Studies on cultured coronary endothelial cells. - Core

I N T E R N A T I O N A L

J O U R N A L

OF

C L I N I C A L

A N G I O L O G Y

E d i t o r e s : Prof. Dr. M a r c e l l o C o m e l , Milano - Prof, Prof. h. c . Dr. Dr. h. c . N o r b e r t K l ü k e n , E s s e n

Ε D I Τ O R Ε S : Prof. Dr. M a r c e l l o COMEL (Milano-Italia) - Prof. Prof. h. c. Dr. Dr. h. c. Norbert KLÜKEN (Essen-Germania) C O - E D I T O R E S : M. COLLARD ( M o n t i g n i e s - I e - T i l l e u l - B e l g i u m ) U. DEMBOWSKI (Bad Neuheim-Germania) - M. FÖLDI (Salzgitter BadGermania) - R. FONTAINE f (Straßburg-Gallia) - W. H. HAUSS (MünsterGermania) - K. ISCHIKAWA (Tokio-Japan) - M. JACQUET (Paris-Gallia) F. MARTORELL (Barcelona-Hispania) - J . MERLEN (Lille-Gallia) E. MIAN (Pisa-Italia) - F. PRATESI (Firenze-Italia) C O N S U L T O R E S : K. ALEXANDER (Hannover-Germania) - J . L. AMBRUS (Buffalo-USA) - C. C. ARNOLDI (Lund-Schweden) - Μ. E. de BAKEY (Houston-Texas-USA) - P. BALAS (Athenae-Graecia) - G l . BASSI (Triest-Italia) - A. BERNSMEIER (Kiel-Germania) - Ε. BETZ (TübingenGermania) - C. BÖHM (München-Germania) - E. CARIATI (Genua-Italia) J. CASLEY-SMITH (Adelaide-Australia) - V. CEPELAK (Pilsen-Bohemia) E. DEUTSCH (Wien-Austria) - H. FRANKE (Würzburg-Germania) - J . GOMEZ-MARQUEZ (Tegucigalpa-Honduras) - H. GRIMMER (WiesbadenGermania) - K. HAEGER (Malmö-Schweden) - R. HILD (Heidelberg-Ger­ mania) - H.-E. HOFFMEISTER (Tübingen-Germania) - W. HORT ( M a r b u r g Germania) - J . W. IRWIN (Boston-USA) - L. LASZT (Freiburg-Helvetia) Κ. Ε. LOOSE (Hamburg-Germania) - R. C. MAYALL (Rio de J a n e i r o Brasilia) - Y. M I S C H I M A (Tokio-Japan) - P. DE NICOLA (Pavia-Italia) C. OLIVIER (Paris-Gallia) - E. PALMA (Montevideo-Uruguay) - J . PFLUG (Göppingen-Germania) - F. PIRNER (München-Germania) - H. POLIWODA (Hannover-Germania) - Z. REINIS (Prag-Bohemia) - M. A. SANTOSGASTON (Huesca-Hispania) - K. W. SCHNEIDER (Würzburg-Germania) W. SCHNEIDER (Tübingen-Germania) - A. SENN (Bern-Helvetia) - Μ. SPERLING (Würzburg-Germania) - R. STEMMER (Straßburg-Gallia) A. STRANO (Palermo-Italia) - R. TOURNAY (Paris-Gallia) - L. WIDMER (Basel-Helvetia) - Ε. W I T Z L E B (Kiel-Germania)

I

O r i g i n a l Reports Originalien Article originaux

E.

Betz:

Introductory R e m a r k s — V o r w o r t — Avant propos

3

S. Björkerud: M o d u l a t i o n of S m o o t h M u s c l e G r o w t h

5

J. Staubesand, G. Adelmann and F. Steel: Matrix L y s o s o m e s a n d M e d i a l D y s p l a s i a L. Raeymaekers, G. Droogmans and R. Does Na-Ca E x c h a n g e Play a Role in the T e n s i o n D e v e l o p m e n t

9

Casteels: Regulating '

H. Heine and H. Henrich: Reactive B e h a v i o u r of M y o c y t e s

22

E. Betz, J. Roth a n d W. Schlote: Proliferation of S m o o t h M u s c l e Cells R. W. Wissler, Chen and B.

K. Fischer-Dzoga, Eisele:

28

S. R. Bates,

R. M.

The Interaction of the Arterial S m o o t h M u s c l e Cells

32

A. K. Horsch, W. D. Kuhlmann and J . C. Early A t h e r o s c l e r o t i c Lesions in Rats

37

H. Sinzinger, Κ. Silberbauer Prostacyclin F o r m a t i o n E. Buddecke

a n d /.

and W.

Salomon:

Auerswald: 40

Filipovic:

Sialic A c i d C o n t e n t of Low Density L i p o p r o t e i n . .

44

H. Heinle and F. Stowasser: The Glycolytic Pathway

47

M. A. Reidy: B l o o d and Arterial Endothelial Cells G. Schmid,

W. Eitel,

Ε. Betz and W.

50 Schlote:

The D e v e l o p m e n t of Early A r t e r i o s c l e r o t i c Changes

54

P. Clopath, J. Suter a n d W. Stäubli: M o r p h o l o g i c a l a n d B i o c h e m i c a l Studies

58

S. Nees, and E.

B. Willershausen-Zönnchen, Gerlach:

A. L.

Gerbes

Studies on C u l t u r e d C o r o n a r y Endothelial Celis . . K.-U.

Herausgeber: Prof. Dr. med. M. Comel N. Klüken

Prof. Prof. h. c. Dr. m e d . Dr. h. c.

Verleger: VERLAG A · F · KOSKA Schaperstraße 18 · D-1000 Berlin 15 · T e l . (030) 8 83 90 41

Thiedemann,

Η. Heinle,

A. Show-Klett

64

and

U. Drews: Electrical S t i m u l a t i o n of Vascular S m . M u s c l e Cells

69

H. C. Stary: Changes in Intimal S m o o t h Muscle Phenotype

72

W. Schlote, J. W. Boellaard and E. Experimental A t h e r o s c l e r o s i s

76

Betz:

M. Marshall: Experimental a n d C l i n i c a l Results

80

F. Seuter, R. Sitt a n d W. D. Busse: Experimentally I n d u c e d T h r o m b o a t h e r o s c l e r o s i s . .

85

I 2 yj^

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Vol.

xxvni

1/2/3/80

Studies on Cultured Coronary Endothelial Cells S. NEES, B. W I L L E R S H A U S E N - Z Ö N N C H E N , A. L G E R B E S , E. G E R L A C H

Physiologisches Institut d e r Universität München

ZUSAMMENFASSUNG

RESUME

Untersuchungen an Endothelzellen a u s Koronargefäßen

Recherches sur d e s cellules endothe­ liales d e s vaisseaux coronaires du coeur

Es w u r d e eine M e t h o d e z u r Isolierung und Kultivierung v o n Endothelzellen aus Koronargefäßen v o n Meerschweinchen ausgearbeitet. Die in d e r G e w e b e k u l t u r gezüchteten Zellen zeigten d i e gleichen m o r p h o l o g i s c h e n Eigenschaften w i e E n ­ dothelzellen in situ. Durch quantitative HPLC-Anaiysen w u r d e nachgewiesen, daß Endothelzellen aus konfluenten K u l ­ turen außergewöhnlich hohe Spiegel an ATP, A D P und A M P e n t h i e l t e n ; auch die Konzentrationen v o n A d e n o s i n , Inosin, Hypoxanthin u n d A d e n i n waren relativ groß. Der A d e n i n - N u c l e o t i d - G e h a l t ä n ­ derte sich nur w e n i g in A b h ä n g i g k e i t vom Wachstumszustand d e r Z e l l k u l t u ­ r e n ; auch nach einer Inkubation v o n 3 T a g e n in Purin-freien N ä h r m e d i e n blieben d i e A d e n i n - N u c l e o t i d - S p i e g e l konstant. Es konnte gezeigt w e r d e n , daß K o r o n a r e n d o t h e l z e l l e n sehr e m p ­ findlich auf Sauerstoffmangel mit einem raschen A T P - A b b a u und einer g e s t e i ­ gerten Freisetzung v o n A d e n o s i n in das K u l t u r m e d i u m reagieren. Durch A k t i v i ­ t ä t s m e s s u n g e n verschiedener kataboler und a n a b o l e r Enzyme des A d e n i n - N u cleotid-Stoffwechsels e r g a b e n sich w e i ­ tere Hinweise, daß sich gezüchtete K o ­ ronarendothelzellen durch einen b e s o n ­ ders aktiven Adenin-Nucleotid-Stoffwechsel auszeichnen.

Une m e t h o d e d'isolation et d ' e l e v a g e des cellules endotheliales d ' o r i g i n e c o ronaire d u cobaye a ete d e v e l o p p e e . Toutes les cellules, elevees en c u l t u r e d e tissus, montraient les p r o p r i e t e s m o r p h o l o g i q u e s typiques des c e l l u l e s en situ. L'analyse quantitative H P L C des cultures c o n f l u e n t e s prouvait un c o n t e n u e x t r a o r d i n a i r e m e n t £lev£ d e s n u c l e o t i d e s d ' a d e n i n e et de leurs p r o duits de d e g r a d a t i o n , q u i ne d e p e n d a i t que peu d e l'otat d e croissance et d u c o n t e n u de purines d u milieu a l i m e n taire. Une sensibilite remarquable d e s cellules endotheliales c o r o n a i r e s au m a n q u e d'oxygene, une d e g r a d a t i o n s i multanee rapide d u t r i p h o s p h a t e d'adonosine et une liberation a u g m e n t e e d ' a d o n o s i n e pouvaient etre m o n t r e e s . Le mesurage d e s activites e n z y m a t i ques d e plusieures enzymes c a t a b o liques et anaboliques d u m e t a b o l i s m e des n u c l e o t i d e s d ' a d £ n i n e supporte notre o p i n i o n , q u e les cellules e n d o ­ theliales d ' o r i g i n e c o r o n a i r e , cultivees en culture d e tissus, soient c a r a c t e r i sees par un metabolisme tres actif d e s nucleotides d'adonine.

SUMMARY Studies on Cultured thelial Cells

Coronary

Endo­

A method was developed f o r isolation and cultivation of endothelial cells f r o m coronary vessels of guinea pig hearts. The cultured cells revealed typical mor­ phological criteria of endothelial cells in vivo. Exceptionally high c o n t e n t s of adenine nucleotides (ATP, A D P , A M P ) and of their degradatives (adenosine, inosine, hypoxanthine) w e r e f o u n d in confluent endothelial cell cultures as determined by specially elaborated HPLC techniques. A d e n i n e nucleotide levels were slightly i n f l u e n c e d by the g r o w t h state of the cell cultures and remained unchanged w h e n cells w e r e incubated for 3 days in purine-free m e ­ dium. The cultured coronary endo­ thelial cells proved to be sensitive to lack of oxygen, as e v i d e n c e d by t h e fast b r e a k d o w n of A T P c o n c o m i t a n t with an e n h a n c e d release of adenosine. Measurements of specific activities of enzymes involved in d e g r a d a t i o n a n d synthesis of adenine nucleotides lend additional support to t h e view that an active adenine n u c l e o t i d e metabolism is a characteristic feature of coronary endothelial cells.

larger vessels of v a r i o u s a n i m a l s ( 8 - 9 ) . T o o u r k n o w l ­

Introduction

e d g e suitable m e t h o d s f o r isolation and c u l t i v a t i o n

of

A l t h o u g h it is g e n e r a l l y a c c e p t e d t h a t e n d o t h e l i a l cells

coronary

are involved in several b i o l o g i c a l p r o c e s s e s (1—6) o u r

so far.

k n o w l e d g e a b o u t their i n t e r m e d i a r y m e t o b o l i s m is r a t h ­

Our interest in the c o r o n a r y e n d o t h e l i a l cell type o r i g i ­

er limited. The possibility to c u l t u r e e n d o t h e l i a l

endothelial

cells

have

not

been

elaborated

cells

nated f r o m recent f i n d i n g s of Schräder a n d Gerlach (10),

provides the o p p o r t u n i t y to set up in vitro systems f o r

w h o s h o w e d that in g u i n e a pig hearts at least t w o dif­

s t u d y i n g certain a s p e c t s of their m e t a b o l i c a n d

ferent

func­

adenine

nucleotide

compartments

tional properties. However, as yet e x p e r i e n c e w i t h c u l ­

these c o m p a r t m e n t s can c o n t r i b u t e t o the

tured

of v a s o a c t i v e a d e n o s i n e , w h i c h

endothelial

cells

was

only

obtained

with

cells

isolated f r o m h u m a n u m b i l i c a l vein (7) as w e l l as f r o m

64

exist.

Both

production

is c o n s i d e r e d to

play

an i m p o r t a n t role in t h e m e t a b o l i c regulation of c o r o -

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FOLIA A N G I O L O G I C A / Vol. XXVIII / 1/2/3/80

nary b l o o d flow. Since the c o r o n a r y e n d o t h e l i u m might c o n s t i t u t e o n e of the adenine n u c l e o t i d e c o m p a r t m e n t s it s e e m e d p r o m i s i n g to develop adequate methods for the i s o l a t i o n a n d cultivation of c o r o n a r y endothelial cells a n d to study s o m e pertinent features of their adenine nucleotide metabolism.

Materials and Methods Culture m e d i u m Μ 199, feta! calf serum (mycoplasma tested a n d virus s c r e e n e d ) , streptomycine-penicillines o l u t i o n a n d L-glutamine w e r e purchased from Serom e d , M ü n c h e n . C r u d e collagenase (CI. histolyticum) type II a n d trypsin w e r e obtained f r o m W o r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n , Freehold, N.J., USA. Reagents for f i x a t i o n a n d e m b e d d i n g tissue samples prior to e l e c t r o n m i c r o s c o p y (glutaraldehyde; o s m i u m t e t r o x i d e ; e p o n 812; 2,4,6-tri(dimethylaminomethyl)phenol; d o d e cenyl s u c c i n i c a c i d a n h y d r i d e ; m e t h y l n a d i c anhydride) w e r e o b t a i n e d f r o m Serva, Heidelberg. Nucleotides, n u c l e o s i d e s and bases for calibration w e r e purchased from B o e h r i n g e r M a n n h e i m , all other chemicals of highest available purity f r o m Merck, Darmstadt. Sub­ stituted silica c o l u m n packings ( N u c l e o s i l - N H and Nucleosil-Cia) for HPLC w e r e obtained f r o m Macherey und N a g e l , D ü r e n . 2

Preparation of cells and cell culture: Guinea pig hearts w e r e c a n n u l a t e d t h r o u g h the aorta and the c o r o n a r y system w a s h e d free of blood was filled with an isotonic buffer s o l u t i o n c o n t a i n i n g collagenase and trypsin (0.1 % each). After an exposure of 20 min perfusion was started a g a i n and all endothelial cells which had been detached w e r e c o l l e c t e d f r o m the perfusate by c e n t r i f u g a t i o n . Subsequently, the cells w e r e w a s h e d w i t h c u l ­ ture m e d i u m (M 199, c o n t a i n i n g 60 mg penicilline and 120 mg s t r e p t o m y c i n e per liter, s u p p l e m e n t e d with 2 0 % fetal calf s e r u m [FCS] and 2 m M glutamine and s e e d e d in c u l t u r e dishes. Cultivation was performed at 4 0 ° C in a humidified air a t m o s p h e r e c o n t a i n i n g 3 % C 0 . C o n f l u e n c y of the cell cultures was reached after 2 to 4 weeks. 2

Transmission electron microscopy: Cultured cells w e r e fixed f o r 1 h in 2 . 5 % glutaraldehyde, 0.09 Μ c a c o d y l a t e buffer (pH 7.4), 5 % s u c r o s e , postfixed in 1 % O s 0 for 1 h, stained w i t h a saturated solution of tungsten phos­ phoric a c i d and uranylacetate in ethanol-water 75 : 2 5 (v/v), d e h y d r a t e d with ethanol and e m b e d d e d in epon 812. 4

Scanning electron microscopy: Cells w e r e fixed as d e c r i b e d before. After dehydration in ethanol they w e r e c r i t i c a l - p o i n t dried and s h a d o w e d w i t h g o l d . Analysis of nucleotides, nucleosides and b a s e s : M o n o ­ layers of endothelial cells in culture dishes w e r e extrac­ ted w i t h 0.4 Ν perchloric a c i d . Separation of the dif­ ferent purine c o m p o u n d s in the neutralized cell extracts w a s c a r r i e d out by application of HPLC (Varian 8500) using w e a k anion exchange c o l u m n s for the separation

et a l . : Studies on C u l t u r e d C o r o n a r y Endothelial Cells

of nucleotides and reverse phase c o l u m n s for nucle­ osides and bases. The c o n c e n t r a t i o n s of the individual metabolites in the eluates w e r e continuously r e c o r d e d using a sensitive UV-detection device (Varian UV-Vis detector „ V a r i s c a n " ) . Determination of enzyme activities: Specific activities of enzymes involved in nucleotide metabolism w e r e measured in a 200 χ g m e m b r a n e preparation as well as in a soluble 200 000 g supernatant fraction of e n d o ­ thelial cells. Enzyme assays w e r e performed using s t a n d a r d p r o c e d u r e s . Substrates and p r o d u c t s of the enzyme catalyzed reaction were separated by HPLC.

Results and Discussion Growth behaviour and cytology: As was revealed by s c a n n i n g electron m i c r o s c o p y the endothelial cells w e r e selectively detached f r o m their basement m e m b r a n e s during the exposure of the c o r o n a r i e s to t r y p s i n collagenase solution. T h e c o r o n a r y perfusate s u b ­ sequently c o l l e c t e d c o n t a i n e d small c l u m p s of rounded cells. After seeding in c o m p l e x culture m e d i u m the cells attached to the substratum w i t h i n 24 h. The clusters of cells increased in size and gradually c o a l e s c e d to f o r m monolayers after 2 to 4 w e e k s . Primary cultures c o n ­ sisted of about 9 8 % endothelial cells, c o n t a m i n a t e d with about 2 % fibroblasts and s m o o t h muscle cells. Selected confluent cultures of guinea pig c o r o n a r y endothelial cells c o u l d be serially s u b c u l t u r e d a n d m a i n ­ tained for at least 4 months. Primary and s u b c u l t u r e d cells appeared similar with respect to shape and typical tight packing in a strict monolayer. The generation time of c o r o n a r y endothelial cells in culture during the logarithmic phase of g r o w t h (G-i phase) t u r n e d out to be 18 h, and was thus very similar to that of aortic endothelial cells in vivo after artificial w o u n d i n g of the intima (11). A d d i t i o n of t h r o m b i n in physiological c o n ­ centrations to the culture m e d i u m (1 μg/ml) resulted in a m a r k e d reduction of the generation time to 16 h, and accelerated DNA-synthesis in resting cells ( G phase) by more than 3 0 % . 0

Phase contrast and interference m i c r o s c o p i c pictures of endothelial cultures (Figure 1a and 1b) demonstrate that the individual cells w e r e of uniform a p p e a r a n c e : elongated ( 2 0 - 3 0 μηη w i d e , 5 0 - 8 0 μΐη long), with single ovoid nuclei c o n t a i n i n g 2 to 3 nucleoli and s u r r o u n d e d by a perinuclear granular region and a broad peripheral cytoplasm with distinct borders. The granules consisted mainly of mitochondria, as c o u l d be s h o w n by use of specific staining techniques (Janus g r e e n ) ; they did not contain neutral lipids (no staining with oil red O). Ultrastructural analysis of the cytoplasm of cultured and in vivo coronary endothelial cells by use of elec­ t r o n m i c r o s c o p y revealed a high degree of conformity. As can be seen f r o m the transmission electron m i c r o ­ g r a p h in Figure 1c the cytoplasm of cultured cells c o n ­ tained large numbers of vesicles, clusters of long and unbranched mitochondria, s m o o t h and rough e n d o 65

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et a l . : Studies on C u l t u r e d Coronary Endothelial Cells

Figure 1 : Cultured coronary endothelial c e l l s : a) Phase contrast m i c r o ­ g r a p h ; b) interference m i c r o g r a p h a c c o r d i n g to N G M A R S K I ;

c) t r a n s m i s s i o n micrograph.

electron

m i c r o g r a p h ; d)

scanning

electron

plasmic reticulum, and abundant numbers of fine and coarse filaments. Weibel-Palade bodies, typical markers of endothelial cells f r o m greater blood vessels, w e r e seldomly f o u n d ; therefore, the cells in culture originat­ ed mainly f r o m small vessels.

Some features of adenine nucleotide metabolism in coronary endothelial cells: In Table 1 mean values f r o m three individual series of analyses c o n c e r n i n g contents

In scanning electron m i c r o g r a p h s (Figure 1d) the c u l ­ tured endothelial cells appeared as h o m o g e n o u s flat cells with prominent nuclei and with numerous blebs and pits in or near the surface, especially in the perinclear region. Many bundles of thick filaments c o u l d be detected which c o n n e c t different parts of the cytoplasm with the cell mebrane and which might be involved in the regulation of size and shape of the cells. All these m o r p h o l o g i c a l findings indicate that the c u l ­ tured coronary endothelial cells do not differ from the respective cells in vivo. This view c o u l d be further s u b ­ stantiated by studies of the gelelectrophoretic cytosolic protein pattern of freshly isolated c o r o n a r y e n d o ­ thelium and of cultured cells, which proved to be very similar.

Table 1 : Content of a d e n i n e n u c l e o t i d e s and their d e p h o s p h o r y l a t e d degradatives in c o n f l u e n t c o r o n a r y e n d o t h e l i a l cells a n d in m y o c a r d i a l tissue of g u i n e a pigs. Mean values f r o m three ana­ lyses, in each of which 30 mg endothelial cells w e r e e x t r a c t e d .

66

Endothelial cells nmoles/g v

ATP ADP AMP Σ ATP, ADP, A M P Adenosine Inosine Adenine Hypoxanthine

11 960 2 760 630 15 350 87 100 60 50

Myocardium nmoles/g 4 280 1 050 160 5 490 2 1.2 0.5 0.9

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FOLIA A N G I O L O G I C A / Vol. XXVIII / 1/2/3/80

of a d e n i n e n u c l e o t i d e s and their dephosphorylated deg r a d a t i v e s in n o n - g r o w i n g confluent endothelial cell c u l t u r e s ( G phase) are listed. For reasons of c o m p a r i ­ son r e s p e c t i v e data d e t e r m i n e d in n o r m o x i c myocardial tissue are also given. It is obvious that endothelial cells c o n t a i n e x t r a o r d i n a r i l y high amounts of ATP, ADP and A M P . T h e s u m of the adenine nucleotides ( Σ ATP, ADP, A M P ) reaches w i t h m o r e than 15 μηηοΙβ8^ a value which is a b o u t t h r e e times higher than the total adenine n u c l e o t i d e c o n t e n t of c a r d i a c tissue. It shall be e m p h a ­ sized t h a t to our k n o w l e d g e c o m p a r a b l y high values of a d e n i n e n u c l e o t i d e s have not been reported so far for any o t h e r m a m m a l i a n tissue. 0

A n o t h e r interesting feature of endothelial cells c o n c e r n s the h i g h levels of adenine nucleotide degradatives. As can be seen the c o n t e n t s of adenosine, inosine, adenine and h y p o x a n t h i n e are about 1 to 2 orders of m a g n i t u d e h i g h e r t h a n the respective values for these c o m p o u n d s in t h e m y o c a r d i u m . A d d i t i o n a l e x p e r i m e n t s revealed that g r o w t h state of the cell c u l t u r e s did not profoundly influence the total c o n t e n t of a d e n i n e nucleotides. As c o u l d further be s h o w n , i n c u b a t i o n of confluent cell cultures in purine free m e d i a for three days did not result in any detect­ able r e d u c t i o n of the adenine nucleotide content. On the o t h e r h a n d , endothelial cells proved to be sensitive to lack of o x y g e n . Brief periods of anoxic incubation (1.5 a n d 3 m i n , respectively) caused a p r o n o u n c e d d e c r e a s e of A T P ( 1 8 % and 2 7 % , respectively) and an increase in ADP and A M P levels. Simultaneously, re­ m a r k a b l e a m o u n t s of adenosine w e r e f o r m e d and re­ leased f r o m the cells into the incubation m e d i u m (107 n m o l e s / g and 218 n m o l e s / g , respectively). Obvious­ ly, e n e r g y p r o d u c t i o n f r o m anaerobic glycolytic pro­ cesses w a s not sufficient to prevent the b r e a k d o w n of nucleotides. In T a b l e 2 activity values of enzymes are listed which are d i r e c t l y or indirectly involved in d e g r a d a t i o n and

et a l . : Studies on C u l t u r e d Coronary Endothelial Cells

synthesis of adenine nucleotides. Again for reasons of c o m p a r i s o n respective figures are included for myo­ cardial tissue. The f o l l o w i n g findings deserve particular a t t e n t i o n : While 5'-nucleotidase activity in endothelial cells e x c e e d e d by far that of myocardial tissue, the activity of adenosine deaminase proved to be much higher in the m y o c a r d i u m . This pattern of enzyme activities may reasonably explain that adenosine is pre­ sent in endothelial cells in rather high amounts c o m ­ pared w i t h the small quantities of this nucleoside in the m y o c a r d i u m . Surthermore it is evident f r o m the data in T a b l e 2 that the activities of giucose-6-phosphate-dehydrogenase (Glu-6-PDH) and ribose phosphate pyrop h o s p h o n k i n a s e (PRPP-synthease) are much higher in endothelial cells than in cardiac tissue. Lince these e n ­ zymes are k n o w n to be involved in the generation and utilization of ribose -5-phosphate, an essential interme­ diate in nucleotide synthesis ,it can be c o n c l u d e d that c o r o n a r y endothelial cells possess a p r o n o u n c e d p o ­ t e n c y for nucleotide synthesis. As a matter of fact, w e c o u l d demonstrate in preliminary experiments using 1 C - g l y c i n e and C - l a b e l e d purine bases that nucleotide synthesis in endothelial cells proceeds via salvage and de novo pathways. 14

14

From all our findings it is evident that coronary e n d o ­ thelial cells are characterized by a very active adenine n u c l e o t i d e metabolism. It is not yet possible, however, to d e c i d e whether the c o r o n a r y endothelium in vivo can be actually regarded as important s o u r c e for the release of adenosine from the heart.

Acknowledgements T h e skilful technical assistance of Mrs. Marie-Luise A r n h o f e r and of Mr. Hans Sima is gratefully a c k n o w l ­ e d g e d . We are very much indebted to Prof. Dr. J . S t a u b e s a n d , Department of Anatomy, University of Frei­ burg i. B., for the preparation of the transmission elec­ tron micrograph.

Table 2: Enzyme activities in c u l t u r e d c o r o n a r y e n d o t h e l i a l cells and in c a r d i a c tissue f r o m guinea pigs.

Specific activity (nmoles/min-mg) Endothelial Cardiac cells tissue 5'-Nucleotidase (E.C. 3.1.3.5) Alkaline phosphatase (E.C. 3.1.3.1) AMP deaminase (E.C. 3.5.4.6) Adenosine deaminase (E.C. 3.5.4.4) Glu-6-P-dehydrogenase (E.C. 1.1.1.49) PRPP s y n t h e t a s e (E.C. 2.7.6.6) APR-transferase (E.C. 2.4.2.7) GPR-transferase (E.C. 2.4.2.8)

95

13.6

14.6

24

1.1 3.4 12.7

2.2 28 4.2

6.58

1.9

0.7

0.2

0.3

0.1

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et a l . : Studies on C u l t u r e d Coronary Endothelial C e l l s

7. JAFFE, Ε. Α., R. L N A C H M A N , C. G. BECKER and C. R. M I N I C K : Culture of h u m a n endothelial cells d e r i v e d f r o m umbilival veins. Identification by m o r p h o l o g i c and i m m u n o ­ logic criteria, J . Clin. Invest. 52, 2745 (1973). 8. SLATER, D. N., a n d J . M. S L O A N : T h e p o r c i n e e n d o t h e l i a l cells in tissue c u l t u r e , A r t e r i o s c l e r o s i s 21, 259 (1975). 9. WECHEZAK, Α., and P. B. M A N S F I E L D : Isolation a n d g r o w t h characteristics of cell lines f r o m bovine v e n o u s e n d o t h e l i u m , In vitro 9, 39 (1973). 10. SCHRÄDER, J., and E. G E R L A C H : C o m p a r t m e n t a t i o n of cardiac adenine n u c l e o t i d e s a n d f o r m a t i o n of a d e n o s i n e , Pflügers A r c h . 367, 129 (1976). 11. REIDY, Μ. Α.: B l o o d f l o w and arterial endothelial c e l l s . Lecture held d u r i n g the S y m p o s i u m " P a t h o p h y s i o l o g y of the vascular s m o o t h m u s c l e " , T u b i n g e n , July 22—24, 1979.

S. NEES Physiologisches Institut der Universität München Pettenkofer Straße 12 D-8000 M ü n c h e n 2

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FOLIA A N G I O L O G I C A / V o l . XXVIII / 1/2/3/80