Brain Research, 124 (1977) 305-315

305

© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands

INDEPENDENT RECEPTORS FOR PRESSOR AND DRINKING RESPONSES TO CENTRAL INJECTIONS OF ANGIOTENSIN II AND 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 July 14th, 1976)

SUMMARY Angiotensin II and carbachol when injected in the brain ventricles of the rat produce similar responses of an increase in blood pressure and drinking behavior. The question of whether these effects are produced by independent receptors or via a cholinergic circuit is debatable for the drinking behavior and evidence is lacking for the blood pressure effect. We have used a chronic rat preparation for recording blood pressure and drinking at the same time during intraventricular injections (i.v.t.) of both angiotensin and carbachol and i.v.t, or intravenous infusions of appropriate antagonists. The results show that drinking and blood pressure response to angiotensin II can be blocked by P113 (500 ng i.v.t.) an angiotensin antagonist; they are not blocked by atropine (10/~g i.v.t.) a cholinergic antagonist; carbachol effects, however, are not blocked by P113, but are totally blocked by atropine (10 #g i.v.t.). At high doses of atropine there is inhibition of both agents but this probably represents a general inhibition. The hormone and cholinomimetic administered together interact and both are inhibited by adrenergic stimulation. We conclude from these experiments that angiotensin and carbachol act upon independent receptors in the brain to produce blood pressure and drinking responses but at some point they share common, central effector pathways.

INTRODUCTION Intracranial administration of both angiotensin II and carbachol has been shown to produce drinking behaviorT,5,12,~s,al and ADH releaseZl,zg,a0 in the rat 1s-2°, 2e. Recently we have demonstrated that carbaehol, like angiotensin, also produces a blood pressure increase when it is injected into the brain ventricles. However, carbachol, unlike angiotensin, has no pressor action when it is injected into the vascular system. The pressor effect that we found with carbachol in rats is very similar to the

306 pressor response to intraventricular injections (i.v.t.) of angiotensin II (All) la,'~5. The question these data raise is whether the similarities between the effects of the peptide and the cholinomimetic are due to an action on the same receptor system or to an independent effect. Both agents could activate independent receptors or alternatively, angiotensin II might act upon a receptor which has neural inputs across a cholinergic synapse. If either of these two possibilities are correct both agents should be blocked by appropriate antagonists. Atropine, which antagonizes cholinergic transmission, should block the effects of central carbachol and if there is only one system for reception, this antagonist should also block angiotensin. If the hormone works independently of cholinergic activity then angiotensin should continue to be effective after atropine. Similarly, antagonists for angiotensin 11, such as sarl-alaS-angiotensin I1 (P113), should block the action of the peptide but leave the cholinergic activity unaltered if the effects which both agents produce are independent of one another. Evidence for this kind of separation is lacking for blood pressure effects of the two agents and is contradictory for the drinking effects that they produce s-l°,25'a~. By studying blood pressure in chronic rats during intraventricular injections we present here evidence for separation of the carbachol and angiotensin responses. The results of these agents on drinking recorded during the blood pressure add to the data reported by others and show that the blood pressure and dipsogenic responses are closely related. In addition, if the hormone and cholinomimetic act positively on the same system their effect in combination should be greater than their individual effects. We have tested this by administering both agents in the same chronic rat preparations. Lastly, it has been reported that central norepinephrine has a differential response on central drinking effects of angiotensin and carbacho124. Since this could be another way of dissecting the mechanism for centrally induced thirst and pressor effects, we have examined the effect of central norepinephrine infusions. METHODS Sixty-seven male Sprague-Dawley rats (300-400 g) were used in these experiments. Thirty-five animals were anesthetized with Nembutal and implanted with one 14 ram, 22-gauge lateral ventricular cannula. Thirty-two rats had 2 bilateral lateral ventricle cannulae implanted 3 days before the start of the experiment. Thirtygauge styli maintained the cannulae patent during recovery. Each animal received 50,000 U of penicillin after surgery. On the morning of the experiment each subject was anesthetized with ether and implanted with a femoral artery catheter. The PE-50 catheter tubing, filled with heparinized saline was led subcutaneously out through an incision in the back where it was cemented to a wound clip. A short length of 22 g rod served a stylus for the cathether during recovery .Thirty rats were also implanted with a femoral vein catheter at the same time. All subjects were allowed 2 h for recovery before testing began. Intraventricular injections in these tests were given through a 30 g injection cannula constructed to fit into and sit flush with the end of the guide cannula. The injection cannula was connected by a section of PE-10 tubing to a remote micro-

307 injection system (25/~1 Hamilton syringe) outside the cage. The arterial catheter was connected by an extended piece of PE-50 tubing to a Statham P23 Gb blood pressure transducer located outside the cage. These methods allowed blood pressure recording in a freely moving animal and testing sessions in which the animal was not handled. Heart rate was measured with a Beckman 9857B small animal cardiotachometer. The signal was triggered by pressure pulses. Blood pressure and heart rate were recorded on a Beckman R411 dynograph recorder. Water intake was measured from calibrated drinking tubes to the nearest 0.1 ml. Values are reported as means :k S.E. and comparisons were made with Student's t-tests. Since it has been shown that All injected into the brain is active dipsogenically when it has access to the ventricles 17, confirmation of the cannulae being in the ventricles was given by a positive drinking response after All injections. Only when the response was below 2 ml was a dye injected through the cannula and histological verification made at the end of the experiment.

Procedure To compare central carbachol and angiotensin induced responses, the 67 animals were tested for their blood pressure and drinking responses to separate 50 ng All and 25 ng carbachol i.v.t, injections given in the same ventricular cannula. These tests were separated by 1 h. The doses were based on previously established dose-response curves showing equivalent responses for drinking la,~8,31. Responses of blood pressure, heart rate and water intake for each test were recorded for 30 min. Both All and carbachol were injected in a 5 #1 artificial cerebrospinal fluid (CSF) vehicle (Elliott's B solution, Travenol Laboratories). All central injections were given over a 15 sec interval. Random selection determined whether the first test injection was carbachol or All. To determine the effect of the various blocking agents on central All and carbachol induced responses, animals tested above were allowed a 1 h rest interval, after which time either i.v.t, or intravenous (i.v.) infusions of the antagonists were given. Intraventricular infusions were administered in a 5 #1 CSF vehicle into the second of the bilateral ventricular cannula. The i.v. infusions were administered in approximately a 0.5 ml saline vehicle given over 30 sec. Ten minutes later drinking and pressor responses to i.v.t, carbachol (25 ng) or All (50 ng) were recorded. One hour later the same procedure, using the same blocking agent, was followed with the second of the two drugs (All or carbachol). Atropine sulfate was injected i.v. at 5 mg/kg in 11 rats and i.v.t, at 100/~g/5/zl in 4 rats, and 10/zg/5/~1 in 7 rats. Atropine methyl nitrate does not penetrate the blood-brain barrier as well as atropine 1. Therefore, in order to show the central action of atropine, methyl-atropine was infused i.v. at 5 mg/kg in 6 rats. Saralasin acetate (sarl-alaS-angiotensin II, P113, Norwich Pharmacal) was infused i.v.t, at a dose level of 500 ng/5/zl in 7 rats for 5 min and the test injection was given at the end of this infusion. Norepinephrine was infused i.v.t, at 1 #g/5/~1 in 9 rats and phentolamine was infused i.v. at 10 mg/kg in 8 animals. Five rats served as controls for the above procedures. Each animal received one 50 ng All and one 25 ng carbachol i.v.t, test separated by 1 h with drinking and pressor

308 responses recorded. This procedure was repeated in the next 2 tests except that 0.5 ml of isotonic saline was infused i.v. 10 min before each central injection. A third set of separate All and carbachol tests was then performed in these animals so that a total of 6 tests was carried out over 6 h. Ten minutes before each drug injection in these tests the animals received 5 #1 artificial CSF, infused into the opposite bilateral ventricular cannula. In 10 subjects the test for additivity in response was measured by injecting 50 ng All and 25 ng carbachol simultaneously in the same 5 #1 CSF vehicle, 1 h after pretesting with each agent separately. The responses to the combined dosages were recorded for 30 rain. RESULTS In all of the 67 rats tested the average drinking response to 50 ng All given i.v.t, was 3.8 4- 0.5 ml and with 25 ng of carbachol by the same route the average water intake was 3.7 :k 0.6 ml. The correlation observed between these two responses was significant (n = 0.48, P < 0.001). Between rats, however, there was variability. Eight cases were seen where i.v.t, angiotensin produced drinking but i.v.t, carbachol did not. Six instances were observed where the opposite was true. The pressor responses with Sprague-Dawley rats to 50 ng All and 25 ng carbachol i.v.t, were 18 :k 1 mm Hg and 23 ± 2 mm Hg, respectively. The correlation between the two responses was n ~- 0.59, P < 0.001. Although there was variability in All versus carbachol induced pressor responses there were no cases observed where either i.v.t. All or carbachol did not produce a pressor response. The latency to the initiation of a pressor response was 0.28 ± 0.08 min for carbachol and 0.41 ± 0.11 min for All. Latencies to drink were 1.27 ~k 0.31 min and 1.66 ~k 0.48 min respectively. Heart rate changes observed with the pressor responses to i.v.t, carbachol and AII were --38 -~ 13 beats/min and --26 ± 11 beats/min respectively.

Atropine and P113 Intravenous atropine infusions alone resulted in no significant change in mean blood pressure (113 ± 6 mm Hg to 105 i 6 mm Hg) or heart rate (385 :k 10 beats/ min (BPM) to 457 :k 13 BPM). The effect of the atropine treatment on central All and carbachol responses is shown in Fig. 1. After i.v. atropine sulfate, drinking and pressor responses to injections of 25 ng carbachol i.v.t, were abolished. Drinking to All (i.v.t.) was reduced 62 ~ but the pressor responses were not changed significantly. After pretreatment by atropine methyl nitrate i.v. at 5 mg/kg we found that both drinking and pressor responses to carbachol i.v.t, were significantly reduced. The responses to angiotensin i.v.t, were not affected by this treatment, however. Centrally, 10 #g of atropine sulfate abolished both carbachol i.v.t, induced drinking and blood pressure increases while having no significant effect on central All induced responses (Fig. 2). However with 100/ag atropine given i.v.t, both carbachol and All effects were abolished. The central atropine infusions alone had no effect on heart rate or blood pressure.

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Fig. 1. Mean and standard error of pressor and drinking responses to i.v.t. AII and carbachol before and 10 min after i.v. atropine treatment. The i.v.t, infusion of 500ng Pl13 produced the opposite effects to those of atropine. The results shown in Fig. 2 indicate that the competitive All antagonist abolished the drinking and pressor effects of angiotensin i.v.t, while having no significant effect on these responses to carbachol. Central infusions of P113 alone had no effect on blood pressure or heart rate.

Adrenergic blockade The effect of peripheral a-adrenergic blockade on central A I I and carbachol induced responses was tested in 8 rats. With i.v. phentolamine infusions alone, mean blood pressure decreased from 120 -4- 2 mm Hg to 73 q- 5 mm Hg and heart rate increased from 364 -4- 15 to 464 q- 9 BPM. After i.v. phentolamine treatment drinking was decreased to both A I I and carbachol i.v.t, tests (Pre-AII = 3.4 -1- 1.1 ml, carbachol = 3.4 -4- 1.0 ml; Post-AII = 1.1 q- 0.5 ml, P < 0.01, carbachol = 1.5 -f- 0.8 ml, P < 0.05). Blood pressure responses were not significantly changed (PreA I I = 18 -4- 3 mm Hg, carbachol = 25 -4- 4 mm Hg; Post-AII = 15 4- 2 mm Hg,

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Independent receptors for pressor and drinking responses to central injections of angiotensin II and carbachol.

Brain Research, 124 (1977) 305-315 305 © Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands INDEPENDENT RECEPTORS FOR PRE...
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