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Natriuretic action of central angiotensin II in conscious rats
Brain Research, 486 (1989) 33-38 Elsevier
33
BRE 14432
Natriuretic action of central angiotensin II in conscious rats Thomas Unger, Peter J. Horst, Martin Bauer, Gudrun Demmert, Rainer Rettig and Peter Rohmeiss Department of Pharmacology and German Institute for High Blood Pressure Research, University of Heidelberg, Heidelberg (F.R. G. ) (Accepted 11 October 1988) Key words: Angiotensin II; Brain; Natriuresis; Saralasin
The effects of intracerebroventricular (i.c.v.) injections of angiotensin II (ANG II, 10 pg, 100 pg and 10 ng) on renal sodium excretion were investigated in conscious rats instrumented with a chronic urethral catheter. ANG II increased renal sodium excretion dose-dependently with a threshold i.c.v, dose of 10 pg. Only after the highest dose was a concomitant increase in arterial blood pressure and urinary flow observed. The ANG II-induced natriuresis began within 5 min of the i.c.v, injection and lasted for more than 1 h. The angiotensin receptor antagonist saralasin (1 ng, i.c.v.) largely prevented the natriuretic effect of i.c.v, injected ANG II (100 pg). Our results lend further support to the hypothesis that brain ANG II by its potent natriuretic actions may be instrumental in central osmotic control.
INTRODUCTION Over the last 20 years the effects of brain angiotensin II ( A N G II) receptor stimulation in various species have been a matter of intensive investigation (for recent reviews see Phillips 16, and Unger et al.2~). Most of the central actions of the peptide, such as increase in blood pressure, drinking, release of vasopressin from the pituitary gland or suppression of renin release from the kidney conform with those exerted by hormonal A N G II when acting on its peripheral receptors with one notable exception, namely the effect on renal salt excretion. While circulating A N G II tends to retain sodium by a direct renal action 15 as well as through aldosterone release from the adrenal gland, stimulation of brain A N G II receptors has been reported to induce natriuresis 1"2"4'5'9"11A8. In the dog, this central natriuretic action of A N G II appears to be independent of the hemodynamic effects of the peptide 5, whereas in the rat this relationship has not yet been investigated. Moreover, in previous inves-
tigations in rats A N G II was infused intracerebroventricularly (i.c.v.) over relatively long periods of time, and urine was collected by placing the animals in metabolic cages. Therefore, these studies did not provide information as to the onset, duration and dose-responSe relationship of the natriuresis in response to acute stimulation of brain A N G II receptors. We have recently developed a new method for urine collection in conscious, unrestrained rats through a chronic indwelling ureteral catheter which allows differential sampling of small urine fractions over periods as small as one minute le. We have employed this method in the present study to investigate the acute centrally induced natriuretic effect of A N G II in rats. Our results demonstrate that i.c.v, injections of A N G II in the picogram range produce a sustained natriuresis of rapid onset which is independent of changes in arterial blood pressure and sensitive to central A N G II receptor blockade.
Correspondence: T. Unger, Department of Pharmacology, Im Neuenheimer Feld 366, 6900 Heidelberg, F.R.G. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
34 MATERIALS AND METHODS Experiments were performed in conscious, unrestrained, sodium replete male Wistar rats (Dr. Karl Thomae, Biberach, E R . G . ) weighing between 350 and 450 g. All animals were subjected to a unilateral right nephrectomy two months prior to the study. They were fed a standard diet (Altromin) and were allowed free access to water. One or two weeks before the experiment a cannula (PP 20, Portex, U.K.) was inserted into the left lateral brain ventricle under ether anaesthesia as described before 19. To verify correct cannula placement, animals were challenged with an i.c.v, injection of 10 ng A N G II 3 days after surgery, and the drinking response was recorded. Only those animals that reacted with an immediate response and drank more than 3 ml of water within 10 rain upon the challenge were used in the study. On the day before the experiment, rats were anaesthetized with ether and instrumented with femoral artery catheters (PP 10 in PP 50) for direct measurements of arterial blood pressure and with ureterai catheters for urine collection, Construction, implantation and verification of the ureteral catheter have been described elsewhere in detail 12. Briefly, a silicone tube (LHD, Heidelberg, E R . G . ) of 25 cm length (i.d. 0.3 mm, o.d. 0.5 mm) was used to serve as ureter catheter. The abdomen was opened via a midline incision extending from the bladder to about 2 cm caudal to the sternum. The catheter was then tunneled under the skin from the abdomen up to the left ear through a trochar perforating the lateral abdominal wall. A transverse incision was made in the ureter with microsurgery scissors. The tip of the catheter was advanced cranially 2 mm into the ureter and tied into place with a suture. The catheter was anchored to the psoas muscle with two additional sutures. The end of the catheter extending from the orifice at the nape of the neck was partly covered with PE 160 tubing (Clay Adams, U.S.A.). The PE 160 was wrapped with tape and sewed to the skin in a way that urine dripping onto the animal was avoided. After the operation the animals were injected i.p. with 0.1 g ampicillin (Binotal, Bayer Leverkusen, F.R.G.). All surgery was done under aseptical conditions. Twenty-four hours were allowed for recovery from surgery until experiments
were commenced. The operative success rate was about 80%. Reasons for failure were obstruction of the catheter due to crystallization of urine, bleeding, detachment of the catheter from the ureter and removal of the catheter by the animal. Kidney function as analyzed by measurements of GFR, plasma urea, urine concentration capacity after water deprivation, excretion of i.v. sodium loads and by microscopical examination was not disturbed by the ureter catheter for up to one week after
implantation 12. Experimental protocol Experiments were performed while the animals were quietly resting in their home cages. Rats were allowed to accustom themselves to the experimental setting for 1 h. The ureter catheter was then connected to an extension tube (PP 25) via a stainless steel cannula. The extension tube was wrapped with tape and the end inserted into a collection vial, which was placed on level with the kidney. The arterial catheter was connected to a Statham 23 dB pressure transducer coupled to a Gould Brush pressure computer and a Gould Brush 2400 recorder for continuous monitoring of mean arterial blood pressure (MAP). An i.v. infusion of isotonic saline (1.2 ml/h) was commenced to promote urinary flow, and 2 h were allowed for equilibration. Rats were divided in groups and injected i.c.v, with 5 ~1 of isotonic saline (control group, n = 6), 10 pg A N G II (1/al peptide solution flushed with 4/~1 isotonic saline, n = 9), 100 pg A N G II (n = 8), or 10 ng A N G II (n = 6). An additional group (n = 6) received an i.c.v, injection of the ANG II receptor antagonist saralasin (1 ng in 1/~1) 2 rain before the i.c.v, injection of 100 pg A N G II. Each rat was only injected once with A N G II. Urine was collected in 5-min fractions 30 rain before and 60 min after the i.c.v, injections. Mean arterial blood pressure was monitored throughout the experiment. Urinary volume was determined gravimetrically. Urinary sodium and potassium concentrations were measured with a Beckman flame photometer.
Drugs Angiotensin II and the angiotensin II receptor antagonist Sarl-Ala8-Angiotensin 1! (Saralasin)
35 ÷
25
(um01/Smin)
~
20 15
~.
Urinory volume
No excrehon Hmoz ANG II lOOpg
30min
i.C.V.
..~..
vehicle
i
I000~
100'
i
t
5
I 0 : -30
:
: : -15
:
: 0
:
:
', : +15
:
: : +30
:
: : : : +60 +45 time (min)
500!
50.
Fig. 1. Time course of the natriuretic effect of an i.c.v. injection of 100 pg ANG II. Arrow indicates bolus injection of the peptide (experimental group, n = 8) or vehicle (0.9% NaCI, control group, n = 6). Urine was collected in 5-min intervals. After 60 rain renal sodium excretion was still elevated in the majority of the ANG II-injected animals. For standard errors and statistical differences between the two groups refer to Fig. 2.
were p u r c h a s e d from Bachem, Bubendoff, Switzerland. Peptides were dissolved in sterile isotonic saline. Solutions were m a d e up on the day of the e x p e r i m e n t and k e p t on ice.
!
10.
100before ~fler
before after
AN5 II AN5 II Fig. 3. Sodium excretion and urinary volume following i.c.v. injections of 100 pg ANG II in rats pretreated with saralasin (1 ng i.c.v.). Natriuresis did still occur, but was greatly reduced when compared to the group treated with ANG II alone (see Fig. 2). *P < 0.05 between pre- and post-ANG II.
Statistics Means _+ S . E . M . are r e p o r t e d . D a t a were analyzed by A N O V A and follow-up comparisons between the cumulative 30-min values pre- and posti.c.v, injection in each group were m a d e by paired t-tests. A statistical significance of P < 0.05 was accepted.
[umol/3Omln) urinary SOdiUm excretion
200 t60 120 80 40 0 vehicle
(mmH9)
t50
PlooO pressure
100
lOpg
iOOpg
~Ong
50 0 vehicle
iOpg
tOOpg
tong
Fig. 2. Dose-dependency of the central natriuretic effects of ANG II. Groups consisted of 6-9 animals. White columns indicate 30-min periods before, and black columns 30-rain periods after i.c.v. ANG II or vehicle (0.9% NaCl) injection. Blood pressure values represent mean arterial blood pressure. ANOVA of the sodium excretion data yielded 'withinsubjects' values of FI,29 = 80.7, P < 0.001 and 'interaction' values of F3,29 = 16.2, P < 0.001. **P < 0.01, ***P < 0.001 between pre- and post-ANG II. Note that the natriuretic threshold was at least one order of magnitude below the pressor threshold.
RESULTS Intracerebroventricular injections of A N G II induced a d o s e . d e p e n d e n t increase in urinary sodium excretion of i m m e d i a t e onset and, in most animals, of m o r e than 1 h duration (Figs. 1 and 2). Following the lowest dose of 10 pg, natriuresis was o b s e r v e d in 7 out of 9 animals, whereas after the higher doses, this effect occurred in all animals tested. Urinary volume was only increased after the highest dose (Table I), but to a much lesser degree than the sodium excretion. M e a n arterial b l o o d pressure r e m a i n e d unchanged following 10 and 100 pg but increased following the highest dose of 10 ng A N G II (Fig. 2). P r e t r e a t m e n t with saralasin (1 ng i.c.v.) m a r k e d l y r e d u c e d the natriuretic effect of 100 pg A N G II (Fig. 3). Following c o m b i n e d saralasin and A N G II t r e a t m e n t , urinary sodium increased from 89.3 _+ 9.2 to 101.0 _+ 10.5 #tool/30 min ( P < 0.05). W h e n c o m p a r e d to the group t r e a t e d with A N G II alone, this corresponds to a 74% reduction of the A N G II-induced natriuresis. H i g h e r doses of saralasin could not be used, since they had p r o v e n to be antagonistic, i.e. natriuretic, in pilot experiments.
36 TABLE I Urinarv volume (/~l/rnin/kg b. wt.) be/ore and alter i.c.v. infections o/A NG 11
Data are means + S.E.M. Values represent averages during 30-min periods before and after ANG II injections.
Control 10pg 100pg 10ng
Be]bre
After
53.3 + 9.0 59.5_+ 9.1 59.3 + 13.8 51.0_+ 10.3
55.8 + 10.5 61.8 + 8.3 69.8 _+16.5 70.5 _+8.3*
*P < 0.05.
DISCUSSION Angiotensin I1 injected into the lateral brain ventricle of conscious rats produced a marked dose-dependent and receptor-specific natriuretic response. There are 3 main features of our study which extend previous observations with regard to the ANG II-induced natriuresis. First, the time course, second the dose range, third the independency of the pressor effect. The technique of urine collection in 5-min fractions from the ureter allowed us to monitor the time course of the response. The urinary sodium excretion was already increased in the first 5-min urine fraction collected after the stimulus. Sixty minutes after the ANG I1 injection, at the end of our observation period, urinary sodium excretion was still considerably increased in the majority of the animals while tending to return to baseline levels in the remainder. The rapid onset of the natriuresis sheds a new light on previous reports concerning the ANG II-induced drinking response 3. In these studies, ANG lI was observed to induce first water drinking and, more delayed~ a preferential sodium appetite. The latter was reported to occur also in anephric animals 3. Although this finding would suggest that the ANG II-induced sodium appetite is not secondary to increased sodium loss, a possible central effect of the peptide on intestinal fluid and electrolyte balance 6 was not considered in these experiments. Additionally, uremia may have had an impact on the data obtained in this study. Our results suggest, that in the animal with intact renal function the immediate renal sodium loss following central ANG II
receptor stimulation may be a primary event which then triggers the sodium appetite as a compensatory reaction. The question of a threshold sodium loss to induce salt appetite cannot be answered on the basis of our present data and warrants further investigation. The natriuretic response was already induced by i.c.v, injections of the peptide in picogram amounts, i.e. well below the doses required to elicit drinking, blood pressure increases or release of hypophyseal hormones under the same experimental conditions (see reviews cited above). In a study by Schelling et al. 17, ANG II concentrations in the cerebrospinal fluid (CSF) of rats were reported as 29.5 -+ 5.9 fmol/ml. Assuming a distribution volume between 100 and 300/A, the ANG It concentrations in the CSF produced by natriuretic i.c.v, doses of the peptide in our study approach the physiological range. The natriuretic response could be clearly separated from the pressor response.to central A N G II. This observation confirms earlier findings by Brooks and Malvin 5 in dogs, although their experimental conditions (infusion of the peptide at a rate of 6 ng/min over a period of 2 h under barbiturate anaesthesia) were different from ours. Only after the highest dose of 10 ng did we see an increase in blood pressure associated with the A N G II-induced natriuretic response. Under these conditions, an additional pressure diuresis ~° may have contributed to the natriuretic effects of the peptide. The generalization of our findings could be limited by the experimental design of our study, with the use of chronically uninephrectomized animals, and an i.v. saline infusion to promote diuresis. Concerning the first point, we feel that the absence of a contralateral kidney did not have a major influence on the outcome of the study, since, in previous experiments, we tested this experimental model with respect to renal function and found no impairment in basal function or renal response to salt loading that could be attributed to e.g. a loss of reno-renal reflexes 12. With respect to the i.v. saline infusion, the amount of saline infused during our experiments was small, i.e. ten-fold lower than the sodium load used in a study by Hoffman et al. H in which the authors investigated the effect of sodium loading on the ANG II-induced natriuresis. More-
37 over, without addressing this question systematically, we have observed a similar natriuretic response upon the A N G II challenge in animals without i.v. saline infusions. Therefore, it is unlikely that these two experimental factors would preclude a generalization of our data. The mechanism by which central A N G II induces its natriuretic effects remains to be elucidated. A reduction of sympathetic nerve traffic to the kidney could be involved, since tubular sodium reabsorption can be increased by stimulation of renal nerves 8. We have previously reported that in conscious rats i.c.v, injections of A N G II at doses between 1 and 100 ng induce an immediate reduction in efferent splanchnic and renal nerve activity 2°. Although this effect persisted after baroreceptor deafferentation, A N G II in this dose range consistently caused a pressor response. So far, we have been unable to detect a decrease in renal nerve activity after central A N G II injections of suppressor doses below 1 ng. However, this may be due to an insufficient sensitivity of multifiber nerve recordings employed. Thus, a contribution of the renal nerves cannot be ruled out at present. A N G II could also induce natriuresis by altering the concentration of circulating hormones, such as vasopressin H or aldosterone, since the former can be stimulated and the latter suppressed by central A N G II 4"13. Against vasopressin speaks the fact, that the i.c.v, doses of A N G II required to release this
hormone are in the nanogram range, and against aldosterone is our observation that the A N G lIinduced natriuresis occurs too rapidly to be accounted for by aldosterone suppression. A third hormone which needs to be considered in this regard is atrial natriuretic peptide (ANP), although there are no reports so far that central A N G II releases A N P from the heart. Finally, according to studies by Buckley et al. 7 in dogs, central A N G II may release a natriuretic sodium/potassium ATPase inhibiting factor. Although the A N G II doses used in these studies were much higher than ours, this natriuretic mechanism may be important and needs further investigation. In conclusion, our results confirm earlier reports on the potent natriuretic effects of central A N G II receptor stimulation. A N G II-induced natriuresis is rapid in onset, receptor-specific and occurs at i.c.v. doses of the peptide below the threshold for pressor effects, vasopressin release and drinking. O u r findings lend further support to the idea that A N G II in the CNS is instrumental in the regulation of osmotic and body fluid homoeostasis.
REFERENCES
Pharmacol., 148 (1988) 411-418. 7 Buckley, J.P., Doursout, M.-E, Liang, Y.-Y. and Chelly, J.E., Central angiotensin II mechanisms and the sodium pump, J. Hypertens., 4, Suppl. 6 (1986) $465-$467. 8 DiBona, B.E, The functions of the renal nerves, Rev. Physiol. Biochem. Pharmacol., 94 (1982)75-181. 9 Fluharty, S.J. and Manaker, S., Sodium appetite elicited by intracerebroventricular infusion of angiotensin II in the rat. I. Relation to urinary sodium excretion, Behav. Neurosci., 97 (1983) 738-745. 10 Hall, J.E., Guyton, A.C., Coleman, T.G., Mizelle, H.L. and Woods, L.L., Regulation of arterial pressure: role of pressure natriuresis and diuresis, Fed. Proc., 45 (1986) 2897-2903. 11 Hoffman, W.E., Weet, J.E, Phillips, M.I. and Schmid, P.G., Central effects of angiotensin II in water and saline loaded rats, Neuroendocrinology, 28 (1979) 289-296. 12 Horst, P.-J., Bauer, M., Veelken, R. and Unger, T., A new method for collecting urine directly from the ureter in conscious unrestrained rats, Renal Physiol., in press. 13 Keil, L.C., Summy-Long, J. and Severs, W.B., Release of vasopressin by angiotensin II, Endocrinology, 96 (1975)
1 Andersson, B. and Westbye, O., Synergistic action of sodium and angiotensin on brain mechanisms controlling fluid balance, Life Sci., 9 (1970) 601-608. 2 Andersson, B., Eriksson, L., Fernandez, O., Kolmodin, C.-G. and Oltner, R., Centrally mediated effects of sodium and angiotensin II on arterial blood pressure and fluid balance, Acta Physiol. Scand., 85 (1972) 398-407. 3 Avrith, D.B. and Fitzsimons, J.T., Increased sodium appetite in the rat induced by intracranial administration of components of the renin-angiotensin system, J. Physiol. (Lond.), 301 (1980) 349-364. 4 Brooks, V.L. and Malvin, R.L., lntracerebroventricular infusion of angiotensin II inhibits aldosterone secretion, Am. J. Physiol., 239 (1980) E447-E453. 5 Brooks, V.L. and Malvin, R.L., Intracerebroventricular infusion of angiotensin II increases sodium excretion, Proc. Soc. Exp. Biol. Med., 169 (1982) 532-537. 6 Brown, D.R. and GiUespie, M.A., Actions of centrally administered neuropeptides on rat intestinal transport: enhancement of ileal absorption by angiotensin II, Eur. J.
ACKNOWLEDGEMENT This study was supported by a grant in aid to T.U. from the Deutsche Forschungsgemeinschafl (Un 47/2-1).
38 1063-1065. 14 Lokhandwala, M.F., Buckley, J.P. and Jandhyala, B.S,, Reduction of plasma renin activity by centrally administered angiotensin II in anesthetized cats, Clin. Exp. Hypertens., 1 (1978) 167-175. 15 OIsen, M.E., Hall, J.E., Montani, J.P., Guyton, A.C., Langford, H.G. and Cornell, J.E., Mechanisms of angiotensin II natriuresis and antinatriuresis, Am. J. Physiol., 249 (1985) F299. 16 Phillips, M.I., Functions of angiotensin in the central nervous system, Annu. Rev. Physiol., 49 (1987) 413-435. 17 Schelling, P., Ganten, U., Sponer, G., Unger, T. and Ganten, D., Components of the renin-angiotensin system in the cerebrospinal fluid of rats and dogs with special consideration of the origin and the fate of angiotensin II, Neuroendocrinology, 31 (1980) 297-308. 18 Severs, W.B., Daniels-Severs, A.E., Summy-Long, J. and
Radio, G.F., Effects of centrally administered angiotensm on salt and water excretion, Pharmacology, 6 (1971) 242-252. 19 Unger, T., Rascher, W., Schuster, C., Pavlovitch, R., Sch6mig, A., Dietz, R. and Ganten, D., Central blood pressure effects of substance P and angiotensin !I: role of the sympathetic nervous system and vasopressin, Eur. J. Pharmacol., 71 (1981) 33-42. 20 Unger, T., Becket, H., Petty, M., Demmert, G., Schneider, B., Ganten, D. and Lang, R.E., Differential effects of central angiotensin II and substance P on sympathetic nerve activity in conscious rats: implications for cardiovascular adaptation to behavioral responses, Circ. Res., 56 (1985) 563-575. 21 Unger, T., Badoer, E., Ganten, D., Lang, R.E. and Rettig, R., Brain angiotensin: pathways and pharmacology, Circulation, 77 (1988) I40-I54.
33
BRE 14432
Natriuretic action of central angiotensin II in conscious rats Thomas Unger, Peter J. Horst, Martin Bauer, Gudrun Demmert, Rainer Rettig and Peter Rohmeiss Department of Pharmacology and German Institute for High Blood Pressure Research, University of Heidelberg, Heidelberg (F.R. G. ) (Accepted 11 October 1988) Key words: Angiotensin II; Brain; Natriuresis; Saralasin
The effects of intracerebroventricular (i.c.v.) injections of angiotensin II (ANG II, 10 pg, 100 pg and 10 ng) on renal sodium excretion were investigated in conscious rats instrumented with a chronic urethral catheter. ANG II increased renal sodium excretion dose-dependently with a threshold i.c.v, dose of 10 pg. Only after the highest dose was a concomitant increase in arterial blood pressure and urinary flow observed. The ANG II-induced natriuresis began within 5 min of the i.c.v, injection and lasted for more than 1 h. The angiotensin receptor antagonist saralasin (1 ng, i.c.v.) largely prevented the natriuretic effect of i.c.v, injected ANG II (100 pg). Our results lend further support to the hypothesis that brain ANG II by its potent natriuretic actions may be instrumental in central osmotic control.
INTRODUCTION Over the last 20 years the effects of brain angiotensin II ( A N G II) receptor stimulation in various species have been a matter of intensive investigation (for recent reviews see Phillips 16, and Unger et al.2~). Most of the central actions of the peptide, such as increase in blood pressure, drinking, release of vasopressin from the pituitary gland or suppression of renin release from the kidney conform with those exerted by hormonal A N G II when acting on its peripheral receptors with one notable exception, namely the effect on renal salt excretion. While circulating A N G II tends to retain sodium by a direct renal action 15 as well as through aldosterone release from the adrenal gland, stimulation of brain A N G II receptors has been reported to induce natriuresis 1"2"4'5'9"11A8. In the dog, this central natriuretic action of A N G II appears to be independent of the hemodynamic effects of the peptide 5, whereas in the rat this relationship has not yet been investigated. Moreover, in previous inves-
tigations in rats A N G II was infused intracerebroventricularly (i.c.v.) over relatively long periods of time, and urine was collected by placing the animals in metabolic cages. Therefore, these studies did not provide information as to the onset, duration and dose-responSe relationship of the natriuresis in response to acute stimulation of brain A N G II receptors. We have recently developed a new method for urine collection in conscious, unrestrained rats through a chronic indwelling ureteral catheter which allows differential sampling of small urine fractions over periods as small as one minute le. We have employed this method in the present study to investigate the acute centrally induced natriuretic effect of A N G II in rats. Our results demonstrate that i.c.v, injections of A N G II in the picogram range produce a sustained natriuresis of rapid onset which is independent of changes in arterial blood pressure and sensitive to central A N G II receptor blockade.
Correspondence: T. Unger, Department of Pharmacology, Im Neuenheimer Feld 366, 6900 Heidelberg, F.R.G. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
34 MATERIALS AND METHODS Experiments were performed in conscious, unrestrained, sodium replete male Wistar rats (Dr. Karl Thomae, Biberach, E R . G . ) weighing between 350 and 450 g. All animals were subjected to a unilateral right nephrectomy two months prior to the study. They were fed a standard diet (Altromin) and were allowed free access to water. One or two weeks before the experiment a cannula (PP 20, Portex, U.K.) was inserted into the left lateral brain ventricle under ether anaesthesia as described before 19. To verify correct cannula placement, animals were challenged with an i.c.v, injection of 10 ng A N G II 3 days after surgery, and the drinking response was recorded. Only those animals that reacted with an immediate response and drank more than 3 ml of water within 10 rain upon the challenge were used in the study. On the day before the experiment, rats were anaesthetized with ether and instrumented with femoral artery catheters (PP 10 in PP 50) for direct measurements of arterial blood pressure and with ureterai catheters for urine collection, Construction, implantation and verification of the ureteral catheter have been described elsewhere in detail 12. Briefly, a silicone tube (LHD, Heidelberg, E R . G . ) of 25 cm length (i.d. 0.3 mm, o.d. 0.5 mm) was used to serve as ureter catheter. The abdomen was opened via a midline incision extending from the bladder to about 2 cm caudal to the sternum. The catheter was then tunneled under the skin from the abdomen up to the left ear through a trochar perforating the lateral abdominal wall. A transverse incision was made in the ureter with microsurgery scissors. The tip of the catheter was advanced cranially 2 mm into the ureter and tied into place with a suture. The catheter was anchored to the psoas muscle with two additional sutures. The end of the catheter extending from the orifice at the nape of the neck was partly covered with PE 160 tubing (Clay Adams, U.S.A.). The PE 160 was wrapped with tape and sewed to the skin in a way that urine dripping onto the animal was avoided. After the operation the animals were injected i.p. with 0.1 g ampicillin (Binotal, Bayer Leverkusen, F.R.G.). All surgery was done under aseptical conditions. Twenty-four hours were allowed for recovery from surgery until experiments
were commenced. The operative success rate was about 80%. Reasons for failure were obstruction of the catheter due to crystallization of urine, bleeding, detachment of the catheter from the ureter and removal of the catheter by the animal. Kidney function as analyzed by measurements of GFR, plasma urea, urine concentration capacity after water deprivation, excretion of i.v. sodium loads and by microscopical examination was not disturbed by the ureter catheter for up to one week after
implantation 12. Experimental protocol Experiments were performed while the animals were quietly resting in their home cages. Rats were allowed to accustom themselves to the experimental setting for 1 h. The ureter catheter was then connected to an extension tube (PP 25) via a stainless steel cannula. The extension tube was wrapped with tape and the end inserted into a collection vial, which was placed on level with the kidney. The arterial catheter was connected to a Statham 23 dB pressure transducer coupled to a Gould Brush pressure computer and a Gould Brush 2400 recorder for continuous monitoring of mean arterial blood pressure (MAP). An i.v. infusion of isotonic saline (1.2 ml/h) was commenced to promote urinary flow, and 2 h were allowed for equilibration. Rats were divided in groups and injected i.c.v, with 5 ~1 of isotonic saline (control group, n = 6), 10 pg A N G II (1/al peptide solution flushed with 4/~1 isotonic saline, n = 9), 100 pg A N G II (n = 8), or 10 ng A N G II (n = 6). An additional group (n = 6) received an i.c.v, injection of the ANG II receptor antagonist saralasin (1 ng in 1/~1) 2 rain before the i.c.v, injection of 100 pg A N G II. Each rat was only injected once with A N G II. Urine was collected in 5-min fractions 30 rain before and 60 min after the i.c.v, injections. Mean arterial blood pressure was monitored throughout the experiment. Urinary volume was determined gravimetrically. Urinary sodium and potassium concentrations were measured with a Beckman flame photometer.
Drugs Angiotensin II and the angiotensin II receptor antagonist Sarl-Ala8-Angiotensin 1! (Saralasin)
35 ÷
25
(um01/Smin)
~
20 15
~.
Urinory volume
No excrehon Hmoz ANG II lOOpg
30min
i.C.V.
..~..
vehicle
i
I000~
100'
i
t
5
I 0 : -30
:
: : -15
:
: 0
:
:
', : +15
:
: : +30
:
: : : : +60 +45 time (min)
500!
50.
Fig. 1. Time course of the natriuretic effect of an i.c.v. injection of 100 pg ANG II. Arrow indicates bolus injection of the peptide (experimental group, n = 8) or vehicle (0.9% NaCI, control group, n = 6). Urine was collected in 5-min intervals. After 60 rain renal sodium excretion was still elevated in the majority of the ANG II-injected animals. For standard errors and statistical differences between the two groups refer to Fig. 2.
were p u r c h a s e d from Bachem, Bubendoff, Switzerland. Peptides were dissolved in sterile isotonic saline. Solutions were m a d e up on the day of the e x p e r i m e n t and k e p t on ice.
!
10.
100before ~fler
before after
AN5 II AN5 II Fig. 3. Sodium excretion and urinary volume following i.c.v. injections of 100 pg ANG II in rats pretreated with saralasin (1 ng i.c.v.). Natriuresis did still occur, but was greatly reduced when compared to the group treated with ANG II alone (see Fig. 2). *P < 0.05 between pre- and post-ANG II.
Statistics Means _+ S . E . M . are r e p o r t e d . D a t a were analyzed by A N O V A and follow-up comparisons between the cumulative 30-min values pre- and posti.c.v, injection in each group were m a d e by paired t-tests. A statistical significance of P < 0.05 was accepted.
[umol/3Omln) urinary SOdiUm excretion
200 t60 120 80 40 0 vehicle
(mmH9)
t50
PlooO pressure
100
lOpg
iOOpg
~Ong
50 0 vehicle
iOpg
tOOpg
tong
Fig. 2. Dose-dependency of the central natriuretic effects of ANG II. Groups consisted of 6-9 animals. White columns indicate 30-min periods before, and black columns 30-rain periods after i.c.v. ANG II or vehicle (0.9% NaCl) injection. Blood pressure values represent mean arterial blood pressure. ANOVA of the sodium excretion data yielded 'withinsubjects' values of FI,29 = 80.7, P < 0.001 and 'interaction' values of F3,29 = 16.2, P < 0.001. **P < 0.01, ***P < 0.001 between pre- and post-ANG II. Note that the natriuretic threshold was at least one order of magnitude below the pressor threshold.
RESULTS Intracerebroventricular injections of A N G II induced a d o s e . d e p e n d e n t increase in urinary sodium excretion of i m m e d i a t e onset and, in most animals, of m o r e than 1 h duration (Figs. 1 and 2). Following the lowest dose of 10 pg, natriuresis was o b s e r v e d in 7 out of 9 animals, whereas after the higher doses, this effect occurred in all animals tested. Urinary volume was only increased after the highest dose (Table I), but to a much lesser degree than the sodium excretion. M e a n arterial b l o o d pressure r e m a i n e d unchanged following 10 and 100 pg but increased following the highest dose of 10 ng A N G II (Fig. 2). P r e t r e a t m e n t with saralasin (1 ng i.c.v.) m a r k e d l y r e d u c e d the natriuretic effect of 100 pg A N G II (Fig. 3). Following c o m b i n e d saralasin and A N G II t r e a t m e n t , urinary sodium increased from 89.3 _+ 9.2 to 101.0 _+ 10.5 #tool/30 min ( P < 0.05). W h e n c o m p a r e d to the group t r e a t e d with A N G II alone, this corresponds to a 74% reduction of the A N G II-induced natriuresis. H i g h e r doses of saralasin could not be used, since they had p r o v e n to be antagonistic, i.e. natriuretic, in pilot experiments.
36 TABLE I Urinarv volume (/~l/rnin/kg b. wt.) be/ore and alter i.c.v. infections o/A NG 11
Data are means + S.E.M. Values represent averages during 30-min periods before and after ANG II injections.
Control 10pg 100pg 10ng
Be]bre
After
53.3 + 9.0 59.5_+ 9.1 59.3 + 13.8 51.0_+ 10.3
55.8 + 10.5 61.8 + 8.3 69.8 _+16.5 70.5 _+8.3*
*P < 0.05.
DISCUSSION Angiotensin I1 injected into the lateral brain ventricle of conscious rats produced a marked dose-dependent and receptor-specific natriuretic response. There are 3 main features of our study which extend previous observations with regard to the ANG II-induced natriuresis. First, the time course, second the dose range, third the independency of the pressor effect. The technique of urine collection in 5-min fractions from the ureter allowed us to monitor the time course of the response. The urinary sodium excretion was already increased in the first 5-min urine fraction collected after the stimulus. Sixty minutes after the ANG I1 injection, at the end of our observation period, urinary sodium excretion was still considerably increased in the majority of the animals while tending to return to baseline levels in the remainder. The rapid onset of the natriuresis sheds a new light on previous reports concerning the ANG II-induced drinking response 3. In these studies, ANG lI was observed to induce first water drinking and, more delayed~ a preferential sodium appetite. The latter was reported to occur also in anephric animals 3. Although this finding would suggest that the ANG II-induced sodium appetite is not secondary to increased sodium loss, a possible central effect of the peptide on intestinal fluid and electrolyte balance 6 was not considered in these experiments. Additionally, uremia may have had an impact on the data obtained in this study. Our results suggest, that in the animal with intact renal function the immediate renal sodium loss following central ANG II
receptor stimulation may be a primary event which then triggers the sodium appetite as a compensatory reaction. The question of a threshold sodium loss to induce salt appetite cannot be answered on the basis of our present data and warrants further investigation. The natriuretic response was already induced by i.c.v, injections of the peptide in picogram amounts, i.e. well below the doses required to elicit drinking, blood pressure increases or release of hypophyseal hormones under the same experimental conditions (see reviews cited above). In a study by Schelling et al. 17, ANG II concentrations in the cerebrospinal fluid (CSF) of rats were reported as 29.5 -+ 5.9 fmol/ml. Assuming a distribution volume between 100 and 300/A, the ANG It concentrations in the CSF produced by natriuretic i.c.v, doses of the peptide in our study approach the physiological range. The natriuretic response could be clearly separated from the pressor response.to central A N G II. This observation confirms earlier findings by Brooks and Malvin 5 in dogs, although their experimental conditions (infusion of the peptide at a rate of 6 ng/min over a period of 2 h under barbiturate anaesthesia) were different from ours. Only after the highest dose of 10 ng did we see an increase in blood pressure associated with the A N G II-induced natriuretic response. Under these conditions, an additional pressure diuresis ~° may have contributed to the natriuretic effects of the peptide. The generalization of our findings could be limited by the experimental design of our study, with the use of chronically uninephrectomized animals, and an i.v. saline infusion to promote diuresis. Concerning the first point, we feel that the absence of a contralateral kidney did not have a major influence on the outcome of the study, since, in previous experiments, we tested this experimental model with respect to renal function and found no impairment in basal function or renal response to salt loading that could be attributed to e.g. a loss of reno-renal reflexes 12. With respect to the i.v. saline infusion, the amount of saline infused during our experiments was small, i.e. ten-fold lower than the sodium load used in a study by Hoffman et al. H in which the authors investigated the effect of sodium loading on the ANG II-induced natriuresis. More-
37 over, without addressing this question systematically, we have observed a similar natriuretic response upon the A N G II challenge in animals without i.v. saline infusions. Therefore, it is unlikely that these two experimental factors would preclude a generalization of our data. The mechanism by which central A N G II induces its natriuretic effects remains to be elucidated. A reduction of sympathetic nerve traffic to the kidney could be involved, since tubular sodium reabsorption can be increased by stimulation of renal nerves 8. We have previously reported that in conscious rats i.c.v, injections of A N G II at doses between 1 and 100 ng induce an immediate reduction in efferent splanchnic and renal nerve activity 2°. Although this effect persisted after baroreceptor deafferentation, A N G II in this dose range consistently caused a pressor response. So far, we have been unable to detect a decrease in renal nerve activity after central A N G II injections of suppressor doses below 1 ng. However, this may be due to an insufficient sensitivity of multifiber nerve recordings employed. Thus, a contribution of the renal nerves cannot be ruled out at present. A N G II could also induce natriuresis by altering the concentration of circulating hormones, such as vasopressin H or aldosterone, since the former can be stimulated and the latter suppressed by central A N G II 4"13. Against vasopressin speaks the fact, that the i.c.v, doses of A N G II required to release this
hormone are in the nanogram range, and against aldosterone is our observation that the A N G lIinduced natriuresis occurs too rapidly to be accounted for by aldosterone suppression. A third hormone which needs to be considered in this regard is atrial natriuretic peptide (ANP), although there are no reports so far that central A N G II releases A N P from the heart. Finally, according to studies by Buckley et al. 7 in dogs, central A N G II may release a natriuretic sodium/potassium ATPase inhibiting factor. Although the A N G II doses used in these studies were much higher than ours, this natriuretic mechanism may be important and needs further investigation. In conclusion, our results confirm earlier reports on the potent natriuretic effects of central A N G II receptor stimulation. A N G II-induced natriuresis is rapid in onset, receptor-specific and occurs at i.c.v. doses of the peptide below the threshold for pressor effects, vasopressin release and drinking. O u r findings lend further support to the idea that A N G II in the CNS is instrumental in the regulation of osmotic and body fluid homoeostasis.
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