Naunyn-Schmiedeberg'sArch Pharmacol (1990) 342:436 - 440

Naunyn-Schmiedeberg's

Archivesof Pharmacology © Springer-Verlag 1990

Quantitative evidence of peripheral conversion of angiotensin within the human leg: effects of local angiotensin-I administration and angiotensin-converting enzyme inhibition on regional blood flow and angiotensin-II balance across the leg S. Gasic 1, G. Heinz 1, and C. Kleinbloesem 2 1 I. MedizinischeUniversit/itsklinik,Divisionof ClinicalPharmacology,Lazarettgasse14, A-1090 Vienna, Austria 2 ResearchDepartment, Hoffmann-LaRoche, Basel, Switzerland Received January 16, 1990/Accepted April 24, 1990

Summary. The renin-angiotensin system relevantly contributes to the maintenance of systemic vascular tone and there is experimental evidence that large amounts of angiotensin-converting enzyme (ACE) are present in peripheral vascular tissues, including resistance vessels. To determine and quantify peripheral vascular conversion of angiotensin-I (ANG-I) to angiotensin-II (ANGII) across the human leg, the response of regional blood flow to local regional intra-arterial infusion of ANG-I and changes in associated ANG-II balance were evaluated during ANG-I infusion and following additional ACE inhibition. Ten sodium-loaded healthy men were enrolled in the study. Following cannulation of both femoral arteries and the right femoral vein, leg blood flow was determined (indocyanine-green dye-dilution method) at baseline conditions and during constant intra-arterial infusion of haemodynamically ineffective doses of ANG-I as well as following concomitant intra-arterial administration of low doses of the non-sulfhydril ACE inhibitor cilazapril. From the transfemoral arterio-venous differences in ANG-II plasma concentrations and the corresponding regional blood (plasma) flow, the ANG-II balance across the leg was calculated. Systemic blood pressure did not change throughout the trial, indicating that no major systemic effects were present during ANG-I infusion or concomitant ACE inhibition. Moreover, arterial ANG-II plasma concentrations were not significantly changed by ANG-I infusion. Leg blood flow decreased to below baseline values following ANG-I infusion, increasing again then in a dose-dependent manner during concomitant cilazapril administration. The calculated baseline ANG-II balance across the leg revealed a net extraction in 6 out of 10 subjects and a net ANG-II formation in 4. Following ANG-I, a shift towards net ANG-II formation or decrease in extraction was seen in 8 subjects, while 2 had no change in ANG-II balance. Send offprint requests to S. Gasic at the above address

During concomitant ACE inhibition, ANG-II balance was again shifted towards net extraction or reduced formation. Our results confirm that, in man, considerable regional arterio-venous differences in ANG-II plasma concentrations are present, resulting in either net transfemoral extraction or net formation of the peptide. It is suggested that systemic vascular conversion of circulating ANG-I might contribute to the maintenance of peripheral vasuclar tone in man.

Key words: Vascular angiotensin conversion - Angiotensin-converting enzyme (ACE) inhibition - Regional angiotensin-II balance

Introduction It has been suggested that the renin-angiotensin system contributes to the maintenance of peripheral vascular resistance and thus might play an important role in the pathophysiology of arterial hypertension (Swales 1979; Miyazaki et al. 1986). From experimental studies it is well recognized that the vascular walls are suitably equipped with enzymes to form angiotensin-II (ANG-II) from circulating angiotensin-I (ANG-I). It has even been demonstrated that this property is common to many vascular tissues and probably accounts for generation of most circulating ANG-II (Kreye and Gross 1971; Aiken and Vane 1972; Fei et al. 1981 ; Campbell 1985). Studies in several species suggest that large amounts of angiotensin-converting enzyme (ACE) are present in systemic vascular tissues, including resistance vessels (Oparil et al. 1979; Miyauchi et al. 1981). However, as yet, little information is available indicating the presence of ACE activity within human resistance vessels (Webb and Collier 1986), and to our knowledge no data have

437 yet been collected o n the q u a n t i t a t i v e assessment o f peripheral regional a n g i o t e n s i n - c o n v e r s i o n rates in m a n . I n order to assess a n d q u a n t i f y the peripheral vascular c o n v e r s i o n o f A N G - I to A N G - I I , we investigated the regional h a e m o d y n a m i c responses to local i n t r a - a r t e r i a l i n f u s i o n o f A N G - I a n d calculated the net A N G - I I balance across the h u m a n leg. Moreover, changes in these responses to A N G - I i n f u s i o n d u r i n g c o n c o m i t a n t A C E i n h i b i t i o n were studied.

@ ARTERIAL A N G I O T E N S I N - I I INTRAARTERIAL

Subjects. Ten healthy volunteers (26.9 _+ 1.5 years, 74.5 + 1.7 kg,

Protocol design (Fig. 1). After local anaesthesia (lidocaine) plastic cannulae were inserted percutaneously into both femoral arteries and into the right femoral vein. The right femoral artery was used for infusion of indocyanine-greendye (ICG) and ANG-I as well as ofcilazaprilat (see below). Arterial blood samples for determination of systemic ICG and ANG-II plasma concentrations were obtained from the left femoral artery. After catheterization a primed-constantinfusion of ICG into the right femoral artery was started (bolus: 12 mg; constant infusion: 0.6 mg/min). The method for determination of estimated leg blood (ptasma) flow by ICG-dilution has been described previously (Jorfeldt and Wahren 1971; Lochs et al. 1988). In order to avoid turbidity, 5% sodium desoxyeholate (Fluka, Buchs, Switzerland) was passed through a millipore filter (0.45 gin) and added to plasma before measurement of ICG concentrations in plasma from the left femoral artery and the right femoral vein (Pasden et al. 1981). Arterial blood pressure was measured in the left femoral artery (Statham P23 ID/3 Transducer; Gould Inc., Oxford, Calif., USA). Peripheral leg vascular resistance (PLVR) was calculated as: PLVR = mean arterial blood pressure (mmHg) x 80/estimated leg blood flow (1/min) and expressed as dyn - s • cm s. 10-2. After determination of baseline values (4 measurements over 30 rain) and following a saline infusion, dose-dependent blood pressure responses to intra-arterially administered ANG-I (Senn Chemicals, Buchs, Switzerland) were assessed. ANG-I was infused at constant rates in a fixed dose sequence of 0.05, 0.1, 0.2, 0.3, 0.4, 0.6, 0.8 gg/ rain over 3 rain each, until a slight rise in intra-arterial mean blood pressure (left femoral artery) of 5 - 1 0 mmHg was achieved. An interval of 30 min was allowed to elapse after the last of these ANG[ infusions, and a constant intra-arterial ANG-I infusion was then started with individuallydetermined infusion rates; for each subject, the infusion rate selected was two dose steps below the critical ANOI rate at which slight increases in systemic blood pressure had been observed. This systemically ineffective infusion rate was then used throughout the trial ( 0 - 9 0 min). From 0 to 30 rain ANG-I was infused alone. Concomitant constant intra-arterial (right femoral artery) infusion of cilazaprilat - the active diaeid form of the nonsulfhydril ACE inhibitor cilazapril (Natoff et al. 1985) - was given at a dose rate of 0.1 rag/30 rain from 30 to 60 min and of 0.2 mg/ 30 min from 60 to 90 min. These cilazaprilat doses were 10 and 20 times lower than the systematically effective doses of the drug (unpublished data). Haemodynamic parameters and the ICG and ANG-II plasma concentrations were determined during the baseline period (4 measurements over 30 min) and throughout the trial: triple determinations of all data were performed for each infusion period at 10,

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Methods 180.3 _+2.5 cm) were enrolled in the study after giving written informed consent. None was taking any drugs at the time of the study. The study protocol was approved by Ethical Committee of the hospital. In order to standardize the sodium balance, all subjects received, in addition to their usual salt intake, 3 g per day of sodium chloride for 3 days before the study. Trials were performed with subjects supine after an overnight fast (10-12 h).

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20, 30 rain, 40, 50, 60 rain, and 70, 80, 90 min, respectively, and mean values of these triple measurements were evaluated for each observation period. The regional net ANG-II balance was assessed by multiplyingregional leg plasma flow (ml/min) with the transfemoral arterio-venous plasma concentration differences (pg/ml), a positive product indicating net ANG-II extraction and a negative product, net ANG-II formation (production). The pressure responses to ANG-I were tested in each individual prior to the study, and in each subject tachyphylaxis in response to intra-arterially administered ANG-I and concomitant saline (= cilazapril-placebo) could be excluded by assessing leg blood flow over a period of 60 min. ANG-II plasma concentrations were assessed after extraction from plasma by bonded-phase silica cartridges (Sep Park C18) and measured by radioimmunoassay with ANG-I as recovery-marker (Natoff et al. 1985).

Statistics. Data in the text, tabIes and figures are presented as means + SEM. Student's t-tests (as appropriate) were employed for statistical evaluation, and P < 0.05 was considered significant. Bonferroni corrections for multiple comparisons were made as necessay, and all P-values represent results after that correction.

Results The titrated A N G - I dose for c o n s t a n t systematically ineffective i n f u s i o n was 0.05 g g / m i n in subject 3; 0.1 ~tg/min in subjects 1, 2, 4, 7, 8, 9, a n d 10, a n d 0.2 g g / m i n in subjects 5 a n d 6.

438 Table 1. Angiotensin-IIplasma concentrations and estimated leg plasma flow at baseline conditions and during drug infusion Subject

Angiotensin-I infusion Baseline

Cilazaprilat 0.1 mg/30 min

Cilazabrilat 0.2 rag/30 min

ANG-IIa ANG-IIv ELPF

ANG-IIa ANG-Itv ELPF

ANG-IIa ANG-IIv ELPF

ANG-IIa ANG-IIv ELPF

1 2 3 4 5 6 7 8 9 ~0

26.6 14.6 23,4 29,7 80A 62.8 37.2 26.7 61,7 25,0

19.5 5.4 10.2 ~9.3 14.0 77.9 53.5 36.3 27.7 38.2

351.0 530.3 262.3 295.6 187.5 162.5 168.8 ~12.3 285.3 184.0

30.4 12.1 8.3 42.2 74.3 40.4 26.7 30.5 64.1 32.0

26,5 13.7 30,4 34.9 76.6 79.9 77.1 97.4 84.4 50.9

291.8 229.3 125,8 254.0 79.0 107,5 135.0 68,0 172.8 156,0

22.6 74.4 63.3 26.8 33.0 69.1 45.0

16,3 8,8 13.5 73.8 71.4 35.3 14,6 68,7 29.7

393,0 396.7 t61,7 274.7 104.0 202.0 195.7 95.7 225.3 158.3

19,7 6.8 6.2 14.2 74.9 135.5 29.9 36.6 66.0 26.8

12.0 9.8 16.8 -72.0 62.1 21.7 13.9 45.0 28.5

439.3 526.7 183.0 286.3 132.0 356.7 271.3 105.0 254.7 167.3

Mean ___SEM

38.8 6.8

32.2 6.8

254.0 8.5

36.1 6.5

57.2 9.3

161.9 23.7

47.7 8.0

36.9 9.0

220.7 33.5

41~7 12.7

31.4 7.7

272.2 43.1

t _

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