In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP

Biochiraicaet BWphysicaActa. 1074(1991)302-311 © 199I Elsevier Science Publishers B.V. 0304-4165/9t/$03.50 ADONIS 030441659100193B

302

BBAGEN 23547

In viva regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracelhdar diffusion of ADP Valdur A. Saks, Yulia O. Belikova and Andrey V. Kuznetsov Laboratory of Bioenergeti~, U.S.S.R. Cardiolo~ Research Center, Moscow (U.S.S.R.) (Received 1 August 1990)

Key words: Mitochondriai oxidative phosphor]arian; Cardiomyocyl¢; ADP diffusion" ADP compartmentation: Creatine: Crealine kinase ~ t e m ; Energ~ transport

Relative diffusivities of ADP and creatine in eardiomyocytes were studied. The isalated rat cardiomyocytes ~ r e iysed with saponin (40 # g / m l ) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenasc. In these cardiomyocytes mltochondria were used as 'enwmatle probes' to determine the average local concentration of snbstrates exerling accepter control of respiration - ADP or ereatine (the latter activates respiration via mitochondrial creatine kinase reaction) - when their eoneentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control ef respiration of saponin-treated cardiomyocytes were compared with those determined in isoin;ed mitochondda and skinned cardiac fibers. The apparent K= for ereatlue (at 0,2 raM ATP) was very close and in a rans¢ of 6.0-6.9 mM in all systems studied, showing the absence ef diffusion difficulties for this substrata. On the contrary, the apparent K= for ADP increased from ",8 + 1 /zM far isolated mitochondria to 2,50 _+59 ;tM for cardiomyoc~es with the lysed sarcolemma and to 264 _+57 #M for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with dladenosine pentaphosphate- When 25 mM creatine was present, the apparent Km fur ADP decreased to 36:1:6 p M. These data are taken to indicate specific restrictions of diffosien of ADP most probably due to its interaction with intermediate binding sites in cnrdlnmyocytes. The important role of phesphocreatinecreatine kinase system of energy transport is to overcome the restrictions in regulation of ener~ fluxes due to decreased diffusivity of ADP.

Introduction Intracellular transport of energy-rich phosphates from mitochondria to the sites of energy utilization (myofibrils, ion pumps, etc.) by phosphocreatinecreatinc kinase system is based on compartmentatinn of ereatine kinase isoenzymes: binding of a specific mitochondria[ form of ereatine kinase to the outer

surface of inner mitochondrial membraneand connection of other isoen~mes to myofibrils (mostly MMcreatine kinase) and to cellular membraes [1,2]. About half of total creatine kinase activity is found in tyro-

Abbreviations: IM, isolated medium; APsA, dizdcnosine penlaphos-

phate Correspondcncc: Dr. V.A. Saks, Laboratory of Bioenergeticg, U.S.S.R. Cardiology Research Center, 2-rd Chcrepko~,'skaya,15 A Moscow 121552, U.S.S.R.

plasm in cardiac cells [3]. It is assumed that such a distribution of creatine kinas¢ reflects, or results in a compartmentation of adenine nueleotides, ATP and ADP [3-6]. However. an alternative theory of mu,s¢lo energetics considers phosphocreatine as an energy store which is used via equilibrium creatine kinase reaction for replenishment of ATP but is not obligatory for imracellular energy supply [7]. This theory assumes high diffusivil~;of both ATP and ADP in the cells. For ATP this conclusion was made on the basis of direct measurements of diffusion coefficients with radioisotopic methods by gushmorick and Podolsky [8] and with the use of 31p pulsed gradient NMR ny Yoshizaki et al. [9-10] and recently by Yoshizaki et al. [7]. The latter authors, however, posed an important question of whether ADP molecules have similar diffusivity to that of ATP or not [7]. They concluded that: "Most ADP molecules in muscle cells are assumed to be bound to macromolecules, such as actin. Free ADP molecules might interact with the binding sites in the

3~ macromolecules to reduce the diffasivity of free ADP in muscle cells as reported for Ca 2÷ ion. In this case the phosphoereatine-creatine kinase system must have an important role for energy transport in muscle cells. However, there are no data on the diffusivity of ADP in muscle cells and this remains to be elucidated in the future". The present work was performed in response to this appeal. Kinetics of regulation of mitochondrial oxidative phosphorylalion by ADP or crealine (through erealine kinase reaction) in isolated mitochnndria~ isolated cardiomyoeytes with saponin-lysed sarcolcmma and saponin-lreated cardiac muscle bundles was studied. The value of apparent K,~ for creatine in the process of activation of respiration was similar in all these systems. However, the apparent K m for ADP was increased more than by an order from 18 _ I ,o.M for isolated mitochondria to 250+39 gM ['or cardiomyocytes. These data may be taken to indicate directly the decreased diffusivity of ADP in cardiomyocytes most probably due to interaction with intermediate binding sites within cardiomyo¢'ytc~. Diffusion difficulties for ADP were significantly overcome by activation of the creatine kinase system: apparent K~n for ADP was decreased to 36 + 6/zM in the presence of 25 mM creatine as a result of am~llflcation ~)f weak ADP flux in the coupled mitochondrial ereatine kinase.oxidative phosphorylation reactions. Materials and Methods

Isolation procedures Cardiomyocytes were isolated from rat hearts. Wistar line rats, 200-300 g, were narcotized by pentobarbital solution (1 ml, 40 mg/ml) with the addition of 500 units of heparin. The heart was quickly excised preserving a part of aorta and placed into aerated isolation medium (IM) of the following composition: 21.1 mM Hepes (pH 7.2 at 20°C), 117 ram NaCI, 5.7 mM KCI, 4.4 mM NaHCO 3, 1.5 mM KI-12PO4, 1.7 mM MgC1z, 11.7 mM glucose, 11 mM creatine, 20 mM taurine, 10 mM phosphocreatine, and 21 mU/ml of insulin. IM was filtered through MilIipore filters (0.45 /.tin). The heart was cannulated and perfused with the flow rate of 6 ml/min during 5 rain with the calcium-free IM which was continuously aerated. The heart was then perfused during I h with IM, containing 1.25 mg/ml of collagenase. Perfusion was performed v,ith ¢ollagcnas¢ solution recirculating in a f i o r d system with the perfusate volume of 20 ml. Perfusat¢ was continuously aerated and its temperature was kept at 37°C, After treatment with collaenase, the heart was placed into ]M containing 20 # M Ca z+ and bovine serum albumin (5 mg/ml). The heart was disrupted mechanically by tbrceps to release cardiomytg'ytes. Crude suspension of cardiomyocytes was fdtered through Dacron textile with

rectangular cells. The fiitrated suspension was transferred into test tubes and kept for 3-5 mir~for precipitation of a major part of cardiomyocyte.g. The upper part of solution above the eardiohlyocytes was digcarded and precipitated cardiomyocytes were resuspended in IM containing 20 gM Ca -'+ and bovine serum albumin (5 mg/ml). After sedimentation during 3-5 rain the cells were resuspended in small volume and stored in thin layer of solution in Erlenmeyer flasks for better aeration of the cells. Isolated cells' population contained 50-60% of rod-llke cells when observed under the light microscope. The cells preserved their shape in the solution containing 300 g M Ca-" (Ca" +-tolerant cells). For electron microscopic observation the cardiomyocy.tcs were fixed for 1 h at room temperature in IM containing 2,5% glutaraldehyde and dehydrated in alcohol solutions with ascending coneentrat!on. Namely the cells, about 1{30000 per ml, were deposited ,at Nuelepore filters (filters diameter 25 ram, pore diameter 1 #m), washed with IM to remove glutaraldehydc, each wash time for 7-l0 rain with 30.. 50, 70, 80 and 96% ethyl alcohol (5 ml) and then three times with 5 ml of absolute ethyl alcohol and dried a day at room temperature. Isolation of rat heart mitochondrie. Isolation of mitochondria was carried out at 4°C. Eight rats of Wistar line were narcotized with diethyl ether, their hearts were excised and placed into a cold solation of 0.3 M sucrose. The atria were removed, ventricles were carefully washed and minced by scissors. The minced cardiac tissue was repeatedly washed with cold 0.3 M sucrose solution and incubated 5 min in 20 ml of medium containing 0.3 M sucrose~ 10 mM Tris-HCI (pH 7.2). 0.2 mM I/[DTA and trypsin, 125 gg per ml. The mixture was lightly homogenized in Iosely fitted Teflon-glass homogenizer and incubated for 10 more rain. After that 20 ml of the same buffer conlaining trypsin inhibitor (650 t~g per ml) were added. The tissue was then homogenized first in loosely and then in tightly fitted Teflon-glass l~omogenizer, Resulting suspension was e-,trifuged for l0 min at 1000 × g. Supematant was eentrifug;*.d fur 15 rain at 10000 × g. Supernatant and upper lif~,htlayer of mitochondria (light, damaged mitoehondria) were discarded. Sedimented milochondria were resuspended in medium containing 0.3 M sucrose, 10 mM Tris-HCl (OH 7.4), 0.2 mM EDTA, bovine serum albumin (1 mg/ml) and recentrifuged as described. This procedure of resuspnes[on/resedimentation was repeated twice, The final mitochondrial preparation was resuspended in a minimal volume of isolation medium (100200 ill, gi,~ing final concentration of protein of 50-80 mg/ml) with bovine serum albumin and stored in ice. lsolalion of bundles of cardiac muscle skinned fibers. Bundles of cardiac muscle, 0.3-0.4 mm in diameter,

304 2-3 mm in length were isolated from endocardial surface of left ventricle and transferred ixi~orelaxing solution A (see below). A group of seven to eight bundles was incubated for 30 rain in I ml of solution A coztaininz 50 #g/ml of saponin. After that bundles were washed for 10 rain to remove .~;aponin in a solution B without high energy phosphates (see below). All procedures were carried out at 4°C with vigorous mixing of solutions [11]. Solutions. Alt solutions which were used in studies with skinned cardiac fibers contained 10 raM EGTACaEGTA buffer (free Ca 2÷ concentration 0.1 p,M), 8 mM free Mg 2÷, 20 mM taurine, 9.5 mM dithiothreitol, 20 mM imidazoIe [pH 7.0). Ionic strength (around 0.16 M) was adjusted by adding potassium 2-(N-morpholino)ethansulfonate. Free Ca2. and Mg 2+ concentrations were calculated on the basis of equations described by Fabiato and Fabiato [12] by using known values of dissociation constants [13]. Relaxin~ solutions A, in which fibers were skinned by saponin contained also 5 mM MgATP and 15 mM phosphoereatine. Solution B in which respiration rates were determined contained, in addition to the main coraponents, 5 mM glutamate, 2 ram malate, 3 mM phosphate and 5 mg/ml of bovine serum albumin (fatty acid free) instead of high energy phosphates. The solutions A and B were composed on the basis of information of composition of muscle cells cytoplasm [14].

Determinations Respiration rates of isolated mitochondria, skinned fibers and cardiomyocytes were determined by using Yellow Spring Instruments (U.S.A.) oxygrapb and Clark electrode. The medium for respiration rate determination for mitochondria contained 0.25 M sucrose, 20 mM HepesK (pH 7.4), 3 mM magnesium acetate, 5 mM glutamate, 2 raM malate, 0.37 mM dithiothreitol, 4 mM KH2PO4, 0.3 mM EGTA and 1 mg/mi bovine set-to albumin. Oxygen solubility was taken to be equal to 400 ng atoms per rat at 30°C [15]. In special experiments 100 /~M diadenoslne pentaphosphate (APsA) for complete inhibition of rayokinase was added. Respiration of skinned fibers was determined in a medium B described above. Respiration rate of isolated eardiomyocytes was determined in both media at 30 ~c. It was shown that the presence of 0.1 ~M Ca2÷ did not affect the respiration rate. ADP solutions used were calibrated by an enzymatic method [15]. Activities of creatine kinase of cardiomyoeytes were determined at 30°C in a medium containing 0.2% Triton X-100 for solubillzation of raembranes~ 25 mM sodium-Hepes (pH 7,4), 5 mM MgCI2, 0.5 ram dithiothreitol, 10 mM phosphocreatine, 1 mM ADP, 1 I.U./ml hexokinase, 1 LU./ral glucosc-6-phusphate

dehydrogenase, 0.6 mM NADP, 20 mM glucose, 13 mM AMP and 15-40 #g of protein of cardiomyoc~es studied. Mtcr addition of cardiomyocytes char.ges in optical density at 340/xm were measured. Activities of lactate dehydrogenase were determined in a medium containing 20 mM pota~ium phosphate (pH 7.4), 120 mM KCI, 0.5 mM dlthiothreitol, 0.2 mM NADH, 10 raM pyruvate, 0.2% Triton X-100 and 50150 ~g of protein of cardiomyocytes. After the addition of pyruvate, the changes in absorbance at 340 ~tra were determined. Protein concentration was determined by a biuret method [16].

Lysis (perforation) of the sarcoterama of cardiomy. ocytes. For determination of the % of cells with intact sarcolemma, the cardiorayocytes were treated with saponin (40 ~.g/ml) in the oxygraph cells after addition of ADP (1 mM). In kinetic experiments saponin was added in the same concentration to the oxygraph medium containing cardiomyocytes (0.5-1 mg/ml) and after 1-2 rain of recording of basal respiration ADP was added to final concentration of 0.05-1.0 ram and creatine to the final concentration of 2-20 mM and the steady.state rates of oxygen uptake were recorded. In the experiments in which the marker enzyme (lactate dehydrogenase and ereatine kinase) leakage was determined, the iysis of sarcolemma was carried out in the following way. 0.2 ml of suspension of cardiorayocytes, 20-30 rag/ml protein concentration, were sedimented in the oxygraph medium in Eppendoff centrifuge for 4-5 rain at 11C00 rpm. The eardiomyocytes were resuspended in 0.4 ml of the same medium and divided into two equal parts. To one part of cardiomyoeytes 3.2 ~1 of saponin solution (23 mg/ml) was added and incubated 3 rain, .tt room temperature and then sediraented in the Eppendorf e ntrifuge. Supernatant was taken away and deposited cardiomyocytes resuspended in 0,2 ral of oxygraph medium, In the supernatant, the resuspe,nded lysed eardiomyoelaes and the control samples the enzyme activities were determined as described above. Statistic analyses of data in double.reciprocal plots were made by using methods of linear regression. Mean Km values and their standard deviations are given. Reagents, Coupled enzyme systems, ereatine, phosphocreatine, nucleotides, Hepes, bovine serum albumin, sucrose, dithiothreitol, raalate, glutamate, EGTA, trypsin, trypsin inhibitor, magnesium acetate, saponin, collagenase, insulin (Sigma, U.S.A.) and EDTA, Triton X-IO0, Tris (Serva, F.R.G.) were used. Results Usually 50-60% (sometimes more) of isolated eardiomyoc~les were of rod-like shape when studied u~-

305 der the light microscope and tolerant (not contracting) in response to the addition of 300 # M Ca ~'+. The remaining cardiomyocytes either contracted in Ca" ~ containing medium or acquired spheric shape. Since all experiments in this work were made in the absence of Ca 2+ and all kinetic seria were carried out in identical conditions, this preparation of cardiom3,ocyles was considered to be adequate and it was not purified further. Electron micrograph~ demonstrated in Fig_ 1. A and B taken by scanning electron microscope show the intact card,.'omyocytes at different magnifications, and the characteristic sarcolemraal striations are seen especially at high magnification (Fig. 1BL Fig. IC and D show the cardiomyocyles in which sarcolemma was lysed by saponin. This picture agrees with earlier data by Allschuld et al. [17] and shows destruction of sarcolemma and the appearance of surface (suhsareolemmai) mitoehondria which stay attached to cellular structures. They are very clealry seen in cardiomyocytes supercontracted in the presence of 1 mM of Ca 2÷. Earlier Altsehuld et al. [18] have shown that the mitochondrial fraction is not altered and that the overall morphology of the myocytes can be retained under conditions of ocmplete solution of sareolemma D81. Further analysis confirmed that sareolemma is entirely disrupted uttder given conditions, in the control (intact) cardiomyocyles the activity of ereatine kinase and lactate dehydrogenase were 1-2 I.U. and 0.3 I.U. per mg of protein, respectively, in accordance with the clara by other authors [19,20]. After saponin treatment eardiomyoeytes were deprived of lactate dehydrogenas~ (which is a marker e n ~ m e of cytoplasm) by 86% and lost about half of the creatine kinase activity (Table !). This is also in accordance with earlier data: about 30-40% of ereatine kinase is bound to inner mitochondrial membrane as a specific isoenzyrne, 10% is bound to myofibrils and consequently, an average half of cellular creatine kinase is in bound form. Thus, TABLE I

Leakage of lactate dehydrogenase and creatine kinase from myocytes treated with saponin Mean values and staftdard de, Julian arc given for four experiments. Activities

LDH specific activib' tO/rag

Control m~cytes 0.29 Permcabiliscdm~aq~tcs: Sulmrnatant Pellet

0.65 0.026

CK percentage of total activity

specific activity lU'/raj

pelcenluge of talal activity

100

1.64

100

85.5:t:5.4 7 4-2,3

2.09 l,~

49.,;-I.4 55±3.5

soluble enzymes leave the cardiomyocytes after lysis of sarcotemma. That means that intracettular systems become accessible to substrates such as ADP added extracellularly. This is illustrated in Fig. 2. ADP added in high concentration (1.2 raM) to cardiomyocytes virtually doe~ not stimulate respiration in intact catdiomyocyt,~s (trace I) because of impermeability of the cellular membrane. However, after addition of saponin the re~plration rate is increased more than 5-times due to rapid lysis of sarcolemma, and oxygen consumption is linear with time. Recordings 2 and 3 in Fig. 2 show the method of determination of kinetic parameters, when saponin was added 2 rain prior ADP. Recording 3 was made in the presence of diadent~ine pentaphosphate to inhibit myokinase. In all cases respiration was linear with time after ADP addition, and no log period was observed. In cardiomyocytes mitoehondria are located between myofibrils throughout the cell. The respiration rate is determined by the local concentration of ADP or creatine available to adenine nueteotide lranslocase or mitochondrial creatine kinase, respectively [14,15~21]. That means that mitochondria inside eardiomyocytes can be considered as 'enzymatic probes" for the determination of average values of local concentrations of these substrates. Therefore, in this work we have determined the responses e! mitochondria in the isolated state or inside ¢ardiomyotstes to changes of ADP or croatine (at 0.2 mM ATP) concentrations in medium. If there are some intracellular restrictions for diffusion of some substances, the K= value for it may be significantly enhanced in cardiomyocytes [221. Detailed kinetic analysis of this situations is given below. Fig. 3 shows in double-reciprocal (Lineweaver-Burk) plots the results for isolated rat heart mltochondria in the absence and presence of saponin. These data gave the K= value for AdP equal to 17.6 _+ 1 ,aM that is in excellent accordance with the classical data by Chance and Williams [23], and the respiration parameters are not changed by saponin, showing good preservation of these structures, in aeeocdance with earlier data by Altsehuld et al. [18]. Fig. 4 shows the results of similar experiments done on isolated mitochondria and saponin-lysed eardiomyocytes when the creatine concentration was changed in the medium in the presence of 0.2 ram ATP and the respiration was activated through mitocbondrial creatine kinas¢ reaction, In both cases the dependonc~ ate vet,,/similar (Fig. 4A), giving the same K m value for ereatine equal of 6.0-6.9 mM (Fig. 4B). The same value for this ldnetie parameter was obtained when skinned fibers of cardiac muscle were studied (Table I!). This shows the absence of any significant restrictions in the concentration range used for diffusion of ereatine molecules from the surrounding medium to the sites of mitochondrial location in eardiomyocytes or in skinned fibers.

306

~=

°~ ~

NuN o

~

307 CM

CM

-

'~P 'It CM

I~ -

-1

1 2

/

rain ~

I~,..,i~. 1. m.

~85,7)

\ Fig, 2, O~graph traces of respiratory activities of mitochoncifia in permeabi|ised cardiac myocytes, Rates of respiration were measured as. described in Materials and Methods. (I. 7.) At the indicated lime points, eardinm~.,o~tes. CM {0.5 mg of protein/rot): salxmin. SAP (40 v,g/ml|; and ADP were added, (3) In addilion, the oX~'graph medium contained O,t ram AP.~A. The numbers radical,: the respiratory rates in n$ atoms O~ per kin per mg of pr, rein.

Fig. 5 shows the deoendence of the rate of ox3,gcn uptake by mitochondria inside saponin-treated cardiomyocytes on the ADP concentration in the medium. This dependence is surprisingiy slow if compared with data shown in Fig. 3. Maximal activation of respiration is seen at ADP concentrations around 0.7-1.0 raM, and the K= value for this substrate was found to be 250:1:38 pM (line i, Fig. 5B). This value exceeds the Km for ADP for isolated mitoehondria by more than an order (Table II). A very similar value for this parameter was found for skinned fibers (see Table ll). Thus, there is no difference in the K m for ADP between eardiomyocytes and skinned fibers, showing that intercellular connections after lysis of snrcolemma are not of too much importance for movement of ADP,

I

E

,

:("

a,

1

E

1/[~reatine], mM"~ Fi~. 4. Determination of K m for the creatine for saponin-treated earidac mot3des (I) aml mitod~ondria (21. IA) the dependence of the respiration rates on the concentration o[ creadne at 0,2 mM ATP, 031 Kinetic data in duuHe-reeiproca~, picots.

7~ 6~

o

i

o.,i

to

o12

o.~3

o:.4

o.5~ o'.~ -~.~

fAoP].~ .

il

g o.1-

o,:~

0.3

Fig. 3. Determination of K m for ADP for i~laled rat heart milfrehondria. Respiration velocities were measured by the u,'.~,graph method (see Materials and Methods). (D The dependenet: of the respiration rates on the ucneemration of ADP. (2) The same dependence in the presence of 40 F g / m l saT~onin.

>

! __

I

5

"i

1D

i

1~

[

20

25

t/IAD~, mM 1 Fi8. 5. Delerrnination of K~ for ADP for saponin-treated cardiac myopias. (A) The dependence of the Rspiration rat~
308 TA[]LE n

Kim'¢ic Furuplrt'a'r,~ ff)r ADP und C'r~r'¢ltiliZ"m prfluU~5 g2f ~tinudatiem o]"ox:duOI e phospht~o,latic~l

Me:in v:lltles :lrld S,D fire giv~ll |'|~r |our experiments. Prt'paralion ~,

Kt~ (/~M)

K~ (raM)

ADP

crculine (at fl.2 r,M ATP)

no ~dditions MJt,)chondria S~lpc~nin-rre~tted cardiomyacytes Skinned fibers

+AF~A

17.6,+ IJI 250.2 ± 38,5 263.7÷57.~

256.5 + 31.8

the diffusion of which seems to be restricted inside cardiomyocytes. The curve 2 in Fig. 5 shows the results of similar experiments to those done for curve 1, but performed in the presence of 25 mM ereafine. There is a sharp increase in the dependence of the respiration rate on the ADP concentration in the medium, and the Kr, value for ADP is decreased to 35.6_ 5.6 tzM. Very obviously, this effect is due to coupling of ereatine kinase and oxidative phosphorylation reactions in mitoehondria, and increased turnover of adenine nucleotides in these coupled reactions as it has been described in detail earlier [21,15]. An alternative explanation of the changes in K m by increased rate of ADP degradation through myokinase and subsequent deaminase reactions is excluded due to lack of the effect of myokinase inhibition by Ap~A on the values of kinetic parameters (Table I1). Discussion The results of this work confirm and clarify the early results by Kummel [24] and our previous work [25] and are in concnrd with some othcr date in the literature ~17,26]. Recently, Kommel [24] has shown that in isolated digitonin-lysed cardiomyocytes the apparent K m for ADP for oxidative phosphorylation decreased from 152 ~M to 45 ~.M when ADP was produced in intracellular ATPasc reactions that were explained by spatial proximity of mitoehondrial oxidative phosphorylation system and sarcoplasmie reticulum ATPasc. In our recent work with skinned fibers we found that the apparent K~ for ADP in activation of mitGchondrial respiration was unusually high, 300 #M, as compared to this parameter of isolated mitochondria [25]. However, the preparation of skinned fibers contains several hundred of cardiorny~cytes and the data may be equally interpreted as showing a decrease of diffusivity due to intercellular connections (and penetration from cell to cell) as it has been discussed by many authors [27-29]. Cooke and Pate have studied this question by using mathematical modeling and diffusion coefficients for

+(¢reat[ae) 13.65:4,4

fi,0 _+0,14

35.6_+5.6 79 -+8

5.67 ± 0.l I 6~25-+0.21

ADP and ATP in water solution [28]. According to their data, even without restricted diffusion ADP produced by ATPase appeared to accumulate in the fibers due to their significant sizes (fiber diameter was 70 ~m). Venlura-Clapier et al. tried to overcome this problem by decreasing the diameter of fihers and extrapolating the data to the sizes of eardiomyocytes [29]. However, the problem can be solved principally by using isolated eardiomyocytes to eliminate any difficulties related to intercellular contacts. In this work we have expanded the approach used by Knmmel [24] by comparing the kinetics of activation of mitoehondrial respirations by ADP and creatine. Both these suhstrates have similar diffusion distances from the medium into eardiomyocytes to the sites of mitochondrial location. However, ereatine is known to interact only with one enzyme, creatine kinase, with the apparent K,, around 6 raM and in lysed cardiomyocytes the bound ereatine kinase is mostly in mitoehondria [3]. Therefore, one may think that creatine should not have any diffusion difficulties in the physiological range of its concentrations because of the absence of intermediate binding inside the cell. This is exactly what was observed in this work: in all systems studied - isolated mitoehoudria, saponin-lysed myocyles and skinned fibers - the gm for creatine was equal to about 6 mM (see Table 1 and Fig. 4), On the contrary, there are abundant sites for binding of ADP within cardiomyocytes. Most part of the cellular ADP is firmly bound to aetin and the free ADP concentration in the cell has been estimated to be below 50 t~M [28.30]. The results of this work show that in saponin-treated cardiomyocytes the K m for ADP in the reaction of activation of mitoehondrial respiration is elevated by more than au order in comparison with the value of this parameter for isolated mitochondria. One of possible and rather trivial explanations of elevation of the K,~ for ADP is that it is rapidly used up by peripheral mitoehondria and, therefore, at low ADP concentrations in the medium this substrate does not reach mitoehondria in the *core' of the cells. Such a 'geometrical' explanation has earlier been considered

3119

by Mainwood and Rakusan [32]~ who proposed that in cardiac muscle cells mitochondria may be predominantly clustered peripherally closely to the capillaries to decrease the diffusion distance for oxygen. They supposed further that the energy for myofibrils in the cells' core is supplied via phosphocreatine pathway [32], That may be true in some cases of hypertrophied heart and may have a sense for oxygen diffusion which is limited by capillaries [33]; however, usually mitochondria are localized between and along myofibrils and in the cross-sectional plane they are distributed randomly but not concentrically [34]. If we suppose that ADP diffusivity is close to that in a water solution and equal in ail directions, the probability that it rcaches each mitochondrion, peripheral or inside cardiomyocytes is more or less equal. Also, this geometrical explanation predicts that in skinned fibers consisting of at least 20-50 cardiac cells in cross section K,~ for ADP should be significantly increased as ctm~parcd to saponin-treated eardiomyocytes. That is not true (TaMe II). Also, this hypothesis does not explain the effect of creatine on K m for ADP (see below), Thus, it is more probable that the increased K,. for ADP for saponin-treated cardiomyoeytes reflects significantly decreased diffusivity of this substrate due to its intermediate binding to the intraeellular structures. The dependence of K m art diffusion distance has been de~ribed directly by Dietschy for the effect of diffusion barriers on solute uptake, in our ease this is ADP uptake by mitochondria I221. The diffusion bartier in cardiomyocytes is most probably related to the intracellular organization and geometry of ADP binding structures. The K,. for ADP in saponin-trcated ca~diomyocytes is cLearly an apparent parameter since the diffusion distance for cxtracellularly added ADP is in a range from practically zero {or subsarcolemmal mitnchondria (see Fig. ID) to about tO v.m for mitoehondria in the center of cardiomyocytes [3I]. Therefore. the change in K~ reflects an averaged accessibility of ADP to all mitoehondria, that means that this effect may show an average local ADP concentration around mitoehondria at the distance of 5 t.tm from the surface. In the center ('core') of the cells the changes in apparent K,, for ADP might be even more remarkable. In the above-cited work by Cooke and Pate ADP has been found to inhibit the muscle fiber contraet;,m due to binding to the nucleotide sites of myosin with an effective K~ of 0.2-0.3 mM {28]. The similarities of the value of this kinetic constant and apparent Km for ADP for cardiomyoeytes reported in this work may be a coincidence or it may reflect the binding of ADP to the same sites under these two experimental conditions. Also, in numerous studies rapid association of ADP with myosin SI fragment has been directly shown, and Siemankowski and White have proposed that the

rate constant of ADP dissociation from acto-S]-ADP complex may be slow enough to bc the mol,:cutar step that limits the unloaded shortening velociq in cardiac muscle [35,36], AI~, glycoiytic enzymes with ADP bi:,ding properties may be associated with the myofibrillar structures [27.38]. The interaction of ADP with some other cellular components, such as cytoskeleton, is also not excluded. The retarded diffusivity of ADP by such an intermediate binding to myofibrillar proteins may lead to compartmentation of this substrate that is in concord with the data by McLellan c t a l . [39] on skinned muscle fibers who ,@owed the existence of ADP pool not accessible for mitochondria but acces.sible for rephosphorylation by myofibrillar creatine kinasc. An interesting sample of iahmnogeneity of adenine nucleotides (ATPI in cytoplasm has been described by Miller and Horowitz [40] for Rana papiens oocytes, It is most remarkable that in the presence of creatint in its physiological concentration. 25 raM. the value of tl~c K,. for ADP for saponin-treated myocytes is decre~scd again and approaches that for isolated mitochondria. Fig. 6 explains the effect of creatine on the apparent K., for ADP on the basis of earlier data at signiiicaat amplification of the ADP fluxes by coupled mitoehondrial creatine kinase-adenine nucleofide translocase-oxidative phosphorylation system in which adenine nucleotides are neoossz, ry ceractors but the end-product of energy producing reactions is phosphocreatine with P C r / O ratio dose to 3 [1-5,15.25]. This amplification ;s obviously an important intracellular factor for overcoming the decreased diffusivity of ADP C; AOP

"

--

&OF'

AOP

!!

011@

Fig~ ,t~ gehemati¢ pre~enlatinn or the interaction of ADP with itucrmediate binding site~ in cardic~mytX~les, permeabi]iscd by sitpnnin. In the presence of creatine amptificalion of tho sigmd nf ADP ('a'curs due to ils raanifoTd use in coupled ere aline kinase4~idalive phu.~pht~rylalion reat:tkm [e. rnilocho,dria (as it shown in the

frame).

310 in cardiomyocytes: only weak signal (ADP flux) from myofibri[s may be enough to significantly stimulate oxygen uptake in mitochondria, This result may show the importance of the treat(he kinase system in regulation of energy metabolism of the cells. This amplification (decrease of K~ for ADP) is difficult to explain on the basis of 'geometrical' hypothesis (see above): if all ADP is used up by maximally activated peripheral mitochondria, the effect of creatine should not be observable since creatine does not change state 3 rate of respiration [2,15] but stimulates respiration at submaximal ADP concentration [2,25]. In the intact cardiomyocytes with well-packed and crowded cytoplasm the ADP diffusion may be even more restricted due to increased number of binding sites and enhanced viscosity of the cell interior. However, two additional components of the creatine kinase system should be accounted for in vivo: facilitated diffusion of high-energy phosphate bound by the cytoplasmic equi!ibrktm creatine kinase reaction elegantly described by Meyer el al. [~,;.] blJt ignored by most other authors [7,42], and myofibriIlar treat(he kinase [29] which rephosphorylates ADP in situ in myofibrils at the expense of pho~phocreatine thus avoiding accumulation of ADP in these structures due to its production by actomyosin ATPase and its decreased dlffusivity. All these three components of the phosphocreatine pathway have been recently reviewed and described in details by Wall(mann and Eppenberger [43], and earlier by Jacobus [4], Bessman et al. [5], Saks et al. [1] and others [44-48]. Very recently, it has been shown [49] that complete and more or less selective inhibition of the croat(he kinase system of perfused rat heart by ioduacctamide resulted in decreased relaxation and contraction rates and elevatio~ of end-dlastolic pressure that deteriorated first the pumping function of the heart due to disturbances in the heart filling. Similar results were reported for ureafine-depleted hearts due to guanidine propionate diet, but the degree of disturbance was less because of preservation of some activi~ of the phosphocreatine pathway [50], and some adaptation of muscles to this state of energy deficiency was observed [51]. From these data and the results reported in this study one can conclude that the intraeellular energy transport by ereatine kinase system in heart, skeletal muscle and other types of cells [52,53] was developed during evolution, in particular, to match the necessity of rapid removal of ADP and to overcome its decreased diffusivity that is important for maintaining high energy fluxes and their effective regulation, especially at increased energy demand. (The old Japanese lady described by Ref. 54 reported to have only 2% of intact treat(he kinase preserved might not be ab[e to perform too much of work any mole and might had suffered from decreased re-

taxation; however, such a case should be verified by more numerous studies to be taken seriously.) In many recent works the steady state levels of phosphocreatine and AT]' have been found to be stable during workload increase (see Ref. 55) and decrease only when Ca activation of dehydrogenase reactions was inhibited in the aerobic heart by ruthenium red [56]. These data, however, do not argue against the importance of the creatine kinase system which seems to be very efficient in intracellular energy channeling (it is not a limiting step in these conditions to be observed), and intracellular metabolite levels seem to depend only on the balance of energy producing and consuming reactions. On the contrary, functional compartmentation of adenine nucleotides which local levels are controlled by the creatine kinase reactions including that in mitochondria most probably explains such a relationship between energy fluxes (oxygen consumption, heart work) and average metabolite levels in cytoplasm. References

I Saks,V.A+,Rosenshtraukh,L.V., $mirnov+V,N. and Chazov,E.L (1977) Can+L Phyfiol. Pharmacol. 56+69]-706. 2 Jacobum,W/2+ and Ingwall, J.S. t1980) Hearl CmealineIOnase, Williams,Wilkins Co., Baltimore. 3 Salts, V,A, Chemousova,G,B., Voronkov, lu.I., Smknov, V.N. and Chazov,E,I. 41974)Cir. Res. 34, (Suppl. liD, 138-149. 4 ]acobus, W.E. ([985) Annu. Roy. Phys[ol.47, 7115-725. 5 Bessman,S.P. and Geiger, PJ. (l~St) Science 211,44g-452. 6 Salts,V.A,, Ventura-Clapier,R, Khuchua,Z.A,, Pr¢obrazhensk'y, A.N. and Emdin, I.V. (1984) Biochim. Biophys,Acta 803, 254264. 7 Yo~hizaki, K., Watari, H, and Radda, G.K (1990) Bi0chim. Biophys, Acta 1051. 144-150. 8 Kushn~criek,M,J~ and Podolsky. RJ. (1963) Science 166, 12~'/1298.

9 Yoshizaki,K,, Radda. G.K., Inuboshi,T. and Chance, g. 41987) Biochim. Biophys,Acla 928, 36-44. 10 Yoshizaki, K.. Sup, Y.. blishikawa,H. and Morimoto,T. 41982) Biophys.J. 38, 209-211. It Veksler. V.I., Kuznutmv,A.V., Sharov,V.G, KapeSko,V.I. and Saks, V.A. (1987) Biochim.Biophys.Acla 892, t91-196. 12 Fob(alp, A, and Fob(alp, F, (1~)79)J, Physiol.(Paris) 75, 463-505. 13 Fabiato, A, 41981)J. Gun. Physiol, 78, 457-497. I4 Maughan,D. and Recchia,C. {t985) J. Physiol.368, 545=563. 15 Saks,V.A,, Kuznets~, A.V.. Kupriyanov.V.V., Miceli, M.V.and Jacohus. W.E. 0985) J. nioL Chem. 260, 7757-7764.. 16 Jaeobs, E,E, Jacob, M., Sanadi, D.R and Bradley. L.B. (1956)1. BiuL Chem. 223, 147-156. 17 Allschuid,R,, wenger, W.C, Lamka,K.G., Kindle,O.R, Capen, C.C., M.izuhira,V., Vander He(de, R.S. and Brierl~y,G~P.(1985) I. Biol. Chem. 260, 14325-14334~ 18 Ahschuld.R~,Hohl, C, Ansel,A. and nricrley,G.P. (1981)Alch, B[ochem. niuphys.209, 175-184. t9 Haworth, R.A., Hunter, D.R. and nerkoff, H,A. (198t) Cir. Res, 49) It 19-1128. 20 Saks.V.A., Seppel, E.K. and Liulina.N.V. 4197"/)Biokhimiya42. 579-$88 (in Russian), 21 Jaeobt~s,W,E, and Sake. V.A. (1982) Arch. Bioehem. Biophys. 219. 167-178.

311 22 Dielschy, JM. (19781 Mieroenvironments and Metabolic Cumlyartmentation, pp. 401-418, Academic Press. New York. 23 Chance, B. and Williams, G.R. (1956) Adv. Enz~mol. 17, 65-134. 24 Kommel, L. (1988) Cardiovasc. Res, 22, 359-~67, 25 Saks, V,A., Kapelko, V,I,, Kupriyanov. V.V., Kuznetsov, A.~., Laeomklu, V.L, Veksler, V.I., Sharov, V.G.. Ja~adov, S.A. and Seppet, EK. (19,q9)J. Mol. Cell. Cardiot 21, (Suppl. Ii, 67-78. 26 Levitsky, D.O. (1984) Methods in Studying Cardiol. Membranes, Vol. I, pp. 19 32, CRC Pfess, ~ c a Raton, FL 27 Seraydarian, K., Mommaerl~, W.F.HM. and Wallner, A. 11962) Bioehim. Biophvs. Acta 65~ 443-460. 28 Cooke, R. and Pale, E. (1085} Biophys. J. 48. 789-798. 29 Vcntura-Clapier, R., Mekhfi, H. and Vassorl, G- {1987) J. Gem Physiul, 89, 815-83;'. 30 Veeeh, R.L., Lawson, J.W,R., Cornell, N,W. and Krebs, HA. (1979) J. Biol. Chem. 254, 6538-654Z 3t Opie, L.H. {lg84) The Heart. pp. 136-1S3. Grune and Stralton. New York. 32 Mainwood, G.W. and Rakosan. K, t 1982) Can. J. Physiol. Pharmaeol. 60, 98 102. 33 Honig. C.R. and Gayeski, T.E.J. (t987) Adv. Exp. Med. Biol. 215, 309-32l. 34 Summer, J.R. and Jennings. R.B. (1986) in The heart and cardiovascular system 4H. Fozzard, el al.. eds.), Vol. 1, pp. 61-100), Raven Press, New York, 35 Siemankowski, R.F. and White, H.d. (1984) J. Biol. Chem, 259, 50*5-5053. 36 Siemankowski, R.F, Wiseman, M.O. and White, H.D. (1~5) Pron. Nat. #.cad. Sei. USA 82, 658-662. 37 Masters, C.J. (1978) Trends. Biochem. Sci. 3, 206-208. 38 Kueganov, B,L (1983) Oxganized Mullienzyme Systems: Catalytic properties, pp. 241~270, Academic lhess. New York. 39 Mdxllan, G,, Welshers, .4. and Winegrad, S. (1983) Am. I. Physiol. 245, 423-427. 40 Miller, D,S. and HorowilZ, S.B. 41986).!. Biol. Chem. 261,139ll13915. 41 Meyer, RA,, Lee Sweehey, Ft, and Kushmerick. MJ. 41984)Am. J. PMsiol. 246, C365-C377.

42 Katz. L.A.. Swain, J.A., Portma~. M.A. and Balaban, R.S. (lgB0) Am. J. Physiol. 256, H265-H27,.1, 43 Wallimann, T. and IFppenbexgcr, H.M. (191¢5)Cell and Muscle Motility, VOI. 6, pp. 239-285, Plenum Publishing, New York. 44 Gudbjarnason, S., Maches, P,G. and Ravens. K.G. 419"/0)L MoL Cell. Cardiol. L 325 339. 45 Seraydarian, M.W. and Abbott, B.C. (1976) J. Mol. Ce!l Uardiol. 8, 74l 746, 46 Ing,wall, l.S. and Fussel, E.T. 41983) Perspectives in Cardiovascular Research. Vol. 7. pp. 601-6t7. Raven press, New York, 47 Ingwall, J,S. 0983) Pelspeclives in Cardiov~cular Research, Vol. 8, pp. 145-155, Raven Press, New York. 48 Vial, C., Marcillat, O.. Goldsehmidt, D., Font, B. and Eichenbergcr, D. (1986} Arehi,~. Bkxhem. Blophys. 251,558-566. 49 Kaprlyanov, V.V~ Lakomkin, V.L., Steinschneide~, A,Ya., V~:ksler, VI., Korcbazhkina, O,V,, Kapelko. V,I,, and Saks. V.A. (1989) J. MoL Cell. Cordial. 21+ (SuPpl. 11) $30. ,50 Kapdku, V.I., Kupriyanou, VN., Novikova, NA.. Lakomkio. V.L., Sleinschoeider, A.Ya., Severina, M.Yu., Veksler, v.r. and Saks, V.A. (1988"1',I, Mol, Cert. Cardiol. 20, 465-479. ..51 Mekhfi. H., Hocrter, J.. Lauer, C,. Wisaewsky, C., Schwartz, g and Vemara-Clapier, R. (19901 Am. J. Ph~iol. 258. HllSlIq158. 52 Wallimann, T., Moser. H-. Zurbrlggen, H , Wegmann, G. and Eppenberger. H,M. (!986) J. Muscle Rex. Cell. Motil. 7, ~-34. 53 Wallimaan, T., Wegmann, G., Mnzer, H., Hnbcr, R, and Eppenberger, H.M. (19~6) Plot. Nat, Aead, S¢i. USA 83, 3816 3819. 54 Oita, T., Imoto+ S., Soma, M., Sakizono, K., Nakamuta, K, Hosomi, K., Fukuda, IC, Yamamiehi. H.. Shiraae, H,, Uehida, H , Kazakura. S., Koizumi, K. and Yoshikawa, 1. 41988) Jpn. J. Cllo. Pathol. 36, 1045-1050 (in Japanese). 55 Balaban. R. (19gO) Am. J. Physiol, 258 (Cell physiol. 27) ~377C389. 56 Katz, L,A,, Koretslqt, A,P, and Balaban, R,S. (1988) Am. J. Physiol. 24, HI85-H18&