On the central hypertensive effect of angiotensin II

Inr. J. Neuropharmacol.,

1961, 6.199-205

Pergamon Press.

Printed in Gt. Britain.

ON THE CENTRAL HYPERTENSIVE ANGIOTENSIN II*

EFFECT OF

WALTERB. SEVERS,~ ANNE E. DANDXS and JOSEPH P. BUCKLZY Cardiovascular

Research Laboratories, Department of Pharmaco logy, University of Pittsburgh School of Pharmacy, Pittsburgh, Pennsylvania (Accepted 15 November 1966)

Summary-It was the purpose of this study to investigate further the mechanism by which angiotensin II produces central hypertensive effects. When administered into the perfused lateral ventricle of the cat, the peptide consistently produced neural pressor effects which were essentially abolished by lesions at high midbrain levels. Pressor activity was apparently not generated by bulbar efferent or afferent mechanisms, as topical application of the peptide at the termination of the cerebral aqueduct during perfusion produced little or no change in femoral blood pressure when compared with the intraventricular responses in the same animals. Similar pressor activity was observed in perfused dog lateral ventricle experiments but was not detected in experiments in which only subarachnoid structures of the dog brain were perfused. INTRODUCTION

SEVERAL investigators have reported that angiotensin II produces pressor effects via central mechanisms (BICKERTONand BUCKLEY,1961; BENETATOet al., 1964; LAVERTY,1963; SMV~OOKLW et al., 1966; SEVERS et al., 1966a). MCCUBBINet al. (1965) observed that the infusion of subpressor doses of angiotensin II to dogs caused increased excitabrlity of the sympathetic nervous system which was influenced by the degree of environmental stimulation, and closely resembled the clinical picture of labile hypertension. In addition, JOHNSSONet al. (1965) obtained evidence that angiotensin II increases adrenergic neuron activity in humans. This latter observation was confirmed by SCROOPand WHELAN(1966) who felt that the increased sympathetic activity was due to a specific central vasomotor effect. An earlier study in this laboratory (SEVERSet al., 1966a) provided evidence that central angiotensin pressor effects originate at midbrain levels. The present report provides further evidence for this conclusion and demonstrates that angiotensin-sensitive neurons in the dog brain can be activated when the peptide is exposed to ventricular, but not subarachnoid, areas. METHODS

Ventricle perfusion in cuts.

Adult cats of either sex were anesthetized with chloralose (60 mg/kg, i.v.). Supplemental doses of the anesthetic were administered as needed during the experiments. The c’ose of chloralose was dissolved in 2-3 ml of hot 95% ethanol and diluted approximately five-fold with warm saline solution just prior to injection. Female *Supported by U.S. Public Health Service training grant GM-01217 from the National Institute of General Medical Sciences. tPresent Address: Laboratory of Chemical Pharmaco logy, National Heart Institute, National Institutes of Health, Bethesda, Md. 199

200

W. B. SEVERS,ANNE E. DANIELS and J. P. BUCKLEY

cats which were pregnant or lactating were not used in this study, as pregnancy markedly inhibits the pressor response to the intraventricular administration of angiotensin to cats (SEVERSet al., 1966b). The ventricles were perfused according to the method of BHATTACHARYA and FELDBERG(1958). A 22 gauge unbeveled hypodermic needle was stereotaxically implanted in the left lateral ventricle (anterior = 13 mm, lateral left = 2.75 mm, and horizontal = +7 mm) and cemented to the skull with dental cement. Artificial cerebrospinal fluid (CSF), prepared as described by MERLIS(1940), was warmed to 38°C and perfused through the lateral ventricle at a rate of 0.1 ml/mm using a Sigmamotor pump. Effluent CSF was allowed to drain through an incision in the dura at the cisterna magna. Blood pressure was recorded from a femoral artery using a Statham pressure transducer and Offner dynograph. Intermittent positive pressure respiration was maintained throughout the experiment. At least 1 hr was allowed to elapse upon completion of the surgical procedures before angiotensin II (Hypertensin, Ciba Pharmaceutical Products, Inc.) was administered into the lateral ventricle using a Baltimore microinjection unit during a brief interruption of the perfusion. The dose of the peptide was dissolved in O-1 ml artificial CSF, a volume which produces little or no effect on blood pressure. At the termination of all experiments, dye was perfused through the ventricles, and the entire brain was removed and fixed in 10 per cent formalin solution for examination. Midbrain lesions. Cats were prepared as described above, except that a piece of skull bounded by the approximate coordinates, anterior = 4.5-65 mm and bilateral = 7 mm, was removed. After obtaining control angiotensin responses, and without disrupting the perfusion, two needles, insulated except for 0.5 mm at their tips and separated by 2 mm, were lowered into the brain at the anterior 5.5 plane. Tissue was coagulated 6 mm bilateral to the saggital suture and from the skull base upwards to include about 2 mm of cortical tissue. In the experiments reported, brain section indicated that this procedure did not affect the distribution of dye through the ventricular system. When viewed from a midsaggital section, the coagulated tissue in these experiments extended from the origin of the aqueduct to a plane no more than 2 mm caudal into the superior colliculus. Bulbar application. Cats were prepared for ventricular perfusion as described above. A small portion of the supraoccipital bone was removed and the medial cerebellum aspirated until the inferior colliculi were visible. The cerebellum was gently elevated with a curved glass spatula during aspiration to prevent damage to the floor of the fourth ventricle. Angiotensin was administered by intraventricular injection and by directly adding the peptide to the perfusate at the termination of the cerebral aqueduct. Ventricle perfusion in dogs. Mongrel dogs weighing 8-l 1 kg were anesthetized with pentobarbital sodium (35 mg/kg, iv.). A sufficient amount of pentobarbital was injected before administering angiotensin to depress diastolic blood pressure to less than 100 mm Hg (see Discussion). Intermittent positive pressure respiration was initiated and maintained throughout the experiment. Using a series of clamps, the skull was rigidly secured to approximate the stereotaxic position described by LIM et al. (1960). After exposing a sufhcient area of the skull, a hole was drilled approximately 8 mm posterior and 7 mm lateral to the bregma. A 22 gauge unbeveled hypodermic needle with a tight fitting stylet was lowered into the brain with a vernier-controlled electrode carrier until CSF could be seen at the needle tip when the stylet was withdrawn. Needles were cemented to the skull and the ventricles or subarachnoid spaces were perfused as described for cats, except that the perfusion rate was O-2 ml/min. Blood pressure was recorded from a femoral

Central effects of angiotensin

201

II

artery. At the termination of the experiments, dye was perfused through the cannula, the brain was removed and fixed in formalin solution. The fixed brain was examined to determine the position of the cannula and flow pattern of the dye, which was used as an index of the flow pattern of the perfusate. In the experiments reported, perfusion through the ventricles caused intense staining of the ventricular system, the ventral surface of bulbar structures, parts of the cortex (mainly occipital) and cerebellum. In some experiments, the needle was misplaced and it was found that the CSF observed was of subarachnoid origin. In these experiments, the ventricular system was entirely devoid of dye, but intense staining of most of the cortex, outer cerebellum, and ventral surface of the brain was observed. RESULTS

EfSect of midbrain lesioning.

The effects of midbrain lesions on the pressor response to intraventricular administration of angiotensin II are summarized in Table 1. The mean control pressor response obtained upon injection of 4 pg angiotensin into the lateral TABLE 1. THE EFFECT OF MIDBRAIN LESIONS ON THE RESPONSE TO ANGIOTENSIN (4 pg), ADMINISTERED INTO THELATERALVENTRICLEOFTHECAT

Post-lesion

Pre-lesion

Sex F M F F F

Weight (kg) 2.3 2.4 1.5 2.4 1.4

Peak angiotensin response (mm Hg)

Blood pressure* (mm Hg) 95165 140/87 90/&l 115/70 75150

+45/+40 +50/+43 t-60/+57 +40/+27 $115/+80

Blood pressure* (mm Hg) 93163 100/58 80150 95155 70145

Peak angiotensin response (mm Hg) +9/+10 O/O O/O O/O

+20/+20

*Blood pressures expressed as: mm Hg systolic/mm Hg diastolic

ventricle of five cats was 62/49 f 14/9 mm Hg. * Following placement of the lesion in the midbrain, pressor activity was reduced to 6/6 f 4/4 mm Hg, which was significantly different from the control responses (P=
8 systolic B diastolic

*

S.E. systolic S.E. diastolic

W. B. SEVERS, ANNEE. DANIEISand J. P.BUCKLEY

202 TABLE 2.

THEEmEcTOF BULBARAPPLICATION OF

ANGIOTeNSIN

(lo

&,

ON THE BU)(ID

PRJWURE

OF THE CAT

Control Weight (Ks)

Blood pressure* (mm H8)

Peak angiotensin response? (mm H8)

Bulbar application Peak angiotensin Blood pressure* response (inin H8) (mm H8)

F M F

2.9 3.3 2.1

75140 90155 95150

+80/+72 +45/+40 +110/+100

75140 90155 105/55

Ll

3.0 2.6

170135 lo/so

+100/+80 t-45/+35

80140 115/55

+15/+15 O/O +15/+20 O/O

*Bloodpressuresexpressedas: mm Hg systolic/mmHg diastolic tPressor responseto 4 pg angiotensinadministeredintraventricularly Central activity in dogs. The effects of angiotensin II, administered into the perfused lateral venfricle of the dog on femoral arterial pressure prior to and following C-l section, are summarized in Table 3. In four dogs in which brain section indicated cannula placement in a lateral ventricle and proper perfusion, 4 ,ug angiotensin administered intraventricularly produced a mean pressor response of 73/52f 15/7 mm Hg. Doses of angiotensin up to 10 pg administered intraventricularly to the same animals were non-pressor following section of the spinal cord at C-l. In three additional dogs, the cannula was positioned so tha! only subarachnoid spaces were perfused and dye did not appear in the ventricular spaces. In these animals, doses up to 20 pg angiotensin were non-pressor when added to the subarachnoid CSF. TABLE 3.

THEEFFECT

OF SPINAL

SECTION

ON THE PRESSOR

RESPONSE

DOG LATERALVENTRICLE

TO

Pre-section

Sex

Weight o(B)

Blood pressure* (mm H8)

F F

11.6 10.3 13.7 7.8

75140 120180 105/55 130/80

ANGIOTENSIN (4 &,

INTHE PERFUSED

PRBPARA’TTON

Peak angiotensin response (mm H8) +110/+64 +37/+35 +75/+ao +70/+48

Post-section Peak Blood angiotensin response pressure* (mm Hs> (mm H8) 75140 70140 60140 65135

O/O O/O O/O O/O

*Bloodpressuresexpressedas: mm Hg systolic/mmHg diastolic DISCUSSION The chronic phase of renal hypertension appears to be mediated by increased sympathetic nervous system activity (MCCUBBIN and PAGE, 1963). MCCUBBIN et al. (1965) have observed that angiotensin exerts indirect pressor activity associated with sympathetic hyperactivity and that the magnitude of the response is associated with the degree of environmental stimuli. Other investigators (JOHNSSON et al., 1965; and SCROOP and WHELAN, 1966) have obtained evidence that angiotensin increases adrenergic neuron

Central effectsof angiotensin II

203

discharge in humans, and the latter group felt that activation of a specific vasomotor center in the central nervous system occurred rather than ganglionic stimulation, an effect which has been attributed to angiotensin (LEWIS and REIT, 1966). SEVERSet al. (1966a) reported that the administration of angiotensm into the perfused lateral ventricle of cats produced marked neural hypertensive effects which were abolished or markedly attenuated by electrolytic cerveuu isold section as well as by catheterization of the cerebral aqueduct. Also in this study it was observed that injection of angiotensin into the posterior hypothalamus did not alter blood pressure. It was concluded that the peptide influenced peripheral blood pressure by an effect on ascending and/or descending midbrain pathways or by bulbar afferent mechanisms. Addition of angiotensin to the perfusing CSF at the termination of the cerebral aqueduct in the present report was essentially non-pressor. This finding indicates that exposure to angiotensin does not activate ascending or descending cardiovascular pathways of bulbar origin. Moreover, since intraventricular administration of angiotensin in these experiments produced pressor responses, whereas more than twice the intraventricular dose applied at the aqueduct terminal did not, it is highly unlikely that the central pressor effect results from angiotensin reaching any subarachnoid structures bathed by the perfusate. The earher observations that the central pressor activity is not due to a direct action on hypothalamic structures and that aqueduct catheterization markedly attentuated this pressor effect (SEVERSet al., 1966a) suggested that the midbrain was the site of action. The results of midbrain lesion studies reported here confirms this hypothesis and suggests that the pressor mechanism requires neuronal structures of the anterior mesencephalon. An afferent mechanism, originating from structures exposed to angiotensin as it traverses the entire aqueduct, must stdl be considered a possibility, but the present study indicates that the ultimate activation of efferent cardiovascular neurons occurs at anterior midbrain levels or higher structures. LINDGREN(1955) reported that mesencephalospinal fibers originating just below the superior colliculus probably merge with descending outflow from the hypothalamus. These pathways were capable of increasing vasoconstrictor outflow to the skin and intestines and also of releasing adrenomedullary catecholamines. NAUTA and KUYPER~(1958) have shown that afferent fiber systems arising from the midbrain could, on an anatomical basis, activate neural and endocrine pathways involved in the stress response. RAICIULESCU and BITTMAN(1966a) demonstrated, by stimulation of the mesencephalic reticular formation, that this area appeared to modulate reciprocally peripheral sympathetic and parasympathetic activity and that the level of carotid baroreceptor excitability was also influenced. Moreover, pre-collicular section attenuated the baroreceptor excitability (RAICIULESCUand BHTMAN, 1966b), which suggests that an afferent mechanism was operant. This observation is consistent with the findings of REIS and CUENOD (1964) who showed that brain structures rostra1 to the classic bulbar vasomotor centers tonically influenced brain-stem mechanisms subserving baroreceptor reflex excitability, rather than maintaining normal blood pressure. Other investigators have also demonstrated that supramedullary structures are required for baroreceptor activity (MANNING, 1965 and ALEXANDERand DECUIR, 1966). Thus, midbrain structures appear to be involved in mechanisms which modify both the level of sympathetic outflow and baroreceptor activity. As pointed out by MCCUBBINet al. (1965), an indirect action of angiotensin on the sympathetic nervous system, “along with an upward resetting of the carotid sinus buffering mechanisms, might logically account for the neural component of chronic renal hypertension.” The pressor activity of angiotensin originating at midbrain levels, considering

204

W. B. SEVERS, ANNEE. DANEL~and J. P. BUCKLEY

the influences of this region on the cardiovascular system, could account for the neural component of renal hypertensive disease. Conflicting evidence in the literature exists with regard to the presence of a central hypertensive effect of angiotensin in dogs. BUCKLEYet al. (1963) and BENJZTATO et al. (1964) have detected central hypertensive effects in the dog cross circulation preparation. BIANCHI et al. (1960) administered 7 pg/kg of synthetic angiotensin into subarachnoid CSF in dogs and did not observe changes in blood pressure. This finding is consistent with data obtained in our experiments in which an~otensin failed to produce pressor responses when only subarachnoid structures were exposed to the perfusing medium. KANEICOet al. (1960) administered 5-100 units of angiotensin contained in volumes of 0.2 to 05 ml into the lateral ventricles of dogs but did not obtain a pressor effect. As in cats (SM~OKLERet al,, 1966), we have observed that in dogs the magnitude of central pressor responses to angiotensin is dependent on resting blood pressure. Angiotensin (4 tcg>, administered to three c~or~ose-anes~eti~d dogs having high basal blood pressures (diastolic pressure of 150 mm Hg or higher) produced comparatively mild hypertensive effects (20-40 mm Hg) (unpublished observations). The high blood pressures are in agreement with the observations of BASSand BUCKL.EY (1966) who reported that chloralose-anesthetized dogs maintain a stable hypertensive blood pressure and an elevated heart rate. In the dog lateral ventricle perfusion experiments reported in this communication, the animals were given pentobarbital until a stable blood pressure was maintained with a diastolic pressure of less than 100 mm Hg. Under these conditions centrally administered angiotensin exerted greater pressor effects than in the chloraloseanesthetized animals. The information available suggests that angiotensin does produce central hypertensive effects in dogs when ventricular, but not subarachnoid, structures are exposed to the peptide. AcknowledgemePl&--The authors wish to thank Mr. Gust J, Markis for his technical assistance.

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