Lafe Sciences, Vol. Printed in the USA

51, pp.

2079-2087

Pergamon

Press

A C U T E E F F E C T S OF A N G I O T E N S I N - C O N V E R T I N G E N Z Y M E I N H I B I T O R ON E R Y T H R O C Y T E S O D I U M ION T R A N S P O R T IN E S S E N T I A L H Y P E R T E N S I O N

Kazutaka

Fujito, M a m o r u Yokomatsu, Hlroo Numahata, Nozoml Ishlguro and Hlkaru Kolde

D i v i s i o n of Nephrology, D e p a r t m e n t of Medlclne, J u n t e n d o U n i v e r s i t y School of M e d l c l n e Tokyo, J a p a n (Recelved

in flnal

form October

22,

1992)

Summary

The acute effects of a n g l o t e n s l n - c o n v e r t l n g enzyme inhlbltor, captoprll, on s o d i u m ion t r a n s p o r t systems were investigated in essential hypertensive and normotenslve sub3ects The p a s s l v e s o d i u m e f f l u x t h r o u g h the e r y t h r o c y t e m e m b r a n e was s l g n l f l c a n t l y higher and erythrocyte sodlum-potassium cotransport was l o w e r in patients with essential hypertension when compared with normal sub3ects. However, s o d l u m - p o t a s s l u m p u m p a c t i v i t y and sodium-lithium countertransport did not dlffer s i g n i f i c a n t l y b e t w e e n the h y p e r t e n s l v e p a t l e n t s a n d the normal subjects. Immediately after captoprll adminlstratlon, erythrocyte passive sodium efflux and sodiump o t a s s i u m c o t r a n s p o r t r e t u r n e d to n o r m a l l e v e l s in the h y p e r t e n s i v e sub3ects. A l t h o u g h the p l a s m a r e n i n a c t i v i t y and plasma aldosterone concentration were altered by captopril, t h e y d i d not c o r r e l a t e w i t h c h a n g e s in any s o d i u m t r a n s p o r t system. T h e s e r e s u l t s s u g g e s t that the c h a n g e s in s o d i u m t r a n s p o r t s y s t e m s w h i c h o c c u r i m m e d i a t e l y a f t e r c a p t o p r l l a d m i n i s t r a t i o n m a y c o n t r i b u t e , at least in part, to its a n t l h y p e r t e n s l v e action.

Recently, genetic abnormalities in t r a n s m e m b r a n e sodlum t r a n s p o r t w e r e f o u n d to be a s s o c l a t e d strongly with essentlal hypertension [1-5]. An increased sodium concentratlon of the arterlal wall in the h y p e r t e n s l v e patients [6] h a s a t t r a c t e d a t t e n t i o n as a m e c h a n i s m by w h l c h h y p e r t e n s l o n c o u l d be i n d u c e d a n d maintained as this w o u l d be e x p e c t e d to increase the r e s i s t a n c e of p e r i p h e r a l b l o o d vessels. Previously, we m e a s u r e d s o d l u m t r a n s p o r t in the e r y t h r o c y t e s of s p o n t a n e o u s l y h y p e r t e n s i v e rats (SHR) a n d found transmembrane sodium influx to be enhanced due to cell m e m b r a n e a b n o r m a l i t i e s [7] If a b n o r m a l l t l e s in t r a n s m e m b r a n e sodium transport are i n v o l v e d in m a i n t a i n i n g h y p e r t e n s i o n , the Corresponding author: Dr. H 1 k a r u D e p a r t m e n t of M e d l c l n e , Juntendo Honge, T o k y o 113, J a p a n

Copyrlght

Kolde, D l V l S l O n of N e p h r o l o g y , U n l v e r s l t y S c h o o l of M e d l c l n e ,

0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rlghts

reserved.

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c h a n g e s in t r a n s m e m b r a n e s o d i u m t r a n s p o r t m i g h t be a s s o c i a t e d w i t h changes in blood p r e s s u r e w h l c h are c a u s e d by a n t l h y p e r t e n s i v e drugs. The a n g i o t e n s l n - c o n v e r t I n g e n z y m e Inhlbltor, captopril, is thought to exert its hypotenslve effect m a l n l y via the renlnangiotensln system. S i n c e c a p t o p r l l h a s b e e n r e p o r t e d to h a v e vasodllatory effects even in s p o n t a n e o u s l y h y p e r t e n s i v e rats in w h i c h the p l a s m a r e n l n a c t l v l t y is n o r m a l [8], it is p o s s i b l e that s o d i u m t r a n s p o r t s y s t e m s are i n v o l v e d in the m e c h a n i s m by w h l c h c a p t o p r i l r e d u c e s the r e s i s t a n c e of p e r i p h e r a l b l o o d v e s s e l s . In the p r e s e n t study, the a c u t e e f f e c t s of c a p t o p r l l a d m l n l s t r a t l o n on transmembrane sodium transport were studied in p a t l e n t s with essential hypertenslon a n d c o m p a r e d to s o d i u m t r a n s p o r t a c r o s s e r y t h r o c y t e m e m b r a n e s in h e a l t h y s u b 3 e c t s

Subjects

and

Methods

Subjects: A c a p t o p r l l l o a d test was c a r r l e d out in i0 p a t l e n t s w i t h e s s e n t l a l h y p e r t e n s i o n in w h o m s e c o n d a r y h y p e r t e n s i o n c o u l d be e x c l u d e d a f t e r a c o m p l e t e m e d l c a l e v a l u a t i o n . S e v e n m a l e s and three f e m a l e s of this g r o u p r a n g e d in age f r o m 24 to 64 y e a r s a n d h a d an a v e r a g e age of 4 2 . 5 ± 1 6 . 6 y e a r s . The b l o o d p r e s s u r e at the time of hospital VlSlt was 136-194 mmHg (systollc) and 95-122 mmHg ( d i a s t o l i c ) , for a m e a n b l o o d p r e s s u r e of 125 3f9 7 mmHg. A f t e r h o s p i t a l i z a t i o n , d i e t a r y salt i n t a k e was r e s t r i c t e d to I0 g per day a n d the c a p t o p r i l l o a d test was c a r r i e d out at least 1 w e e k a f t e r all a n t l h y p e r t e n s i v e a g e n t s h a d b e e n d l s c o n t i n u e d . At the time of the test, the s y s t o l i c b l o o d p r e s s u r e was 1 2 6 - 1 6 6 mmHg, d i a s t o l l c blood pressure 72-104 mmHg, a n d the m e a n b l o o d p r e s s u r e was 1 0 8 ± 1 1 . 7 mmHg, l o w e r than p r i o r to h o s p i t a l i z a t i o n S e v e n m a l e s and t h r e e f e m a l e s w e r e s e l e c t e d for a c o n t r o l g r o u p of n o r m o t e n s i v e sub3ects. T h e y r a n g e d in age from 26 to 45 y e a r s and h a d an a v e r a g e age of 3 4 . 8 f 1 2 . 8 y e a r s . This g r o u p ' s s y s t o l l c b l o o d p r e s s u r e was 1 1 6 - 1 3 4 mmHg, d A a s t o l l c b l o o d p r e s s u r e was 68-86 mmHg, and the m e a n b l o o d p r e s s u r e was 9 4 . 3 f 6 . 4 mmHg. C a D t o D r i l l o a d test: Five h y p e r t e n s i v e p a t l e n t s w l t h a m e a n b l o o d p r e s s u r e of m o r e t h a n ii0 m m H g w e r e g i v e n o r a l l y 50 m g of c a p t o p r l l w h i l e the r e m a i n i n g five p a t i e n t s w i t h a m e a n b l o o d p r e s s u r e of less t h a n ii0 m m H g r e c e i v e d 25 m g of c a p t o p r l l . The b l o o d p r e s s u r e of e a c h p a t i e n t w a s m e a s u r e d In the s u p i n e p o s i t i o n e v e r y 30 m i n u t e s for 120 m i n u t e s a f t e r c a p t o p r i l w a s a d m i n i s t e r e d . B l o o d samples were collected before a n d 60 m i n u t e s after captoprll a d m l n l s t r a t l o n and the f o l l o w i n g p a r a m e t e r s w e r e d e t e r m i n e d ; p l a s m a renAn activlty, plasma aldosterone concentration, sodium and potassium concentrations in e r y t h r o c y t e s , a n d the a c t i v i t y of e r y t h r o c y t e s o d i u m t r a n s p o r t s y s t e m s i n c l u d i n g the s o d l u m - p o t a s s i u m pump, s o d i u m - p o t a s s l u m c o t r a n s p o r t , s o d l u m - l i t h i u m c o u n t e r t r a n s p o r t and sodlum permeablllty. Plasma renAn actlVlty and plasma aldosterone concentration: Both p l a s m a r e n A n a c t i v i t y [9] a n d p l a s m a a l d o s t e r o n e c o n c e n t r a t i o n [i0] w e r e m e a s u r e d by r a d ~ o l m m u n o a s s a y . M e a s u r e m e n t of s o d i u m and p o t a s s i u m c o n c e n t r a t i o n in e r y t h r o c y t e s : S o d l u m a n d p o t a s s i u m l e v e l s of e r y t h r o c y t e s w e r e m e a s u r e d by the m e t h o d of Sachs et al. [ii]. A f t e r w a s h i n g the e r y t h r o c y t e s w i t h I0 v o l u m e s of i s o t o n i c m a g n e s l u m c h l o r i d e s o l u t i o n (4°C), an a p p r o x l -

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of C a p t o p r i l

on Na+ T r a n s p o r t

2081

m a t e l y 50% s u s p e n s i o n of e r y t h r o c y t e s was p r e p a r e d by the a d d i t i o n of i s o t o n i c m a g n e s i u m c h l o r i d e solution. The s u s p e n s l o n was d i l u t e d in 50 v o l u m e s of d i s t i l l e d w a t e r to p r o d u c e h e m o l y s i s . I n t r a c e l lular sodium and potassium concentration was estimated by determlnlng sodium, p o t a s s i u m a n d h e m o g l o b i n c o n c e n t r a t i o n in a h e m o l y s a t e of e r y t h r o c y t e s . H e m a t o c r i t and h e m o g l o b i n c o n c e n t r a t i o n were also determined in a separate suspension of cells. E r y t h r o c y t e c o n c e n t r a t i o n in m l l l l m o l e s per llter e r y t h r o c y t e s was c a l c u l a t e d from the relatlon, [Nab] (or [Nae]

(or

[Ke])

[Kh]) × Hgbs × i0

:

Hgbh × HCts w h e r e [Nae] or [Ke] iS the s o d i u m or p o t a s s i u m c o n c e n t r a t i o n in milllmoles per liter erythrocytes, [Nah] or [Kh] lS the s o d i u m or p o t a s s i u m c o n c e n t r a t l o n in the h e m o l y s a t e in m l l l l m o l e s p e r llter, Hgbh is the h e m o l y s a t e h e m o g l o b l n c o n c e n t r a t l o n In g r a m s p e r 100 ml, Hgbs is the h e m o g l o b i n c o n c e n t r a t i o n in the s u s p e n s l o n in g r a m s p e r 100 ml, a n d HCts is the h e m a t o c r l t of s u s p e n s l o n in m i l l i l i t e r s erythrocytes p e r 100 ml s u s p e n s l o n . The sodlum and potassium concentration was estlmated by the H l t a c h l atomic absorption s p e c t r o p h o t o m e t e r Model 208. M e a s u r e m e n t of the e r v t h r o c y t e s o d l u m t r a n s p o r t svstems: The rate c o n s t a n t s for s o d i u m e f f l u x w e r e m e a s u r e d by the m e t h o d of W a l t e r et al. [12]. 3.5 ml of e r y t h r o c y t e s w l t h some resldual p l a s m a w e r e p r e l n c u b a t e d w l t h 0.5 ~Cl 22Na at 3 7 ° C for 2 hours. S u b s e q u e n t l y , the erythrocytes were washed three times in a n o n r a d l o a c t l v e i n c u b a t i o n m e d l u m - i c o n t a i n i n g ii0 mmol 1 -I NaCl, 30 mmol 1 -I sodlum acetate, 1.2 mmol 1 -I MgCI2, 1.2 mmol 1 -I CaCI2, 2.5 mmol 1 -I K2HPO4, 20.0 mmol 1 -I glucose, a d 3 u s t e d to pH 7.4 at 37°C. A f t e r the last washlng, 1.0 ml of e r y t h r o c y t e s was added to e a c h of t h r e e d i f f e r e n t i n c u b a t i o n media: (a) i n c u b a t i o n medlum-l, (b) i n c u b a t i o n medium-2 [incubation medlum-i + 10 -4 m o l 1 -I ouabaln] and (c) incubation medium-3 [incubation medlum-2 + 10 -3 mmol 1 -I f u r o s e m l d e ] . The r e s u l t l n g s u s p e n s l o n was i n c u b a t e d in a s h a k l n g b a t h at 37°C. At the b e g l n n l n g of the e f f l u x studies and after 30, 60, and 90 m i n u t e s of incubation, 1.4 ml samples w e r e taken, c o o l e d i m m e d l a t e l y in an i c e - w a t e r bath, and c e n t r i f u g e d for 5 m l n u t e s at 2,000 g at 4°C; 1.0 ml p o r t i o n s of the s u p e r n a t a n t w e r e t a k e n to measure the radloactlvlty (Nt). The total rad±oactlvlty was determlned in 1.0 ml of uncentrlfuged medlum (No). The r a d i o a c t i v ± t y of 22Na was d e t e r m i n e d by s c i n t i l l a t i o n c o u n t e r (LKB W a l l a c 1282 C o m p u g a m m a , Turku, Finland) . The r a t e c o n s t a n t for s o d i u m e f f l u x (k) was c a l c u l a t e d from the slope of the r e g r e s s i o n line, w h i c h w a s o b t a l n e d by p l o t t i n g the l o g a r ± t h m of r e s i d u a l r a d l o a c t l V l t y w i t h i n the e r y t h r o c y t e s in (i- Nt/No) a g a l n s t the incubation time. Llnear~ty was ascertained throughout the i n c u b a t i o n p e r i o d of 90 minutes. In this way, (a) o u a b a l n - s e n s l t i v e 22Na e f f l u x r a t e c o n s t a n t (sodium-potassium pump actlvlty), (b) o u a b a l n - l n s e n s l t l v e f u r o s e m l d e - s e n s l t l v e 22Na e f f l u x rate c o n s t a n t (sodium-potassium cotransport) and (c) ouabaln-insensltlve furosemlde-lnsensitive 22Na e f f l u x rate c o n s t a n t (sodium p a s s i v e permeab111ty) were determined. S o d l u m - i i t h l u m c o u n t e r t r a n s p o r t was measured according to the Canessa et al. [13]. 2.5 ml of

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erythrocytes were washed three times with an isotonic solutlon (washing solution-l) containing 75 m m o l 1 -I MgCI2, 95 m m o l 1 -I sucrose, and i0 mmol 1 -I Trls (hydroxymethyl) aminomethanemorphollnopropane sulfonlc acld (Trls-MOPS), a d 3 u s t e d to p H 7.4 at 4°C. 2 ml of e r y t h r o c y t e s w e r e i n c u b a t e d for 4 h o u r s at 3 7 ° C in 8 ml of m e d i u m c o n t a i n i n g 150 m m o l 1 -I LACl, i0 m m o l 1 -I T r l s - M O P S , p H 7.4 at 3 7 ° C . The cells were washed with washing solution-2 (75 m m o l 1 -I MgCI2, 95 m m o l 1 -I s u c r o s e , i0 m m o l 1 -I T r l s - M O P S , a n d i0 4 m o l 1 -I o u a b a l n ) f i v e t l m e s to e l l m l n a t e external lithium. A 50% suspension of w a s h e d c e l l s in w a s h i n g solution-2 was made up and k e p t cold. A n a l i q u o t of t h e s e l l t h l u m - l o a d e d c e l l s w a s t a k e n for a n a l y s i s of h e m o g l o b l n a n d l i t h i u m , a n d t h e r e m a i n d e r w a s u s e d for a s s a y of l l t h i u m - s o d l u m countertransport. 0.6 ml of t h i s s u s p e n s i o n w a s a d d e d to 6 ml of s o d l u m - f r e e medium (75 m m o l 1 -I MgCI2 a n d 85 m m o l 1 -I s u c r o s e ) or to s o d l u m - e n r l c h e d medlum (150 m m o l 1 -I NaCl) c o n t a ± n l n g i0 m m o l 1 -I g l u c o s e , 10 -4 m o l 1 -I o u a b a l n , a n d i0 m m o l 1 -I Trls-MOPS, and then incubated at 3 7 ° C . Samples of 1.5 ml w e r e t a k e n at the b e g l n n l n g a n d a f t e r 30, 60, 90 m i n u t e s of I n c u b a t i o n The llthlum concentration in t h e s u p e r n a t a n t was measured w l t h an atomic absorption spectrophotometer The lithium efflux in mllllmoles (llter of e r y t h r o c y t e s x hour) -I w a s d e t e r m l n e d f r o m the llnear regresslon of llthium loss (milllmoles per llter of erythrocytes) as a f u n c t l o n tlme Sodium-llthlum countertransport activlty was calculated from the dlfference between the llthlum e f f l u x r a t e in t h e s o d i u m - e n r l c h e d m e d i u m a n d t h a t in t h e s o d i u m free medium. Results

B l o o d D r e s s u r e : T h e a c u t e c h a n g e s in b l o o d p r e s s u r e a f t e r c a p t o p r l l administration a r e s h o w n in F i g u r e i. P r l o r to c a p t o p r l l a d m i n l s tration, t h e m e a n b l o o d p r e s s u r e of t h e f i v e p a t i e n t s in the 50 m g g r o u p w a s 1 1 7 . 8 ± 4 . 8 m m H g w h i l e t h a t of the five p a t i e n t s in t h e 25 mg group was 98.2±6.8 mmHg. However, at 60 m i n u t e s a f t e r c a p t o p r l l adminlstration, the mean blood pressure dropped slgniflcantly to 1 0 7 . 0 ± 7 . 8 m m H g in t h e 50 m g g r o u p (p