Volume 85, number 1

FLUORESCENT

FEBS LETTERS

VISUALIZATION

OF fl-ADRENERGIC

January 1978

RECEPTORS

ON CELL SURFACES

Daphne A T L A S and Alexander L E V I T Z K I Department o f Biological Chemistry, The Hebrew University o]'Jerusalem, Jerusalem, Israel Received 12 September 1977 Revised version received 31 October 1977

1. I n t r o d u c t i o n N u m e r o u s cell types were shown to possess ~adrenergic receptors coupled to adenylate cyclase. Using radiolabeled high affinity/3-adrenergic blockers, it became possible to determine the exact n u m b e r o f .t3-receptors per cell. Most cells [1--11] were f o u n d to possess a small niimber o f receptors which varies between a few h u n d r e d to a few thousand receptors per cell. Recently it was d e m o n s t r a t e d that rat skeletal myoblasts g r o w n in culture (L6P cells) [12] and a strain o f HeLa cells [13] possess a large n u m b e r o f /3-adrenerDc receptors. In this c o m m u n i c a t i o n we report on the visualization of/3-adrenoreceptor sites by the use o f the fluorescent/3-adrenergic blocker 9m~L~oacridino-propanolol (9-AAP) [ 1 4 , 1 5 ] . It will be shown that the capability t o visualize the/3-receptors, using a fluorescent/3-blocker, is limited by receptor density on the cell surface. ~i'he fluorescent/3-blockers 9 - A S P and DAPN [14] have been used successfully to map .B-adrenergic receptors in vSvo [ i 5 - - 2 2 ] . In th%e studies the fluorescent B-blockers were first injected in vivo into the tail vein o f rats or mice; then the organs wer~ frozen, cut in a ' cryostat, and subsequently the sections were subjected t o fluorescence microscopy. Using thi3 technique distinct cells within the tissues examined which possess/3-adrenergic receptors on their surface were identified [ 1 5 - - 2 2 ] . These findings indicate that r_he

Abbreviations: 9-AAP, DL-N-(2-hydroxy-3-naphthyloxypropyl)-N*-9-acridino-isopropylenediamine or 9-aminoacridinopropranolot; DAPN, DL-N-(2-hvdroxy-3-naphthyloxypropyl)-N'-dansylethylenediamine or dansyt analogae of propranolol

158

receptor density o f these cells is high e n o u g h to enable their visualization. An a t t e m p t will be made to correlwte zhe findings in vivo with those reported in this s t u d y on cells grown in culture. 2. Experimental 2.1. G r o w t h o f cells 2.1 .i. L6P Cells Skeletal muscle cells (L6P cells) were g r o w n in culture, as described [ 1 ] . Cells were removed from the growth bottles using E D T A and c o u n t e d , as described [ 1 2 ] . Protein was determined in order to established no. cells/rag protein. Since trypsin is often used to remove cells from tissue culture dishes, it was also used to remove L6P cells f r o m the tissue culture bottles in another set o f experiments, according to the foUowing procedure: The g r o w t h m e d i u m was rem o v e d from the tissue culture bottle and the cells were incubated with 50 ml 130 mM saline, containing 20 mM Tris--HCl buffer, p H 7.4, and 500/2g/ml trypsin for 30 rain at 37°C. The cells were detached from the bott!e, centrifuged at 500 × g, and washed twice with trypsin-free sMine containing buffer. 2.1.2. HeLa Cells Cells were grown for 3 days at 37°C in M-199 medium, containing 10% calf serum inactivated at 56°C for 30 rain. Ceils were removed from the growth bottle using a solution o f 140 mM NaCI, 10 mM Tris--HCl, pH 7.4, 10 mM Nucose, and 2 told E D T A . 2.1.3. T u r k e y e r y t h r o c y t e s and $49 l y m p h o m a cells T u r k e y e r y t h r o c y t e s were prepared as described [12,23].

I:lserier/North-lto!!and Biomedical Press

Volume 85, number 1

FEBS LETTERS

January 1978

2.2. Binding o f [ ~ : : I ] t t r T [~zS[]HYP bh~dhag t o i n t a c t cells a n d t o cell m e m b r a n e s was c o n d u c t e d as d e s c r i b e d [ 7 , 1 2 ] .

0 X !

2.3. Protein determination P r o t e i n was d e t e r m i n e d u s i n g the m e t h o d [ 2 4 ] . A c c o r d i n g t o the p r o t e i n d e t e r m i n a t i o n , it was f o u n d t h a t 1.0 m g t o t a l p r o t e i n = 2 . 8 8 × 10 ~ L 6 P cells a n d 7.1 X I 0 s H e L a cells, r e s p e c t i v e l y .

4.5

w~th o r w i t h o u t 3.3 X 10 -6 M L - p r o p r a n o ! o i o r D - p r o p r a n o l o l . F l u o r e s c e n c e was v i e w e d a n d p h o t o graphs were t a k e n u s i n g a N i k o n f l u o r e s c e n c e m~croscope e q u i p p e d w i t h a N i k o n M-355 c a m e r a . "['he f-din, K o d a k 4 0 0 A S A , was d e v e l o p e d w i t h t h e corres p o n d i n g 4 0 0 A S A d e v e l o p e r . E x p o s u r e t i m e was 2 r a i n u s i n g the B fdters as f l u o r e s c e n c e e x c i t a t i o a fdter. P i c t u r e s w e r e t a k e n w i t h i n 10 m i n a f t e r m i x i n g t h e cells w i t h t h e f l u o r e s c e n t d y e a n d t h e p r o p r a n o l o l s t e r e o i s o m e r s . T h e i n t e n s i t y o f f l u o r e s c e n c e was f o u n d t o d e c a y u p o n cell d e a t h , w h i c h m a y o c c u r w i z h i n 3 0 r a i n s u b s e q u e n t t o the e x p o s u r e o f the cells t o f l u o r e s c e n t d y e u n d e r the cover slide.

3. R e s u l t s

3 . 1 . B i n d i n g o f [12SI]HYP to intact cetls and the e f f e c t o f trypsin L 6 P ceils a n d H e L a cells possess 8 0 0 0 9 r e c e p t o r s and 37 000 receptors, respectively (data n o t shown). I n b o t h cases t h e cells possess a single class o f [~zs!]_ I-I~'P r e c e p t o r - b i n d i n g sites. R e m o v a l o f L 6 P cells f r o m t h e tissue c u l t u r e b o t t l e u s i n g t r y p s i n results ':;~a 5-fold r e d u c t i o n i n t h e c o n t e n t o f ~ - r e c e p t o r s as c o m p a r e d t o ceUs removed. by EDTA treatment (fig.l).

i | i ~j

S.O 2.5 2.0

2 A . Fluorescence m i c r o s c o p y o f intact cells W a s h e d L 6 P cells o r H e L a cells were s u s p e n d e d i n 130 m M NaC1, 2 0 rnN1Tr~s--HCI, pFi 7 . 4 a n d 1O m M glucose. Cells w e r e m ~ e d w i t h 1.3 X l 0 -s M 9 - A A P

i

4.0 5.5

,o

I..5 i.O

21 ~...L

0

°

%.,

r

r

I

2

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F

5 4

)i I

~

5

6

-

7

8 9

12"5[-{~)-H'rP BOUND, (sffes per cell) XlO-4

Fig.I. The binding of [~2S[]HYP to intact L6P ceils_ The binding data is represented in the form o f a Scatchard plot. The non-specific binding was measured in the prese~ce of 1.0 × 10 -s M L-propranolol, as described [ 12]. Cel!s removed by trypsin treatment exhibited identical extent of non-specific [~asl]HYP b i n d h g as cells removed by EDTA. ( e - - e ) L6P cells removed by EDTA treatment. ( o - - o ) L6P .'=ellaremoved ['y trypsin treatment.

r e s c e n c e p a t t e r n o b s e r v e d w h e n tile cells are m i x e d w i t h 9 - A A P in t h e p r e s e n c e o f L - p r o p r a n o l o l is r a t h e r f a i n t . O n t h e o t h e r h a n d , w h e n t h e cells are m i x e d w i t h 9 - A A P i n t h e p r e s e n c e o f t h e i n a c t i v e stereo= isomer D-propanolol, the intense fluorescence pattern remaLns e s s e n t i a l l y u n c h a u g e d . T h e h a z y f l u o r e s c e n c e o b s e r v e d / n the presence or absence of L-proprano!ol iS e v e n l y d i s t r i b u t e d o n the cell s u r f a c e , a n d p r o b a b l y r e p r e s e n t s n o n - s p e c i f i c s o t u b i l i z a t i o n o f 9 - A A P i n the cell m e m b r a n e . S i m ~ a r e x p e r i m e n t s u s i n g $ 4 9 g l i o m a cells, w h / c h possess a b o u t 2 0 0 - - 3 0 0 r e c e p t o r s / c e l l [ 8 ] , a n d t u r k e y e r y t h r o c y t e gJaosts, w h i c h possess a b o u t 1000 receptors/cell [ t , i 2 ] , did n o t reveal fluorescent dots except for the non-specific, evenlyd i s t r / b u t e d f a i n t fluorescence.

3.2. Stereospecific fluorescence visualization o f B-receptors I n fig.2 it c a n b e seen t h a t L 6 P a n d H e L a cells b/rid 9 - A A P , p r e s u m a b l y t o t h e / ~ - r e c e p t o r s . T h e appearance o f / n t e n s e fluorescence dots can be inhibited by L-propranolol b u t n o t by D-propranolol. The fluo-

4. Discussion

4_ 1 _N u m b e r o f B-reeepto~'s p e r cell I n this s t u d y we have s h o w n t h a t the f l u o r e s c e n t

15 °

V o l u m e 85, n u m b e r I

January 1978

FEBS LETTERS

Fig.2. Fluorescence visualization o f ~-receptors o n L6P cells and on HeLa cel~s. (a) L6P cells in the presence o f 1.0 X 10 -s M 9-AAP. (b) L6P cells in the presence o f 1.0 × I0 -s M 9-AAP and 3.0 × [0 -6 M L-propranoloL (e) HeLa cells in the presence o f 1.0 X I0 -s M 9-AAP. The bar in a - c represents 40 #m. analogue of propranolo!, 9-AAP, which binds stereos p e c i f i c a l l y to, t h e 1 3 - a d r e n o r e c e p t o r s , a l l o w s t h e

tors per/lrn 2 are treated with 9-AAP, no fluorescence cap. b e d e t e c t e d , T h u s , f o r example, turkey erythro-

~sualization of the/3-adrenergic receptors, provided t h a t t h e d e n s i t y o f t h e r e c e p t o r s is h i g h e n o u g h , When ceils or membranes possessing only a few recep-

cyte membranes, native turkey erythrocytes, or $49 lyrnphoma cells do not reveal specific fluorescence sta'_'ning u s i n g 9 - A A P ( s e e a l s o t a b l e 1). Skel_etal m y o -

Table 1 T h e density of/3-adrenergic receptors in a n u m b e r o f cell types Cell type

Receptors/cell

Averago density (recep t o t s / g i n 2)

Turkey e r y t h r o c y t e $49 l y m p h o m a L6P (skeletal muscle) HeLa

a F r o m [1,12] b F r o m [8.1 c This study and [12] d T h i s study 160

1100 a 200-300 b 80 000 37 000 d

8