Life Sciences Vol . 20, pp . 1855-1862, 1977 . Printed in the U .S .A .

Pergamon Press

EFFECTS OF TWO ANGIOTENSIN II ANALOGUES ON BLOOD PRESSURE IN NORMAL SUBJECTS WITH VARIOUS SODIUM BALANCES Toshio Ogihara, Takeshi Hata, Hiroshi Mikami, Takashi Mandai and Yuichi Kumahara Department of Medicine and Geriatrics, Osaka University Medical School, Fukushima-ku, Osaka, 553, Japan (Received in final form May 2, 1977) Summary The antagonistic potencies and agonistic effects of two clinically available angiotensin II analogues, 1-sarcosine,8-isoleucine angiotensin II and 1-sarcosine,8-alanine angiotensin II, were compared in normal subjects in various sodium balances . Both compounds had an agonistic pressor effect and this was affected by the sodium balance ; in the low sodium state, the agonistic effect of both compounds was decreased significantly and that of 1-Sar,8-Ala angiotensin II nut seen . The agonistic effect of 1-Sar,B-Ile angiotensin II was greater than that of 1-Sar,B-Ala angiotensin II in all sodium balances . The antagonistic effects of both compounds were also affected by the sodium balance, and were greatest in the low sodium state . In subjects on low sodium diet or regular diet 1-Sar,8-Ile angiotensin II had the same or more antagonistic action than 1-San, 8-Ala angiotensin II , but in those on high sodium diet the reverse was observed . In clinical and pharmacological studies, the sodium balance of patient should always monitored . Preteatment of patients for sodium depletion is necessary to prevent side effects due to the agonistic pressor effects of these compounds . Agents that block the renin-angiotensin system should provide valuable information on the role of this system in the maintenance or pathogeneis of hypertension and the edematous state in humans . Two kinds of peptides are available clinically as blocking agents, a competitive angiotensin II analogue and a bradykinin potentiator (converting enzyme inhibitor) . There are reports on the clinical use of angiotensin II analogues . 1-Sarcosine,8-alanine angiotensin II (1-Sar,B-Ala A II) was first tested in humans by Brunner et al . (1), and since then the merits and limitations of this analogue have beendiscussed (2,3) . The other analogue, 1-sarcosine,8-isoleucine angiotensin II (1-Sar,8-Ile A II) was first tested on humans by our group (4) . Both analogues had an antagonistic effect in the high reninemic state and an agonistic effect in the low reninemic state (1,2,3,5), but the pressor response to these compounds limits their use in diagnosis . A less agonistic and more antagonistic compound would be better for detecting renin-dependency in hypertensives . In the present study, we compared the antagonistic potencies and agonistic effects of these two analogues of angiotensin II in normal volunteers with 1855

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various sodium balances . Materials and Methods Five healthy male volunteers aged at 25 to 33 years were chosen for this study . The following experiments were done in three different sodium balances, i .e ., i) normal sodium state ; unrestricted diet, ü) high sodium state ; normal diet supplemented with 6g sodium chloride and iii) low sodium state ; sodium free diet with 40mg of furosemide given orally on the first day . Experiments were done on the 3rd and 4th days in each sodium state . Urinary sodium excretion (UN~V), basal plasma renin activity (PRA) and the plasma aldosterone (PAC) were monitored each day, and subjects were kept supine for at least one hour before and during the experiments . Both 1-Sar,8-Ile A II and 1-5ar,8-Ala A II were synthetized by Dr . Sakaki bars, Peptide Institute, Protein Research Foundation, Osaka . Synthetic angiotensin II (Hypertensin, Ciba) and angiotensin II analogûes were diluted with normal saline and infused intravenously with a Harvard infusion pump . Blood pressure was measured every five minutes with a sphygmomanometer . Studies on the effects of 1-Sar,8-Ile A II and 1-Sar,8-Ala A II were done on the 3rd and 4th days,respectively, on each sodium balance. The rates of infusion of these compounds were increased gradually after the 10 minutes' infusion . Experiment 1 , Evaluation of agonistic effect . 1-Sar,8-Ile A II and 1-Sar,B-Ala A II was infused at the rates of 120,240, 600, 1200, 2400 ng/kg/min ., and 0 .1, 1, 5, 10 ug/kg/min ., respectively . Experiment 2 , Evaluation of antagonistic effect (A) . Angiotensin II was infused throughout the experiment at a rate sufficient to maintain the mean blood pressure at 30 mmHg above the basal level . After the first 10 minutes of infusion, the infusion of the analogues was also start ed, using the same infusion rates as in Experiment 1 . Experiment 3 , Evaluation of antagonistic effect (B) . The pressor effects of Angiotensin II infused at the rates of 10, 20, 40, 80 ng/kg/min . were compared to those with 120 ng/kg/min . of 1-Sar,B-Ile A II or 500 ng/kg/min . of 1-Sar,8-Ala A II . The infusion of either analogue was started 10 minutes before that of Angiotensin II and was continued at a constant rate throughout the experiment . Analytical methods PRA was measured by radioimmunoassay of angiotensin I generated during incubation for two hours at pH 5 .5 using a commercial kit . PAC was measured by radioimmunoassay using a commercial kit . UNaV was measured on a Tecknikon autoanalyzer . Statistical analysis was done by analysis of variance (6) . Results The values for UNaV, PRA and PAC of normal subjects on each sodium balance are summarized in Table 1 . The mean values were calculated from the values obtained in duplicate determinations on two separate days . TABLE 1 UNaV, PRA and PAC on each sodium balance (mean ± S .E .M .) Sodium Balance Unrestricted state High sodium state Low sodium state

UNaV(mEq/day) 157 ± 11 256 ± 24 18 ±

3

PRA(ng/ml/hr) 1 .5 ± 0 .4 0 .5 ± 0 .1

10 .4 ± 1 .5

PAC(pg/ml) 49 ± 11 18 ± 3

223 ± 22

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Experiment 1 . The blood pressure responses to infusions of 1-Sar,8-Ile A II end 1-Sar, 8-Ala A II in five normal volunteers on three different sodium balances are shown in Figs . 1 . and 2 . 1-Sar,B-Ile A II evoked pressor responses in all three sodium balance states ; the changes in the mean blood pressure (ABP) ranged from 5-9 mmHg, 12-18 mmHg and 18-26 mmHg in low sodium, normal sodium and high sodium states, respectively . 1-Sar,8-Ala A II evoked pressor responses in the normal and high sodium states, but at a high dose it evoked slight reduction in blood pressure in the low sodium state; the mean blood pressure changes (ABP) were 1--7 mmHg, 8-15 mmHg and 12-18 mmHg in the low, normal and high sodium states, respectively . FIG . 1 ~I~

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Experiment 2 . The results are summarized in Figs . 3 and 4. Both analogues caused dose dependent reduction in the blood pressure in all three sodium balances, and the reductions by both analogues were significant in the low sodium state . With both analogues, the extents of blood pressure reduction in the normal and high sodium states were very similar . 'l'o compare the potencies of the antagonistic actions of the two analogues in the three sodium balance states, we raised the blood pressure by infusion of sitgiotensin II and then measured the doses of the analogues necessary for its 50 $ reduction. The results are shown in Table 2 .

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Angiotensin Analogues on Blood Pressure

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Vol . 20, No . 11, 1977

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TABLE 2 Comparison of antagonistic potencies Sodium balance Unrestricted state High sodium state Low sodium state

1-Sar,8-Ile A II 1 .0 1 .0 1 .0

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Dose ratio causing 50 ~ reduction of the mean blood pressure, raised about 30 mmHg by exogenous angiotensin II . Values are expressed taking the dosis of 1-Sar,8-Ile A II as 1 .0 . ExP eriment 3 . The pressor effect of angiotensin II was significantly suppressed by infus ion of either of the analogues and the suppression was greatest in the low sodium state (Figs . 5 and 6) .

AnRiotensin Analogues on Blood Pressure

Vol . 20, No . 11, 1977

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Discussion Both currently available angiotensin II analogues ; 1-Sar,B-Ala A II and 1-Sar,8-Ile A II , have intrinsic agonistic activity . In the report of Case et al ., dûring a normal sodium intake, 1-Sar,B-Ala A II (Saralasin) produced â mild pressor response in 50 per cent of the patients with normal FRA and a pressor response in all of the patients with low PRA value . They suggested this drug is a partial competitive agonist of angiotensin II and provide only a rough physiologic validation for renin profiling (3) . Clinical superiority of the converting enzyme inhibitor over Saralasin was also suggested because Saralasin may underestimate renin dependency (7) . We also found that 1-Sar,B-Ile A II usually evoked a pressor response in the normoreninemic and hyporeninemic patients we tested, and especially in patients with primary aldosteronism who were greatly depleted of endogenous angiotensin II (5,8) . These analogues of angiotensin II are known to act agonistic on the adrenal medulla and release catecholamines (9,10) . A marked and rapid rise of blood pressure was observed in patients with pheochromocytoma on 1-Sar,8-Ile A II infusion (8) . A hypertensive crisis was also been observed when a patient with pheochromocytoma and neurofibromatosis was given Saralasin (11) . The present study showed that both analogues had an agonistic pressor effect in normal subjects, and that this effect was influenced by the sodium balance ; in the low sodium state, the agonistic effect of both analogues was reduced significantly and there was no pressor response to 1-Sar,8-Ala A II . 1-Sar,B-Ile A II had a greater agonistic effect than 1-Sar,8-Ala A II in all sodium balances . The antagonistic effects of both compounds were also affected by the sodium balance, being greatest in the low sodium state . Furthermore, the ratio of~the antagonistic potencies of the two compounds also varied with the sodium balance ; with low sodium and regular diets the antagonistic action of 1-Sar,8Ile A II was equol to, or more than that of 1-Sar,8-Ala A II , but with high sodium diet the reverse was observed . It is well known that the sensitivity of the vascular receptors to angiotensin II is modified by the level of endogenous angiotensin II , â.nd that this in turn is affected by the sodium balance(12,13) . Recently it was found that the vascular and adrenal receptors for angiotensin II changed in number and affinity with the sodium balance (14), and that the level of circulating endogenous angiotensin II, rather than sodium ion per se, was the chief cause of these changes (15) . The changes in the agonistic effects and relative antagonistic potencies of the two compounds which we observed in this work with different sodium balances also seems to involve a change in reactivity of angiotensin II receptors in the vascular smooth muscle . In clinical and pharmacological studies, the sodium balance of patients should always be monitored, and pretreatment for sodiwn depletion may be necessary to prevent cardiovascular accidents caused by the agonistic effect of these compounds . However, Case et al . claimed that prior sodium depletion does not measure intrinsic renin dependency of the blood pressure but the reactivity of the renin-angiotensin system to sodium depletion (3) . Ac knowledgments We are grateful to Dr . and Mrs . A . Ichihara for their help in preparing this manuscript, and also to Miss Tomiko Kawano for secretarial assistance . This work was supported by a grant from the Ministry of Education, Japan .

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References 1 . H. D . BRUNNER, H . GAVRAS and J . H . LARAGH, Lancet 2 1045-1048, 1973 . 2 . D. H . P . STREEREN, G . H . ANDERSON, J . M . FREIBERG ând T . G . DALAKOS, _N . Engl . J . Med . 292 657-662, 1975 . 3. D. B . CASE,J . M . WALLACE, H . J . KEIM, J . E. SEALEY and J . H . LARAGH, _Am. J. Med . _60 825-836, 1976 . 4 . T. OGIHARA, T . YAMAMOTO and Y . KUMA1-L4RA, Lancet _1 219, 1974 . 5 . T. OGIHARA, T . YAMAMOTO and Y . KUMAHARA, Ja Circul . J . 38 997-1003, 1974. 6. R . C . CAMPBELL, Statistics for Biologist 2nd Edition) p 188, Cambridge University Press, England, 1974 . 7. D . B . CASE, J . M. WALLACE, H. J . KEIM, M . A . WEBER, J . I . M . DRAYER, R . P . WHITE, J . E . SEALEY and J. H. LARAGH, Am . J_. Med . 61 790-796, 1976 . . Jam. _Soc . _Int . M_ed . 8. T . YAMAMOTO, T . OGIHARA, Y . KUMAIIARA and T HATA, _J 65 464-470, 1976 . (In Japanese) 9 . S. SEN, R. R . SMEBY and F . M. BUMPUS, Proc . _Soc . Expl . Biol . M_ed . _147 847849, 1974 . 10 . M . T . PEACH, Circul . Res. Suppl . 28 $ 29, _II 107-116, 1971 . . C . KEM, J . R. HIGGINS and E . D. 11 . F . G . DUNN, J . G . R. DÉCARBALHO, D FRÖHLICH, N. Engl . J . Med. 29 5 605-607, 1976 . 12 . N . M . KAPLNN and J . 3. SILAH,J . Clin . Invest . 43 656-669, 1964 . 13 . N . K . HÖLLENBERG, H. S . SOLOMON D . F . ADAMS, H . L. ABRAMS and J . P. MERRILL, Circul . Res . _31 750-757, 1972 . 14 . H . THURSTON, Am . J .Med . 61 768-778, 1976 . .D . BRIGGS, J .J . BROWN, R. FRASER, A . F . LEVER, 15 . J . DEHENEFFE, V. CUESTA, J J . J . MORTON, J . I . S . ROBERTSON and M . TREE, Circul . _Res . _39 183-190, 1976 .