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Tetrahydroisoquinolines in vivo. I. Rat brain formation of salsolinol, a condensation product of dopamine and acetaldehyde, under certain conditions during ethanol intoxication
Pergamon Press
Life Sciences Vol. 16, pp. 573-584 Printed in the U.S.A.
PARTICIPATION OF CHOLINERGIC CIRCUITS IN RENIN INDUCED DRINKING Paul Casner, H. Warren Goldman and David lehr Department of Pharmacology New York Medical College Valhalla, New York 10595 (Received in final form January 13, 1975)
Summary Pharmacological analysis of the drinking response produced by the intraperitoneal (i .p.) injection of renin in the intact and bilaterally nephrectomized rat revealed that peripherally administered atropine sulfate ( 5 mg/kg s.c. ) produced a highly significant block of renin-induced drinking, while alpha and beta-adrenergic blockade with tolazoline (8.5 mg/kg s.c. ) and propranolol ( 6.0 mg/kg s.c. ), respectively, were without effect. In the nephrectomized rat inhibition of the synthesis of norepinephrine and dopamine with i .p., alpha methyl-p-tyrosine hod likewise no effect on renin-induced water intake, whereas injection of the dopominergic blocking agent, haloperidol ( 0.05 mg/kg s.c. ), reduced the drinking response by about one third. However, doubling the dose of haloperidol caused no further reduction. It is concluded that cholinergic pathways are importantly involved in renin-induced drinking behavior. Recent investigations of mammalian drinking behavior suggests an important role for the renin-angiotensin system in the mediation of thirst. Intravenous infusion of renin or synthetic angiotensin II have been shown to elicit copious drinking in the water-sated rat ( 1,2 ). Evidence has been presented indicating that angiotensin evokes water consumption by an action on the central nervous system (CNS) (3). Characterization of the neurotransmitters involved in the drinking behavior produced by intra-cerebral injection of angiotensin indicated cholinergic (4) anq/or dopaminergic (5,6) mediation of the central angiotensin response. Pharmacological analysis of renin-angiotensin drinking behavior has employed almost exclusively the central route of drug administration, despite the fact that it has been shown that peripheral angiotensin II can elicit a drinking response by acting at sites in the 573
Cholinergic Circuits in Angiotensin Thirst
574
Vol. 16, No. 4
CNS that are outside the blood-brain-barrier (7). The recent discovery of an independent renin-angiotensin system in the brain (8,9), whose role in drinking behavior is as yet unknown, has further complicated the study of systemic renin-angiotensin thirst. The present report was undertaken to provide a pharmacologic ana Iysis of peripherallyinduced angiotensin thirst and to use this information as a means of comparison with the pharmacology of centrallrinduced angiotensin drinking. Methods Intact Animals Male Wister albino rats weighing 240-300 grams were used as experimental animals. Individual metabolism units fitted with 100 ml graduated drinking tubes were utilized in all experiments. Food was withheld during the experimental period. Water intake was measured at 1,2, and 3 hours after drug treatment. In experiments with intact rats, 24 hour water intake was measured before and after the test in order to confirm stable baseline drinking. Bilaterally Nephrectomized Animals Male Wistar albino rats weighing 250-300 grams were used in these experiments, The testing procedure was identical to that for intact animals, except that 24 hour drinking was not recorded. Rats were bilaterally nephrectomized under ether anesthesia. The kidneys were removed through a midline abdominal incision after decapsulation leaving the adrenal glands intact. The animals were allowed a two hour recovery period before an experiment was begun, except when a-methyl-p-tyrosine was given, in which case the animals were allowed a four hour recovery period. It was found that the additional two hour recovery period did not have a significant effect on the renin-drinking response. Intraperitoneal injections were mode lateral to the incision site and absorbent paper was placed on the floor of the cage to detect possible leakage of the drug. Each nephrectomized rot was used only once for a particular experiment.
Vol. 16, No. 4
Cholinergic Circuits in Angiotensin Thirst
575
Drugs Lyophilized hog renin (Nutritional Biochemicals) was injected intraperitoneally (i .p.) in doses of 1.0, 3.0 and 6.0 units per 100 grams body weight (gbw) for intact rats and 1.0, 2.0 and 3.0 units per 100 gbw for nephrectomized rats. The dose to be injected was dissolved in 0.50 ml of isotonic saline for the nephrectomized rat or 1.0 ml of isotonic saline for the intact rat. Controls were injected i .p. with 0.50 and 1.0 ml of isotonic saline for the nephrectomized and intact rat, respectively. Pharmacologic blocking agents were atropine sulfate (5.0 mg./kg), tolazoline hydrochloride (8.5 mg./kg), propranolol hydrochloride, (6.2 mg./kg), haloperidol {0.1 mg./kg and 0.05 mg./kg) and alpha methyl-p-tyrosine methyl ester (300 mg./kg). These agents were injected subcutaneously (s,c.) in 1.0 ml of isotonic saline approximately 15 minutes before the administration of renin, except for alpha methyl-p-tyrosine which was injected i .p. immediately after nephrectomy. In this instance renin was injected after a period of four hours had elapsed. Controls in all blocking studies were injected s.c, with 1.0 ml of isotonic saline. Data were analyzed by the Student's t-test and were considered statistically significant when P was< 0.05 and highly significant when P was<. 0.001. Results are shown in the graphs as the mean± S.E. Results Intact Rat The thirst-inducing effect of renin administered i .p. to the intact, water replete rat was modest and inconsistent unless excessive dosage was employed (Figure I ) • One as well as three units per 100 gbw produced effects which were statistical! y not significant. A reliable drinking effect was observed only with the administration of 6.0 units per 100 gbw. The onset of the main water intake at this dose was delayed for about 30-40 minutes after injection with sporadic drinking continuing for approximately 3 hours. Thus, a reliable dose-response relationship could not be demonstrated at the dose range employed.
576
Cholinergic Circuits in Angiotensin Thirst
Vol. 16, No. 4
RENIN 6 0 U/toOqbw
IQO
8.0
WATER INTAKE
6.0
RENrN 3.0 u/100 ~b·
{mls} RENfN
1.0 UIIOOgbw
4.0 CONTROL
2.
12
12
18
FIGURE 1 Drinking response of the intact rat after i .p. administration of hog renin. Renin is expressed in units per 100 grams of bodyweight (gbw). Water intake was measured over a 3 hour period for this and, all subsequent figures. Figures inside bars indicate numbers of animals in each group. Only response at 6.0 u/100 gbw is significantly different from Control ( P <. 0.001). The effect of autonomic blockade on this response was studie:l with the dose of i .p. renin which was found to elicit significant water intake (6.0 units per 100 gbw). The doses of blocking agents utilized are based on a study by Lehr et al. (10) on isoproterenol induced drinking. As is shown in Figure 2, blockade of either alpha or beta-adrenergic receptors had no statistically significant effect on renin drinking. Pretreatment with atropine sulfate, however, completely abolished the dipsogenic action of renin. It is interesting to note that beta-adrenergic thirst, which has been claimed to be completely dependent on the renin-angiotensin system, (II), is obviated by propranolol yet unaffected by atropine,(IO) an observation which is the exact reverse of our findings with renin-induced thirst. Bi latera II y Nephrectomized Rat Bilateral nephrectomy greatly potentiates the effects of renin on water intake when compared to its actions in the presence of the kidneys, as first demonstrated by Fitzsimons (1). Renin in amounts of I, 2, and 3 units per 100 gbw produced highly significant and
dose related drinking responses (Figure 3). Animals injected with 3 units per 100 gbw drank over 15 ml in the three hour observation period following nephrectomy whereas the
Cholinergic Circuits in Angiotensin Thirst
Vol. 16, No. 4
intact animal, given the same dose, drank less than 4.0 ml in the same period ( see Figure I). 12.0
RENIN
10.0
6 OU/I009bw
8.0
RE~IN
INTAKE
R:;:!'JN
TOLAZOLNE 5m'l)/kQ
e
WATER
PROPRANOLOL 6.Zmqlkg
T
60
(mit)
.
40
RENIN
ATROPINE· 50 4 ~Om;lk9
CON.TROL
~0
18
20
10
FIGURE 2 The effect of autonomic blocking agents on the drinking response produced by i •P• injection of 6.0 units of renin per 100 gbw in the intact rat. Blocking agents were administered 15 min. prior to renin, Only atropine produced a significant block of the drinking produced by 6.0 u/IOOgbw of renin ( P < 0.001).
~00
RENIN 3.0 U /IOOgbw
16.0
RENIN
12.0
2.0 U/IOOQbw
WATER INTAKE
lmltl
RENIN 1.0 U/IOOgbw
8.0
4.0 CONTROL
10
10
19
FJGURE"3 Drinking response of the bilaterally nephrectomized rat after i.p. administration of hog renin. Response at all doses was statistically significant. ( P <. 0.001 ).
577
578
Cholinergic Circuits in Angiotensin Thirst
Vol. 16, No. 4
The effects of adrenergic and cholinergic blockade in this series of experiments were similar to our findings with the intact animal (Figure 4). Neither tolazoline nor propranolol interfered with the copious drinking responses produced by 3 units per [00 gbw of renin, whereas blockade of cholinergic receptors with atropine significantly antagonized renin drinking. In fact, cholinergic blockade lowered the renin-induced drinking to about 20% of the expected response.
20.0 RENIN 3.0U/100qbw
16.
WATER INTAI
.
RENIN RENt<
•
TOLAZOLIN£ 8_5mo/kg
PROPRANOLOL
6.2mgllto
12 0
{mls)
8.0
4.0
19
6
FIGURE 4 The effect of autonomic blocking agents on the drinking response produced by i.p. injection of 3.0 units per 100 gbw in the bilaterally nephrectomized rat. Blocking agents were administered 15 min. prior to renin. Only atropine produced a statistically significant reduction of the drinking induced by 3.0 u/IOOgbw of renin.( P <. 0.001). To determine the role of dopamine in peripheral renin-angiotensin drinking, o dopaminereceptor blocking agent, haloperidol (12), and an inhibitor of catecholamine synthesis alpha methyl-p-tyrosine (13) were employed. The latter agent prevents formation of norepinephrine as well as dopamine. Subcutaneous injection of 0.05 mg./kg of haloperidol reduced the drinking response elicited by 3 unit¥'100 gbw of renin by 300,1,. However doubling the dose of haloperidol did not enhance this blocking effect
( Figure 5 ).
Pretreatment of the nephrectomized rat with 300 mg./kg (i .p.) of alpha methyl-p-tyrosine had no effect on the renin drinking response.
CHolinergic Circuits in Angiotensin Thirst
Vol. 16, No. 4
579
o<.-MpT 300mg/kg
RENIN 30u/100gbw
15
~
r+
+
RENIN HALOPERIDOL 005mg/kg
HALOPERIDOL O.IOmg/kg
+
+
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. RENIN
RENIN
.,
.5
-
10 r-
w
f-
- 1-
~
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w
1
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12
12
12
12
FIGURE 5 The effect of haloperidol and alpha-methyl-p-tyrosine on the drinking response produced by i •P• administration of 3.0 units of renin per 100 gbw in the bilaterally nephrectomized rat. Haloperidol was given 15 min. prior to renin while alpho-MpT was given 4 hours before renin. Reduction of water intake by both doses of haloperidol is statistically significant compared to the drinking response produced by renin alone ( P 0.05 ).
<
Removal of the kidneys is known to significantly prolong the half-life of many agents, particularly if they are inactivated by renal biotransformation or their elimination is strongly dependent upon renal integrity. This may explain not only the far more potent effect of exogenous renin, but also the lack of difference in blocking activity observed with the high and low dose of haloperidol. Discussion Drinking behavior produced by actual components of the renin-angiotensin system should be distinguished from that produced by agents that release renin from the kidney (e.g. isoproterenol, diazoxide). Thirst produced by renin or angiotensin can be attributed to these agents alone, while the thirst produced by isoproterenol or diazoxide (reninreleasers) is not necessarily a result of their ability to release renin from the kidney. Indeed, recent studies have suggested the independence of these drinking behaviors from the renin-angiotensin system (14, 15, 16),
580
Cholinergic Circuits in Angiotensin Thirst
Vol. 16, No. 4
If exogenous renin is used to induce thirst in the rat, the drinking response is unimpressive (see Figure I ) unless the animal is bilaterally nephrectomized (I ), The increased sensitivity of the reniprival rat to renin is nlustrated in Figure 3. The reasons for the enhanced drinking response after nephrectomy are not known, although several possibilities have been suggested, such as enhanced production of substrate due to removal of an inhibitory loop (17), absence of renin degradation by the kidneys (I), or enhanced enzyme kinetics (18).
It should be pointed out that these explanations were
originally advanced as possible reasons for the increased pressor responses seen after injections of renin in the bilaterally nephrectomized rat. These explanations could apply equally well to the enhanced drinking response, Among pharmacological studies of the systemic role of the renin-angiotensin system in drinking behavior, employing renin-releasing agents { 19, 20, 21), the study by Blass and Chapman (22) seems to be the only investigation of renin-angiotensin thirst which employed the peripheral administration of an actual component of the renin-angiotensin system, These authors investigated the possible involvement of cholinergic mechanisms in thirst produced by intraperitoneal
administration of renin in the intact rat. They concluded
that cholinergic pathways were not involved in renin-angiotensin drinking behavior, since atropine sulfate produced a block only at very high concentrations {IS mgjkg). Blass and Chapman used a dose of 2.0 units of renin/100 gbw in the intact rat, which elicited a total water intake of approximately 3 mi.
It is conceivable that their failure
to observe a block with lower doses of atropine is attributable to the small baseline drinking response evoked by this dose of renin in the intact animal, Most investigators have found that drinking elicited by the injection of angiotensin into the prefrontal area of the hypothalamus could not be blocked by pretreatment of this~ site with intracerebrally administered atropine ( 5,23,24,25 ).
Vol. 16, No. 4
Cholinergic Circuits in Angiotensin Thirst
This observation was fully confirmed in our laboratory (4).
581
However, when atropine is
administered peripherally ( 4, 22,26 ), or intraventricularly { 26, unpublished observations from this laboratory ) the central angiotensin drinking response can be significantly inhibited, suggesting that the cholinergic neurons involved are not in close proximity to the angiotensin injection site. Recent evidence has pointed to dopaminergic involvement in central angiotensin drinking { 5,6 ). However, in the present study, inhibiting dopamine synthesis with alpha methyl-p-tyrosine had no effect on the renin drinking response, .while systemic haloperidol in doses which did not induce obvious sedation reduced the drinking response by about 30% suggesting a possible involvement of dopaminergic pathways in this thirst behavior, although a non-specific effect cannot be ruled out. Indeed our finding that a higher dose of haloperidol does not increase the effectiveness of the block speaks for a non-specific effect. Moreover, we have been unable to block the drinking response produced by hypothalamic injection of angiotensin with haloperidol applied at the same site, although peripheral haloperidol does block centrally-induced angiotensin drinking
( 27 ). In summary, our findings do not support substantial involvement of dopaminergic circuits in central angiotensin drinking, but are in line with earlier observations from this laboratory { 4, 28, 29 ) and those of Severs and Daniels-Severs { 26) with regard to cholinergic mediation of angiotensin induced thirst. The cholinergic block by atropine of peripherally and centrally-induced angiotensin drinking behavior indicate that when given peripherally, renin or angiotensin most likely elicit thirst by acting at sites in the CNS. These sites might well be outside the blood-brain-barrier allowing angiotensin to act centrally without crossing the ependymal layer { 7). Our results, however, do not rule out possible participation of the peripheral nervous system and its transmitters in renin-
1 • t ens1n • dnn • k.1ng be hav1or. . ang1o
582
Cholinergic Circuits in Angiotensin Thirst
Vol. 16, No. 4
In recent preliminary studies we have found that the quaternized analogue of atropine (atropine methyl nitrate ) produced a significant although less substantial block of renin-induced drinking. Whether this indicates peripheral involvement in the drinking response or simply penetration of the quoternized analogue into the CNS cannot be determined at this time since the previously accepted tenet of CNS-exclusion of this quaternized analogue hos recently been challenged ( 30, 31 ). References I.
J. T. Fitzsimons, J. Physiol., (Lond.) 201, 349-368 (1969).
2.
J. T. Rtzsimons, and B.J. Simons, J. Physiol., (Land.) 203, 45-57 {1969).
3.
A. N. Epstein, J. T. Rtzsimons, and B.J. Rolls (nee Simons), J. Physiol, (Land.) 210, 457-474 (1970).
4.
D. Lehr, and W. Goldman, Eur. J. Pharmac., 23, 197-210 (1973),
5.
J. T. Rtzsimons and P.E. Setler, J. Physiol., (Lond) 218, 43-44 (1971),
6.
P. E. Setler, in The Neuropsychology of Thirst: New Findings and Advances in Concepts, (A. N. Epstein, H. R. K1SSeleff and E. Stellar, Editors) pp 279-291, Winston/Wiley, New York, 1973.
7.
J. Simpson and A. Routtenberg, Science, 181, 1172-1175 (1973)
8.
D. Gonten, J. L. Minnich, P. Granger, K. Hayduk, H.M. Brecht, A. Barbeau, R. Boucher and J. Genest, Science, 173, 64-65 {1971).
9.
C. Fischer- Ferraro, V. E. Nahmod, D. J. Goldstein, and S. Finkiel man, J. Exptl, Med., 133, 353-361 ( 1971).
10.
D. Lehr, J. Mallow and M. Krukowski, J. Pharmac, Exp. Ther., 158, 150-163 {1967).
II.
K. A. Houpt and A. N. Epstein, Physiol. Behav., 7, 897-902 {1971),
12.
N. E. Anden, A. Carlsson and J. Haggendal, A. Rev. Pharmac., 9, 119-134 (1969).
13.
S. Spector, A. Sjoerdsma and S. Udenfriend, J. Pharmac. Exp. Ther., 147, 86-95 {1965).
14.
D. Lehr, H. W. Goldman and P. Casner, Science, 182, 1031-1034 (1973).
15.
M. Tang and J. L. Folk, Pharmac, Biochem. Behav., 2, 401-408 (1974).
16.
J. L. Folk, M. Tang and R.W. Bryant, J. Pharmac. Exp. Ther., 190, 154-164 (1974).
Vol. 16, No. 4
Cholinergic Circuits in Angiotensin Thirst
583
17.
0. Carretero and F. Gross, Am. J. Physiol., 213, 695-700 (1967).
18.
J. C. Romero, and S. W. Hoobler, Am. J. Physiol., 223, 1076-1080, (1972).
19.
B. Peskar, D. K. Meyer, U.Tauchmann and G. Hertting, Eur. J. Pharmac<, 9, 394-396 (1970).
20.
D. K. Meyer, W. Rauscher, B. Peskar and G. Hertting, Naunyn-Schmiedeberg's Arch. Pharmac., 276, 13-24 (1973).
21.
D. K. Meyer, and G. Hertting, Naunyn-Schmiedeberg's Arch. Phormac., 280, 191-200 (1973).
22.
E. M. Blass and H. W. Chapman, Physiol. Behav., 7, 679-686 {1971).
23.
A. R. Giardina and A. E. Fisher, Physiol. Behav., 7, 653-655 (1971).
24.
M. R. Covian, C. G. Gentil, and J. Antunes-Rodrigues, Physiol. Behav., 9, 373-377 (1972).
25.
L. W. Swanson, G. R. Marshall, P. Needleman, L. G. Sharpe, Brain Res., 49, 441-446 (1973).
26.
W. B. Severs and A. E. Daniels-Severs, Pharmac. Rev., 25, 415-449 (1973).
27.
P. Casner, H. W. Goldman, and D. Lehr, paper presented at the XXVI International Congress of Physiological Sciences, Jerusalem Satellite Symposium On Regulation of Food ond Water Intake, October, 1974.
28.
P. Casner, W. Goldman, and D. Lehr, Fed. Proc., 32, 786 (1973).
29.
D. Lehr, in The Neuropsychology of Thirst: New Findings and Advances in Concepts, (A. N. Eptstein, H. R. Kisseleff and E. Stellar, Editors) pp. 307-314, Winston/ Wiley, New York, 1973.
30.
I. Izquierdo and J. A. Izquierdo, A. Rev. Pharmac., II, 189-208 (1971).
31.
G. K. Terpstra and J. L. Slangen, Neuropharmacology, II, 807-818 (1972).
Life Sciences Vol. 16, pp. 573-584 Printed in the U.S.A.
PARTICIPATION OF CHOLINERGIC CIRCUITS IN RENIN INDUCED DRINKING Paul Casner, H. Warren Goldman and David lehr Department of Pharmacology New York Medical College Valhalla, New York 10595 (Received in final form January 13, 1975)
Summary Pharmacological analysis of the drinking response produced by the intraperitoneal (i .p.) injection of renin in the intact and bilaterally nephrectomized rat revealed that peripherally administered atropine sulfate ( 5 mg/kg s.c. ) produced a highly significant block of renin-induced drinking, while alpha and beta-adrenergic blockade with tolazoline (8.5 mg/kg s.c. ) and propranolol ( 6.0 mg/kg s.c. ), respectively, were without effect. In the nephrectomized rat inhibition of the synthesis of norepinephrine and dopamine with i .p., alpha methyl-p-tyrosine hod likewise no effect on renin-induced water intake, whereas injection of the dopominergic blocking agent, haloperidol ( 0.05 mg/kg s.c. ), reduced the drinking response by about one third. However, doubling the dose of haloperidol caused no further reduction. It is concluded that cholinergic pathways are importantly involved in renin-induced drinking behavior. Recent investigations of mammalian drinking behavior suggests an important role for the renin-angiotensin system in the mediation of thirst. Intravenous infusion of renin or synthetic angiotensin II have been shown to elicit copious drinking in the water-sated rat ( 1,2 ). Evidence has been presented indicating that angiotensin evokes water consumption by an action on the central nervous system (CNS) (3). Characterization of the neurotransmitters involved in the drinking behavior produced by intra-cerebral injection of angiotensin indicated cholinergic (4) anq/or dopaminergic (5,6) mediation of the central angiotensin response. Pharmacological analysis of renin-angiotensin drinking behavior has employed almost exclusively the central route of drug administration, despite the fact that it has been shown that peripheral angiotensin II can elicit a drinking response by acting at sites in the 573
Cholinergic Circuits in Angiotensin Thirst
574
Vol. 16, No. 4
CNS that are outside the blood-brain-barrier (7). The recent discovery of an independent renin-angiotensin system in the brain (8,9), whose role in drinking behavior is as yet unknown, has further complicated the study of systemic renin-angiotensin thirst. The present report was undertaken to provide a pharmacologic ana Iysis of peripherallyinduced angiotensin thirst and to use this information as a means of comparison with the pharmacology of centrallrinduced angiotensin drinking. Methods Intact Animals Male Wister albino rats weighing 240-300 grams were used as experimental animals. Individual metabolism units fitted with 100 ml graduated drinking tubes were utilized in all experiments. Food was withheld during the experimental period. Water intake was measured at 1,2, and 3 hours after drug treatment. In experiments with intact rats, 24 hour water intake was measured before and after the test in order to confirm stable baseline drinking. Bilaterally Nephrectomized Animals Male Wistar albino rats weighing 250-300 grams were used in these experiments, The testing procedure was identical to that for intact animals, except that 24 hour drinking was not recorded. Rats were bilaterally nephrectomized under ether anesthesia. The kidneys were removed through a midline abdominal incision after decapsulation leaving the adrenal glands intact. The animals were allowed a two hour recovery period before an experiment was begun, except when a-methyl-p-tyrosine was given, in which case the animals were allowed a four hour recovery period. It was found that the additional two hour recovery period did not have a significant effect on the renin-drinking response. Intraperitoneal injections were mode lateral to the incision site and absorbent paper was placed on the floor of the cage to detect possible leakage of the drug. Each nephrectomized rot was used only once for a particular experiment.
Vol. 16, No. 4
Cholinergic Circuits in Angiotensin Thirst
575
Drugs Lyophilized hog renin (Nutritional Biochemicals) was injected intraperitoneally (i .p.) in doses of 1.0, 3.0 and 6.0 units per 100 grams body weight (gbw) for intact rats and 1.0, 2.0 and 3.0 units per 100 gbw for nephrectomized rats. The dose to be injected was dissolved in 0.50 ml of isotonic saline for the nephrectomized rat or 1.0 ml of isotonic saline for the intact rat. Controls were injected i .p. with 0.50 and 1.0 ml of isotonic saline for the nephrectomized and intact rat, respectively. Pharmacologic blocking agents were atropine sulfate (5.0 mg./kg), tolazoline hydrochloride (8.5 mg./kg), propranolol hydrochloride, (6.2 mg./kg), haloperidol {0.1 mg./kg and 0.05 mg./kg) and alpha methyl-p-tyrosine methyl ester (300 mg./kg). These agents were injected subcutaneously (s,c.) in 1.0 ml of isotonic saline approximately 15 minutes before the administration of renin, except for alpha methyl-p-tyrosine which was injected i .p. immediately after nephrectomy. In this instance renin was injected after a period of four hours had elapsed. Controls in all blocking studies were injected s.c, with 1.0 ml of isotonic saline. Data were analyzed by the Student's t-test and were considered statistically significant when P was< 0.05 and highly significant when P was<. 0.001. Results are shown in the graphs as the mean± S.E. Results Intact Rat The thirst-inducing effect of renin administered i .p. to the intact, water replete rat was modest and inconsistent unless excessive dosage was employed (Figure I ) • One as well as three units per 100 gbw produced effects which were statistical! y not significant. A reliable drinking effect was observed only with the administration of 6.0 units per 100 gbw. The onset of the main water intake at this dose was delayed for about 30-40 minutes after injection with sporadic drinking continuing for approximately 3 hours. Thus, a reliable dose-response relationship could not be demonstrated at the dose range employed.
576
Cholinergic Circuits in Angiotensin Thirst
Vol. 16, No. 4
RENIN 6 0 U/toOqbw
IQO
8.0
WATER INTAKE
6.0
RENrN 3.0 u/100 ~b·
{mls} RENfN
1.0 UIIOOgbw
4.0 CONTROL
2.
12
12
18
FIGURE 1 Drinking response of the intact rat after i .p. administration of hog renin. Renin is expressed in units per 100 grams of bodyweight (gbw). Water intake was measured over a 3 hour period for this and, all subsequent figures. Figures inside bars indicate numbers of animals in each group. Only response at 6.0 u/100 gbw is significantly different from Control ( P <. 0.001). The effect of autonomic blockade on this response was studie:l with the dose of i .p. renin which was found to elicit significant water intake (6.0 units per 100 gbw). The doses of blocking agents utilized are based on a study by Lehr et al. (10) on isoproterenol induced drinking. As is shown in Figure 2, blockade of either alpha or beta-adrenergic receptors had no statistically significant effect on renin drinking. Pretreatment with atropine sulfate, however, completely abolished the dipsogenic action of renin. It is interesting to note that beta-adrenergic thirst, which has been claimed to be completely dependent on the renin-angiotensin system, (II), is obviated by propranolol yet unaffected by atropine,(IO) an observation which is the exact reverse of our findings with renin-induced thirst. Bi latera II y Nephrectomized Rat Bilateral nephrectomy greatly potentiates the effects of renin on water intake when compared to its actions in the presence of the kidneys, as first demonstrated by Fitzsimons (1). Renin in amounts of I, 2, and 3 units per 100 gbw produced highly significant and
dose related drinking responses (Figure 3). Animals injected with 3 units per 100 gbw drank over 15 ml in the three hour observation period following nephrectomy whereas the
Cholinergic Circuits in Angiotensin Thirst
Vol. 16, No. 4
intact animal, given the same dose, drank less than 4.0 ml in the same period ( see Figure I). 12.0
RENIN
10.0
6 OU/I009bw
8.0
RE~IN
INTAKE
R:;:!'JN
TOLAZOLNE 5m'l)/kQ
e
WATER
PROPRANOLOL 6.Zmqlkg
T
60
(mit)
.
40
RENIN
ATROPINE· 50 4 ~Om;lk9
CON.TROL
~0
18
20
10
FIGURE 2 The effect of autonomic blocking agents on the drinking response produced by i •P• injection of 6.0 units of renin per 100 gbw in the intact rat. Blocking agents were administered 15 min. prior to renin, Only atropine produced a significant block of the drinking produced by 6.0 u/IOOgbw of renin ( P < 0.001).
~00
RENIN 3.0 U /IOOgbw
16.0
RENIN
12.0
2.0 U/IOOQbw
WATER INTAKE
lmltl
RENIN 1.0 U/IOOgbw
8.0
4.0 CONTROL
10
10
19
FJGURE"3 Drinking response of the bilaterally nephrectomized rat after i.p. administration of hog renin. Response at all doses was statistically significant. ( P <. 0.001 ).
577
578
Cholinergic Circuits in Angiotensin Thirst
Vol. 16, No. 4
The effects of adrenergic and cholinergic blockade in this series of experiments were similar to our findings with the intact animal (Figure 4). Neither tolazoline nor propranolol interfered with the copious drinking responses produced by 3 units per [00 gbw of renin, whereas blockade of cholinergic receptors with atropine significantly antagonized renin drinking. In fact, cholinergic blockade lowered the renin-induced drinking to about 20% of the expected response.
20.0 RENIN 3.0U/100qbw
16.
WATER INTAI
.
RENIN RENt<
•
TOLAZOLIN£ 8_5mo/kg
PROPRANOLOL
6.2mgllto
12 0
{mls)
8.0
4.0
19
6
FIGURE 4 The effect of autonomic blocking agents on the drinking response produced by i.p. injection of 3.0 units per 100 gbw in the bilaterally nephrectomized rat. Blocking agents were administered 15 min. prior to renin. Only atropine produced a statistically significant reduction of the drinking induced by 3.0 u/IOOgbw of renin.( P <. 0.001). To determine the role of dopamine in peripheral renin-angiotensin drinking, o dopaminereceptor blocking agent, haloperidol (12), and an inhibitor of catecholamine synthesis alpha methyl-p-tyrosine (13) were employed. The latter agent prevents formation of norepinephrine as well as dopamine. Subcutaneous injection of 0.05 mg./kg of haloperidol reduced the drinking response elicited by 3 unit¥'100 gbw of renin by 300,1,. However doubling the dose of haloperidol did not enhance this blocking effect
( Figure 5 ).
Pretreatment of the nephrectomized rat with 300 mg./kg (i .p.) of alpha methyl-p-tyrosine had no effect on the renin drinking response.
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o<.-MpT 300mg/kg
RENIN 30u/100gbw
15
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+
RENIN HALOPERIDOL 005mg/kg
HALOPERIDOL O.IOmg/kg
+
+
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. RENIN
RENIN
.,
.5
-
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FIGURE 5 The effect of haloperidol and alpha-methyl-p-tyrosine on the drinking response produced by i •P• administration of 3.0 units of renin per 100 gbw in the bilaterally nephrectomized rat. Haloperidol was given 15 min. prior to renin while alpho-MpT was given 4 hours before renin. Reduction of water intake by both doses of haloperidol is statistically significant compared to the drinking response produced by renin alone ( P 0.05 ).
<
Removal of the kidneys is known to significantly prolong the half-life of many agents, particularly if they are inactivated by renal biotransformation or their elimination is strongly dependent upon renal integrity. This may explain not only the far more potent effect of exogenous renin, but also the lack of difference in blocking activity observed with the high and low dose of haloperidol. Discussion Drinking behavior produced by actual components of the renin-angiotensin system should be distinguished from that produced by agents that release renin from the kidney (e.g. isoproterenol, diazoxide). Thirst produced by renin or angiotensin can be attributed to these agents alone, while the thirst produced by isoproterenol or diazoxide (reninreleasers) is not necessarily a result of their ability to release renin from the kidney. Indeed, recent studies have suggested the independence of these drinking behaviors from the renin-angiotensin system (14, 15, 16),
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If exogenous renin is used to induce thirst in the rat, the drinking response is unimpressive (see Figure I ) unless the animal is bilaterally nephrectomized (I ), The increased sensitivity of the reniprival rat to renin is nlustrated in Figure 3. The reasons for the enhanced drinking response after nephrectomy are not known, although several possibilities have been suggested, such as enhanced production of substrate due to removal of an inhibitory loop (17), absence of renin degradation by the kidneys (I), or enhanced enzyme kinetics (18).
It should be pointed out that these explanations were
originally advanced as possible reasons for the increased pressor responses seen after injections of renin in the bilaterally nephrectomized rat. These explanations could apply equally well to the enhanced drinking response, Among pharmacological studies of the systemic role of the renin-angiotensin system in drinking behavior, employing renin-releasing agents { 19, 20, 21), the study by Blass and Chapman (22) seems to be the only investigation of renin-angiotensin thirst which employed the peripheral administration of an actual component of the renin-angiotensin system, These authors investigated the possible involvement of cholinergic mechanisms in thirst produced by intraperitoneal
administration of renin in the intact rat. They concluded
that cholinergic pathways were not involved in renin-angiotensin drinking behavior, since atropine sulfate produced a block only at very high concentrations {IS mgjkg). Blass and Chapman used a dose of 2.0 units of renin/100 gbw in the intact rat, which elicited a total water intake of approximately 3 mi.
It is conceivable that their failure
to observe a block with lower doses of atropine is attributable to the small baseline drinking response evoked by this dose of renin in the intact animal, Most investigators have found that drinking elicited by the injection of angiotensin into the prefrontal area of the hypothalamus could not be blocked by pretreatment of this~ site with intracerebrally administered atropine ( 5,23,24,25 ).
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Cholinergic Circuits in Angiotensin Thirst
This observation was fully confirmed in our laboratory (4).
581
However, when atropine is
administered peripherally ( 4, 22,26 ), or intraventricularly { 26, unpublished observations from this laboratory ) the central angiotensin drinking response can be significantly inhibited, suggesting that the cholinergic neurons involved are not in close proximity to the angiotensin injection site. Recent evidence has pointed to dopaminergic involvement in central angiotensin drinking { 5,6 ). However, in the present study, inhibiting dopamine synthesis with alpha methyl-p-tyrosine had no effect on the renin drinking response, .while systemic haloperidol in doses which did not induce obvious sedation reduced the drinking response by about 30% suggesting a possible involvement of dopaminergic pathways in this thirst behavior, although a non-specific effect cannot be ruled out. Indeed our finding that a higher dose of haloperidol does not increase the effectiveness of the block speaks for a non-specific effect. Moreover, we have been unable to block the drinking response produced by hypothalamic injection of angiotensin with haloperidol applied at the same site, although peripheral haloperidol does block centrally-induced angiotensin drinking
( 27 ). In summary, our findings do not support substantial involvement of dopaminergic circuits in central angiotensin drinking, but are in line with earlier observations from this laboratory { 4, 28, 29 ) and those of Severs and Daniels-Severs { 26) with regard to cholinergic mediation of angiotensin induced thirst. The cholinergic block by atropine of peripherally and centrally-induced angiotensin drinking behavior indicate that when given peripherally, renin or angiotensin most likely elicit thirst by acting at sites in the CNS. These sites might well be outside the blood-brain-barrier allowing angiotensin to act centrally without crossing the ependymal layer { 7). Our results, however, do not rule out possible participation of the peripheral nervous system and its transmitters in renin-
1 • t ens1n • dnn • k.1ng be hav1or. . ang1o
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In recent preliminary studies we have found that the quaternized analogue of atropine (atropine methyl nitrate ) produced a significant although less substantial block of renin-induced drinking. Whether this indicates peripheral involvement in the drinking response or simply penetration of the quoternized analogue into the CNS cannot be determined at this time since the previously accepted tenet of CNS-exclusion of this quaternized analogue hos recently been challenged ( 30, 31 ). References I.
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