Inhibition of drinking in water-deprived by combined central angiotensin II and cholinergic receptor blockade
rats
WILLIAM E. HOFFMAN, URSULA GANTEN, MICHAEL IAN PHILLIPS, PHILLIP G. SCHMID, PIERRE SCHELLING, AND DETLEV GANTEN Department of Pharmacology, University of Heidelberg, 6900 Heidelberg, Germany; and Cardiovascular Center, Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242 HOFFMAN, WILLIAM E., URSULA GANTEN, MICHAEL IAN PHILLIPS, PHILLIP G. SCHMID, PIERRE SCHELLING, AND DETLEV GANTEN. Inhibition of drinking in water-deprived rats by combined central angiotensin II and cholinergic receptor blockade. Am. J. Physiol. 234(l): F41-F47, 1978 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 3(l): F41F47, 1978. -The effect of blockade of central angiotensin II (AII) receptors and cholinergic receptors on thirst induced by water deprivation was studied in Sprague-Dawley rats and rats with hereditary hypothalamic diabetes insipidus (DI). Neither central AI1 nor cholinergic blockade alone affected drinking. Antagonism of both receptors simultaneously, however, significantly inhibited water intake of both SpragueDawley and DI rats. This inhibitory effect was not observed in water-deprived, nephrectomized rats. The combined antagonism on water intake was specific, since milk intake in hungry rats was not affected by simultaneous AI1 and cholinergic blockade. Isorenin concentrations in brain tissue were at control levels in water-deprived, nephrectomized, and nonnephrectomized Sprague-Dawley rats but were increased in water-deprived DI rats. The results suggest that angiotensin and cholinergic receptors in the brain have a physiological role in thirst. Thirst is maintained when either receptor is intact, but reduced when both receptors are inhibited by antagonists. They are independently capable of maintaining thirst. thirst; hereditary renin-angiotensin
hypothalamic diabetes system; nephrectomy
insipidus;
brain
iso-
SPECIFIC RECEPTORSFORANGIOTENSIN II (AII)havebeen characterized in the brain tissue (2, 10, 26). Stimulation of these receptors by low doses of AI1 results in a short latency drinking response (7, 27). There is some evidence, recently presented, that these receptors are involved in physiological thirst (22). While exogenously administered AI1 can readily be blocked by specific AI1 antagonists (16, 20), attempts to block physiological thirst challenges and endogenous angiotensin by AI1 antagonists have not been in agreement (1, 22). Drinking can also be elicited by intracranial injections of the cholinergic agonist carbachol (24, 30). Cholinergic receptors mediating short latency drinking are independent of central AI1 receptors pharmacologically (8, 17) and behaviorally (5). It was the purpose of these
experiments to investigate the possible involvement of central AI1 receptors in physiological thirst such as water deprivation and to determine whether the peptidergic (AH) and choline& thirst pathways are functionally interrelated. If the latter were the case then combined blockade of both receptors should be more effective in inhibiting thirst and the failure of previous experiments to block endogenous AII-induced thirst might be explained by auxiliary cholinergic thirst pathways. METHODS
Experimental groups. Male Sprague-Dawley (SD) rats, 250-300 g, and Battleboro rats with hereditary hypothalamic diabetes insipidus (DI), 150-200 g, were used in these experiments. The test groups were as follows. 1) Forty-eight hour water-deprived SD rats. Three separate groups were tested: one group with intraventricular (IVT) saralasin, an AI1 antagonist (Norwich Pharmaceutical) (n, 8); one group with IVT atropine sulfate (n, lo), and the third group with Saralasin and atropine infused simultaneously (n, 8). Each group served as its own control with IVT cerebrospinal fluid (CSF) infusions. 2) DI rats were water deprived for 14 h (n, 12). They were tested with combined IVT infusions of saralasin plus atropine. These animals served as their own controls with IVT CSF infusions. 3) Forty-eight hour water-deprived, 24-h nephrectomized SD rats. Four groups were tested with the following drugs: a) IVT saralasin (n, 9); b) IVT atropine sulfate (n, 8); c) saralasin plus atropine IVT (n, 17); and d) IVT CSF (n, 9). 4) Twenty-four hour food-deprived SD rats (n, 7) were tested with IVT saralasin plus atropine infusions and given milk to drink. Each animal served as its own control with IVT CSF infusions. One infusion dose of saralasin (1.4 pg/ min) and atropine (0.7 p/min) was used in all of the experiments described above. 5) Brain isorenin concentrations (iso-RC) were measured in the following experimental groups: a) control SD (n, 6); b) 48-h waterdeprived SD (n, 6); c) 24-h nephrectomized SD (control) (n, 8); d) 48-h water-deprived DI (n, 8); e) DI control (n, 5); f3 14-h water-deprived DI (n, 5); g) 2-h IVT
F41
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.059.095.115) on January 13, 2019.
F42 saralasin-infusion SD rats (n, ll), and h) Z-h IVT CSF control SD ( n , 8). Surgery. Rats receiving intracranial infusions were implanted with 14.mm, 22.gauge chronic cerebral cannulas under Narcoren anesthesia (Merieux-Rentschler GmbH) (1 .O ml/kg). The coordinates for cannula implantation with respect to bregma (flat skull) were 0.5 mm posterior, 1.O mm lateral, 6.0 mm deep from the skull. The cannulas were th us designed to end in the media 1 preoptic region .of the brain and to pass through the cerebral ventricular system. All surgery was performed at least 6 days before testing. The animals received 50,000 U penicillin i.m. following surgery. Drinking experiments. For intake experiments, all Sprague-Dawley rats were water deprived for 48 h but had food available during this period. Nonnephrectomized rats were tested under two conditions, with CSF control and drug infusions. All infusions continued for 30 min and were started 10 min before access to water was given. Water intake was meas ured at 20-min 9 1-h 90.min, and 2-h time periods. The drug infusion rates used in these experiments were: atropine, 0.7 pg/min, saralasin, 1.4 pg/min, or a combination of both. The animals were randomly selected to begin the first test with CSF vehicle or with drug infusions. For the second test in each case water deprivation was started 2 days after the end of the first test. Nephrectomized animals were water deprived for 48 h and nephrectomized for 24 h. These rats only received one test infusion. Twelve rats with diabetes insipidus (DI) were randomly tested twice after 14 h of water deprivation, once with a CSF control infusion and once with an atropine plus saralasin infusion. Water deprivation periods were separated by 3 days. All rats were tested in their home cages and during d rinking tests food was not avai lable. All in fusions were carried out i .n unanesthetized unrestrained animals with an infusion pump. The volume rate was 1.4 pl/min for intracranial infusions given through a 30-gauge infusion stylus designed to fit inside the chronically implanted guide cannula. This cannula was connected by PE-10 tubing to the infusion pump positioned outside the home cage. Food depriuation. In order to determine whether the combined IVT infusion of atropine plus saralasin would inhibit other ingestive behaviors besides drinking, seven rats were -tested after food deprivation. Each animal was first deprived of all solid food, and condensed milk diluted -1:2 with water was substituted as the diet. Each rat was regimented for 3 days to consume this diet over a l-h period each morning. Water was not withheld during this regimentation. On the fourth day these rats were tested under similar conditions as with water deprivation, with either saralasin plus atropine or CSF infusions. Milk intake was measured for the first 20 min. The following day the procedure was repeated with each rat receiving the second of the two IVT infusion treatments. Brain isorenin concentration. Brain isorenin concentration was measured according to the method of Ganten et al. (12). Briefly, the rats were decapitated by guillotine, and the specific brain regions dissected out and immediately weighed, frozen, and kept at -80°C.
HOFFMAN
ET
AL.
The tissues were then homogenized, refrozen until incubated for enzyme analysis at 37OC and pH 5.5 with excess dog plasma renin substrate (11). Angiotensin I was purified on ion excnange resin as described by Boucher et al. (4) and measured by radioimmunoassay. Cross-reactivity of the AI antibody with AII, angiotensin fragments, and saralasin was less than 0.1%. The brain regions assayed were hypothalamus, frontal cortex, and choroid plexus. Hypothalamic tissue was assayed in 14-h water-deprived DI rats and compared to DI control animals. Plasma AII, osmolality, sodium, and potassium levels were measured from samples taken by aortic puncture in separate groups of animals. These groups of animals were anesthetized with Narcoren (1.0 ml/kg). Plasma AI1 was measured by radioimmunoassay. Cross-reactivity of the AI1 antibody with AI was less than 0.1%. For brain iso-RC measurement after intracranial saralasin infusions all the animals were chronically implanted with brain cannulas as described above. These rats and their controls were unanesthetized and food and water sated at the time of testing. Following a 2-h infusion of saralasin at a rate of 7 pg/min or artificial CSF, each animal was immediately decapitated and specific brain regions dissected for later assay. All data are given as means t SE, and comparisons were made with the Student t test. RESULTS
Drinking experiments. The effect of intracranial infusions of saralasin and atropine on water intake in 48-h water-deprived nonnephrectomized Sprague-Dawley rats is shown in Fig. 1. With saralasin infusions at a rate of 1.4 pg/min there was no difference in water intake during the initial 20 min infusion period or during the 60 min period. At 90 min (70 min after the drug infusion was terminated) there was a significant increase in water intake of the saralasin-treated rats as compared to controls. With intracranial infusions of atropine at a rate of 0.7 pg/min the only difference when compared to controls was an increase in water intake at the 60.min time period. The combined infusion of atropine and saralasin produced a significant decrease in water intake in water-deprived rats during the infusion period and at the l-h interval when compared to control CSF infused animals. A similar increase in water intake to the saralasin-treated rats was observed at the 90.min interval in saralasin-plus-atropine-treated rats. A different view of the effect of the various drug treatments on drinking can be obtained if cumulative water intakes are plotted (Fig. 2). There are no obvious differences in total intake between control CSF infusions and either saralasin or atropine treatments alone. However, cumulative water intake is depressed with saralasin plus atropine infusions both during the infusion period and for 40 min after. There is then observed a recovery of intake in these rats over the next hour which leaves the cumulative water intake in the saralasin-plus-atropine-treated rats not significantly different from controls at the 120.min period. Latencies were measured in terms of the number of
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.059.095.115) on January 13, 2019.
CENTRAL
BLOCKADE
F43
OF THIRST SARALASIN
10 I
AlROPlNE
Effect of saralasin and atropine on water intake after 48 h of water deprivation. Each graph represents water-intake values with drug infusion and CSF control infusions, with each rat serving as its own control. All intracranial infusions were given at a rate of 1.4 Fl/min starting 10 min before access to water and continued for 30 min. Top: saralasin (1.4 pglmin), n, 8; middle: atropine (0.7 pg/min), n, 10; bottom: atropine (0.7 pglmin) plus saralasin (1.4 pg/min), n, 8. Vertical bars indicate +l SE. FIG.
z8,
1.
g f
6,
---
Atrapine
b ov
infusion
I 8
r
SA RAL A St N
r
l
4
.
ATROPINE -CSF
oJ/
idusion
v
20 *ptoos
A I
A 1
60
90
8 I
I 120
TIME (min)
**p 0.05). The effect of simultaneous atropine plus saralasin infusions on water deprivation thirst was similar in both DI and Sprague-Dawley rats. After 14 h of overnight water deprivation these animals drank less with saralasin plus atropine infusions as compared to CSF control infusions (Fig. 3). The difference was significant at both the 20. and 60.min intake measurements. In nephrectomized SD rats the inhibitory effect of saralasin plus atropine infusions on thirst was not present (Fig. 4). None of the drug-infusion regimes including the saralasin plus atropine treatment had any significant effect on water intake in these animals as compared to CSF infusions. Food deprivation. Intracranial infusions of atropine plus saralasin did not significantly afKect milk intake in food-deprived rats. The intake of milk at 20 min was
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.059.095.115) on January 13, 2019.
HOFFMAN
F44 19.2 t 3.0 ml with CSF control infusions and 15.2 t 3.7 ml with atropine plus saralasin treatments (n, 7, P > 0.05). Brain isorenin concentration. Forty-eight hours of water deprivation produced no significant effect in isoRC in frontal cortex, hypothalamus, or choroid plexus in Sprague-Dawley rats as compared to their control animals (Table 1). Forty-eight hours of water deprivation also had no effect on brain isorenin in 24-h nephrectomized rats. Increased hypothalamic iso-RC was measured in Sprague-Dawley rats which had been infused intracranially with 7 pg/min if saralasin for 2 h compared to CSF-infused animals. The choroid plexus and &o&al cortex in these rats were not significantly af-
CSF cant rol P n3 (1,4~g/min) + ATR (0,7pg/min 1
*---I=
#I-- -St
*= **
\
I s I
\ \
A
II 0 y I
Infusion
\
0
\** ,’ \1’0 A
0
. 0
\T
i
I
I
I
20
60
90
time (min)
3. Effect of saralasin (P 113) and atropine on water intake in 14-h water-deprived DI rats (n, 12). Each rat was given intracranial saralasin (1.4 pg/min) plus atropine (0.7 pglmin) infusion and served as its own control with CSF infusions. All infusions were given for 30 min, starting 10 min before access to water. Vertical bars indicate SE. FIG.
Water
Intake
fected. Changes in brain iso-RC were also observed after water deprivation in DI rats. Fourteen hours of water deprivation produced an average hypothalamic iso-RC of 50.89 t 3.83 rig/g per h in five DI rats as compared to a value of 32.73 t 2.98 rig/g per h in four DI controls (P < 0.01). Other brain regions were not analyzed in these rats. Water deprivation produced increases in plasma AI1 levels in Sprague-Dawley rats together with increases in plasma sodium levels (Table 2). Plasma potassium levels were decreased and plasma osmolarity was not significantly changed. In nephrectomized rats water deprivation resulted in increased plasma potassium and osmolarity. Plasma sodium was slightly but not significantly increased. Plasma AI1 levels were below those of control nonnephrectomized rats. Increased plasma sodium levels and osmolarity also marked 14 h of water deprivation in the DI rats (Table 2). High plasma AI1 levels were apparent in DI rats under control conditions and following water deprivation. DISCUSSION
pc .05 = p
rats
WILLIAM E. HOFFMAN, URSULA GANTEN, MICHAEL IAN PHILLIPS, PHILLIP G. SCHMID, PIERRE SCHELLING, AND DETLEV GANTEN Department of Pharmacology, University of Heidelberg, 6900 Heidelberg, Germany; and Cardiovascular Center, Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242 HOFFMAN, WILLIAM E., URSULA GANTEN, MICHAEL IAN PHILLIPS, PHILLIP G. SCHMID, PIERRE SCHELLING, AND DETLEV GANTEN. Inhibition of drinking in water-deprived rats by combined central angiotensin II and cholinergic receptor blockade. Am. J. Physiol. 234(l): F41-F47, 1978 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 3(l): F41F47, 1978. -The effect of blockade of central angiotensin II (AII) receptors and cholinergic receptors on thirst induced by water deprivation was studied in Sprague-Dawley rats and rats with hereditary hypothalamic diabetes insipidus (DI). Neither central AI1 nor cholinergic blockade alone affected drinking. Antagonism of both receptors simultaneously, however, significantly inhibited water intake of both SpragueDawley and DI rats. This inhibitory effect was not observed in water-deprived, nephrectomized rats. The combined antagonism on water intake was specific, since milk intake in hungry rats was not affected by simultaneous AI1 and cholinergic blockade. Isorenin concentrations in brain tissue were at control levels in water-deprived, nephrectomized, and nonnephrectomized Sprague-Dawley rats but were increased in water-deprived DI rats. The results suggest that angiotensin and cholinergic receptors in the brain have a physiological role in thirst. Thirst is maintained when either receptor is intact, but reduced when both receptors are inhibited by antagonists. They are independently capable of maintaining thirst. thirst; hereditary renin-angiotensin
hypothalamic diabetes system; nephrectomy
insipidus;
brain
iso-
SPECIFIC RECEPTORSFORANGIOTENSIN II (AII)havebeen characterized in the brain tissue (2, 10, 26). Stimulation of these receptors by low doses of AI1 results in a short latency drinking response (7, 27). There is some evidence, recently presented, that these receptors are involved in physiological thirst (22). While exogenously administered AI1 can readily be blocked by specific AI1 antagonists (16, 20), attempts to block physiological thirst challenges and endogenous angiotensin by AI1 antagonists have not been in agreement (1, 22). Drinking can also be elicited by intracranial injections of the cholinergic agonist carbachol (24, 30). Cholinergic receptors mediating short latency drinking are independent of central AI1 receptors pharmacologically (8, 17) and behaviorally (5). It was the purpose of these
experiments to investigate the possible involvement of central AI1 receptors in physiological thirst such as water deprivation and to determine whether the peptidergic (AH) and choline& thirst pathways are functionally interrelated. If the latter were the case then combined blockade of both receptors should be more effective in inhibiting thirst and the failure of previous experiments to block endogenous AII-induced thirst might be explained by auxiliary cholinergic thirst pathways. METHODS
Experimental groups. Male Sprague-Dawley (SD) rats, 250-300 g, and Battleboro rats with hereditary hypothalamic diabetes insipidus (DI), 150-200 g, were used in these experiments. The test groups were as follows. 1) Forty-eight hour water-deprived SD rats. Three separate groups were tested: one group with intraventricular (IVT) saralasin, an AI1 antagonist (Norwich Pharmaceutical) (n, 8); one group with IVT atropine sulfate (n, lo), and the third group with Saralasin and atropine infused simultaneously (n, 8). Each group served as its own control with IVT cerebrospinal fluid (CSF) infusions. 2) DI rats were water deprived for 14 h (n, 12). They were tested with combined IVT infusions of saralasin plus atropine. These animals served as their own controls with IVT CSF infusions. 3) Forty-eight hour water-deprived, 24-h nephrectomized SD rats. Four groups were tested with the following drugs: a) IVT saralasin (n, 9); b) IVT atropine sulfate (n, 8); c) saralasin plus atropine IVT (n, 17); and d) IVT CSF (n, 9). 4) Twenty-four hour food-deprived SD rats (n, 7) were tested with IVT saralasin plus atropine infusions and given milk to drink. Each animal served as its own control with IVT CSF infusions. One infusion dose of saralasin (1.4 pg/ min) and atropine (0.7 p/min) was used in all of the experiments described above. 5) Brain isorenin concentrations (iso-RC) were measured in the following experimental groups: a) control SD (n, 6); b) 48-h waterdeprived SD (n, 6); c) 24-h nephrectomized SD (control) (n, 8); d) 48-h water-deprived DI (n, 8); e) DI control (n, 5); f3 14-h water-deprived DI (n, 5); g) 2-h IVT
F41
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.059.095.115) on January 13, 2019.
F42 saralasin-infusion SD rats (n, ll), and h) Z-h IVT CSF control SD ( n , 8). Surgery. Rats receiving intracranial infusions were implanted with 14.mm, 22.gauge chronic cerebral cannulas under Narcoren anesthesia (Merieux-Rentschler GmbH) (1 .O ml/kg). The coordinates for cannula implantation with respect to bregma (flat skull) were 0.5 mm posterior, 1.O mm lateral, 6.0 mm deep from the skull. The cannulas were th us designed to end in the media 1 preoptic region .of the brain and to pass through the cerebral ventricular system. All surgery was performed at least 6 days before testing. The animals received 50,000 U penicillin i.m. following surgery. Drinking experiments. For intake experiments, all Sprague-Dawley rats were water deprived for 48 h but had food available during this period. Nonnephrectomized rats were tested under two conditions, with CSF control and drug infusions. All infusions continued for 30 min and were started 10 min before access to water was given. Water intake was meas ured at 20-min 9 1-h 90.min, and 2-h time periods. The drug infusion rates used in these experiments were: atropine, 0.7 pg/min, saralasin, 1.4 pg/min, or a combination of both. The animals were randomly selected to begin the first test with CSF vehicle or with drug infusions. For the second test in each case water deprivation was started 2 days after the end of the first test. Nephrectomized animals were water deprived for 48 h and nephrectomized for 24 h. These rats only received one test infusion. Twelve rats with diabetes insipidus (DI) were randomly tested twice after 14 h of water deprivation, once with a CSF control infusion and once with an atropine plus saralasin infusion. Water deprivation periods were separated by 3 days. All rats were tested in their home cages and during d rinking tests food was not avai lable. All in fusions were carried out i .n unanesthetized unrestrained animals with an infusion pump. The volume rate was 1.4 pl/min for intracranial infusions given through a 30-gauge infusion stylus designed to fit inside the chronically implanted guide cannula. This cannula was connected by PE-10 tubing to the infusion pump positioned outside the home cage. Food depriuation. In order to determine whether the combined IVT infusion of atropine plus saralasin would inhibit other ingestive behaviors besides drinking, seven rats were -tested after food deprivation. Each animal was first deprived of all solid food, and condensed milk diluted -1:2 with water was substituted as the diet. Each rat was regimented for 3 days to consume this diet over a l-h period each morning. Water was not withheld during this regimentation. On the fourth day these rats were tested under similar conditions as with water deprivation, with either saralasin plus atropine or CSF infusions. Milk intake was measured for the first 20 min. The following day the procedure was repeated with each rat receiving the second of the two IVT infusion treatments. Brain isorenin concentration. Brain isorenin concentration was measured according to the method of Ganten et al. (12). Briefly, the rats were decapitated by guillotine, and the specific brain regions dissected out and immediately weighed, frozen, and kept at -80°C.
HOFFMAN
ET
AL.
The tissues were then homogenized, refrozen until incubated for enzyme analysis at 37OC and pH 5.5 with excess dog plasma renin substrate (11). Angiotensin I was purified on ion excnange resin as described by Boucher et al. (4) and measured by radioimmunoassay. Cross-reactivity of the AI antibody with AII, angiotensin fragments, and saralasin was less than 0.1%. The brain regions assayed were hypothalamus, frontal cortex, and choroid plexus. Hypothalamic tissue was assayed in 14-h water-deprived DI rats and compared to DI control animals. Plasma AII, osmolality, sodium, and potassium levels were measured from samples taken by aortic puncture in separate groups of animals. These groups of animals were anesthetized with Narcoren (1.0 ml/kg). Plasma AI1 was measured by radioimmunoassay. Cross-reactivity of the AI1 antibody with AI was less than 0.1%. For brain iso-RC measurement after intracranial saralasin infusions all the animals were chronically implanted with brain cannulas as described above. These rats and their controls were unanesthetized and food and water sated at the time of testing. Following a 2-h infusion of saralasin at a rate of 7 pg/min or artificial CSF, each animal was immediately decapitated and specific brain regions dissected for later assay. All data are given as means t SE, and comparisons were made with the Student t test. RESULTS
Drinking experiments. The effect of intracranial infusions of saralasin and atropine on water intake in 48-h water-deprived nonnephrectomized Sprague-Dawley rats is shown in Fig. 1. With saralasin infusions at a rate of 1.4 pg/min there was no difference in water intake during the initial 20 min infusion period or during the 60 min period. At 90 min (70 min after the drug infusion was terminated) there was a significant increase in water intake of the saralasin-treated rats as compared to controls. With intracranial infusions of atropine at a rate of 0.7 pg/min the only difference when compared to controls was an increase in water intake at the 60.min time period. The combined infusion of atropine and saralasin produced a significant decrease in water intake in water-deprived rats during the infusion period and at the l-h interval when compared to control CSF infused animals. A similar increase in water intake to the saralasin-treated rats was observed at the 90.min interval in saralasin-plus-atropine-treated rats. A different view of the effect of the various drug treatments on drinking can be obtained if cumulative water intakes are plotted (Fig. 2). There are no obvious differences in total intake between control CSF infusions and either saralasin or atropine treatments alone. However, cumulative water intake is depressed with saralasin plus atropine infusions both during the infusion period and for 40 min after. There is then observed a recovery of intake in these rats over the next hour which leaves the cumulative water intake in the saralasin-plus-atropine-treated rats not significantly different from controls at the 120.min period. Latencies were measured in terms of the number of
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.059.095.115) on January 13, 2019.
CENTRAL
BLOCKADE
F43
OF THIRST SARALASIN
10 I
AlROPlNE
Effect of saralasin and atropine on water intake after 48 h of water deprivation. Each graph represents water-intake values with drug infusion and CSF control infusions, with each rat serving as its own control. All intracranial infusions were given at a rate of 1.4 Fl/min starting 10 min before access to water and continued for 30 min. Top: saralasin (1.4 pglmin), n, 8; middle: atropine (0.7 pg/min), n, 10; bottom: atropine (0.7 pglmin) plus saralasin (1.4 pg/min), n, 8. Vertical bars indicate +l SE. FIG.
z8,
1.
g f
6,
---
Atrapine
b ov
infusion
I 8
r
SA RAL A St N
r
l
4
.
ATROPINE -CSF
oJ/
idusion
v
20 *ptoos
A I
A 1
60
90
8 I
I 120
TIME (min)
**p 0.05). The effect of simultaneous atropine plus saralasin infusions on water deprivation thirst was similar in both DI and Sprague-Dawley rats. After 14 h of overnight water deprivation these animals drank less with saralasin plus atropine infusions as compared to CSF control infusions (Fig. 3). The difference was significant at both the 20. and 60.min intake measurements. In nephrectomized SD rats the inhibitory effect of saralasin plus atropine infusions on thirst was not present (Fig. 4). None of the drug-infusion regimes including the saralasin plus atropine treatment had any significant effect on water intake in these animals as compared to CSF infusions. Food deprivation. Intracranial infusions of atropine plus saralasin did not significantly afKect milk intake in food-deprived rats. The intake of milk at 20 min was
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.059.095.115) on January 13, 2019.
HOFFMAN
F44 19.2 t 3.0 ml with CSF control infusions and 15.2 t 3.7 ml with atropine plus saralasin treatments (n, 7, P > 0.05). Brain isorenin concentration. Forty-eight hours of water deprivation produced no significant effect in isoRC in frontal cortex, hypothalamus, or choroid plexus in Sprague-Dawley rats as compared to their control animals (Table 1). Forty-eight hours of water deprivation also had no effect on brain isorenin in 24-h nephrectomized rats. Increased hypothalamic iso-RC was measured in Sprague-Dawley rats which had been infused intracranially with 7 pg/min if saralasin for 2 h compared to CSF-infused animals. The choroid plexus and &o&al cortex in these rats were not significantly af-
CSF cant rol P n3 (1,4~g/min) + ATR (0,7pg/min 1
*---I=
#I-- -St
*= **
\
I s I
\ \
A
II 0 y I
Infusion
\
0
\** ,’ \1’0 A
0
. 0
\T
i
I
I
I
20
60
90
time (min)
3. Effect of saralasin (P 113) and atropine on water intake in 14-h water-deprived DI rats (n, 12). Each rat was given intracranial saralasin (1.4 pg/min) plus atropine (0.7 pglmin) infusion and served as its own control with CSF infusions. All infusions were given for 30 min, starting 10 min before access to water. Vertical bars indicate SE. FIG.
Water
Intake
fected. Changes in brain iso-RC were also observed after water deprivation in DI rats. Fourteen hours of water deprivation produced an average hypothalamic iso-RC of 50.89 t 3.83 rig/g per h in five DI rats as compared to a value of 32.73 t 2.98 rig/g per h in four DI controls (P < 0.01). Other brain regions were not analyzed in these rats. Water deprivation produced increases in plasma AI1 levels in Sprague-Dawley rats together with increases in plasma sodium levels (Table 2). Plasma potassium levels were decreased and plasma osmolarity was not significantly changed. In nephrectomized rats water deprivation resulted in increased plasma potassium and osmolarity. Plasma sodium was slightly but not significantly increased. Plasma AI1 levels were below those of control nonnephrectomized rats. Increased plasma sodium levels and osmolarity also marked 14 h of water deprivation in the DI rats (Table 2). High plasma AI1 levels were apparent in DI rats under control conditions and following water deprivation. DISCUSSION
pc .05 = p
