Cefl Bm~gy In~rnaNonal Repor~ VoL 1~ No. 1~ 1992 SUBCELLULAR DISTRIBUTION IN R A T S I N G L E V A S C U L A R
J.R.J.
Baker,
P.R.
Hedwall,
1023
OF HYDRALAZINE MUSCLE CELLS
and
*K.
Hermsmeyer
O r e g o n R e g i o n a l P r i m a t e R e s e a r c h Center, D e p a r t m e n t s of M e d i c i n e and Cell B i o l o g y and Anatomy, O r e g o n H e a l t h S c i e n c e s University, Portland, OR 97213 *Corresponding
author
ABSTRACT
H i g h s p e c i f i c a c t i v i t y (20 Ci/mmol) t r i t i a t e d h y d r a l a z i n e (3Hyd) d i s t r i b u t i o n in isolated, c u l t u r e d v a s c u l a r m u s c l e cells was determined to i d e n t i f y the sites of Hyd binding. 3Hyd d o s e - d e p e n d e n t l y bound to e x t r a c e l l u l a r p r o t e i n and to the area of o r g a n e l l e s w h i c h s e c r e t e these proteins. I n c r e a s e d e x t r a c e l l u l a r b i n d i n g after Hyd p r e - e x p o s u r e s u g g e s t s new b i n d i n g sites may be exacerbated as a result of Hyd interactions. These e x p e r i m e n t s s u g g e s t a p o t e n t i a l l y i m p o r t a n t f e a t u r e of the m e c h a n i s m of a c t i o n of this d i r e c t l y acting v a s o d i l a t o r . INTRODUCTION
The q u e s t i o n of the m e c h a n i s m s and sites of a c t i o n of the d i r e c t l y a c t i n g vasodilator, h y d r a l a z i n e (Hyd), at the v a s c u l a r m u s c l e cell level has b e e n d i f f i c u l t to answer. A m o n g the p u z z l e s o f f e r e d by this agent, w h i c h is being used in l~rge n u m b e r s of h y p e r t e n s i v e p a t i e n t s (Frohlich, 1985), is how the mechanism differs from other peripherally acting antihypertensives, e.g., calcium antagonists or a l p h a - a d r e n e r g i c antagonists, and the p r e c i s e sites of action. Hyd is n o t a b l e b e c a u s e it preferentially inhibits norepinephrine, c o m p a r e d with p o t a s s i u m induced, c o n t r a c t i o n s ( C r i s c i o n e and Hedwall, 1979). The antihypertensive effectiveness of the c o m b i n a t i o n of Hyd w i t h b e t a - a d r e n e r g i c b l o c k i n g agents (Zacest et al., 1972) has c a u s e d a renaissance of i n t e r e s t and led to its w i d e s p r e a d use for t r e a t m e n t of h y p e r t e n s i o n (Frohlich, 1985). How and w h e r e Hyd acts in v a s c u l a r m u s c l e cells poses a c h a l l e n g i n g puzzle. We sought the sites of s u b c e l l u l a r l o c a l i z a t i o n of Hyd as a clue to the m e c h a n i s m of action. The p u r p o s e s of the p r e s e n t study were 2-fold along this rationale. Firstly, we used contractile, previously defined, c u l t u r e d v a s c u l a r m u s c l e cells (Marvin, et al., 1979; H e r m s m e y e r and Mason, 1982; S t u r e k and Hermsmeyer, 1986) to m i n i m i z e the c o m p l i c a t i o n of c o n n e c t i v e tissue J
0309-1651/92/101023-17/$08.00/0
© 1992 Academic Press Ltd
Cell Biology International Reports, Vol. 16, No. 10, 1992
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b i n d i n g of 3H-Hyd in vivo (Baker, et al., 1985). And secondly, we s t u d i e d the time c o u r s e of l o c a l i z a t i o n in r e l a t i o n to the slow onset of Hyd v a s o r e l a x a t i o n . In vivo studies (Baker et al., 1985) had shown that Hyd, w h e t h e r o r a l l y or i n t r a v e n o u s l y a d m i n i s t e r e d to rats, e x h i b i t s more than 50% of its b i n d i n g to e l a s t i n and collagen. M u l t i p l e m e c h a n i s m s are to be e x p e c t e d for Hyd. M e m b r a n e (Hermsmeyer et al., 1983) and i n t r a c e l l u l a r actions (Diamond and Janis, 1980; D i a m o n d and Shaikh, 1980) for Hyd have been proposed, but the s i g n i f i c a n c e of these m e c h a n i s m s has not been established. L o c a l i z a t i o n of Hyd is likely to be an i m p o r t a n t clue b e c a u s e the time c o u r s e of v a s o d i l a t i o n c o r r e l a t e s with slow, p e r s i s t e n t Hyd b i n d i n g and not w i t h the c o n c e n t r a t i o n c i r c u l a t i n g in the blood p l a s m a (Baker et al., 1985). MATERIALS
AND
METHODS
P r i m a r y c u l t u r e s of a z y g o u s vein, dorsal aorta, and portal vein m u s c l e cells were used in this study as p u b l i s h e d ( H e r m s m e y e r and Mason, 1982). Radiolabelled hydralazine was prepared by Dr. D.E. Brundish, R e s e a r c h Centre, C i b a - G e i g y P h a r m a c e u t i c a l s , Horsham, U.K. and had a specific radioactivity 20 Ci/mmol. A c i r c u l a r sample (491 mm 2) of e a c h s u r f a c e was e x p o s e d for 60 min to 1 pM 3H-Hyd p r e p a r e d from an a m p o u l e w i t h a c o n c e n t r a t i o n of 260 n m o l e s / m l w h i c h gave 11.828 m i l l i o n c o u n t s / m i n (measured with c o r r e c t i o n for loss at high counting rates) in a Beckman LS-250 s c i n t i l l a t i o n counter. Each sample was t h o r o u g h l y rinsed i0 times with distilled water, placed in a glass s c i n t i l l a t i o n vial c o n t a i n i n g i0 ml of R e s e a r c h P r o d u c t s International complete counting cocktail 3a70, and c o u n t e d for I0 minutes. T h r e e - d a y cultures: C u l t u r e d cells (from d o r s a l aorta, a z y g o u s vein, or h e p a t i c portal vein) w e r e w a s h e d free of c u l t u r e m e d i u m w i t h seven c h a n g e s of ISM and i n c u b a t e d for 3, 30 or 60 min in 1 ml of 2 ~M 3H-Hyd in ISM (40 vCi) or I ml of 0.06 ~M 3H-Hyd in ISM (1.33 vCi). The cells were grown on p o l y l y s i n e - c o a t e d discs. In some c u l t u r e d cells, 30 and 60 min s a m p l e s w e r e p r e - i n c u b a t e d with 1 DM u n l a b e l l e d HP for 60 min prior to the i n t r o d u c t i o n of the t r i t i a t e d drug. Duplicate samples were prepared for autoradiographic and radiometric processing. T r i p l i c a t e samples of aorta cells i n c u b a t e d for 60 min w i t h 2 NM ~H-Hyd w e r e p r e p a r e d and p r o c e s s e d in p a r a l l e l w i t h all s a m p l e s for EM a u t o r a d i o g r a p h y using identical reagent aliquots (5 ml) to q u a n t i t a t i v e l y
Cell Biology International Reports, Vol. 16, No. 10, 1992
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assess the elution of r a d i o l a b e l at e a c h stage of processing. F o r m o r p h o l o g i c a l e x a m i n a t i o n , o n e w e l l for e a c h c e l l t y p e w a s r e s e r v e d for 60 m i n i n c u b a t i o n w i t h ISM only. Five-day cultures: C e l l s f r o m d o r s a l a o r t a and a z y g o u s v e i n w h i c h h a d b e e n in c u l t u r e for 5 d a y s w e r e i n c u b a t e d for 3, 30, or 60 m i n w i t h 3H-Hyd (0.06 ~M, 1 p M or 2 pM) w i t h or w i t h o u t preincubation for 60 m i n w i t h 1 pM u n l a b e l l e d H y d (30 and 60 m i n o n l y ) . These samples were u s e d for r a d i o m e t r y only. In a s e p a r a t e e x p e r i m e n t , 5-day cultures of dorsal aorta cells underwent an i d e n t i c a l t r e a t m e n t r e g i m e for the p u r p o s e s of r a d i o m e t r y a n d EM a u t o r a d i o g r a p h y , a n d w e r e g r o w n d i r e c t l y o n the f l o o r of the p o l y s t y r e n e c u l t u r e w e l l s . S a m p l e s w e r e f i x e d in a m e d i u m of g l u t a r a l d e h y d e (2.5%) and f r e s h l y p r e p a r e d formaldehyde (4%) in c a c o d y l a t e buffer (0.i M, pH 7.0) at r o o m t e m p e r a t u r e for 1 h. R i n s e s in i d e n t i c a l c a c o d y l a t e b u f f e r (15, 15 and 30 min) w e r e f o l l o w e d b y p o s t - f i x a t i o n in o s m i u m t e t r o x i d e (1% in 0.i M c a c o d y l a t e , pH 7.0, 1 h). Following a rapid cacodylate rinse the samples were dehydrated through a g r a d e d e t h a n o l s e r i e s and f i n a l l y t r a n s f e r r e d to E p o n for infiltration via a 50/50 Epon/absolute ethanol mixture. F i n a l e m b e d d i n g w a s in E p o n w i t h p o l y m e r i z a t i o n at 6 0 ° C for 48 h. L e s s t h a n 1% of t h e t r i t i u m w a s e l u t e d in the e n t i r e s e r i e s of p r e p a r a t i v e s o l u t i o n s f o l l o w e d by 24 hrs in IN N a O H at 22°C, w h i c h s h o w s t h e r e m a r k a b l y t i g h t b i n d i n g of H y d t h a t w a s h i g h l y f a v o r a b l e for s u b c e l l u l a r localization. In t h e c a s e w h e r e s a m p l e s w e r e p r o c e s s e d f o r a s s e s s m e n t of t r i t i u m e l u t i o n , f o l l o w i n g d e h y d r a t i o n t h e y w e r e t r a n s f e r r e d to N a O H (i M, r o o m t e m p e r a t u r e , 24 h) a n d t h e n to f r e s h m o l a r N a O H for f u r t h e r d i g e s t i o n for s e v e n d a y s at 55°C. The method u s e d to p r e p a r e autoradiographs has b e e n d e s c r i b e d p r e v i o u s l y (Baker, et al., 1977). E x p o s u r e of t h e f i l m w a s for f o u r w e e k s (aorta cells from 5-day cultures) o r 18 w e e k s (portal vein cells from 3-day cultures). T h e s p e c i m e n s w e r e d e v e l o p e d in K o d a k DI9 or Microdol X depending upon the grain density required. The rationale and methodology for a n a l y s i s h a v e b e e n d e s c r i b e d e l s e w h e r e (Baker, et al., 1985; B l a c k e t t and Parry, 1973, 1977). For a o r t a cells, t h e n u m b e r s of r e a l g r a i n s c o u n t e d r a n g e d f r o m 1 9 9 - 7 1 2 a n d t h e n u m b e r s of hypothetical grains from 948-1108. In the single e x p e r i m e n t o n p o r t a l v e i n cells, t h e n u m b e r of r e a l and h y p o t h e t i c a l g r a i n s w e r e 137 a n d 432 r e s p e c t i v e l y . For analysis,
we have used the hypothetical
grain method
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of B l a c k e t t and P a r r y (1973, 1977), in w h i c h a c o m p u t e r program is used to compare hypothetical grain distributions with the real grain distribution. H y p o t h e t i c a l s o u r c e s of r a d i o a c t i v i t y are e s t a b l i s h e d by a p p l y i n g to the a u t o r a d i o g r a p h s a t r a n s p a r e n t o v e r l a y screen which contains computer-predicted source-to-site distances, w h i c h g e n e r a t e the h y p o t h e t i c a l s i l v e r g r a i n s e m a n a t i n g from t h e s e sources. The d i r e c t i o n s of t h e s e hypothetical decays are, of course, random. The d i s t a n c e s are d e r i v e d from r a n g e d i s t r i b u t i o n c u r v e s from e x p e r i m e n t a l l y m e a s u r e d line s o u r c e s of a g i v e n i s o t o p e as first d e s c r i b e d by Salpeter, et al. (1969). H e n c e a rectangular m a t r i x for c r o s s - f i r e was created. E s t i m a t e s of r a d i o a c t i v i t y in the v a r i o u s s o u r c e s were d e r i v e d from s y s t e m a t i c m o d i f i c a t i o n of the h y p o t h e t i c a l s o u r c e v a l u e s by a m i n i m i z i n g s u b - r o u t i n e until the h y p o t h e t i c a l and real g r a i n d i s t r i b u t i o n s fit o p t i m a l l y for the d e s i g n a t e d sites, as a s s e s s e d by the c h i - s q u a r e d test. The n u m b e r of d e g r e e s of f r e e d o m a l l o w e d was the d i f f e r e n c e b e t w e e n the n u m b e r of sites and s o u r c e s in the cross-fire matrix. Where the crude location of radiolabel was difficult to interpret by visual i n s p e c t i o n of s i l v e r grains, we c o l l e c t e d h y p o t h e t i c a l g r a i n d a t a by r a n d o m or r e g u l a r p o i n t s a m p l i n g of the m i c r o g r a p h s so as to i n c l u d e all the p o s s i b l e s o u r c e s of r a d i o a c t i v i t y in the analysis. O t h e r c h e m i c a l s used: A d d i t i o n a l m a t e r i a l s u s e d in t h e s e e x p e r i m e n t s i n c l u d e d u n l a b e l l e d h y d r a l a z i n e (Ciba-Geigy, Basel), g l u t a r a l d e h y d e (Taab), p a r a f o r m a l d e h y d e (BDH), s o d i u m c a c o d y l a t e (BDH), E p o n (Gurr), L4 n u c l e a r e m u l s i o n (Ilford), DI9 and M i c r o d o l X d e v e l o p e r s (Kodak), and s o d i u m t h i o s u l p h a t e (BDH). RESULTS
A c t i o n of Hyd on v a s c u l a r m u s c l e c o n t r a c t i o n : Hyd dose dependently decreased spontaneous contraction frequency in s i n g l e a z y g o u s v e n o u s m u s c l e cells. Fig 1 shows that the ECs0 was a b o u t 60 nM, but o n l y w h e n long e x p o s u r e t i m e s to Hyd w e r e allowed. D u r i n g the first 10-15 minutes, t h e r e was a l m o s t no p e r c e p t i b l e a c t i o n of Hyd, f o l l o w e d by a g r a d u a l d e c r e a s e in c o n t r a c t i o n frequency. By 30 minutes, a c l e a r and n e a r l y s t e a d y d e c r e a s e in c o n t r a c t i o n f r e q u e n c y occurred, and this time p o i n t was used for the data in Fig. i. This a c t i o n of Hyd was in m a r k e d c o n t r a s t to the r a p i d (peak a c t i o n w i t h i n 30 sec) increase in contraction frequency to i0-i00 nM n o r e p i n e p h r i n e , serotonin, or h i s t a m i n e , or the d e c r e a s e in contraction frequency to nitroglycerine or nitroprusside.
Cell Biology International Reports, Vol. 16, No. 10, 1992 100n
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F i g i: D e c r e a s e in s p o n t a n e o u s c o n t r a c t i o n f r e q u e n c y of azygos venous muscle cells suffused by Hyd was dosed e p e n d e n t w i t h r e m a r k a b l y s l o w (>i0 min) o n s e t of a c t i o n . The suffusion method was described previously (Hermsmeyer a n d R o b i n s o n , 1976). N u m b e r s in p a r e n t h e s e s a r e n u m b e r of c e l l s (i m i n c o u n t s ) . A
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F i g 2: C o m p a r i s o n of H y d u p t a k e at t w o c o n c e n t r a t i o n s a n d t h r e e t i m e s of e x p o s u r e (3, 30, a n d 60 min) in c e l l s c u l t u r e d for t h r e e d a y s s h o w e d t h a t H y d u p t a k e i n c r e a s e d w i t h t i m e in all c a s e s in h e p a t i c p o r t a l v e i n (n = 3), a z y g o u s v e i n (n = 3), and d o r s a l a o r t a (n = 5). A: Midr a n g e c o n c e n t r a t i o n (0.06 pM), a n d B: h i g h c o n c e n t r a t i o n (2 pM) Hyd. A l l e x p e r i m e n t s w e r e c a r r i e d o u t i n ISM.
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Radiometry: Hyd b i n d i n g in all three types of v a s c u l a r m u s c l e cells was slow and c o n c e n t r a t i o n - d e p e n d e n t , as shown in Figs. 2-4. Fig. 2 allows c o m p a r i s o n of three e x p o s u r e times in each of the t h r e e types of v e s s e l s using e i t h e r 60 nM or 2 ~M 3H-Hyd. The b i n d i n g p r o c e s s was slow in all three types of v a s c u l a r m u s c l e cell cultures, and i n c r e a s e d w i t h time a l t h o u g h r e a c h i n g a p l a t e a u b e t w e e n 30 and 60 minutes. The a m o u n t of c o n n e c t i v e t i s s u e s e c r e t e d was n e g l i g i b l e in these 3 - d a y cultures. The i n c r e a s e w i t h time is also shown in Fig. 3, w h i c h shows data from the 5-day cultures, in w h i c h at least ten times as m u c h c o n n e c t i v e t i s s u e p r o t e i n had b e e n secreted. The total 3H-Hyd b i n d i n g at 5 days was slow (Fig. 3) and d o s e - d e p e n d e n t (Fig. 4). Furthermore, the p r e v i o u s e x p o s u r e to n o n - r a d i o a c t i v e Hyd i n c r e a s e d s u b s e q u e n t l a b e l l i n g with 3H-Hyd (Fig. 4). The Hyd b i n d i n g thus gave no e v i d e n c e of being s a t u r a t e d with i n c r e a s e in c o n c e n t r a t i o n or by p r e - e x p o s u r e , a l t h o u g h the b i n d i n g rate was always o b s e r v e d to d e c r e a s e a f t e r 30 min (Fig. 3). Hyd bound not o n l y to cells, but also to the cell c u l t u r e wells, even after ten t h o r o u g h r i n s i n g procedures. We tried to find a l a b o r a t o r y m a t e r i a l s u b s t a n t i a l l y less r e a c t i v e in a s e p a r a t e Hyd b i n d i n g study, but found that Hyd binds s t r o n g l y to c u l t u r e s u r f a c e s tested, i n c l u d i n g the glass, p o l y s t y r e n e , and aklar s u b s t r a t e s used in this study (Table i). In e a c h case, the test m a t e r i a l c o n s i s t e d of 3 c o v e r s l i p like s a m p l e s of m a t e r i a l of the same s u r f a c e area (491 2 mm ) w h i c h w e r e e x p o s e d to 1 pM Hyd for 60 minutes. The test s a m p l e s were s u b s e q u e n t l y w a s h e d at least i0 times for > 2 min (each wash) w i t h d i s t i l l e d w a t e r to r e m o v e any free Hyd. R e p e a t c o u n t i n g after a d d i t i o n a l w a s h e s g a v e the same result. Autoradiography: At the e l e c t r o n m i c r o s c o p i c level, 5-day c u l t u r e s (Fig. 5) p r o d u c e d e x t r a c e l l u l a r c o n n e c t i v e tissue while 3-day cultures (Fig. 6) did not. Intracellularly, the 5-day cultures contained more e x t e n s i v e r e g i o n s of rough e n d o p l a s m i c r e t i c u l u m and r i b o s o m e s and fewer m y o f i l a m e n t s than the 3-day cultures. Quantitative cross-fire (hypothetical grain) a n a l y s i s s h o w e d that i n c u b a t i o n of 5-day aorta cells w i t h 2 pM ~HHyd for 60 min gave a 3-fold i n c r e a s e in c y t o p l a s m i c label (relative activity) over that found w i t h 1 pM 3HHyd (Table 2). The i n c r e a s e in Hyd b i n d i n g to the c y t o p l a s m in 2 pM v e r s u s 1 pM 3H-Hyd for 60 min is even m o r e d r a m a t i c w h e n e x p r e s s e d as g r a i n s / g r i d point
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Fig
/
"M
Concentration-dependence
of
Hyd u p t a k e
after
60
m i n i n c u b a t i o n of d o r s a l a o r t a a n d a z y g o u s v e i n c e l l s cultured for five days at t h r e e concentrations was similar. P r e i n c u b a t i o n w i t h 1 D M u n l a b e l l e d H y d for 1 h e n h a n c e d ( r a t h e r t h a n i n h i b i t e d ) 3H-Hyd u p t a k e at b o t h 1 p M a n d 2 MM.
Cell Biology lnternational Reports, VoL 16, No. 10, 1992
1030
(relative specific radioactivity), where the i n c r e a s e is over 5-fold (Table 3). The higher 3H-Hyd concentration, or longer i n c u b a t i o n times, also i n c r e a s e d Hyd b i n d i n g to c o n n e c t i v e tissue, matrix, and s u b s t r a t e (Table 3). The hepatic portal vein 3 day c u l t u r e s had the largest c o n c e n t r a t i o n of c y t o p l a s m i c and c o n n e c t i v e tissue Hyd binding of any cells tested. Pretreatment of the cells with non-radioactive Hyd prevented cytoplasmic accumulation and decreased c o n n e c t i v e tissue a c c u m u l a t i o n of Hyd in r e l a t i v e terms (Table 3), a l t h o u g h c o n n e c t i v e tissue b i n d i n g r e m a i n e d considerable. The significant changes in Hyd d i s t r i b u t i o n after u n l a b e l l e d Hyd p r e t r e a t m e n t were the d e c r e a s e in c y t o p l a s m i c and the increase in s u b s t r a t e Hyd (Tables 2 and 3). There was no p r e f e r e n t i a l binding of Hyd at the cell m e m b r a n e ( c y t o p l a s m / m a t r i x interface), either with or without the non-radioactive Hyd pretreatment. The highest c o n c e n t r a t i o n of Hyd was found in the c o n n e c t i v e tissue, either e x t r a c e l l u l a r l y or in v e s i c l e s p r o b a b l y filled with nascent (pre-exocytotic) c o n n e c t i v e tissue p r o t e i n (Fig. 7).
TABLE 3H-HYDRALAZINE
(HYD)
1
BINDING
TO E X P O S E D
SURFACES
Counts/ min io
Glass
2.
Epon
(embedding
3.
Aklar
(plastic
4.
Polystyrene (tissue culture
5.
(coverslip)
Polypropylene (scintillation
% of total
45,216
0.04
medium)
178,508
1.51
coverslip)
187,846
1.59
291-608
2.47
478,264
4.04
dish) vial)
6.
Polycarbonate (Nucleopore filter)
750,501
6.35
7.
C e l l u l o s e acetate (Millipore filter)
832,584
7.04
CellBiology lnternational Reports, Vol. 16, No. 10, 1992
1031
F i g 5: E M a u t o r a d i o g r a p h of 5 - d a y c u l t u r e d a o r t a c e l l s f o l l o w i n g i n c u b a t i o n f o r 3 m i n w i t h 3H-Hyd (2 pM). Most of t h e s i l v e r g r a i n s w e r e a s s o c i a t e d w i t h c o n n e c t i v e tissue. S = polystyrene substrate. S c a l e b a r = 1 ~M. E l u t i o n t e s t i n g ( T a b l e 4) s h o w s t h a t o n l y a b o u t 1% o f t h e H y d w a s r e m o v a b l e d u r i n g t h e EM p r e p a r a t i v e p r o c e d u r e , i n d i c a t i n g t h a t it w a s t i g h t l y b o u n d to t h e c u l t u r e d cells sufficiently to a l l o w s u b c e l l u l a r localization ( T a b l e s 2 a n d 3). Autoradiographic and radiometric data indicated slow, s t r o n g b i n d i n g of H y d to c o n n e c t i v e t i s s u e p r o t e i n s , w i t h increasing a s s o c i a t i o n w i t h time, e v e n if t h e c e l l s w e r e f i r s t e x p o s e d to n o n - r a d i o a c t i v e Hyd. The term matrix d e s c r i b e s t h e a r e a in a u t o r a d i o g r a p h s w h e r e n o s t r u c t u r e s a r e a p p a r e n t a n d c o r r e s p o n d s s p a t i a l l y to t h e c u l t u r e fluid volume. It is o b v i o u s l y impossible for the r a d i o l a b e l t o b e f i x e d i n a s t r u c t u r e - f r e e s p a c e a n d it
1032
Cell Biology lnternational Reports, Vol. 16, No. 10, 1992
is t h e r e f o r e p r o b a b l e t h a t s i l v e r g r a i n s in t h i s r e g i o n are almost all a s s o c i a t e d with amorphous connective tissue. C o n s e q u e n t l y , t h e r e is s o m e j u s t i f i c a t i o n for r e g a r d i n g t h e m a t r i x v a l u e s in t h e h y p o t h e t i c a l grain a n a l y s e s in c o m b i n a t i o n w i t h t h o s e for c o n n e c t i v e t i s s u e . T h e v e r y h i g h c o n c e n t r a t i o n of r a d i o l a b e l d e s i g n a t e d to v e s i c l e s w i t h f i l a m e n t o u s c o n t e n t s in t h e 3 - d a y p o r t a l v e i n c e l l s l e a d s us to the s u g g e s t i o n t h a t t h e s e v e s i c l e s contain newly forming connective tissue with which radiolabelled Hyd can react after uptake into the cytoplasm.
F i g u r e 6: E l e c t r o n m i c r o g r a p h o f a 3 - d a y c u l t u r e d a o r t a c e l l f o l l o w i n g i n c u b a t i o n for 1 h o u r w i t h m e d i u m only. Note the absence of secreted connective tissue. S = p o s i t i o n of a k l a r s u b s t r a t e . L = lipid droplets. RER = rough endoplasmic reticulum. Scale bar = 1NM.
Cell Biology lnternational Reports, Vol. 16, No. 10, 1992
1033
F i g u r e 7: E M a u t o r a d i o g r a p h of a 3 - d a y c u l t u r e d p o r t a l v e i n c e l l f o l l o w i n g i n c u b a t i o n for 1 h o u r w i t h 3H-Hyd (2 ~M). Silver grains were most concentrated where associated with a cytoplasmic vesicle with flocculent contents. S = position of aklar substrate. L = lipid droplets. V = cytoplasmic vesicle. S c a l e b a r = 1 NM.
,
S
F i g u r e 8: E M a u t o r a d i o g r a p h of 5 - d a y a o r t a c e l l c u l t u r e preincubated with unlabelled Hyd (i ~M) for 1 h o u r f o l l o w e d b y ~H-Hyd (i pM) f o r 1 h o u r . Connective tissue was well labelled. S = polystyrene substrate. Scale bar =
i
~M.
Aorta
Aorta
Aorta
Aorta
Portal
i pM 60 min
i ~M 60 min*
2 NM 3 min
2 ~M 60 min
2 ~M 60 min
3
significant
v
5
5
5
5
5
°Day
I ± I
0 ± i
1 ± I
I ± 3
0 ± I
Nucl
9 ± I
19 ± 2
12 ± i
34 ± 2
24 ± 3
14 ± 2
Subst
53
90
145
125
100
140
n
n i s the number o f c e l l s .
9 ± 4
13 ± 9
20 ± 8
11 ± 5
11 ± 5
Matrix
(p > 0 . 0 5 ) ;
58 ± 22 (vesicle)
46 ± 4
70 ± 7
45 ± 5
56 ± 8
58 ± 6
C.T.
at the 95% c o n f i d e n c e l e v e l
33 ± 23
25 ± 5
5 ± 2
0 ± 0
8 ± 5
17 ± 9
Cyto
4.8
2.4
3.4
7.4
0.6
1.0
Xz
4
3
3
3
2
1
df
* = p l u s p r e t r e a t m e n t w i t h n o n - r a d i o a c t i v e Hyd. Relative activity is % total radioactivity. A b b r e v i a t i o n s : Hyd tmt = h y d r a l a z i n e t r e a t m e n t , Cyto = cytoplasm, Nucl = n u c l e u s , C.T. : c o n n e c t i v e t i s s u e , Subst = s u b s t r a t e , n = number, XL = chi squared, and d f = degrees of freedom. M a t r i x i s d e f i n e d as the space between c e l l s where c o n n e c t i v e t i s s u e cannot be i d e n t i f i e d . S u b s t r a t e ( s u b s t ) is d e f i n e d as the p o l y s t y r e n e or A k l a r m a t e r i a l on which the c e l l s were grown.
X2 are s t a t i s t i c a l l y
Aorta
I pM 3 min
All
Cells
Hyd tmt
EM AUTORADIOGRAPHIC ANALYSIS SHOWINGDISTRIBUTION OF RELATIVE ACTIVITY IN VASCULARMUSCLE CELLS
TABLE 2
Aorta Aorta Aorta Aorta Aorta
Portal
I pM 3 min
1 pM 60 min
i ~M 60 min*
2 pM 3 min
2 pM 60 min
2 pM 60 min
v
0.03 (57) 0.28
5 5 3
0.00 (50)
5
0.37 (82)
(57)
0.05 (50)
5
(5)
(9)
0.09
(9)
0.00
(vesicle)
(7)
7.71
(7)
4.00
(7)
3.10 0.24 (24)
(24)
0.18
(28)
0.21
2.55
(9)
(11)
0.78
(2)
5.04
C2)
1.59
(8)
1.19
(8)
0.02
(5)
0.90
(9)
0.05
0.13 (28)
0.35
Subst
3.42
0.08
Matrix
(8)
(5)
2.37
C.T.
(28)
(9)
0.00
0.07
5 (50)
Nucl
Cyto
Day
* = plus pretreatment w i t h n o n - r a d i o a c t i v e Hyd. A b b r e v i a t i o n s : Hyd tmt = h y d r a l a z i n e treatment, Cyto = cxtoplasm, Nucl = nucleus, C.T. = connective t i s s u e , Subst = s u b s t r a t e , n = number, X~ = chi squared, and df = degrees of freedom. M a t r i x is defined as the space between c e l l s where connective t i s s u e cannot be i d e n t i f i e d . Substrate (Subst) is defined as the p o l y s t y r e n e or A k l a r material on which the c e l l s were grown. Results were obtained from 53-145 c e l l s and are expressed as g r a i n s / p o i n t and r e l a t i v e areas ( ) . G r a i n s / g r i d p o i n t ( r e l a t i v e s p e c i f i c r a d i o a c t i v i t y ) is % t o t a l r a d i o a c t i v i t y % t o t a l area.
Cells
Hyd tmt
RELATIVE TRITIUM SPECIFIC RADIOACTIVITY
EM AUTORADIOGRAPHIC ANALYSIS SHOWING
TABLE 3
o oo Ol
o
Cb
c~
1036
CefB~gyln~rnationalReports, VoL 1~ N o . l ~ 1992 TABLE 4 ELUTION OF 3H FROM 3-DAY CULTURED DORSAL AORTIC MUSCLE CELLS FOLLOWING INCUBATION FOR 60 MIN WITH 2 pM 3H-HYD
Solution
Mean counts/min
Percentage
(n = 3)
Glutaraldehyde (2.5%) and formaldehyde (4.0%) Cacodylate (15 min)
1,100
0.37
86
0.03
69
0.02
108
0.04
74
0.03
70% EtOH
295
0.10
90% EtOH
130
0.04
Abs EtOH (I)
167
0.06
Abs EtOH (2)
113
0.04
1,063
0.40
290,917
98.89
294,189
100.04
Cacodylate (15 min) Os04 (1% w/v) Cacodylate
IM NaOH (room temp
24 h)
IM NaOH (55 ° - 7 days) TOTAL DISCUSSION
The present results reinfoce the conclusions of the previous in vivo study (Baker et a l . , 1985) that 3H-Hyd, whether administered intravenously or per os, binds to vascular muscle c e l l s . Although the majority of label is associated with connective tissue at all times, s i g n i f i c a n t quantities of Hyd are taken up into the myocyte sarcoplasm. Based on Hyd l o c a l i z a t i o n , c e l l u l a r mechanisms of action would occur at intracellular sites and, e v e n more predominantly, e x t r a c e l l u l a r sites. The cell membrane was not s i g n i f i c a n t l y labelled. These experiments to determine where Hyd binds to isolated, single vascular c e l l s have allowed three main conclusions. F i r s t , Hyd binds p r i m a r i l y to connective tissue proteins, whether secreted or nascent, and the binding is strong. Second, Hyd enters the cell in s u f f i c i e n t quantity to allow for i n t r a c e l l u l a r actions, including on
Cell Biology International Reports, Vol. 16, No. 10, 1992
1037
proteins that decrease contraction. Third, pre-exposure of cells to non-radioactive Hyd increases, rather than decreases, subsequent e x t r a c e l l u l a r Hyd binding, perhaps by reacting with collagen and e l a s t i n to expose new binding sites on these large polymeric proteins. Even in young cells isolated from neonates, copious amounts of connective tissue proteins were present in the 5-day cultures, and evident as precursor proteins in the 3-day cultures, as has also been biochemically documented (Abraham et a l . , 1974, 1975). Most of the Hyd binding was to secreted collagen and e l a s t i n in all cells observed (Tables 2 and 3; Figs 5, 7, and 8). These connective tissue f i b e r s were less of a b a r r i e r to Hyd reaching vascular muscle c e l l s than might have been anticipated (Baker et a l . , 1985). When Hyd has access to both cells and connective tissue proteins, the proteins are more densely labelled. I t is overwhelmingly Hyd, rather than a metabolite, which is measured in these experiments because there was no plasma environment where enzymes could metabolize the drug. Furthermore, metabolites of Hyd have recently been shown to be 5-25X less potent than Hyd (or inactive) in i n h i b i t i n g contraction (Criscione et a l . , 1986), suggesting that Hyd is the active form. The entry of Hyd into the cytoplasm shows that access of Hyd should be s u f f i c i e n t to allow actions on membrane regulatory or c o n t r a c t i l e proteins and enzymes w i t h i n the c e l l . The mechanism of action of Hyd probably involves i n t r a c e l l u l a r , as well as membrane actions (Criscione and Hedwall, 1979; Hermsmeyer et a l . , 1983). Other sites might be important as w e l l , as Hyd binds i r r e v e r s i b l y to an amine oxidase associated with muscle membranes (Lyles and Callingham, 1982), and with lysyl oxidase, which is important for function of e l a s t i n and collagen (Numata et a l . , 1981). The f a c i l i t a t i o n of Hyd binding by previous exposure to I pM non-radioactive Hyd for 1 hour is opposite to the prediction of detecting non-specific binding. The hypothesis that Hyd has such a prolonged onset and duration of action because of slow binding to the blood vessel wall (Reece, 1981) would be supported by these data. The unlabelled Hyd might have been expected to occupy the s p e c i f i c Hyd binding sites, making them unavailable to 'H-Hyd, j u s t as appears to have occurred with cytoplasmic l a b e l l i n g . However, e x t r a c e l l u l a r l a b e l l i n g s i g n i f i c a n t l y increased a f t e r pre-exposure to Hyd, suggesting formation of new sites for i n t e r a c t i o n , perhaps by a d i r e c t chemical reaction. Furthermore, labelled Hyd was p r a c t i c a l l y t o t a l l y r e s i s t a n t to elution during EM processing. These l o c a l i z a t i o n studies show Hyd to be a remarkably reactive compound, probably changing some of the proteins with which i t interacts.
1038
Cell Biology lnternational Reports, VoL 16, No. 10, 1992
Since total Hyd binding was always equal or greater after unlabelled Hyd pretreatment (see Fig. 4), the data suggest a new property for Hyd, namely, the sequential, slow reaction with e x t r a c e l l u l a r proteins which f a c i l i t a t e s subsequent binding of 3H-Hyd. Since the binding increased beyond i (or even 2) hours, the process is slow and continuous, and might well explain the slow onset of action Hyd. No metabolite of Hyd would be needed, and none is l i k e l y (Criscione et a l . , 1986), to explain the paradoxical 30-150 hour h a l f - l i f e for efficacy when plasma h a l f - l i f e is only 30-60 minutes. I t would appear that, at least under the conditions of culture, non-radioactive Hyd eliminated cytoplasmic labelling while connective tissue labelling was increased. Notably, i t was these secreted proteins which contained most of the radiolabel and in the most concentrated form, just as in in vivo studies. These observations suggest both i n t r a c e l l u l a r and e x t r a c e l l u l a r sites of action, probably involving direct chemical reaction with Hyd, in arterial and venous vascular muscle cells. REFERENCES
Abraham, P.A., Smith, D.W. and Carnes, W.H. (1974). Synthesis of soluble elastin by aortic medial cells in culture. Biochem. Biophys. Res. Comm., 58: 597-604. Abraham, P.A., Smith, D.W. and Carnes, W.H. (1975). Biosynthesis of lysine-derived elastin crosslinks in aortic cell culture. Biochem. Biophys. Res. Comm., 67: 723-727. Baker, J.R.J., Bullock, G.R., Crawford, N. and TaylorED.G. (1977). Localization in platelets of s o d i u m J~Cr-chromate, 12Syl-antibody to whole membrane, and 3H-diisopropylfluorophosphate using electron microscopic autoradiography. Am. J. Pathol., 88: 277-290. Baker, J.R.J., Bullock, G . R . and Williamson, I.H.M. (1985). Autoradiographic study of the d i s t r i b u t i o n of 3~_ and 14C-hydralazine in the rat. B r i t . J. Pharmacol., 84: 07-120. Blackett, N.M. and Parry, D.M. (1973). A new method for analyzing electron microscope autoradiographs using.hypothetical grain distributions. J. Cell. Biol., 57: 9-15. Blackett, N.M. and Parry, D.M. (1977). A simplified method of 'hypothetical grain' analysis of electron microscope autoradiographs. J. Histochem. Cytochem., 25: 206-214. Criscione, L., Eichenberger, K., Hedwall, P.R. and Schmid, K. (1986). Hydralazine and its metabolites: in v i t r o and in vivo a c t i v i t y in the rat. J. Cardiovasc. Pharmacol. 8:420-427. Criscione, L. and Hedwall, P.R. (1979). Does hydralazine have a dual site of action? Naunyn-Schmeid. Arch. of Pharmacol., 307: (Suppl.), R45. Frohlich, E.D. (1985). Antihypertensive therapy: newer concepts and agents. Cardiology 72: 349-365.
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Hermsmeyer, K. and Mason, R. (1982). Norepinephrine sensitivity and desensitization of cultured single vascular muscle cells. Circ. Res., 50: 627-632. Hermsmeyer, K., Trapani, A., Abel, P.W. and Worcel, M. (1983). Effect of hydralazine on tension and membrane potential in the rat caudal artery. J. Pharmacol. Exp. Ther., 227: 322-326. Lyles, G.A. and Callingham, B.A. ( 1 9 8 2 ) . Hydralazine is an irreversible inhibitor of semicarbazide-sensitive, cqorgyline-resistant amineoxidase in rat aorta homogenates. J. Pharm. Pharmacol., 34: 139-140. Marvin, W., Robinson, R. and Hermsmeyer, K. (1979). Correlation of function and morphology of neonatal rat and embryonic chick cultured cardiac and vascular muscle cells. Circ. Res., 45: 528-540. Numata, Y., Takei, T. and Hayakawa, T. (1981). Hydralazine as an inhibitor of lysyl oxidase activity. Biochem. Pharmacol. 30: 3125-3126. Reece, P.A. (1981). Hydralazine and related compounds: chemistry, metabolism, and mode of action. Medicinal Res. Rev. I: 73-96. Sturek, M. and Hermsmeyer, K. (1986). Calcium and sodium channels in spontaneously contracting vascular muscle cells. Science 233: 475-478. Zacest, R., Gilmore, E. and Koch-Weser, J. (1972). Treatment of essential hypertension with combined vasodilation and betaadrenergic blockade. New Engl. J. Med. 286: 617-622.
This research was HL38537, HL38645 Switzerland
supported by NIH grants HL16328, and by Ciba-Geigy Ltd., Basel,
Paper received 18.11.91.
Revised paper accepted 14.08.92.
J.R.J.
Baker,
P.R.
Hedwall,
1023
OF HYDRALAZINE MUSCLE CELLS
and
*K.
Hermsmeyer
O r e g o n R e g i o n a l P r i m a t e R e s e a r c h Center, D e p a r t m e n t s of M e d i c i n e and Cell B i o l o g y and Anatomy, O r e g o n H e a l t h S c i e n c e s University, Portland, OR 97213 *Corresponding
author
ABSTRACT
H i g h s p e c i f i c a c t i v i t y (20 Ci/mmol) t r i t i a t e d h y d r a l a z i n e (3Hyd) d i s t r i b u t i o n in isolated, c u l t u r e d v a s c u l a r m u s c l e cells was determined to i d e n t i f y the sites of Hyd binding. 3Hyd d o s e - d e p e n d e n t l y bound to e x t r a c e l l u l a r p r o t e i n and to the area of o r g a n e l l e s w h i c h s e c r e t e these proteins. I n c r e a s e d e x t r a c e l l u l a r b i n d i n g after Hyd p r e - e x p o s u r e s u g g e s t s new b i n d i n g sites may be exacerbated as a result of Hyd interactions. These e x p e r i m e n t s s u g g e s t a p o t e n t i a l l y i m p o r t a n t f e a t u r e of the m e c h a n i s m of a c t i o n of this d i r e c t l y acting v a s o d i l a t o r . INTRODUCTION
The q u e s t i o n of the m e c h a n i s m s and sites of a c t i o n of the d i r e c t l y a c t i n g vasodilator, h y d r a l a z i n e (Hyd), at the v a s c u l a r m u s c l e cell level has b e e n d i f f i c u l t to answer. A m o n g the p u z z l e s o f f e r e d by this agent, w h i c h is being used in l~rge n u m b e r s of h y p e r t e n s i v e p a t i e n t s (Frohlich, 1985), is how the mechanism differs from other peripherally acting antihypertensives, e.g., calcium antagonists or a l p h a - a d r e n e r g i c antagonists, and the p r e c i s e sites of action. Hyd is n o t a b l e b e c a u s e it preferentially inhibits norepinephrine, c o m p a r e d with p o t a s s i u m induced, c o n t r a c t i o n s ( C r i s c i o n e and Hedwall, 1979). The antihypertensive effectiveness of the c o m b i n a t i o n of Hyd w i t h b e t a - a d r e n e r g i c b l o c k i n g agents (Zacest et al., 1972) has c a u s e d a renaissance of i n t e r e s t and led to its w i d e s p r e a d use for t r e a t m e n t of h y p e r t e n s i o n (Frohlich, 1985). How and w h e r e Hyd acts in v a s c u l a r m u s c l e cells poses a c h a l l e n g i n g puzzle. We sought the sites of s u b c e l l u l a r l o c a l i z a t i o n of Hyd as a clue to the m e c h a n i s m of action. The p u r p o s e s of the p r e s e n t study were 2-fold along this rationale. Firstly, we used contractile, previously defined, c u l t u r e d v a s c u l a r m u s c l e cells (Marvin, et al., 1979; H e r m s m e y e r and Mason, 1982; S t u r e k and Hermsmeyer, 1986) to m i n i m i z e the c o m p l i c a t i o n of c o n n e c t i v e tissue J
0309-1651/92/101023-17/$08.00/0
© 1992 Academic Press Ltd
Cell Biology International Reports, Vol. 16, No. 10, 1992
1024
b i n d i n g of 3H-Hyd in vivo (Baker, et al., 1985). And secondly, we s t u d i e d the time c o u r s e of l o c a l i z a t i o n in r e l a t i o n to the slow onset of Hyd v a s o r e l a x a t i o n . In vivo studies (Baker et al., 1985) had shown that Hyd, w h e t h e r o r a l l y or i n t r a v e n o u s l y a d m i n i s t e r e d to rats, e x h i b i t s more than 50% of its b i n d i n g to e l a s t i n and collagen. M u l t i p l e m e c h a n i s m s are to be e x p e c t e d for Hyd. M e m b r a n e (Hermsmeyer et al., 1983) and i n t r a c e l l u l a r actions (Diamond and Janis, 1980; D i a m o n d and Shaikh, 1980) for Hyd have been proposed, but the s i g n i f i c a n c e of these m e c h a n i s m s has not been established. L o c a l i z a t i o n of Hyd is likely to be an i m p o r t a n t clue b e c a u s e the time c o u r s e of v a s o d i l a t i o n c o r r e l a t e s with slow, p e r s i s t e n t Hyd b i n d i n g and not w i t h the c o n c e n t r a t i o n c i r c u l a t i n g in the blood p l a s m a (Baker et al., 1985). MATERIALS
AND
METHODS
P r i m a r y c u l t u r e s of a z y g o u s vein, dorsal aorta, and portal vein m u s c l e cells were used in this study as p u b l i s h e d ( H e r m s m e y e r and Mason, 1982). Radiolabelled hydralazine was prepared by Dr. D.E. Brundish, R e s e a r c h Centre, C i b a - G e i g y P h a r m a c e u t i c a l s , Horsham, U.K. and had a specific radioactivity 20 Ci/mmol. A c i r c u l a r sample (491 mm 2) of e a c h s u r f a c e was e x p o s e d for 60 min to 1 pM 3H-Hyd p r e p a r e d from an a m p o u l e w i t h a c o n c e n t r a t i o n of 260 n m o l e s / m l w h i c h gave 11.828 m i l l i o n c o u n t s / m i n (measured with c o r r e c t i o n for loss at high counting rates) in a Beckman LS-250 s c i n t i l l a t i o n counter. Each sample was t h o r o u g h l y rinsed i0 times with distilled water, placed in a glass s c i n t i l l a t i o n vial c o n t a i n i n g i0 ml of R e s e a r c h P r o d u c t s International complete counting cocktail 3a70, and c o u n t e d for I0 minutes. T h r e e - d a y cultures: C u l t u r e d cells (from d o r s a l aorta, a z y g o u s vein, or h e p a t i c portal vein) w e r e w a s h e d free of c u l t u r e m e d i u m w i t h seven c h a n g e s of ISM and i n c u b a t e d for 3, 30 or 60 min in 1 ml of 2 ~M 3H-Hyd in ISM (40 vCi) or I ml of 0.06 ~M 3H-Hyd in ISM (1.33 vCi). The cells were grown on p o l y l y s i n e - c o a t e d discs. In some c u l t u r e d cells, 30 and 60 min s a m p l e s w e r e p r e - i n c u b a t e d with 1 DM u n l a b e l l e d HP for 60 min prior to the i n t r o d u c t i o n of the t r i t i a t e d drug. Duplicate samples were prepared for autoradiographic and radiometric processing. T r i p l i c a t e samples of aorta cells i n c u b a t e d for 60 min w i t h 2 NM ~H-Hyd w e r e p r e p a r e d and p r o c e s s e d in p a r a l l e l w i t h all s a m p l e s for EM a u t o r a d i o g r a p h y using identical reagent aliquots (5 ml) to q u a n t i t a t i v e l y
Cell Biology International Reports, Vol. 16, No. 10, 1992
1025
assess the elution of r a d i o l a b e l at e a c h stage of processing. F o r m o r p h o l o g i c a l e x a m i n a t i o n , o n e w e l l for e a c h c e l l t y p e w a s r e s e r v e d for 60 m i n i n c u b a t i o n w i t h ISM only. Five-day cultures: C e l l s f r o m d o r s a l a o r t a and a z y g o u s v e i n w h i c h h a d b e e n in c u l t u r e for 5 d a y s w e r e i n c u b a t e d for 3, 30, or 60 m i n w i t h 3H-Hyd (0.06 ~M, 1 p M or 2 pM) w i t h or w i t h o u t preincubation for 60 m i n w i t h 1 pM u n l a b e l l e d H y d (30 and 60 m i n o n l y ) . These samples were u s e d for r a d i o m e t r y only. In a s e p a r a t e e x p e r i m e n t , 5-day cultures of dorsal aorta cells underwent an i d e n t i c a l t r e a t m e n t r e g i m e for the p u r p o s e s of r a d i o m e t r y a n d EM a u t o r a d i o g r a p h y , a n d w e r e g r o w n d i r e c t l y o n the f l o o r of the p o l y s t y r e n e c u l t u r e w e l l s . S a m p l e s w e r e f i x e d in a m e d i u m of g l u t a r a l d e h y d e (2.5%) and f r e s h l y p r e p a r e d formaldehyde (4%) in c a c o d y l a t e buffer (0.i M, pH 7.0) at r o o m t e m p e r a t u r e for 1 h. R i n s e s in i d e n t i c a l c a c o d y l a t e b u f f e r (15, 15 and 30 min) w e r e f o l l o w e d b y p o s t - f i x a t i o n in o s m i u m t e t r o x i d e (1% in 0.i M c a c o d y l a t e , pH 7.0, 1 h). Following a rapid cacodylate rinse the samples were dehydrated through a g r a d e d e t h a n o l s e r i e s and f i n a l l y t r a n s f e r r e d to E p o n for infiltration via a 50/50 Epon/absolute ethanol mixture. F i n a l e m b e d d i n g w a s in E p o n w i t h p o l y m e r i z a t i o n at 6 0 ° C for 48 h. L e s s t h a n 1% of t h e t r i t i u m w a s e l u t e d in the e n t i r e s e r i e s of p r e p a r a t i v e s o l u t i o n s f o l l o w e d by 24 hrs in IN N a O H at 22°C, w h i c h s h o w s t h e r e m a r k a b l y t i g h t b i n d i n g of H y d t h a t w a s h i g h l y f a v o r a b l e for s u b c e l l u l a r localization. In t h e c a s e w h e r e s a m p l e s w e r e p r o c e s s e d f o r a s s e s s m e n t of t r i t i u m e l u t i o n , f o l l o w i n g d e h y d r a t i o n t h e y w e r e t r a n s f e r r e d to N a O H (i M, r o o m t e m p e r a t u r e , 24 h) a n d t h e n to f r e s h m o l a r N a O H for f u r t h e r d i g e s t i o n for s e v e n d a y s at 55°C. The method u s e d to p r e p a r e autoradiographs has b e e n d e s c r i b e d p r e v i o u s l y (Baker, et al., 1977). E x p o s u r e of t h e f i l m w a s for f o u r w e e k s (aorta cells from 5-day cultures) o r 18 w e e k s (portal vein cells from 3-day cultures). T h e s p e c i m e n s w e r e d e v e l o p e d in K o d a k DI9 or Microdol X depending upon the grain density required. The rationale and methodology for a n a l y s i s h a v e b e e n d e s c r i b e d e l s e w h e r e (Baker, et al., 1985; B l a c k e t t and Parry, 1973, 1977). For a o r t a cells, t h e n u m b e r s of r e a l g r a i n s c o u n t e d r a n g e d f r o m 1 9 9 - 7 1 2 a n d t h e n u m b e r s of hypothetical grains from 948-1108. In the single e x p e r i m e n t o n p o r t a l v e i n cells, t h e n u m b e r of r e a l and h y p o t h e t i c a l g r a i n s w e r e 137 a n d 432 r e s p e c t i v e l y . For analysis,
we have used the hypothetical
grain method
1026
Cell Biology lnternational Reports, Vol. 16, No. 10, 1992
of B l a c k e t t and P a r r y (1973, 1977), in w h i c h a c o m p u t e r program is used to compare hypothetical grain distributions with the real grain distribution. H y p o t h e t i c a l s o u r c e s of r a d i o a c t i v i t y are e s t a b l i s h e d by a p p l y i n g to the a u t o r a d i o g r a p h s a t r a n s p a r e n t o v e r l a y screen which contains computer-predicted source-to-site distances, w h i c h g e n e r a t e the h y p o t h e t i c a l s i l v e r g r a i n s e m a n a t i n g from t h e s e sources. The d i r e c t i o n s of t h e s e hypothetical decays are, of course, random. The d i s t a n c e s are d e r i v e d from r a n g e d i s t r i b u t i o n c u r v e s from e x p e r i m e n t a l l y m e a s u r e d line s o u r c e s of a g i v e n i s o t o p e as first d e s c r i b e d by Salpeter, et al. (1969). H e n c e a rectangular m a t r i x for c r o s s - f i r e was created. E s t i m a t e s of r a d i o a c t i v i t y in the v a r i o u s s o u r c e s were d e r i v e d from s y s t e m a t i c m o d i f i c a t i o n of the h y p o t h e t i c a l s o u r c e v a l u e s by a m i n i m i z i n g s u b - r o u t i n e until the h y p o t h e t i c a l and real g r a i n d i s t r i b u t i o n s fit o p t i m a l l y for the d e s i g n a t e d sites, as a s s e s s e d by the c h i - s q u a r e d test. The n u m b e r of d e g r e e s of f r e e d o m a l l o w e d was the d i f f e r e n c e b e t w e e n the n u m b e r of sites and s o u r c e s in the cross-fire matrix. Where the crude location of radiolabel was difficult to interpret by visual i n s p e c t i o n of s i l v e r grains, we c o l l e c t e d h y p o t h e t i c a l g r a i n d a t a by r a n d o m or r e g u l a r p o i n t s a m p l i n g of the m i c r o g r a p h s so as to i n c l u d e all the p o s s i b l e s o u r c e s of r a d i o a c t i v i t y in the analysis. O t h e r c h e m i c a l s used: A d d i t i o n a l m a t e r i a l s u s e d in t h e s e e x p e r i m e n t s i n c l u d e d u n l a b e l l e d h y d r a l a z i n e (Ciba-Geigy, Basel), g l u t a r a l d e h y d e (Taab), p a r a f o r m a l d e h y d e (BDH), s o d i u m c a c o d y l a t e (BDH), E p o n (Gurr), L4 n u c l e a r e m u l s i o n (Ilford), DI9 and M i c r o d o l X d e v e l o p e r s (Kodak), and s o d i u m t h i o s u l p h a t e (BDH). RESULTS
A c t i o n of Hyd on v a s c u l a r m u s c l e c o n t r a c t i o n : Hyd dose dependently decreased spontaneous contraction frequency in s i n g l e a z y g o u s v e n o u s m u s c l e cells. Fig 1 shows that the ECs0 was a b o u t 60 nM, but o n l y w h e n long e x p o s u r e t i m e s to Hyd w e r e allowed. D u r i n g the first 10-15 minutes, t h e r e was a l m o s t no p e r c e p t i b l e a c t i o n of Hyd, f o l l o w e d by a g r a d u a l d e c r e a s e in c o n t r a c t i o n frequency. By 30 minutes, a c l e a r and n e a r l y s t e a d y d e c r e a s e in c o n t r a c t i o n f r e q u e n c y occurred, and this time p o i n t was used for the data in Fig. i. This a c t i o n of Hyd was in m a r k e d c o n t r a s t to the r a p i d (peak a c t i o n w i t h i n 30 sec) increase in contraction frequency to i0-i00 nM n o r e p i n e p h r i n e , serotonin, or h i s t a m i n e , or the d e c r e a s e in contraction frequency to nitroglycerine or nitroprusside.
Cell Biology International Reports, Vol. 16, No. 10, 1992 100n
1027
(~
z u~ CI
N~
t~ (m
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F i g i: D e c r e a s e in s p o n t a n e o u s c o n t r a c t i o n f r e q u e n c y of azygos venous muscle cells suffused by Hyd was dosed e p e n d e n t w i t h r e m a r k a b l y s l o w (>i0 min) o n s e t of a c t i o n . The suffusion method was described previously (Hermsmeyer a n d R o b i n s o n , 1976). N u m b e r s in p a r e n t h e s e s a r e n u m b e r of c e l l s (i m i n c o u n t s ) . A
o.oe t~M H y d r s I I z l n e
2 uM Hyclrllizlne 16-
.
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F i g 2: C o m p a r i s o n of H y d u p t a k e at t w o c o n c e n t r a t i o n s a n d t h r e e t i m e s of e x p o s u r e (3, 30, a n d 60 min) in c e l l s c u l t u r e d for t h r e e d a y s s h o w e d t h a t H y d u p t a k e i n c r e a s e d w i t h t i m e in all c a s e s in h e p a t i c p o r t a l v e i n (n = 3), a z y g o u s v e i n (n = 3), and d o r s a l a o r t a (n = 5). A: Midr a n g e c o n c e n t r a t i o n (0.06 pM), a n d B: h i g h c o n c e n t r a t i o n (2 pM) Hyd. A l l e x p e r i m e n t s w e r e c a r r i e d o u t i n ISM.
1028
Cell Biology lntemational Reports, VoL 16, No. 10, 1992
Radiometry: Hyd b i n d i n g in all three types of v a s c u l a r m u s c l e cells was slow and c o n c e n t r a t i o n - d e p e n d e n t , as shown in Figs. 2-4. Fig. 2 allows c o m p a r i s o n of three e x p o s u r e times in each of the t h r e e types of v e s s e l s using e i t h e r 60 nM or 2 ~M 3H-Hyd. The b i n d i n g p r o c e s s was slow in all three types of v a s c u l a r m u s c l e cell cultures, and i n c r e a s e d w i t h time a l t h o u g h r e a c h i n g a p l a t e a u b e t w e e n 30 and 60 minutes. The a m o u n t of c o n n e c t i v e t i s s u e s e c r e t e d was n e g l i g i b l e in these 3 - d a y cultures. The i n c r e a s e w i t h time is also shown in Fig. 3, w h i c h shows data from the 5-day cultures, in w h i c h at least ten times as m u c h c o n n e c t i v e t i s s u e p r o t e i n had b e e n secreted. The total 3H-Hyd b i n d i n g at 5 days was slow (Fig. 3) and d o s e - d e p e n d e n t (Fig. 4). Furthermore, the p r e v i o u s e x p o s u r e to n o n - r a d i o a c t i v e Hyd i n c r e a s e d s u b s e q u e n t l a b e l l i n g with 3H-Hyd (Fig. 4). The Hyd b i n d i n g thus gave no e v i d e n c e of being s a t u r a t e d with i n c r e a s e in c o n c e n t r a t i o n or by p r e - e x p o s u r e , a l t h o u g h the b i n d i n g rate was always o b s e r v e d to d e c r e a s e a f t e r 30 min (Fig. 3). Hyd bound not o n l y to cells, but also to the cell c u l t u r e wells, even after ten t h o r o u g h r i n s i n g procedures. We tried to find a l a b o r a t o r y m a t e r i a l s u b s t a n t i a l l y less r e a c t i v e in a s e p a r a t e Hyd b i n d i n g study, but found that Hyd binds s t r o n g l y to c u l t u r e s u r f a c e s tested, i n c l u d i n g the glass, p o l y s t y r e n e , and aklar s u b s t r a t e s used in this study (Table i). In e a c h case, the test m a t e r i a l c o n s i s t e d of 3 c o v e r s l i p like s a m p l e s of m a t e r i a l of the same s u r f a c e area (491 2 mm ) w h i c h w e r e e x p o s e d to 1 pM Hyd for 60 minutes. The test s a m p l e s were s u b s e q u e n t l y w a s h e d at least i0 times for > 2 min (each wash) w i t h d i s t i l l e d w a t e r to r e m o v e any free Hyd. R e p e a t c o u n t i n g after a d d i t i o n a l w a s h e s g a v e the same result. Autoradiography: At the e l e c t r o n m i c r o s c o p i c level, 5-day c u l t u r e s (Fig. 5) p r o d u c e d e x t r a c e l l u l a r c o n n e c t i v e tissue while 3-day cultures (Fig. 6) did not. Intracellularly, the 5-day cultures contained more e x t e n s i v e r e g i o n s of rough e n d o p l a s m i c r e t i c u l u m and r i b o s o m e s and fewer m y o f i l a m e n t s than the 3-day cultures. Quantitative cross-fire (hypothetical grain) a n a l y s i s s h o w e d that i n c u b a t i o n of 5-day aorta cells w i t h 2 pM ~HHyd for 60 min gave a 3-fold i n c r e a s e in c y t o p l a s m i c label (relative activity) over that found w i t h 1 pM 3HHyd (Table 2). The i n c r e a s e in Hyd b i n d i n g to the c y t o p l a s m in 2 pM v e r s u s 1 pM 3H-Hyd for 60 min is even m o r e d r a m a t i c w h e n e x p r e s s e d as g r a i n s / g r i d point
Cell Biology lnternational Reports, Vol. 16, No. 10, 1992 Dorsal O 0141 I M
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F i g 3: T i m e c o u r s e ( 3 - 6 0 min) of H y d u p t a k e b y A) d o r s a l a o r t a (n = 3) a n d B) a z y g o u s v e i n (n = 3) c e l l s c u l t u r e d for f i v e d a y s at 60 nM, 1 pM, a n d 2 p M Hyd. 600• 500'
velnopralraalad//7
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m i n i n c u b a t i o n of d o r s a l a o r t a a n d a z y g o u s v e i n c e l l s cultured for five days at t h r e e concentrations was similar. P r e i n c u b a t i o n w i t h 1 D M u n l a b e l l e d H y d for 1 h e n h a n c e d ( r a t h e r t h a n i n h i b i t e d ) 3H-Hyd u p t a k e at b o t h 1 p M a n d 2 MM.
Cell Biology lnternational Reports, VoL 16, No. 10, 1992
1030
(relative specific radioactivity), where the i n c r e a s e is over 5-fold (Table 3). The higher 3H-Hyd concentration, or longer i n c u b a t i o n times, also i n c r e a s e d Hyd b i n d i n g to c o n n e c t i v e tissue, matrix, and s u b s t r a t e (Table 3). The hepatic portal vein 3 day c u l t u r e s had the largest c o n c e n t r a t i o n of c y t o p l a s m i c and c o n n e c t i v e tissue Hyd binding of any cells tested. Pretreatment of the cells with non-radioactive Hyd prevented cytoplasmic accumulation and decreased c o n n e c t i v e tissue a c c u m u l a t i o n of Hyd in r e l a t i v e terms (Table 3), a l t h o u g h c o n n e c t i v e tissue b i n d i n g r e m a i n e d considerable. The significant changes in Hyd d i s t r i b u t i o n after u n l a b e l l e d Hyd p r e t r e a t m e n t were the d e c r e a s e in c y t o p l a s m i c and the increase in s u b s t r a t e Hyd (Tables 2 and 3). There was no p r e f e r e n t i a l binding of Hyd at the cell m e m b r a n e ( c y t o p l a s m / m a t r i x interface), either with or without the non-radioactive Hyd pretreatment. The highest c o n c e n t r a t i o n of Hyd was found in the c o n n e c t i v e tissue, either e x t r a c e l l u l a r l y or in v e s i c l e s p r o b a b l y filled with nascent (pre-exocytotic) c o n n e c t i v e tissue p r o t e i n (Fig. 7).
TABLE 3H-HYDRALAZINE
(HYD)
1
BINDING
TO E X P O S E D
SURFACES
Counts/ min io
Glass
2.
Epon
(embedding
3.
Aklar
(plastic
4.
Polystyrene (tissue culture
5.
(coverslip)
Polypropylene (scintillation
% of total
45,216
0.04
medium)
178,508
1.51
coverslip)
187,846
1.59
291-608
2.47
478,264
4.04
dish) vial)
6.
Polycarbonate (Nucleopore filter)
750,501
6.35
7.
C e l l u l o s e acetate (Millipore filter)
832,584
7.04
CellBiology lnternational Reports, Vol. 16, No. 10, 1992
1031
F i g 5: E M a u t o r a d i o g r a p h of 5 - d a y c u l t u r e d a o r t a c e l l s f o l l o w i n g i n c u b a t i o n f o r 3 m i n w i t h 3H-Hyd (2 pM). Most of t h e s i l v e r g r a i n s w e r e a s s o c i a t e d w i t h c o n n e c t i v e tissue. S = polystyrene substrate. S c a l e b a r = 1 ~M. E l u t i o n t e s t i n g ( T a b l e 4) s h o w s t h a t o n l y a b o u t 1% o f t h e H y d w a s r e m o v a b l e d u r i n g t h e EM p r e p a r a t i v e p r o c e d u r e , i n d i c a t i n g t h a t it w a s t i g h t l y b o u n d to t h e c u l t u r e d cells sufficiently to a l l o w s u b c e l l u l a r localization ( T a b l e s 2 a n d 3). Autoradiographic and radiometric data indicated slow, s t r o n g b i n d i n g of H y d to c o n n e c t i v e t i s s u e p r o t e i n s , w i t h increasing a s s o c i a t i o n w i t h time, e v e n if t h e c e l l s w e r e f i r s t e x p o s e d to n o n - r a d i o a c t i v e Hyd. The term matrix d e s c r i b e s t h e a r e a in a u t o r a d i o g r a p h s w h e r e n o s t r u c t u r e s a r e a p p a r e n t a n d c o r r e s p o n d s s p a t i a l l y to t h e c u l t u r e fluid volume. It is o b v i o u s l y impossible for the r a d i o l a b e l t o b e f i x e d i n a s t r u c t u r e - f r e e s p a c e a n d it
1032
Cell Biology lnternational Reports, Vol. 16, No. 10, 1992
is t h e r e f o r e p r o b a b l e t h a t s i l v e r g r a i n s in t h i s r e g i o n are almost all a s s o c i a t e d with amorphous connective tissue. C o n s e q u e n t l y , t h e r e is s o m e j u s t i f i c a t i o n for r e g a r d i n g t h e m a t r i x v a l u e s in t h e h y p o t h e t i c a l grain a n a l y s e s in c o m b i n a t i o n w i t h t h o s e for c o n n e c t i v e t i s s u e . T h e v e r y h i g h c o n c e n t r a t i o n of r a d i o l a b e l d e s i g n a t e d to v e s i c l e s w i t h f i l a m e n t o u s c o n t e n t s in t h e 3 - d a y p o r t a l v e i n c e l l s l e a d s us to the s u g g e s t i o n t h a t t h e s e v e s i c l e s contain newly forming connective tissue with which radiolabelled Hyd can react after uptake into the cytoplasm.
F i g u r e 6: E l e c t r o n m i c r o g r a p h o f a 3 - d a y c u l t u r e d a o r t a c e l l f o l l o w i n g i n c u b a t i o n for 1 h o u r w i t h m e d i u m only. Note the absence of secreted connective tissue. S = p o s i t i o n of a k l a r s u b s t r a t e . L = lipid droplets. RER = rough endoplasmic reticulum. Scale bar = 1NM.
Cell Biology lnternational Reports, Vol. 16, No. 10, 1992
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F i g u r e 7: E M a u t o r a d i o g r a p h of a 3 - d a y c u l t u r e d p o r t a l v e i n c e l l f o l l o w i n g i n c u b a t i o n for 1 h o u r w i t h 3H-Hyd (2 ~M). Silver grains were most concentrated where associated with a cytoplasmic vesicle with flocculent contents. S = position of aklar substrate. L = lipid droplets. V = cytoplasmic vesicle. S c a l e b a r = 1 NM.
,
S
F i g u r e 8: E M a u t o r a d i o g r a p h of 5 - d a y a o r t a c e l l c u l t u r e preincubated with unlabelled Hyd (i ~M) for 1 h o u r f o l l o w e d b y ~H-Hyd (i pM) f o r 1 h o u r . Connective tissue was well labelled. S = polystyrene substrate. Scale bar =
i
~M.
Aorta
Aorta
Aorta
Aorta
Portal
i pM 60 min
i ~M 60 min*
2 NM 3 min
2 ~M 60 min
2 ~M 60 min
3
significant
v
5
5
5
5
5
°Day
I ± I
0 ± i
1 ± I
I ± 3
0 ± I
Nucl
9 ± I
19 ± 2
12 ± i
34 ± 2
24 ± 3
14 ± 2
Subst
53
90
145
125
100
140
n
n i s the number o f c e l l s .
9 ± 4
13 ± 9
20 ± 8
11 ± 5
11 ± 5
Matrix
(p > 0 . 0 5 ) ;
58 ± 22 (vesicle)
46 ± 4
70 ± 7
45 ± 5
56 ± 8
58 ± 6
C.T.
at the 95% c o n f i d e n c e l e v e l
33 ± 23
25 ± 5
5 ± 2
0 ± 0
8 ± 5
17 ± 9
Cyto
4.8
2.4
3.4
7.4
0.6
1.0
Xz
4
3
3
3
2
1
df
* = p l u s p r e t r e a t m e n t w i t h n o n - r a d i o a c t i v e Hyd. Relative activity is % total radioactivity. A b b r e v i a t i o n s : Hyd tmt = h y d r a l a z i n e t r e a t m e n t , Cyto = cytoplasm, Nucl = n u c l e u s , C.T. : c o n n e c t i v e t i s s u e , Subst = s u b s t r a t e , n = number, XL = chi squared, and d f = degrees of freedom. M a t r i x i s d e f i n e d as the space between c e l l s where c o n n e c t i v e t i s s u e cannot be i d e n t i f i e d . S u b s t r a t e ( s u b s t ) is d e f i n e d as the p o l y s t y r e n e or A k l a r m a t e r i a l on which the c e l l s were grown.
X2 are s t a t i s t i c a l l y
Aorta
I pM 3 min
All
Cells
Hyd tmt
EM AUTORADIOGRAPHIC ANALYSIS SHOWINGDISTRIBUTION OF RELATIVE ACTIVITY IN VASCULARMUSCLE CELLS
TABLE 2
Aorta Aorta Aorta Aorta Aorta
Portal
I pM 3 min
1 pM 60 min
i ~M 60 min*
2 pM 3 min
2 pM 60 min
2 pM 60 min
v
0.03 (57) 0.28
5 5 3
0.00 (50)
5
0.37 (82)
(57)
0.05 (50)
5
(5)
(9)
0.09
(9)
0.00
(vesicle)
(7)
7.71
(7)
4.00
(7)
3.10 0.24 (24)
(24)
0.18
(28)
0.21
2.55
(9)
(11)
0.78
(2)
5.04
C2)
1.59
(8)
1.19
(8)
0.02
(5)
0.90
(9)
0.05
0.13 (28)
0.35
Subst
3.42
0.08
Matrix
(8)
(5)
2.37
C.T.
(28)
(9)
0.00
0.07
5 (50)
Nucl
Cyto
Day
* = plus pretreatment w i t h n o n - r a d i o a c t i v e Hyd. A b b r e v i a t i o n s : Hyd tmt = h y d r a l a z i n e treatment, Cyto = cxtoplasm, Nucl = nucleus, C.T. = connective t i s s u e , Subst = s u b s t r a t e , n = number, X~ = chi squared, and df = degrees of freedom. M a t r i x is defined as the space between c e l l s where connective t i s s u e cannot be i d e n t i f i e d . Substrate (Subst) is defined as the p o l y s t y r e n e or A k l a r material on which the c e l l s were grown. Results were obtained from 53-145 c e l l s and are expressed as g r a i n s / p o i n t and r e l a t i v e areas ( ) . G r a i n s / g r i d p o i n t ( r e l a t i v e s p e c i f i c r a d i o a c t i v i t y ) is % t o t a l r a d i o a c t i v i t y % t o t a l area.
Cells
Hyd tmt
RELATIVE TRITIUM SPECIFIC RADIOACTIVITY
EM AUTORADIOGRAPHIC ANALYSIS SHOWING
TABLE 3
o oo Ol
o
Cb
c~
1036
CefB~gyln~rnationalReports, VoL 1~ N o . l ~ 1992 TABLE 4 ELUTION OF 3H FROM 3-DAY CULTURED DORSAL AORTIC MUSCLE CELLS FOLLOWING INCUBATION FOR 60 MIN WITH 2 pM 3H-HYD
Solution
Mean counts/min
Percentage
(n = 3)
Glutaraldehyde (2.5%) and formaldehyde (4.0%) Cacodylate (15 min)
1,100
0.37
86
0.03
69
0.02
108
0.04
74
0.03
70% EtOH
295
0.10
90% EtOH
130
0.04
Abs EtOH (I)
167
0.06
Abs EtOH (2)
113
0.04
1,063
0.40
290,917
98.89
294,189
100.04
Cacodylate (15 min) Os04 (1% w/v) Cacodylate
IM NaOH (room temp
24 h)
IM NaOH (55 ° - 7 days) TOTAL DISCUSSION
The present results reinfoce the conclusions of the previous in vivo study (Baker et a l . , 1985) that 3H-Hyd, whether administered intravenously or per os, binds to vascular muscle c e l l s . Although the majority of label is associated with connective tissue at all times, s i g n i f i c a n t quantities of Hyd are taken up into the myocyte sarcoplasm. Based on Hyd l o c a l i z a t i o n , c e l l u l a r mechanisms of action would occur at intracellular sites and, e v e n more predominantly, e x t r a c e l l u l a r sites. The cell membrane was not s i g n i f i c a n t l y labelled. These experiments to determine where Hyd binds to isolated, single vascular c e l l s have allowed three main conclusions. F i r s t , Hyd binds p r i m a r i l y to connective tissue proteins, whether secreted or nascent, and the binding is strong. Second, Hyd enters the cell in s u f f i c i e n t quantity to allow for i n t r a c e l l u l a r actions, including on
Cell Biology International Reports, Vol. 16, No. 10, 1992
1037
proteins that decrease contraction. Third, pre-exposure of cells to non-radioactive Hyd increases, rather than decreases, subsequent e x t r a c e l l u l a r Hyd binding, perhaps by reacting with collagen and e l a s t i n to expose new binding sites on these large polymeric proteins. Even in young cells isolated from neonates, copious amounts of connective tissue proteins were present in the 5-day cultures, and evident as precursor proteins in the 3-day cultures, as has also been biochemically documented (Abraham et a l . , 1974, 1975). Most of the Hyd binding was to secreted collagen and e l a s t i n in all cells observed (Tables 2 and 3; Figs 5, 7, and 8). These connective tissue f i b e r s were less of a b a r r i e r to Hyd reaching vascular muscle c e l l s than might have been anticipated (Baker et a l . , 1985). When Hyd has access to both cells and connective tissue proteins, the proteins are more densely labelled. I t is overwhelmingly Hyd, rather than a metabolite, which is measured in these experiments because there was no plasma environment where enzymes could metabolize the drug. Furthermore, metabolites of Hyd have recently been shown to be 5-25X less potent than Hyd (or inactive) in i n h i b i t i n g contraction (Criscione et a l . , 1986), suggesting that Hyd is the active form. The entry of Hyd into the cytoplasm shows that access of Hyd should be s u f f i c i e n t to allow actions on membrane regulatory or c o n t r a c t i l e proteins and enzymes w i t h i n the c e l l . The mechanism of action of Hyd probably involves i n t r a c e l l u l a r , as well as membrane actions (Criscione and Hedwall, 1979; Hermsmeyer et a l . , 1983). Other sites might be important as w e l l , as Hyd binds i r r e v e r s i b l y to an amine oxidase associated with muscle membranes (Lyles and Callingham, 1982), and with lysyl oxidase, which is important for function of e l a s t i n and collagen (Numata et a l . , 1981). The f a c i l i t a t i o n of Hyd binding by previous exposure to I pM non-radioactive Hyd for 1 hour is opposite to the prediction of detecting non-specific binding. The hypothesis that Hyd has such a prolonged onset and duration of action because of slow binding to the blood vessel wall (Reece, 1981) would be supported by these data. The unlabelled Hyd might have been expected to occupy the s p e c i f i c Hyd binding sites, making them unavailable to 'H-Hyd, j u s t as appears to have occurred with cytoplasmic l a b e l l i n g . However, e x t r a c e l l u l a r l a b e l l i n g s i g n i f i c a n t l y increased a f t e r pre-exposure to Hyd, suggesting formation of new sites for i n t e r a c t i o n , perhaps by a d i r e c t chemical reaction. Furthermore, labelled Hyd was p r a c t i c a l l y t o t a l l y r e s i s t a n t to elution during EM processing. These l o c a l i z a t i o n studies show Hyd to be a remarkably reactive compound, probably changing some of the proteins with which i t interacts.
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Cell Biology lnternational Reports, VoL 16, No. 10, 1992
Since total Hyd binding was always equal or greater after unlabelled Hyd pretreatment (see Fig. 4), the data suggest a new property for Hyd, namely, the sequential, slow reaction with e x t r a c e l l u l a r proteins which f a c i l i t a t e s subsequent binding of 3H-Hyd. Since the binding increased beyond i (or even 2) hours, the process is slow and continuous, and might well explain the slow onset of action Hyd. No metabolite of Hyd would be needed, and none is l i k e l y (Criscione et a l . , 1986), to explain the paradoxical 30-150 hour h a l f - l i f e for efficacy when plasma h a l f - l i f e is only 30-60 minutes. I t would appear that, at least under the conditions of culture, non-radioactive Hyd eliminated cytoplasmic labelling while connective tissue labelling was increased. Notably, i t was these secreted proteins which contained most of the radiolabel and in the most concentrated form, just as in in vivo studies. These observations suggest both i n t r a c e l l u l a r and e x t r a c e l l u l a r sites of action, probably involving direct chemical reaction with Hyd, in arterial and venous vascular muscle cells. REFERENCES
Abraham, P.A., Smith, D.W. and Carnes, W.H. (1974). Synthesis of soluble elastin by aortic medial cells in culture. Biochem. Biophys. Res. Comm., 58: 597-604. Abraham, P.A., Smith, D.W. and Carnes, W.H. (1975). Biosynthesis of lysine-derived elastin crosslinks in aortic cell culture. Biochem. Biophys. Res. Comm., 67: 723-727. Baker, J.R.J., Bullock, G.R., Crawford, N. and TaylorED.G. (1977). Localization in platelets of s o d i u m J~Cr-chromate, 12Syl-antibody to whole membrane, and 3H-diisopropylfluorophosphate using electron microscopic autoradiography. Am. J. Pathol., 88: 277-290. Baker, J.R.J., Bullock, G . R . and Williamson, I.H.M. (1985). Autoradiographic study of the d i s t r i b u t i o n of 3~_ and 14C-hydralazine in the rat. B r i t . J. Pharmacol., 84: 07-120. Blackett, N.M. and Parry, D.M. (1973). A new method for analyzing electron microscope autoradiographs using.hypothetical grain distributions. J. Cell. Biol., 57: 9-15. Blackett, N.M. and Parry, D.M. (1977). A simplified method of 'hypothetical grain' analysis of electron microscope autoradiographs. J. Histochem. Cytochem., 25: 206-214. Criscione, L., Eichenberger, K., Hedwall, P.R. and Schmid, K. (1986). Hydralazine and its metabolites: in v i t r o and in vivo a c t i v i t y in the rat. J. Cardiovasc. Pharmacol. 8:420-427. Criscione, L. and Hedwall, P.R. (1979). Does hydralazine have a dual site of action? Naunyn-Schmeid. Arch. of Pharmacol., 307: (Suppl.), R45. Frohlich, E.D. (1985). Antihypertensive therapy: newer concepts and agents. Cardiology 72: 349-365.
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Hermsmeyer, K. and Mason, R. (1982). Norepinephrine sensitivity and desensitization of cultured single vascular muscle cells. Circ. Res., 50: 627-632. Hermsmeyer, K., Trapani, A., Abel, P.W. and Worcel, M. (1983). Effect of hydralazine on tension and membrane potential in the rat caudal artery. J. Pharmacol. Exp. Ther., 227: 322-326. Lyles, G.A. and Callingham, B.A. ( 1 9 8 2 ) . Hydralazine is an irreversible inhibitor of semicarbazide-sensitive, cqorgyline-resistant amineoxidase in rat aorta homogenates. J. Pharm. Pharmacol., 34: 139-140. Marvin, W., Robinson, R. and Hermsmeyer, K. (1979). Correlation of function and morphology of neonatal rat and embryonic chick cultured cardiac and vascular muscle cells. Circ. Res., 45: 528-540. Numata, Y., Takei, T. and Hayakawa, T. (1981). Hydralazine as an inhibitor of lysyl oxidase activity. Biochem. Pharmacol. 30: 3125-3126. Reece, P.A. (1981). Hydralazine and related compounds: chemistry, metabolism, and mode of action. Medicinal Res. Rev. I: 73-96. Sturek, M. and Hermsmeyer, K. (1986). Calcium and sodium channels in spontaneously contracting vascular muscle cells. Science 233: 475-478. Zacest, R., Gilmore, E. and Koch-Weser, J. (1972). Treatment of essential hypertension with combined vasodilation and betaadrenergic blockade. New Engl. J. Med. 286: 617-622.
This research was HL38537, HL38645 Switzerland
supported by NIH grants HL16328, and by Ciba-Geigy Ltd., Basel,
Paper received 18.11.91.
Revised paper accepted 14.08.92.
