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excretion and blood pressure
Physiology&Behavior.Vol. 53, pp. 291-299, 1993
0031-9384/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
Printed in the USA.
Converting Enzyme Inhibition in Rabbits: Effects on Sodium and Water Intake/Excretion and Blood Pressure E. TAR JAN, l T. FERRARO, C. M A Y A N D R. S. W E I S I N G E R Howard Florey Institute o f Experimental Physiology and Medicine, University o f Melbourne, Parkville, Victoria, 3052 Australia R e c e i v e d 12 D e c e m b e r 1992 TARJAN, E., T. FERRARO, C. MAY AND R. S. WEISINGER. Converting enzyme inhibition in rabbits: Effects on sodium and waterintake~excretion and bloodpressure. PHYS1OL BEHAV 53(2) 291-299, 1993.--Earlier studies in rabbits revealed that in this species, in contrast to most other species studied, water intake was not influenced by injection or infusion of angiotensin II (ANG II). In order to establish whether ANG II has any role in the regulation of water intake of rabbits, a comprehensive study of the effect of converting enzyme inhibition was undertaken. Enalaprilat was given systemically in various doses to sodium- and water-replete, sodium-deplete, and water-deprived rabbits, and the intake and excretion of water and sodium was measured. In replete rabbits systemic injection of enalaprilat, 8 mg/kg and 8 #g/kg, but not 0.8 mg/kg, was followed by increased daily water intake. In sodium-deplete rabbits injection of enalaprilat, 80 mg/kg, was followed by water drinking within l h, and daily sodium intake was reduced. Systemic administration of ANG Il increased, but did not restore to control level the sodium appetite of sodium-deplete rabbits attenuated by 80 mg/kg enalaprilat. Rabbits deprived of water for 24 h, however, drank the same amount of water after injection of vehicle or enalaprilat, 80 and 8 mg/kg. The efficacy of converting enzyme inhibition was also tested by measuring the blood pressure response to ANG I. Blood pressure responses revealed that in replete animals converting enzyme activity was depressed below control levels for 30 h after injection of 80 mg/kg enalaprilat. In sodium-deplete rabbits blood pressure fell following injection of 80 mg/kg enalaprilat and did not return to control level until 48 h after the injection. These results indicate that in rabbits, similar to other species studied, ANG II plays a role in the regulation of water intake, though probably through mechanisms that are not as well developed or used in other species. Further results suggest that rabbits react to the combination of converting enzyme inhibition and Na-depletion similar to hypertensive patients in regards to renal complications. Angiotensin II Sodium appetite
Blood pressure Converting enzyme inhibitor Sodium depletion Water intake
A N G I O T E N S I N II, acting both peripherally and centrally, plays a major role in the regulation of water and sodium homeostasis in rats, sheep, mice, and cattle [cf., (6)]. In rabbits, evidence of the dipsogenic effect of A N G II is equivocal: following intracerebroventricular (ICV) injection of A N G II ingestion of water was (38), or was not (16,31) observed. In our previous study prolonged ICV or intravenous (IV) infusion of A N G II did not influence water intake, although prolonged ICV, but not IV, infusion resulted in an increase in sodium intake (31). The lack of dipsogenic effect of A N G II was surprising, as the presence and distribution of A N G II binding sites in the rabbit brain (22) was only subtly different from that observed in the central nervous system of other species, including rat and human [cf., (22)]. If rabbits did not drink water following administration of A N G II, this was due to a functional, rather than anatomical, difference from other species.
Enalaprilat
Furosemide
Rabbits
Administration of exogenous A N G II may not be the most appropriate method to demonstrate the role of A N G II in regulatory processes, as A N G II binding sites in various tissues, including the brain, vary with the physiological condition (21). Inhibition o f A N G II formation by administration of converting enzyme inhibitor (CEI) provided an alternative avenue for the investigation of the role of A N G II in physiological regulatory processes. The present studies were undertaken to further investigate the role o f A N G II in the regulation of water and sodium intake and excretion of rabbits. The effect of acute systemic administration of enalaprilat, a converting enzyme inhibitor, in various doses was studied in wild rabbits. Enalaprilat was chosen because it did not influence taste as reported for captopril, the most extensively used CEI (10). Enalaprilat in vitro inhibited converting enzyme with a
l Requests for reprints should be addressed to Dr. Eva Tarjan.
291
291
IARJ,\N til \ t
potency similar to caplopril (19) and influenced water and sodium intake of rats in concentrations nearly or equimolar with captopril (28,36). The stimuli selected were water deprivation and sodium depletion, both conditions known to involve ANG II in the correction of deficit in rats (28), and to a lesser degree, in sheep (35). The efficacy of converting enzyme inhibition following enalaprilat was determined by measuring the effect of ANG I on mean arterial blood pressure (MAP). Also, changes in MAP were recorded after administration of enalaprilat in sodium-deplete rabbits. Although the effect of CEI on the ingestive behaviour of rabbits has not been reported before, its effect on blood pressure is well documented: systemic administration of captopril did not influence MAP in sodium-replete or sodium-loaded rabbits, but caused a sharp fall in MAP in sodium-deplete rabbits (24).
was tested in replete (n 6) rabbits. On the morning ufthe first experimental day, food. water, and NaCI were removed and saline (0.1 ml/kg) was injected into the marginal ear vein. Two hours later urine was collected for the measuremenl of sodium and potassium concentration. Water was returned and intake was measured at hourly intervals for the next 2 h. [:ood was returned 4 h after the injection of saline. Twenty-tour hours after the administration of saline, enalaprilat (MK422. Merk Sharpe & Dohme, Australia), dissolved in saline, was given intramuscularly (IM) in three different doses: 8 mg/kg, 80 ug/kg, and 8 ~g/kg. Two of these doses (8 mg/kg and 80 ~g/kg) were selected as the upper and lower limits at which captopril was reported to stimulate water intake in rats ( 13,14} and to these a third, lower dose was added. One hour after the injection of enalaprilat, 0.5 M NaCI was returned, and intake of water and NaCl was measured at hourly intervals during the next 4 h.
METHOD
Sodilon-deplete rabbit.s: Effect ~!I enalaprilat on waler and sodilon intake and ~:~:cretion. The effect of enalaprilat was tested
ttousing and Maintenance q[ A nimaL~ Wild rabbits, both male and female, were trapped in Central Victoria and after a quarantine period of 3 weeks were housed in individual metabolism cages at 20°C with natural light-dark cycles. Dry food pellets (sodium content 8.5-32.5 mmol/kg, potassium content 274-290 mmol/kg dry weight) and water were available continuously, except immediately after furosemide treatment. Altogether 37 rabbits (body weight 1.4-2.3 kg) were used. The care and maintenance of animals conformed with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
Measurement q[' Water and Sodium Intake and Excretion Ingestion of water and 0.5 M NaCl--when offered--was determined by loss from graduated pipettes. Urine was collected in graduated cylinders and any urine trapped in the feces or adherent to the funnel was removed by washing with a measured amount of distilled water. Measurements of daily intakes and urinary excretions were made between 0900 and 1000 h. Food consumption was measured by the weight loss of containers. Normally wild rabbits do not eat and drink during the light hours: ingestion starts about 1 h before dark, at sunset. Sodium and potassium concentration of urine and plasma was measured by Technicon autoanalyser.
Measurement ql Blood Pressure Blood pressure was measured in conscious rabbits via a cannula implanted into the left carotid artery under general anaesthesia (ketamine hydrochloride, 15 mg/kg, and xylazine, 2 mg/ kg, IV). The catheter consisted of a 3.5 cm length of silastic tubing (0.012 inch i.d. 0.025 inch o.d., Dow Coming) sleeved over the thinned end of polythene P47 tubing. The silicone tube was inserted into the artery up to the junction with the polythene tube and sutured to the vessel wall leaving the artery patent. The catheter was fed under the skin and fixed to the horizontal end of an L-shaped hypodermic tube attached to the skull by dental cement, anchored with stainless steel screws. A luer-lock fitting was embedded into the cement around the vertical end of the hypodermic tube to which a short length of polythene tubing was attached. The catheter was filled with heparinised saline (5000 IU/ml) and the end heat sealed until use. The rabbits were allowed 7 days to recover after surgery.
Experimental Groups Sodium- and water-replete rabbits: Effect of enalaprilat on water and sodium intake and excretion. The effect ofenalaprilat
in sodium-deplete rabbits (n 7), which received furosemide. 20 mg/kg, intravenously (IV) instead of saline in the above protocol. Vehicle or enalaprilat was injected intramuscularly 24 h later in doses 80 mg/kg, 8 mg/kg, and 8 ug/kg. The two lower doses of enalaprilat were selected as they stimulated water intake in the experiment above, and the highest dose was chosen on the basis of reported effects of enalaprilat or captopril on the sodium intake of rats (25,36). Blood samples for plasma creatinine concentration were collected from 10 rabbits at the end of the control period and 1 day after enalaprilat treatment. Additionally, sequential samples were collected 24 h after furosemide, 5 h after enalaprilat, and daily for 3 more consecutive days from 5 of the 10 animals. Plasma creatinine concentration was measured by Jaffa's method with a Technicon autoanalyser. In another series of experiments the effect of ANG II on sodium-deplete, enalaprilat-treated rabbits was studied in 13 animals. They were given IM the two higher enalaprilat doses used in the experiment above: 80 mg/kg (n = 6) or 8 mg/kg (n = 7), following furosemide treatment, as above. On a second occasion, at least I week later, ANG II (Peninsula Laboratories lnc), 1 mg/kg, was injected subcutaneously (SC) 10 min before the injection of the same dose of enalaprilat. Intakes of water. 0.5 M NaCI, and food were measured as above.
IQtter-deprived rabbits: EIC/~'ct ~?/ enalaprilat on water and sodium intake and e~cretion. The effect of enalaprilat was tested on eight rabbits following removal of water for 24 h. In the morning of the first experimental day, a blood sample was taken from the marginal ear vein and water was removed. Twentyfour hours later another blood sample was taken and vehicle or enalaprilat, 80 or 8 mg/kg, was injected 1M. Water was returned 1 h later, and intake was recorded following 1, 2. and 4 h.
Sodium- and water-replete rabbits. Effect ojenalaprilat on thepressor action q/ANG I. On the day of experiment a polythene tube (P47), protected in a counterbalanced flexible wire spring (31), was fixed to the hypodermic tube on the skull and held in place by a luer-lock fitting. The catheter was led out of the cage via a swivel (Harvard Apparatus Ltd.) to a pressure transducer (Statham), and MAP and heart rate (HR) were recorded on a Gould recorder. The cannula was filled with heparinised saline (50 IU/ml). The time course of the effect of enalaprilat was examined by measuring the pressor response to ANG I, 3 #g, at set times after IM administration of enalaprilat. Two control responses to ANG I injected into the arterial cannula (IA) were obtained in two groups of five wild rabbits prior to injection of enalaprilat, 80 or 0.8 mg/kg, IM. The responses to ANG I were measured up
CONVERTING ENZYME INHIBITION IN RABBITS
293
to 6 h after the lower dose of enalaprilat and for 4 days after the higher dose. The responses to ANG I are presented as a percentage of the mean of the control responses.
Sodium-deplete rabbits: Effect of enalaprilat on blood pressure. The effect of enalaprilat on MAP was examined in two groups of five Na-deplete rabbits 24 h after furosemide. After a 30-rain control period, furosemide, 20 mg/kg, was injected via the arterial cannula and MAP was measured for 5 h. Enalaprilat, 80 or 0.8 mg/kg, IM, was given 24 h later, and MAP was monitored up to 30 h after the lower dose of enalaprilat and up to 78 h after the higher dose.
Statistical Analysis Statistical analysis of daily intakes, excretions, MAP, and plasma creatinine concentration was performed by analysis of variance, repeated measures design, with subsequent NewmanKeuls' tests for multiple comparisons (3). The effect of ANG II treatment on the sodium and water intake/excretion of enalaprilat-treated, sodium-deplete rabbits was evaluated by paired t-test. Values are presented as mean _+ SEM. RESULTS
Sodium- and Water-Replete Rabbits: Effect of Enalaprilat on Water and Sodium Intake and Excretion Injection of saline, 0.1 ml/kg IM, did not influence daily water and sodium intake and excretion in rabbits (data not presented). After injection of enalaprilat, 8 mg/kg, 80 #g/kg, or 8 ug/kg, 24 h after IV injection of saline, daily intake of water was significantly higher following injections of enalaprilat, 8 mg/kg and 8 ug/kg, than the mean intake during the preceding control days, F(3, 15) = 10.49, p < 0.05 (Table 1). Daily intake of 0.5 M NaC1 and sodium and potassium excretion were not different from control after injection ofenalaprilat (Table 1). Enalaprilat, at these doses, did not influence daily food intake; the mean value for the control days preceding the experiments was 60 _+ 3.1 g, and ingestion was not different from this after any dose ofenalaprilat, F(3, 15) = 2.63, p > 0.05.
Sodium-Deplete Rabbits: Effect of Enalaprilat on Water and Sodium Intake and Excretion Furosemide induced similar sodium, potassium, and volume losses before enalaprilat treatment in the seven animals on all four occasions: the range of mean sodium loss was 4.8-6.1 mmol, potassium loss 1.9-3.8 mmol, and volume loss 42.0-51.6 ml.
During the 22 h after furosemide treatment, before injection of enalaprilat, rabbits drank significantly more water than during the preceding control days: intake ranged from 95 to 128 ml, whereas mean intake during the control days was 77.3 _ 4.4, F(4, 24) = 10.4, p < 0.05. This range of sodium, potassium, and volume loss, and water intake corresponds to our earlier experience with furosemide-treated rabbits (8). On the next day, after injection of enalaprilat or vehicle, consumption of water was observed during the first hour after treatment with enalaprilat, 80 mg/kg. Rabbits so treated drank 18.6 _+ 16.9 ml within the hour, and five out of the seven animals consumed some water. With the other doses, no water was drunk during the same period; therefore, statistical comparison could not be made. However, during the 24 h following enalaprilat treatment rabbits drank amounts of water not different from the mean of control days, F(4, 24) = 2.93, p > 0.05, or following injection of vehicle, F(3, 18) = 0.81, p > 0.05) (Fig. 1, bottom panel). When 0.5 M NaCI was offered 25 h after injection of furosemide, 1 h after injection ofenalaprilat or vehicle, rabbits commenced consumption on presentation at each occasion. During the next 23 h rabbits drank less 0.5 M NaCI after enalaprilat than vehicle treatment, F(3, 18) = 4.93, p < 0.05 (Fig. 1, top panel). The mortality rate of wild rabbits following combined furosemide and enalaprilat treatment was high: 24 percent (11/ 46) in all the experiments where this treatment combination was used. Similar experiments carried out in laboratory rabbits, using furosemide, 20 mg/kg IM, followed by SQ 14,225, 8 mg/rabbit intraperitoneally (about 2.5 mg/kg), resulted in a 83 percent (5/ 6) mortality rate. In contrast, no experimental mortality was observed after administration of enalaprilat to sodium-replete rabbits. In the present report, data from rabbits that completed the studies and survived the combined furosemide-enalaprilat treatment by at least 3 days, at which time they consumed food and water in close to control quantities, were included. Plasma creatinine concentration did not change significantly in the blood samples taken 24 h after the administration ofenalaprilat, 80 mg/kg, 48 h after furosemide treatment: control 104 + 5 ~ M a n d after enalaprilat 152 + 28 #M (NS, n = 10). In 5 of the 10 rabbits an increase of greater than 30 #M was observed between the two time intervals. Serial samples were taken from five rabbits daily before and after furosemide and enalaprilat, 80 mg/kg, treatment. Plasma creatinine concentration tended to rise from the control 97 + 6 #M to a maximum of 253 _+ 80 tsM 2 days after the furosemide and 1 day after the enalaprilat treatment, and decreased to 142 + 23 u M 2 days
TABLE 1 EFFECT OF ENALAPRILAT ON WILD RABBITS
Water intake (ml/day) 0.5 M NaCI intake (ml/day) Urine volume (ml/day) UN,tV (mmol/day) UKtV (mmol/day)
Control*
8 ug/kg
80 #g/kg
8 mg/kg
68.4 + 7.0 4.31 _+2.31 32.3 + 4.9 1.93 _+0.87 13.3 _+ 1.32
82.2 _+5.6t 2.35 _+ 1.24 29.5 + 2.7 1.27 + 0.37 12.06 _+2.78
70.3 _+4.0 5.38 + 3.80 29.7 + 3.8 3.21 _+ 1.66 11.71 _+9.44
83.7 _+6.It 0.69 + 0.27 29.5 + 1.9 1.25 _+0.28 11.89 _+0.86
n=6.
* Mean of the 3 days preceding administration of saline 24 h before injection of enalaprilat. t P < 0.05, ANOVA, repeated measures design, with subsequent Neuman-Keul's test.
294
[ , \ R . I k N t!1 \1 Intake of 0.5M NaCI solution after furosemide and enalaprilat in wild rabbits (n=7)
20
t
i
'
J i
ANOVA
!
p<005
I
f
0.5M I NaCI consumed 10 ml/24h
~
iiiii: iiii!i ii!
";:;;:211::;2;2::
Enalaprilat mg/kg
0
0.008
80
8
Water intake after furosemide and enalaprilat in wild rabbits (n=7). ANOVA
NS
120100I
Water
80-
consumed ml/24h
604020"
Enalaprilat mg/kg
0
0.008
8
80
FIG. 1. (Top panel) Intake of 0.5 M NaCI solution by wild rabbits during the 23 h after enalaprilat treatment. The animals received furosemide, 20 mg/kg, 24 h before the injection of enalaprilat, 0.008, 8, and 80 mg/kg, or saline, and the NaC1 solution was withheld until 1 h after enalaprilat. Values are mean _+SEM, ANOVA, F(3, 18) = 4.93,p < 0.05. (Bottom panel) Intake of water during the 23 h after enalaprilat treatment. The animals received furosemide, 20 mg/kg, 24 h before the injection of enalaprilat, 0.008, 8, or 80 mg/kg, or saline; water was available from 2 h after furosemide. Values are mean _+SEM, ANOVA, F(3, 18) = 0.80, p > 0.05• later. Although the post hoc test showed significant difference between the control and the highest value, the overall change was not statistically significant, F(5, 20) = 3.29, p > 0.05 (Fig. 2). The effect o f A N G II, injected SC l0 min before enalaprilat, 22 h after injection of furosemide, on water and sodium intake was compared with the intakes of the same rabbits after enalaprilat and furosemide treatment. A N G II, given to rabbits before injection of enalaprilat, 80 mg/kg, significantly increased the intake of 0.5 M NaCl above the suppressed level that followed treatment with enalaprilat alone (Fig. 3, top panel). A N G II did not influence sodium intake after enalaprilat, 8 mg/kg. Water
intake was not different between A N G lI-treated and untreated conditions (Fig. 3, bottom panel).
Water-Deprived Rabbits: Effect of Enalaprilat on Water and Sodium Intake and Excretion Withdrawal of water for 24 h was accompanied by a rise in plasma sodium concentration from 141.7 + 0.6 to 149.2 _+ 0.9 mM, (p < 0.05), although plasma potassium and protein concentration and osmolality did not change. During water deprivation, a reduction in urine volume was noted and food intake and daily sodium balance was negative, which is similar to our
C O N V E R T I N G E N Z Y M E INHIBITION IN RABBITS
300 -
295 but there was a significant reduction after 4 and 5 h (p < 0.05) (Fig. 6). The higher dose of enalaprilat, 80 mg/kg, given 24 h after furosemide, reduced MAP from 81 -_. 5 m m H g to 64 + 6 m m H g (p < 0.05) after 10 min, and to a m i n i m u m of 51 + 5 m m H g after 6 h. MAP remained significantly below control for 24 h, F(13, 52) --- 7.37, p < 0.05 (Fig. 6). In another group of five wild rabbits the control MAP was 94 __- 3 (mean of five readings at 5-min intervals), and administration of furosemide, 20 mg/kg IA, did not significantly alter MAP. At 24 h after furosemide, MAP was 95 -+ 5 m m H g (mean
. p
Enalaprilat
.
/
pmol 200"
Furosemide +
m
100"
1
2
3
4
I1 5 days
FIG. 2. Plasma concentration of creatinine before and after sodium depletion with furosemide, 20 mg/kg, and additional treatment with enalaprilat, 80 mg/kg. Values are mean _+SEM, ANOVA, F(5, 20) = 3.29, p > 0.05, with Newman-Keuls' test.
previous experience (30). Before the administration of vehicle or enalaprilat, 80 or 8 mg/kg, the rabbits were in a similar state of water and sodium deficit. Upon presentation of water, 1 h after injection of vehicle or enalaprilat, 80 or 8 mg/kg, the rabbits drank eagerly (Fig. 4). The cumulative intake of water was significantly lower following injection of enalaprilat, 8 mg/kg, during the second and fourth hour of access, compared with that following injection of vehicle or enalaprilat, 80 mg/kg (Fig. 4). There was, however, no difference in the amounts of water consumed during the first hour of access or during the 23 h after the presentation of water. During the 23 h after treatment, food intake, urine volume, and body weight were not different from control (Table 2). A reduction in sodium excretion was observed in the vehicle-treated rabbits, but not following enalaprilat treatment. On the other hand, enalaprilat-treated rabbits had a lower potassium excretion during the same period.
Sodium- and Water-Replete Rabbits: Effect of Enalaprilat on the Pressor Action of ANG I In five rabbits IA A N G I, 3 ~zg, increased MAP by 55.6 + 6.4 mmHg. The higher dose of enalaprilat, 80 mg/kg, reduced the ANG I-induced pressor response to 2 ___2% of the control response within 15 min (p < 0.05) and it remained suppressed at 17 + 11% of control after 8 h, F(14, 56) = 21.4, p < 0.05. At 48 h after enalaprilat, the response to A N G I (71 + 7%) was not significantly different from control (Fig. 5). In a second group of five rabbits, IA injection of A N G I, 3 #g, increased MAP by 62.5 + 6.9 mmHg. Following administration ofenalaprilat, 0.8 mg/kg IM, the pressor response to A N G I was reduced to 2 ___ 1% of the control value within 15 m i n (p < 0.05), and by 6 h the response to A N G I had returned to control levels, F(7, 28) = 114.6, p < 0.05 (Fig. 5).
Sodium-Deplete Rabbits: Effect of Enalaprilat on Blood Pressure In five rabbits the control MAP was 89 ___6 mmHg. There was no fall in MAP for the first 3 h after furosemide, 20 mg/kg,
* p
paired t-test
8mg/kg enelaprilet, i.m. • 80mg/kg o 8mg/kg •
1210"
a 80mg/kg
enalaprilat, i.m. plus l i n g anglotensi t 11, s.c.
Cumulative 8intake of 0.5 M 6-
NaCl
(ml)
42,
,
1
2
iiP'''~
4
6
214 hours
~ 7
80-
7
7060-
Cumulative
6
50-
intake of 40water (ml)
6
302010-
~
~,
I
2'4 hours
FIG. 3. (Top panel) Intake of 0.5 MNaCI solution by wild rabbits during the 23 h after enalaprilat treatment, or combination of enalaprilat and ANG II. The animals received furosemide, 20 mg/kg, 24 h before the injection of enalaprilat, and the NaCI solution was withheld until 1 h after enalaprilat. Values are mean _+SEM, paired t-tests. (Bottom panel) Intake of water during the 23 h after enalaprilat treatment or combination of enalaprilat and ANG II. The animals received furosemide, 20 mg/kg, 24 h before the injection of enaiaprilat, and water was available from 2 h after furosemide. Values are mean _+SEM, paired t-tests.
296
I,~,RJAN t'1 A I
I 170150-
* 0<0.05 o-o im saline H im Enalaprilat, 8mg/kg ~--r, im Enalaorilat, 80mg/kg
j/~ ,/ /
I
130-
Cumulate
110-
intake of water ml
9070-
5030|
I
I
1
2
4
/
24 hours
after presentation of water
FIG. 4. Cumulative water intake of rabbits deprived of water for the 24 h preceding injection ofenalaprilat. Water was offered 1 h after enalaprilat. Values are mean _+ SEM, ANOVA, with Newman-Keuls' test.
of four readings at 5-min intervals, not different from control). Subsequent administration of enalaprilat, 0.8 mg/kg IM, given 24 h after furosemide, significantly reduced MAP to 79 +_ 7 m m H g (p < 0.05) after 10 min and to 54 _+ 8 m m H g (p < 0.05) after 6 h. By 24 h after enalaprilat MAP (74 + 8 m m H g ) was not significantly different from control, F(20, 80) = 14.24, p < 0.05. DISCUSSION
The effect of converting enzyme inhibition on the ingestive behaviour of rabbits has not been investigated before. Comparison with other species will highlight regulatory processes that are probably present not only in rabbits but other species as well, but are expressed more in rabbits than in other species, including Sprague-Dawley rats.
In sodium- and water-replete rabbits increased water intake tbllowed the highest (8 mg/kg) and lowest (8 ug/kg) doses of enalaprilat. Over a thousand-fold dose range of enalaprilat the changes in water intake were not dose dependent. In studies conducted on rats, acute systemic administration of CEI was demonstrated to cause a dual effect on water intake: low doses (captopril < 5 mg/kg, enalapril < 1 mg/kg, ramipril .: 0.1-It.?, mg/kg) increased the water intake of satiated rats (9,11,28), en-hanced water intake following ureteric ligation (13), adrenalectomy (12), systemic injection of A N G II [(28), Ramipril only], and following peritoneal dialysis with glucose solution (4). Doms higher than those detailed above that did not influence (11,28) or reduce the water intake in satiated rats (36), attenuated water drinking following systemic injection of A N G II (28) or ICV injection o f A N G II (14). The increased intakes in rats [bllowing low doses of CEl was attributed to high circulating ANG l concentration, from which A N G II is produced in brain areas accessible to circulating A N G I, namely in the circumventricular organs, by the local, not yet blocked, converting enzyme (20). The high dose of CEl was thought to inhibit drinking by reducing the availability o r A N G ll in the brain (13) as converting enzyme activity and local production o f A N G II decreased in the brain (30). The dichotomy in the drinking behaviour of rats, increased intake following low dose of CEI, and reduced consumption after high dose of CEl, may be explained by the above hypotheses. In replete rabbits, however, no such dichotomy was observed, which could be due to inadequate doses used or to involvement of different mechanisms. A N G I plasma concentration probably rises in replete rabbits alter inhibition of converting enzyme, as reported in dogs (26) and humans (5). The increased water intake by replete rabbits after enalaprilat, however, could not be attributed to the increased A N G I concentration, because in our previous experiments injection of renin or A N G II did not induce water intake in rabbits (31). Although enalaprilal influences other systems, e.g., bradykinin or endorphins [cf., (5)], there are no data 1o suggest a role for these systems in the regulation of ingestive behaviour of rabbits. It is, however, possible that in rabbits one of the effects of increased plasma A N G II concentration is inhibition of water intake, and this inhibition is released by enalaprilat. If this hypothetical mechanism, which inhibited water drinking when circulating A N G lI concentration increased, existed, its receptors could be in brain areas devoid of blood-brain
TABLE 2 EFFECT OF ENALAPRILAT. ON WILD RABBITS Following Water Deprivation (24 h)
Water intake (ml/day) Urine volume (ml/day) Body weight (g) UNatV (mmol/day) UKtV (mmol/day) //=8.
Control*
Saline
8 mg/kg
80 mg/kg
102.4 _+ 25.1 52.1 _+ 13.6 1684 _+ 123 1.63 +_ 0.31 14.04 _+ 2.09
168.0 _+ 17.8t 45.6 _+ 1 t.7 1681 _+ 121 0.59 +_ 0.22t 11.27 _+ 1.62
159.0 _+ 13.7+ 56.0 _+ 11.5 1678 +_ 122 1.02 + 0.18 9.23 _+ 1.15t
158.6 + 22.6t 48.5 _+ 6.8 1693 _+ 128 1.25 +_ 0.34 8.87 _+ 2.15t
* Mean of the 3 days preceding water deprivation for 24 h. Values for the day of water deprivation are not shown. Saline or enalaprilat, 8 or 80 mg/h was injected 1 h before presentation of water, following 24 h of water deprivation. t P < 0.05 compared to control, ANOVA, repeated measures design, with subsequent Neuman-Keul's test.
CONVERTING ENZYME INHIBITION IN RABBITS
297
120
2 °o 1 0 0
8O
._c
6O *
C ¢1
o
10 ¢b 0
7"
40-
"10
•-¢ 2 0 I
<~
II -20
1.25
0.5
1
2
3
4
6
8
24
30
48
54
72
78
Time after Enalaprilat (hour)
FIG. 5. Percent reduction of the pressor effect of ANG l, 3 #g, in Na-replete rabbits following injection of enalaprilat 80 mg/kg (filled bars) or 0.8 mg/kg (empty bars). Values are mean _+SEM, ANOVA, with Newman-Keuls' test. *p < 0.05.
barrier, such as the subfornical organ, organum vasculosum laminae terminalis, or area postrema. A further support to the existence of this inhibitory system can be found in sodium-deplete rabbits, where a very high dose of enalaprilat stimulated water intake. This very high dose of enalaprilat nearly totally blocked the pressor action of ANG I, indicating a very low converting enzyme activity in the areas where enalaprilat gets access to, including the circumventricular organs. In rabbits deprived of water for 24 h the very high dose of enalaprilat did not influence water intake, although a somewhat lower dose, 8 mg/kg, caused a temporary reduction in water drinking. This result would also fit into the hypothesis outlined above. In water-deprived rabbits it is possible that converting enzyme blockade with the lower dose of enalaprilat was insufficient to counteract the inhibitory effect of the higher ANG I concentration after CEI on the increased number of ANG II binding sites in the circumventricular organs, which have been demonstrated in rats after water deprivation (18,20). The higher dose of enalaprilat would block conversion close to these binding sites and thereby disable the inhibitory effect o f A N G II binding on water drinking in rabbits. In rats, a small reduction in waterdeprivation-induced water intake was observed (2) following systemic administration of captopril or enalapril in doses similar to those used in the present studies in rabbits. Bamey et al. (2) interpreted this finding as a demonstration of the involvement of ANG II in water-deprivation-induced drinking in rats. The present experiments support our previous finding that ANG II is involved in the generation of sodium appetite in rabbits (8,31). The sodium intake of sodium-deplete rabbits was significantly inhibited by the highest dose of enalaprilat; lower doses also tended to reduce sodium intake at all time intervals. The specific effect of enalaprilat on sodium intake is supported by the observation that systemic injection of ANG II raised the sodium intake of sodium-deplete rabbits, attenuated by enala-
prilat. In sodium-deplete rabbits the inhibition of converting enzyme lasted long enough after single injection to influence daily sodium intake. The dose-dependent reduction of sodium intake of sodium-deplete rabbits contrasts sharply with the findings by others in rats. In sodium-deplete rats CEI had a dosedependent dual effect: low doses enhanced and high doses inhibited sodium intake. In detail: the sodium intake of rats sodium deplete after injection of furosemide (25,36), or peritoneal dialysis with glucose solution (4), was enhanced by low doses of CEI, although higher doses inhibited furosemide-induced sodium intake (36), though further enhancement was noted after peritoneal dialysis (4). In rabbits there was no evidence for stimulation at any dose levels indicating, perhaps, that the sodium appetite following furosemide treatment is fully expressed in rabbits and cannot be enhanced, as in rats, by other mechanisms, e.g., a rise in plasma ANG I concentration following CEI treatment. Systemic injection of ANG II enhanced the Na appetite of rabbits reduced by enalaprilat, as was found in sheep (35), rats (36), and mice (34). As systemically administered ANG II is thought not to cross the blood-brain barrier, the restoration of the depressed sodium intake of Na-deplete rabbits by ANG II suggests that the active site is in the circumventricular organs. Alternately, ANG II may cross the blood-brain barrier in sufficient quantities to be effective at ANG II binding sites that have been demonstrated in other areas of the rabbit brain (22). Urinary sodium and potassium excretion in the main depended on the experimental condition and the amount of sodium or water ingested, and was not influenced by enalaprilat treatment. The present findings demonstrate that in conscious, sodiumdeplete wild rabbits converting enzyme inhibition resulted in a large, sustained fall in blood pressure. Similar effects of converting enzyme inhibition have been reported in sodium-deplete laboratory rabbits (24), and in sodium-deplete rats (23). In sodium-
298
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FIG. 6. Mean arterial blood pressure of rabbits after injection of furosemide, 20 mg/kg, and 24 h later enalaprilat, 80 mg/kg. Values are mean _+ SEM, ANOVA, F(13, 52) = 7.37, p < 0.05, with Newman-Keuls' test (+p < 0.05 vs. -24 h, *p < 0.05 vs. 0 h).
restricted humans, chronic treatment with enalapril resulted in a moderate fall in blood pressure (33). The smaller fall in pressure in humans may reflect a larger degree of sodium depletion in the studies with rabbits and rats, resulting in their increased dependence on A N G II to maintain peripheral resistance. The large fall in pressure following enalapril reduced renal function, as evidenced by the increase in plasma creatinine, and in extreme cases, this may have caused renal failure and account for the mortality that was observed. Angiotensin-mediated efferent arteriolar constriction normally plays a major role in the autoregulation ofglomerular filtration rate, which is of particular importance when perfusion pressure is reduced (17,27). Pharmacological blockade of this system reduced renal function in sodium-deplete patients and in those with renal artery stenosis, thus warranting cautious use of this class of drugs in these situations (1,29). In summary, inhibition of converting enzyme by enalaprilat revealed a role for A N G II in the regulation of water intake in
rabbits that was difficult to demonstrate in earlier studies by administration of A N G II. The previously uncovered role of A N G II in the regulation of sodium intake of rabbits was confirmed by the use ofenalaprilat. The effect ofenalaprilat on both water and sodium intake of rabbits differs from that of rats: the opposite effects following low and high doses of CEI, reported in rats, were not demonstrated in rabbits. The rabbits, similar to hypertensive patients, proved sensitive to the renal complications of combined sodium depletion and converting enzyme inhibition. ACKNOWLEDGEMENTS This work was supported by the National Health and Medical Research Council of Australia and the Victorian Health Promotion Foundation. The valuable discussions with Professor Derek A. Denton and Dr. John R. Blair-West during the preparation of the manuscript are gratefully acknowledged.
REFERENCES 1. Andreucci, V. E.; Conte, G.; Dal Canton, A.; Di Minno G.; Usberi, M. The causal role of salt depletion in acute renal failure due to captopril in hypertensive patients with a single functioning kidney and renal artery stenosis. Ren. Fail. 10:9-20; 1987. 2. Barney, C. C.; Katovich, M. J.; Fregly, M. J. The effect of acute administration of an angiotensin converting enzyme inhibitor, captopril (SQ 14,225), on experimentally included thirst in rats. J Pharmacol. Exp. Ther. 212:53-57; 1980. 3. Bruning, J. L.; Kintz, B. L. Computational handbook of statistics. Glenview, IL.: Scott, Foresman and Co.; 1977.
4. Buggy, J.; Jonklass, J. Sodium appetite decreased by central angiotensin blockade. Physiol. Behav. 32:749-753; 1984. 5. Cushman, D. W.; Ondetti, M. A. lnhibitors ofangiotensin-converting enzyme for treatment of hypertension. Biochem. Pharmacol. 29: 1871-1877; 1980. 6. Denton, D. A. The hunger for salt--An anthropological, physiological and medical analysis. Berlin: Springer; 1982. 7. Denton, D. A.; Blair-West, J. R.; McBurnie, M. I.; Osborne, P. G.; Tarjan, E.; Williams, R. M.; Weisinger, R. S. Angiotensin and salt appetite of BALB/c mice. Am. J. Physiol. 259:R729-R735; 1990.
C O N V E R T I N G E N Z Y M E I N H I B I T I O N IN RABBITS 8. Denton, D. A.; Nelson, J. F.; Tarjan, E. Water and salt intake of wild rabbits (Oryctolagus cuniculus (L)) following dipsogenic stimuli. J. Physiol. (Lond.) 362:285-301; 1985. 9. DiNicolantonio, R.; Weisinger, R. S. Feeding and drinking behaviour following angiotensin converting enzyme blockade: Role of injectant pH. Pharmacol. Biochem. Behav. 29:547-551; 1988. 10. Dollery, C. T. Safety and et~cacy of enalapril. Summing up the evidence. J. Hypertens. l(Suppl 1):155-157; 1983. 11. Elfont, R. M.; Epstein, A. N.; Fitzsimons, J. T. Involvement of the renin-angiotensin system in captopril-induced sodium appetite in the rat. J. Physiol. (Lond.) 354:11-27; 1984. 12. Elfont, R. M.; Fitzsimons, J. T. The role ofangiotensin in increased sodium appetite after adrenalectomy. J. Physiol. (Lond.) 320:70P; 1981. 13. Elfont, R. M.; Fitzsimons, J. T. Renin dependence of captoprilinduced drinking after ureteric ligation in the rat. J. Physiol. (Lond.) 343:17-30; 1983. 14. Evered, M. D.; Robinson, M. M. Increased or decreased thirst caused by inhibition of angiotensin-converting enzyme in the rat. J. Physiol. (Lond.) 348:573-588; 1984. 15. Findlay, A. R. L.; Epstein, A. N. Increased sodium intake is somehow induced in rats by intravenous angiotensin II. Horm. Behav. 14:8692; 1980. 16. Fink, G. D.; Bryan, W. G.; Mokler, D. J. Effects of chronic intracerebroventricular infusion of angiotensin II on arterial pressure and fluid homeostasis. Hypertension 4:312-319; 1982. 17. Hall, J. E. Control of sodium excretion by angiotensin II: Intrarenal mechanisms and blood pressure regulation. Am. J. Physiol. 250: R960-R972; 1986. 18. Israel, A.; Saavedra, J. M.; Plunkett, L. Water deprivation upregulates angiotensin I1 receptors in rat anterior pituitary. Am. J. Physiol. 248:E264-E267; 1985. 19. Johnston, C. 1.; Cubela, R.; Jackson, B. Relative inhibitory potency and plasma drug levels of angiotensin converting enzyme inhibitors in the rat. Clin. Exp. Pharmacol. Physiol. 15:123-129; 1988. 20. Lehr, D.; Goldman, H. W.; Casner, P. Renin-angiotensin role in thirst: Paradoxical enhancement of drinking by angiotensin converting enzyme inhibitor. Science 182:1031-1032; 1973. 21. Mendelsohn, F. A. O.; Aguilera, G.; Saavedra, J. M.; Quirion, R.; Catt, K. J. Characteristics and regulation of angiotensin II receptors in pituitary, circumventricular organs and kidney. Clin. Exp. Hypertens. A5:1081-1097; 1983. 22. Mendelsohn, F. A. O.; Allen, A. M.; Clevers, J.; Denton, D. A.; Tarjan, E.; McKinley, M. J. Localization of angiotensin 11 receptor binding in rabbit brain by in vitro autoradiography. J. Comp. Neurol. 270:372-384; 1988. 23. Mento, P. F.; Wilkes, B. M. Azotemia during chronic converting enzyme inhibition with enalapril in sodium-depleted rats: Role of renal circulatory changes. J. Cardiovasc. Pharmacol. 8:670-675; 1986.
299 24. Mimran, A.; Guiod, L.; Hollenberg, N. K. The role of angiotensin in the cardiovascular and renal response to salt restriction. Kidney Int. 5:348-355; 1974. 25. Moe, K. E.; Weiss, M. L.; Epstein, A. N. Sodium appetite during captopril blockade of endogenous angiotensin II formation. Am. J. Physiol. 247:R356-R365; 1984. 26. Morton, J. J.; Tree, M.; Casals-Stenzel, J. The effect ofcaptopril on blood pressure and angiotensins 1, II and III in sodium-depleted dogs: Problems associated with the measurement of angiotensin I1 after inhibition of converting enzyme. Clin. Sci. 58:445-450; 1980. 27. Rosivall, L.; Navar, L. G. Effects on renal hemodynamics ofintraarterial infusions of angiotensins I and II. Am. J. Physiol. 245:FI81F187; 1983. 28. Rowland, N. E.; Fregly, M. J. Comparison of the effects of the dipiptidyl peptidase inhibitors captopril, ramipril, and enalapril on water intake and sodium appetite of Spague-Dawley rats. Behav. Neurosci. 102:953-960; 1988. 29. Speirs, C. J.; Dollery, C. T.; Inman, W. H.; Rawson, N. S.; Wilton, L. V. Postmarketing surveillance of enalapril. II: Investigations of the potential role ofenalapril in deaths with renal failure. Brit. Med. J. 297:830-832; 1988. 30. Tarjan, E.; Denton, D. A.; Weisinger, R. S. Atrial natriuretic peptide inhibits water and sodium intake in rabbits. Regul. Pept. 23:63-75; 1988. 31. Tarjan, E.; Denton, D. A.; McBurnie, M. I.; Weisinger, R. S. Water and sodium intake of wild and New Zealand rabbits following angiotensin. Peptides 9:677-679; 1988. 32. Unger, T.; Ganten, D.; Lang, R. E. Effect of converting enzyme inhibitors on tissue converting enzyme and angiotensin II: Therapeutic implications. Am. J. Cardiol. 59:I8D-22D; 1987. 33. Volpe, M.; Lembo, G.; Morganti, A.; Condorelli, M.; Trimarco, B. Contribution of the renin-angiotensin system and of the sympathetic nervous system to blood pressure homeostasis during chronic restriction of sodium intake. Am. J. Hypertens. 1:353-358; 1988. 34. Weisinger, R. S.; Blair-West, J. R.; Denton, D. A.; McBurnie, M.; Ong, F.; Tarjan, E.; Williams, R. M. Effect of angiotensin-converting enzyme inhibitor on salt appetite and thirst of BALB/c mice. Am. J. Physiol. 259:R736-R740; 1990. 35. Weisinger, R. S.; Denton, D. A.; Di Nicolantonio, R.; McKinley, M. J.; Muller, A. F.; Tarjan, E. Role ofangiotensin in sodium appetite of sodium-deplete sheep. Am. J. Physiol. 253:R482-R488; 1987. 36. Weisinger, R. S.; Denton, D. A.; Di Nicolantonio, R.; McKinley, M. J. The effect of captopril or enalaprilic acid on the Na appetite of Na-deplete rats. Clin. Exp. Pharmacol. Physiol. 15:55-65; 1988. 37. Weisinger, R. S.; Denton, D. A.; McKinley, M. J.; Muller, A. F.; Tarjan, E. Angiotensin and Na appetite of sheep. Am. J. Physiol. 25 l:R690-R699; 1986. 38. Wright, J. W.; Sullivan, M. J.; Petersen, E. P.; Harding, J. W. Brain angiotensin II and Ill binding and dipsogenicity in the rabbit. Brain Res. 358:376-379; 1985.
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Converting Enzyme Inhibition in Rabbits: Effects on Sodium and Water Intake/Excretion and Blood Pressure E. TAR JAN, l T. FERRARO, C. M A Y A N D R. S. W E I S I N G E R Howard Florey Institute o f Experimental Physiology and Medicine, University o f Melbourne, Parkville, Victoria, 3052 Australia R e c e i v e d 12 D e c e m b e r 1992 TARJAN, E., T. FERRARO, C. MAY AND R. S. WEISINGER. Converting enzyme inhibition in rabbits: Effects on sodium and waterintake~excretion and bloodpressure. PHYS1OL BEHAV 53(2) 291-299, 1993.--Earlier studies in rabbits revealed that in this species, in contrast to most other species studied, water intake was not influenced by injection or infusion of angiotensin II (ANG II). In order to establish whether ANG II has any role in the regulation of water intake of rabbits, a comprehensive study of the effect of converting enzyme inhibition was undertaken. Enalaprilat was given systemically in various doses to sodium- and water-replete, sodium-deplete, and water-deprived rabbits, and the intake and excretion of water and sodium was measured. In replete rabbits systemic injection of enalaprilat, 8 mg/kg and 8 #g/kg, but not 0.8 mg/kg, was followed by increased daily water intake. In sodium-deplete rabbits injection of enalaprilat, 80 mg/kg, was followed by water drinking within l h, and daily sodium intake was reduced. Systemic administration of ANG Il increased, but did not restore to control level the sodium appetite of sodium-deplete rabbits attenuated by 80 mg/kg enalaprilat. Rabbits deprived of water for 24 h, however, drank the same amount of water after injection of vehicle or enalaprilat, 80 and 8 mg/kg. The efficacy of converting enzyme inhibition was also tested by measuring the blood pressure response to ANG I. Blood pressure responses revealed that in replete animals converting enzyme activity was depressed below control levels for 30 h after injection of 80 mg/kg enalaprilat. In sodium-deplete rabbits blood pressure fell following injection of 80 mg/kg enalaprilat and did not return to control level until 48 h after the injection. These results indicate that in rabbits, similar to other species studied, ANG II plays a role in the regulation of water intake, though probably through mechanisms that are not as well developed or used in other species. Further results suggest that rabbits react to the combination of converting enzyme inhibition and Na-depletion similar to hypertensive patients in regards to renal complications. Angiotensin II Sodium appetite
Blood pressure Converting enzyme inhibitor Sodium depletion Water intake
A N G I O T E N S I N II, acting both peripherally and centrally, plays a major role in the regulation of water and sodium homeostasis in rats, sheep, mice, and cattle [cf., (6)]. In rabbits, evidence of the dipsogenic effect of A N G II is equivocal: following intracerebroventricular (ICV) injection of A N G II ingestion of water was (38), or was not (16,31) observed. In our previous study prolonged ICV or intravenous (IV) infusion of A N G II did not influence water intake, although prolonged ICV, but not IV, infusion resulted in an increase in sodium intake (31). The lack of dipsogenic effect of A N G II was surprising, as the presence and distribution of A N G II binding sites in the rabbit brain (22) was only subtly different from that observed in the central nervous system of other species, including rat and human [cf., (22)]. If rabbits did not drink water following administration of A N G II, this was due to a functional, rather than anatomical, difference from other species.
Enalaprilat
Furosemide
Rabbits
Administration of exogenous A N G II may not be the most appropriate method to demonstrate the role of A N G II in regulatory processes, as A N G II binding sites in various tissues, including the brain, vary with the physiological condition (21). Inhibition o f A N G II formation by administration of converting enzyme inhibitor (CEI) provided an alternative avenue for the investigation of the role of A N G II in physiological regulatory processes. The present studies were undertaken to further investigate the role o f A N G II in the regulation of water and sodium intake and excretion of rabbits. The effect of acute systemic administration of enalaprilat, a converting enzyme inhibitor, in various doses was studied in wild rabbits. Enalaprilat was chosen because it did not influence taste as reported for captopril, the most extensively used CEI (10). Enalaprilat in vitro inhibited converting enzyme with a
l Requests for reprints should be addressed to Dr. Eva Tarjan.
291
291
IARJ,\N til \ t
potency similar to caplopril (19) and influenced water and sodium intake of rats in concentrations nearly or equimolar with captopril (28,36). The stimuli selected were water deprivation and sodium depletion, both conditions known to involve ANG II in the correction of deficit in rats (28), and to a lesser degree, in sheep (35). The efficacy of converting enzyme inhibition following enalaprilat was determined by measuring the effect of ANG I on mean arterial blood pressure (MAP). Also, changes in MAP were recorded after administration of enalaprilat in sodium-deplete rabbits. Although the effect of CEI on the ingestive behaviour of rabbits has not been reported before, its effect on blood pressure is well documented: systemic administration of captopril did not influence MAP in sodium-replete or sodium-loaded rabbits, but caused a sharp fall in MAP in sodium-deplete rabbits (24).
was tested in replete (n 6) rabbits. On the morning ufthe first experimental day, food. water, and NaCI were removed and saline (0.1 ml/kg) was injected into the marginal ear vein. Two hours later urine was collected for the measuremenl of sodium and potassium concentration. Water was returned and intake was measured at hourly intervals for the next 2 h. [:ood was returned 4 h after the injection of saline. Twenty-tour hours after the administration of saline, enalaprilat (MK422. Merk Sharpe & Dohme, Australia), dissolved in saline, was given intramuscularly (IM) in three different doses: 8 mg/kg, 80 ug/kg, and 8 ~g/kg. Two of these doses (8 mg/kg and 80 ~g/kg) were selected as the upper and lower limits at which captopril was reported to stimulate water intake in rats ( 13,14} and to these a third, lower dose was added. One hour after the injection of enalaprilat, 0.5 M NaCI was returned, and intake of water and NaCl was measured at hourly intervals during the next 4 h.
METHOD
Sodilon-deplete rabbit.s: Effect ~!I enalaprilat on waler and sodilon intake and ~:~:cretion. The effect of enalaprilat was tested
ttousing and Maintenance q[ A nimaL~ Wild rabbits, both male and female, were trapped in Central Victoria and after a quarantine period of 3 weeks were housed in individual metabolism cages at 20°C with natural light-dark cycles. Dry food pellets (sodium content 8.5-32.5 mmol/kg, potassium content 274-290 mmol/kg dry weight) and water were available continuously, except immediately after furosemide treatment. Altogether 37 rabbits (body weight 1.4-2.3 kg) were used. The care and maintenance of animals conformed with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.
Measurement q[' Water and Sodium Intake and Excretion Ingestion of water and 0.5 M NaCl--when offered--was determined by loss from graduated pipettes. Urine was collected in graduated cylinders and any urine trapped in the feces or adherent to the funnel was removed by washing with a measured amount of distilled water. Measurements of daily intakes and urinary excretions were made between 0900 and 1000 h. Food consumption was measured by the weight loss of containers. Normally wild rabbits do not eat and drink during the light hours: ingestion starts about 1 h before dark, at sunset. Sodium and potassium concentration of urine and plasma was measured by Technicon autoanalyser.
Measurement ql Blood Pressure Blood pressure was measured in conscious rabbits via a cannula implanted into the left carotid artery under general anaesthesia (ketamine hydrochloride, 15 mg/kg, and xylazine, 2 mg/ kg, IV). The catheter consisted of a 3.5 cm length of silastic tubing (0.012 inch i.d. 0.025 inch o.d., Dow Coming) sleeved over the thinned end of polythene P47 tubing. The silicone tube was inserted into the artery up to the junction with the polythene tube and sutured to the vessel wall leaving the artery patent. The catheter was fed under the skin and fixed to the horizontal end of an L-shaped hypodermic tube attached to the skull by dental cement, anchored with stainless steel screws. A luer-lock fitting was embedded into the cement around the vertical end of the hypodermic tube to which a short length of polythene tubing was attached. The catheter was filled with heparinised saline (5000 IU/ml) and the end heat sealed until use. The rabbits were allowed 7 days to recover after surgery.
Experimental Groups Sodium- and water-replete rabbits: Effect of enalaprilat on water and sodium intake and excretion. The effect ofenalaprilat
in sodium-deplete rabbits (n 7), which received furosemide. 20 mg/kg, intravenously (IV) instead of saline in the above protocol. Vehicle or enalaprilat was injected intramuscularly 24 h later in doses 80 mg/kg, 8 mg/kg, and 8 ug/kg. The two lower doses of enalaprilat were selected as they stimulated water intake in the experiment above, and the highest dose was chosen on the basis of reported effects of enalaprilat or captopril on the sodium intake of rats (25,36). Blood samples for plasma creatinine concentration were collected from 10 rabbits at the end of the control period and 1 day after enalaprilat treatment. Additionally, sequential samples were collected 24 h after furosemide, 5 h after enalaprilat, and daily for 3 more consecutive days from 5 of the 10 animals. Plasma creatinine concentration was measured by Jaffa's method with a Technicon autoanalyser. In another series of experiments the effect of ANG II on sodium-deplete, enalaprilat-treated rabbits was studied in 13 animals. They were given IM the two higher enalaprilat doses used in the experiment above: 80 mg/kg (n = 6) or 8 mg/kg (n = 7), following furosemide treatment, as above. On a second occasion, at least I week later, ANG II (Peninsula Laboratories lnc), 1 mg/kg, was injected subcutaneously (SC) 10 min before the injection of the same dose of enalaprilat. Intakes of water. 0.5 M NaCI, and food were measured as above.
IQtter-deprived rabbits: EIC/~'ct ~?/ enalaprilat on water and sodium intake and e~cretion. The effect of enalaprilat was tested on eight rabbits following removal of water for 24 h. In the morning of the first experimental day, a blood sample was taken from the marginal ear vein and water was removed. Twentyfour hours later another blood sample was taken and vehicle or enalaprilat, 80 or 8 mg/kg, was injected 1M. Water was returned 1 h later, and intake was recorded following 1, 2. and 4 h.
Sodium- and water-replete rabbits. Effect ojenalaprilat on thepressor action q/ANG I. On the day of experiment a polythene tube (P47), protected in a counterbalanced flexible wire spring (31), was fixed to the hypodermic tube on the skull and held in place by a luer-lock fitting. The catheter was led out of the cage via a swivel (Harvard Apparatus Ltd.) to a pressure transducer (Statham), and MAP and heart rate (HR) were recorded on a Gould recorder. The cannula was filled with heparinised saline (50 IU/ml). The time course of the effect of enalaprilat was examined by measuring the pressor response to ANG I, 3 #g, at set times after IM administration of enalaprilat. Two control responses to ANG I injected into the arterial cannula (IA) were obtained in two groups of five wild rabbits prior to injection of enalaprilat, 80 or 0.8 mg/kg, IM. The responses to ANG I were measured up
CONVERTING ENZYME INHIBITION IN RABBITS
293
to 6 h after the lower dose of enalaprilat and for 4 days after the higher dose. The responses to ANG I are presented as a percentage of the mean of the control responses.
Sodium-deplete rabbits: Effect of enalaprilat on blood pressure. The effect of enalaprilat on MAP was examined in two groups of five Na-deplete rabbits 24 h after furosemide. After a 30-rain control period, furosemide, 20 mg/kg, was injected via the arterial cannula and MAP was measured for 5 h. Enalaprilat, 80 or 0.8 mg/kg, IM, was given 24 h later, and MAP was monitored up to 30 h after the lower dose of enalaprilat and up to 78 h after the higher dose.
Statistical Analysis Statistical analysis of daily intakes, excretions, MAP, and plasma creatinine concentration was performed by analysis of variance, repeated measures design, with subsequent NewmanKeuls' tests for multiple comparisons (3). The effect of ANG II treatment on the sodium and water intake/excretion of enalaprilat-treated, sodium-deplete rabbits was evaluated by paired t-test. Values are presented as mean _+ SEM. RESULTS
Sodium- and Water-Replete Rabbits: Effect of Enalaprilat on Water and Sodium Intake and Excretion Injection of saline, 0.1 ml/kg IM, did not influence daily water and sodium intake and excretion in rabbits (data not presented). After injection of enalaprilat, 8 mg/kg, 80 #g/kg, or 8 ug/kg, 24 h after IV injection of saline, daily intake of water was significantly higher following injections of enalaprilat, 8 mg/kg and 8 ug/kg, than the mean intake during the preceding control days, F(3, 15) = 10.49, p < 0.05 (Table 1). Daily intake of 0.5 M NaC1 and sodium and potassium excretion were not different from control after injection ofenalaprilat (Table 1). Enalaprilat, at these doses, did not influence daily food intake; the mean value for the control days preceding the experiments was 60 _+ 3.1 g, and ingestion was not different from this after any dose ofenalaprilat, F(3, 15) = 2.63, p > 0.05.
Sodium-Deplete Rabbits: Effect of Enalaprilat on Water and Sodium Intake and Excretion Furosemide induced similar sodium, potassium, and volume losses before enalaprilat treatment in the seven animals on all four occasions: the range of mean sodium loss was 4.8-6.1 mmol, potassium loss 1.9-3.8 mmol, and volume loss 42.0-51.6 ml.
During the 22 h after furosemide treatment, before injection of enalaprilat, rabbits drank significantly more water than during the preceding control days: intake ranged from 95 to 128 ml, whereas mean intake during the control days was 77.3 _ 4.4, F(4, 24) = 10.4, p < 0.05. This range of sodium, potassium, and volume loss, and water intake corresponds to our earlier experience with furosemide-treated rabbits (8). On the next day, after injection of enalaprilat or vehicle, consumption of water was observed during the first hour after treatment with enalaprilat, 80 mg/kg. Rabbits so treated drank 18.6 _+ 16.9 ml within the hour, and five out of the seven animals consumed some water. With the other doses, no water was drunk during the same period; therefore, statistical comparison could not be made. However, during the 24 h following enalaprilat treatment rabbits drank amounts of water not different from the mean of control days, F(4, 24) = 2.93, p > 0.05, or following injection of vehicle, F(3, 18) = 0.81, p > 0.05) (Fig. 1, bottom panel). When 0.5 M NaCI was offered 25 h after injection of furosemide, 1 h after injection ofenalaprilat or vehicle, rabbits commenced consumption on presentation at each occasion. During the next 23 h rabbits drank less 0.5 M NaCI after enalaprilat than vehicle treatment, F(3, 18) = 4.93, p < 0.05 (Fig. 1, top panel). The mortality rate of wild rabbits following combined furosemide and enalaprilat treatment was high: 24 percent (11/ 46) in all the experiments where this treatment combination was used. Similar experiments carried out in laboratory rabbits, using furosemide, 20 mg/kg IM, followed by SQ 14,225, 8 mg/rabbit intraperitoneally (about 2.5 mg/kg), resulted in a 83 percent (5/ 6) mortality rate. In contrast, no experimental mortality was observed after administration of enalaprilat to sodium-replete rabbits. In the present report, data from rabbits that completed the studies and survived the combined furosemide-enalaprilat treatment by at least 3 days, at which time they consumed food and water in close to control quantities, were included. Plasma creatinine concentration did not change significantly in the blood samples taken 24 h after the administration ofenalaprilat, 80 mg/kg, 48 h after furosemide treatment: control 104 + 5 ~ M a n d after enalaprilat 152 + 28 #M (NS, n = 10). In 5 of the 10 rabbits an increase of greater than 30 #M was observed between the two time intervals. Serial samples were taken from five rabbits daily before and after furosemide and enalaprilat, 80 mg/kg, treatment. Plasma creatinine concentration tended to rise from the control 97 + 6 #M to a maximum of 253 _+ 80 tsM 2 days after the furosemide and 1 day after the enalaprilat treatment, and decreased to 142 + 23 u M 2 days
TABLE 1 EFFECT OF ENALAPRILAT ON WILD RABBITS
Water intake (ml/day) 0.5 M NaCI intake (ml/day) Urine volume (ml/day) UN,tV (mmol/day) UKtV (mmol/day)
Control*
8 ug/kg
80 #g/kg
8 mg/kg
68.4 + 7.0 4.31 _+2.31 32.3 + 4.9 1.93 _+0.87 13.3 _+ 1.32
82.2 _+5.6t 2.35 _+ 1.24 29.5 + 2.7 1.27 + 0.37 12.06 _+2.78
70.3 _+4.0 5.38 + 3.80 29.7 + 3.8 3.21 _+ 1.66 11.71 _+9.44
83.7 _+6.It 0.69 + 0.27 29.5 + 1.9 1.25 _+0.28 11.89 _+0.86
n=6.
* Mean of the 3 days preceding administration of saline 24 h before injection of enalaprilat. t P < 0.05, ANOVA, repeated measures design, with subsequent Neuman-Keul's test.
294
[ , \ R . I k N t!1 \1 Intake of 0.5M NaCI solution after furosemide and enalaprilat in wild rabbits (n=7)
20
t
i
'
J i
ANOVA
!
p<005
I
f
0.5M I NaCI consumed 10 ml/24h
~
iiiii: iiii!i ii!
";:;;:211::;2;2::
Enalaprilat mg/kg
0
0.008
80
8
Water intake after furosemide and enalaprilat in wild rabbits (n=7). ANOVA
NS
120100I
Water
80-
consumed ml/24h
604020"
Enalaprilat mg/kg
0
0.008
8
80
FIG. 1. (Top panel) Intake of 0.5 M NaCI solution by wild rabbits during the 23 h after enalaprilat treatment. The animals received furosemide, 20 mg/kg, 24 h before the injection of enalaprilat, 0.008, 8, and 80 mg/kg, or saline, and the NaC1 solution was withheld until 1 h after enalaprilat. Values are mean _+SEM, ANOVA, F(3, 18) = 4.93,p < 0.05. (Bottom panel) Intake of water during the 23 h after enalaprilat treatment. The animals received furosemide, 20 mg/kg, 24 h before the injection of enalaprilat, 0.008, 8, or 80 mg/kg, or saline; water was available from 2 h after furosemide. Values are mean _+SEM, ANOVA, F(3, 18) = 0.80, p > 0.05• later. Although the post hoc test showed significant difference between the control and the highest value, the overall change was not statistically significant, F(5, 20) = 3.29, p > 0.05 (Fig. 2). The effect o f A N G II, injected SC l0 min before enalaprilat, 22 h after injection of furosemide, on water and sodium intake was compared with the intakes of the same rabbits after enalaprilat and furosemide treatment. A N G II, given to rabbits before injection of enalaprilat, 80 mg/kg, significantly increased the intake of 0.5 M NaCl above the suppressed level that followed treatment with enalaprilat alone (Fig. 3, top panel). A N G II did not influence sodium intake after enalaprilat, 8 mg/kg. Water
intake was not different between A N G lI-treated and untreated conditions (Fig. 3, bottom panel).
Water-Deprived Rabbits: Effect of Enalaprilat on Water and Sodium Intake and Excretion Withdrawal of water for 24 h was accompanied by a rise in plasma sodium concentration from 141.7 + 0.6 to 149.2 _+ 0.9 mM, (p < 0.05), although plasma potassium and protein concentration and osmolality did not change. During water deprivation, a reduction in urine volume was noted and food intake and daily sodium balance was negative, which is similar to our
C O N V E R T I N G E N Z Y M E INHIBITION IN RABBITS
300 -
295 but there was a significant reduction after 4 and 5 h (p < 0.05) (Fig. 6). The higher dose of enalaprilat, 80 mg/kg, given 24 h after furosemide, reduced MAP from 81 -_. 5 m m H g to 64 + 6 m m H g (p < 0.05) after 10 min, and to a m i n i m u m of 51 + 5 m m H g after 6 h. MAP remained significantly below control for 24 h, F(13, 52) --- 7.37, p < 0.05 (Fig. 6). In another group of five wild rabbits the control MAP was 94 __- 3 (mean of five readings at 5-min intervals), and administration of furosemide, 20 mg/kg IA, did not significantly alter MAP. At 24 h after furosemide, MAP was 95 -+ 5 m m H g (mean
. p
Enalaprilat
.
/
pmol 200"
Furosemide +
m
100"
1
2
3
4
I1 5 days
FIG. 2. Plasma concentration of creatinine before and after sodium depletion with furosemide, 20 mg/kg, and additional treatment with enalaprilat, 80 mg/kg. Values are mean _+SEM, ANOVA, F(5, 20) = 3.29, p > 0.05, with Newman-Keuls' test.
previous experience (30). Before the administration of vehicle or enalaprilat, 80 or 8 mg/kg, the rabbits were in a similar state of water and sodium deficit. Upon presentation of water, 1 h after injection of vehicle or enalaprilat, 80 or 8 mg/kg, the rabbits drank eagerly (Fig. 4). The cumulative intake of water was significantly lower following injection of enalaprilat, 8 mg/kg, during the second and fourth hour of access, compared with that following injection of vehicle or enalaprilat, 80 mg/kg (Fig. 4). There was, however, no difference in the amounts of water consumed during the first hour of access or during the 23 h after the presentation of water. During the 23 h after treatment, food intake, urine volume, and body weight were not different from control (Table 2). A reduction in sodium excretion was observed in the vehicle-treated rabbits, but not following enalaprilat treatment. On the other hand, enalaprilat-treated rabbits had a lower potassium excretion during the same period.
Sodium- and Water-Replete Rabbits: Effect of Enalaprilat on the Pressor Action of ANG I In five rabbits IA A N G I, 3 ~zg, increased MAP by 55.6 + 6.4 mmHg. The higher dose of enalaprilat, 80 mg/kg, reduced the ANG I-induced pressor response to 2 ___2% of the control response within 15 min (p < 0.05) and it remained suppressed at 17 + 11% of control after 8 h, F(14, 56) = 21.4, p < 0.05. At 48 h after enalaprilat, the response to A N G I (71 + 7%) was not significantly different from control (Fig. 5). In a second group of five rabbits, IA injection of A N G I, 3 #g, increased MAP by 62.5 + 6.9 mmHg. Following administration ofenalaprilat, 0.8 mg/kg IM, the pressor response to A N G I was reduced to 2 ___ 1% of the control value within 15 m i n (p < 0.05), and by 6 h the response to A N G I had returned to control levels, F(7, 28) = 114.6, p < 0.05 (Fig. 5).
Sodium-Deplete Rabbits: Effect of Enalaprilat on Blood Pressure In five rabbits the control MAP was 89 ___6 mmHg. There was no fall in MAP for the first 3 h after furosemide, 20 mg/kg,
* p
paired t-test
8mg/kg enelaprilet, i.m. • 80mg/kg o 8mg/kg •
1210"
a 80mg/kg
enalaprilat, i.m. plus l i n g anglotensi t 11, s.c.
Cumulative 8intake of 0.5 M 6-
NaCl
(ml)
42,
,
1
2
iiP'''~
4
6
214 hours
~ 7
80-
7
7060-
Cumulative
6
50-
intake of 40water (ml)
6
302010-
~
~,
I
2'4 hours
FIG. 3. (Top panel) Intake of 0.5 MNaCI solution by wild rabbits during the 23 h after enalaprilat treatment, or combination of enalaprilat and ANG II. The animals received furosemide, 20 mg/kg, 24 h before the injection of enalaprilat, and the NaCI solution was withheld until 1 h after enalaprilat. Values are mean _+SEM, paired t-tests. (Bottom panel) Intake of water during the 23 h after enalaprilat treatment or combination of enalaprilat and ANG II. The animals received furosemide, 20 mg/kg, 24 h before the injection of enaiaprilat, and water was available from 2 h after furosemide. Values are mean _+SEM, paired t-tests.
296
I,~,RJAN t'1 A I
I 170150-
* 0<0.05 o-o im saline H im Enalaprilat, 8mg/kg ~--r, im Enalaorilat, 80mg/kg
j/~ ,/ /
I
130-
Cumulate
110-
intake of water ml
9070-
5030|
I
I
1
2
4
/
24 hours
after presentation of water
FIG. 4. Cumulative water intake of rabbits deprived of water for the 24 h preceding injection ofenalaprilat. Water was offered 1 h after enalaprilat. Values are mean _+ SEM, ANOVA, with Newman-Keuls' test.
of four readings at 5-min intervals, not different from control). Subsequent administration of enalaprilat, 0.8 mg/kg IM, given 24 h after furosemide, significantly reduced MAP to 79 +_ 7 m m H g (p < 0.05) after 10 min and to 54 _+ 8 m m H g (p < 0.05) after 6 h. By 24 h after enalaprilat MAP (74 + 8 m m H g ) was not significantly different from control, F(20, 80) = 14.24, p < 0.05. DISCUSSION
The effect of converting enzyme inhibition on the ingestive behaviour of rabbits has not been investigated before. Comparison with other species will highlight regulatory processes that are probably present not only in rabbits but other species as well, but are expressed more in rabbits than in other species, including Sprague-Dawley rats.
In sodium- and water-replete rabbits increased water intake tbllowed the highest (8 mg/kg) and lowest (8 ug/kg) doses of enalaprilat. Over a thousand-fold dose range of enalaprilat the changes in water intake were not dose dependent. In studies conducted on rats, acute systemic administration of CEI was demonstrated to cause a dual effect on water intake: low doses (captopril < 5 mg/kg, enalapril < 1 mg/kg, ramipril .: 0.1-It.?, mg/kg) increased the water intake of satiated rats (9,11,28), en-hanced water intake following ureteric ligation (13), adrenalectomy (12), systemic injection of A N G II [(28), Ramipril only], and following peritoneal dialysis with glucose solution (4). Doms higher than those detailed above that did not influence (11,28) or reduce the water intake in satiated rats (36), attenuated water drinking following systemic injection of A N G II (28) or ICV injection o f A N G II (14). The increased intakes in rats [bllowing low doses of CEl was attributed to high circulating ANG l concentration, from which A N G II is produced in brain areas accessible to circulating A N G I, namely in the circumventricular organs, by the local, not yet blocked, converting enzyme (20). The high dose of CEl was thought to inhibit drinking by reducing the availability o r A N G ll in the brain (13) as converting enzyme activity and local production o f A N G II decreased in the brain (30). The dichotomy in the drinking behaviour of rats, increased intake following low dose of CEI, and reduced consumption after high dose of CEl, may be explained by the above hypotheses. In replete rabbits, however, no such dichotomy was observed, which could be due to inadequate doses used or to involvement of different mechanisms. A N G I plasma concentration probably rises in replete rabbits alter inhibition of converting enzyme, as reported in dogs (26) and humans (5). The increased water intake by replete rabbits after enalaprilat, however, could not be attributed to the increased A N G I concentration, because in our previous experiments injection of renin or A N G II did not induce water intake in rabbits (31). Although enalaprilal influences other systems, e.g., bradykinin or endorphins [cf., (5)], there are no data 1o suggest a role for these systems in the regulation of ingestive behaviour of rabbits. It is, however, possible that in rabbits one of the effects of increased plasma A N G II concentration is inhibition of water intake, and this inhibition is released by enalaprilat. If this hypothetical mechanism, which inhibited water drinking when circulating A N G lI concentration increased, existed, its receptors could be in brain areas devoid of blood-brain
TABLE 2 EFFECT OF ENALAPRILAT. ON WILD RABBITS Following Water Deprivation (24 h)
Water intake (ml/day) Urine volume (ml/day) Body weight (g) UNatV (mmol/day) UKtV (mmol/day) //=8.
Control*
Saline
8 mg/kg
80 mg/kg
102.4 _+ 25.1 52.1 _+ 13.6 1684 _+ 123 1.63 +_ 0.31 14.04 _+ 2.09
168.0 _+ 17.8t 45.6 _+ 1 t.7 1681 _+ 121 0.59 +_ 0.22t 11.27 _+ 1.62
159.0 _+ 13.7+ 56.0 _+ 11.5 1678 +_ 122 1.02 + 0.18 9.23 _+ 1.15t
158.6 + 22.6t 48.5 _+ 6.8 1693 _+ 128 1.25 +_ 0.34 8.87 _+ 2.15t
* Mean of the 3 days preceding water deprivation for 24 h. Values for the day of water deprivation are not shown. Saline or enalaprilat, 8 or 80 mg/h was injected 1 h before presentation of water, following 24 h of water deprivation. t P < 0.05 compared to control, ANOVA, repeated measures design, with subsequent Neuman-Keul's test.
CONVERTING ENZYME INHIBITION IN RABBITS
297
120
2 °o 1 0 0
8O
._c
6O *
C ¢1
o
10 ¢b 0
7"
40-
"10
•-¢ 2 0 I
<~
II -20
1.25
0.5
1
2
3
4
6
8
24
30
48
54
72
78
Time after Enalaprilat (hour)
FIG. 5. Percent reduction of the pressor effect of ANG l, 3 #g, in Na-replete rabbits following injection of enalaprilat 80 mg/kg (filled bars) or 0.8 mg/kg (empty bars). Values are mean _+SEM, ANOVA, with Newman-Keuls' test. *p < 0.05.
barrier, such as the subfornical organ, organum vasculosum laminae terminalis, or area postrema. A further support to the existence of this inhibitory system can be found in sodium-deplete rabbits, where a very high dose of enalaprilat stimulated water intake. This very high dose of enalaprilat nearly totally blocked the pressor action of ANG I, indicating a very low converting enzyme activity in the areas where enalaprilat gets access to, including the circumventricular organs. In rabbits deprived of water for 24 h the very high dose of enalaprilat did not influence water intake, although a somewhat lower dose, 8 mg/kg, caused a temporary reduction in water drinking. This result would also fit into the hypothesis outlined above. In water-deprived rabbits it is possible that converting enzyme blockade with the lower dose of enalaprilat was insufficient to counteract the inhibitory effect of the higher ANG I concentration after CEI on the increased number of ANG II binding sites in the circumventricular organs, which have been demonstrated in rats after water deprivation (18,20). The higher dose of enalaprilat would block conversion close to these binding sites and thereby disable the inhibitory effect o f A N G II binding on water drinking in rabbits. In rats, a small reduction in waterdeprivation-induced water intake was observed (2) following systemic administration of captopril or enalapril in doses similar to those used in the present studies in rabbits. Bamey et al. (2) interpreted this finding as a demonstration of the involvement of ANG II in water-deprivation-induced drinking in rats. The present experiments support our previous finding that ANG II is involved in the generation of sodium appetite in rabbits (8,31). The sodium intake of sodium-deplete rabbits was significantly inhibited by the highest dose of enalaprilat; lower doses also tended to reduce sodium intake at all time intervals. The specific effect of enalaprilat on sodium intake is supported by the observation that systemic injection of ANG II raised the sodium intake of sodium-deplete rabbits, attenuated by enala-
prilat. In sodium-deplete rabbits the inhibition of converting enzyme lasted long enough after single injection to influence daily sodium intake. The dose-dependent reduction of sodium intake of sodium-deplete rabbits contrasts sharply with the findings by others in rats. In sodium-deplete rats CEI had a dosedependent dual effect: low doses enhanced and high doses inhibited sodium intake. In detail: the sodium intake of rats sodium deplete after injection of furosemide (25,36), or peritoneal dialysis with glucose solution (4), was enhanced by low doses of CEI, although higher doses inhibited furosemide-induced sodium intake (36), though further enhancement was noted after peritoneal dialysis (4). In rabbits there was no evidence for stimulation at any dose levels indicating, perhaps, that the sodium appetite following furosemide treatment is fully expressed in rabbits and cannot be enhanced, as in rats, by other mechanisms, e.g., a rise in plasma ANG I concentration following CEI treatment. Systemic injection of ANG II enhanced the Na appetite of rabbits reduced by enalaprilat, as was found in sheep (35), rats (36), and mice (34). As systemically administered ANG II is thought not to cross the blood-brain barrier, the restoration of the depressed sodium intake of Na-deplete rabbits by ANG II suggests that the active site is in the circumventricular organs. Alternately, ANG II may cross the blood-brain barrier in sufficient quantities to be effective at ANG II binding sites that have been demonstrated in other areas of the rabbit brain (22). Urinary sodium and potassium excretion in the main depended on the experimental condition and the amount of sodium or water ingested, and was not influenced by enalaprilat treatment. The present findings demonstrate that in conscious, sodiumdeplete wild rabbits converting enzyme inhibition resulted in a large, sustained fall in blood pressure. Similar effects of converting enzyme inhibition have been reported in sodium-deplete laboratory rabbits (24), and in sodium-deplete rats (23). In sodium-
298
rla. R.IAN [! I \1
FRUSEMIDE
ENALAPRILAT (80mg/kg)
(20mg/kg)
-IE
[
8o_
1
70a. < 60-
50-
40 , , , -24-23-22-21-20-19
I
I
//
r 0
I 1
I 2
TIME
I 3
4
, 5
f 6
//
I 24
//
418
// 712
(hour)
FIG. 6. Mean arterial blood pressure of rabbits after injection of furosemide, 20 mg/kg, and 24 h later enalaprilat, 80 mg/kg. Values are mean _+ SEM, ANOVA, F(13, 52) = 7.37, p < 0.05, with Newman-Keuls' test (+p < 0.05 vs. -24 h, *p < 0.05 vs. 0 h).
restricted humans, chronic treatment with enalapril resulted in a moderate fall in blood pressure (33). The smaller fall in pressure in humans may reflect a larger degree of sodium depletion in the studies with rabbits and rats, resulting in their increased dependence on A N G II to maintain peripheral resistance. The large fall in pressure following enalapril reduced renal function, as evidenced by the increase in plasma creatinine, and in extreme cases, this may have caused renal failure and account for the mortality that was observed. Angiotensin-mediated efferent arteriolar constriction normally plays a major role in the autoregulation ofglomerular filtration rate, which is of particular importance when perfusion pressure is reduced (17,27). Pharmacological blockade of this system reduced renal function in sodium-deplete patients and in those with renal artery stenosis, thus warranting cautious use of this class of drugs in these situations (1,29). In summary, inhibition of converting enzyme by enalaprilat revealed a role for A N G II in the regulation of water intake in
rabbits that was difficult to demonstrate in earlier studies by administration of A N G II. The previously uncovered role of A N G II in the regulation of sodium intake of rabbits was confirmed by the use ofenalaprilat. The effect ofenalaprilat on both water and sodium intake of rabbits differs from that of rats: the opposite effects following low and high doses of CEI, reported in rats, were not demonstrated in rabbits. The rabbits, similar to hypertensive patients, proved sensitive to the renal complications of combined sodium depletion and converting enzyme inhibition. ACKNOWLEDGEMENTS This work was supported by the National Health and Medical Research Council of Australia and the Victorian Health Promotion Foundation. The valuable discussions with Professor Derek A. Denton and Dr. John R. Blair-West during the preparation of the manuscript are gratefully acknowledged.
REFERENCES 1. Andreucci, V. E.; Conte, G.; Dal Canton, A.; Di Minno G.; Usberi, M. The causal role of salt depletion in acute renal failure due to captopril in hypertensive patients with a single functioning kidney and renal artery stenosis. Ren. Fail. 10:9-20; 1987. 2. Barney, C. C.; Katovich, M. J.; Fregly, M. J. The effect of acute administration of an angiotensin converting enzyme inhibitor, captopril (SQ 14,225), on experimentally included thirst in rats. J Pharmacol. Exp. Ther. 212:53-57; 1980. 3. Bruning, J. L.; Kintz, B. L. Computational handbook of statistics. Glenview, IL.: Scott, Foresman and Co.; 1977.
4. Buggy, J.; Jonklass, J. Sodium appetite decreased by central angiotensin blockade. Physiol. Behav. 32:749-753; 1984. 5. Cushman, D. W.; Ondetti, M. A. lnhibitors ofangiotensin-converting enzyme for treatment of hypertension. Biochem. Pharmacol. 29: 1871-1877; 1980. 6. Denton, D. A. The hunger for salt--An anthropological, physiological and medical analysis. Berlin: Springer; 1982. 7. Denton, D. A.; Blair-West, J. R.; McBurnie, M. I.; Osborne, P. G.; Tarjan, E.; Williams, R. M.; Weisinger, R. S. Angiotensin and salt appetite of BALB/c mice. Am. J. Physiol. 259:R729-R735; 1990.
C O N V E R T I N G E N Z Y M E I N H I B I T I O N IN RABBITS 8. Denton, D. A.; Nelson, J. F.; Tarjan, E. Water and salt intake of wild rabbits (Oryctolagus cuniculus (L)) following dipsogenic stimuli. J. Physiol. (Lond.) 362:285-301; 1985. 9. DiNicolantonio, R.; Weisinger, R. S. Feeding and drinking behaviour following angiotensin converting enzyme blockade: Role of injectant pH. Pharmacol. Biochem. Behav. 29:547-551; 1988. 10. Dollery, C. T. Safety and et~cacy of enalapril. Summing up the evidence. J. Hypertens. l(Suppl 1):155-157; 1983. 11. Elfont, R. M.; Epstein, A. N.; Fitzsimons, J. T. Involvement of the renin-angiotensin system in captopril-induced sodium appetite in the rat. J. Physiol. (Lond.) 354:11-27; 1984. 12. Elfont, R. M.; Fitzsimons, J. T. The role ofangiotensin in increased sodium appetite after adrenalectomy. J. Physiol. (Lond.) 320:70P; 1981. 13. Elfont, R. M.; Fitzsimons, J. T. Renin dependence of captoprilinduced drinking after ureteric ligation in the rat. J. Physiol. (Lond.) 343:17-30; 1983. 14. Evered, M. D.; Robinson, M. M. Increased or decreased thirst caused by inhibition of angiotensin-converting enzyme in the rat. J. Physiol. (Lond.) 348:573-588; 1984. 15. Findlay, A. R. L.; Epstein, A. N. Increased sodium intake is somehow induced in rats by intravenous angiotensin II. Horm. Behav. 14:8692; 1980. 16. Fink, G. D.; Bryan, W. G.; Mokler, D. J. Effects of chronic intracerebroventricular infusion of angiotensin II on arterial pressure and fluid homeostasis. Hypertension 4:312-319; 1982. 17. Hall, J. E. Control of sodium excretion by angiotensin II: Intrarenal mechanisms and blood pressure regulation. Am. J. Physiol. 250: R960-R972; 1986. 18. Israel, A.; Saavedra, J. M.; Plunkett, L. Water deprivation upregulates angiotensin I1 receptors in rat anterior pituitary. Am. J. Physiol. 248:E264-E267; 1985. 19. Johnston, C. 1.; Cubela, R.; Jackson, B. Relative inhibitory potency and plasma drug levels of angiotensin converting enzyme inhibitors in the rat. Clin. Exp. Pharmacol. Physiol. 15:123-129; 1988. 20. Lehr, D.; Goldman, H. W.; Casner, P. Renin-angiotensin role in thirst: Paradoxical enhancement of drinking by angiotensin converting enzyme inhibitor. Science 182:1031-1032; 1973. 21. Mendelsohn, F. A. O.; Aguilera, G.; Saavedra, J. M.; Quirion, R.; Catt, K. J. Characteristics and regulation of angiotensin II receptors in pituitary, circumventricular organs and kidney. Clin. Exp. Hypertens. A5:1081-1097; 1983. 22. Mendelsohn, F. A. O.; Allen, A. M.; Clevers, J.; Denton, D. A.; Tarjan, E.; McKinley, M. J. Localization of angiotensin 11 receptor binding in rabbit brain by in vitro autoradiography. J. Comp. Neurol. 270:372-384; 1988. 23. Mento, P. F.; Wilkes, B. M. Azotemia during chronic converting enzyme inhibition with enalapril in sodium-depleted rats: Role of renal circulatory changes. J. Cardiovasc. Pharmacol. 8:670-675; 1986.
299 24. Mimran, A.; Guiod, L.; Hollenberg, N. K. The role of angiotensin in the cardiovascular and renal response to salt restriction. Kidney Int. 5:348-355; 1974. 25. Moe, K. E.; Weiss, M. L.; Epstein, A. N. Sodium appetite during captopril blockade of endogenous angiotensin II formation. Am. J. Physiol. 247:R356-R365; 1984. 26. Morton, J. J.; Tree, M.; Casals-Stenzel, J. The effect ofcaptopril on blood pressure and angiotensins 1, II and III in sodium-depleted dogs: Problems associated with the measurement of angiotensin I1 after inhibition of converting enzyme. Clin. Sci. 58:445-450; 1980. 27. Rosivall, L.; Navar, L. G. Effects on renal hemodynamics ofintraarterial infusions of angiotensins I and II. Am. J. Physiol. 245:FI81F187; 1983. 28. Rowland, N. E.; Fregly, M. J. Comparison of the effects of the dipiptidyl peptidase inhibitors captopril, ramipril, and enalapril on water intake and sodium appetite of Spague-Dawley rats. Behav. Neurosci. 102:953-960; 1988. 29. Speirs, C. J.; Dollery, C. T.; Inman, W. H.; Rawson, N. S.; Wilton, L. V. Postmarketing surveillance of enalapril. II: Investigations of the potential role ofenalapril in deaths with renal failure. Brit. Med. J. 297:830-832; 1988. 30. Tarjan, E.; Denton, D. A.; Weisinger, R. S. Atrial natriuretic peptide inhibits water and sodium intake in rabbits. Regul. Pept. 23:63-75; 1988. 31. Tarjan, E.; Denton, D. A.; McBurnie, M. I.; Weisinger, R. S. Water and sodium intake of wild and New Zealand rabbits following angiotensin. Peptides 9:677-679; 1988. 32. Unger, T.; Ganten, D.; Lang, R. E. Effect of converting enzyme inhibitors on tissue converting enzyme and angiotensin II: Therapeutic implications. Am. J. Cardiol. 59:I8D-22D; 1987. 33. Volpe, M.; Lembo, G.; Morganti, A.; Condorelli, M.; Trimarco, B. Contribution of the renin-angiotensin system and of the sympathetic nervous system to blood pressure homeostasis during chronic restriction of sodium intake. Am. J. Hypertens. 1:353-358; 1988. 34. Weisinger, R. S.; Blair-West, J. R.; Denton, D. A.; McBurnie, M.; Ong, F.; Tarjan, E.; Williams, R. M. Effect of angiotensin-converting enzyme inhibitor on salt appetite and thirst of BALB/c mice. Am. J. Physiol. 259:R736-R740; 1990. 35. Weisinger, R. S.; Denton, D. A.; Di Nicolantonio, R.; McKinley, M. J.; Muller, A. F.; Tarjan, E. Role ofangiotensin in sodium appetite of sodium-deplete sheep. Am. J. Physiol. 253:R482-R488; 1987. 36. Weisinger, R. S.; Denton, D. A.; Di Nicolantonio, R.; McKinley, M. J. The effect of captopril or enalaprilic acid on the Na appetite of Na-deplete rats. Clin. Exp. Pharmacol. Physiol. 15:55-65; 1988. 37. Weisinger, R. S.; Denton, D. A.; McKinley, M. J.; Muller, A. F.; Tarjan, E. Angiotensin and Na appetite of sheep. Am. J. Physiol. 25 l:R690-R699; 1986. 38. Wright, J. W.; Sullivan, M. J.; Petersen, E. P.; Harding, J. W. Brain angiotensin II and Ill binding and dipsogenicity in the rabbit. Brain Res. 358:376-379; 1985.