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A pressor response to intraventricular injections of carbachol
Brain Research, 105 (1976) 157-162
157
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Short Communications
A pressor response to intraventricular injections of carbachol
W. E. HOFFMAN AND M. IAN PHILLIPS Neurobehavior Laboratory, Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242 (U.S.A.)
(Accepted December 10th, 1975)
Carbachol is a cholinergic agonist which is resistant to hydrolysis by acetylcholine esterase. It has mostly muscarinic, but some nicotonic activityL Angiotensin II is an octapeptide which is a physiologically active hormonal component of the renin-angiotensin system. Both carbachol and angiotensin II produce similar effects when injected into the brain. Carbachol produces drinking 2,a,5,9,1~,la, A D H release s,9 and natriuresisl, 9 when injected intracranially in the rat. Angiotensin II also yields drinking s,6,11,13, A D H release7,11 and natriuresis 1°. Swanson and Sharpe la have found that carbachol and angiotensin II induced drinking in the rat was obtained through mostly similar cannulae sites in the brain. In addition, intraventricular (i.v.t.) injections of angiotensin II produced a short latency pressor response 6,11. It has not been reported, however, whether carbachol produces a similar blood pressure effect. In an attempt to establish similarities and differences of the two dipsogenic agents we are carrying out a series of experiments. This paper focuses on the action of carbachol (i.v.t.) on rats in which drinking and blood pressure were recorded simultaneously. We find that carbachol produces a pressor response and this response appears to be mediated by central sympathetic stimulation and vasopressin release. The subjects were 22 male Sprague-Dawley rats (350-450 g), including 5 hypophysectomized male animals (Hormone Assay Co., Chicago). Three days before the start of the experiment each subject was implanted with a 14 mm, 22 g stainless steel cannula in the lateral ventricle. On the morning of the experiment each subject was anesthetized with ether and implanted with femoral artery and vein catheters. These catheters were led subcutaneously to the back and out through an incision where they were cemented to a wound clip. The animal was then returned to his home cage and allowed to recover from the anesthesia. Testing was begun 2 h later when the rat had fully recovered from anesthesia. A 30 g injector cannula, constructed so that the tip was flush with the implanted guide cannula, was used for i.v.t, injections. This injector cannula was connected via PE 10 tubing to a 25/A Hamilton syringe positioned close to the cage. For blood
158 pressure recording, the arterial c a t h e t e r t u b i n g was led outside the cage to a S t a t h a m P 2 3 G b b l o o d pressure transducer. H e a r t rate was m e a s u r e d with a B e c k m a n type 9857B c a r d i o t a c h o m e t e r t h a t was triggered by pressure pulses. M e a n b l o o d pressure, pulse pressure a n d h e a r t rate were all r e c o r d e d on a B e c k m a n R411 D y n o g r a p h recorder. D r i n k i n g was m e a s u r e d f r o m d r i n k i n g tubes, c a l i b r a t e d to 0.1 ml, a t t a c h e d to the h o m e cage. The chronic a n i m a l p r e p a r a t i o n a n d e x p e r i m e n t a l m e t h o d s allowed us to m a k e all the testing p r o c e d u r e s on unanesthetized, u n r e s t r a i n e d rats that were able to move freely a r o u n d their h o m e cage. N o r m a l , t h a t is n o n - h y p o p h y s e c t o m i z e d animals, were tested centrally with a dose range o f c a r b a c h o l ( c a r b a m y l c h o l i n e chloride, Sigma) from 0.025 ng to 250 ng, injected in a 5/zl artificial C S F vehicle (Elliott's B, T r a v e n o l L a b o r a t o r i e s ) . C o n t r o l 5 #1 C S F injections were also given to each animal. Blood pressure changes a n d d r i n k ing were r e c o r d e d for 30 min. A l l tests were s e p a r a t e d by 90 min. In an i n d e p e n d e n t experiment, 5 n o r m a l rats received 250 ng o f c a r b a c h o l infused intravenously to test for d r i n k i n g a n d pressor responses by this alternative route. H y p o p h y s e c t o m i z e d rats (n =: 5) were tested with a single i.v.t, dose o f 25 ng c a r b a c h o l . F o l l o w i n g c o m p l e t i o n o f the p r o t o c o l , rats were anesthetized with N e m b u t a l (0.2 ml/100 g) a n d 5/~1 methylene blue dye was injected t h r o u g h the ventricular cannulae. The chest cavity was then o p e n e d a n d the vascular system perfused with iso-
TABLE I EFFECTS OF CARBACHOL
Carbachol dose (ng)
i.v.t.
INJECTIONS
Blood pressure increase (ram Hg ) *
5 #1 CSF control 1~ 0 0,025 4 :{: 1 0.25 15 ± 3 2.5 18 ± 4 25 34±3 250 33 ± 4
P
< < < < <
Water intake (ml) *
0.01"* 0.01"* 0.01"* 0,001"* 0.001'*
0 0 0.2 -3:0.2 1.4±0.6 3.4±0.6 5.4 ~ 0.9
P
it
: 0.50** :-- 0.50** ~-. 0.05** ~. 0.01"* -: 0.001 **
12 8 10 10 12 10
Non-hypophysectomized phentolamine Pre-phentolamine 28 ± 6 Post-phentolamine 27 ± 5
4.4 ± 1.5 0.50§
t.8 ± 1.3
,: 0.05§
< 0.05§§
1.8 -L 1.5
:, 0.05§§
< 0.01§
0
> 0.05§
Hypophysectomized phentolamine Pre-phentolamine 12 i 2 Post-phentolamine 0± 0
± 0
* Values given 4- S.E.M. ** Paired t-test compared to CSF controls. § Paired t-test compared to pre-phentolamine test. §§ Unpaired t-test compared to non-hypophysectomized rats.
159 tonic saline, followed by 1 0 ~ formalin through the heart. Subjects were debrained and the brains were stored in sugar formalin until sectioned on a freezing microtome to determine that there had been ventricular access by the dye injectate. The pressor and drinking effects of i.v.t, injections of different doses of carbachol are shown in Table I. Carbachol produced dose-response related increases in both blood pressure and drinking in the animals tested (see Fig. 1). With all doses tested, pressor responses were initiated in 10-30 sec after injection and drinking occurred within 0.5-5 min. The peak pressor response was reached within 2 min with blood pressure returning to baseline levels in 5-30 min, depending on dose. The lower the dose the shorter the time to recovery. Absolute pulse pressure increased with all i.v.t. carbachol induced pressor responses but because of the capacitance of the tubing system these values were not quantitated. Heart rate changes were inversely related to the magnitude of the pressor responses observed. Fig. 2 illustrates a typical example of the recorded data. Intravenous infusions on the other hand produced no blood pressure change or
40
?
-~20
c~ 10 O
jt .025
.25
2.5
25
2.50
.025
.25
2.5
25
250
8
6
~4
2
0
,~,fo
CARBACHOL DOSE Cng.)
Fig. 1. Dose response curve for blood pressure changes and drinking to i.v.t, injections of carbachol.
160
i°2°°1 i,oo
-~ ~ i r i n k l n g 01 2,5ngCarbachoJ
! ~- 80 /
4801
360[.k~ n: 240 0
5 Time[mln.)
10
15
Fig. 2. Representative raw data of polygraph recordings of mean blood pressure (top) differentiated pulse pressure (middle) and heart rate (bottom) from a rat (No. 1072) before and after injection of 25 ng carbachol (in 5/~1) into the lateral ventricle. drinking behavior. Five animals were tested with intravenous infusions of 250 ng of carbachol and none showed responses to the cholinergic agonist. In addition we found that Nembutal anesthesia abolished the blood pressure increase to 25 ng carbachol i.v.t. Taken together these two observations indicate that the pressor effects of carbachol are of central origin and are dependent on the integrity of the central nervous system. In order to examine the role of the sympathetic nervous system in the pressor response elicited by i.v.t, carbachol, another group of 5 rats were tested for drinking and pressor responses with 25 ng of carbachol i.v.t, only. One hour later they were infused intravenously with 10 mg/kg phentolamine, which blocks sympathetic activity by antagonizing a-adrenergic receptors 4. Blood pressure decreased to an average of 75 ± 5 mm Hg with this treatment from control levels of 124 4~ 2 mm Hg. After 10 min the phentolamine infusion blood pressure had stabilized and the animals were again tested with 25 ng carbachol i.v.t. The results of this experiment are shown in Table I where it can be seen that although the drinking response to carbachol i.v.t, was decreased, the pressor response was not changed. This experiment suggests that a component other than sympathetic activation was involved in the blood pressure increase to central carbachol injections. To test for the role that pituitary hormones might play in the responses to i.v.t, carbachol, 5 hypophysectomized rats were tested. In these animals the pressor
161 and drinking responses to a 25 ng dose were decreased by 58 ~o. This reduction in the pressor response was significant (see Table I). This indicates that a humoral pressor agent released from the pituitary is one component of the blood pressure increase. It seems probable that this agent is vasopressin s. Heart rate decreases and increases in pulse pressure were also associated with the pressor response to i.v.t, carbachol in these rats. The hypophysectomized rats were then infused with 10 mg/kg phentolamine intravenously. Blood pressure decreased from 98 ± 5 mm Hg to 39 i 5 mm Hg. This treatment abolished the remaining drinking and pressor response to carbachol i.v.t. injections (Table I). Thus it appears that the remaining pressor response observed after hypophysectomy is the result of central activation of sympathetic pressor mechanisms. The results of these experiments lead us to make the following conclusions. First, carbachol injected i.v.t, in rats, but not intravenously, is capable of producing a blood pressure increase. This is in addition to the previously reported effects of drinking and A D H release2,8,5,s,9,12,1L Second, there are two components of the blood pressure increase to i.v.t carbachol infusions: one a pituitary humoral component, probably A D H release, and the second a central activation of sympathetic pressor mechanisms. Third, central carbachol injections will cause release of pressor amounts of A D H from the pituitary. At first we found it difficult to explain why non-hypophysectomized rats treated with phentolamine did not have at least a partial reduction in the pressor response seen with carbachol i.v.t, injections. We now have evidence, however, which suggests the pressor effect of A D H is potentiated by hypotensive treatments such as that used here (unpublished results). The finding of a pressor response to i.v.t, carbachol injections demonstrates that the cholinomimetic drug produces virtually identical responses to those elicited by angiotensin II when given i.v.t, in the rat. Whether both drugs act through the same mechanism or not remains to be elucidated. The pressor response with i.v.t, injections of carbachol or angiotensin 1111, however, is probably due to similarly mediated A D H release and sympathetic activity. We are grateful for the advice of A. K. Johnson and the technical assistance of Judy Phipps. This work was supported by NSF Grant BNS75-16346 to M. I. Phillips, NSF Neurobehavioral Sciences Predoctoral Training Program Grant to W. E. Hoffman and Research Scientist Development Award, 3K02-MH70983-0151 to M. I. Phillips.
1 DORN, J., ANTUNES-RODRIGUES, J., AND MCCANN, S. M., Natiuresis in the rat following intraventricular carabachol, ,4mer. J. PhysioL, 219 (1970) 1292-1298. 2 FISHER, A. E., Chemical stimulation of the brain. In J. L. McGAUGH, N. M. WEINBERGER AND R. E. WHATLAN (Eds.), Psychobiology: The Biological Basis of Behavior, Freeman, San Francisco, Calif., 1967, pp. 67-74. 3 FITZSIMONS, J. T., AND SETLER, P. E., Catecholaminergic mechanisms in angiotensin-induced drinking, J. PhysioL, 218 (1971) 43P-44P. 4 GOODMAN, L. S., AND GILMAN, A., The Pharmacological Basis of Therapeutics, MacMillan, London, 1970.
162 5 GROSSMAN,J. P., Eating or drinking elicited by direct adrenergic or chotinergic stimulation of the hypothalamus, Science, 132 0960) 301-302. 6 HOFFMAN,W. E., AND PHILLIPS, M. I., Evidence for Sarl-alaS-angiotensin crossing the blood cerebrospinal fluid barrier to antagonize central effects of angiotensin II, Brain Research, in press. 7 KEIL, L. C., SUMMY-LONG, J., AND SEVERS, W. B., Release of vasopressin by angiotensin II, Endocrinology, 96 (1975) 1063-1064. 8 KUHN, E. R., Cholinergic and adrenergic release mechanism for vasopressin in the male rat: a study with injections of neurotransmitters and blocking agents into the third ventricle, Neuroendocrinology, 16 (1974) 255-264. 9 MILLER, N. E., Chemical coding of behavior in the brain, Science, 148 0965) 328-338. 10 SEVERS,W. B., DANIELS-SEVERS,A. E., SUMMY-LONG,J., AND RADIO, G. J., Effects of centrally administered angiotensin II on salt and water excretion, Pharmacologist, 6 (1971) 242-252. 11 SEVERS,W. B., SUMMY-LONG,J., TAYLOR,J. S., AND CONNOR,J. D., A central effect of angiotensin: release of pituitary pressor material, J. Pharmacol. exp. Ther., 174 (1970) 27-34. 12 SIMPSON, J. B., AND ROUTTENaERG,A., The subfornical organ and carbachol induced drinking, Brain Research, 45 (1972) 135-152. 13 SWANSON,L. W., AND SnARPE, L. G., Centrally induced drinking: comparison of angiotensin IIand carbachol-sensitive sites in rats, Amer. J. PhysioL, 225 (1973) 566-572.
157
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Short Communications
A pressor response to intraventricular injections of carbachol
W. E. HOFFMAN AND M. IAN PHILLIPS Neurobehavior Laboratory, Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242 (U.S.A.)
(Accepted December 10th, 1975)
Carbachol is a cholinergic agonist which is resistant to hydrolysis by acetylcholine esterase. It has mostly muscarinic, but some nicotonic activityL Angiotensin II is an octapeptide which is a physiologically active hormonal component of the renin-angiotensin system. Both carbachol and angiotensin II produce similar effects when injected into the brain. Carbachol produces drinking 2,a,5,9,1~,la, A D H release s,9 and natriuresisl, 9 when injected intracranially in the rat. Angiotensin II also yields drinking s,6,11,13, A D H release7,11 and natriuresis 1°. Swanson and Sharpe la have found that carbachol and angiotensin II induced drinking in the rat was obtained through mostly similar cannulae sites in the brain. In addition, intraventricular (i.v.t.) injections of angiotensin II produced a short latency pressor response 6,11. It has not been reported, however, whether carbachol produces a similar blood pressure effect. In an attempt to establish similarities and differences of the two dipsogenic agents we are carrying out a series of experiments. This paper focuses on the action of carbachol (i.v.t.) on rats in which drinking and blood pressure were recorded simultaneously. We find that carbachol produces a pressor response and this response appears to be mediated by central sympathetic stimulation and vasopressin release. The subjects were 22 male Sprague-Dawley rats (350-450 g), including 5 hypophysectomized male animals (Hormone Assay Co., Chicago). Three days before the start of the experiment each subject was implanted with a 14 mm, 22 g stainless steel cannula in the lateral ventricle. On the morning of the experiment each subject was anesthetized with ether and implanted with femoral artery and vein catheters. These catheters were led subcutaneously to the back and out through an incision where they were cemented to a wound clip. The animal was then returned to his home cage and allowed to recover from the anesthesia. Testing was begun 2 h later when the rat had fully recovered from anesthesia. A 30 g injector cannula, constructed so that the tip was flush with the implanted guide cannula, was used for i.v.t, injections. This injector cannula was connected via PE 10 tubing to a 25/A Hamilton syringe positioned close to the cage. For blood
158 pressure recording, the arterial c a t h e t e r t u b i n g was led outside the cage to a S t a t h a m P 2 3 G b b l o o d pressure transducer. H e a r t rate was m e a s u r e d with a B e c k m a n type 9857B c a r d i o t a c h o m e t e r t h a t was triggered by pressure pulses. M e a n b l o o d pressure, pulse pressure a n d h e a r t rate were all r e c o r d e d on a B e c k m a n R411 D y n o g r a p h recorder. D r i n k i n g was m e a s u r e d f r o m d r i n k i n g tubes, c a l i b r a t e d to 0.1 ml, a t t a c h e d to the h o m e cage. The chronic a n i m a l p r e p a r a t i o n a n d e x p e r i m e n t a l m e t h o d s allowed us to m a k e all the testing p r o c e d u r e s on unanesthetized, u n r e s t r a i n e d rats that were able to move freely a r o u n d their h o m e cage. N o r m a l , t h a t is n o n - h y p o p h y s e c t o m i z e d animals, were tested centrally with a dose range o f c a r b a c h o l ( c a r b a m y l c h o l i n e chloride, Sigma) from 0.025 ng to 250 ng, injected in a 5/zl artificial C S F vehicle (Elliott's B, T r a v e n o l L a b o r a t o r i e s ) . C o n t r o l 5 #1 C S F injections were also given to each animal. Blood pressure changes a n d d r i n k ing were r e c o r d e d for 30 min. A l l tests were s e p a r a t e d by 90 min. In an i n d e p e n d e n t experiment, 5 n o r m a l rats received 250 ng o f c a r b a c h o l infused intravenously to test for d r i n k i n g a n d pressor responses by this alternative route. H y p o p h y s e c t o m i z e d rats (n =: 5) were tested with a single i.v.t, dose o f 25 ng c a r b a c h o l . F o l l o w i n g c o m p l e t i o n o f the p r o t o c o l , rats were anesthetized with N e m b u t a l (0.2 ml/100 g) a n d 5/~1 methylene blue dye was injected t h r o u g h the ventricular cannulae. The chest cavity was then o p e n e d a n d the vascular system perfused with iso-
TABLE I EFFECTS OF CARBACHOL
Carbachol dose (ng)
i.v.t.
INJECTIONS
Blood pressure increase (ram Hg ) *
5 #1 CSF control 1~ 0 0,025 4 :{: 1 0.25 15 ± 3 2.5 18 ± 4 25 34±3 250 33 ± 4
P
< < < < <
Water intake (ml) *
0.01"* 0.01"* 0.01"* 0,001"* 0.001'*
0 0 0.2 -3:0.2 1.4±0.6 3.4±0.6 5.4 ~ 0.9
P
it
: 0.50** :-- 0.50** ~-. 0.05** ~. 0.01"* -: 0.001 **
12 8 10 10 12 10
Non-hypophysectomized phentolamine Pre-phentolamine 28 ± 6 Post-phentolamine 27 ± 5
4.4 ± 1.5 0.50§
t.8 ± 1.3
,: 0.05§
< 0.05§§
1.8 -L 1.5
:, 0.05§§
< 0.01§
0
> 0.05§
Hypophysectomized phentolamine Pre-phentolamine 12 i 2 Post-phentolamine 0± 0
± 0
* Values given 4- S.E.M. ** Paired t-test compared to CSF controls. § Paired t-test compared to pre-phentolamine test. §§ Unpaired t-test compared to non-hypophysectomized rats.
159 tonic saline, followed by 1 0 ~ formalin through the heart. Subjects were debrained and the brains were stored in sugar formalin until sectioned on a freezing microtome to determine that there had been ventricular access by the dye injectate. The pressor and drinking effects of i.v.t, injections of different doses of carbachol are shown in Table I. Carbachol produced dose-response related increases in both blood pressure and drinking in the animals tested (see Fig. 1). With all doses tested, pressor responses were initiated in 10-30 sec after injection and drinking occurred within 0.5-5 min. The peak pressor response was reached within 2 min with blood pressure returning to baseline levels in 5-30 min, depending on dose. The lower the dose the shorter the time to recovery. Absolute pulse pressure increased with all i.v.t. carbachol induced pressor responses but because of the capacitance of the tubing system these values were not quantitated. Heart rate changes were inversely related to the magnitude of the pressor responses observed. Fig. 2 illustrates a typical example of the recorded data. Intravenous infusions on the other hand produced no blood pressure change or
40
?
-~20
c~ 10 O
jt .025
.25
2.5
25
2.50
.025
.25
2.5
25
250
8
6
~4
2
0
,~,fo
CARBACHOL DOSE Cng.)
Fig. 1. Dose response curve for blood pressure changes and drinking to i.v.t, injections of carbachol.
160
i°2°°1 i,oo
-~ ~ i r i n k l n g 01 2,5ngCarbachoJ
! ~- 80 /
4801
360[.k~ n: 240 0
5 Time[mln.)
10
15
Fig. 2. Representative raw data of polygraph recordings of mean blood pressure (top) differentiated pulse pressure (middle) and heart rate (bottom) from a rat (No. 1072) before and after injection of 25 ng carbachol (in 5/~1) into the lateral ventricle. drinking behavior. Five animals were tested with intravenous infusions of 250 ng of carbachol and none showed responses to the cholinergic agonist. In addition we found that Nembutal anesthesia abolished the blood pressure increase to 25 ng carbachol i.v.t. Taken together these two observations indicate that the pressor effects of carbachol are of central origin and are dependent on the integrity of the central nervous system. In order to examine the role of the sympathetic nervous system in the pressor response elicited by i.v.t, carbachol, another group of 5 rats were tested for drinking and pressor responses with 25 ng of carbachol i.v.t, only. One hour later they were infused intravenously with 10 mg/kg phentolamine, which blocks sympathetic activity by antagonizing a-adrenergic receptors 4. Blood pressure decreased to an average of 75 ± 5 mm Hg with this treatment from control levels of 124 4~ 2 mm Hg. After 10 min the phentolamine infusion blood pressure had stabilized and the animals were again tested with 25 ng carbachol i.v.t. The results of this experiment are shown in Table I where it can be seen that although the drinking response to carbachol i.v.t, was decreased, the pressor response was not changed. This experiment suggests that a component other than sympathetic activation was involved in the blood pressure increase to central carbachol injections. To test for the role that pituitary hormones might play in the responses to i.v.t, carbachol, 5 hypophysectomized rats were tested. In these animals the pressor
161 and drinking responses to a 25 ng dose were decreased by 58 ~o. This reduction in the pressor response was significant (see Table I). This indicates that a humoral pressor agent released from the pituitary is one component of the blood pressure increase. It seems probable that this agent is vasopressin s. Heart rate decreases and increases in pulse pressure were also associated with the pressor response to i.v.t, carbachol in these rats. The hypophysectomized rats were then infused with 10 mg/kg phentolamine intravenously. Blood pressure decreased from 98 ± 5 mm Hg to 39 i 5 mm Hg. This treatment abolished the remaining drinking and pressor response to carbachol i.v.t. injections (Table I). Thus it appears that the remaining pressor response observed after hypophysectomy is the result of central activation of sympathetic pressor mechanisms. The results of these experiments lead us to make the following conclusions. First, carbachol injected i.v.t, in rats, but not intravenously, is capable of producing a blood pressure increase. This is in addition to the previously reported effects of drinking and A D H release2,8,5,s,9,12,1L Second, there are two components of the blood pressure increase to i.v.t carbachol infusions: one a pituitary humoral component, probably A D H release, and the second a central activation of sympathetic pressor mechanisms. Third, central carbachol injections will cause release of pressor amounts of A D H from the pituitary. At first we found it difficult to explain why non-hypophysectomized rats treated with phentolamine did not have at least a partial reduction in the pressor response seen with carbachol i.v.t, injections. We now have evidence, however, which suggests the pressor effect of A D H is potentiated by hypotensive treatments such as that used here (unpublished results). The finding of a pressor response to i.v.t, carbachol injections demonstrates that the cholinomimetic drug produces virtually identical responses to those elicited by angiotensin II when given i.v.t, in the rat. Whether both drugs act through the same mechanism or not remains to be elucidated. The pressor response with i.v.t, injections of carbachol or angiotensin 1111, however, is probably due to similarly mediated A D H release and sympathetic activity. We are grateful for the advice of A. K. Johnson and the technical assistance of Judy Phipps. This work was supported by NSF Grant BNS75-16346 to M. I. Phillips, NSF Neurobehavioral Sciences Predoctoral Training Program Grant to W. E. Hoffman and Research Scientist Development Award, 3K02-MH70983-0151 to M. I. Phillips.
1 DORN, J., ANTUNES-RODRIGUES, J., AND MCCANN, S. M., Natiuresis in the rat following intraventricular carabachol, ,4mer. J. PhysioL, 219 (1970) 1292-1298. 2 FISHER, A. E., Chemical stimulation of the brain. In J. L. McGAUGH, N. M. WEINBERGER AND R. E. WHATLAN (Eds.), Psychobiology: The Biological Basis of Behavior, Freeman, San Francisco, Calif., 1967, pp. 67-74. 3 FITZSIMONS, J. T., AND SETLER, P. E., Catecholaminergic mechanisms in angiotensin-induced drinking, J. PhysioL, 218 (1971) 43P-44P. 4 GOODMAN, L. S., AND GILMAN, A., The Pharmacological Basis of Therapeutics, MacMillan, London, 1970.
162 5 GROSSMAN,J. P., Eating or drinking elicited by direct adrenergic or chotinergic stimulation of the hypothalamus, Science, 132 0960) 301-302. 6 HOFFMAN,W. E., AND PHILLIPS, M. I., Evidence for Sarl-alaS-angiotensin crossing the blood cerebrospinal fluid barrier to antagonize central effects of angiotensin II, Brain Research, in press. 7 KEIL, L. C., SUMMY-LONG, J., AND SEVERS, W. B., Release of vasopressin by angiotensin II, Endocrinology, 96 (1975) 1063-1064. 8 KUHN, E. R., Cholinergic and adrenergic release mechanism for vasopressin in the male rat: a study with injections of neurotransmitters and blocking agents into the third ventricle, Neuroendocrinology, 16 (1974) 255-264. 9 MILLER, N. E., Chemical coding of behavior in the brain, Science, 148 0965) 328-338. 10 SEVERS,W. B., DANIELS-SEVERS,A. E., SUMMY-LONG,J., AND RADIO, G. J., Effects of centrally administered angiotensin II on salt and water excretion, Pharmacologist, 6 (1971) 242-252. 11 SEVERS,W. B., SUMMY-LONG,J., TAYLOR,J. S., AND CONNOR,J. D., A central effect of angiotensin: release of pituitary pressor material, J. Pharmacol. exp. Ther., 174 (1970) 27-34. 12 SIMPSON, J. B., AND ROUTTENaERG,A., The subfornical organ and carbachol induced drinking, Brain Research, 45 (1972) 135-152. 13 SWANSON,L. W., AND SnARPE, L. G., Centrally induced drinking: comparison of angiotensin IIand carbachol-sensitive sites in rats, Amer. J. PhysioL, 225 (1973) 566-572.