AJH
1991; 4.67S-72S
Kenji Mizuno, Susumu Niimura, Makio Tant, Hiroshi Haga, Tadashi Inagami, and Soitsu
Fukuchii
Previously w e reported that immunoreactive a n giotensin II (Ang II) release from isolated perfused h u m a n umbilical v e i n s w a s inhibited b y t h e a n giotensin-converting e n z y m e inhibitor C a p t o p r i l . To further investigate t h e m e c h a n i s m b y w h i c h A n g II is generated i n the blood v e s s e l s of h u m a n s , w e examined the effects of various inhibitors of the r e n i n - a n g i o t e n s i n system (Captopril, delapril, Nacetyl-pepstatin, a n d h u m a n renin inhibitor KRI1314) o n A n g II release from perfused h u m a n u m bilical cord v e i n s i n vitro. Isolated h u m a n umbilical v e i n s w e r e perfused w i t h Krebs-Ringer solution, a n d immunoreactive Ang II released into the perfusate w a s measured di rectly b y u s i n g a Sep-Pak C cartridge connected to the perfusion system. Both Captopril a n d delapril diacid (10" to 5 Χ 10" m o l / L ) , an active metabolite of delapril hydrochloride, suppressed t h e A n g II release i n a dose-dependent fashion; t h e maximal percent suppression of A n g II release e v o k e d b y these inhibitors (5 Χ 10" m o l / L ) w a s 56% a n d 64%,
respectively, for Captopril a n d delapril. Both Nacetyl-pepstatin (10" to 10" m o l / L ) a n d KRI-1314 ( 1 0 to 1 0 m o l / L ) suppressed A n g II release i n a dose-related manner. At a 1 0 m o l / L concentration, KRI-1314 produced a 74% reduction i n t h e basal rate of A n g II release, a n d a reduction threefold greater than t h e maximal reduction i n basal A n g II release produced b y N-acetyl-pepstatin. T h e s e results provide direct e v i d e n c e for local generation a n d subsequent release of A n g II b y p e ripheral h u m a n vascular tissues a n d t h u s suggest that suppression of vascular A n g II generation b y renin inhibitors a n d converting e n z y m e inhibitors might contribute to t h e antihypertensive mecha n i s m s responsible for h u m a n hypertension. A m J Hypertens 1991;4:67S-72S
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tensin receptors, has been d e m o n s t r a t e d . Although earlier reports of renin in homogenates of vascular tis sues ' ' ' may be explained largely by adherence of plasma renin of renal origin to vascular beds, rigorous studies in recent years have revealed the presence of specific immunoreactive renin in arterial t i s s u e , cul tured smooth muscle cells, and endothelial cells. In addition, angiotensinogen messenger RNA could be de tected in vascular tissues, and angiotensin converting enzyme and angiotensin II (Ang II) receptors are present in the vasculature. Thus A n g II probably is formed lo cally in the vascular tissues, is released from it, and binds to A n g II receptors in vascular smooth muscle. Indeed, w e previously demonstrated that sizable
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he presence of essential components renin-angiotensin system in vascular such as renin enzyme, angiotensinogen, tensin converting enzyme (ACE), and
of the tissues, angio angio-
KEY WORDS: Renin, angiotensin II, h u m a n vascular tissue, angiotensin converting e n z y m e inhibitor, renin inhibitor.
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From the Third Department of Internal Medicine, Fukushima Medi cal College, Fukushima, Japan (KM, S N , MT, H H , SF), and The Depart ment of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee (TI). Address correspondence to Kenji Mizuno, The Third Department of Internal Medicine, Fukushima Medical College, 1 Hikari-ga-oka, Fu kushima 9 6 0 - 1 2 Japan. This paper w a s abstracted in J Hypertens 1990;(suppl 3):S36.This study w a s supported by a Grant-in-Aid for General Scientific Re search from the Ministry of Education, Science and Culture of Japan (B01480251 and C10570500).
© 1991 by the American Journal of Hypertension, Inc.
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Direct Proof for Local Generation and Release of Angiotensin II in Peripheral Human Vascular Tissue
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P e r f u s i o n of U m b i l i c a l V e i n W h o l e h u m a n umbilical cords w e r e kept in ice-cold saline immediately after they w e r e obtained at delivery. A polyethylene cannula (in side diameter, 2.3 m m ) w a s inserted into the umbilical vein, w h i c h w a s t h e n perfused with Krebs-Ringer solu tion (112 m m o l / L NaCl, 5 m m o l / L KCl, 1 m m o l / L N a H P 0 , 1.2 m m o l / L M g S 0 , 2.5 m m o l / L C a C l , 25 m m o l / L N a H C 0 , a n d 11.2 m m o l / L D(+)-glucose) in a water-jacketed container m a i n t a i n e d at 3 7 ° C . The solu tion w a s aerated with a mixture of 5 % C 0 a n d 9 5 % 0 to obtain a p H of 7.4. The tissues w e r e perfused at a constant flow rate of 6 m L / m i n with a roller p u m p (Polystaltic P u m p , Buchler, Fort Lee, NJ). The perfusion pressure w a s recorded with a pressure transducer (Model C P - 0 1 , C e n t u r y Technology Co., Inglewood, CA) connected to a polygraph (Model S-0411, N i h o n Koden Co., Tokyo). During perfusion of the umbilical cord, the perfusion pressure w a s approximately 25 to 30 m m Hg. To avoid the contamination of plasma angio tensins, the preparations w e r e thoroughly w a s h e d with Krebs-Ringer solution for 20 to 30 m i n before the exper iments w e r e started. 2
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D e t e r m i n a t i o n of A n g i o t e n s i n s Angiotensins that w e r e released from the perfused umbilical cord w e r e isolated with a solid-phase octadecasilyl-silica cartridge (Sep-Pak C , Waters Associates, Milford, MA) as d e scribed p r e v i o u s l y . In brief, the cartridge w a s con nected to the water-jacketed container to allow the per 1 8
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fusate to pass t h r o u g h the cartridge. Cartridges, w h i c h w e r e exchanged at 30 m i n intervals, h a d b e e n moist e n e d with 5 mL of m e t h a n o l a n d pre w a s h e d with 10 m L of Krebs-Ringer solution just before use. After w a s h i n g w i t h 10 m L of 0 . 1 % trifluoroacetic acid (TFA), angioten sins w e r e eluted with the mixture of m e t h a n o l : distilled water : TFA ( 8 0 : 1 9 . 9 : 0 . 1 volume ratio). The eluate w a s evaporated to dryness in a v a c u u m centrifuge (Model M C - 9 0 , Taiyo Scientific Instruments Co., Tokyo) a n d r e s u s p e n d e d in 0.1 m o l / L Tris-acetate buffer containing 2.6 m m o l / L N a E D T A , 1 m m o l / L p h e n y l m e t h y l s u l fonyl fluoride (PMSF), a n d 0 . 1 % bovine serum albumin, p H 7.4, for radioimmunoassay. The A n g II antiserum s h o w e d less t h a n 1% crossreactivity with A n g I, b u t 100% crossreactivity with angiotensin III (Ang III; A n g (2-8) heptapeptide), Ang-(3-8) hexapeptide, a n d A n g (4-8) p e n t a p e p t i d e . The detectability of the i m m u n o r e active A n g II (irAng II) determination w a s 1 p g / t u b e . The blank values of A n g I a n d A n g II w e r e always less than 1 pg/tube. 2
D r u g s Captopril (Sankyo Pharmaceutical Co., Tokyo, Japan) a n d delapril diacid 16 (Takeda Chemical I n d u s tries Co., Osaka, Japan) w e r e dissolved in isotonic saline a n d s o d i u m bicarbonate solution ( 1 % , w / v ) , respec tively. N-acetyl-pepstatin (generously provided b y Prof. M. Miyazaki, Osaka Medical College, Japan) a n d KRI1314 (isopropyl(2R, 2S)-3-[N-[2-(l-naphtyl-methyl)-3(morpholino carbonyl) propionyl]-L-histidyl]amino-4cyclohexyl-2-hydroxy butyrate; Kissei Pharmaceutical Co., Tokyo, Japan) w e r e dissolved in 0.2 m o l / L p h o s p h a t e buffer, p H 7.4, a n d 10~ Ν HCl, respectively. The physiochemical characteristics a n d biological efficacy of KRI-1314 w e r e described e l s e w h e r e . These drugs were diluted to the approximate concentrations with KrebsRinger buffer just before use, freshly p r e p a r e d each day, a n d w e r e infused continuously for a 30 min period at each concentration. 3
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Statistical A n a l y s i s The values are expressed as m e a n s ± SE. Statistical analysis of data w a s performed by a o n e - w a y of analysis of variance a n d S t u d e n t ' s t test w h e n appropriate. A Ρ < .05 w a s accepted as t h e level of significance. RESULTS The release of angiotensins from isolated perfused u m bilical veins w a s examined u p to 3 h. To equilibrate the preparations a n d to eliminate the possible contamina tion of plasma angiotensins, the tissues w e r e thoroughly w a s h e d with Krebs-Ringer solution (about 200 mL) over a period of 20 to 30 m i n before sample collection was started. U n d e r these conditions, the rate of release of angiotensins w a s 41.9 ± 7 . 4 a n d 6 3 . 4 ± 1 2 . 0 p g f o r A n g I a n d A n g II, respectively, during the first perfusion period of 30 min. A l t h o u g h t h e release of angiotensins b e t w e e n different umbilical cords w a s considerable,
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a m o u n t s of immunoreactive A n g II w a s released contin uously for several h o u r s from diverse vascular beds such as mesenteric a r t e r i e s a n d h i n d l e g s ' of rats w h e n these vascular tissues w e r e isolated a n d t h e n per fused with angiotensinogen-free m e d i u m . It w a s also s h o w n in these studies that such release of A n g II is not d u e to m e r e leakage from the vascular b e d s b u t d u e to regulated m e c h a n i s m , findings providing experi m e n t a l evidence for production a n d subsequent secre tion of A n g II from vascular tissue. More recently, w e h a v e s h o w n in isolated perfused h u m a n umbilical veins that readily detectable a m o u n t s of immunoreactive angiotensin I (Ang I) a n d A n g II w e r e released continuously in the absence of a n external supply of a n g i o t e n s i n o g e n . In addition, the release of A n g II w a s suppressed by C a p t o p r i l with reciprocal in crease of A n g I r e l e a s e , indicating the presence of es sential c o m p o n e n t s for angiotensin generation in h u m a n vascular tissues. In this study, w e examined the effect of ACE inhibi tors a n d renin inhibitors o n A n g II release in isolated perfused h u m a n umbilical veins to further delineate mechanism(s) by w h i c h A n g II is generated in periph eral h u m a n vascular beds.
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the basal rate of A n g II release, w h i c h w a s about three fold greater t h a n that induced by N-acetyl-pepstatin (10~ m o l / L ) .
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The notable findings from the present study are that 1) sizable a m o u n t s of A n g II could be detected in t h e per fusate from isolated perfused h u m a n umbilical veins, 2) the ACE inhibitors C a p t o p r i l a n d delapril, w h e n a d d e d to the perfusion m e d i u m , decreased A n g II release in a d o s e - d e p e n d e n t fashion, a n d 3) the renin inhibitors N Basal 10 " 10 " 10 " 5x10" acetyl-pepstatin a n d KRI-1314 also suppressed t h e A n g (n=5) (n=5) (n=5) (n=4) (n=4) II release. Captopril (M) A l t h o u g h several studies h a v e d e m o n s t r a t e d t h e pres ence of A n g II-like immunoreactivity in various tis FIGURE 1. Effect of Captopril on immunoreactive Ang I and s u e s , t h e methodology of the present study (using a Ang II release from isolated perfused human umbilical veins; *P < Sep-Pak C cartridge connected to the perfusion sys .05, **P < .01 compared with basal values. tem) permitted us to directly detect sizable a m o u n t s of A n g II released from t h e isolated perfused h u m a n umbil ical veins. W e did not identify the A n g II-like i m m u n o r e activity in the perfusate as A n g II in this study, b u t ranging from 15.8 to 108.5 p g / 3 0 min for A n g I a n d 28.1 previously w e w e r e able to confirm that t h e i m m u n o r e to 128.2 p g / 3 0 min for A n g II for u n k n o w n reasons, active A n g II in the perfusate is A n g II octapeptide by individual cords examined s h o w e d a stable rate of the using high performance liquid c h r o m a t o g r a p h y . In peptides release at least u p to 3 h; at the e n d of the 3 h these past a n d present studies, special care w a s taken to perfusion peripd, the rate of release of A n g I a n d A n g II avoid t h e contamination of plasma angiotensins; t h e was 46.8 ± 1.3 p g / 3 0 m i n a n d 60.8 ± 9.8 p g / 3 0 min, umbilical vein preparations w e r e thoroughly w a s h e d with about 200 mL of Krebs-Ringer solution for at least respectively. To further address the m e c h a n i s m of angiotensin gen 20 m i n before t h e experiments w e r e started. U n d e r eration, t h e effect of converting e n z y m e inhibitors on these conditions, the basal release of A n g II w a s con the immunoreactive A n g II release w a s examined. As stant u p to 3 h. Thus, the possibility is negligible that t h e shown in Figure 1, Captopril, a d d e d to the perfusion A n g II determined in this study w a s d u e to contamina medium ( 1 0 to 5 Χ 1 0 " m o l / L ) , caused a significant tion or sequestration of circulating plasma peptide. decrease of the A n g II release in a d o s e - d e p e n d e n t fash While some studies h a v e d e m o n s t r a t e d biochemically ion (F = 10.96, Ρ < .001), with a significant, reciprocal a n d immunohistochemically the presence of i m m u n o r e increase of A n g I release (F = 3.65, Ρ < .05). At the active renin in vascular tissues, it is still controversial highest concentration of Captopril (5 X 10~ m o l / L ) , it w h e t h e r t h e renin is e n d o g e n o u s or taken u p from induced a n approximately 5 6 % decrease in the basal plasma renin by the vascular wall. Vascular s m o o t h rate of A n g II release (P < .01). Similarly, as s h o w n in muscle cells in tissue culture d o synthesize renin, w h i c h Figure 2, delapril diacid ( 1 0 " to 5 X 10~ m o l / L ) elicited strongly supports the contention that there is local syn a significant decrease of the A n g II release in a dose-de thesis of the e n z y m e in vascular s m o o t h muscle. O n the pendent m a n n e r (F = 12.14, Ρ < .001), at the highest other h a n d , the finding that vascular renin-like activity concentration of delapril diacid (5 X 10~ m o l / L ) , a disappeared after n e p h r e c t o m y in parallel w i t h plasma 64% decrease of A n g II in the basal rate of release w a s renin activity supports the concept that renin activity in shown (P < .005). vascular tissues is d u e to the a d h e r e n c e of, or contamina Figure 3 s h o w s the effect of N-acetyl-pepstatin on tion by, plasma renin of renal origin. As for angiotensin Ang II release from isolated perfused umbilical veins. ogen, its messenger RNA h a s recently b e e n detected in Ang II w a s significantly suppressed by N-acetyl-pepsta rat a o r t a e , providing evidence for local synthesis of tin in a dose-related fashion (F = 3.15, Ρ < .05). The angiotensinogen in vascular tissue. Thus, w h a t e v e r the maximal percent inhibition of A n g II release evoked by source of the vascular renin m a y be, the r e n i n the inhibitor (10~ m o l / L ) w a s approximately 2 4 % (P < angiotensinogen reaction can occur locally in the vascu .05). O n the other h a n d , another renin inhibitor (KRI- lar tissue to p r o d u c e angiotensin. 1314, 1 0 " to 10~ m o l / L ) elicited m a r k e d suppression O u r results provide experimental evidence for such a of Ang II release (F = 16.24, Ρ < .001), as s h o w n in mechanism, because A n g II w a s p r o d u c e d continuously Figure 4. The maximal percent inhibition induced by in t h e absence of a n external supply of angiotensinogen this inhibitor (10~ m o l / L ) w a s about 7 4 % (P < .001) in in the perfusion m e d i u m , a finding that is consistent
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with our previous observations in isolated perfused rat vascular t i s s u e s . " H o w e v e r , since it is n o t clear at present w h e t h e r the umbilical vein synthesizes t h e es sential c o m p o n e n t s (including angiotensinogen) for a n giotensin generation, further studies are n e e d e d to clar ify these pivotal problems. In the present study, ACE inhibitors suppressed A n g II release from t h e umbilical veins. Captopril, w h e n a d d e d to the perfusion m e d i u m , decreased A n g II re lease in a d o s e - d e p e n d e n t fashion, a n d , m o r e impor tantly, it caused a n a p p a r e n t increase in A n g I release. T h u s , these data clearly indicate that A n g I is converted to A n g II either intracellularly or extracellularly, because ACE is localized n o t only in the luminal surface of the 12
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vascular e n d o t h e l i u m b u t also in the subcellular parti cles of various t i s s u e s ' including arterial w a l l . W e d o not k n o w w h i c h m e c h a n i s m is m o r e responsible for vas cular A n g II generation. The extent of suppression of A n g II release w a s s o m e w h a t greater with delapril t h a n Captopril. Thus, in view of the greater lipophilicity of delapril, c o m p a r e d with Captopril (about 5 times, u n published observation), it might b e possible that delapril is readily taken u p b y vascular tissue to suppress A n g II generation in the tissue. Indeed, in a previous study, w e d e m o n s t r a t e d that a n o t h e r lipophilic ACE inhibitor (SA 446) suppressed A n g II release from isolated perfused rat h i n d legs m o r e effectively t h a n did Captopril. Taken together, intracellular m e c h a n i s m seems most 21
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FIGURE 3. Effect of N-acetyl-pepstatin on immunoreactive Ang II re lease from isolated perfused human um bilical veins; *P < .05 compared with basal values.
N-acetyl-pepstatin (M)
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FIGURE 2. Effect of delapril diacid on immunoreactive Angll release fromisolatedperfusedhumanumbilical veins; *P < .05, **P < .002, fP < .001 compared with basal values.
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likely responsible for the generation of A n g II in t h e vascular b e d s . In addition to the ACE inhibitors, renin inhibitors were f o u n d to suppress A n g II release from isolated perfused h u m a n umbilical veins in a d o s e - d e p e n d e n t fashion. It s h o u l d b e n o t e d that a specific h u m a n renin inhibitor, KRI-1314, w a s m u c h m o r e effective in A n g II release c o m p a r e d w i t h N-acetyl-pepstatin. T h u s , these findings not only provide direct evidence for t h e pres ence of t h e specific r e n i n - a n g i o t e n s i n system in t h e vascular b e d s in h u m a n s b u t also suggest the impor tance of renin in A n g II formation in t h e vasculature. In t h e present study using isolated perfused h u m a n umbilical veins, w e did not address t h e role of t h e vascu lar r e n i n - a n g i o t e n s i n system in blood pressure regula tion. H o w e v e r , it is possible that secreted A n g II b i n d s to receptors o n t h e s a m e or neighboring cells in a n a u t o crine or paracrine fashion a n d t h e n exerts its biological effects, such as stimulation of prostaglandin b i o s y n t h e s i s , activation of vascular s m o o t h muscle t o n e , a n d potentiation of sympathetic activity. Indeed, w e previously p r e s e n t e d data suggesting that t h e isoproterenol-induced facilitation of vascular noradrenergic neurotransmission is d u e to the activation of t h e vascu lar renin - angiotensin s y s t e m . In s u p p o r t of this physi ological relevance, it w a s d e m o n s t r a t e d that Captopril resulted in prejunctional a n d postjunctional inhibition of vascular sympathetic function in spontaneously h y pertensive r a t s . These findings suggest that A n g II re leased from t h e vascular tissue h a s an i m p o r t a n t influ ence o n blood pressure control. 23
cate t h e existence of a functional r e n i n - a n g i o t e n s i n system in peripheral h u m a n vascular tissues, w h i c h m i g h t explain, at least partially, t h e h y p o t e n s i v e effect of these inhibitors in h y p e r t e n s i v e h u m a n subjects w i t h little renin activity in blood t h r o u g h suppression of t h e vascular renin - angiotensin system.
ACKNOWLEDGEMENTS We thank Takeda Chemical Industries Co., Ltd., Osaka, Japan, and Kissei Pharmaceutical Co., Tokyo, Japan for the gift of delapril and KRI-1314, respectively.
REFERENCES 1.
Gould AB, Skeggs LT, Kahn JR: The presence of renin activity in blood vessel walls. J Exp Med 1964; 119:389399.
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Ganten D, Schelling P, Vecsei P, et al: Iso-renin of extra renal origin: "the tissue angiotensinogenase system/' Am J Med 1976;60:760-772.
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Ohkubo H, Nakayama K, Tanaka T, et al: Tissue distri bution of rat angiotensinogen mRNA and structural anal ysis of its heterogeneity. J Biol Chem 1986;261:319-323.
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Mizuno K, Hata S, Fukuchi S: Effect of sodium intake on angiotensin-converting enzyme activity of aorta in rats. Clin Sei 1981;61:249-251.
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Lin S-Y, Goodfriend TL: Angiotensin receptors. Am J Physiol 1970;218:1319-1328.
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Rosenthal J, Boucher R, Rojo-Ortega JM, et al: Renin activity in aortic tissue of rat. Can J Physiol Pharmacol 1969;47:53-56.
7.
Thurston H, Swales J, Bing RF, et al: Vascular renin-like activity and blood pressure maintenance in the rat: stud ies of the effect of changes in sodium balance, hyperten sion and nephrectomy. Hypertension 1979;1:643-649.
8.
Dzau VJ: Vascular renin-angiotensin: a possible auto-
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In s u m m a r y , t h e present studies h a v e d e m o n s t r a t e d that sizable a m o u n t s of A n g II w e r e released from iso lated perfused h u m a n umbilical veins. The release of Ang II w a s inhibited markedly b y either ACE inhibitors or renin inhibitors. Taken together, these findings indi
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FIGURE 4. Effect of human renin inhibitor KRI-1314 on immunoreac tive Ang II release from isolated perfused human umbilical veins; * P < . 0 5 , **P
1991; 4.67S-72S
Kenji Mizuno, Susumu Niimura, Makio Tant, Hiroshi Haga, Tadashi Inagami, and Soitsu
Fukuchii
Previously w e reported that immunoreactive a n giotensin II (Ang II) release from isolated perfused h u m a n umbilical v e i n s w a s inhibited b y t h e a n giotensin-converting e n z y m e inhibitor C a p t o p r i l . To further investigate t h e m e c h a n i s m b y w h i c h A n g II is generated i n the blood v e s s e l s of h u m a n s , w e examined the effects of various inhibitors of the r e n i n - a n g i o t e n s i n system (Captopril, delapril, Nacetyl-pepstatin, a n d h u m a n renin inhibitor KRI1314) o n A n g II release from perfused h u m a n u m bilical cord v e i n s i n vitro. Isolated h u m a n umbilical v e i n s w e r e perfused w i t h Krebs-Ringer solution, a n d immunoreactive Ang II released into the perfusate w a s measured di rectly b y u s i n g a Sep-Pak C cartridge connected to the perfusion system. Both Captopril a n d delapril diacid (10" to 5 Χ 10" m o l / L ) , an active metabolite of delapril hydrochloride, suppressed t h e A n g II release i n a dose-dependent fashion; t h e maximal percent suppression of A n g II release e v o k e d b y these inhibitors (5 Χ 10" m o l / L ) w a s 56% a n d 64%,
respectively, for Captopril a n d delapril. Both Nacetyl-pepstatin (10" to 10" m o l / L ) a n d KRI-1314 ( 1 0 to 1 0 m o l / L ) suppressed A n g II release i n a dose-related manner. At a 1 0 m o l / L concentration, KRI-1314 produced a 74% reduction i n t h e basal rate of A n g II release, a n d a reduction threefold greater than t h e maximal reduction i n basal A n g II release produced b y N-acetyl-pepstatin. T h e s e results provide direct e v i d e n c e for local generation a n d subsequent release of A n g II b y p e ripheral h u m a n vascular tissues a n d t h u s suggest that suppression of vascular A n g II generation b y renin inhibitors a n d converting e n z y m e inhibitors might contribute to t h e antihypertensive mecha n i s m s responsible for h u m a n hypertension. A m J Hypertens 1991;4:67S-72S
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tensin receptors, has been d e m o n s t r a t e d . Although earlier reports of renin in homogenates of vascular tis sues ' ' ' may be explained largely by adherence of plasma renin of renal origin to vascular beds, rigorous studies in recent years have revealed the presence of specific immunoreactive renin in arterial t i s s u e , cul tured smooth muscle cells, and endothelial cells. In addition, angiotensinogen messenger RNA could be de tected in vascular tissues, and angiotensin converting enzyme and angiotensin II (Ang II) receptors are present in the vasculature. Thus A n g II probably is formed lo cally in the vascular tissues, is released from it, and binds to A n g II receptors in vascular smooth muscle. Indeed, w e previously demonstrated that sizable
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he presence of essential components renin-angiotensin system in vascular such as renin enzyme, angiotensinogen, tensin converting enzyme (ACE), and
of the tissues, angio angio-
KEY WORDS: Renin, angiotensin II, h u m a n vascular tissue, angiotensin converting e n z y m e inhibitor, renin inhibitor.
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From the Third Department of Internal Medicine, Fukushima Medi cal College, Fukushima, Japan (KM, S N , MT, H H , SF), and The Depart ment of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee (TI). Address correspondence to Kenji Mizuno, The Third Department of Internal Medicine, Fukushima Medical College, 1 Hikari-ga-oka, Fu kushima 9 6 0 - 1 2 Japan. This paper w a s abstracted in J Hypertens 1990;(suppl 3):S36.This study w a s supported by a Grant-in-Aid for General Scientific Re search from the Ministry of Education, Science and Culture of Japan (B01480251 and C10570500).
© 1991 by the American Journal of Hypertension, Inc.
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Direct Proof for Local Generation and Release of Angiotensin II in Peripheral Human Vascular Tissue
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P e r f u s i o n of U m b i l i c a l V e i n W h o l e h u m a n umbilical cords w e r e kept in ice-cold saline immediately after they w e r e obtained at delivery. A polyethylene cannula (in side diameter, 2.3 m m ) w a s inserted into the umbilical vein, w h i c h w a s t h e n perfused with Krebs-Ringer solu tion (112 m m o l / L NaCl, 5 m m o l / L KCl, 1 m m o l / L N a H P 0 , 1.2 m m o l / L M g S 0 , 2.5 m m o l / L C a C l , 25 m m o l / L N a H C 0 , a n d 11.2 m m o l / L D(+)-glucose) in a water-jacketed container m a i n t a i n e d at 3 7 ° C . The solu tion w a s aerated with a mixture of 5 % C 0 a n d 9 5 % 0 to obtain a p H of 7.4. The tissues w e r e perfused at a constant flow rate of 6 m L / m i n with a roller p u m p (Polystaltic P u m p , Buchler, Fort Lee, NJ). The perfusion pressure w a s recorded with a pressure transducer (Model C P - 0 1 , C e n t u r y Technology Co., Inglewood, CA) connected to a polygraph (Model S-0411, N i h o n Koden Co., Tokyo). During perfusion of the umbilical cord, the perfusion pressure w a s approximately 25 to 30 m m Hg. To avoid the contamination of plasma angio tensins, the preparations w e r e thoroughly w a s h e d with Krebs-Ringer solution for 20 to 30 m i n before the exper iments w e r e started. 2
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D e t e r m i n a t i o n of A n g i o t e n s i n s Angiotensins that w e r e released from the perfused umbilical cord w e r e isolated with a solid-phase octadecasilyl-silica cartridge (Sep-Pak C , Waters Associates, Milford, MA) as d e scribed p r e v i o u s l y . In brief, the cartridge w a s con nected to the water-jacketed container to allow the per 1 8
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fusate to pass t h r o u g h the cartridge. Cartridges, w h i c h w e r e exchanged at 30 m i n intervals, h a d b e e n moist e n e d with 5 mL of m e t h a n o l a n d pre w a s h e d with 10 m L of Krebs-Ringer solution just before use. After w a s h i n g w i t h 10 m L of 0 . 1 % trifluoroacetic acid (TFA), angioten sins w e r e eluted with the mixture of m e t h a n o l : distilled water : TFA ( 8 0 : 1 9 . 9 : 0 . 1 volume ratio). The eluate w a s evaporated to dryness in a v a c u u m centrifuge (Model M C - 9 0 , Taiyo Scientific Instruments Co., Tokyo) a n d r e s u s p e n d e d in 0.1 m o l / L Tris-acetate buffer containing 2.6 m m o l / L N a E D T A , 1 m m o l / L p h e n y l m e t h y l s u l fonyl fluoride (PMSF), a n d 0 . 1 % bovine serum albumin, p H 7.4, for radioimmunoassay. The A n g II antiserum s h o w e d less t h a n 1% crossreactivity with A n g I, b u t 100% crossreactivity with angiotensin III (Ang III; A n g (2-8) heptapeptide), Ang-(3-8) hexapeptide, a n d A n g (4-8) p e n t a p e p t i d e . The detectability of the i m m u n o r e active A n g II (irAng II) determination w a s 1 p g / t u b e . The blank values of A n g I a n d A n g II w e r e always less than 1 pg/tube. 2
D r u g s Captopril (Sankyo Pharmaceutical Co., Tokyo, Japan) a n d delapril diacid 16 (Takeda Chemical I n d u s tries Co., Osaka, Japan) w e r e dissolved in isotonic saline a n d s o d i u m bicarbonate solution ( 1 % , w / v ) , respec tively. N-acetyl-pepstatin (generously provided b y Prof. M. Miyazaki, Osaka Medical College, Japan) a n d KRI1314 (isopropyl(2R, 2S)-3-[N-[2-(l-naphtyl-methyl)-3(morpholino carbonyl) propionyl]-L-histidyl]amino-4cyclohexyl-2-hydroxy butyrate; Kissei Pharmaceutical Co., Tokyo, Japan) w e r e dissolved in 0.2 m o l / L p h o s p h a t e buffer, p H 7.4, a n d 10~ Ν HCl, respectively. The physiochemical characteristics a n d biological efficacy of KRI-1314 w e r e described e l s e w h e r e . These drugs were diluted to the approximate concentrations with KrebsRinger buffer just before use, freshly p r e p a r e d each day, a n d w e r e infused continuously for a 30 min period at each concentration. 3
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Statistical A n a l y s i s The values are expressed as m e a n s ± SE. Statistical analysis of data w a s performed by a o n e - w a y of analysis of variance a n d S t u d e n t ' s t test w h e n appropriate. A Ρ < .05 w a s accepted as t h e level of significance. RESULTS The release of angiotensins from isolated perfused u m bilical veins w a s examined u p to 3 h. To equilibrate the preparations a n d to eliminate the possible contamina tion of plasma angiotensins, the tissues w e r e thoroughly w a s h e d with Krebs-Ringer solution (about 200 mL) over a period of 20 to 30 m i n before sample collection was started. U n d e r these conditions, the rate of release of angiotensins w a s 41.9 ± 7 . 4 a n d 6 3 . 4 ± 1 2 . 0 p g f o r A n g I a n d A n g II, respectively, during the first perfusion period of 30 min. A l t h o u g h t h e release of angiotensins b e t w e e n different umbilical cords w a s considerable,
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a m o u n t s of immunoreactive A n g II w a s released contin uously for several h o u r s from diverse vascular beds such as mesenteric a r t e r i e s a n d h i n d l e g s ' of rats w h e n these vascular tissues w e r e isolated a n d t h e n per fused with angiotensinogen-free m e d i u m . It w a s also s h o w n in these studies that such release of A n g II is not d u e to m e r e leakage from the vascular b e d s b u t d u e to regulated m e c h a n i s m , findings providing experi m e n t a l evidence for production a n d subsequent secre tion of A n g II from vascular tissue. More recently, w e h a v e s h o w n in isolated perfused h u m a n umbilical veins that readily detectable a m o u n t s of immunoreactive angiotensin I (Ang I) a n d A n g II w e r e released continuously in the absence of a n external supply of a n g i o t e n s i n o g e n . In addition, the release of A n g II w a s suppressed by C a p t o p r i l with reciprocal in crease of A n g I r e l e a s e , indicating the presence of es sential c o m p o n e n t s for angiotensin generation in h u m a n vascular tissues. In this study, w e examined the effect of ACE inhibi tors a n d renin inhibitors o n A n g II release in isolated perfused h u m a n umbilical veins to further delineate mechanism(s) by w h i c h A n g II is generated in periph eral h u m a n vascular beds.
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the basal rate of A n g II release, w h i c h w a s about three fold greater t h a n that induced by N-acetyl-pepstatin (10~ m o l / L ) .
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The notable findings from the present study are that 1) sizable a m o u n t s of A n g II could be detected in t h e per fusate from isolated perfused h u m a n umbilical veins, 2) the ACE inhibitors C a p t o p r i l a n d delapril, w h e n a d d e d to the perfusion m e d i u m , decreased A n g II release in a d o s e - d e p e n d e n t fashion, a n d 3) the renin inhibitors N Basal 10 " 10 " 10 " 5x10" acetyl-pepstatin a n d KRI-1314 also suppressed t h e A n g (n=5) (n=5) (n=5) (n=4) (n=4) II release. Captopril (M) A l t h o u g h several studies h a v e d e m o n s t r a t e d t h e pres ence of A n g II-like immunoreactivity in various tis FIGURE 1. Effect of Captopril on immunoreactive Ang I and s u e s , t h e methodology of the present study (using a Ang II release from isolated perfused human umbilical veins; *P < Sep-Pak C cartridge connected to the perfusion sys .05, **P < .01 compared with basal values. tem) permitted us to directly detect sizable a m o u n t s of A n g II released from t h e isolated perfused h u m a n umbil ical veins. W e did not identify the A n g II-like i m m u n o r e activity in the perfusate as A n g II in this study, b u t ranging from 15.8 to 108.5 p g / 3 0 min for A n g I a n d 28.1 previously w e w e r e able to confirm that t h e i m m u n o r e to 128.2 p g / 3 0 min for A n g II for u n k n o w n reasons, active A n g II in the perfusate is A n g II octapeptide by individual cords examined s h o w e d a stable rate of the using high performance liquid c h r o m a t o g r a p h y . In peptides release at least u p to 3 h; at the e n d of the 3 h these past a n d present studies, special care w a s taken to perfusion peripd, the rate of release of A n g I a n d A n g II avoid t h e contamination of plasma angiotensins; t h e was 46.8 ± 1.3 p g / 3 0 m i n a n d 60.8 ± 9.8 p g / 3 0 min, umbilical vein preparations w e r e thoroughly w a s h e d with about 200 mL of Krebs-Ringer solution for at least respectively. To further address the m e c h a n i s m of angiotensin gen 20 m i n before t h e experiments w e r e started. U n d e r eration, t h e effect of converting e n z y m e inhibitors on these conditions, the basal release of A n g II w a s con the immunoreactive A n g II release w a s examined. As stant u p to 3 h. Thus, the possibility is negligible that t h e shown in Figure 1, Captopril, a d d e d to the perfusion A n g II determined in this study w a s d u e to contamina medium ( 1 0 to 5 Χ 1 0 " m o l / L ) , caused a significant tion or sequestration of circulating plasma peptide. decrease of the A n g II release in a d o s e - d e p e n d e n t fash While some studies h a v e d e m o n s t r a t e d biochemically ion (F = 10.96, Ρ < .001), with a significant, reciprocal a n d immunohistochemically the presence of i m m u n o r e increase of A n g I release (F = 3.65, Ρ < .05). At the active renin in vascular tissues, it is still controversial highest concentration of Captopril (5 X 10~ m o l / L ) , it w h e t h e r t h e renin is e n d o g e n o u s or taken u p from induced a n approximately 5 6 % decrease in the basal plasma renin by the vascular wall. Vascular s m o o t h rate of A n g II release (P < .01). Similarly, as s h o w n in muscle cells in tissue culture d o synthesize renin, w h i c h Figure 2, delapril diacid ( 1 0 " to 5 X 10~ m o l / L ) elicited strongly supports the contention that there is local syn a significant decrease of the A n g II release in a dose-de thesis of the e n z y m e in vascular s m o o t h muscle. O n the pendent m a n n e r (F = 12.14, Ρ < .001), at the highest other h a n d , the finding that vascular renin-like activity concentration of delapril diacid (5 X 10~ m o l / L ) , a disappeared after n e p h r e c t o m y in parallel w i t h plasma 64% decrease of A n g II in the basal rate of release w a s renin activity supports the concept that renin activity in shown (P < .005). vascular tissues is d u e to the a d h e r e n c e of, or contamina Figure 3 s h o w s the effect of N-acetyl-pepstatin on tion by, plasma renin of renal origin. As for angiotensin Ang II release from isolated perfused umbilical veins. ogen, its messenger RNA h a s recently b e e n detected in Ang II w a s significantly suppressed by N-acetyl-pepsta rat a o r t a e , providing evidence for local synthesis of tin in a dose-related fashion (F = 3.15, Ρ < .05). The angiotensinogen in vascular tissue. Thus, w h a t e v e r the maximal percent inhibition of A n g II release evoked by source of the vascular renin m a y be, the r e n i n the inhibitor (10~ m o l / L ) w a s approximately 2 4 % (P < angiotensinogen reaction can occur locally in the vascu .05). O n the other h a n d , another renin inhibitor (KRI- lar tissue to p r o d u c e angiotensin. 1314, 1 0 " to 10~ m o l / L ) elicited m a r k e d suppression O u r results provide experimental evidence for such a of Ang II release (F = 16.24, Ρ < .001), as s h o w n in mechanism, because A n g II w a s p r o d u c e d continuously Figure 4. The maximal percent inhibition induced by in t h e absence of a n external supply of angiotensinogen this inhibitor (10~ m o l / L ) w a s about 7 4 % (P < .001) in in the perfusion m e d i u m , a finding that is consistent
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with our previous observations in isolated perfused rat vascular t i s s u e s . " H o w e v e r , since it is n o t clear at present w h e t h e r the umbilical vein synthesizes t h e es sential c o m p o n e n t s (including angiotensinogen) for a n giotensin generation, further studies are n e e d e d to clar ify these pivotal problems. In the present study, ACE inhibitors suppressed A n g II release from t h e umbilical veins. Captopril, w h e n a d d e d to the perfusion m e d i u m , decreased A n g II re lease in a d o s e - d e p e n d e n t fashion, a n d , m o r e impor tantly, it caused a n a p p a r e n t increase in A n g I release. T h u s , these data clearly indicate that A n g I is converted to A n g II either intracellularly or extracellularly, because ACE is localized n o t only in the luminal surface of the 12
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vascular e n d o t h e l i u m b u t also in the subcellular parti cles of various t i s s u e s ' including arterial w a l l . W e d o not k n o w w h i c h m e c h a n i s m is m o r e responsible for vas cular A n g II generation. The extent of suppression of A n g II release w a s s o m e w h a t greater with delapril t h a n Captopril. Thus, in view of the greater lipophilicity of delapril, c o m p a r e d with Captopril (about 5 times, u n published observation), it might b e possible that delapril is readily taken u p b y vascular tissue to suppress A n g II generation in the tissue. Indeed, in a previous study, w e d e m o n s t r a t e d that a n o t h e r lipophilic ACE inhibitor (SA 446) suppressed A n g II release from isolated perfused rat h i n d legs m o r e effectively t h a n did Captopril. Taken together, intracellular m e c h a n i s m seems most 21
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FIGURE 3. Effect of N-acetyl-pepstatin on immunoreactive Ang II re lease from isolated perfused human um bilical veins; *P < .05 compared with basal values.
N-acetyl-pepstatin (M)
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FIGURE 2. Effect of delapril diacid on immunoreactive Angll release fromisolatedperfusedhumanumbilical veins; *P < .05, **P < .002, fP < .001 compared with basal values.
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likely responsible for the generation of A n g II in t h e vascular b e d s . In addition to the ACE inhibitors, renin inhibitors were f o u n d to suppress A n g II release from isolated perfused h u m a n umbilical veins in a d o s e - d e p e n d e n t fashion. It s h o u l d b e n o t e d that a specific h u m a n renin inhibitor, KRI-1314, w a s m u c h m o r e effective in A n g II release c o m p a r e d w i t h N-acetyl-pepstatin. T h u s , these findings not only provide direct evidence for t h e pres ence of t h e specific r e n i n - a n g i o t e n s i n system in t h e vascular b e d s in h u m a n s b u t also suggest the impor tance of renin in A n g II formation in t h e vasculature. In t h e present study using isolated perfused h u m a n umbilical veins, w e did not address t h e role of t h e vascu lar r e n i n - a n g i o t e n s i n system in blood pressure regula tion. H o w e v e r , it is possible that secreted A n g II b i n d s to receptors o n t h e s a m e or neighboring cells in a n a u t o crine or paracrine fashion a n d t h e n exerts its biological effects, such as stimulation of prostaglandin b i o s y n t h e s i s , activation of vascular s m o o t h muscle t o n e , a n d potentiation of sympathetic activity. Indeed, w e previously p r e s e n t e d data suggesting that t h e isoproterenol-induced facilitation of vascular noradrenergic neurotransmission is d u e to the activation of t h e vascu lar renin - angiotensin s y s t e m . In s u p p o r t of this physi ological relevance, it w a s d e m o n s t r a t e d that Captopril resulted in prejunctional a n d postjunctional inhibition of vascular sympathetic function in spontaneously h y pertensive r a t s . These findings suggest that A n g II re leased from t h e vascular tissue h a s an i m p o r t a n t influ ence o n blood pressure control. 23
cate t h e existence of a functional r e n i n - a n g i o t e n s i n system in peripheral h u m a n vascular tissues, w h i c h m i g h t explain, at least partially, t h e h y p o t e n s i v e effect of these inhibitors in h y p e r t e n s i v e h u m a n subjects w i t h little renin activity in blood t h r o u g h suppression of t h e vascular renin - angiotensin system.
ACKNOWLEDGEMENTS We thank Takeda Chemical Industries Co., Ltd., Osaka, Japan, and Kissei Pharmaceutical Co., Tokyo, Japan for the gift of delapril and KRI-1314, respectively.
REFERENCES 1.
Gould AB, Skeggs LT, Kahn JR: The presence of renin activity in blood vessel walls. J Exp Med 1964; 119:389399.
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Ohkubo H, Nakayama K, Tanaka T, et al: Tissue distri bution of rat angiotensinogen mRNA and structural anal ysis of its heterogeneity. J Biol Chem 1986;261:319-323.
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Lin S-Y, Goodfriend TL: Angiotensin receptors. Am J Physiol 1970;218:1319-1328.
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Rosenthal J, Boucher R, Rojo-Ortega JM, et al: Renin activity in aortic tissue of rat. Can J Physiol Pharmacol 1969;47:53-56.
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Thurston H, Swales J, Bing RF, et al: Vascular renin-like activity and blood pressure maintenance in the rat: stud ies of the effect of changes in sodium balance, hyperten sion and nephrectomy. Hypertension 1979;1:643-649.
8.
Dzau VJ: Vascular renin-angiotensin: a possible auto-
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In s u m m a r y , t h e present studies h a v e d e m o n s t r a t e d that sizable a m o u n t s of A n g II w e r e released from iso lated perfused h u m a n umbilical veins. The release of Ang II w a s inhibited markedly b y either ACE inhibitors or renin inhibitors. Taken together, these findings indi
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FIGURE 4. Effect of human renin inhibitor KRI-1314 on immunoreac tive Ang II release from isolated perfused human umbilical veins; * P < . 0 5 , **P
