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In vivo autoradiographic demonstration of β-adrenergic binding sites in adult rat type II alveolar epithelial cells
Life Sciences, Vol. 34, pp. 519-527 Printed in the U.S.A.
Pergamon Press
IN VIVO AUTORADIOGRAPHIC DEMONSTRATION OF ~-ADRENERGIC BINDING SITES IN ADULT RAT TYPE I I ALVEOLAR EPITHELIAL CELLS D e n n i s M. S m i t h and N a v j e e t K. S i d h u Department of B i o l o g i c a l S c i e n c e s , W e l l e s l e y C o l l e g e , W e l l e s l e y , MA 02181
(Received in final form November 17, 1984) Summary Adult male rats were injected intravenously with the muscarinic binding probe 3H-Quinuclidinyl benzilate (O~) or the ~ - a d r e n e r g i c p r o b e 3 H - d i h y d r o a l p r e n o l o l (DHA). Other rats were pre-treated w i t h an i n t r a p e r i t o n e a l i n j e c t i o n of a 5 0 0 - f o l d e x c e s s of L-isoproterenol p r i o r t o t h e DHA. L i g h t m i c r o s c o p i c a u t o r a d i o g r a p h y o f 0 . 5 ~ s e c t i o n s o f l u n g f r o m t h e QNB g r o u p demonstrated very little labelling even after 6 months of exposure. In contrast, trachealis smooth m u s c l e from t h e s e a n i m a l s c o n t a i n e d substantial labelling. A u t o r a d i o g r a p h s o f l u n g from r a t s i n j e c t e d w i t h DHA d e m o n s t r a t e d l a b e l l i n g w h i c h was w e l l l o c a l i z e d o v e r a l v e o l a r s e p t a and c o n c e n t r a t e d o v e r t h e c y t o p l a s m o f t y p e I I cells. O u a n t i t a t i v e a n a l y s i s o f l a b e l l i n g i n t h e DHA g r o u p s indicated a significant reduction of labelling in animals treated with L-isoproterenol p r i o r t o DBA, i n b o t h t h e a l v e o l a r p a r e n c h y m a i n g e n e r a l and o v e r t y p e I I c e l l s . The r e s u l t s o f t h i s s t u d y p r o v i d e m o r p h o l o g i c e v i d e n c e f o r t h e u p t a k e and s p e c i f i c b i n d i n g o f ~ - a d r e n e r g i c a n t a g o n i s t s by t h e a d u l t l u n g i n r i v e , w h i l e f a i l i n g to demonstrate similar binding of a muscarinic probe. In addition, t h e r e s u l t s d e m o n s t r a t e s p e c i f i c ~ - a d r e n e r g i c r e c e p t o r s on t y p e I I c e l l s i n v i v o and s u b s t a n t i a t e the view of a direct effect of ~ - a d r e n e r g i c a g o n i s t s on a l v e o l a r t y p e I I c e l l s .
T h e r e i s e v i d e n c e t h a t p u l m o n a r y s u r f a c t a n t s y n t h e s i s and s e c r e t i o n by t h e type II alveolar epithelial c e l l s o f t h e mammalian I n n s may be a t l e a s t partially c o n t r o l l e d by t h e a u t o n o m i c n e r v o u s s y s t e m , a l t h o u g h t h e d i v i s i o n having a direct effect remains problematical. A number o f a u t h o r s ( 1 - 4 ) h a v e d e m o n s t r a t e d t h e s t i m u l a t o r y e f f e c t o f p i l o c a r p i n e and o t h e r m u s c a r i n i c a g e n t s on t h e s u r f a c t a n t s y s t e m . However, b a s e d on s t u d i e s u t i l i z i n g appropriate b l o c k i n g a g e n t s , C o r b e t and c o - w o r k e r s c o n c l u d e d t h a t t h e a c t i o n o f p i l o c a r p i n e was m e d i a t e d t h r o u g h ~ - a d r e n e r g i c r e c e p t o r s , w h i c h t h e y t h e o r i z e d w o u l d be l o c a t e d on t h e s u r f a c e o f t y p e I I c e l l s ( 5 , 6 ) . ~-adrenergic agonists have also been demonstrated to have a stimulatory e f f e c t on s u r f a c t a n t secretion in fetal lung type II cells, both in rive (7,8) and i n v i t r o ( 9 ) . I n a d d i t i o n , M a r i n e and R o o n e y , u t i l i z i n g t h e s l i c e model (10), have described a stimulatory effect in newbor~ rat lung. Although there is less information regarding their effect in the adult lung, Mettler et el. (11) h a v e r e c e n t l y d e m o n s t r a t e d a a t i m u l a t o r y e f f e c t on c u l t u r e d a d u l t t y p e I I
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
520
~-adrenergic Binding to Type II Cells
cells. In addition, Brown and L o n g m o r e (12) secretion following ~-adrenergic stimulation
Vol. 34, No. 6, 1984
reported increased surfactant of the i s o l a t e d - p e r f u s e d rat
lung.
The t h e o r y t h a t ~ - a d r e n e r g i c agonists elicit t h e i r e f f e c t on t y p e I I c e l l s directly, t h r o u g h r e c e p t o r s on t h e c e l l s t h e m s e l v e s , h a s b e e n s u b s t a n t i a t e d by the demonstration of ~-adrcnergic receptors i n i s o l a t e d membrane p r e p a r a t i o n s f r o m f e t a l and a d u l t w h o l e l u n g s ( 1 3 , 1 4 ) . R e c e n t l y S m i t h and G i a n n o p o u l o s (15) have similarly demonstrated such receptors in enriched type II cell populations from f e t a l , n e o n a t a l and a d u l t l u n g s . The a d d i t i o n a l theory that cholinergic a g o n i s t s a l s o m e d i a t e t h e i r e f f e c t on s u r f a c t a n t s e c r e t i o n and s y n t h e s i s through ~-adrenergic r e c e p t o r s r e m a i n s t o be p r o v e n , a l t h o u g h t h i s t h e o r y was g i v e n s u p p o r t by t h e r e c e n t e v i d e n c e o f M a s s a r o e t a l . ( 1 6 ) . R e c e n t l y , B a r n e s et a l . (17) have u t i l i z e d light microscopic autoradiography of frozen lung sections reacted with ~-adrenergic binding p r o b e s to d e m o n s t r a t e s p e c i f i c ~ - r e c e p t o r s in peripheral lung. However, w h i l e they observed a great deal of binding in the alveolar walls, the resolution of t h e t e c h n i q u e ( d u e t o t h e t h i c k n e s s o f t h e f r o z e n s e c t i o n s and t h e e m u l s i o n layer) did not permit the specific observation of type II cells. In addition, t h e b i n d i n g was c a r r i e d o u t i n v i t r o w h i c h , w h i l e b e i n g t h e m o s t commonly u s e d method of demonstrating ~-receptors, does not take into account the interplay of circumstances in the in vivo milieu. The p u r p o s e of t h e p r e s e n t s t u d y i s t o m o r p h o l o g i c a l l y demonstrate uptake and b i n d i n g of l a b e l l e d o h o l i n e r g i c or ~-adrenergic antagonists in the adult lung and, specifically, in alveolar type II cells in vivo. An a d d i t i o n a l p u r p o s e of t h i s s t u d y i s t o d e m o n s t r a t e s p e c i f i c r e c e p t o r s on t h e t y p e I I c e l l s by u t i l i z i n g a large excess of unlabelled agonist.
Methods Adult male Sprague-Dawley rats (250-3008, Charles River Farms, Wilmington M a s s ) w e r e m a i n t a i n e d on a d i e t o f s t a n d a r d l a b chow and w a t e r ad l i b i t u m . In a l l e x p e r i m e n t s t h e l a b e l l e d b i n d i n g p r o b e was i n j e c t e d , under light chloroform anesthesia, into the left jugular vein. The v a r i o u s e x p e r i m e n t a l p r o t o c o l s utilized in this study are summarized in Table I. 3H-quinuclidinyl benzilate (3H--OJ~B; New E n g l a n d N u c l e a r , s p e c i f i c activity 33 C i / m m o l ) o r 3 H - d i h y d r o alprenolol hydrochloride (3H-DHA; New E n g l a n d N u c l e a r , s p e c i f i c a c t i v i t y 41 Ci/mmol) were u t i l i z e d as the binding probes. The a l c o h o l v e h i c l e , as s u p p l i e d , was e v a p o r a t e d o f f o v e r n i t r o g e n a n d r e p l a c e d w i t h an e q u a l v o l u m e o f isotonic saline immediately prior to use. I n some e x p e r i m e n t s u t i l i z i n g JH-DHA, t h e a n i m a l s r e c e i v e d a n a d d i t i o n a l injection of a 500-fold excess of L-isoproterenol hydrochloride ( L - i s o ; Sigma) i n t r a - p e r i t o n e a l l y , prior to the 3H-DHA ( u p t a k e i n h i b i t i o n controls). Either immediately after the injection of t h e l a b e l or i 0 m i n u t e s p o s t injection ( T a b l e I) t h e a n i m a l s were k i l l e d and l u n g t i s s u e o b t a i n e d from t h e u p p e r , l o w e r and h i l a r r e g i o n s o f t h e r i g h t l u n g . In addition, in the experiments utilizing ~H-C~B, t i s s u e was o b t a i n e d f r o m t h e t r a c h e a l i s smooth muscle. The t i s s u e s w e r e c h o p p e d i n t o a p p r o x i m a t e l y 1 mm c u b e s a n d f i x e d i n c o l d 2% p h o s p h a t e - b u f f e r e d glutaraldehyde. The t i s s u e s w e r e t h e n w a s h e d i n buffer, post-fixed i n 1% osmium t e t r o x i d e for 1.5 hours, dehydrated through a g r a d e d e t h a n o l s e r i e s and p r o p y l e n e o x i d e , i n f i l t r a t e d and embedded i n e p o n ( P o l y b e d 812, P o l y s c i e n o e s I n c . ) . In order to shorten exposure times, scintillation autoradiographic techniques were utilized. E a s e d on t h e r e s u l t s o f K o p r i w a ( 1 8 ) , t h e p r o p y l e n e o x i d e u s e d f o r d e h y d r a t i o n and i n £ i l t r a t i o n c o n t a i n e d 10% PPO a n d 0.05% d i m e t h y l POPOP. The e p o n u t i l i z e d for infiltration
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B-adrenergic Binding to Type II Cells
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and embedding contained 3% PPO. Following polymerization, sections were cut at 0.5tim, mounted on clean glass slides and coated with Kodak NTB-3 nuclear track emulsion diluted 1:2 with distilled water. Following exposure at 4 ° C for varying lengths of time (Table I), the autoradiographs were developed in Kodak Dektol developer, fixed, stained with toluidine blue and examined by bright field light microscopy, where the amount and distribution of labelling were noted. TABLE I Summary of Experimental
Group #
Injections
Protocols
Time Protocol
Exposure
1.
130 pCi 3H-O~B
Animals killed at 10 minutes
6 months
2.
150 ~Ci 3H-DHA
Animals killed at 10 minutes
g weeks
DHA i n j e c t e d immediately L-iso, animals killed at
8 weeks
3.
4. 5.
500-fold excess of L-iso 1 5 0 ~ C i 3H-DHA
200 pCi
5H-DHA
500-fold excess of L-iso 200 ~Ci JH-DHA
Animals
killed
immediately
after 10 m i n .
6 weeks
DHA injected I0 minutes after 6 weeks L-iso, animals killed immediately
In the experiments utilizing 3H-DHA, t h e l a b e l l i n g was quantified in a blind study which determined the grain density per unit area of alveolar parenchyma. In order to accomplish this a 100 mesh reticle was inserted into the ocular of the microscope and the autoradiographs were studied at 1250x magnification. The n u m b e r o f a u t o r a d i o g r a p h i c grains observed over tissue in each of i0 randomly selected fields from each lung region of each animal was then determined, a field being defined by the limits of the reticle. In order to correct for the numerous and variable spaces in the lungs, a morphometric determination of the parenchymal density was performed for each field studied. Using the intersections of the reticle lines as test points, the number of points falling over pareuchymal tissue (not alveolar spaces or capillary lumina) was determined. Utilizing the equations: (I); tissue points / total points = tissue density (2); grains counted / tissue density = grain density per unit area of hypothetical 100% a l v e o l a r parenchyma, the data were standardized. Grain density analyses over individual type II cells were also performed. In these studies 10 t y p e I I c e l l s from each of the upper, lower and hilar regions of each lung were randomly selected and the number of autoradiographic grains occuring over the nucleus and cytoplasm of each cell was determined. All data were analyzed using Student's t-test. Results
The light microscopic autoradiographs utilized in this study had very little background and the labelling was distinct. The use of 0.5 tim sections
522
B-adrenergic
and a v e r y t h i n autoradiographic
Binding to Type II Cells
Vol.
emulsion layer resulted in excellent microscopic resolution o f a p p r o x i m t e l y 0 . 5 ~n ( 1 9 ) .
34, No. 6, 1984
resolution
and
Light microscopic observation of autoradiographs of lung from animals i n j e c t e d w i t h ~H-QNB d e m o n s t r a t e d v e r y l i t t l e autoradiographic labelling, despite the 6 month exposure time (fig. 1). In contrast, autoradiographs of tracheal smooth muscle from these animals demonstrated substantial labelling w h i c h was w e l l - l o c a l i z e d over cellular elements (fig. 2). A u t o r a d i o g r a p h s o f l u n g f r o m a n i m a l s i n j e c t e d w i t h 3H-DHA and k i l l e d i 0 minutes post-injection revealed substantial labelling after only eight weeks. D i f f u s e l a b e l l i n g was o b s e r v e d o v e r t r a c h e o - b r o n c h i a l epithelial cells, but very little was o b s e r v e d o v e r t h e a s s o c i a t e d smooth muscle. The l a b e l l i n g was well localized over alveolar parenchymal tissues. In addition, t h e g r a i n s were concentrated over the alveolar type II cells, particularly in the area of cytoplasm, with only occasional grains falling over the nucleus (fig. 3). The uptake inhibition controls corresponding to these animals appeared to contain labelling which followed a similar pattern, b u t a t a l o w e r o v e r a l l l e v e l ( f i g . 4) A u t o r a d i o g r a p h s o f l u n g f r o m a n i m a l s t r e a t e d w i t h SH-DHA and k i l l e d i m m e d i a t e l y were d e v e l o p e d a t 6 weeks and d e m o n s t r a t e d a s u b s t a n t i a l amount of labelling ( f i g . 5) w h i c h a g a i n f o l l o w e d t h e p a t t e r n o f c o n c e n t r a t i o n over type II cell cytoplasm. In addition, t h e r e a p p e a r e d t o be l a b e l l i n g over blood s p a c e s and r e d b l o o d c e l l s . The u p t a k e i n h i b i t i o n controls corresponding to these demonstrated substantially less labelling. I n many c a s e s t h e r e was v e r y little labelling present (fig. 6).
o
Fig.
1
Fig. 2
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B-adrenergic Binding to Type II Cells
523
Fig. 4
Fig. 3
1
tl
Fig. 5
Fig. 6
524
B-adrenergic Binding to Type II Cells
Vol.
34, No. 6, 1984
The i m p r e s s i o n s g a i n e d by r o u t i n e o b s e r v a t i o n s w e r e s u b s t a n t i a t e d and quantified by t h e g r a i n d e n s i t y a n a l y s i s o f r a n d o m f i e l d s in each g r o u p , as summarized in Table II. T h e s e a n a l y s e s r e v e a l e d no u n i f o r m d i f f e r e n c e s between l u n g s a m p l e s f r o m t h e t h r e e r e g i o n s o f t h e l u n g , and t h e y h a v e b e e n p o o l e d . The h e a v i e s t l a b e l l i n g w a s f o u n d i n t h e a u t o r a d i o g r a p h s of lung from animals t r e a t e d w i t h 3H-DHA a n d k i l l e d i m m e d i a t e l y . T h e r e was s i g n i f i c a n t l y less labelling present in the uptake inhibition controls corresponding to these. Less labelling was p r e s e n t i n t h e a u t o r a d i o g r a p h s of lung from animals treated w i t h 3H-DHA a n d k i l l e d a t 10 m i n u t e s p o s t - i n j e c t i o n d e s p i t e a n 8 week e x p o s u r e period. The u p t a k e i n h i b i t i o n controls corresponding to these again contained significantly less labelling. TABLE I I
Results of Grain Density Analysis of Lung Fields
Group #
Summary of Protocol
Parenchymal
Grains
2,
DHA only, killed at 10 minutes
519.40 ~ 20.11
3.
DHA plus L-iso, killed at 10 min.
341.28 ~ 8.50
4.
DHA only, killed immediately
5.
DHA plus L-iso, killed immediately
1449.60 ± 92.08 p<.001 425.93 ± 58.47
p<.O01
Variation
= SEM.
The results of the analysis of cytoplasmic labelling of individual type II cells is presented in Table III. The nuclei of these cells in all groups contained only occasional grains and no significant differences existed between any of the groups. As demonstrated by Table III, there was significantly less labelling in the uptake inhibition control groups when compared with the groups injected with SH-DHA only. TABLE I I I Labelling
Group #
o f Type I I
Cells
W i t h aH-DHA
Summary of Protocol
Cytoplasmic
2.
DHA
3.
DHA plus L-Iso, killed at 10 minutes
11.35 ~ 0.71 p<.001 8.05 ~ 0.5
4.
DHA only, killed immediately
5.
DHA p l u s
Variation = SEM
only, killed at 10 minutes
L-Iso,
killed
immediately
Grains
14.47 Z 1.~8 p<.01 9.57 ~ 1.06
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Discussion The r e s u l t s o f t h e p r e s e n t s t u d y p r o v i d e a u t o r a d i o g r a p h i c e v i d e n c e f o r t h e u p t a k e and b i n d i n g o f t h e ~ - a d r e n e r g i c a n t a g o n i s t S H - d i h y d r o a l p r e n o l o l by t h e adult lung, In addition, they provide a quantitative, morphologic in vivo d e m o n s t r a t i o n o f s p e c i f i c ~ - a d r e n e r g i c r e c e p t o r s i t e s on a d u l t r a t l u n g t y p e I I alveolar epithelial cells. A u t o r a d i o g r a p h s of lung from animals i n j e c t e d w i t h the m u s c a r i n i c b i n d i n g probe 3 H - Q u i n u e l i d i n y l b e n z i l a t e showed v e r y l i t t l e labelling despite a 6 month e x p o s u r e tim e. However, t r a c h e a l i s smooth m u s c l e from t h e s e a n i m a l s d i d show l a b e l l i n g w h i c h was w e l l l o c a l i z e d o v e r smooth m u s c l e c e l l s . This substantiates m u s c a r i n i c b i n d i n g t o t r a c h e a l smooth m u s c l e ( 2 0 ) , w h i l e f a i l i n g to demonstrate similar binding to type II cells. The d e m o n s t r a t i o n o f QNB b i n d i n g t o t r a c h e a l smooth m u s c l e a g r e e s w e l l w i t h t h e f i n d i n g s o f B a r n e s e t a l . (21) who u t i l i z e d light microscopic autoradiography of frozen sectioned tissue. The r e s u l t s o f t h e p r e s e n t s t u d y a l s o c o r r o b o r a t e t h o s e o f M a r q u a r d t e t a l . (22) who f o u n d 14x more ~ - a d r e n e r g i c r e c e p t o r s t h a n m u s c a r i n i c r e c e p t o r s i n a w h o l e l u n g membrane p r e p a r a t i o n . I t i s a p p r o p r i a t e t o p o i n t o u t t h a t t h e r e was a s u b s t a n t i a l difference b e t w e e n t h e r e s u l t s o f t h e two g r o u p s i n j e c t e d w i t h aH-DHA. The a u t o r a d i o g r a p h s of lung from animals k i l l e d i m m e d i a t e l y a f t e r i n j e c t i o n of the l a b e l c o n t a i n e d more l a b e l l i n g t h a n t h o s e f r o m a n i m a l s k i l l e d 10 m i n u t e s a f t e r i n j e c t i o n o f t h e l a b e l . The q u a n t i t a t i v e r e s u l t s o f t h i s s t u d y would s u g g e s t a more s t a b l e l e v e l o f b i n d i n g a t 10 m i n u t e s p o s t - i n j e c t i o n , since the standard e r r o r s o f t h e d a t a a r e q u i t e low a t t h i s p o i n t . 3H-DHA h a s b e e n shown t o be t a k e n up r a p i d l y by t h e i s o l a t e d - p e r f u s e d l u n g , w i t h 84~ b e i n g c l e a r e d i n t h e f i r s t p a s s , and t h e r e m a i n d e r b e i n g v i r t u a l l y c l e a r e d by 10 m i n u t e s ( 2 3 ) . H o w e v e r , d i f f e r e n c e s b e t w e e n t h i s model and t h e i n r i v e s y s t e m a r e s u b s t a n t i a l and t h e c o m p l e x i n t e r a c t i o n o f f a c t o r s i n v i v o make c o m p a r i s o n s d i f f i c u l t , For e x a m p l e , i t i s known t h a t serum p r o t e i n s b i n d ~ - a d r e n e r g i c a g o n i s t s i n r i v e ( r e v i e w e d i n 24 ) and r e d b l o o d c e l l s p o s s e s s ~ - a d r e n e r g i c r e c e p t o r s ( 2 5 ) . The p o s s i b i l i t y also exists that the autoradiographic technique itself resulted in the diffusion of the labels in the present study. However, t h e autoradiographic grains appeared well-localized o v e r t h e a l v e o l a r s e p t a and, particularly, the type II cells. S i m i l a r p r o c e s s i n g t e c h n i q u e s as t h o s e utilized in the p r e s e n t s t u d y have been used f o r a u t o r a d i o g r a p h i c l o c a l i z a t i o n o f S H - n o r e p i n e p h r i n e (26) w i t h o u t a p p r e c i a b l e d i f f u s i o n o f t h e l a b e l . In addition, scintillation counting of the discarded processing fluids in the present study revealed negligible loss of radioactivity. The h i s t o l o g i c d e m o n s t r a t i o n o f ~ - a d r e n e r g i c b i n d i n g s i t e s on t y p e I I cells, in the present study, agrees well with previous studies utilizing i s o l a t e d membrane p r e p a r a t i o n s o f w h o l e l u n g ( 1 3 , 1 4 ) o r e n r i c h e d t y p e I I c e l l populations (15). The l o c a l i z a t i o n of the bindin 8 to the cytoplasmic compartment of the cell also argues for the applicability of the autoradiographic technique. In contrast to the localization o f t h y r o i d hormone b i n d i n g s i t e s t o t h e n u c l e i , a s w e l l as t h e c y t o p l a s m , o f t y p e I I c e l l s ( 2 7 ) , no b i n d i n g t o n u c l e i was o b s e r v e d i n t h e p r e s e n t s t u d y . It might have been e x p e c t e d , however, t h a t s p e c i f i c b i n d i n g s i t e s f o r ~ - a d r e n e r g i c r e c e p t o r s would be c o n c e n t r a t e d a t t h e c e l l membrane. The d e m o n s t r a t i o n o f l a b e l l i n g w i t h i n t h e c y t o p l a s m i c c o m p a r t m e n t , i n t h e p r e s e n t s t u d y , may be due t o t h e r e l a t i v e l y poor resolution of light microscopic autoradiography (0.59). However, t h e rapid internalization of ~-receptors following isoproterenol binding has recently been demonstrated (28). I n a d d i t i o n , iH-DHA i s c a p a b l e o f d i f f u s i n g i n t o i n t a c t c e l l s and b i n d i n g t o t h e s e r e c e p t o r s , w h i l e i s o p r o t e r e n o l is not (reviewed in 28). T h e s e f i n d i n g s may, i n p a r t , e x p l a i n t h e b i n d i n g p a t t e r n s
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observed in the present study. Electron microscopic autoradiography is currently being performed in order to further morphologically localize the observed binding. Many s u b s t a n c e s h a v e b e e n d e m o n s t r a t e d t o h a v e an e f f e c t on a l v e o l a r t y p e II cells, particularly in the f e t a l lung where the m a j o r i t y of such s t u d i e s have been performed (for review see 29). I t w o u l d a p p e a r t h a t a t l e a s t some o f these substances elicit t h e i r e f f e c t t h r o u g h an i n d i r e c t m e c h a n i s m . The failure, i n t h e p r e s e n t s t u d y , t o d e m o n s t r a t e m u s c a r i n i c b i n d i n g s i t e s on t y p e I I c e l l s p r o v i d e s f u r t h e r e v i d e n c e of the p o s s i b l e i n d i r e c t mechanism of a c t i o n theorized for muscarinic agonists (5,6,16). The d e m o n s t r a t i o n o f s p e c i f i c b i n d i n g s i t e s f o r a s u b s t a n c e , on t h e o t h e r h a n d , would s u g g e s t a d i r e c t e f f e c t on t y p e I I c e l l s . The r e c e n t r e p o r t d e m o n s t r a t i n g t h y r o i d hormone b i n d i n g s i t e s on t y p e I I c e i l n u c l e i and c y t o p l a s m i s an e x a m p l e ( 2 7 ) . The p r e s e n t s t u d y p r o v i d e s i__~ny i v o m o r p h o l o g i c e v i d e n c e o f t h e p r e s e n c e o f s p e c i f i c ~ - a d r e n e r g l c b i n d i n g s i t e s on t y p e I I c e l l s , thus further substantiating the d i r e c t e f f e c t of ~ - a d r e n e r g i c a g o n i s t s . The e x i s t e n c e o f s p e c i f i c b i n d i n g s i t e s f o r b o t h t h y r o i d h o r m o n e s and ~ - a d r e n e r g i c a g o n i s t s on t y p e I I c e l l s i s n o t u n u s u a l f o r a s e c r e t o r y c e l l , and i s o f p a r t i c u l a r interest in l i g h t of the known i n t e r a c t i o n b e t w e e n t h e s e two c l a s s e s o f compounds ( 3 0 ) . AcknowledKements This study was supported by grant #1359 from the Council R e s e a r c h , USA I n c .
for Tobacco
References i. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
M°M. ABDELLATIF and M, BOLLINGSWORTB, Brit. J. Pharmacol. 6 1 5 0 2 p (1977). A J . CORBET, P. FLAX and A. RUDOLPH, J. Appl. Physiol. 4_!17-14 (1976). D.M. SMITH, S.A. SHELLEY and J.U. BALIS, Amer. Y. Anat 1 5 4 1 6 3 - 1 7 8 (1979). D.M. SMITH, S.A. SHELLAY and J.U. BALIS, Anat. Rec. 2 0 2 2 3 - 3 1 (1982). A.J. CORBET, P. FLAX and A. RUDOLPH, 7. Appl. Physiol. 4 3 1 0 3 9 - 1 0 4 5 (1977). A.J. CORBET, P. FLAX and A. RUDOLPH, Aust. Ped. J. 1 5 2 3 8 - 2 4 1 (1979). L. Eg~I.AND, R. BURGOYNE, D. BRYMER and G. ENHORNING, Scand. 7. Clin. Lab. Invest, 4_11237-245 (1981). I. WSZOGRODSKI, H,W. TAEUSCH and M.E. AV]~Y, Am. J. Obstet. Gynecol. 1 1 9 1 1 0 7 (1974). L. DOBBS and R. MASON, J. Clin. Invest. 6_33378-387 (1979). P.A. MARINO and S.A. ROONEY, Biochim. Biophys. Acta 66_,_44389-396 (1981). N.R. METTLER, M.E. GRAY, S. SCHUFFMAN and V.S. LF~UIRE, Lab. Invest. 45 575-586 (1981). L.A. BROWN and W . J . LONGMORE, J . B i o l , Chem. 256 6 6 - 7 2 ( 1 9 8 1 ) . G. GIANNOPOULOS, B i o c h e m . B i o p h y s . R e s . Comm. 95 388-395 ( 1 9 8 0 ) . J . A . Y~HITSBTT, M.A. blANTON, C. DOROVEC-BECKblAN and K. ADAMS, L i f e S c i . 2_88 339-345 ( 1 9 8 1 ) . S.K.S. SMITH and G. GIANNOPOULOS, Life Sci. (in press). D. MASSARO, L. CLERCH and G.D. MASSARO, Am. J. Physiol. 243 C39-C45 (1982). P. BARNES C.B. BASBAUM, J.A. NADEL and J.M. ROBERTS, Nature 2 9 9 4 4 4 - 4 4 7 (1982). B.M. KOPRIWA, H i s t o c h e m . 6_88265-279 ( 1 9 8 0 ) . A.W. ROGERS, T e c h n i o u e s o_ff A u t o r a d i o g r a v h y , p. 49, E l s e v i e r , New York (1967). J . B . RICHARDSON and C.C. FERGUSON, F e d . P x o c . 3_88202-208 ( 1 9 7 9 ) . P . J . BARNES, J . A . NADEL, I . M . ROBERTS and C.B, BASBAUM, E u r . $. P h a r m a c o l 866103-106. D.L. MARQUARDT, H . J . MOTULSk'Y and S . I . WASSEJtMAN, J . A p p l . P h y s i o l . 5/3731-736 (1982). J . J . BLANCK and C.H. GILLIS, B i o c h e m . P h a r m a c o l . 2_881903-1909 ( 1 9 7 9 ) .
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24. 25, 26. 27. 28. 29. 30.
B-adrenergic Binding to Type II Cells
M. LEbiAIRE and J . P . TILLEMENT, B i o c h e m . P h a r m a c o l . 3_! 3 5 9 - 3 6 5 ( 1 9 8 2 ) . G. SAGER, B i o c h e m P h a r m a c o l 3 1 9 9 - 1 0 4 ( 1 9 8 2 ) , L. bL~NBER and M.D. GERSRON, Amer. 2. P h y s i o l , 23_~6 E738-E745 ( 1 9 7 9 ) . D.M. SMITH and K.R. HITEItCOCK, Exp, Lung R e s . 5 1 4 1 - 1 5 3 ( 1 9 8 3 ) . hi. STAEItLIN and P. SIbIONS, EMBO 3 . ! 1 8 7 - 1 9 5 ( 1 9 8 2 ) . B . T . SMITH a n d W.G. BOGUES, Pharm. T h e r . 9 5 1 - 7 4 ( 1 9 8 0 ) . 3 . A . WHITSETT, C. DAROVEC-BECKERbtAN, 2 . POLLINGER and J J . MOORE, P e d . R e s . 166 3 8 1 - 3 8 7 ( 1 9 8 2 ) .
527
Pergamon Press
IN VIVO AUTORADIOGRAPHIC DEMONSTRATION OF ~-ADRENERGIC BINDING SITES IN ADULT RAT TYPE I I ALVEOLAR EPITHELIAL CELLS D e n n i s M. S m i t h and N a v j e e t K. S i d h u Department of B i o l o g i c a l S c i e n c e s , W e l l e s l e y C o l l e g e , W e l l e s l e y , MA 02181
(Received in final form November 17, 1984) Summary Adult male rats were injected intravenously with the muscarinic binding probe 3H-Quinuclidinyl benzilate (O~) or the ~ - a d r e n e r g i c p r o b e 3 H - d i h y d r o a l p r e n o l o l (DHA). Other rats were pre-treated w i t h an i n t r a p e r i t o n e a l i n j e c t i o n of a 5 0 0 - f o l d e x c e s s of L-isoproterenol p r i o r t o t h e DHA. L i g h t m i c r o s c o p i c a u t o r a d i o g r a p h y o f 0 . 5 ~ s e c t i o n s o f l u n g f r o m t h e QNB g r o u p demonstrated very little labelling even after 6 months of exposure. In contrast, trachealis smooth m u s c l e from t h e s e a n i m a l s c o n t a i n e d substantial labelling. A u t o r a d i o g r a p h s o f l u n g from r a t s i n j e c t e d w i t h DHA d e m o n s t r a t e d l a b e l l i n g w h i c h was w e l l l o c a l i z e d o v e r a l v e o l a r s e p t a and c o n c e n t r a t e d o v e r t h e c y t o p l a s m o f t y p e I I cells. O u a n t i t a t i v e a n a l y s i s o f l a b e l l i n g i n t h e DHA g r o u p s indicated a significant reduction of labelling in animals treated with L-isoproterenol p r i o r t o DBA, i n b o t h t h e a l v e o l a r p a r e n c h y m a i n g e n e r a l and o v e r t y p e I I c e l l s . The r e s u l t s o f t h i s s t u d y p r o v i d e m o r p h o l o g i c e v i d e n c e f o r t h e u p t a k e and s p e c i f i c b i n d i n g o f ~ - a d r e n e r g i c a n t a g o n i s t s by t h e a d u l t l u n g i n r i v e , w h i l e f a i l i n g to demonstrate similar binding of a muscarinic probe. In addition, t h e r e s u l t s d e m o n s t r a t e s p e c i f i c ~ - a d r e n e r g i c r e c e p t o r s on t y p e I I c e l l s i n v i v o and s u b s t a n t i a t e the view of a direct effect of ~ - a d r e n e r g i c a g o n i s t s on a l v e o l a r t y p e I I c e l l s .
T h e r e i s e v i d e n c e t h a t p u l m o n a r y s u r f a c t a n t s y n t h e s i s and s e c r e t i o n by t h e type II alveolar epithelial c e l l s o f t h e mammalian I n n s may be a t l e a s t partially c o n t r o l l e d by t h e a u t o n o m i c n e r v o u s s y s t e m , a l t h o u g h t h e d i v i s i o n having a direct effect remains problematical. A number o f a u t h o r s ( 1 - 4 ) h a v e d e m o n s t r a t e d t h e s t i m u l a t o r y e f f e c t o f p i l o c a r p i n e and o t h e r m u s c a r i n i c a g e n t s on t h e s u r f a c t a n t s y s t e m . However, b a s e d on s t u d i e s u t i l i z i n g appropriate b l o c k i n g a g e n t s , C o r b e t and c o - w o r k e r s c o n c l u d e d t h a t t h e a c t i o n o f p i l o c a r p i n e was m e d i a t e d t h r o u g h ~ - a d r e n e r g i c r e c e p t o r s , w h i c h t h e y t h e o r i z e d w o u l d be l o c a t e d on t h e s u r f a c e o f t y p e I I c e l l s ( 5 , 6 ) . ~-adrenergic agonists have also been demonstrated to have a stimulatory e f f e c t on s u r f a c t a n t secretion in fetal lung type II cells, both in rive (7,8) and i n v i t r o ( 9 ) . I n a d d i t i o n , M a r i n e and R o o n e y , u t i l i z i n g t h e s l i c e model (10), have described a stimulatory effect in newbor~ rat lung. Although there is less information regarding their effect in the adult lung, Mettler et el. (11) h a v e r e c e n t l y d e m o n s t r a t e d a a t i m u l a t o r y e f f e c t on c u l t u r e d a d u l t t y p e I I
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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cells. In addition, Brown and L o n g m o r e (12) secretion following ~-adrenergic stimulation
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reported increased surfactant of the i s o l a t e d - p e r f u s e d rat
lung.
The t h e o r y t h a t ~ - a d r e n e r g i c agonists elicit t h e i r e f f e c t on t y p e I I c e l l s directly, t h r o u g h r e c e p t o r s on t h e c e l l s t h e m s e l v e s , h a s b e e n s u b s t a n t i a t e d by the demonstration of ~-adrcnergic receptors i n i s o l a t e d membrane p r e p a r a t i o n s f r o m f e t a l and a d u l t w h o l e l u n g s ( 1 3 , 1 4 ) . R e c e n t l y S m i t h and G i a n n o p o u l o s (15) have similarly demonstrated such receptors in enriched type II cell populations from f e t a l , n e o n a t a l and a d u l t l u n g s . The a d d i t i o n a l theory that cholinergic a g o n i s t s a l s o m e d i a t e t h e i r e f f e c t on s u r f a c t a n t s e c r e t i o n and s y n t h e s i s through ~-adrenergic r e c e p t o r s r e m a i n s t o be p r o v e n , a l t h o u g h t h i s t h e o r y was g i v e n s u p p o r t by t h e r e c e n t e v i d e n c e o f M a s s a r o e t a l . ( 1 6 ) . R e c e n t l y , B a r n e s et a l . (17) have u t i l i z e d light microscopic autoradiography of frozen lung sections reacted with ~-adrenergic binding p r o b e s to d e m o n s t r a t e s p e c i f i c ~ - r e c e p t o r s in peripheral lung. However, w h i l e they observed a great deal of binding in the alveolar walls, the resolution of t h e t e c h n i q u e ( d u e t o t h e t h i c k n e s s o f t h e f r o z e n s e c t i o n s and t h e e m u l s i o n layer) did not permit the specific observation of type II cells. In addition, t h e b i n d i n g was c a r r i e d o u t i n v i t r o w h i c h , w h i l e b e i n g t h e m o s t commonly u s e d method of demonstrating ~-receptors, does not take into account the interplay of circumstances in the in vivo milieu. The p u r p o s e of t h e p r e s e n t s t u d y i s t o m o r p h o l o g i c a l l y demonstrate uptake and b i n d i n g of l a b e l l e d o h o l i n e r g i c or ~-adrenergic antagonists in the adult lung and, specifically, in alveolar type II cells in vivo. An a d d i t i o n a l p u r p o s e of t h i s s t u d y i s t o d e m o n s t r a t e s p e c i f i c r e c e p t o r s on t h e t y p e I I c e l l s by u t i l i z i n g a large excess of unlabelled agonist.
Methods Adult male Sprague-Dawley rats (250-3008, Charles River Farms, Wilmington M a s s ) w e r e m a i n t a i n e d on a d i e t o f s t a n d a r d l a b chow and w a t e r ad l i b i t u m . In a l l e x p e r i m e n t s t h e l a b e l l e d b i n d i n g p r o b e was i n j e c t e d , under light chloroform anesthesia, into the left jugular vein. The v a r i o u s e x p e r i m e n t a l p r o t o c o l s utilized in this study are summarized in Table I. 3H-quinuclidinyl benzilate (3H--OJ~B; New E n g l a n d N u c l e a r , s p e c i f i c activity 33 C i / m m o l ) o r 3 H - d i h y d r o alprenolol hydrochloride (3H-DHA; New E n g l a n d N u c l e a r , s p e c i f i c a c t i v i t y 41 Ci/mmol) were u t i l i z e d as the binding probes. The a l c o h o l v e h i c l e , as s u p p l i e d , was e v a p o r a t e d o f f o v e r n i t r o g e n a n d r e p l a c e d w i t h an e q u a l v o l u m e o f isotonic saline immediately prior to use. I n some e x p e r i m e n t s u t i l i z i n g JH-DHA, t h e a n i m a l s r e c e i v e d a n a d d i t i o n a l injection of a 500-fold excess of L-isoproterenol hydrochloride ( L - i s o ; Sigma) i n t r a - p e r i t o n e a l l y , prior to the 3H-DHA ( u p t a k e i n h i b i t i o n controls). Either immediately after the injection of t h e l a b e l or i 0 m i n u t e s p o s t injection ( T a b l e I) t h e a n i m a l s were k i l l e d and l u n g t i s s u e o b t a i n e d from t h e u p p e r , l o w e r and h i l a r r e g i o n s o f t h e r i g h t l u n g . In addition, in the experiments utilizing ~H-C~B, t i s s u e was o b t a i n e d f r o m t h e t r a c h e a l i s smooth muscle. The t i s s u e s w e r e c h o p p e d i n t o a p p r o x i m a t e l y 1 mm c u b e s a n d f i x e d i n c o l d 2% p h o s p h a t e - b u f f e r e d glutaraldehyde. The t i s s u e s w e r e t h e n w a s h e d i n buffer, post-fixed i n 1% osmium t e t r o x i d e for 1.5 hours, dehydrated through a g r a d e d e t h a n o l s e r i e s and p r o p y l e n e o x i d e , i n f i l t r a t e d and embedded i n e p o n ( P o l y b e d 812, P o l y s c i e n o e s I n c . ) . In order to shorten exposure times, scintillation autoradiographic techniques were utilized. E a s e d on t h e r e s u l t s o f K o p r i w a ( 1 8 ) , t h e p r o p y l e n e o x i d e u s e d f o r d e h y d r a t i o n and i n £ i l t r a t i o n c o n t a i n e d 10% PPO a n d 0.05% d i m e t h y l POPOP. The e p o n u t i l i z e d for infiltration
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and embedding contained 3% PPO. Following polymerization, sections were cut at 0.5tim, mounted on clean glass slides and coated with Kodak NTB-3 nuclear track emulsion diluted 1:2 with distilled water. Following exposure at 4 ° C for varying lengths of time (Table I), the autoradiographs were developed in Kodak Dektol developer, fixed, stained with toluidine blue and examined by bright field light microscopy, where the amount and distribution of labelling were noted. TABLE I Summary of Experimental
Group #
Injections
Protocols
Time Protocol
Exposure
1.
130 pCi 3H-O~B
Animals killed at 10 minutes
6 months
2.
150 ~Ci 3H-DHA
Animals killed at 10 minutes
g weeks
DHA i n j e c t e d immediately L-iso, animals killed at
8 weeks
3.
4. 5.
500-fold excess of L-iso 1 5 0 ~ C i 3H-DHA
200 pCi
5H-DHA
500-fold excess of L-iso 200 ~Ci JH-DHA
Animals
killed
immediately
after 10 m i n .
6 weeks
DHA injected I0 minutes after 6 weeks L-iso, animals killed immediately
In the experiments utilizing 3H-DHA, t h e l a b e l l i n g was quantified in a blind study which determined the grain density per unit area of alveolar parenchyma. In order to accomplish this a 100 mesh reticle was inserted into the ocular of the microscope and the autoradiographs were studied at 1250x magnification. The n u m b e r o f a u t o r a d i o g r a p h i c grains observed over tissue in each of i0 randomly selected fields from each lung region of each animal was then determined, a field being defined by the limits of the reticle. In order to correct for the numerous and variable spaces in the lungs, a morphometric determination of the parenchymal density was performed for each field studied. Using the intersections of the reticle lines as test points, the number of points falling over pareuchymal tissue (not alveolar spaces or capillary lumina) was determined. Utilizing the equations: (I); tissue points / total points = tissue density (2); grains counted / tissue density = grain density per unit area of hypothetical 100% a l v e o l a r parenchyma, the data were standardized. Grain density analyses over individual type II cells were also performed. In these studies 10 t y p e I I c e l l s from each of the upper, lower and hilar regions of each lung were randomly selected and the number of autoradiographic grains occuring over the nucleus and cytoplasm of each cell was determined. All data were analyzed using Student's t-test. Results
The light microscopic autoradiographs utilized in this study had very little background and the labelling was distinct. The use of 0.5 tim sections
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and a v e r y t h i n autoradiographic
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emulsion layer resulted in excellent microscopic resolution o f a p p r o x i m t e l y 0 . 5 ~n ( 1 9 ) .
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resolution
and
Light microscopic observation of autoradiographs of lung from animals i n j e c t e d w i t h ~H-QNB d e m o n s t r a t e d v e r y l i t t l e autoradiographic labelling, despite the 6 month exposure time (fig. 1). In contrast, autoradiographs of tracheal smooth muscle from these animals demonstrated substantial labelling w h i c h was w e l l - l o c a l i z e d over cellular elements (fig. 2). A u t o r a d i o g r a p h s o f l u n g f r o m a n i m a l s i n j e c t e d w i t h 3H-DHA and k i l l e d i 0 minutes post-injection revealed substantial labelling after only eight weeks. D i f f u s e l a b e l l i n g was o b s e r v e d o v e r t r a c h e o - b r o n c h i a l epithelial cells, but very little was o b s e r v e d o v e r t h e a s s o c i a t e d smooth muscle. The l a b e l l i n g was well localized over alveolar parenchymal tissues. In addition, t h e g r a i n s were concentrated over the alveolar type II cells, particularly in the area of cytoplasm, with only occasional grains falling over the nucleus (fig. 3). The uptake inhibition controls corresponding to these animals appeared to contain labelling which followed a similar pattern, b u t a t a l o w e r o v e r a l l l e v e l ( f i g . 4) A u t o r a d i o g r a p h s o f l u n g f r o m a n i m a l s t r e a t e d w i t h SH-DHA and k i l l e d i m m e d i a t e l y were d e v e l o p e d a t 6 weeks and d e m o n s t r a t e d a s u b s t a n t i a l amount of labelling ( f i g . 5) w h i c h a g a i n f o l l o w e d t h e p a t t e r n o f c o n c e n t r a t i o n over type II cell cytoplasm. In addition, t h e r e a p p e a r e d t o be l a b e l l i n g over blood s p a c e s and r e d b l o o d c e l l s . The u p t a k e i n h i b i t i o n controls corresponding to these demonstrated substantially less labelling. I n many c a s e s t h e r e was v e r y little labelling present (fig. 6).
o
Fig.
1
Fig. 2
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B-adrenergic Binding to Type II Cells
523
Fig. 4
Fig. 3
1
tl
Fig. 5
Fig. 6
524
B-adrenergic Binding to Type II Cells
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The i m p r e s s i o n s g a i n e d by r o u t i n e o b s e r v a t i o n s w e r e s u b s t a n t i a t e d and quantified by t h e g r a i n d e n s i t y a n a l y s i s o f r a n d o m f i e l d s in each g r o u p , as summarized in Table II. T h e s e a n a l y s e s r e v e a l e d no u n i f o r m d i f f e r e n c e s between l u n g s a m p l e s f r o m t h e t h r e e r e g i o n s o f t h e l u n g , and t h e y h a v e b e e n p o o l e d . The h e a v i e s t l a b e l l i n g w a s f o u n d i n t h e a u t o r a d i o g r a p h s of lung from animals t r e a t e d w i t h 3H-DHA a n d k i l l e d i m m e d i a t e l y . T h e r e was s i g n i f i c a n t l y less labelling present in the uptake inhibition controls corresponding to these. Less labelling was p r e s e n t i n t h e a u t o r a d i o g r a p h s of lung from animals treated w i t h 3H-DHA a n d k i l l e d a t 10 m i n u t e s p o s t - i n j e c t i o n d e s p i t e a n 8 week e x p o s u r e period. The u p t a k e i n h i b i t i o n controls corresponding to these again contained significantly less labelling. TABLE I I
Results of Grain Density Analysis of Lung Fields
Group #
Summary of Protocol
Parenchymal
Grains
2,
DHA only, killed at 10 minutes
519.40 ~ 20.11
3.
DHA plus L-iso, killed at 10 min.
341.28 ~ 8.50
4.
DHA only, killed immediately
5.
DHA plus L-iso, killed immediately
1449.60 ± 92.08 p<.001 425.93 ± 58.47
p<.O01
Variation
= SEM.
The results of the analysis of cytoplasmic labelling of individual type II cells is presented in Table III. The nuclei of these cells in all groups contained only occasional grains and no significant differences existed between any of the groups. As demonstrated by Table III, there was significantly less labelling in the uptake inhibition control groups when compared with the groups injected with SH-DHA only. TABLE I I I Labelling
Group #
o f Type I I
Cells
W i t h aH-DHA
Summary of Protocol
Cytoplasmic
2.
DHA
3.
DHA plus L-Iso, killed at 10 minutes
11.35 ~ 0.71 p<.001 8.05 ~ 0.5
4.
DHA only, killed immediately
5.
DHA p l u s
Variation = SEM
only, killed at 10 minutes
L-Iso,
killed
immediately
Grains
14.47 Z 1.~8 p<.01 9.57 ~ 1.06
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525
Discussion The r e s u l t s o f t h e p r e s e n t s t u d y p r o v i d e a u t o r a d i o g r a p h i c e v i d e n c e f o r t h e u p t a k e and b i n d i n g o f t h e ~ - a d r e n e r g i c a n t a g o n i s t S H - d i h y d r o a l p r e n o l o l by t h e adult lung, In addition, they provide a quantitative, morphologic in vivo d e m o n s t r a t i o n o f s p e c i f i c ~ - a d r e n e r g i c r e c e p t o r s i t e s on a d u l t r a t l u n g t y p e I I alveolar epithelial cells. A u t o r a d i o g r a p h s of lung from animals i n j e c t e d w i t h the m u s c a r i n i c b i n d i n g probe 3 H - Q u i n u e l i d i n y l b e n z i l a t e showed v e r y l i t t l e labelling despite a 6 month e x p o s u r e tim e. However, t r a c h e a l i s smooth m u s c l e from t h e s e a n i m a l s d i d show l a b e l l i n g w h i c h was w e l l l o c a l i z e d o v e r smooth m u s c l e c e l l s . This substantiates m u s c a r i n i c b i n d i n g t o t r a c h e a l smooth m u s c l e ( 2 0 ) , w h i l e f a i l i n g to demonstrate similar binding to type II cells. The d e m o n s t r a t i o n o f QNB b i n d i n g t o t r a c h e a l smooth m u s c l e a g r e e s w e l l w i t h t h e f i n d i n g s o f B a r n e s e t a l . (21) who u t i l i z e d light microscopic autoradiography of frozen sectioned tissue. The r e s u l t s o f t h e p r e s e n t s t u d y a l s o c o r r o b o r a t e t h o s e o f M a r q u a r d t e t a l . (22) who f o u n d 14x more ~ - a d r e n e r g i c r e c e p t o r s t h a n m u s c a r i n i c r e c e p t o r s i n a w h o l e l u n g membrane p r e p a r a t i o n . I t i s a p p r o p r i a t e t o p o i n t o u t t h a t t h e r e was a s u b s t a n t i a l difference b e t w e e n t h e r e s u l t s o f t h e two g r o u p s i n j e c t e d w i t h aH-DHA. The a u t o r a d i o g r a p h s of lung from animals k i l l e d i m m e d i a t e l y a f t e r i n j e c t i o n of the l a b e l c o n t a i n e d more l a b e l l i n g t h a n t h o s e f r o m a n i m a l s k i l l e d 10 m i n u t e s a f t e r i n j e c t i o n o f t h e l a b e l . The q u a n t i t a t i v e r e s u l t s o f t h i s s t u d y would s u g g e s t a more s t a b l e l e v e l o f b i n d i n g a t 10 m i n u t e s p o s t - i n j e c t i o n , since the standard e r r o r s o f t h e d a t a a r e q u i t e low a t t h i s p o i n t . 3H-DHA h a s b e e n shown t o be t a k e n up r a p i d l y by t h e i s o l a t e d - p e r f u s e d l u n g , w i t h 84~ b e i n g c l e a r e d i n t h e f i r s t p a s s , and t h e r e m a i n d e r b e i n g v i r t u a l l y c l e a r e d by 10 m i n u t e s ( 2 3 ) . H o w e v e r , d i f f e r e n c e s b e t w e e n t h i s model and t h e i n r i v e s y s t e m a r e s u b s t a n t i a l and t h e c o m p l e x i n t e r a c t i o n o f f a c t o r s i n v i v o make c o m p a r i s o n s d i f f i c u l t , For e x a m p l e , i t i s known t h a t serum p r o t e i n s b i n d ~ - a d r e n e r g i c a g o n i s t s i n r i v e ( r e v i e w e d i n 24 ) and r e d b l o o d c e l l s p o s s e s s ~ - a d r e n e r g i c r e c e p t o r s ( 2 5 ) . The p o s s i b i l i t y also exists that the autoradiographic technique itself resulted in the diffusion of the labels in the present study. However, t h e autoradiographic grains appeared well-localized o v e r t h e a l v e o l a r s e p t a and, particularly, the type II cells. S i m i l a r p r o c e s s i n g t e c h n i q u e s as t h o s e utilized in the p r e s e n t s t u d y have been used f o r a u t o r a d i o g r a p h i c l o c a l i z a t i o n o f S H - n o r e p i n e p h r i n e (26) w i t h o u t a p p r e c i a b l e d i f f u s i o n o f t h e l a b e l . In addition, scintillation counting of the discarded processing fluids in the present study revealed negligible loss of radioactivity. The h i s t o l o g i c d e m o n s t r a t i o n o f ~ - a d r e n e r g i c b i n d i n g s i t e s on t y p e I I cells, in the present study, agrees well with previous studies utilizing i s o l a t e d membrane p r e p a r a t i o n s o f w h o l e l u n g ( 1 3 , 1 4 ) o r e n r i c h e d t y p e I I c e l l populations (15). The l o c a l i z a t i o n of the bindin 8 to the cytoplasmic compartment of the cell also argues for the applicability of the autoradiographic technique. In contrast to the localization o f t h y r o i d hormone b i n d i n g s i t e s t o t h e n u c l e i , a s w e l l as t h e c y t o p l a s m , o f t y p e I I c e l l s ( 2 7 ) , no b i n d i n g t o n u c l e i was o b s e r v e d i n t h e p r e s e n t s t u d y . It might have been e x p e c t e d , however, t h a t s p e c i f i c b i n d i n g s i t e s f o r ~ - a d r e n e r g i c r e c e p t o r s would be c o n c e n t r a t e d a t t h e c e l l membrane. The d e m o n s t r a t i o n o f l a b e l l i n g w i t h i n t h e c y t o p l a s m i c c o m p a r t m e n t , i n t h e p r e s e n t s t u d y , may be due t o t h e r e l a t i v e l y poor resolution of light microscopic autoradiography (0.59). However, t h e rapid internalization of ~-receptors following isoproterenol binding has recently been demonstrated (28). I n a d d i t i o n , iH-DHA i s c a p a b l e o f d i f f u s i n g i n t o i n t a c t c e l l s and b i n d i n g t o t h e s e r e c e p t o r s , w h i l e i s o p r o t e r e n o l is not (reviewed in 28). T h e s e f i n d i n g s may, i n p a r t , e x p l a i n t h e b i n d i n g p a t t e r n s
526
B-adrenergic
Binding to Type II Cells
Vol.
34, No. 6, 1984
observed in the present study. Electron microscopic autoradiography is currently being performed in order to further morphologically localize the observed binding. Many s u b s t a n c e s h a v e b e e n d e m o n s t r a t e d t o h a v e an e f f e c t on a l v e o l a r t y p e II cells, particularly in the f e t a l lung where the m a j o r i t y of such s t u d i e s have been performed (for review see 29). I t w o u l d a p p e a r t h a t a t l e a s t some o f these substances elicit t h e i r e f f e c t t h r o u g h an i n d i r e c t m e c h a n i s m . The failure, i n t h e p r e s e n t s t u d y , t o d e m o n s t r a t e m u s c a r i n i c b i n d i n g s i t e s on t y p e I I c e l l s p r o v i d e s f u r t h e r e v i d e n c e of the p o s s i b l e i n d i r e c t mechanism of a c t i o n theorized for muscarinic agonists (5,6,16). The d e m o n s t r a t i o n o f s p e c i f i c b i n d i n g s i t e s f o r a s u b s t a n c e , on t h e o t h e r h a n d , would s u g g e s t a d i r e c t e f f e c t on t y p e I I c e l l s . The r e c e n t r e p o r t d e m o n s t r a t i n g t h y r o i d hormone b i n d i n g s i t e s on t y p e I I c e i l n u c l e i and c y t o p l a s m i s an e x a m p l e ( 2 7 ) . The p r e s e n t s t u d y p r o v i d e s i__~ny i v o m o r p h o l o g i c e v i d e n c e o f t h e p r e s e n c e o f s p e c i f i c ~ - a d r e n e r g l c b i n d i n g s i t e s on t y p e I I c e l l s , thus further substantiating the d i r e c t e f f e c t of ~ - a d r e n e r g i c a g o n i s t s . The e x i s t e n c e o f s p e c i f i c b i n d i n g s i t e s f o r b o t h t h y r o i d h o r m o n e s and ~ - a d r e n e r g i c a g o n i s t s on t y p e I I c e l l s i s n o t u n u s u a l f o r a s e c r e t o r y c e l l , and i s o f p a r t i c u l a r interest in l i g h t of the known i n t e r a c t i o n b e t w e e n t h e s e two c l a s s e s o f compounds ( 3 0 ) . AcknowledKements This study was supported by grant #1359 from the Council R e s e a r c h , USA I n c .
for Tobacco
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