Journal of the Autonomic Nervous System, 39 (1992) 119-126 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1838/92/$05.00

119

JANS 01282

Chronic central administration of enalaprilat lowers blood pressure in stroke-prone spontaneously hypertensive rats L i n a T . J a b l o n s k i s a,b, P a u l F. R o g e r s a Y v o n n e K. L u n g e r s h a u s e n

a and Peter R.C. Howe a

a Division of Human Nutrition, Commonwealth Scientific and Industrial Research Organisation, and h Department of Physiology, University of Adelaide, Adelaide, Australia

(Received 25 November 1991) (Revision received 18 February 1992) (Accepted 13 March 1992)

K e y words: A C E I n h i b i t o r ; Blood pressure; Sympathetic n e r v o u s system; A n g i o t e n s i n II; S p o n t a n e o u s l y hypertensive rat

Abstract Earlier studies on the cardiovascular effects of intracerebroventricular (i.c.v.) administration of angiotensin converting enzyme (ACE) inhibitors implicate angiotensin II (AII) present in the central nervous system in the pathogenesis of hypertension. We have now examined whether central All contributes to the maintenance of established hypertension in adult stroke-prone spontaneously hypertensive rats (SHRSP). The ACE inhibitor, enalaprilat, was infused i.c.v, for two weeks at a rate of 5 /zg/h via osmotic minipumps. Control rats were either untreated or infused with saline. Mean arterial pressure (MAP), measured via an indwelling catheter, fell within 24 h in the enalaprilat-treated rats and remained at least 30 mmHg lower than in controls. This difference persisted after intravenous (i.v.) administration of a vasopressin (AVP) antagonist but was eliminated by subsequent ganglion blockade with i.v. pentolinium. Without prior administration of the AVP antagonist, however, the reductions of MAP after pentolinium were smaller. The reduction was still attenuated in treated rats compared with controls but there was a significant difference in the residual MAP. Circulating catecholamine levels were reduced by central ACE inhibition. However, pressor responsiveness to i.v. phenylephrine was unaffected. The results suggest that, in SHRSP, central ACE inhibition lowers blood pressure by reducing sympathetic outflow, implying that central All has a tonic sympathoexcitatory effect in this strain.

Introduction A n g i o t e n s i n II ( A l l ) is a p o t e n t pressor a g e n t f o u n d in the circulation a n d in various cardiovascular structures i n c l u d i n g the heart, kidneys, blood vessels a n d a d r e n a l glands [15]. However, there is c o n s i d e r a b l e e v i d e n c e that A l l may par-

Correspondence to." P.R.C. Howe, CSIRO, Division of Human Nutrition, P.O. Box 10041 Gouger Street, Adelaide, SA 5000, Australia.

ticipate in blood pressure r e g u l a t i o n by acting not only p e r i p h e r a l l y b u t also in the central nervous system. A n i n d e p e n d e n t r e n i n - A I I system has b e e n shown to exist in the b r a i n [14]. A c u t e i.c.v. a d m i n i s t r a t i o n of A l l elicits pressor responses which have b e e n a t t r i b u t e d to increased sympathetic activation, increased release of A V P a n d c o r t i c o t r o p h i n ( A C T H ) or d e c r e a s e d reflex b r a d y c a r d i a [14]. Moreover, acute i.c.v, administ r a t i o n of A C E inhibitors [10,16,17] or A l l antagonists [12] has b e e n shown to lower blood pres-

120

sure in spontaneously hypertensive rats (SHR), suggesting that central AII may be contributing to the elevation of blood pressure in this strain. This possibility is further supported by experiments in which chronic i.c.v, administration of ACE inhibitors, in doses too small to act peripherally, attenuated the development of hypertension in young SHR [2-4,13]. This attenuation was attributed to a depressed responsiveness of the vasculature to either vasoconstrictors or centrally evoked sympathetic nerve stimulation. However, the mechanism for translating the action of an ACE inhibitor at a central site into a peripheral effect has not yet been elucidated. The aims of this study were to determine whether chronic i.c.v, administration of an ACE inhibitor can lower blood pressure in adult stroke-prone SHR (SHRSP) with established hypertension and to further examine mechanisms by which a central action of the drug might affect peripheral determinants of blood pressure. In a previous investig~/tion of the effects of peripheral ACE inhibition on hypertension development in SHRSP [8], we gave enalapril orally. However, because of uncertainty regarding the extent to which enalapril can be hydrolysed to its active diacid form, enalaprilat, in the central nervous system [1,18], enalaprilat was used for the present study, although we note recent evidence indicating that enalapril is active when infused i.c.v, in normotensive Wistar Kyoto rats (WKY) [6].

Materials and M e t h o d s

Experimental animals Male SHRSP obtained from our breeding colony were used in two experiments: 18 aged 5 - 7 months and weighing 330-390 g were used in experiment 1; 20 aged 7 months and weighing 300-360 g were used in experiment 2; ages were dependent on availability. Rats were caged individually and given commercial chow and drinking water ad libitum. The latter contained Clavulox antibiotic (Beecham Veterinary Products, Victoria, Australia) at a final concentration of 0.2 mg/ml, as a prophylactic measure.

Blood pressure measurements Catheters were implanted for direct measurement of blood pressure, as described previously [9]. Rats were injected intraperitoneally with a short-acting anaesthetic comprising sodium methihexitone (Eli Lily, NSW, Australia, 40 mg/kg) and sodium pentobarbitone (Bomac Laboratories, NSW, Australia, 30 mg/kg). A vinyl catheter with a fine, non-occluding teflon tip was inserted into the lower abdominal aorta. In experiment 1 only, a Silastic catheter was also inserted proximally in the right jugular vein for drug administration. Catheters were exteriorised intrascapularly and sealed. After allowing two days for recovery, blood pressure readings were taken daily from conscious, unrestrained rats by connecting arterial catheters to a Statham P23D pressure transducer and monitoring continuous pulsatile traces on a Neotrace physiological recorder (Neomedix Systems, Australia). The resting value for MAP was taken when heart rate (HR) had settled to a steady minimum level.

Effects of chronic intracerebroventricular drug infusion After resting MAP and H R had been recorded on three consecutive days, rats were re-anaesthetised and infusion cannulae were implanted as follows. The flat tip of a bent 27-gauge stainless steel needle was stereotaxically located in the right lateral cerebral ventricle (1.5 mm lateral, 4.5 mm ventral to bregma) and secured with dental cement to a stainless steel screw in the skull. This cannula was then connected with vinyl tubing to an osmotic minipump (Aizet model 2002, Alza Corporation, Palo Alto, CA) which was implanted subcutaneously in the interscapular region. In half of the rats used in each experiment, the pumps and connecting tubes were filled with a solution of enalaprilat maleate (MK-422, Merck, Sharpe and Dohme, Australia) which had been prepared by dissolving the salt in a small volume of ethanol then diluting to 10 m g / m l in sterile saline. The nominal reservoir volume and pumping rate of the pumps were 220 izl and 0.5 p,l/h respectively, which provided continuous infusion of the drug at a rate of 0.12 m g / d a y for at least

121 and sodium E G T A (1% w / v ) then frozen at - 8 0 ° C for subsequent radioenzymic assay of catecholamines [9].

two weeks. In experiment 1, rats which served as controls did not receive minipumps; in experiment 2, the controls were infused with saline. Daily measurements of resting MAP continued for approximately two weeks. To test for the possibility of peripheral ACE inhibition, pressor responses to a 25 /xg i.v. bolus dose of angiotensin I (AI, Sigma, St. Louis, MO) were tested in a few treated and control rats taken at random in the first experiment. Finally, i.c.v, infusions were verified in each rat by observing the intracerebral distribution of Evans blue dye injected via the connecting tube.

Effects of acute peripheral drug admin&tration To see whether any antihypertensive effect of central ACE inhibition could be attributed to centrally-mediated changes in autonomic nerve activity, after taking the final daily reading of resting MAP, peripheral autonomic transmission was blocked by i.v. or intra-arterial injection of pentolinium tartrate (Sigma, St. Louis, MO, 2 m g / k g ) and the resultant basal level of MAP was then recorded. In experiment 1 only, vasopressor responsiveness was tested by measuring the peak rises in MAP elicited by bolus i.v. injection of graded doses of phenylephrine. Sigmoidal curves were fitted logistically to responses of individual animals and derivations of the maximum response and EDs0 were averaged for rats in each treatment group. In experiment 2, the possible influence of vasopressin on blood pressure in the treated rats

Plasma catecholamine measurements In experiment 2, at both 7 and 14 days after commencement of the i.c.v, infusions, blood samples were taken under resting conditions (immediately after recording the resting value of MAP). A 300 p.l blood sample was withdrawn from the arterial catheter into a chilled, heparinised tube and rapidly centrifuged at 4°C. The plasma was mixed with dithiothreitol (1% w / v )

Experiment

250

MAP (mm Hg)

• 0

1

Experiment

250

untreated ( n = 7 ) icv e n a l a p r i l a t ( n = 9 )

• icy saline ( n = l O ) 0 icv e n a l a p r i l a t ( n = 9 )

T

200

200

1,50

150

PRETR£ATN£NT

PIIETREATMI~NT

100

I

0

2

I

10 15 DAYS AFTER CANNULATION

I

20

100

0

5 10 15 DAYS AFTER CANNULATION

20

Fig. 1. Effect of chronic i.c.v, infusion of enalaprilat on repeated daily measurements of MAP in conscious SHRSP. Control rats were untreated (experiment 1) or infused with saline i.c.v. (experiment 2). Points represent mean + S.E.M.; numbers of rats are shown in parentheses.

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was examined by giving an intra-arterial injection (66 /xg/kg) of [1-(B-Mercapto-B,B-cyclopentamethylene propionic acid), 2-(O-Methyl)-tyrosine] ArgS-Vasopressin (Peninsula Laboratories, CA), a specific antagonist of the pressor effect of AVP, after the measurement of resting MAP. This treatment effectively blocked pressor responses to intra-arterial administration of AVP. When MAP had reached a new resting level (5-10 min later), pentolinium was then administered.

Results Arterial catheters functioned for at least two weeks in 29 of the 38 rats. Placement of i.c.v. cannulae was satisfactory in all cases. Figure 1 shows the resting levels of MAP in enalaprilattreated and control rats from both experiments which were recorded daily (excepting weekends), starting two days after catheterisation. The pretreatment level of MAP (averaged over three

consecutive days) was similar in treated and control rats (176 + 4 and 177 + 7 mmHg respectively in experiment 1; 184 + 4 and 177 + 4 mmHg respectively in experiment 2; n = 9, 7, 9 and 10 respectively). One day after commencing i.c.v, infusion, MAP in the enalaprilat-treated rats had fallen significantly compared with either untreated controls (experiment 1 : - 2 5 _+ 4 vs. - 4 + 6 mmHg, P < 0.05) or saline-infused controls (experiment 2: - 42 + 8 vs. - 12 + 6 mmHg, P < 0.01). After the initial fall, MAP rose gradually in all rats but to a slightly lesser extent in those receiving enalaprilat. Thus, in both experiments, MAP remained on average more than 30 mmHg less in the treated rats than in either control group ( P < 0 . 0 0 1 , split-plot analysis of variance for repeated measures). Using the same statistical treatment, there was no significant difference between the two experiments in the post-infusion measurements of resting MAP in either the enalaprilat-treated groups or the control groups. Acute pressor re-

PLASMA NORADRENALINE 4OO (pg/ml) 350

PLASMA ADRENALINE 400

I ~ ] icy saline

(pg/mD 350

icy enalaprilat T i

3O0

300

I

T

2OO

I

250

250

i

II

200

I I I

150

150

100

100

I

, \\N

i

50

i I

7 DAYS

14 DAYS

50

7 DAYS

14 DAYS

Fig. 2. Plasma catecholamine concentrations in arterial blood sampled under resting conditions in SHRSP at 7 and 14 days after commencing i.c.v, infusions of enalaprilat or saline. Asterisks denote significant differences (* P < 0.05; ** P < 0.01, Student's t-test).

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sponses to i.v. administration of AI were similar in treated (23 + 3 mmHg, n = 7) and control (25 + 8 mmHg, n = 4) rats. Figure 2 shows plasma catecholamine concentrations in arterial blood sampled from rats in experiment 2 under resting conditions at 7 and 14 days after commencing infusions. The small volume of blood withdrawn had no effect on the resting levels of MAP or HR. Central ACE inhibition for 7 days reduced both noradrenaline and adrenaline to less than half of their respective control levels ( P < 0.01 and P < 0.05 respectively). After 14 days, the plasma noradrenaline concentration was still significantly lower in the treated group (P < 0.01). Table I shows the effects of acute drug administration on the final day of blood pressure recording. In both experiments central ACE inhibition had a sustained hypotensive effect, averaging 42 mmHg after 14 days. However, HR was unaffected. Injection of an AVP antagonist in experiment 2 caused MAP to decline slightly ( < 10 mmHg) and to a similar extent in treated and control rats ( P < 0.01 in each case, paired t-test). After ganglion blockade, however, MAP fell rapidly in all rats ( P < 0.01, paired t-test) but, in both experiments, the reduction of MAP was about 30 mmHg less in treated rats than in controls. In experiment 1 there was still a significant residual difference in MAP between treated and

140

untreated

A MAP 120

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(ram Hg)

100 8O 6O 40 2O

-2

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LOG DOSE PHENYLEPHRINE(pg/kg) Fig. 3. Acute rises in MAP induced by bolus i.v. injection of phenylephrine in untreated and i.c.v, enalaprilat-treated SHRSP in experiment 1. Sigmoidal dose-response curves are derived from averaged parameters obtained for individual rats in each group. Points denote m e a n values ± S.E.M. for maxim u m pressor response and EO50.

control rats. In experiment 2, however, where ganglion blockade was preceded by administration of the AVP antagonist, MAP reached an even lower basal level which was the same for treated and control groups. Bolus administration of phenylephrine to autonomically-blocked rats in experiment 1 elicited acute dose-dependent increases of MAP. Under

TABLE I

Effects o f an A VP antagonist and ganglion blockade on M A P Experiment 1

Resting M A P (mmHg) Resting H R (bpm)

Experiment 2

Control (6)

Enalaprilat (9)

2214- 7 333 _ 15

173±7"* 313 4. 9

M A P (mmHg) after AVPantagonist Reduction of M A P M A P (mmHg) after ganglion blockade Reduction of M A P

Control (6) 211 322

± 8 4. 20

204 4. 8 7.3 4- 0.7 140± 81±

8 8

120±4 * 52+6 *

75 128

5:4 ±10

Enalaprilat (8) 185 304

4- 5 * ± 10

175 ± 5 * * 9.9 ± 1.5 77 96

± 2 4. 6 " *

Values represent m e a n ± S.E.M. for groups of rats (numbers shown in parentheses). Asterisks.denote significant differences from corresponding value for control group (* P < 0.05; * * P < 0.01, Student's t-test).

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these conditions, the responses are not accompanied by changes in H R or cardiac output (unpublished observations) but represent alpha-adrenoceptor mediated vasoconstriction in vivo. The sigmoidal dose-response curves derived from the treated and control groups by averaging the curve-fitting parameters for individual rats are superimposed in Fig. 3. The treated group displayed a slight decrease in sensitivity. However, neither the EDs0 nor the maximum pressor response differed significantly from the control group.

Discussion

Acute i.c.v, injection of captopril has been shown to lower the blood pressure of SHR with established hypertension in some [10,16,17] but not all [1,7] cases. In the latter studies, single i.c.v, injections of enalaprilat also failed to lower blood pressure. Our results, however, show that i.c.v, infusion of enalaprilat in SHRSP can lower blood pressure within 24 h and that this antihypertensive effect can be sustained by chronic treatment. This extends earlier observations by Berecek et al. [2-4] that chronic i.c.v, infusions of both enalaprilat and captopril can attenuate the development of hypertension in young SHR. The ability of central ACE inhibition to lower blood pressure in adult SHRSP, without affecting the pressor activity of AI peripherally, implies that endogenous All in the central nervous system must be exerting a tonic pressor effect in this strain which contributes to the maintenance of hypertension. One possible explanation for the variable effects seen with single i.c.v, injections of ACE inhibitors is that All stored intraneuronally in pressor pathways in the brain is likely to be depleted less readily after synthesis inhibition than All in the circulation. Hence a longer period of inhibition may be necessary to achieve a functional deficit. Alternatively, the antihypertensive effect of the ACE inhibitor may be masked initially by an acute pressor effect which has been attributed to transient elevation of endogenous brain kinin levels [11]. The mechanisms which might mediate a pres-

sor effect of central AII are yet to be elucidated but must involve efferent neural or hormonal processes. Since hypothalamic All can stimulate release of AVP from the pituitary [5] which can then act at vasoconstrictor receptors to raise blood pressure, we tested whether the blood pressure lowering effect of central ACE inhibition was due to decreased secretion of AVP by administering an antagonist of the pressor effect of AVP. However, the antagonist had little effect on MAP in either the ACE-inhibited or the control rats. In fact, the reduction of MAP by the AVP antagonist tended to be greater in the treated rats. This is consistent with an observed increase in the circulating level of AVP in SHR after i.c.v, infusion of captopril [13]. Alternatively, the present results provide two lines of evidence suggesting that the antihypertensive effect of i.c.v, enalaprilat may be due to a decrease in central sympathetic nerve outflow, viz. the accompanying reduction of circulating catecholamines and the smaller fall in blood pressure after ganglion blockade. The reduced plasma concentrations of both noradrenaline and adrenaline indicate suppression of both sympathetic neuroeffector and sympathoadrenal function. These results contrast with those of Okuno et al. who found that plasma catecholamines in SHR infused i.c.v, with captopril from an early age were unaffected [13]. The reason for this discrepancy is not apparent. The fall in blood pressure after ganglion blockade represents withdrawal of sympathetic drive to the heart and blood vessels. This crude index of sympathetic pressor activity was also evaluated by Okuno et al., who found that the percentage reduction of blood pressure following administration of hexamethonium was similar in their captopril-treated and control SHR [13]. However, in absolute terms, approximately 8 mmHg of their observed 23 mmHg difference in resting MAP between treated and control rats was eliminated by ganglion blockade. The residual difference (approximately 15 mmHg) was similar to the residual difference which we observed after ganglion blockade in experiment 1. We attribute this residual difference to an effect of AVP, as it was not evident in the presence of the AVP antago-

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nist in experiment 2. Although AVP appears to make little contribution to the resting level of MAP, we find that it compensates rapidly for the loss of blood pressure following ganglion blockade in SHRSP and to a far greater extent than in WKY (unpublished observations). This should be taken into account when using this approach to assess differences in sympathetic tone between strains. Berecek et al. [2] have shown that chronic i.c.v. captopril administration in SHR attenuates vascular responsiveness to i.v. noradrenaline and to sympathetic nerve stimulation. The nature of this decrease in sensitivity is unclear, although loss of vascular-sensitising effects of AVP and ACTH due to central inhibition of their release was suggested as a possible mechanism. Moreover, the extent to which such changes of resistance in isolated vascular beds could influence arterial pressure is uncertain. In our first experiment we found that pressor responsiveness to phenylephrine was not significantly altered by central ACE inhibition. This appears contrary to the attenuation of pressor responses in vivo observed by Berecek et al. In their case, however, autonomic reflexes were not blocked so that at least part of the attenuation may have been due to the increase in baroreflex sensitivity conferred by ACE inhibition [4]. Moreover, the difference in baseline blood pressure may have influenced the magnitude of their pressor responses. In any case, it is likely that any decrease in pressor sensitivity, in either the whole animal or an isolated vascular bed, is secondary to the sustained reduction of blood pressure. In our experiments it is clear that the ability of chronic central ACE inhibition to lower blood pressure in SHRSP with established hypertension is attributable primarily to a reduction of sympathetic vasomotor outflow, rather than a change in AVP secretion. Attenuation of efferent sympathetic activity also contributes to the enhancement of baroreflex sensitivity by chronic central ACE inhibition, even in normotensive rats [6]. Thus central ACE inhibition can influence blood pressure by suppressing sympathetic cardiovascular regulatory mechanisms. The role of specific All-containing nerve pathways in this process is yet to be elucidated.

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arterial pressure of the spontaneously hypertensive rat, Brain Res., 186 (1980) 499-503. 17 Unger, T., Kaufmann-Buhler, I., Scholkens, B. and Ganten, D., Brain converting enzyme inhibition: a possible mechanism for the antihypertensive action of captopril in spontaneously hypertensive rats, Eur. J. Pharmacol., 70 (1981) 467-478. 18 Unger, T., Schull, B., Rascher, W., Lang, R.E. and Ganten, D., Selective activation of the converting enzyme inhibitor MK421 and comparison of its active diacid form with captopril in different tissues of the rat, Biochem. Pharmacol., 19 (1982) 3063-3070.