Araraquara, S.P., Brazil, and Department of Surgery, School of Medicine, University of Sdo Paulo, S.P., Brazil

Transcription

1 J. Physiol. (1984), 350, pp With 4 text-figures Printed in Great Britain INTERACTION BETWEEN AREAS OF THE CENTRAL NERVOUS SYSTEM IN THE CONTROL OF WATER INTAKE AND ARTERIAL PRESSURE IN RATS BY L. A. A. CAMARGO, J. V. MENANI, WILLIAM A. SAAD AND WILSON A. SAAD From the Department of Physiology and Pathology, School of Dentistry, UNESP, Araraquara, S.P., Brazil, and Department of Surgery, School of Medicine, University of Sdo Paulo, S.P., Brazil (Received 4 January 1983) SUMMARY 1. Water intake induced by injection of 0-2 M-NaCl into the lateral preoptic area was increased by the injection of angiotensin II into the subfornical organ of rats. The injection of hypertonic saline solution into the subfornical organ increased water intake. However, the increase was lower than when the solution was injected into the lateral preoptic area. The injection of 4,sg angiotensin II into the lateral preoptic area further augmented this effect. 2. Injection of angiotensin II into the subfornical organ caused a rise in blood pressure which preceded the thirst-inducing effect. The injection of 0-2 M-NaCl into the subfornical organ caused no changes in blood pressure, whereas the injection of angiotensin II into the lateral preoptic area caused some increase. 3. Dehydration of the lateral preoptic area by means of 0-2 M-NaCl in combination with intravenous infusion of angiotensin II caused a summation of effects in terms of the water intake, without changing cardiovascular alterations induced by the infusion of angiotensin II. A summation of effects in the water intake, but not in blood pressure, was also observed when 0-5 M-NaCl was infused intravenously in combination with the injection of angiotensin II into the subfornical organ and into the lateral preoptic area. 4. The results indicate that there are interactions between the subfornical organ and lateral preoptic area in the regulation of cardiovascular and thirst mechanisms. INTRODUCTION Administration of angiotensin II either directly into the cerebral ventricles or intravenously, causes a rise in blood pressure and polydipsia (Epstein, Fitzsimons & Rolls, 1970; Mouw, Bonjour, Malvin & Vander, 1971; Severs & Daniel-Severs, 1973; Addo & Isom, 1979; Hoffman, Weet, Phillips & Schmid, 1979; Gronan & York, 1979; Phillips, Kalter, Lewis & Rosenbaum, 1979). Hypertonic NaCl solutions injected intracranially or systemically have similar effects (Blass & Epstein, 1971; Andersson, 1977; Buggy, Hoffman, Phillips, Fisher & Johnson, 1979; Saad & Camargo, 1980). 1 PHY 350

2 2 L. A. A. CAMARGO AND OTHERS It is known that there are connexions between the brain sites which are sensitive to angiotensin II and hypertonic NaCl solutions. Two areas closely involved in the regulation of the water intake and in the blood pressure are the lateral preoptic area and the subfornical organ. A bilateral lesion of the lateral preoptic area eliminates thirst caused by cellular dehydration (Peck & Novin, 1971; Saad & Camargo, 1980). Lesions in the subfornical organ reduce or abolish the water intake induced by administration of angiotensin II into the lateral preoptic area (Simpson & Routtenberg, 1973) or lateral ventricle (Phillips, Leavitt & Hoffman, 1974). In this paper we describe the interactions of two dipsogenic stimuli (angiotensin II and hypertonic NaCl solution) applied systemically, into the lateral preoptic area or into the subfornical organ. Observations were also made on blood pressure and heart rate. METHODS Adult male Holtzman rats weighing 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 h light-dark cycle with lights on at daily. Room temperature was maintained at approximately 24 0C. Following an acclimatization period of 7 days animals were anaesthetized with ether and two cannulae were implanted in the same animal, one into the lateral preoptic area and one into the subfornical organ. The procedures were carried out using a Model 900 stereotaxic apparatus (David Kopf Instruments) and the coordinates of the Konig & Klippel (1963) atlas: AP, 7-4; L, 0-0; and V, 2-8 below the dura mater, for the subfornical organ, and AP, 8-7; L, 1-6; V, 6-0 below the dura mater, for the lateral preoptic area. After brain surgery, a prophylactic dose of u. of penicillin was administered to all animals. On the morning of the 5th day after the cannula implantation, animals were anaesthetized with ether, a PE-20 polyethylene catheter was introduced into the jugular vein, and a PE-50 catheter was introduced into the femoral artery. Both catheters were filled with heparinized saline solution and fixed to the animal's back with suture through a skin incision. Tests were started after a 3 h period of recovery from surgery. A stainless-steel 30-gauge needle was used for injection into the lateral preoptic area and a 34-gauge needle was used for injection into the subfornical organ. The needle was connected by PE-10 tubing to a Hamilton-type syringe with 5 #1 markings kept outside the cage. All drugs were injected in 1-0,1 volumes over s. The arterial pressure was recorded from the femoral artery and heart rate was recorded using the pressure wave to trigger a rate-meter. The catheter in the jugular vein was connected by PE-20 polyethylene tubing to a 5 ml plastic syringe coupled with an infusion pump. The water intake was measured using burettes with 0-1 ml markings. The injections were: Ile5 angiotensin II, and different NaCl solutions. At the end of the experimental period animals were anaesthetized with ether and perfused with 10 % saline and formol solution. The brains were removed, frozen sections cut, and routine histology performed. This permitted rapid location of the cannula pathway. Some brain sections were later stained by the Pal-Weigert method modified by Erhart (1951). The results were analysed statistically by analysis of variance. All comparisons between the control values and the values after injections, were made using the paired t test. Experimental procedures (a) In thirty-six rats different doses of angiotensin II ( ng IP-1) were injected into the subfornical organ and water intake measured. Since the highest dose (4 0 ng sl-1) had the greatest effect it was used in all further experiments. In twenty-four rats 0-2 M-NaCl was injected into the lateral preoptic area, and 20 min later angiotensin II was injected into the subfornical region. The reverse experiment, i.e. 0-2 M-NaCl into

3 CENTRAL CELLULAR DEHYDRATION AND ANGIOTENSIN the subfornical organ followed by angiotensin II into the preoptic area, was performed in twelve animals. Water intake, arterial pressure and heart rate were recorded for 20 min prior to injection of saline, during the 20 min after saline injection and during the 20 min after angiotensin II injection. (b) In twenty-eight animals, different doses of angiotensin II (10, 20 and 40 ng 10 ul-' min-) were infused intravenously, and water intake, arterial pressure, and heart rate were measured. The highest dose of angiotensin used (40 ng min-), which gave the highest water intake was then infused into the thirteen other animals for a period of 40 min. During the last 20 min of this period 0-2 M-NaCl was injected into the lateral preoptic area. (c) In twenty-one animals different doses of hypertonic NaCl solution were infused intravenously (0-25, 0-5 and 1-0 M at a rate of 100,ul min-) and water intake, arterial pressure, and heart rate were measured. The 0-5 M-NaCl was then infused into seven other animals for a period of 60 min. During the last 40 min of this period 4-0 ng of angiotensin II was injected into the subfornical organ and later followed by an injection of 4-0 ng of angiotensin II into the lateral preoptic area. The interval between these injections was 20 min. 4l A C 3 TA E :R I.L Fig. 1. Effect of injection of 0-2 M-NaCl solution into the lateral preoptic area (IM) and of angiotensin II (4 ng) into the subfornical organ (U) on water intake, arterial pressure and heart rate. Data are means+±s.e. of mean of twenty-four animals. Control; O. RESULTS Effects ofinjection ofhypertonic NaCi solution and angiotensin II into the lateral preoptic area and into the 8ubfornical organ The intracerebral injection of ng angiotensin II into the subfornical organ caused a dose-dependent increase in water intake as previously described (Simpson, Epstein & Camardo, 1978). The injection of 0-2 M-NaCl into the lateral preoptic area caused a significant increase in water intake compared with the control values (P < 0-001). The injection of 4-0 ng angiotensin II into the subfornical organ caused a significant increase in water intake compared with the values obtained with the injection of NaCl into the lateral preoptic area (P < 0-05) (Fig. 1 A). The injection of 0-2 M-NaCl into the lateral preoptic area caused an increase in arterial pressure compared with the control values (P < 0-001). After the injection of angiotensin II into the subfornical organ there was a greater increase in arterial pressure (P < 0-001) than that obtained after injection of NaCl into the lateral preoptic area (Fig. 1 B). The injection of 0-2 M-NaCl into the lateral preoptic area caused a significant increase in the heart rate compared with the control values (P < 0-001). On the other hand, the injection of angiotensin II into the subfornical organ reduced the heart rate compared to the values obtained by injecting 0-2 M-NaCl into the lateral preoptic area (P<0-001) (Fig. IC). 1-2

4 4 L. A. A. CAMARGO AND OTHERS In the inverse experiment, the injection of 0-2 M-NaCl into the subfornical organ caused a significant increase in water intake compared with the control values (P < 01001). The injection of4 0 ng angiotensin II into the lateral preoptic area caused a greater increase in water intake when compared to the values obtained after injecting NaCl into the subfornical organ (P < 0-05) (Fig. 2A). There were no significant changes in arterial pressure when 0-2 M-NaCl was injected into the subfornical organ when compared with the control values, but pressure increased when 4 ng of angiotensin II was injected into the lateral preoptic area (P < 0 05) (Fig. 2B). The heart rate showed no significant change when 0-2 M-NaCl was injected into the subfornical organ or angiotensin II (4 ng) was injected into the lateral preoptic area (Fig. 2C). - 3 M ~~~ E E ~~~~EE 2 E o _..100 ~~~~~~~~~~~300- A B C 0~~~~~~~~~~~~ Fig. 2. Effect of injection of 0-2 m-nacl solution into the subfornical organ (Mi) and of angiotensin li(4 ng) into the lateral preoptic area (U) on water intake, arterial pressure and heart rate. Data are means + s.e. of mean of twelve animals. Control; E]. A BC E The 2 n of 1 ngangotes120 o t 300 0~~~~~~~~~~~~ intravenous infusion of angiotensin II (40 Fig. 3. ng;mi) and Effect of of and heart rate. Data are means + s.e. of mean of thirteen animals~. Control; [:]. injection of m-naci solution (E) into the lateral preoptic area on water intake, arterial pressure Effect8 of intravenous infusion of angiotensin II and the injection of hypertonic NaCi 8olutton into the lateral preoptic area The infusion of ng angiotensin II caused dose-dependent increases in water intake and arterial pressure. No dose-dependent changes in the heart rate occurred. The intravenous infusion of angiotensin 11 (40 ng 10,ul11 min-") caused a significant increase in water intake compared with the control values (P < 0-01). The injection of 0-2 m-nacl into the lateral preoptic area caused a water intake significantly different from that obtained with intravenous infusion of angiotensin II (P < 0-001) (Fig. 3A).

5 CENTRAL CELLULAR DEHYDRATION AND ANGIOTENSIN 5 The intravenous infusion ofangiotensin II (40 ng 10 1l-1 min-) caused a significant increase in the arterial pressure compared with control values (P < 01001), and these values remained unchanged after the injection of 0-2 M-NaCl into the lateral preoptic area (Fig. 3 B). The heart rate decreased after the intravenous infusion of angiotensin II (40 ng 10 #1-' min-) compared with the control values (P <0-001) and these values remained unchanged after the injection of 0-2 M-NaCl into the lateral preoptic area (Fig. 3C). Effects of intravenous infusion of hypertonic NaCt solution and the injection of angiotensin II into the 8ubfornical organ and into the lateral preoptic area The intravenous infusion of 0-25, 0-5 and 10 M-NaCl (100 #1 min-") caused a dose-dependent increase in water intake. No dose-dependent changes were observed in the arterial pressure or in the heart rate C X E E E 0 3- ~~~90 V, ~350-3:~~~~~~~~~~~( 1 t ) Fig. 4. Effect of intravenous infusion of 05 M-NaCl solution (MD) and of injection of angiotensin II (4 ng) into the subfornical organ (M) and into the lateral preoptic area (U) on water intake, arterial pressure and heart rate. Data are means + S.E. of mean of seven animals. Control; EO. The intravenous infusion of 0-5 M-NaCl caused a significant increase in water intake when compared with the control values (P < 0-01). The injection of angiotensin II (4 ng) into the subfornical organ also caused water intake. The additional injection of angiotensin II (4 ng) into the lateral preoptic area also caused water intake (Fig. 4A). The intravenous infusion of 0 5 M-NaCl caused a significant increase in the arterial pressure in comparison with the control values (P < 0 05). There were no significant changes in arterial pressure when angiotensin II (4 ng) was injected into the subfornical organ and lateral preoptic area in comparison with the values after intravenous infusion of 0-5 M-NaCl (Fig. 4B). The intravenous infusion of 0-5 M-NaCl caused a significant increase in the heart rate in comparison with the control values (P < 0-05). No significant difference occurred when compared to the values obtained after intravenous infusion of 0-5 M-NaCl with those after the injection of angiotensin II (4 ng) into the subfornical organ and into the lateral preoptic area (Fig. 4C).

6 6 L. A. A. CAMARGO AND OTHERS Histological analysis Location of the cannulae in the lateral preoptic area. The cannulae passed through the anterior commissure and came to rest bilaterally in the head of the lateral preoptic area. The ends were bilaterally symmetrical and terminated in or just above the anteromedial portion of the lateral preoptic area. Only the animals whose areas were reached symmetrically by the cannulae were included in the statistical analysis of the data. The results obtained confirmed those reported by Blass & Epstein (1971), who, using electrolytic lesions and osmotic stimulation of the lateral preoptic area, obtained changes in water intake. Location of the cannulae in the mubfornical organ. In general, the cannulae reached the middle portion of the body of the subfornical organ, and also reached the entire anteroposterior extension of the organ. DISCUSSION The injection of hypertonic saline into the subfornical organ ofrats resulted in lower drinking responses than that produced when the same solution was injected into the lateral preoptic area. The drinking induced by application of hypertonic saline was reduced by the lesion in the subfornical organ (Saad & Camargo, 1980). When hypertonic saline was injected into the subfornical organ the water intake was not accompanied by change in arterial pressure and heart rate. It suggests that, in the rat, drinking in response to application of hypertonic saline into the subfornical organ is not the result of cardiovascular changes. At least four kinds of neurones are known to exist in the subfornical organ of many mammalian species and they differ at the ultrastructural level (Simpson et al. 1978). The functions of some of these are still unknown. It is possible that a hypertonic solution may cause thirst by dehydrating some of the neurones of the subfornical organ or the pathways that connect the subfornical organ and the hypothalamus (Miselis, Hand & Berger, 1977; Shapiro & Miselis, 1978). Injection of hypertonic saline into the lateral preoptic area increased drinking caused by the intravenous infusion of angiotensin II. However, the injection of hypertonic saline into the lateral preoptic area caused no change in the arterial pressure in contrast to the intravenous infusion of angiotensin II. We presume that the dose of angiotensin II needed to induce thirst is much lower when a second stimulus (in this case, dehydration of the lateral preoptic area) is applied. It has been demonstrated previously that very small doses of angiotensin II are required when the hypertonic saline solutions are infused concomitantly (Hsiao, Epstein & Camardo, 1977). These data were confirmed when the hypertonic NaCl solution was infused intravenously before angiotensin II was injected into the subfornical organ. The present results corroborate those obtained by Blass & Epstein (1971), who succeeded in blocking thirst caused by subcutaneous injection of 2 M-NaCl through destruction of the lateral preoptic area. It was found that when angiotensin II was injected into the subfornical organ in combination with an intravenous infusion of NaCl, a summation of effects on water intake occurred, with values similar to those obtained in the second experiment. This fact again demonstrates that when a thirst-inducing stimulus is used, the thirst threshold for the second is reduced.

7 CENTRAL CELLULAR DEHYDRATION AND ANGIOTENSIN When a third stimulus, i.e. injection of angiotensin II into the lateral preoptic area, was used in combination with the intravenous NaCl infusion, a summation of effects again occurred. The intravenous infusion of hypertonic saline solution per se caused an increase in arterial pressure. However, there was no increase when angiotensin II was subsequently injected into the subfornical organ and lateral preoptic area. The pressor and dipsogenic effects induced by intravenous and intracranial administration of angiotensin II occurred simultaneously, but there was a clear dissociation between these effects (Fitzsimons, Kucharczyk & Richards, 1978; Camacho & Phillips, 1981; Evered & Fitzsimons, 1981; Di Nicolantonio, Mendelsohn, Hutchinson, Takata & Doyle, 1982; Phillips, Hoffman & Bealer, 1982). In conclusion these results demonstrate that angiotensin II and hypertonic NaCl solution, administered systemically, induce thirst, and cardiovascular alterations. The fact that angiotensin II and cellular dehydration elicits the drinking behaviour, when injected systemically or intracranially, suggests that thirst can be provoked by one or more stimuli at the same time. These results also suggest that there are interactions between the subfornical organ and lateral preoptic area in the regulation of cardiovascular and thirst mechanisms. This work has been supported by grants from the FAPESP-76/1308, Sao Paulo, Brazil. The authors greatly appreciate the technical aid of Aparecida C. Marcelino. They also thank Reginaldo C. Queiroz for the preparation of the histological material and Silvana A. Derobio for assistance with the manuscript. REFERENCES ADDO, M. F. & ISOM, G. E. (1979). Chronic intracerebroventricular infusion of angiotensin II. Proc. West Pharmacol. Soc. 22, ANDERSSON, B. (1977). Central sodium-angiotensin interaction. In Symposium on Central Actions of Angiotensin and Related Hormones, ed. BUCKLEY, J. P. & FERRARIO, C. M., pp New York: Pergamon Press. BLASS, E. M. & EPSTEIN, A. N. (1971). A lateral preoptic osmosensitive zone for thirst in the rat. J. comp. physiol. Psychol. 76, BUGGY, J., HOFFMAN, W. E., PHILLIPS, M. I., FISCHER, A. E. & JOHNSON, A. K. (1979). Osmosensitivity of rat third ventricle and interactions with angiotensin. Am. J. Physiol. 236, R CAMACHO, "A.- -& PHILLIPS, M. I. (1981). Separation of drinking and pressor responses to central angiotensin by monoamines. Am. J. Physiol. 240, DI NICOLANTONIO, R., MENDELSOHN, 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. Am. J. Physiol. 242, R EPSTEIN, A. N., FITZSIMONS, J. T. & ROLLS, B. J. (1970). Drinking induced by injection of angiotensin into the brain of the rat. J. Physiol. 210, ERHART, E. A. (1951). Modificaqao simples e rapida do metodo de Pal-Weighert para a coloragao das bainhas de mielina. Arquiv. Neuro-Psiquiat. 9, EVERED, M. D. & FITZSIMONS, J. T. (1981). Drinking and changes in blood pressure in response to angiotensin II in the pigeon Columba Livia. J. Physiol. 310, FITZSIMONS, J. T., KUCHARCZYK, J. & RICHARDS, G. (1978). Systemic angiotensin-induced drinking in the dog: a physiological phenomenon. J. Physiol. 276, GRONAN, R. J. & YORK, D. H. (1979). Effects of chronic intraventricular administration of angiotensin II on drinking behaviour and blood pressure. Pharmacol. Biochem. & Behav. 10, HSIAO, S., EPSTEIN, A. N. & CAMARDO, J. S. (1977). The dipsogenic potency of peripheral angiotensin II. Horm. & Behav. 8,

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