- Email: [email protected]
Plasma from cirrhotic patients induces inotropic changes on cultured rat heart cells
Life Sciences, Vol. 41. pp. 2177-2184 Printed in the U.S.A.
PLASMA
FROM
Lidia Catherine
Pergamon Journals
CIRRHOTIC PATIENTS INDUCES INOTROPIC ON C U L T U R E D R A T H E A R T C E L L S E. P. de M i k u l i c , Vernimmen, Didier
CHANGES
Marie-Claude Auclair, Lebrec, Esteban Mikulic.
I n s t i t u t de P h a r m a c o l o g i e - U n i v e r s i t ~ C u r i e - P a r i s VI, and H o p i t a l B e a u j o n ,
P i e r r e et M a r i e Clichy, France.
(Received in final form September ii, 1987) Summar Z C u l t u r e d m o n o l a y e r s of n e w b o r n r a t h e a r t c e l l s w e r e p e r f u s e d w i t h p l a s m a f r o m p a t i e n t s w i t h a l c o h o l i c l i v e r cirrhosis. The results showed that cirrhotic plasma, when c o m p a r e d to c o n t r o l p l a s m a , d e p r e s s e d c o n t r a c t i l i t y of beating cardiac cells. The in-vitro deleterious effects of p l a s m a f r o m c i r r h o t i c s u b j e c t s on c e l l c u l t u r e s w e r e n e i t h e r r e l a t e d to h e m o d y n a m i c p a r a m e t e r s nor to the lev e l s of p l a s m a t i c c a t e c h o l a m i n e s . T h i s s t u d y s u g g e s t s the p r e s e n c e in t h e p l a s m a of p a t i e n t s w i t h a l c o h o l i c l i v e r c i r r h o s i s , of h u m o r a l f a c t o r ( s ) w h i c h c a u s e d e p r e s s i v e e f f e c t s on r a t h e a r t c e l l s in c u l t u r e . A h y p e r k i n e t i c c i r c u l a t o r y s t a t e is an u n c o m m o n p i c t u r e ~ss o c i a t e d w i t h c h r o n i c d i s e a s e of t h e l i v e r (1,2), a c o n d i t i o n w h ~ h is a l s o a c c o m p a n i e d by h i g h c a r d i a c o u t p u t (3,4) and e l e v a t e d p l a s ma l e v e l s of c a t e c h o l a m i n e s (5). A l s o h e m o d y n a m i c (6,7), m e t a b o l i c (8), and s t r u c t u r a l (9) s t u d i e s in m a n and e x p e r i m e n t a l a n i m a l s h a v e e m p h a s i z e d the e f f e c t s of c h r o n i c a l a l c o h o l i n t a k e on h e a r t . L e f t v e n t r i c u l a r e n d d i a s t o l i c p r e s s u r e (LVEDP) , are u s u a l l y e l e v a t e d at rest, d u r i n g e x e r c i s e (10,11), and w i t h a n g i o t e n s i n i n f u s i o n (12), in c i r r h o t i c p a t i e n t s . T h e o b s e r v a t i o n s of i m p a i r e d left v e n t r i c u l a r f u n c t i o n a r e in a c c o r d a n c e w i t h f i n d i n g s of S p o d i c k et al (13). T h e s e a u t h o r s h a v e a l s o d e m o n s t r a t e d a b n o r m a l s y s t o l i c t i m e i n t e r v a l s i n d i c a t i v e of i m p a i r e d c a r d i a c f u n c t i o n in a l c o h o l i c s u b j e c t s w i t h o u t e v i d e n t s i g n s of h e a r t d i s e a s e , O n the o t h e r hand H e i n e m a n n (14) c o n s i d e r s t h a t c a r d i a c f a i l u r e s are a c o n c o m i t a n t f i n d i n g r e l a t e d to p a t i e n t age and are n o t l i n k e d to a l c o h o l i c liver cirrhosis. T h e p u r p o s e of the p r e s e n t s t u d y w a s to e v a l u a t e the p l a s m a e f f e c t s f r o m a l c o h o l i c c i r r h o t i c p a t i e n t s on the i n o t r o p i c and c h r o n o t r o p i c p r o p e r t i e s of c u l t u r e d c a r d i a c cell. Patients
and M e t h o d s
T w e n t y - s i x p a t i e n t s a d m i t t e d to the B e a u j o n H o p i t a l (Paris) w e r e s t u d i e d . In f o u r t e e n p a t i e n t s c i r r h o s i s w a s h i s t o l o g i c a l l y p r o v e n and w a s a t t r i b u t e d to c h r o n i c a l c o h o l i s m . N o n e of the p a t i e n t s w a s in a n a c u t e a l c o h o l w i t h d r a w a l state. T h e o t h e r t w e l v e s u b j e c t s w e r e u s e d as a c o n t r o l g r o u p . T h e d i a g n o s i s of t h e ~ e p&0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
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tients were: myeloid metaplasia (n=3), i d i o p a t i c a p l a s t i c a n e m i a (n=2), h a i r y c e l l l e u k e m i a (n=l), l y m p h o m a (n=2), s a r c o i d o s i s (n=l) , s c l e r o d e r m a w i t h S j o g r e n ' s s y n d r o m e (n=l), r h e u m a t o i d arthritis with cryoglobulinemia (n=l) c h r o n i c a c t i v e h e p a t i t i s (n=l) . A l l t r e a t m e n t s i n v o l v i n g d r u g s w e r e w i t h h e l d for 48 hrs. p r i o r to the study. H e m o d y n a m i c s t u d i e s w e r e c a r r i e d out on all p a t i e n t s . A S w a n - G a n z c a t h e t e r w a s i n s e r t e d v i a i n t e r n a l j u g u l a r v e i n a n d it w a s a d v a n c e d up to the p u l m o n a r y a r t e r y . T h e p r e s s u r e w a s r e c o r d e d f r o m r i g h t a t r i u m (RA), p u l m o n a r y a r t e r y (PA) and f r o m p u l m o n a r y c a p i l l a r y w e d g e (PCW). H e p a t i c p r e s s u r e w a s o b t a i n e d t h r o u g h a C o r d i s N8 m u l t i p u r p o s e c a t h e t e r , w h i c h w a s a d v a n c e d up to s u p r a h e p a t i c v e i n . The g r a d i e n t b e t w e e n w e d g e d (HWP) a n d free h e p a t i c v e n o u s p r e s s u r e (FHP) w a s m e a s u r e d . C a r d i a c o u t p u t (CO) w a s d e t e r m i n e d in t r i p l i c a t e by t h e r m o d i l u t i o n m e t h o d (15). H e a r t r a t e ~I~HR) w a s a l s o r e c o r d e d d u r i n g the m e a s u r e m e n t s of CO. M e a s u r e m e n t of p l a s m a t i c l e v e l s of n o r a d r e n a l i n e , a d r e n a l i n e and d o p a m i n e w e r e c a r r i e d o u t b y r a d i o e n z y m a t i c m e t h o d (16), u s i n g b l o o d o b t a i n e d f r o m t h e f o r e a r m v e i n of 8 c i r r h o t i c and 8 n o n - c i r r h o t i c patients. P r e p a r a t i o n of c e l l c u l t u r e s C e l l c u l t u r e s w e r e p r e p a r e d a c c o r d i n g to a m o d i f i e d v e r s i o n of the H a r a r y and F a r l e y ' s m e t h o d (17). W h o l e h e a r t s w e r e rem o v e d f r o m one to t h r e e d a y s old r a t s and w e r e k e p t in H a n k ' s solution, a f t e r m i n c i n g and w a s h i n g t h e m in c a l c i u m and m a g n e s i u m f r e e p h o s p h a t e b u f f e r e d s a l i n e (PBS) . T h e h e a r t t i s s u e w a s t h e n d i s s o c i a t e d by t r e a t m e n t w i t h 2.5% of b o v i n e t r y p s i n for ten m i n u t e s at 37°C. A f t e r d i s c a r d i n g the i n i t i a l s u p e r n a t a n t , the t r y p s i n a t i o n p r o c e s s w a s r e p e a t e d 5 to 7 t i m e s and the r e s u l t a n t s u p e r n a t a n t l a y e r w a s c o l l e c t e d i n t o c e n t r i f u g e t u b e s p l a c e d on ice. T h e a c t i o n of t r y p s i n w a s s t o p p e d by a d d i t i o n of c u l t u r e m e d i u m (Eagles m i n i m a l e s s e n t i a l m e d i u m w i t h E a r l e ' s s a l t s u p p l e m e n t e d w i t h 10% c a l f s e r u m ) . T h e p r e p a r a t i o n w a s t h e n c e n t r i f u g e d at 750 g. for 5 m i n u t e s at 4°C. T h e c e l l p e l l e t t h u s o b t a i n e d w a s s u s p e n d e d in c u l t u r e m e d i u m a n d f i l t e r e d t h r o u g h lens p a p e r to r e m o v e c e l l u l a r c l u m p s . A n a l i q u o t w a s t h e n c o u n t e d on a h e m a t o c y t o m e t e r , the c e l l s u s p e n s i o n w a s d i l u t e d to 2 5 0 , 0 0 0 c e l l s / m l and s e e d e d on c o v e r s l i p s p l a c e d in 2 ml L e i g h t o n t u b e s . T h e t u b e s w e r e i n c u b a t e d at 3 7 ° C w i t h air and CO^ as g a s e o u s p h a s e . N o c h a n g e w a s m a d e 24 h o u r s a f t e r the p l a t i n g . T h i s led to restricted fibroblastic proliferation. Plasma samples P l a s m a s a m p l e s f r o m n o n - c i r r h o t i c p a t i e n t s w e r e u s e d as a c o n t r o l g r o u p . C i r r h o t i c p l a s m a w a s o b t a i n e d f r o m the p a t i e n t s d e s c r i b e d in t h i s s t u d y . A l l s a m p l e s w e r e d r a w n f r o m the c a t h e t e r p l a c e d in the p u l m o n a r y a r t e r y in o r d e r to h a v e a m i x e d v e n o u s b l o o d . P l a s m a w a s s e p a r a t e d for e v a l u a t i o n of p l a s m a e l e c t r o l y t e s , b i l i r u b i n , c r e a t i n i n e , c a t e c h o l a m i n e s , u r e a and c o n t r a c t i l e a c t i v ity of c u l t u r e d h e a r t c e l l s . P r o c e s s i n g of c e l l c u l t u r e s E x p e r i m e n t s w e r e p e r f o r m e d on 5 d a y o l d c u l t u r e s , at w h i c h t i m e the c u l t u r e s had r e a c h e d a s t e a d y state. O n the d a y of the e x p e r i m e n t , the c o v e r s l i p s w i t h the c e l l s w e r e p l a c e d i n t o a p l e x i g l a s s c h a m b e r on the s t a g e of an i n v e r t e d m i c r o s c o p e , m a i n -
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t a i n e d at 35°C. The d e s i r e d m e d i u m was i n f u s e d at the rate of 0.1 ml/min, t h r o u g h o u t the c h a m b e r by m e a n s of a force and s u c t i o n pump. Contractile activity A m i c r o s c o p e p i c t u r e (magnified x 400) was t r a n s m i t t e d to a t e l e v i s i o n s c r e e n v i a a t e l e v i s i o n camera. On the screen, v a r i a t i o n in light i n t e n s i t y r e s u l t i n g f r o m cell edge m o v e m e n t d u r i n g c o n t r a c t i o n was t r a n s m i t t e d to a p h o t o c e l l by an o p t i c a l fiber. The o u t p u t signal of the p h o t o c e l l w a s t r a n s m i t t e d to an o s c i l l o scope w h e r e v a r i a t i o n s in light i n t e n s i t y a p p e a r e d as waves. T h e a m p l i t u d e of the w a v e s i n d i c a t e d in a r b i t r a r y u n i t s (mv) , v a r i e d p r o p o r t i o n a l l y w i t h light intensity. T h e r e f o r e , this p r o c e d u r e p r o v i d e s an e v a l u a t i o n of the m o v e m e n t of a cell edge so that cont r a c t i l e a c t i v i t y can be assessed.
Results C o n t r a c t i l e a c t i v i t y of c u l t u r e d h e a r t c e l l s fn the p r e s e n c e of c o n t r o l h u m a n plasma, c o n t r a c t i l e a c t i v i ty o c c u r r e d r e g u l a r l y for one hour. In the p r e s e n c e of p l a s m a f r o m c i r r h o t i c patients, the f r e q u e n c y was s i m i l a r to that obt a i n e d in the p r e s e n c e of c o n t r o l plasma, but the a m p l i t u d e of c e ~ lular edge m o v e m e n t s was r e d u c e d by 33.3% ( 0 . 0 1 > p < 0.001) (Fig. 1,2). T h e s e c h a n g e s in a m p l i t u d e w e r e h i g h l y s i g n i f i c a n t as e a r l y as 10 min. a f t e r start, at w h i c h time a r e d u c t i o n of 20.4 % (p < 0 . 0 3 ) f r o m i n i t i a l v a l u e s w a s observed. All the p r e p a r a t i o n s of c u l t u r e d h e a r t c e l l s u n d e r p e r f u s i o n w i t h c i r r h o t i c p l a s m a dev e l o p e d a r r h y t h m i a s at any time in the c o u r s e of the e x p e r i m e n t (Fig. 3). On the o t h e r hand, the h u m a n c o n t r o l p l a s m a did not m o d ify the r e g u l a r i t y of the b e a t i n g c a r d i a c cells. Hemodynamic parameters T h e h e m o d y n a m i c p a r a m e t e r s , h e a r t rate (HR) and a r t e r i a l p r e s s u r e (AP) w e r e s i m i l a r in b o t h g r o u p s of p a t i e n t s . However, the r i g h t a t r i u m p r e s s u r e (RA) w a s 3.5 + 0.5 m m H g in the c o n t r o l group, w h i l e in the c i r r h o t i c p a t i e n t s was 5.7 + 0.7 m m H g (p <0.02). P u l m o n a r y c a p i l l a r y w e d g e p r e s s u r e (PCW) w a s 6.7 + 0.8 m m H g in n o n - c i r r h o t i c s and 9.3 + 0.9 m m H g (p < 0 . 0 4 ) in t h e c i r r h o t i c group. In addition, h e p a t i c w e d g e p r e s s u r e (HWP) in c i r r h o t i c p a t i e n t s was s i g n i f i c a n t l y g r e a t e r than that of c o n t r o l subjects. The values o b t a i n e d f r o m c i r r h o t i c p a t i e n t s w e r e 28.1 + i.i m m H g and 13.9 + 1.2 m m H g for c o n t r o l s u b j e c t s (p< 0.0001). T h e g r a d i e n t between-h e p a t i c p r e s s u r e (HP) and free h e p a t i c p r e s s u r e (FHP) w a s inc r e a s e d from 5.1 + i.i m m H g in the c o n t r o l g r o u p up to 16.2 + 1.4 m m H g (p < 0 . 0 0 0 1 ) ~n the c i r r h o t i c group. C a r d i a c o u t p u t (CO) a l s o was e l e v a t e d f r o m 5.4 + 0.3 i/m in c o n t r o l s u b j e c t s to 6.9 + 0.5 i/m (p < 0 . 0 3 ) in p a t i e n t s w i t h cirrhosis. S y s t e m i c v a s c u l a r r e s i s t a n c e (SVR) was r e d u c e d in p a t i e n t s w i t h liver c i r r h o s i s (1095 + 56.3 d y n e s - s e c - c m - 5 ) in r e s p e c t of the c o n t r o l g r o u p (1440 + iO3.7 d y n e s - s e c - c m - 5 ) ( p < 0.005). P l a s m a c a t e c h o l a m i n e s levels P l a s m a t i c n o r a d r e n a l i n e levels
in p a t i e n t s w i t h
liver cir-
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Vol. 41, No. 19, 1987
Q
0 %
Fig.
I
C o n t r a c t i l e a c t i v i t y of c u l t u r e d m y o c a r d i a l cells. P i c t u r e s w e r e taken f r o m the same cell (A) after c o n t a c t w i t h p l a s m a f r o m c o n t r o l patient, and (B) after c o n t a c t w i t h p l a s m a f r o m one p a t i e n t w i t h a l c o h o l i c liver cirrhosis. S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h p l a s m a f r o m the 13 other c i r r h o t i c p a t i e n t s .
r h o s i s w a s h i g h e r t h a n in c o n t r o l subjects. O n the other hand, a d r e n a l i n e and d o p a m i n e did not show any d i f f e r e n c e in b o t h groups. (Table I). Table Circulating cathecolamines
I
in p e r i p h e r a l p l a s m a
(Mean + SE) Group
Noradrenaline (pg/ml)
Adrenaline (pg/ml)
Dopamine (pg/m])
Control
188,9 +
40,7
67,9 + 17,5
61,7 + 21,1
Cirrhotic
848,4 + 159,9
161,8 + 42,9
Significance
p < 0.004
NS
63,1 +
6,2
NS
B l o o d s a m p l e s w e r e t a k e n from the f o r e a r m v e i n of e i g h t c i r r h o t i c and eight n o n - c i r r h o t i c patients. C a t h e c o l a m i n e s w e r e d e t e r m i n e d in p l a s m a as d e s c r i b e d on Methods.
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2 0 mm
15
o O
120
/_ o
10 >
o
80
<
Q) In
5 40
Fig.
2
B e a t i n g f r e q u e n c y and c o n t r a c t i l e a c t i v i t y of c u l t u r e h e a r t c e l l s in p r e s e n c e of e i t h e r p l a s m a f r o m t w e l v e c o n t r o l (r--]) or f o u r t e e n c i r r h o t i c p a t i e n t s ( ~ ) . T h e r e s u l t s are e x p r e s s e d as M e a n + SE. D i f f e r e n c e s b e t w e e n m e a n v a l u e s w e r e e v a l u a t e d ~ b y S t u d e n t ' s "t" test. (* p < 0.01) .
t
Fig. Arrhythmia
observed
during
3
cirrhotic
plasma
infusion.
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Vol. 41, No. 19, 1987
P l a s m a e l e c t r o l y t e s , b i l i r u b i n , c r e a t i n i n e and u r e a c o n c e n trations T h e r e a r e no s i g n i f i c a n t d i f f e r e n c e s in p l a s m a c o n c e n t r a t i o n s of s o d i u m , p o t a s s i u m , c a l c i u m , b i l i r u b i n , c r e a t i n i n e and u r e a in b o t h g r o u p s of n a t i e n t s (Table II). T h e p a t i e n t s in the c o n t r o l g r o u p had h i g h b i l i r u b i n l e v e l s and w e r e s t u d i e d and subj e c t e d to l i v e r b i o p s y in o r d e r to e s t a b l i s h t h e i r d e f i n i t i v e diagnosis. Table Na (mEq/l) Control (n=12)
K (mEq/l)
II
Ca (mEq/l)
Bilirubin (mg/l)
Creatinine (mg/dl)
Urea (mg/d])
138.7 6.8
+
4.4 0.2
+
9.5 0.3
+
25.3 3.5
+
1.2 0.i
0,36 + 0.03
Cirrhotic 137.3 (n=14) + 4.2
+
4.2 0.2
+
9.4 0.2
+
28.5 4.8
+
i.i 0.2
0.37 + 0.02
+
Discussion T h e r e s u l t s o b t a i n e d s h o w e d t h a t the c o n t r a c t i l e a c t i v i t y of c u l t u r e d c e l l s w a s a l t e r e d w h e n in c o n t a c t w i t h p l a s m a f r o m c i r r h o t i c p a t i e n t s . T h e s e f i n d i n g s s u g g e s t the p r e s e n c e of a f a c tor(s) c a u s i n g a c a r d i a c d e p r e s s i v e a c t i o n , in t h e p l a s m a of p a tients with alcoholic cirrhosis. These effects have never been r e p o r t e d by o t h e r a u t h o r s and the f i n d i n g s c o u l d be of g r e a t int e r e s t in an a t t e m p t to u n d e r s t a n d the m o d e of a c t i o n of m y o c a r d i a l d e p r e s s a n t f a c t o r s in t h i s p a t h o l o g y . N o n e of the c i r r h o t i c p a t i e n t s i n c l u d e d in t h i s s t u d y w e r e t h o u g h t to h a v e c l i n i c a l l y s i g n i f i c a n t c a r d i a c i m p a i r m e n t . H o w ever, p u l m o n a r y c a p i l l a r y w e d g e , w h i c h is c l o s e l y r e l a t e d to left v e n t r i c u l a r end d i a s t o l i c p r e s s u r e , w a s h i g h e r t h a n our c o n t r o l s u b j e c t s . T h e h i g h r e s t i n g c a r d i a c o u t p u t n o t e d in t h e s e s u b j e c t s is o f t e n v i e w e d as e v i d e n c e t h a t c a r d i a c p e r f o r m a n c e is n o r m a l (3,4). It is w e l l e s t a b l i s h e d t h a t the g r a d i e n t b e t w e e n w e d g e d and f r e e h e p a t i c v e n o u s p r e s s u r e s r e f l e c t s p o r t a l v e n o u s p r e s s u r e in c i r r h o s i s (18). T h e r e f o r e , the h e m o d y n a m i c r e s u l t s i n d i c a t e t h a t the p a t i e n t s s e l e c t e d for t h i s s t u d y had a h y p e r k i n e t i c c i r c u l a t o r y s t a t e and p o r t a l h y p e r t e n s i o n . P l a s m a l e v e l s of c a t e c h o l a m i n e s w e r e a l s o s t u d i e d in t h e s e p a t i e n t s w i t h l i v e r c i r r h o s i s and the v a l u e s o b t a i n e d r e v e a l e d t h a t no s i g n i f i c a n t e l e v a t i o n in a d r e n a l i n e l e v e l s w a s f o u n d in all p a t i e n t s e x a m i n e d , w h e r e a r i s e in n o r a d r e n a l i n e w a s seen, in a c c o r d a n c e w i t h o t h e r a u t h o r s ' f i n d i n g s (5). D e s p i t e the inc r e a s e in c i r c u l a t i n g n o r a d r e n a l i n e , the c o n t r a c t i l e a c t i v i t y of c u l t u r e d c a r d i a c c e l l d e c r e a s e d w h e n the p l a s m a f r o m c i r r h o t i c p a t i e n t s w a s added. T h e s e o b s e r v a t i o n s s u g g e s t t h a t o t h e r h u m o r al f a c t o r s m i g h t be i n v o l v e d in t h i s case. A m o n g t h e s e , the a t r i a l n a t r i u r e t i c f a c t o r m u s t be t a k e n i n t o a c c o u n t , s i n c e it has e v i d e n t c a r d i o v a s c u l a r e f f e c t s . T h i s p e p t i d e c a u s e s h y p o t e n s i o n , b r a d i c a r d i a and f a i l u r e of s t r o k e v o l u m e to r i s e p r o p e r -
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ly. H o w e v e r , it h a s n o t b e e n e l u c i d a t e d if c a r d i o i n h i b i t o r y eff e c t s a r e p r o d u c e d b y d i r e c t a c t i o n on the c a r d i o m y o c y t e s , by inh i b i t i o n or r e f l e x m e c h a n i s m s or b y r e l e a s e of an u n i d e n t i f i e d neg a t i v e l y i n o t r o p i c s u b s t a n c e (20). H y p o c a l c e m i a h a s b e e n n o t e d s p o r a d i c a l l y in p a t i e n t s w i t h c i r r h o s i s w i t h no a p p a r e n t r e l a t i o n s h i p b e t w e e n s e r u m i o n i z e d and t o t a l c a l c i u m c o n c e n t r a t i o n s . A l t h o u g h it h a s b e e n r e p o r t e d an a v e r a g e r e d u c t i o n of 5% in t h e C a + + l e v e l s in c i r r h o t i c s u b j e c t s , s e r u m i o n i z e d c a l c i u m is q u i t e v a r i a b l e in t h i s t y p e of p a t i e n t s (19). O u r d a t a s h o w t h a t t h e r e are no s i g n i f i c a n t d i f f e r e n c e s in t h e c a l c i u m p l a s m a t i c c o n c e n t r a t i o n in b o t h g r o u p s of p a t i e n t s . In a d d i t i o n , n o c h a n g e s w e r e o b s e r v e d in the b i l i r u b i n , s o d i u m , p o t a s s i u m , c r e a t i n i n e and u r e a p l a s m a t i c c o n c e n t r a t i o n s . In c o n c l u s i o n , the p r e s e n t r e s u l t s c l e a r l y s h o w t h a t the p l a s m a f r o m c i r r h o t i c p a t i e n t s d e p r e s s e d the c o n t r a c t i l i t y of c u l t u r e d c e l l s . T h e p l a s m a f a c t o r ( s ) i n v o l v e d in t h i s c a r d i a c d e p r e s s i n g e f f e c t d e s e r v e f u r t h e r s t u d y and t h i s m i g h t p r o v i d e n e w c l u e s for t h e u n d e r s t a n d i n g of c a r d i o v a s c u l a r r e s p o n s e s in cirrhotic patients.
References i. 2. 3. 4. 5. 6.
7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17.
P. W O O D . D i s e a s e of the H e a r t a n d C i r c u l a t i o n , p. 1-25. E y r e 2nd e d i t i o n . S p o t w i d e , L o n d o n (1956). J.G. C L A Y P O O L , M. D E L P and T.K. LIN. A m e r . J. Med. Sci. 234: 4 8 - 5 5 (1957). J.F. M U R R A Y , A . M . D A W S O N and S. S H E R L O C K . A m e r . J. M e d . 24: 3 5 8 - 3 6 7 (1958) . W.H. A B E L M A N , H.J. K O W A L S K I and W.F. M c N E E L Y . J. Clin. I n v e s t . 34: 6 9 0 - 6 9 5 (1955). K. I S H I I , H. S H I B A T A , H. Y A M A D A , H. N A K A S A W A and J. O K A B E . Jap. G a s t r o e n t e r o l . 76: 2 4 1 0 - 2 4 1 5 (1979). T.J. R E G A N , G. K O R O Z E N I D I S , C.B. M O S C H O S , H.A. O L D E W U R T E L , P.H. L E H A N a n d H.K. H E L L E M S . J. C l i n . I n v e s t . 45: 2 7 0 - 2 8 0 (1966) . D.P. RIFF, A . C . J A I N and J.T. D O Y L E . Am. H e a r t J. 78: 5 9 2 - 5 9 7 (1969) . V . E . W E N D T , R. A J L U N I , T.~. B R U C E , A.S. P R A S A D a n d R.J. B I N G . A m e r . J. C a r d i o l . 1 7 : 8 0 4 - 8 1 2 (1966). V.J. F E R R A N S , R.G. H I B B S , G. W E I L B A E C H E R , W.C. B L A C K a n d G.E. B U R C H . A m e r . H e a r t J. 69: 7 4 8 - 7 6 5 (1966). T.J. B A Y L E Y , N. S E G E L a n d J.J. B I S H O P . Clin. Sci. 26: 2 2 7 - 2 3 5 (1964). L. G O U L D , M. S H A R I F F , M. Z A H I R a n d M. DI L I E T O . J. C l i n . Inv e s t . 48: 8 6 0 - 8 6 8 (1969) C.J. L I M A S , H.H. G U I H A , O. L E K A G U L and J.N. COHN. C i r c u l a t i o n 49: 7 5 5 - 7 6 0 (1974). D.H. S P O D I K , V . N . P I G O T T and R. C H I R I F E . N. E n g l . J. M e d . 287: 6 6 7 - 6 8 0 (1972). H.O. H E I N E M A N N . C i r c u l a t i o n 22: 1 5 4 - 1 5 9 (1960). J.S. F O R R E S T E R , W. G A N Z , G. D-~AMOND, T. M c H U G H , D.W. CHONETTE, a n d H . J . C . S W A N . Am. H e a r t J. 83: 3 0 6 - 3 1 1 ( 1 9 7 2 ) . M. D A P R A D A a n d G. Z U R C H E R . Lif--e S c i e n c e s 19: 1 1 6 1 - 1 1 7 1 (1976) I. H A R A R I a n d B. F A R L E Y . Exp. C e l l Res. 29.7--451-465 (1963).
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18. 19. 20.
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T, D. B O Y E R , D.R. T R I G E R , M. H O R I Z A W A , A.G. R E D E K E R and T.B. R E Y M O L D S . G a s t r o e n t e r o l o g y 72: 5 8 4 - 5 8 9 (1977). E.W. M O O R E . G a s t r o e n t e r o l o g y 60: 4 3 - 5 4 (1971). V. A C K E B M A N N . F e d e r a t i o n Proc-~--45: 2 1 1 1 - 2 1 1 4 (1986).
PLASMA
FROM
Lidia Catherine
Pergamon Journals
CIRRHOTIC PATIENTS INDUCES INOTROPIC ON C U L T U R E D R A T H E A R T C E L L S E. P. de M i k u l i c , Vernimmen, Didier
CHANGES
Marie-Claude Auclair, Lebrec, Esteban Mikulic.
I n s t i t u t de P h a r m a c o l o g i e - U n i v e r s i t ~ C u r i e - P a r i s VI, and H o p i t a l B e a u j o n ,
P i e r r e et M a r i e Clichy, France.
(Received in final form September ii, 1987) Summar Z C u l t u r e d m o n o l a y e r s of n e w b o r n r a t h e a r t c e l l s w e r e p e r f u s e d w i t h p l a s m a f r o m p a t i e n t s w i t h a l c o h o l i c l i v e r cirrhosis. The results showed that cirrhotic plasma, when c o m p a r e d to c o n t r o l p l a s m a , d e p r e s s e d c o n t r a c t i l i t y of beating cardiac cells. The in-vitro deleterious effects of p l a s m a f r o m c i r r h o t i c s u b j e c t s on c e l l c u l t u r e s w e r e n e i t h e r r e l a t e d to h e m o d y n a m i c p a r a m e t e r s nor to the lev e l s of p l a s m a t i c c a t e c h o l a m i n e s . T h i s s t u d y s u g g e s t s the p r e s e n c e in t h e p l a s m a of p a t i e n t s w i t h a l c o h o l i c l i v e r c i r r h o s i s , of h u m o r a l f a c t o r ( s ) w h i c h c a u s e d e p r e s s i v e e f f e c t s on r a t h e a r t c e l l s in c u l t u r e . A h y p e r k i n e t i c c i r c u l a t o r y s t a t e is an u n c o m m o n p i c t u r e ~ss o c i a t e d w i t h c h r o n i c d i s e a s e of t h e l i v e r (1,2), a c o n d i t i o n w h ~ h is a l s o a c c o m p a n i e d by h i g h c a r d i a c o u t p u t (3,4) and e l e v a t e d p l a s ma l e v e l s of c a t e c h o l a m i n e s (5). A l s o h e m o d y n a m i c (6,7), m e t a b o l i c (8), and s t r u c t u r a l (9) s t u d i e s in m a n and e x p e r i m e n t a l a n i m a l s h a v e e m p h a s i z e d the e f f e c t s of c h r o n i c a l a l c o h o l i n t a k e on h e a r t . L e f t v e n t r i c u l a r e n d d i a s t o l i c p r e s s u r e (LVEDP) , are u s u a l l y e l e v a t e d at rest, d u r i n g e x e r c i s e (10,11), and w i t h a n g i o t e n s i n i n f u s i o n (12), in c i r r h o t i c p a t i e n t s . T h e o b s e r v a t i o n s of i m p a i r e d left v e n t r i c u l a r f u n c t i o n a r e in a c c o r d a n c e w i t h f i n d i n g s of S p o d i c k et al (13). T h e s e a u t h o r s h a v e a l s o d e m o n s t r a t e d a b n o r m a l s y s t o l i c t i m e i n t e r v a l s i n d i c a t i v e of i m p a i r e d c a r d i a c f u n c t i o n in a l c o h o l i c s u b j e c t s w i t h o u t e v i d e n t s i g n s of h e a r t d i s e a s e , O n the o t h e r hand H e i n e m a n n (14) c o n s i d e r s t h a t c a r d i a c f a i l u r e s are a c o n c o m i t a n t f i n d i n g r e l a t e d to p a t i e n t age and are n o t l i n k e d to a l c o h o l i c liver cirrhosis. T h e p u r p o s e of the p r e s e n t s t u d y w a s to e v a l u a t e the p l a s m a e f f e c t s f r o m a l c o h o l i c c i r r h o t i c p a t i e n t s on the i n o t r o p i c and c h r o n o t r o p i c p r o p e r t i e s of c u l t u r e d c a r d i a c cell. Patients
and M e t h o d s
T w e n t y - s i x p a t i e n t s a d m i t t e d to the B e a u j o n H o p i t a l (Paris) w e r e s t u d i e d . In f o u r t e e n p a t i e n t s c i r r h o s i s w a s h i s t o l o g i c a l l y p r o v e n and w a s a t t r i b u t e d to c h r o n i c a l c o h o l i s m . N o n e of the p a t i e n t s w a s in a n a c u t e a l c o h o l w i t h d r a w a l state. T h e o t h e r t w e l v e s u b j e c t s w e r e u s e d as a c o n t r o l g r o u p . T h e d i a g n o s i s of t h e ~ e p&0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
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tients were: myeloid metaplasia (n=3), i d i o p a t i c a p l a s t i c a n e m i a (n=2), h a i r y c e l l l e u k e m i a (n=l), l y m p h o m a (n=2), s a r c o i d o s i s (n=l) , s c l e r o d e r m a w i t h S j o g r e n ' s s y n d r o m e (n=l), r h e u m a t o i d arthritis with cryoglobulinemia (n=l) c h r o n i c a c t i v e h e p a t i t i s (n=l) . A l l t r e a t m e n t s i n v o l v i n g d r u g s w e r e w i t h h e l d for 48 hrs. p r i o r to the study. H e m o d y n a m i c s t u d i e s w e r e c a r r i e d out on all p a t i e n t s . A S w a n - G a n z c a t h e t e r w a s i n s e r t e d v i a i n t e r n a l j u g u l a r v e i n a n d it w a s a d v a n c e d up to the p u l m o n a r y a r t e r y . T h e p r e s s u r e w a s r e c o r d e d f r o m r i g h t a t r i u m (RA), p u l m o n a r y a r t e r y (PA) and f r o m p u l m o n a r y c a p i l l a r y w e d g e (PCW). H e p a t i c p r e s s u r e w a s o b t a i n e d t h r o u g h a C o r d i s N8 m u l t i p u r p o s e c a t h e t e r , w h i c h w a s a d v a n c e d up to s u p r a h e p a t i c v e i n . The g r a d i e n t b e t w e e n w e d g e d (HWP) a n d free h e p a t i c v e n o u s p r e s s u r e (FHP) w a s m e a s u r e d . C a r d i a c o u t p u t (CO) w a s d e t e r m i n e d in t r i p l i c a t e by t h e r m o d i l u t i o n m e t h o d (15). H e a r t r a t e ~I~HR) w a s a l s o r e c o r d e d d u r i n g the m e a s u r e m e n t s of CO. M e a s u r e m e n t of p l a s m a t i c l e v e l s of n o r a d r e n a l i n e , a d r e n a l i n e and d o p a m i n e w e r e c a r r i e d o u t b y r a d i o e n z y m a t i c m e t h o d (16), u s i n g b l o o d o b t a i n e d f r o m t h e f o r e a r m v e i n of 8 c i r r h o t i c and 8 n o n - c i r r h o t i c patients. P r e p a r a t i o n of c e l l c u l t u r e s C e l l c u l t u r e s w e r e p r e p a r e d a c c o r d i n g to a m o d i f i e d v e r s i o n of the H a r a r y and F a r l e y ' s m e t h o d (17). W h o l e h e a r t s w e r e rem o v e d f r o m one to t h r e e d a y s old r a t s and w e r e k e p t in H a n k ' s solution, a f t e r m i n c i n g and w a s h i n g t h e m in c a l c i u m and m a g n e s i u m f r e e p h o s p h a t e b u f f e r e d s a l i n e (PBS) . T h e h e a r t t i s s u e w a s t h e n d i s s o c i a t e d by t r e a t m e n t w i t h 2.5% of b o v i n e t r y p s i n for ten m i n u t e s at 37°C. A f t e r d i s c a r d i n g the i n i t i a l s u p e r n a t a n t , the t r y p s i n a t i o n p r o c e s s w a s r e p e a t e d 5 to 7 t i m e s and the r e s u l t a n t s u p e r n a t a n t l a y e r w a s c o l l e c t e d i n t o c e n t r i f u g e t u b e s p l a c e d on ice. T h e a c t i o n of t r y p s i n w a s s t o p p e d by a d d i t i o n of c u l t u r e m e d i u m (Eagles m i n i m a l e s s e n t i a l m e d i u m w i t h E a r l e ' s s a l t s u p p l e m e n t e d w i t h 10% c a l f s e r u m ) . T h e p r e p a r a t i o n w a s t h e n c e n t r i f u g e d at 750 g. for 5 m i n u t e s at 4°C. T h e c e l l p e l l e t t h u s o b t a i n e d w a s s u s p e n d e d in c u l t u r e m e d i u m a n d f i l t e r e d t h r o u g h lens p a p e r to r e m o v e c e l l u l a r c l u m p s . A n a l i q u o t w a s t h e n c o u n t e d on a h e m a t o c y t o m e t e r , the c e l l s u s p e n s i o n w a s d i l u t e d to 2 5 0 , 0 0 0 c e l l s / m l and s e e d e d on c o v e r s l i p s p l a c e d in 2 ml L e i g h t o n t u b e s . T h e t u b e s w e r e i n c u b a t e d at 3 7 ° C w i t h air and CO^ as g a s e o u s p h a s e . N o c h a n g e w a s m a d e 24 h o u r s a f t e r the p l a t i n g . T h i s led to restricted fibroblastic proliferation. Plasma samples P l a s m a s a m p l e s f r o m n o n - c i r r h o t i c p a t i e n t s w e r e u s e d as a c o n t r o l g r o u p . C i r r h o t i c p l a s m a w a s o b t a i n e d f r o m the p a t i e n t s d e s c r i b e d in t h i s s t u d y . A l l s a m p l e s w e r e d r a w n f r o m the c a t h e t e r p l a c e d in the p u l m o n a r y a r t e r y in o r d e r to h a v e a m i x e d v e n o u s b l o o d . P l a s m a w a s s e p a r a t e d for e v a l u a t i o n of p l a s m a e l e c t r o l y t e s , b i l i r u b i n , c r e a t i n i n e , c a t e c h o l a m i n e s , u r e a and c o n t r a c t i l e a c t i v ity of c u l t u r e d h e a r t c e l l s . P r o c e s s i n g of c e l l c u l t u r e s E x p e r i m e n t s w e r e p e r f o r m e d on 5 d a y o l d c u l t u r e s , at w h i c h t i m e the c u l t u r e s had r e a c h e d a s t e a d y state. O n the d a y of the e x p e r i m e n t , the c o v e r s l i p s w i t h the c e l l s w e r e p l a c e d i n t o a p l e x i g l a s s c h a m b e r on the s t a g e of an i n v e r t e d m i c r o s c o p e , m a i n -
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t a i n e d at 35°C. The d e s i r e d m e d i u m was i n f u s e d at the rate of 0.1 ml/min, t h r o u g h o u t the c h a m b e r by m e a n s of a force and s u c t i o n pump. Contractile activity A m i c r o s c o p e p i c t u r e (magnified x 400) was t r a n s m i t t e d to a t e l e v i s i o n s c r e e n v i a a t e l e v i s i o n camera. On the screen, v a r i a t i o n in light i n t e n s i t y r e s u l t i n g f r o m cell edge m o v e m e n t d u r i n g c o n t r a c t i o n was t r a n s m i t t e d to a p h o t o c e l l by an o p t i c a l fiber. The o u t p u t signal of the p h o t o c e l l w a s t r a n s m i t t e d to an o s c i l l o scope w h e r e v a r i a t i o n s in light i n t e n s i t y a p p e a r e d as waves. T h e a m p l i t u d e of the w a v e s i n d i c a t e d in a r b i t r a r y u n i t s (mv) , v a r i e d p r o p o r t i o n a l l y w i t h light intensity. T h e r e f o r e , this p r o c e d u r e p r o v i d e s an e v a l u a t i o n of the m o v e m e n t of a cell edge so that cont r a c t i l e a c t i v i t y can be assessed.
Results C o n t r a c t i l e a c t i v i t y of c u l t u r e d h e a r t c e l l s fn the p r e s e n c e of c o n t r o l h u m a n plasma, c o n t r a c t i l e a c t i v i ty o c c u r r e d r e g u l a r l y for one hour. In the p r e s e n c e of p l a s m a f r o m c i r r h o t i c patients, the f r e q u e n c y was s i m i l a r to that obt a i n e d in the p r e s e n c e of c o n t r o l plasma, but the a m p l i t u d e of c e ~ lular edge m o v e m e n t s was r e d u c e d by 33.3% ( 0 . 0 1 > p < 0.001) (Fig. 1,2). T h e s e c h a n g e s in a m p l i t u d e w e r e h i g h l y s i g n i f i c a n t as e a r l y as 10 min. a f t e r start, at w h i c h time a r e d u c t i o n of 20.4 % (p < 0 . 0 3 ) f r o m i n i t i a l v a l u e s w a s observed. All the p r e p a r a t i o n s of c u l t u r e d h e a r t c e l l s u n d e r p e r f u s i o n w i t h c i r r h o t i c p l a s m a dev e l o p e d a r r h y t h m i a s at any time in the c o u r s e of the e x p e r i m e n t (Fig. 3). On the o t h e r hand, the h u m a n c o n t r o l p l a s m a did not m o d ify the r e g u l a r i t y of the b e a t i n g c a r d i a c cells. Hemodynamic parameters T h e h e m o d y n a m i c p a r a m e t e r s , h e a r t rate (HR) and a r t e r i a l p r e s s u r e (AP) w e r e s i m i l a r in b o t h g r o u p s of p a t i e n t s . However, the r i g h t a t r i u m p r e s s u r e (RA) w a s 3.5 + 0.5 m m H g in the c o n t r o l group, w h i l e in the c i r r h o t i c p a t i e n t s was 5.7 + 0.7 m m H g (p <0.02). P u l m o n a r y c a p i l l a r y w e d g e p r e s s u r e (PCW) w a s 6.7 + 0.8 m m H g in n o n - c i r r h o t i c s and 9.3 + 0.9 m m H g (p < 0 . 0 4 ) in t h e c i r r h o t i c group. In addition, h e p a t i c w e d g e p r e s s u r e (HWP) in c i r r h o t i c p a t i e n t s was s i g n i f i c a n t l y g r e a t e r than that of c o n t r o l subjects. The values o b t a i n e d f r o m c i r r h o t i c p a t i e n t s w e r e 28.1 + i.i m m H g and 13.9 + 1.2 m m H g for c o n t r o l s u b j e c t s (p< 0.0001). T h e g r a d i e n t between-h e p a t i c p r e s s u r e (HP) and free h e p a t i c p r e s s u r e (FHP) w a s inc r e a s e d from 5.1 + i.i m m H g in the c o n t r o l g r o u p up to 16.2 + 1.4 m m H g (p < 0 . 0 0 0 1 ) ~n the c i r r h o t i c group. C a r d i a c o u t p u t (CO) a l s o was e l e v a t e d f r o m 5.4 + 0.3 i/m in c o n t r o l s u b j e c t s to 6.9 + 0.5 i/m (p < 0 . 0 3 ) in p a t i e n t s w i t h cirrhosis. S y s t e m i c v a s c u l a r r e s i s t a n c e (SVR) was r e d u c e d in p a t i e n t s w i t h liver c i r r h o s i s (1095 + 56.3 d y n e s - s e c - c m - 5 ) in r e s p e c t of the c o n t r o l g r o u p (1440 + iO3.7 d y n e s - s e c - c m - 5 ) ( p < 0.005). P l a s m a c a t e c h o l a m i n e s levels P l a s m a t i c n o r a d r e n a l i n e levels
in p a t i e n t s w i t h
liver cir-
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Q
0 %
Fig.
I
C o n t r a c t i l e a c t i v i t y of c u l t u r e d m y o c a r d i a l cells. P i c t u r e s w e r e taken f r o m the same cell (A) after c o n t a c t w i t h p l a s m a f r o m c o n t r o l patient, and (B) after c o n t a c t w i t h p l a s m a f r o m one p a t i e n t w i t h a l c o h o l i c liver cirrhosis. S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h p l a s m a f r o m the 13 other c i r r h o t i c p a t i e n t s .
r h o s i s w a s h i g h e r t h a n in c o n t r o l subjects. O n the other hand, a d r e n a l i n e and d o p a m i n e did not show any d i f f e r e n c e in b o t h groups. (Table I). Table Circulating cathecolamines
I
in p e r i p h e r a l p l a s m a
(Mean + SE) Group
Noradrenaline (pg/ml)
Adrenaline (pg/ml)
Dopamine (pg/m])
Control
188,9 +
40,7
67,9 + 17,5
61,7 + 21,1
Cirrhotic
848,4 + 159,9
161,8 + 42,9
Significance
p < 0.004
NS
63,1 +
6,2
NS
B l o o d s a m p l e s w e r e t a k e n from the f o r e a r m v e i n of e i g h t c i r r h o t i c and eight n o n - c i r r h o t i c patients. C a t h e c o l a m i n e s w e r e d e t e r m i n e d in p l a s m a as d e s c r i b e d on Methods.
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2 0 mm
15
o O
120
/_ o
10 >
o
80
<
Q) In
5 40
Fig.
2
B e a t i n g f r e q u e n c y and c o n t r a c t i l e a c t i v i t y of c u l t u r e h e a r t c e l l s in p r e s e n c e of e i t h e r p l a s m a f r o m t w e l v e c o n t r o l (r--]) or f o u r t e e n c i r r h o t i c p a t i e n t s ( ~ ) . T h e r e s u l t s are e x p r e s s e d as M e a n + SE. D i f f e r e n c e s b e t w e e n m e a n v a l u e s w e r e e v a l u a t e d ~ b y S t u d e n t ' s "t" test. (* p < 0.01) .
t
Fig. Arrhythmia
observed
during
3
cirrhotic
plasma
infusion.
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P l a s m a e l e c t r o l y t e s , b i l i r u b i n , c r e a t i n i n e and u r e a c o n c e n trations T h e r e a r e no s i g n i f i c a n t d i f f e r e n c e s in p l a s m a c o n c e n t r a t i o n s of s o d i u m , p o t a s s i u m , c a l c i u m , b i l i r u b i n , c r e a t i n i n e and u r e a in b o t h g r o u p s of n a t i e n t s (Table II). T h e p a t i e n t s in the c o n t r o l g r o u p had h i g h b i l i r u b i n l e v e l s and w e r e s t u d i e d and subj e c t e d to l i v e r b i o p s y in o r d e r to e s t a b l i s h t h e i r d e f i n i t i v e diagnosis. Table Na (mEq/l) Control (n=12)
K (mEq/l)
II
Ca (mEq/l)
Bilirubin (mg/l)
Creatinine (mg/dl)
Urea (mg/d])
138.7 6.8
+
4.4 0.2
+
9.5 0.3
+
25.3 3.5
+
1.2 0.i
0,36 + 0.03
Cirrhotic 137.3 (n=14) + 4.2
+
4.2 0.2
+
9.4 0.2
+
28.5 4.8
+
i.i 0.2
0.37 + 0.02
+
Discussion T h e r e s u l t s o b t a i n e d s h o w e d t h a t the c o n t r a c t i l e a c t i v i t y of c u l t u r e d c e l l s w a s a l t e r e d w h e n in c o n t a c t w i t h p l a s m a f r o m c i r r h o t i c p a t i e n t s . T h e s e f i n d i n g s s u g g e s t the p r e s e n c e of a f a c tor(s) c a u s i n g a c a r d i a c d e p r e s s i v e a c t i o n , in t h e p l a s m a of p a tients with alcoholic cirrhosis. These effects have never been r e p o r t e d by o t h e r a u t h o r s and the f i n d i n g s c o u l d be of g r e a t int e r e s t in an a t t e m p t to u n d e r s t a n d the m o d e of a c t i o n of m y o c a r d i a l d e p r e s s a n t f a c t o r s in t h i s p a t h o l o g y . N o n e of the c i r r h o t i c p a t i e n t s i n c l u d e d in t h i s s t u d y w e r e t h o u g h t to h a v e c l i n i c a l l y s i g n i f i c a n t c a r d i a c i m p a i r m e n t . H o w ever, p u l m o n a r y c a p i l l a r y w e d g e , w h i c h is c l o s e l y r e l a t e d to left v e n t r i c u l a r end d i a s t o l i c p r e s s u r e , w a s h i g h e r t h a n our c o n t r o l s u b j e c t s . T h e h i g h r e s t i n g c a r d i a c o u t p u t n o t e d in t h e s e s u b j e c t s is o f t e n v i e w e d as e v i d e n c e t h a t c a r d i a c p e r f o r m a n c e is n o r m a l (3,4). It is w e l l e s t a b l i s h e d t h a t the g r a d i e n t b e t w e e n w e d g e d and f r e e h e p a t i c v e n o u s p r e s s u r e s r e f l e c t s p o r t a l v e n o u s p r e s s u r e in c i r r h o s i s (18). T h e r e f o r e , the h e m o d y n a m i c r e s u l t s i n d i c a t e t h a t the p a t i e n t s s e l e c t e d for t h i s s t u d y had a h y p e r k i n e t i c c i r c u l a t o r y s t a t e and p o r t a l h y p e r t e n s i o n . P l a s m a l e v e l s of c a t e c h o l a m i n e s w e r e a l s o s t u d i e d in t h e s e p a t i e n t s w i t h l i v e r c i r r h o s i s and the v a l u e s o b t a i n e d r e v e a l e d t h a t no s i g n i f i c a n t e l e v a t i o n in a d r e n a l i n e l e v e l s w a s f o u n d in all p a t i e n t s e x a m i n e d , w h e r e a r i s e in n o r a d r e n a l i n e w a s seen, in a c c o r d a n c e w i t h o t h e r a u t h o r s ' f i n d i n g s (5). D e s p i t e the inc r e a s e in c i r c u l a t i n g n o r a d r e n a l i n e , the c o n t r a c t i l e a c t i v i t y of c u l t u r e d c a r d i a c c e l l d e c r e a s e d w h e n the p l a s m a f r o m c i r r h o t i c p a t i e n t s w a s added. T h e s e o b s e r v a t i o n s s u g g e s t t h a t o t h e r h u m o r al f a c t o r s m i g h t be i n v o l v e d in t h i s case. A m o n g t h e s e , the a t r i a l n a t r i u r e t i c f a c t o r m u s t be t a k e n i n t o a c c o u n t , s i n c e it has e v i d e n t c a r d i o v a s c u l a r e f f e c t s . T h i s p e p t i d e c a u s e s h y p o t e n s i o n , b r a d i c a r d i a and f a i l u r e of s t r o k e v o l u m e to r i s e p r o p e r -
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ly. H o w e v e r , it h a s n o t b e e n e l u c i d a t e d if c a r d i o i n h i b i t o r y eff e c t s a r e p r o d u c e d b y d i r e c t a c t i o n on the c a r d i o m y o c y t e s , by inh i b i t i o n or r e f l e x m e c h a n i s m s or b y r e l e a s e of an u n i d e n t i f i e d neg a t i v e l y i n o t r o p i c s u b s t a n c e (20). H y p o c a l c e m i a h a s b e e n n o t e d s p o r a d i c a l l y in p a t i e n t s w i t h c i r r h o s i s w i t h no a p p a r e n t r e l a t i o n s h i p b e t w e e n s e r u m i o n i z e d and t o t a l c a l c i u m c o n c e n t r a t i o n s . A l t h o u g h it h a s b e e n r e p o r t e d an a v e r a g e r e d u c t i o n of 5% in t h e C a + + l e v e l s in c i r r h o t i c s u b j e c t s , s e r u m i o n i z e d c a l c i u m is q u i t e v a r i a b l e in t h i s t y p e of p a t i e n t s (19). O u r d a t a s h o w t h a t t h e r e are no s i g n i f i c a n t d i f f e r e n c e s in t h e c a l c i u m p l a s m a t i c c o n c e n t r a t i o n in b o t h g r o u p s of p a t i e n t s . In a d d i t i o n , n o c h a n g e s w e r e o b s e r v e d in the b i l i r u b i n , s o d i u m , p o t a s s i u m , c r e a t i n i n e and u r e a p l a s m a t i c c o n c e n t r a t i o n s . In c o n c l u s i o n , the p r e s e n t r e s u l t s c l e a r l y s h o w t h a t the p l a s m a f r o m c i r r h o t i c p a t i e n t s d e p r e s s e d the c o n t r a c t i l i t y of c u l t u r e d c e l l s . T h e p l a s m a f a c t o r ( s ) i n v o l v e d in t h i s c a r d i a c d e p r e s s i n g e f f e c t d e s e r v e f u r t h e r s t u d y and t h i s m i g h t p r o v i d e n e w c l u e s for t h e u n d e r s t a n d i n g of c a r d i o v a s c u l a r r e s p o n s e s in cirrhotic patients.
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7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17.
P. W O O D . D i s e a s e of the H e a r t a n d C i r c u l a t i o n , p. 1-25. E y r e 2nd e d i t i o n . S p o t w i d e , L o n d o n (1956). J.G. C L A Y P O O L , M. D E L P and T.K. LIN. A m e r . J. Med. Sci. 234: 4 8 - 5 5 (1957). J.F. M U R R A Y , A . M . D A W S O N and S. S H E R L O C K . A m e r . J. M e d . 24: 3 5 8 - 3 6 7 (1958) . W.H. A B E L M A N , H.J. K O W A L S K I and W.F. M c N E E L Y . J. Clin. I n v e s t . 34: 6 9 0 - 6 9 5 (1955). K. I S H I I , H. S H I B A T A , H. Y A M A D A , H. N A K A S A W A and J. O K A B E . Jap. G a s t r o e n t e r o l . 76: 2 4 1 0 - 2 4 1 5 (1979). T.J. R E G A N , G. K O R O Z E N I D I S , C.B. M O S C H O S , H.A. O L D E W U R T E L , P.H. L E H A N a n d H.K. H E L L E M S . J. C l i n . I n v e s t . 45: 2 7 0 - 2 8 0 (1966) . D.P. RIFF, A . C . J A I N and J.T. D O Y L E . Am. H e a r t J. 78: 5 9 2 - 5 9 7 (1969) . V . E . W E N D T , R. A J L U N I , T.~. B R U C E , A.S. P R A S A D a n d R.J. B I N G . A m e r . J. C a r d i o l . 1 7 : 8 0 4 - 8 1 2 (1966). V.J. F E R R A N S , R.G. H I B B S , G. W E I L B A E C H E R , W.C. B L A C K a n d G.E. B U R C H . A m e r . H e a r t J. 69: 7 4 8 - 7 6 5 (1966). T.J. B A Y L E Y , N. S E G E L a n d J.J. B I S H O P . Clin. Sci. 26: 2 2 7 - 2 3 5 (1964). L. G O U L D , M. S H A R I F F , M. Z A H I R a n d M. DI L I E T O . J. C l i n . Inv e s t . 48: 8 6 0 - 8 6 8 (1969) C.J. L I M A S , H.H. G U I H A , O. L E K A G U L and J.N. COHN. C i r c u l a t i o n 49: 7 5 5 - 7 6 0 (1974). D.H. S P O D I K , V . N . P I G O T T and R. C H I R I F E . N. E n g l . J. M e d . 287: 6 6 7 - 6 8 0 (1972). H.O. H E I N E M A N N . C i r c u l a t i o n 22: 1 5 4 - 1 5 9 (1960). J.S. F O R R E S T E R , W. G A N Z , G. D-~AMOND, T. M c H U G H , D.W. CHONETTE, a n d H . J . C . S W A N . Am. H e a r t J. 83: 3 0 6 - 3 1 1 ( 1 9 7 2 ) . M. D A P R A D A a n d G. Z U R C H E R . Lif--e S c i e n c e s 19: 1 1 6 1 - 1 1 7 1 (1976) I. H A R A R I a n d B. F A R L E Y . Exp. C e l l Res. 29.7--451-465 (1963).
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18. 19. 20.
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T, D. B O Y E R , D.R. T R I G E R , M. H O R I Z A W A , A.G. R E D E K E R and T.B. R E Y M O L D S . G a s t r o e n t e r o l o g y 72: 5 8 4 - 5 8 9 (1977). E.W. M O O R E . G a s t r o e n t e r o l o g y 60: 4 3 - 5 4 (1971). V. A C K E B M A N N . F e d e r a t i o n Proc-~--45: 2 1 1 1 - 2 1 1 4 (1986).