Phosphorylation and deamination of adenosine by the isolated, perfused rat heart

252 BBA

B I O C H I M I C A ET B I O P H Y S I C A ACTA

26963

P H O S P H O R Y L A T I O N A N D D E A M I N A T I O N OF A D E N O S I N E BY T H E ISOLATED, PERFUSED RAT HEART

J. \v. DE JONG Departme~zt of Biochemistry I, l?oth'rdam Medical School, Rotterdam (The Nelhcrla~ds)

( Received March i6th, 1972) (Revised manuscript received July 24th, 1972)

SUMMARY

I. Adenosine (initial concentration o.5 raM) disappears from the perfusion medium of an isolated, beating rat heart at a rate of 1.4 Fnloles/min per g myocardial protein. About 7 a,o/ / o of the adenosine converted is recovered as inosine. 2. Evidence is presented t h a t the inosine is formed from exogenous adenosine (initial concentration 5 or 5oo FM), without appreciable participation of the nueleotide pool. 3. The isolated rat heart incorporates about I . I }~moles ~t~C]adenosine per g myocardial protein into adenine nucleotides in a 3o-min perfusion experiment. 4. W h e n the heart is perfused with about 5 FM adenosine only a few percent is phost)horvlated , ~ whereas some 45°/..o is deaminated. 5. It is concluded t h a t deamination of adenosine is by far the most important p a t h w a y of adenosine metabolism in the isolated, perfused rat heart, under the conditions employed.

INTRODUCTION

When tile myocardial oxygen concentration is diminished, dephosphorylation of adenine nucleotides to adenosine occurs in the heart t G. Adenosine is able to diffuse out of the myocardial celD, in contrast to its phosphorylated derivativest, 7. The increased cardiac blood flow during hypoxia is probably due to an adenosine-induced vasodilatation~,'~,L In m a n y species the red blood cells contain adenosine deaminase (EC 3.5.4-4) (see, for instance, refs 5 and 8 IO), which catalyzes the conversion of adenosine to inosine. The latter does not possess vasoactive propertiesT,l~, 1~. However, adenosine deaminase activity in rat erythrocytes (and plasma) is absentS, ta or low (refs 9, IO, 14). An alternative important p a t h w a y for adenosine removal via the adenosine kinase (EC 2.7.1.2o ) reaction has been demonstrated in ervthrocvtes f r o m I T l a n t5 is and mouse .6. Inside the myocardial cell adenosine can be either phosphorylated or deaminated vs (other p a t h w a y s being probably relatively inactive). After a single passage of 14iochim. t~iophys, dcta, 286 (1972) 252 -'59

P H O S P H O R Y L A T I O N AND D E A M I N A T I O N OF A D E N O S I N E

253

[x4C]adenosine through an isolated guinea pig heart, Pfleger et al.2°, ~ recovered 3o% of the ~4C activity in the heart. Jacob and Berne'2, 2~ and Liu and Feinberg 2a observed a rapid incorporation of exogenous adenosine into adenine nucleotides of perfused cat and rabbit heart, respectively. We wish to report some experiments performed on the isolated, beating rat heart. Under the conditions employed, the bulk of the adenosine metabolized by the heart is deaminated. EXPERIMENTAL

Heart perfusion Male Wistar rats (2oo-25o g) with free access to food and water, were anesthetized with about 20 mg pentobarbital (Abbott, Saint-R~my-sur-Avre, France) and heparinized with 500 I.U. Thromboliquiue (Organon, Oss, The Netherlands). Langendorff preparations were perfused with Medium A at 37 °C as described by Zimmerman ct al.2L with a pace maker set at 5 pulses/s, 2 ms duration and 0.2 mA. Final concentrations in this Medium A, a modified Tvrode solution, were: 128 mM NaCI, 4.19 mM KC1, 2o.2 inM NaHCOa, 0.42 mM NaH2PO~, I.O mM MgC12, 1.35 mM CaCI,,, 5.0 mM D-glucose, 0.5o mM potassium palmitate (recrystallized ppt. of equimolar ethanolic Cxs acid and K O H solutions) bound to o.17 mM f a t t y acid-free bovine serum albumin (Fluka, Buchs, Switzerland) and 0.5 mM adenosine (puriss., Koch-Light, Colnbrook, Bucks.). A warm solution of palmitate (IO raM) was added dropwise to a concentrated albumin solution, stirred for 30 min and diluted; the salts were subsequently added from stock solutions and the mixture was agitated for another 30 rain. The clear solutk)n was passed through a washed Millipore filter and finally, glucose and adenosine were dissolved. The medium was stored at --20 °C. When equilibrated with 95%/ 0/ 02 3/o C02, the pH was 7.4- Foaming of the mixture was suppressed by application of a minimal amount of Antifoam A Spray (Dow Coming, Midland, Mich.). Several perfusions were performed with Medium B fortified with low adenosine concentrations (I IO FM). Medium B contained: 128 nlM NaC1, 4.7 ° mM KC1, 20.2 mM NaHCOa, o.42 mM NaH.,PO~, I.O mM MgC12, 1.35 mM CaCL and II.O mM D-glucose '-'~. Myocardial extract A perfused, beating rat heart was clamped at 178 °C. Homogenization took place at the temperature of solid CO2. The heart was extracted with 6 ml of 40/0 HC104 at o °C. Protein was removed by centrifugation in the cold, the supernatant fluid was brought to p H 6.5 by the addition of 2 M KOH-KC104, which precipitated at o °C was removed by centrifugation. If necessary, the volume of the supernatant fluid was reduced by lyophilization. The residue was extracted with I.O ml water. Chromatography 5 F1 of the lyophilized extract were applied to a cellulose plate (MN-Polygram Cel 3o0, o.I m m layer, Macherey-Nagel, Diiren, Germany). Development took place for 9 ° rain in 2-methylbutan-2-ol-formic acid-water (3:2:1, by vol.) 2~. Radioactive spots were detected by thin-layer scanning (RTLS-IA, Panax Equipment, Redhill, Surrey) at a rate of 3o ram/h, the time constant being ioo s. References were: ATP, ADP, ~8-14C]AMP, IMP, inosine, hypoxanthine, [8-14C]adenosine and adenine. Biochim. Biophys. Acta, 286 (1972) 252 259

254

j. ~v. DE JOXG

Separation of adenosine, inosine and hypoxanthine in tim perfusate was accomplished at 4 °C on a column (2.5 cm x 25 cm) of Sephadex G-zo (Pharmacia, Uppsala). Elution took place at a rate of z 2 ml/min with 5o mM Tris-HC1 buffer (pH 6.5); in later experiments this buffer was fortified with o.002% Hibitane (chlorhexidine digluconate; I.C.I.) to prevent microbial nucleoside breakdown. Fractions of 5 ml were collected. Determinations

Analvses for ATP, ADP, AMP, inosine, hypoxanthine and adenosine were usually carried out according to standard enzymatical methods '-'~. Values for AMP were corrected for AMP-like material in the NADH (Boehringer, Mannheim). Use was made of a Zeiss PMQII spectrophotometer or a Gilford 2400 recording spectrophotometer. Concentrations of adenosine, inosine and hypoxanthine in the/~e~'i range were estimated with a Perkin-Ehner 356 double-beam/double-wavelength spectrophotometer. Radioactivity was determined in a Packard Tri-Carb liquid scintillation spectrometer. The scintillation mixture consisted of either IO ml Insta-Gel (Packard, Zfirich) or (ref. 27) 5.33 ml toluene 2.00 ml ethanol-2.67 ml Triton X-1oo (Rohm and Haas, Philadelphia, Pa.), containing 21.3 nag PPO and 0.53 nag dimethyl-POPOP (Merck, Darmstadt). Radioactivity in nucleotides, scratched from cellulose plates, was estimated in IO ml toluene containing 4 ° mg PPO, I.O nag dimethyl-POPOP and 0.35 g Thixin (Nuclear Enterprises, Edinburgh). Protein was measured by the biuret method 2~. RESULTS AND DISCUSSION

Fig. I S]lO'WSthe time courses of tim disappearance of adenosine (added in a concentration of 0.5 raM) and tile accumulation of inosine. Adenosine disappearance and inosine accunmlation, measured in the medium, were calculated to be 1.42 ~ o.45 (,z -- 8) and 1.o5 _q o.33 (;l -- 0) fmoles/min per g myocardial protein, respectively. (Tile amounts of nucleoside inside the heart are relatively unimportant.) Since no appreciable adenosine deaminase leakage from the heart into the perfusate could be detected, tile inosine nmst have been formed in tile heart by tile (soluble~4,'-'") adenosine deaminase. Berne c! al.7, ~' reported an el'flux of the enzyme from the isolated, perfused cat heart, whereas other investigators did not find adenosine deaminase in the perfusate of rabbit * and dog '~ heart. The addition of adenosine to a perfusion medium caused the flow through isolated rat hearts to increase 3 4 times (not shown). This observation is in agreement with those from work carried out on the open-chest dog myocardiumT,l~, a"-aa and the isolated, perfused heart of the cata, ~2 and the guinea piga,L Adenosine is a strong inhibitor of pahnitoyl-CoA svnthetase (EC 6.2.1.3); the mitochondrial oxidation of pahnitate is inhibited by tile nucleosidea4,a'L However, the myocardial extraction of palmitate from a perfusion medium is stinmlated e-fold by added adenosine aG. l:urthermore, aH,O production from 9,1o-aH]oleate by the perfused rat heart is stimulated by the addition of adenosine (Hiilsmann, W.C., unpublished). This could indicate that the concentration of the inhibitor adenosine is effectively kept low inside the cardiac cell. The inhibition bv adenosine is competitive with l~iochi~>~. Iqiophys. Acta, e 8 6 (1972) 25e-e59

PHOSPHORYLATION AND DEAMINATION OF ADENOSINE

255

Radioactivity 1.O

o

o eL

=k

~Adenosine

eL

O

0.5

O

t~ O

o

t*

-o

7o f 0

o 10 20 Time (rain)

'

30

Fig. i. Disappearance of adenosine and a c c u m u l a t i o n of inosine, m e a s u r e d in the perfusion m e d i u m of isolated, beating r a t hearts. H e a r t s were perfused (without adenosine) for 3 ° min w i t h Medium B (see Experimental) with 5.o nlM glucose instead of i i .o inM. Perfusion was continued in a recirculating s y s t e m w i t h 5 ° ml of Medium A. o. 5 mM adenosine or (8-x4C]adenosine (o.i67 Ci/mole, Radiochemical Centre, A m e r s h a m , Bucks.) were used. Samples (o. 5 ml) were taken at 5-nlin intervals and analyzed for radioactivity, adenosine and inosine (see Experimental). Average values (based on myocardial protein) are given for 4-6 experiments.

respect to ATP (ref. 35). F a t t y acid activation can only be increased by addition of adenosine if the latter causes the ATP/adenosine ratio to become higher. Since the rate of adenosine disappearance exceeds that of inosine formation, an additional path of adenosine metabolism was investigated. Studies with [t4C]adenosine revealed that isolated rat hearts are able to incorporate this nucleoside into adenine nucleotides; from the perfusion fluid the radioactive label disappeared (Fig. i). Radioactive

-!

3 o

e~

0.

10111111

Fig. 2. D e m o n s t r a t i o n of L8-14C]adenosine (o.334 Ci/mole, initial concentratiou o.5 raM) incorporation into myocardial adenine nucleotides. A r a t h e a r t was perfused as described in the legend to Fig. I a n d clamped at liquid-N 2 t e m p e r a t u r e after 3 ° min of recirculation (with an additional i - m i n perfusion w i t h t14C]adenosine-free medium). Adenine nucleotides and nucleosides were extracted and c h r o m a t o g r a p h e d . The figure shows a thin-layer scan. F o r details a b o u t extraction and c h r o m a t o g r a p h y , see Experinlental. C h r o m a t o g r a p h y in a second direction in satd (NH4)~SO 4 I M s o d i u m a c e t a t e - p r o p a n - 2 - o l (8o:18:2, b y vol.) (ref. 25) and s u b s e q u e n t a u t o r a d i o g r a p h y revealed a trace of radioactivity in the l l y p o x a n t h i n e area.

Biochim. Biophys. Acta, 286 (1972) 252 259

256

J.w.

l i e JONG

TABLE 1 RAT

MYOCARDIAL

N1JCLEOTIDE

CONTENT

AFTER

PERFUSION

WITIt

ADENOSINE

L a n g e n d o r f f p r e p a r a t i o n s were p e r f u s e d as described in t h e legend to Fig. I. H e a r t s were q u i c k l y frozen, a n d e x t r a c t e d w i t h HC10 v In deproteinized, n e u t r a l i z e d e x t r a c t s n u c l e o t i d e s were determ i n e d 2". In s o m e e x p e r i m e n t s IS-nC ladenosine was used. N u c l e o t i d e s were separatcll b y tb.in layer c h r o m a t o g r a p h y a n d localized by a u t o r a d i o g r a p h y or t h i n - l a y e r s c a n n i n g . Spots were s c r a t c h e d from t h e t h i n - l a y e r plates a n d a n a l y z e d for r a d i o a c t i v i t y (see E x p e r i m e n t a l ) . A v e r a g e values } S.D. are p r e s e n t e d with n u m b e r of e x p e r i m e n t s in p a r e n t h e s e s .

Nuch'otidc

ATP ADI' AMP

.lli~ltts ade*,wsim' (/~moles/g myocardial protei12 )

Plus adcm~si~zc (,um,~lcs./~*myocardial prolei@

nC incorporated (Izmolcs/g myocardial p~'otciJz)

22-73 !_ 5.3 O (7) 4.74 :: I-5S (0) o.0o ! o.32 (0)

21-75 ": 5.35 (13) 5.28 _j 1.5o (~i) ~.32 { o.78 (rl)

o.S4 (3) o-~5 (3) o.to(3)

adenine nucleotides were identified in extracts of perfused hearts by means of thinlaver chromatography and thin-laver scanning (lqg. 2). The major product was ATP (Table I). When the extract was incubated with hexokinase (EC 2.7.i.I), adenylate kinase (EC 2.7.4.3), D-glucose and Mg('l.,, nucleotide radioactivity on the thin-layer chromatograrn was predominantly in the area of AMP, confirming the identity of the triphosphate. The isolated rat heart incorporated about I.I y.moles {it4Cladenosine per g myocardial protein into adenine nucleotides in a 3o-min perfusion experiment. There was no significant net synthesis of adenine nucleotides (Table I). Prolonged infusion of adenosine in the rabbit, on the contrary, was reported to increase the ATP content of the heart by approx. 4o°/~ (ref. 37). Whether the adenosine-induced increase in fatty acid oxidation by the isolated, perfused rat heart is due to the higher flow rate, is presently under investigation. Several paths of adenine nucleotide breakdown could give rise to the formation of inosine (lqg. 3). ATp14'as and ADP a'a have been reported to inhibit dephosphorylation of AMP, whereas AMP deaminase (EC 3.5.4.6) is stimulated by ATP as. In order to test, whether the turnover of nucleotides contributed to an appreciable extent to the formation of inosine, a comparison was made of the specific activity of the radioactive adenosine added and the inosine formed. Rather unexpectedly, Sephadex G-IO could be used to separate these nucleosides (Fig. 4). Minor alnounts of the catabolite hvp()xanthine were detected. No at)preciable difference was observed in specific activity of ATP

,ll, ADP AMP adenine

>" 'l 1' ~adenosine

7 --~

IMP

51 d ° h y p o x a n t h i n e - - ~ inosine 4

e

uric acid Fig. 3. Several p a t h w a y s of m y o c a r d i a l a d e n i n e nucleotide s y n t h e s i s a n d b r e a k d o w n . E n z v n l e s i n v o l v e d are: (T) a d e n o s i n e kinase (EC 2.7.1.2o) ; (2) a d e n y l a t e kinase (EC 2.7.4.3) ; (3) e n z y m e s concerned with o x i d a t i v e p h o s p h o r y l a t i o n a n d s u b s t r a t e - l i n k e d p h o s p h o r y l a t i o n ; (4) A T P a s e (EC 3.6.1.3) ; (5) 5 ' - n n c l e o t i d a s e (EC 3.1.3.5) ; (6) a d e n o s i n e d e a m i n a s e (EC 3-5.4-4); (7) A M P d e a m i n a s e (t,/(" 3.5.4.6); (8) p u r i n e nucleoside p h o s p h o r y l a s e (I:.C 2.4.2.1); (9) a d e n i n e p h o s p h o r i b o s y l t r a n s ferase (KC 2.4.2.7); 11o) h y p o x a n t h m e p h o s p h o r i b o s y l t r a n s f e r a s e (EC 2.4.2.8 ).

Bwchim. tJiophys. Acta, 286 (1972) 252-259

PHOSPHORYLATION AND DEAMINATION OF ADENOSINE

257

2.6

ii

2.4

Albumin / palmitate

Adenosine

30

0.6

20

0.4

~k ~t

Inosine

~

0.2

Hypoxanthine •

1t' .D 0

10

20

30 Fraction

io

~o

60

number

Fig. 4. Separation of adenosine and inosine by chromatography on Sephadex G-io. A rat heart was perfused (see legend to Fig. i) with [8-1~C]adenosine (o.334 Ci/mole). A 5.o-ml sample of the perfusion medinnl was taken after 3o min of recircnlation and chromatographed on a column of SephadexG-io(see Experimental).The absorbance at --48 nm and the radioactivitywere measured. Characterization of peaks, see Experimental.

the 0.5 mlXI adenosine added, and the adenosine and inosine recovered in the perfusate. Similar results were obtained when tile adenosine concentration was about 5/J.M. This could indicate that the concentration of adenosine and inosine produced from endogenous nucleotides of the isolated, perfused rat heart is comparatively low. Very recently, Wiedmeier et al. 4°, who perfused isolated guinea pig myocardium with much lower adenosine concentrations (o.oi FM), reported very significant changes in the specific activity of adenosine, inosine and hypoxanthine. I t is c o n c l u d e d t h a t , u n d e r t h e c o n d i t i o n s e m p l o y e d , d e a m i n a t i o n of a d e n o s i n e

is more rapid than phosphorylation. Also, when rat hearts are perfused with low concentrations of adenosine (approx. 5/~M), deamination is tile main fate of this nucleoside (Table II). Only a few percent of the adenosine is phosphorylated, whereas some O/ is deaminated. In contrast to these results obtained with rat heart, isolated cat 45/o (refs I2, 22) and rabbit 2'~ hearts, perfused wittl adenosine, showed a predominant 1-~iochi*n. Biophys. Acta, 286 (r972) 252 259

258

j . W . DE JOXG

T A B L E II METABOLIC F A T E OF [14CI]ADENOSINE IN T H E I SOLATED, P E R F U S E D RAT H E A RT

Isolated rat hearts were perfused in an open systeln for 3 ° rain with Mediunl B (see Experimental). The perfusions were continued with a similar fluid fortified with about 5 l*IxI !8-nC~ adelIosine. After 15 rain samples of the perfusates were collected in liquid N 2. Samples were thawed and analyzed immediately for radioactivity and purine derivatives. The nlethod of Olsson a: was used to determine adenosine, inosine and hypoxanthine coneentratim?s. Expt l

lixpt [[

PeU)~sion fluid Adenosine (/~M) Radioactivity (clpm/nil)

4.2 L 988 t

4.S3 !)765

Pe~fusatc Adenosine (#M) Inosine ([zM) Hypoxanthine (#M) Radioactivity (dpm/ml)

3.57 I..t2 o. 23 92S0

2.50 1.74 o. 21 9714

i n c o r p o r a t i o n of t h e n u c l e o s i d e i n t o a d e n i n e n u c l e o t i d e s . I n l m m a n e r y t h r o c y t e s , p h o s p h o r y l a t i o n of a d e n o s i n e p r o d u c e d f r o m e x o g e n o u s A M P , w a s f o u n d t o b e t h e m a j o r p a t h w a y 1~. Also w o r k i n g w i t h h u m a n r e d cells, L o w y a n d W i l l i a m s 1~ o b s e r v e d the phosphorylation/deamination ratio to be increased when the adenosine concent r a t i o n in t h e i n c u b a t i o n m e d i u m w a s i n c r e a s e d . W h e n a d e n o s i n e k i n a s e is l o c a l i z e d a t t h e l e v e l of t h e c e l l u l a r m e m b r a n e , it m a y h a v e a c c e s s t o e x o g e n o u s a d e n o s i n e p r i o r t o a d e n o s i n e d e a m i n a s e ~-. T h e p r e s e n t e x p e r i m e n t s i n d i c a t e t h a t t h i s d o e s n o t h o l d for r a t h e a r t . H o p k i n s a n d G o l d i e % w h o s t u d i e d t h e i n f l u e n c e of d i p y r i d a m o l e o n t h e m y o c a r d i a l u p t a k e of a d e n o s i n e , s u g g e s t e d recently that rat heart, contrary to guinea pig heart, lacks a membrane-bound adenosine kinase. ACKNOWLEDGEMENTS T h e v a l u a b l e t e c h n i c a l a s s i s t a n c e of Miss C. K a l k m a n is a c k n o w l e d g e d . T h e a u t h o r w o u l d like t o t h a n k P r o f e s s o r W . C. H i i l s m a n n for a d v i c e . REFERENCES I 2 3 4

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P H O S P H O R Y L A T I O N A N D D E A M I N A T I O N OF A D E N O S I N E

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Biochint. Biophys. Acta, 286 (1972) 252 259