl on water intake and cardiovascular responses. We also studied the action of different receptor antagonists of AnglI on these effects which were substituted in position 8. METHODS
Adult male Holtzman rats weighing 250-300 g were housed in individual metabolic cages. Drinking water was available in burettes with standard metal spouts projecting into the cages. Food pellets were available all the time except during an experiment. The animals were maintained on a 12-12 h light-dark cycle with light on at 08.00 h daily. Room temperature was maintained at approximately 24 'C. Following an acclimatization period of 7 days animals were anaesthetized with ether and cannulae were implanted into the lateral ventricle. The procedures were carried out using a Model 900 stereotaxic apparatus (David Kopf Instruments, Tujunga, USA) and the co-ordinates of the Konig & Klippel (1963) atlas. After brain surgery, a prophylactic dose of 50000 i.u. of penicillin was administered to each animal. On the morning of the fifth day after implantation of the cannulae, the animals were anaesthetized with ether and a PE-50 polyethylene catheter was introduced into the femoral artery. The catheter was filled with heparinized saline (0-15 M) and fixed to the animal's back with suture through a skin incision (Camargo, Menani, Saad & Saad, 1984). Tests were started after a 3 h period of recovery from surgery. A stainless-steel 30-gauge needle was used for infusion of the peptides into the lateral ventricle (LV) at a rate of 5,1l h-' using a PE-10 polyethylene tube connected to a 1 ml syringe connected to an infusion pump. Arterial pressure was recorded from the femoral artery and heart rate was recorded using the pressure wave to trigger a rate meter. The water intake was measured using burettes with 0-1 ml markings. The peptides used were the agonists Isoleu5-AngII and des-amine-Angll, and the AngIl antagonists [Leu8]-AngII, des-amine[Leu8]-AngJI and octanoyl-[Leu8]-AngII. The peptides were generously provided by Professor A. C. Paiva from the School of Paulista Medicine. At the end of the experimental period, the animals were anaesthetized with ether and perfused through the heart with 10 % saline and formol solution. The brains were removed, frozen sections cut, and routine histology was performed for rapid location of the cannula pathway. The results were analysed statistically by analysis of variance and unpaired t test with Dunnett's correction for multiple comparisons with a control, as necessary.
Experimental procedures Water intake, arterial pressure and heart rate were measured in different groups of rats submitted to the following experimental protocols:
CENTRAL ANGIOTENSIN AND ANALOGUES
515
Infusion of Isoleu5-AngII (60 ng h-') into the LV (n = 21 rats). Injection of [Leu8]-AngII (60 ng) into the LV+Isoleu5-AngII (60 ng h-1) infusion (n = 21 rats). Injection of des-amine-[Leu8]-AngII (60 ng) into the LV + Isoleu5-AngII (60 ng h-') infusion (n = 22 rats). 130
-
A
450
B
8
C
-T00-6E~~~~~~~~~~~~~~ E100
U)
40
(0~~~~~~~~~~~~~~~*
.3500
Fig. 1. Effect of infusion of Isoleu5-AngII into the lateral ventricle ([WI) on arterial pressure, heart rate and water intake. The antagonists [Leu8]-AngII (m), des-amine[Leu8]-AngII (0) and octanoyl-[Leu8]-AngII (C) were injected 7 min before Isoleu5Angll. Data are means +S.E.M.; (El) control (no infusion). * different from control; ° different from Isoleu5-AngII.
Injection of octanoyl-[Leu8]-AngII (60 ng) into the LV + Isoleu5-AngII (60 ng h-') infusion (n = 25 rats). Infusion of des-amine-AngIl (60 ng h-') into the LV (n = 25 rats). Injection of [Leu8]-AngII (60 ng) into the LV + des-amine-AngIl (60 ng h-1) infusion (n = 24 rats). Injection of des-amine-[Leu8]-AngII (60 ng) into the LV + des-amine-AngIl (60 ng h-1) infusion (n = 24 rats). Injection of octanoyl-[Leu8]-AngII (60ng) into the LV+des-amine-AngII (60ngh-1) infusion
(n = 25 rats). The AngIl antagonists were injected into the LV 7 min before the AngII infusion was started. The volume injected was 1 ,ul and the rate of infusion was 5 /ul h-1. The period of injection was 30 s and the time of infusion was 1 h. The recording period for water intake, arterial pressure and heart rate was 1 h. Each experiment was carried out on a separate day. The minimum period between experiments was 3 days. RESULTS
Effects of Isoleu5-AngII infusion into the lateral ventricle and of previous injection of the antagonists [Leu8]-AngII, des-amine-[Leu8]-AngII and octanoyl-[Leu']-AngII The infusion of Isoleu5-AngII into the lateral ventricle caused an increase in water intake, blood pressure and heart rate compared with control values (P < 0 05; Fig. 1A, B and C). The increase in arterial pressure was gradual and reached a maximum after 10 min of infusion. Previous injection of the antagonists blocked the pressor effect and the increase in heart rate produced by infusion of Isoleu5-Ang1I when compared with the experiment utilizing only the peptide agonist (P < 0 05; Fig. 1A and B). This blocking effect lasted throughout the observation period. The antagonists reduced but did not fully block the water intake induced by Isoleu5AnglI as compared with the experiment using only the agonists and with the control values (P < 0 05); Fig. 1 C). 17-2
516
L. A. A. CAMARGO AND OTHERS
Effects of des-amine-AnglI infusion into the lateral ventricle and of previous injection of the antagonists [Leu8]-AngII, des-amine-[Leu8]-AngII and octanoyl-[Leu8]-AngII The infusion of des-amine-AnglI into the lateral ventricle caused an increase in blood pressure, heart rate and water intake (with the latency around 30 s) compared 130
-
A
450
B
-
*
E
8 ~~
~
~
C ~
~
~
*
1000 E~~~~~~~c
g~~~~~~~~~~~o~c a,~~~~~~~~~~~~C
c*n 70
a)
0 35t
4X
40302
Fig. 2. Effect of infusion of des-amine-AngII into the lateral ventricle (ODh) on arterial pressure, heart rate and water intake. The antagonists [Leu8]-AnglI (n), des-amine[Leu8]-AnglI (0) and octanoyl-[Leu8]-AngII (0) were injected 7 min before des-amineAngll. Data are means + S.E.M.; l control (no infusion). * different from control, 0 different from des-amine-AngII.
with control values (P < 0 05; Fig. 2A, B and C). The increase in arterial pressure started immediately after the infusion of des-amine-AnglI. Arterial pressure and heart rate remained unchanged after previous (7 min) injection of the three antagonists compared with the control values nor was there any effect upon the response to des-amine-AnglI. The water intake induced by des-amine-Angll infusion was significantly decreased by previous injection of [Leu8]-AngIJ and des-amine[Leu8]-AngII when compared with the values obtained after infusion of des-amineAngll alone (P < 0-05). Octanoyl-[Leu8]-AngII did not block the effect of des-amineAnglI (Fig. 2C). DISCUSSION
The infusion of Isoleu5-Ang1I into the lateral ventricle produced an increase in arterial blood pressure, heart rate and water intake. These results agree with those obtained by Hoffman & Phillips (1976) and Colombari, Saad, Camargo, Renzi, De Luca & Menani (1990). A series of observations have indicated that the action of this peptide in inducing a pressor response is due to the activation of the receptors at many sites in the brain (Zimmerman, 1973, 1978; Anderson, 1977; Simpson, Epstein & Camargo, 1978; Brosnihan, Berti & Ferrario, 1979). Another possible action of Isoleu5-AngII is the activation of the release of a cerebral hormone with a prolonged vasopressor effect. Isoleu5-AngIJ may act on more than one type of receptor to induce a thirst response. The present results show that the two agonists studied (Isoleu5-AngII and des-amine-Angll) have both pressor and dipsogenic effect. The
CENTRAL ANGIOTENSIN AND ANALOGUES
517
latency for the pressor and dipsogenic effect of des-amine-AngII is shorter than that for the effect of Isoleu5-AnglI. The duration of the effect of des-amine-AnglI is shorter than that of Isoleu5-AngII. These agree with the results of Saad, Menani, Camargo & Abrao-Saad (1985) and Gongalves, Camargo, Menani, Saad, Saad, Renzi & De Luca (1988). Three AnglI antagonists were injected into the lateral ventricle before the infusion of Isoleu5-AngII and des-amine-Angll. The thirst-inducing effect of Isoleu5Angll was decreased. The three antagonists had no influence on the pressor and tachyeardic effect of des-amine-Angll. The dipsogenic effect was decreased by prior injection of the first two antagonists. In all of the antagonists studied, the amino acid and residue phenylanine at position 8a is substituted by one leucine. Bumpus (1977) reported that for the myotropic and vasopressor activity of the many antagonists of Isoleu5-AngII it has been determined that the phenyl group at position 8 has the information for the biological response. The aromatic groups at positions 4 and 6 are involved with the receptor site (Harding, Stone & Wright, 1981). The dipsogenic effects induced by the two agonists behaved differently when the antagonists were previously injected but the responses followed a similar time course. There was a clear dissociation between the effects of the agonists as also proposed by other investigators (Fitzsimons, Kucharczyk & Richards, 1978; Camacho & Phillips, 1981; Evered & Fitzsimons, 1981; Di Nicolantonio, Mendlesen, Hutchinson, Takata & Doyle, 1982; Phillips, Hoffman & Bealer, 1982; Camargo et al. 1984). In conclusion, the present results, taken together with those of others, demonstrate that the receptors of Isoleu5-AnglI, in the central nervous system, are necessary for the cardiovascular and dipsogenic action of the Isoleu5-AngIJ and its analogues. The study with the antagonists showed that des-amine-AnglI probably binds more strongly to AnglI receptors, with stronger thirst-inducing effects than Isoleu5-AngII. This work was supported by grants from FAPESP 76/1308 and FUNDUNESP 242/89DFP/F/CBS, Sato Paulo, Brazil. The authors greatly appreciate the technical aid of Aparecida C. Luiz, Reginaldo C. Queiroz, Silvia Foglia and Silas P. Barbosa. They also thank Silvana A. D. Malavolta and Elisa Sakae Sinzato for typing the manuscript. REFERENCES
ANDERSON, B. (1977). Central sodium-angiotensin interaction. In Symposium on Central Actions of Angiotensin and Related Hormones, ed. BUCKLEY, J. P. & FERRARIO, C. M, pp. 463-474. Pergamon Press, New York. BEALER, S. L. & VAN HuYssE, J. W. (1989). Axon destruction and adrenergic systems mediate pressor responses after AV3V lesions. American Journal of Physiology 257, R8s-86. BROSNIHAN, K. B., BERTI, G. A. & FERRARIO, C. M. (1979). Hemodynamics of central infusion of angiotensin II in normal and sodium-depleted dogs. American Journal of Physiology 237, H139-145. BUMPUS, F. M. (1977). Mechanisms and sites of action of newer angiotensin agonists and antagonists in terms of activity and receptor. Federation Proceedings 36, 2128-2132. CAMACHO, A. & PHILLIPS, M. I. (1981). Separation of drinking and pressor responses to central angiotensin by monoamines. American Journal of Physiology 240, R106-113. CAMARGO, L. A. A., MENANI, J. V., SAAD, W. A. & SAAD, W. A. (1984). Interaction between areas of the central nervous system in the control of water intake and arterial pressure in rats. Journal of Physiology 350, 1-8.
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COLOMBARI, E., SAAD, W. A., CAMARGO, L. A. A., RENZI, A., DE LuCA, L. A. JR. & MENANI, J. V. (1990). Role of central al- and a2-adrenoceptors on the dipsogenic and cardiovascular effect of angiotensin II. Pharmacology, Biochemistry & Behavior 36, 893-896. Di NIcOLANTONIO, R., MENDELSON, F. A. O., HUTCHINSON, J. S., TAKATA, Y. & DOYLE, A. E. (1982). Dissociation of dipsogenic and pressor responses to chronic central angiotensin II in rats. American Journal of Physiology 242, R498-504. EVERED, M. D. & FITZSIMONS, J. T. (1981). Drinking and changes in blood pressure in response to precursors, fragments and analogues of angiotensin II in the pigeon Columba livia. Journal of Physiology 310, 353-366. FITZSIMONS, J. T. (1971). The effect on drinking of peptide precursors and of shorter chain peptide fragments of angiotensin II injected into the rat's diencephalon. Journal of Physiology 214, 295-303. FITZSIMONS, J. T., KUCHARCZYK, J. & RicHARDS, G. (1978). Systemic angiotensin-induced drinking in the dog: a physiological phenomenon. Journal of Physiology 276, 435-448. GANTEN, D., HUTCHINSON, P., SCHELLING, U., GANTEN, U. & FISCHER, H. (1976). The iso-renin angiotensin system in extrarenal tissue. Clinical Experimental Pharmacology and Physiology 3, 103-126. GILDENBERG, P. L., FERRARIO, C. M. & MCCUBBIN, J. W. (1973). Two sites of cardiovascular action of angiotensin in the dog. Clinical Science 44, 417-420. GoNgALVES, J. R., CAMARGO, L. A. A., MENANI, J. V., SAAD, W. A., SAAD, W. A., RENZI, A. & DE LUCA, L. A. JR. (1988). Effects of intracerebroventricular infusion of angiotensin II and related peptides on water and sodium ingestion and excretion. Brazilian Journal of Medical and Biological Research 21, 615619. HARDING, J. W., STONE, L. P. & WRIGHT, J. W. (1981). The distribution of angiotensin II binding sites in rodent brain. Brain Research 205, 265-274. HAYWOOD, J. R., BALL, N. A., LIFSCHITZ, M. D. & BRENNAN, T. J. (1987). Contribution of AV3V region in anephric NaCl-induced hypertension in the rat. American Journal of Physiology 252, F536-542. HOFFMAN, W. E. & PHILLIPS, M.I. (1976). Regional study of cerebral ventricle sensitive sites to angiotensinII. Brain Research 110, 313-330. KONIG, J. F. R. & KLIPPEL, R. A. (1963). The Rat Brain. Williams and Wilkins, Baltimore, MD, USA. NISHIMURA, H., NORTON, V. M. & BUMPUS, F. M. (1978). Lack of specific inhibition of an angiotensinII on eels by angiotensin antagonists. American Journal of Physiology 235, 95-103. PEACH, M. (1977). Renin angiotensin system. Biochemistry and mechanisms of action. Physiological Reviews 57, 313-370. PHILLIPS, M. I., HOFFMAN, W. E. & BEALER, S. L. (1982). Dehydration and fluid balance: central effects of angiotensin. Federation Proceedings 41, 2520-2527. SAAD, W. A., MENANI, J. V., CAMARGO, L. A. A. & ABRAO-SAAD, W. (1985). Interaction between cholinergic and adrenergic synapses of the rat subfornical organ and the thirst-inducing effect of angiotensin II. Brazilian Journal of Medical and Biological Research 18, 37-46. SIMPSON, J. B., EPSTEIN, A. N. & CAMARGO, J. S. JR. (1978). Localization of receptors for the dipsogenic action of angiotensin II in the subfornical organ of rat. Journal of Comparative and Physiological Psychology 92, 581-601. THRASHER, T. N. (1989). Role of forebraincircumventricular organs in body fluid balance. Acta Physiologica Scandinavica 136, 141-150. UNGER, T., HORST, P. J., VAUER, M., DEMMERT, G., RETTIG, R. & ROHMEISS, P. (1989). Natriuretic action of central angiotensin II in conscious rats. Brain Research 486, 33-38. ZIMMERMAN, B. G. (1973). Blockade of adrenergic potentiating effect of angiotensin by 1-Sar-8Ala-AngiotensinII. Journal of Pharmacology and Experimental Therapeutics 185, 456-492. ZIMMERMAN, B. G. (1978). Actions of angiotensin on adrenergic nerve endings. Federation II
Proceedings 37,
199-202.
Journal of Physiology (1991), 443, pp. 513-518 With 2 figures Printed in Great Britain
THE EFFECT OF ANALOGUES OF ANGIOTENSIN II ON DRINKING AND CARDIOVASCULAR RESPONSES TO CENTRAL ANGIOTENSIN II IN THE RAT
BY L. A. A. CAMARGO, L. A. DE LUCA JR, J. V. MENANI, A. RENZI, W. A. SAAD, WILLIAN A. SAAD* AND J. E. N. SILVEIRA From the Department of Physiology, School of Dentistry, UNESP, 14800, Araraquara, SP, Brazil, and the *Department of Surgery, School of Medicine, University of Sdo Paulo, SP, Brazil
(Received 6 July 1990) SUMMARY
1. Intracerebroventricular (i.c.v.) infusion (60 ng h-1) of Isoleu5-angiotensin II (Isoleu5-AngIl) and des-amine-angiotensin II (des-amine-AngII) in rats caused increased drinking behaviour and an increase in arterial blood pressure. 2. Des-amine-AnglI caused similar increases in heart rate and arterial blood pressure as AnglI. 3. Previous i.c.v. injection of the antagonists [Leu8]-AngII, des-amine-[Leu8]Angll and octanoyl-[Leu8]-AnglI prevented the increases in heart rate and blood pressure produced by i.c.v. infusion of AngII and caused partial reduction of the dipsogenic response.
4. The three antagonists had no effect on the increase in arterial blood pressure and heart rate caused by des-amine-AngII. The drinking response was reduced by previous injection of [Leu8]-Angll and des-amine-[Leu8]-Angll but not by octanoyl-
[Leu8]-AngII. 5. In conclusion, Isoleu5-AngII and des-amine-Angll increase drinking behaviour, arterial blood pressure and heart rate when infused into the cerebral ventricle of rats. The study with the antagonists showed that des-amine-AnglI probably binds more strongly to AnglI-receptors. INTRODUCTION
An abundance of evidence has shown that angiotensin II (AnglI) plays an important role in the forebrain circumventricular organs for the control of blood pressure and body fluid balance (Haywood, Ball, Lifschitz & Brennan, 1987; Bealer & Van Huysse, 1989; Thrasher, 1989; Unger, Horst, Bauer, Demmert, Rettig & Rohmeiss, 1989). Several studies on the renin-angiotensin system and its participation in different functions of the body, have indicated the action of angiotensin and its analogues in terms of structure-activity. Many AnglI analogues have been obtained by substituting one or more amino acids and radicals in the AnglI molecule (Fitzsimons, 1971). In relation to the myotropic and vasopressor MS 8633 17
PHY 443
514
L. A. A. CAMARGO AND OTHERS
activity of a number of antagonists of Angll analogues, it has been determined that the phenyl-group at position 8 has the information for the biological response, while the aromatic groups at positions 4 and 6 (tyrosine and histidine respectively), the guanide group at position 2 and the carboxyl C-ending are involved in the site of receptor binding. Also it has been determined that when the octapeptide Angll is deprived of its N-ending (N-terminal) this causes a progressive reduction in its central dipsogenic activity (Gildenberg, Ferrario & McCubbin, 1973; Ganten, Hutchinson, Schelling, Ganten & Fischer, 1976). The affinity of Angll and AngII analogues for converting enzymes is strongly infltienced by the charge of the NH2terminal amino acid residue. Inhibitory activity is enhanced by central or basic substituents and attenuated by acid NH2-terminal residues (Peach, 1977). In mammals, the structure of position 8 of the AngIl appears to be important in determining the affinity for angiotensin receptors, as well as acting as a part of the molecule which transcribes information to evoke intrinsic activity, presumably through the secondary mechanisms in the receptor (Nishimura, Norton & Bumpus, 1978). The present experiments were undertaken in order to test the effects of Isoleu5-AngII and its analogue des-amine-AngIl on water intake and cardiovascular responses. We also studied the action of different receptor antagonists of AnglI on these effects which were substituted in position 8. METHODS
Adult male Holtzman rats weighing 250-300 g were housed in individual metabolic cages. Drinking water was available in burettes with standard metal spouts projecting into the cages. Food pellets were available all the time except during an experiment. The animals were maintained on a 12-12 h light-dark cycle with light on at 08.00 h daily. Room temperature was maintained at approximately 24 'C. Following an acclimatization period of 7 days animals were anaesthetized with ether and cannulae were implanted into the lateral ventricle. The procedures were carried out using a Model 900 stereotaxic apparatus (David Kopf Instruments, Tujunga, USA) and the co-ordinates of the Konig & Klippel (1963) atlas. After brain surgery, a prophylactic dose of 50000 i.u. of penicillin was administered to each animal. On the morning of the fifth day after implantation of the cannulae, the animals were anaesthetized with ether and a PE-50 polyethylene catheter was introduced into the femoral artery. The catheter was filled with heparinized saline (0-15 M) and fixed to the animal's back with suture through a skin incision (Camargo, Menani, Saad & Saad, 1984). Tests were started after a 3 h period of recovery from surgery. A stainless-steel 30-gauge needle was used for infusion of the peptides into the lateral ventricle (LV) at a rate of 5,1l h-' using a PE-10 polyethylene tube connected to a 1 ml syringe connected to an infusion pump. Arterial pressure was recorded from the femoral artery and heart rate was recorded using the pressure wave to trigger a rate meter. The water intake was measured using burettes with 0-1 ml markings. The peptides used were the agonists Isoleu5-AngII and des-amine-Angll, and the AngIl antagonists [Leu8]-AngII, des-amine[Leu8]-AngJI and octanoyl-[Leu8]-AngII. The peptides were generously provided by Professor A. C. Paiva from the School of Paulista Medicine. At the end of the experimental period, the animals were anaesthetized with ether and perfused through the heart with 10 % saline and formol solution. The brains were removed, frozen sections cut, and routine histology was performed for rapid location of the cannula pathway. The results were analysed statistically by analysis of variance and unpaired t test with Dunnett's correction for multiple comparisons with a control, as necessary.
Experimental procedures Water intake, arterial pressure and heart rate were measured in different groups of rats submitted to the following experimental protocols:
CENTRAL ANGIOTENSIN AND ANALOGUES
515
Infusion of Isoleu5-AngII (60 ng h-') into the LV (n = 21 rats). Injection of [Leu8]-AngII (60 ng) into the LV+Isoleu5-AngII (60 ng h-1) infusion (n = 21 rats). Injection of des-amine-[Leu8]-AngII (60 ng) into the LV + Isoleu5-AngII (60 ng h-') infusion (n = 22 rats). 130
-
A
450
B
8
C
-T00-6E~~~~~~~~~~~~~~ E100
U)
40
(0~~~~~~~~~~~~~~~*
.3500
Fig. 1. Effect of infusion of Isoleu5-AngII into the lateral ventricle ([WI) on arterial pressure, heart rate and water intake. The antagonists [Leu8]-AngII (m), des-amine[Leu8]-AngII (0) and octanoyl-[Leu8]-AngII (C) were injected 7 min before Isoleu5Angll. Data are means +S.E.M.; (El) control (no infusion). * different from control; ° different from Isoleu5-AngII.
Injection of octanoyl-[Leu8]-AngII (60 ng) into the LV + Isoleu5-AngII (60 ng h-') infusion (n = 25 rats). Infusion of des-amine-AngIl (60 ng h-') into the LV (n = 25 rats). Injection of [Leu8]-AngII (60 ng) into the LV + des-amine-AngIl (60 ng h-1) infusion (n = 24 rats). Injection of des-amine-[Leu8]-AngII (60 ng) into the LV + des-amine-AngIl (60 ng h-1) infusion (n = 24 rats). Injection of octanoyl-[Leu8]-AngII (60ng) into the LV+des-amine-AngII (60ngh-1) infusion
(n = 25 rats). The AngIl antagonists were injected into the LV 7 min before the AngII infusion was started. The volume injected was 1 ,ul and the rate of infusion was 5 /ul h-1. The period of injection was 30 s and the time of infusion was 1 h. The recording period for water intake, arterial pressure and heart rate was 1 h. Each experiment was carried out on a separate day. The minimum period between experiments was 3 days. RESULTS
Effects of Isoleu5-AngII infusion into the lateral ventricle and of previous injection of the antagonists [Leu8]-AngII, des-amine-[Leu8]-AngII and octanoyl-[Leu']-AngII The infusion of Isoleu5-AngII into the lateral ventricle caused an increase in water intake, blood pressure and heart rate compared with control values (P < 0 05; Fig. 1A, B and C). The increase in arterial pressure was gradual and reached a maximum after 10 min of infusion. Previous injection of the antagonists blocked the pressor effect and the increase in heart rate produced by infusion of Isoleu5-Ang1I when compared with the experiment utilizing only the peptide agonist (P < 0 05; Fig. 1A and B). This blocking effect lasted throughout the observation period. The antagonists reduced but did not fully block the water intake induced by Isoleu5AnglI as compared with the experiment using only the agonists and with the control values (P < 0 05); Fig. 1 C). 17-2
516
L. A. A. CAMARGO AND OTHERS
Effects of des-amine-AnglI infusion into the lateral ventricle and of previous injection of the antagonists [Leu8]-AngII, des-amine-[Leu8]-AngII and octanoyl-[Leu8]-AngII The infusion of des-amine-AnglI into the lateral ventricle caused an increase in blood pressure, heart rate and water intake (with the latency around 30 s) compared 130
-
A
450
B
-
*
E
8 ~~
~
~
C ~
~
~
*
1000 E~~~~~~~c
g~~~~~~~~~~~o~c a,~~~~~~~~~~~~C
c*n 70
a)
0 35t
4X
40302
Fig. 2. Effect of infusion of des-amine-AngII into the lateral ventricle (ODh) on arterial pressure, heart rate and water intake. The antagonists [Leu8]-AnglI (n), des-amine[Leu8]-AnglI (0) and octanoyl-[Leu8]-AngII (0) were injected 7 min before des-amineAngll. Data are means + S.E.M.; l control (no infusion). * different from control, 0 different from des-amine-AngII.
with control values (P < 0 05; Fig. 2A, B and C). The increase in arterial pressure started immediately after the infusion of des-amine-AnglI. Arterial pressure and heart rate remained unchanged after previous (7 min) injection of the three antagonists compared with the control values nor was there any effect upon the response to des-amine-AnglI. The water intake induced by des-amine-Angll infusion was significantly decreased by previous injection of [Leu8]-AngIJ and des-amine[Leu8]-AngII when compared with the values obtained after infusion of des-amineAngll alone (P < 0-05). Octanoyl-[Leu8]-AngII did not block the effect of des-amineAnglI (Fig. 2C). DISCUSSION
The infusion of Isoleu5-Ang1I into the lateral ventricle produced an increase in arterial blood pressure, heart rate and water intake. These results agree with those obtained by Hoffman & Phillips (1976) and Colombari, Saad, Camargo, Renzi, De Luca & Menani (1990). A series of observations have indicated that the action of this peptide in inducing a pressor response is due to the activation of the receptors at many sites in the brain (Zimmerman, 1973, 1978; Anderson, 1977; Simpson, Epstein & Camargo, 1978; Brosnihan, Berti & Ferrario, 1979). Another possible action of Isoleu5-AngII is the activation of the release of a cerebral hormone with a prolonged vasopressor effect. Isoleu5-AngIJ may act on more than one type of receptor to induce a thirst response. The present results show that the two agonists studied (Isoleu5-AngII and des-amine-Angll) have both pressor and dipsogenic effect. The
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latency for the pressor and dipsogenic effect of des-amine-AngII is shorter than that for the effect of Isoleu5-AnglI. The duration of the effect of des-amine-AnglI is shorter than that of Isoleu5-AngII. These agree with the results of Saad, Menani, Camargo & Abrao-Saad (1985) and Gongalves, Camargo, Menani, Saad, Saad, Renzi & De Luca (1988). Three AnglI antagonists were injected into the lateral ventricle before the infusion of Isoleu5-AngII and des-amine-Angll. The thirst-inducing effect of Isoleu5Angll was decreased. The three antagonists had no influence on the pressor and tachyeardic effect of des-amine-Angll. The dipsogenic effect was decreased by prior injection of the first two antagonists. In all of the antagonists studied, the amino acid and residue phenylanine at position 8a is substituted by one leucine. Bumpus (1977) reported that for the myotropic and vasopressor activity of the many antagonists of Isoleu5-AngII it has been determined that the phenyl group at position 8 has the information for the biological response. The aromatic groups at positions 4 and 6 are involved with the receptor site (Harding, Stone & Wright, 1981). The dipsogenic effects induced by the two agonists behaved differently when the antagonists were previously injected but the responses followed a similar time course. There was a clear dissociation between the effects of the agonists as also proposed by other investigators (Fitzsimons, Kucharczyk & Richards, 1978; Camacho & Phillips, 1981; Evered & Fitzsimons, 1981; Di Nicolantonio, Mendlesen, Hutchinson, Takata & Doyle, 1982; Phillips, Hoffman & Bealer, 1982; Camargo et al. 1984). In conclusion, the present results, taken together with those of others, demonstrate that the receptors of Isoleu5-AnglI, in the central nervous system, are necessary for the cardiovascular and dipsogenic action of the Isoleu5-AngIJ and its analogues. The study with the antagonists showed that des-amine-AnglI probably binds more strongly to AnglI receptors, with stronger thirst-inducing effects than Isoleu5-AngII. This work was supported by grants from FAPESP 76/1308 and FUNDUNESP 242/89DFP/F/CBS, Sato Paulo, Brazil. The authors greatly appreciate the technical aid of Aparecida C. Luiz, Reginaldo C. Queiroz, Silvia Foglia and Silas P. Barbosa. They also thank Silvana A. D. Malavolta and Elisa Sakae Sinzato for typing the manuscript. REFERENCES
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