Specific binding sites in the rabbit kidney for prostaglandin a

SPECIFIC BINDING RABBIT KIDNEY FOR

AhmadA.

SITES IN THE PROSTAGLANDIN

A

Attallah and J a m e s B. Lee

ABSTRACT

C o m p e t i t i v e b i n d i n g s t u d i e s of v a r i o u s p r o s t a g l a n d i n s w i t h h o m o g e n a t e s of r a b b i t r e n a l c o r t e x , o u t e r m e d u l l a and p a p i l l a d e m o n s t r a t e h i g h e r s p e c i f i c i t y f o r p r o s t a g l a n d i n A. T h e r e is h i g h a f f i n i t y f o r PGA b i n d i n g to the e x t r a c t s which is s a t u r a b l e and t i m e and t e m p e r a t u r e d e p e n d e n t . T h e r e s u l t s s u g g e s t the p r e s e n c e of s p e c i f i c b i n d i n g s i t e s in the r a b b i t k i d n e y f o r P G A .

F r o m The D e p a r t m e n t of M e d i c i n e State U n i v e r s i t y of New Y o r k at Buffalo S c h o o l of M e d i c i n e and T h e Buffalo G e n e r a l H o s p i t a l 100 High S t r e e t Buffalo, New Y o r k 14203

T h e s e s t u d i e s w e r e s u p p o r t e d in p a r t b y US PHS G r a n t AM NIH 5 RO1 A M 5 9 8 2 - 0 2 f r o m the N a t i o n a l I n s t i t u t e of A r t h r i t i s and M e t a b o l i c D i s e a s e s and the I r w i n S t r a s b u r g e r M e m o r i a l Foundation.

Accepted September22, 1973

PROSTAGLANDINS N O V E M B E R 1973

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703

PROSTAGLANDINS

INTRODUCTION F o l l o w i n g t h e i s o l a t i o n (1) a n d i d e n t i f i c a t i o n (2) of p r o s t a g l a n d i n s A2, E 2 a n d F2c~ i n r a b b i t r e n a l m e d u l l a , it h a s b e e n o b s e r v e d t h a t c o m p o u n d s of the PGA and PGE series are potent antihypertensive and natriuretic a g e n t s in a n i m a l s (1, 3) a n d h y p e r t e n s i v e h u m a n s (4). A l t h o u g h t h e m e c h a n i s m o f t h e e n h a n c e d s o d i u m e x c r e t i o n h a s b e e n a t t r i b u t e d to the c o i n c i d e n t h e m o d y n a m i c e f f e c t s of a n i n c r e a s e d r e n a l c o r t i c a l b l o o d f l o w (5) i t h a s r e c e n t l y b e e n s h o w n t h a t P G A ~ (but n o t P G E 2) l e a d s to a m a r k e d r e d u c t i o n i n o x y g e n c o n s u m p t i o n a n d N a r - K + A T P a s e i n v i t r o (6). T h i s s u g g e s t s t h a t t h e d e c r e a s e d r e n a l r e a b s o r p t i o n b y P G A i n vitro m a y b e t h e r e s u l t o f m e t a b o l i c i n h i b i t i o n of e n e r g y s o u r c e s n o r m a l l y a p p l i e d to s o d i u m r e a b s o r p tion. Furthermore, it h a s b e e n h y p o t h e s i z e d t h a t N a + - K + A T P a s e m a y a c t a s a r e c e p t o r f o r P G A 2 i n m e d i a t i n g t h e n a t r i u r e t i c e f f e c t s of t h i s c l a s s o f p r o s t a g l a n d i n (6). S t u d i e s w e r e u n d e r t a k e n t h e r e f o r e to d e t e r m i n e t h e presence and characteristics of prostaglandin binding sites in rabbit renal cortex and medulla. METHODS Rabbits were stunned by neck blow exsanguinated and the kidneys removed. Following decapsulation, rabbit renal cortex, outer medulla and papilla were separated by scissor dissection. The various regions of the kidney were separately homogenized in 0. i M Tris IICI, 0.25 M sucrose buffer, pH 7.6, using four 30 second pulses at intervals of one minute. The extracts were centrifuged at i0, 000 x g for 30 minutes and the supernatants assayed for binding activity • Tritium labeled PGAI, PGE I" PGF 2 c~ and arachidonic acid (New England Nuclear Corp., Boston, Mass. ) were repurified on a 1.0 gram silicic acid column (7). 311-PGBI was prepared from 3H-PGEI (8). Each tracer compound was added to 0. i ml of the kidney supernatants (400 CPM/mg tissue wet weight), the total volumes adjusted to 0.5 ml with the buffer and incubated for 8 hours at 37 ° C. Free 3H-PG was separated from bound by absorption to dextran-coated charcoal (I mlof 25 rng dextran T 70, 250 mg Norit A in I00 ml of 0.15 phosphate buffer, pH 7.6). The bound fractions were decanted into scintillation vials containing Aquasol (15 ml) and counted in a liquid scintillation spectrophotometer (Packard Model 3320). RESULTS T a b l e 1 s h o w s t h e p e r c e n t b o u n d of l a b e l e d P G ' s to c o r t i c a l , o u t e r m e d u l l a r y a n d p a p i l l a r y h o m o g e n a t e s of r a b b i t k i d n e y . P G A 1 a n d P G B 1 which is a metabolite of PGA 1 bind significantly higher than PGE 1 and P G F 2 a to t h e 10, 000 x g s u p e r n a t a n t o f a l l t h r e e z o n e s of t h e k i d n e y . S t u d i e s w e r e u n d e r t a k e n to d e t e r m i n e t h e r a n g e o f c o n c e n t r a t i o n s a t which the binding sites were saturated. Figure i illustrates that addition o f n o n - l a b e l e d P G A 1 i n h i b i t s t h e p e r c e n t of 3 H - P G A 1 i n i t i a l l y b o u n d to h o m o g e n a t e s of c o r t e x , o u t e r m e d u l l a and, to a h i g h e r d e g r e e , p a p i l l a . A

704

NOVEMBER

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VOL. 4 NO. 5

¢j1

cj l

©

©

V$

©

Results

PGE

PGF

PGB

PGA

expressed

a s m e a n - S E M n = 12.

Each

0.7

value represents

5.4-

+

6.6 - 0.6

+

+

4.2 - 1.0

+

7.9 - 1.0

20.0 + 0.4

÷

the percentage

MEDULLA

19.0 + 0.3

OUTER

of PG bound.

BY VARIOUS ZONES OF RABBIT KIDNEY

1

19.5 + 0.5

20.7 - 1.0

CORTEX

BINDING OF PROSTAGLANDINS

TABLE

3.6+0.4

2.6 - 0.8

+

19.7 + 1.0

17.4 + 0.1

PA P I L L A

PROSTAGLANDINS

FIGURF

Figure

1:

LEGEND

Effect of increasing concentrations of arachidonic acid (arach.), PGE1, PGF2~ , and PGA 1 on the binding of 3H-PGA 1 to renal cortical, medullary, and papillary hornogenates.

2e 2Q

CoTlex

2,4 2: 2(

~

li

e

o ARACH.

•

• PGAI

16 0! Outer Medulia

~--

|o, ~24

~

poptllo

1: I0 8 6

o~ ' ioo tooQ

! 3ooo

I 6ouo

N m ~

706

N O V E M B E R 1973

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PROSTAGLANDINS

s i m i l a r experiment with PGA2 as an inhibitor of 3H-PGA 1 binding showed comparable competitive interactions as those obtained with PGA1 (data not shown). S u c h m a r k e d i n h i b i t i o n o f t h e b i n d i n g o f 3 H - P G A I i s in c o n t r a s t to t h e complete lack of displacement when non-labeled PGE1, PGF2a or arachidonic a c i d w e r e a d d e d to c o m p e t e f o r b i n d i n g s i t e s w i t h 3 H - P G A 1 ( F i g u r e 1). T h e r e w a s a p p r o x i m a t e l y 30% b i n d i n g o f 3 H - a r a c h i d o n i c a c i d b y h o m o g e n a t e s of renal cortex and medulla. T h i s b i n d i n g c o u l d n o t be d i s p l a c e d b y a d d i t i o n o f n o n - l a b e l e d a r a c h i d o n i c (0. 0 0 1 - 1 0 p g ) o r b y P G A 1 s u g g e s t i n g n o n - s p e c i f i c i t y o f a r a c h i d o n i c a c i d b i n d i n g s i m i l a r to t h a t w h i c h h a s b e e n o b s e r v e d f o r many fatty acids such as palmitate, oleate, etc. In o r d e r to s t u d y t h e a f f i n i t y o f t h e b i n d i n g s i t e s f o r P G A t h e d e g r e e of dissociation of PGA from the kidney preparation was studied. 3H-PGA1 w a s a d d e d in q u a d r u p l i c a t e to 1) 0 . 2 5 M s u c r o s e b u f f e r f o r a b s e n c e o f b i n d i n g , 2 ) 0 . 2 5 M s u c r o s e b u f f e r c o n t a i n i n g 5% b o v i n e g a m m a g l o b u l i n f o r n o n - s p e c i f i c b i n d i n g a n d 3) to e a c h 10, 000 x g s u p e r n a t a n t of t h e t h r e e r e n a l z o n e s . A f t e r i n c u b a t i o n at 37 ° C f o r 8 h o u r s o n e m l o f d e x t r a n c o a t e d c h a r c o a l w a s a d d e d to a l l t u b e s . A t t i m e d i n t e r v a l s , 1 - 3 0 m i n u t e s , t h e s a m p l e s were centrifuged and the bound fractions analyzed for radioactivity. Within t h r e e m i n u t e s , 95% of t h e r a d i o a c t i v i t y a d d e d to b o t h t h e b u f f e r a l o n e a n d t h a t c o n t a i n i n g g a m m a g l o b u l i n w e r e a b s o r b e d by t h e c h a r c o a l . By contrast, 20% o f i n i t i a l l y a d d e d 3 H - P G A 1 w a s s t i l l b o u n d b y t h e r e n a l s u p e r n a t a n t s within three minutes of incubation with charcoal. T h i s d e c r e a s e d to 14% w i t h i n 15 m i n u t e s a n d w a s u n c h a n g e d o n f u r t h e r i n c u b a t i o n to 30 m i n u t e s . These results suggest a relatively high affinity for PGA which did not appreciably differ among the various zones of the kidney. Studies were made on a time-temperature d e p e n d e n c e of t h e b i n d i n g o f P G A 1. B o t h a t 0 ° C a n d 37 ° C a s t e a d y s t a t e w a s r e a c h e d in f i v e h o u r s w i t h approximately 20% d e c r e a s e d b i n d i n g a t t h e l o w e r t e m p e r a t u r e . When the r e n a l h o m o g e n a t e s w e r e h e a t e d a t 95 ° C f o r o n e h o u r p r i o r to i n c u b a t i o n w i t h 3 H - P G A 1 t h e r e w a s a n a p p r o x i m a t e 80% l o s s in s u b s e q u e n t b i n d i n g o f 3 H - P G A 1. H o w e v e r , w h e n 3 H - P G A 1 w a s b o u n d to t h e k i d n e y h o m o g e n a t e s t h e c o m p l e x w a s stabilized against heat denaturation. These results suggest that the binding s i t e s m a y , a t l e a s t in p a r t , b e p r o t e i n in n a t u r e . T h e b i n d i n g a c t i v i t y w a s a l s o p r e s e n t in t h e s u p e r n a t a n t s o f h o m o g e n a t e s c e n t r i f u g e d a t 105, 000 x g a n d a b s e n t in t h e p e l l e t s o f t h e 10, 0 0 0 x g a s w e l l a s t h e 105, 000 x g h o m o g e n a t e s s u g g e s t i n g t h a t t h e b i n d i n g s i t e s a r e m o s t likely a soluble protein. S i n c e N a + - K + A T P a s e in t h e k i d n e y i s p r i m a r i l y m i c r o s o m a l in n a t u r e a n d a b s e n t in t h e 1 0 5 , 0 0 0 x g s u p e r n a t a n t s (9) it w o u l d appear unlikely that our protein is Na+-K + ATPase, DISC USSION The results of the present study demonstrate the existence of PGA binding s i t e s in r e n a l c o r t e x , o u t e r m e d u l l a a n d p a p i l l a o f t h e r a b b i t k i d n e y . T h e

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PROSTAGLANDINS

failure of inhibition of binding of 3H-PGAI to renal extracts by increasing the concentration of PGEI, PGF2c~ and arachidonic acid as well as the decrease in 3H-PGAI binding with increasing the concentration of non-labeled PGA demonstrates specificity for PGAo The compatibility of the concentration of endogenous (4 ng/mg) with that at which inhibition of binding can f i r s t be observed (5-10 ng/mg) suggests biological function(s) for the binding sites. The differences in degree of inhibition of PGA I binding among renal cortex, outer medulla and papilla may be the r e s u l t of the high endogenous levels of P G A in the papilla as contrasted to the cortex or it is possible that there exists within the kidney three distinct sets of binding sites for" P G A , The marked binding of 3H-PGBI t o the binding sites is not surprising. Such competitive interaction between PGB and PGA is probably the r e s u l t of their nearly identical configuration, PGB differs from PGA only in that there is isomerization of the double bond at C10_11 to C8_12 in the cyclopenlane ring. However, the possibility of enzymatic conversion of PGA to PGB prior to its binding is not excluded. T h e demonstration of binding sites with a m u c h higher specificity for P G A than P G E or P G F is in contrast to investigations in rat adipocytes (ii) and s t o m a c h (12) which s h o w P G E binds m o r e effectively than P G A . The biological effects of P G E and P G F (non-vascular s m o o t h m u s c l e contraction and inhibition of lipolysis) are m u c h m o r e pronounced that P G A in these tissues. I~y contrast, the biological effects of P G A are almost entirely manifested in the cardiovascular renal system and the finding of binding sites in the kidney relatively specific for this c o m p o u n d raises the possibility that it m a y be importantly related to the m e c h a n i s m of the antihypertensive and natriuretic effects of the P G A compounds.

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NOVEMBER 1973

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PROSTAGLANDINS

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Lee, J.B°, Covino, 27: 57, 1965.

2.

Lee, J.B., Crowshaw, J. 105: 1251, 1967.

3.

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Lee, J.B., McGiff, J°C., Kannegiesser, H., Aykent, Y.Y., and Frawley, T.F., Annals of Internal Med. 74: 703, 1971.

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Barger, A.C., 1966, p. 174.

6.

Lafferty, J. Jo, Kannegiesser, II., Lee, J,B. and Parker, COW., Advances in the ]3iosciences, International Conference on Prostaglandins, S. Bergstrom, Ed. (Pergamrnon, Vieweg, 1973), p. 293.

7.

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Zusman, R.M.,

9.

Katz, AoI., Epstein, F,H., J. Clin. Invest. 46: 1999, 1967.

AoJo,

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B.G.,

Takman,

K., Takman,

J. Physiol.

Herd,

JoA.,

B.H. B.H.

and Smith, andAttrep,

E.R., KoAo,

Circ. Res. Biochem.

214: 218, 1968.

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Mudd,

J.G.

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I0.

Kuehl, F.A., Jr., and Humes,

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rvliller,O.V. and l~agee, W. Eo, Advances in the Biosciences, International Conference on Prostaglandins, S. Bergstrom, Ed., (Pergammon, Vieweg, 1973), p. 83.

NOVEMBER 1973

J. Lo, Proc. Nat. Aead. Sci. 69:480, 1972.

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