Relationship of phosphorylation potential and oxygen consumption in isolated perfused rat hearts

Journal of Molecular and Cellular Cardiology (1980) 12, 891-907

Relationship of Phosphorylation Potential and Oxygen Consumption in Isolated Perfused Rat Hearts* J. GIESEN

AND H. KAMMERMEIERf

Department of Physiology, Medical Faculty of the ‘Technical University, Aachen, Melatener Strasse 2 11, D-5100 Aachen, Federal Republic of Germany (Received November 1979, accepted in revised form 4 February 1980) Relationship of Phosphorylation Potential and Oxygen Consumption in Isolated Perfused Rat Hearts. Journal of Molecular and Cellular Cardioloa (1980) 12, 891-907. In isolated mitochondria (State 4 to 3) respiration depends linearly on the phosphorylation potential (log [ATP]/[ADP] [Pi] of the medium. Experiments were performed to look for similar relationship in the intact myocardium. The following corrections for metabolite binding or compartmentation were carried out to obtain sarcoplasmic concentrations of the respective metabolites: (1) Correction for constant and proportional values according to literature. (2) Correction for ATP and Pi as (1) calculation of ADP from CPK equilibrium assuming a constant pH. (3) Correction as (2) assuming respiration dependent changes of cytosolic pH according to literature. (4) Calculation of Pi distribution correlated to H+ distribution according to literature. For (1) to (4) increasing coefficients of correlation (0.43 to 0.95) were obtained indicating that in the intact heart mitochondrial respiration is regulated in the same way as in mitochondrial suspensions by the phosphorylation potential or indirectly because of CPK equilibrium by the ratio [CrP] [H+] * KcPK/[Cr] ’ [Pi].

J. GIESEN AND H. KAMMERMEIER.

KEY WORDS : High energy phosphates ; Phosphorylation

potential ; Oxygen consumption ;

Isolated rat hearts.

1. Introduction The

most prominent

metabolism sumption.

are The

tissue

principal

detail in isolated studies a reciprocal phosphorylation oxygen oxygen

parameters

the

for the characterization

content

of high

relationship

energy

of both

of the myocardial phosphates

parameters

and has

been

energy

oxygen

con-

studied

in

mitochondria of heart and other tissues. According to these linear relationship exists between the logarithm of the so called potential,

consumption and substrates

i.e.

. [Pi]

and

provided a State 4 to a State 3 transition is considered, are available in excess [8, 9, 10, 14, 16, 28, 34, 42-461.

extramitochondrial

[ATP]/[ADP]

i.e.

An answer to the question, whether this relationship is valid for whole cells or organs like heart, is hampered by the difficulties of estimating the degree of binding and compartmentalization of the respective compounds when tissue * Preliminary results of this study were published partly in P&ers Archiv. European Journal of Physiology 379, 6 (1979). Journal of Molecular and Cellular Cardiology 11, 18 (1979). t With the co-operation of Mr E. Juengling. 0022-2828/80/090891+

17 $02.00/O

0 1980 Academic

Press Inc. (London)

Limited

892

J. GIESEN

AND

H. KAMMERMEIER

content of metabolites is assayed following tissue extraction. An approach to overcome these obstacles by calculation of the cytosolic concentration of ADP, the parameter most under question in this context, from the creatine kinase equilibrium assuming a constant pHi was published by Nishiki et al. [31, 321. They demonstrated that in principal a low phosphorylation potential is accompanied by increased oxygen consumptions and vice versa in isolated hearts. A similar result was yielded by Hassinen et al. [15] making a constant correction for ADP. The aim of the present study is to evaluate a relationship between the two aforementioned parameters in the isolated perfused rat heart analogous to that of mitochondrial suspensions. 2. Materials

and Methods

The experiments were carried out on isolated perfused rat hearts of female SPF Sprague-Dawley rats (180 to 220 g). The animals were anesthetized by ether and, following thoracotomy the hearts were immediately connected to a thermostated (38”C), non-recirculating perfusion system according to the Langendorff technique. The left ventricular pressure was recorded by a catheter inserted in the left ventricle via the mitral valve using a pressure transducer. The heart rate was evaluated from the pressure signal using an electronic rate meter. Perfusion pressures ranging from 40 to 140 cm H,O were used to alter intraventricular Lowest oxygen consumption values were pressure and oxygen consumption. obtained under cardiac arrest induced by elevated extracellular potassium concentration (15 mM) or low Ca2+ concentration (0.1 mM). Following preparation, the hearts were perfused for 10 min at perfusion pressure of 70 cm H,O. Thereafter, the perfusion pressure or the medium respectively were changed for a further 10 min. Subsequently the hearts were immediately frozen in freonR (- 150°C) and transferred to liquid nitrogen. Auricles and adherent perfusate were removed by dental drill. The perfusion medium was a bicarbonate Krebs-Henseleit-solution gassed by 95% 0, and 5% CO, (pH = 7.4) containing 5.5 mM glucose, 4.7 mM acetate and 2.5 mM pyruvate. In order to prevent oxygen deficiency at high oxygen consumption the perfusion medium contained 15% washed bovine red cells, and 4 g/l albumine was added to the medium to maintain red cell flexibility. Analytical procedure Oxygen consumption was evaluated using two Lex O,-Cona-oxygen analyzers (Lexington Instruments, Waltham, Mass., U.S.A.). These were used to assay the oxygen content of arterial and venous perfusate. For the estimation of oxygen

PHOSPHORYLATION

POTENTIAL

AND

OXYGEN

CONSUMPTION

893

consumption, arterial samples were taken from the storage vessel while venous samples were obtained from the venous perfusate dropping from the heart which was placed in a chamber filled with liquid paraffin, thereby preventing contact of venous perfusate with the oxygen of the ambient air. The volume used for oxygen estimation was 20 ~1. li0, values expressed in ~1 mine’ g-l W.W. were calculated from coronary flow and difference of arterial and venous oxygen content of the perfusate. In order to compare our data with those obtained in experiments with isolated mitochondria published in the literature, our values of VO, were converted to pg ATOM 0 min-* mg-1 mitochondrial protein by the following procedure. (1) The measured values of PO, were converted to the values of li0, expressed in kg ATOM 0 min-l g-l W.W. using the ideal gas equation of state. (2) To calculate li0, in pg ATOM 0 min-1 mg-l mitochondrial protein a content of 65 mg mitochondrial protein per g wet weight of heart [5] was applied. Additionally the oxygen consumption of isolated mitochondria assayed by other investigators [33, 161 which was obtained at different temperatures, was corrected to the condition of 38°C by application of a modified Arrhenius equation [29]. This calculation yields an activation energy of 67.63 kJ mol-l using a Qr,-value of 2.31 between 34 and 38.5% [II]. Metabolites were extracted from deeply frozen tissue powder by 0.3 N perchloric acid. Neutralization was performed by stepwise addition of KOH up to pH of 7.5 following precipitation and centrifugation of KClO,. The extract was analyzed for adenine nucleotides (ATP, ADP, AMP), creatine and phosphocreatine by High Pressure Liquid Chromatography [ 181. All these metabolites are compartmentalized or bound to protein to a different extent (Table 1). For calculation of sarcoplasmic metabolite concentration therefore corrections were performed according to the average data given in literature (Table 2) ~ Sarcoplasmic concentration of creatine and phosphocreatine were calculated assuming a cytosolic space of distribution of 60% wet weight, since compartmentation and binding seem to be negligible [Z]. The cystolic ADP-concentration, in addition to the corrections by constant values, was calculated from creatine-kinase-reaction, since this seems to be near the equilibrium in vivo. The equilibrium constant K

CPK

=

[ATPI PI [ADPl [pcl

[H+l

=

1.51’ 1o8M-1

[32, 391

and the intracellular hydrogen concentration lo-?.’ M [37] were taken from literature. Influence of Mg2+ was omitted though adenine nucleotides seem to be almost completely complexed to it, since influence of possible intracellular changes of Mg”+ concentrati-on can be expected to be small [25, 471.

0.15

et al. [36]

et al. [40]

Liver,

Kidney

Frog muscle

muscle 10%:

0.2-0.25

0.6:

0.25

ADP

8%

8-10%

Pi

to literature

0.65

0.291

0.167

6%

0.26

0.46t

0*44t

23%

Compartmentalized ADP ATP

and Pi according

g-r W.W.

.33*

1.3”

1.3-1.95*

1.95*

0.84-l

0.78*

in mitochondria Pi

(pmol.

* These values were converted from pmol. g-i mitochondrial protein to pmol. g-’ W.W. assuming 65 mg mitochondrial protein g-l W.W. according to Carafoli et al. [5]. t The calculations were made for wet weight assuming a relatively dry weight of 22% corresponding to our experimental conditions. : Bound and compartmentalized metabolities were summarized.

Pfaff

Seraydarian

Skeletal

et al. [25]

Liver

Liver

McGilvery

et al. [38]

Liver

Siess et al. [41]

Reynafarje

et al. [33]

Heart

La Noue et al. [23]

Ogawa

Frog muscle

Janke et al. [17]

et al. [ZZ]

Krause

and Skeletal

0.45

Heart-

0.22t

Skeletal

et al. [,?I]

et al. [ZO]

Krause

Kohn

et al. [ 151

Heart

Bound ADP

of ATP,

0.6:

muscle

ATP

and binding

Liver

muscle

compartmentation

Heart

Liver

concerning

Fony6 et al. [12]

et al. [I]

1. Information or o/0 resp.)

Hassinen

Akerboon

TABLE

PHOSPHORYLATION

TABLE

POTENTIAL

AND

OXYGEN

2. Average values taken from table 1 for calculation of the respective metabolites Compartmentalized pm01 g-r

ATP

0.2

of sarcoplasmic

concentrations

Cytosolic concentration pm01 g-l

Bound pm01 g-r

0.1

895

CONSUMPTION

(x -

0.3) . 5* 3

ADP

0.4

0.2

(x -

0.6) * 5 3

Pi

1.3

8%

[x -

-

(0.08x)

1.7t]

. 5

3 * Factor 5/3 results assuming a cytosolic water content of 60% wet weight. t This value is the sum of mitochondrial Pi (1.3 pmol g-r) and extracellular The parameter x is the analysed tissue content of the respective metabolites.

Pi (0.4 pmol g-l).

3. Results The data of tissue metabolites obtained by analysis were processed in different ways. First corrections were carried out assuming a constant extent of compartmentation and binding for the respective metabolites (cf. Table 2). This approach results in the relationship shown in Figure 1. There is a significant correlation (P < 0.01) between VO, and the phosphorylation potential; however, because of the low coefficient of correlation a meaningful regression function cannot be calculated. Figure 2 demonstrates the relationship which is obtained, if ADP is calculated from the creatine kinase reaction. The coefficient of correlation (r = 0.70) is appreciably higher than in Figure 1. In this case a meaningful regression straight line and function can be evaluated. However, the question remains whether a straight line is the best fit, particularly concerning the uppermost data points. In Figure 2 the cytosolic ADP-concentration was calculated from CPKreaction assuming a constant intracellular pH of 7.1. However, the chemiosmotic theory of oxydative phosphorylation [27] is based on the existence of a proton gradient across the inner mitochondrial membrane. The resulting proton potential depends on the respiration activity [3, 26, 301. Accordingly, the cytosolic pH can be postulated to increase with ADP-induced respiration rate and vice versa. In experimental assays of intracellular pH this fact was not respected until now. But checking the data of literature reveals that the apparent scattering of pHvalues [37] might be partly due to different rates of respiration of the various tissues.

896

J.

GIESEN

AND

H.

KAMMERMEIER

++ + +++ +

r=O.43

+ +r+ y**+ + 3, ++cj;+ ++ + “+++ + ++

FIGURE 1. Relationship The corrections concerning

of the phosphorylation potential and ii0, in isolated perfused rat hearts. the cytosolic concentrations were performed as described in Table 2.

T

.E E

6.0 y = -3.49 I- = 0.83

7

x +14.53

+

f 0

5

4.0

-

2.0

-

5 ? .p

I 1.0

I 3.0

I 2.0

I 4.0

I 5.0

[ATPI log

[ADP].

[Pi]

FIGURE 2. Relationship between phosphorylation potential and li0, of isolated perfused rat hearts. The correction for cytosolic ADP concentration was performed by using the CPK-equilibrium at a constant pH of 7.1; the other factors were corrected as described in Table 2.

PHOSPHORYLATION

According potential

POTENTIAL

to the investigations

of Nicholls

CH’Im =

-

W+lc

AND

59 ApH

OXYGEN

[30]

CONSUMPTION

the electrochemical

897 proton

(at 23°C) = AEn

contributes about 33% to the total membrane potential Ap = AE,+AEH (proton membrane motive force according to Mitchell et al. [26] of th e inner mitochondrial at the State 4. During ADP controlled respiration the electrical potential AE, and the electrochemical proton potential AEn decrease by different extent, resulting in a contribution of AEn of about 60% of the total alteration of Ap (AAp) at the respiration rates in the experiments of Nicholls [30]. These results are in principle consistent with the theory of chemiosmotic coupling developed by Mitchell [ 271. The aforementioned relationship between ADP-induced respiration rate and ApH was used to evaluate the total contribution of AEn to the total alteration of Ap given by Azzone and co-workers [3] during various ADP induced respiration rates This results in the linear relationship between oxygen consumption (PO,) and ApH demonstrated in Figure 3. Assuming a distribution volume of cardiac matrix space of 20% of the cell volume (60% of the mitochondrial volume of 34% of myocardial volume [35])

1.2

0.8 t/ I

/

a

y=-0.245

x Cl.109

I = 0.98

0.4 I

I

I 0.6

I 0.3 i0,

pg

ATOM

0 lmg-’

I 0.9 mine’

FIGURE 3. Relationship between VO, vs. ApH across the inner mitochondrial derived from the data published by Azzone et al. [3] and Nicholls [30].

membrane

898

J.

GIESEN

AND

H.

and an equal buffer capacity of cytosolic which describes the relationship between pH, (y) was evaluated (r = 0.98) :

KAMMERMEIER

and matrix space, the following function the respiration rate (x) and the cytosolic

y = 0.061~ + 6 (1) The zero degree coefficient 6 can be determined most reliably using the estimation of myocardial pH by microelectrodes as published by Ellis et al. [7], since evaluation of pH, by other methods suffers from systematic errors brought about by intracellular compartmentation of protons [37]. The resulting pH of 7.14 [7] for arrested hearts according to our data can be related to an average VO, of 0.87 [*g ATOM 0 mg-1 min-1. These values inserted in function (1) yield the following function for the relationship between VO, and pH,:

(Y = 0.98)

y = 0.061~ + 7.088 (2) The plot in Figure

4 exhibits

this function

and the data given

by Azzone

et al.

[31* Figure 5 demonstrates the relationship between the phosphorylation potential and the oxygen consumption of isolated rat hearts resulting from the calculation of cytosolic ADP from creatine kinase equilibrium using the aforementioned 7.15,

+

;+/ 7. IO +

i I

y = 0.061

a

x t7.088

r=0.98

7.05

I 1 I

0.3 lh,

pg

ATOM

I

I

0.6

0.9

0 mg-’

mine’.

FIGURE 4. Correlation VO, vs. pHe from the data of Azzone corresponds to VO, in our experiments in arrested hearts, the pHi Ellis et al. [7].

et al. [3]. The highest VO, of which is 7.14 according to

PHOSPHORYLATION

7 .c E

6.0

POTENTIAL

y = -3.56 I =

x

AND

OXYGEN

899

CONSUMPTION

+15.29

+

0.70

T

+ + ++

r

+ \t +$ +++ ++++ 3 *+++j ++++,+ ++‘7 + $ I 1.0

I

I

I

2.0

3.0

4.0

log

I 5.0

[ATPI bDP]- [Pi 1

FIGURE 5. Relationship of phosphorylation potential vs. I’O, in isolated perfused hearts. In addition to the data in Figure 2 the cytosolic pH was corrected for its dependence on VO, (for detailed description of correction see text).

correction for cytosolic pH. As can be seen a close correlation results. The coefficient of correlation (I = 0.83) is appreciably higher as compared to that of Figure 2. Thus, correction for a respiration rate dependent pH, improves the relationship markedly indicating that this correction takes in account the real cytosolic pH changes. According to the investigations of several authors [4, 13, 191 the distribution of Pi-concentration is not constant as assumed in the previous sections. These studies indicate rather, that the Pi distribution is dependent on ApH across the inner mitochondrial membrane. Therefore, additionally, a calculation was performed to take in account this ApH-dependence of Pi distribution between sarcoplasm and mitochondrial matrix space in order to estimate the real value of the cytosolic concentration of Pi. The details of this calculation are presented in the Appendix. After performing this correction for [Pile (cytosolic Piiconcentration) the correlation shown in Figure 6 is obtained. As can be seen the data scattering is much less than in Figure 5 yielding a very close correlation (r = 0.95) between the phosphorylation potential and the oxygen consumption in isolated perfused rat hearts. Thus, this correction for ApH dependent cytosolic Pi-concentration apparently reflects the real condition in isolated perfused hearts. Considering the close relationship between the phosphorylation-potential and M.C.C.

20

900

J.

CIESEN

AND

H.

KAMMERMEIER

8.0

T

f

6.0 y = -2.36 I = 0.95

T ?

x +11.30

0

4.0

-

2.0

-

5 k “i *p

I

I 3.0

I 2.0

1.0

log

FIGURE In addition

1 4.0

I 5.0

[ATPI CADPI . Cpi 3

6. Relationship of the phosphorylation potential vs. I’O, in isolated perfused hearts. the cytosolic Pi concentration was corrected foI; it:, ApH dependence (see text).

oxygen consumption, one has to take into account that the ADP concentrations are not estimated directly but calculated from the CPK-equilibrium constant. This, however, includes the phosphorylation potential

[ATPIc 19

CADPl,

after substituting [ADP] CPK-reaction equation.

Ipilc

is

equal

to

log

in the phosphorylation

WI c W+l c KCPK CCrl, [WC potential

by [ADP]

from

the

4. Discussion of this study was the evaluation of a relationship between the >hosThe intention phate potential and oxygen consumption in isolated hearts, which resembles Lhat obtained in suspensions of isolated mitochondria in State 4 to State 3 transition. To overcome difficulties of compartmentation and binding of the respective metabolites various corrections were performed. In a first approach a constant correction was made according to the data given in the literature (c.f. Tables 1 and 2). The resulting data yields a significant correlation, but the low coefficient of correlation (7 = 0.43) may not be used to evaluate a meaningful regression function.

PHOSPHORYLATION

POTENTIAL

AND

OXYGEN

901

CONSUMPTION

The main problem of estimation of cytosolic phosphorylation potential concerns the calculation of the true cytosolic ADP-concentration. This was overcome by calculation of cytosolic ADP-concentration from the CPK-reaction, which this correction for is assumed to be near equilibrium in VZUO.After performing [ADP], a markedly closer correlation was obtained (r = 0.70). The same calculation was carried out by Nishiki et al. [32, 151. Their data show a lowered phosphorylation potential to be accompanied by an increased oxygen consumption, which is in principal agreement with the observed behaviour of isolated mitochondria and our data. However, in their studies a correlation was not calculated. In the calculation of [ADP], from CPK-reaction the cytosolic H+-concentration In the previous calculation the pH, is a factor of CPK equilibrium equation. was taken constant (pH, = 7.1) as in calculations of the afore-mentioned authors [31, 321. However, if the chemiosmotic theory of oxidative phosphorylation [27] is valid in vivo, then the changes of the respiration rate are accompanied by alterations of ApH across the inner mitochondrial membrane and correspondingly by changes of pH,. After performing the respective correction for pH, at various pie, according to Figure 4 a close relationship (r = 0.83) between both parameters of oxidative phosphorylation is achieved (Figure 5). The further correction for cytosolic Pi concentration dependent on ApH across the inner mitochondrial membrane [4, 13, 191 leads to a still closer relationship (r = 0.95 in Figure 6). The function achieved can be compared to that relationship obtained in isolated dog heart mitochondria (Figure 7). It also has a similar slope (-2.3 1 for mitochondria, -2.36 for isolated perfused hearts) and a similar x intercept (f5.12 for mitochondria, $4.79 for isolated hearts). This agreement between in vitro and in viva experiments and the step by step increased degree of correlation, can be considered as evidence that in the intact isolated organ the mechanisms involved in control of respiration are consistent with those elaborated from experiments in isolated mitochondria. Thus, besides our assumption concerning distribution space of cytosolic and matrix volume and the buffer capacity of both, the following conditions seem to be fulfilled in the isolated perfused hearts in our experiments : (a)

State 4 to State 3 transition,

(b)

equilibrium

(c)

control

(d)

coupling of oxydative phosphorylation theory and the pH-gradient involved,

(e)

the distribution of inorganic phosphate inner mitochondrial membrane.

In addition not dependent

conditions

of respiration

for creatine

kinase reaction,

by the phosphorylation

potential,

according dependent

to the chemiosmotic on ApH

these results indicate, that the mitochondrial respiration on an additional respiration stimulating mechanism.

across the in v&o is Especially

202

902

J. GIESEN AND H. KAMMERMEIER

8.0

7 .E E

6.0

y = -2.31 r = 0.96

I 1.0

x + Il.83

I

I

I

2.0

3.0

4.0

log

I 5.0

[ATPI bDPl* [PiI

FIGURE 7. Relationship of the phosphorylation potential and liO, of isolated dog heart mitochondria. The values published by Holian et al. [16] were converted to our experimental conditions according to the description in methods.

the CaZ+ ions are under discussion in this context [S, 241. Apparently the accumulation of calcium by the mitochondria does not affect the respiration in uiuo by a significant extent probably because of its low sarcoplasmic concentration which might be similar to those of EDTA containing mitochondria suspensions. Finally it should be mentioned, however, that the equilibrium theory of respiratory control as well as the chemiosmotic theory, which are basic concepts of this paper do not sufficiently explain all experimental data observed in this field. Both concepts therefore are not unanimously accepted. Nevertheless, our results do not give any reason to question their validity. Acknowledgements

We wish to thank Mrs S. Eshimokhai for the skilful technical assistance. This work was supported by Grant No. Ka 337/4-5 from the Deutsche Forschungsgemeinschaft. REFEREXCES 1.

AKERBOOM, T. P. M., BOOKELMAN, H., ZUURENDONK. P. F., VAN DER MEER, R. & TAGER, J. kI. Intramitochondrial and extramitochondrial concentrations of adenine nucleotides and inorganic phosphate in isolated hepatocytes from fasted rats. European Journal of Biochemistry 84, 4 13420 (1978).

PHOSPHORYLATION 2. 3.

4.

5.

6.

8.

9.

10.

11. 12. 13. 14.

15.

16.

17.

18.

19. 20.

POTENTIAL

AND OXYGEN CONSUMPTION

903

of heart ALTSCHULD, R. A., MEROLA, A. J. & BRIERLEY, G. P. The permeability mitochondria to creatine. journal of Molecular and Cellular Cardiology 7, 1-5l-162 ( 1975). AZZONE, 6. F., POZZAN, T., MASSARI, S. & BRAGADIN, M. Proton electochemical gradient and rate of controlled respiration in mitochondria. Biochimica et biophy.rica acta 501, 296-306 (1978). BOHNENSACK, R. & KUNZ, W. Mathematical model of regulation of oxidative phosphorylation in intact mitochondria. Acta biologica et medica germanica 37, 97-l 12 (1978). CARAFOLI, E., TIOZZO, R., LUGLI, G., CROVETTI, F. & KRATZINC, C. The release of calcium from heart mitochondria by sodium. 2ournal ofhfoolecuiar and Cellular Cardiology 6, 361-371 (1974). CHANCE, B. The energy-linked reaction of calcium with mitochondria. Journal of Biological Chemistry 240, 2729-2748 ( 1965). ELLIS, D. & THOMAS, R. C. Direct measurement of the intracellular pH of mammalian cardiac muscle. Journal of Physiology 262, 755-77 1 (1976). ERECINSKA, M., KULA, T. & WILSON, D. F. Regulation of energy metabolism: Evidence against a primary role of adenine nucleotide translocase. FEBS Letters 87, 139-111 (1978). ERECINSKA, M., VEECH, R. L. & WILSON, D. F. Thermodynamic relationships between the oxidation-reduction reactions and the ATP synthesis in suspensions of isolated pigeon heart mitochondria. Archives of Biochemistry and Biophysics 160, 41%-12 1 (1974). FERGUSON, S. J. & SORGATO, M. C. The Phosphorylation potential generated by respiring bovine heart submitochondrial particles. Biochemical Journal 168, 299-303 (1977). FISHER, R. B. & WILLIAMSON, J. R. The oxygen uptake of the perfused rat heart. Journal of Physiology 158, 86-101 (1961). FONY~, A. Sr LIGETI, E. The Role of intramitrochondrial Pi in stimulation of respiration by calcium and strontium. FEBS Letters 93, 289-292 (1978). FONY~, A. Sr LIGETI, E. Intramitochondrial phosphate is the source of protons in the response of liver mitochondrial to cations. FEBS Letters 96, 343-3-1-5 (1978). GARFINKEL, D., ACHS, M. J. & DZUBOW, L. Simulation of biological systems at the level of biochemistry and physiology. Federation Proceedings. Federation of American Societies for Experimental Biology 33, 176-182 (1974). HASSINEN, I. E. & HILTUNEN, K. Respiratory control in isolated perfused rat heart. Role of the ecluilibrium relations between the mitochondrial electron carriers and the Xdenylate system. Biochimica et biophysics acta 408, 319-330 (1975). HOLIAN, A., OINEN, C. S. & WILSON, D. F. Control of respiration in isolated mitochondria: Quantitative evaluation of the dependence of respiratory rates on (ATP), (ADP), and (Pi). Archir~esofBiochemistryandBiophysics 181, 164-171 (1977). JANKE, J. Die Aufteilung des intrazellularen ADP, ATP und Orthophosphats in der ruhenden tatanisch gereizten Froschmuskulatur mittels einter fraktionierten. Extraktion. PJcgers Archiu ftu die gesamte Physiologie des Menschen und der Tiere 300. 1-22 (1968). J~:NGLINC, E. & KAMMERMEIER, H. Rapid assay of adenine nucleotides or creatine compounds in extracts of cardiac tissue by paired-ion reverse-phase high-performance liquid chromatography. Analytical Biochemistry 102, 358-36 1 ( 1980). KLINGENBERG, M. Metabolic transport in mitochondria: An example for intracellular membrane function. Essays of Biochemistry 6, 119-159 (1970). KOHN, M. C., ACHS, M. J. & GARFINKEL, D. Distribution of adenine nucleotides in the perfused rat heart. American Journal of Phy.&ou 232, 158-163 (1977).

904 21. 22.

23.

24. 25.

26.

27. 28.

29. 30.

31.

32.

33.

34. 35.

36.

37. 38.

39.

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AND

H. KAMMERMEIER

E. G. & WOLLENBERGER, A. Auftrennung des Adenosindiphosphatpools des Skelettmuskels bie Kaninchen. Acta biologica et medica germanica 13, 7-12 (1964). KRAUSE, E. G. & WOLLENBERGER, A. Extraktionsprozedur zur Bestimmung des wahren Orthophosphats in Herz- und Skelettmuskel von Warmbltitern. Biochemische zeitschr;ft 339, 3 15-326 (1964). LA NOUE, K. F., BRYLA, J. & WILLIAMSON, J. R. Feedback interactions in the control of citric acid cycle activity in rat heart mitochondria. Journal of Biological Chemistry 247, 667-679 (1972). LEHNINGER, A. L., CARAFOLI, E. & ROSSI, C. S. Energy-linked ion movement in mitochondrial systems. Advances in EnrymoloQ 29, 259-320 (1967). MCGILVERY, R. W. & MURRAY, TH. W. Calculated equilibria of phosphocreatine and adenosine phosphates during utilization of high energy phosphate by muscle. Journal of Biological Chemistry 249, 5845-5850 (1974). MITCHELL, P. & MOYLE, J. Estimation of membrane potential and pH-difference across the Cristae membrane of rat liver mitochondria. European Journal of Biochemistry 7, 471-484 (1969). MITCHELL, P. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. .Nature 191, 144-148 (1961). MURAOKA, S. & SLATER, E. C. The redox states of respiratory-chain components in rat-liver mitochondria. II. The “crossover” on the transition from state 3 to state 4. Biochimica et biophysics acta 180, 227-236 (1969). NETTER, H. Theoretical Biochemistry, pp. 608-610. Edinburgh: Oliver & Boyd (1969). NICHOLLS, D. G. The influence of respiration and ATP hydrolysis on the protonelectrochemical gradient across the inner membrane of rat liver mitochondria as determined by ion distribution. European Journal of Biochemistry 50, 305-3 15 ( 1974). NISHIKI, K., ERECINSKA, M. & WILSON, D. F. Energy relationship between cytosolic metabolism and mitochondrial respiration in rat heart. American Journal of Physiology 234, 73-81 (1978). NISHIKI, K., ERECINSKA, M., WILSON, D. F. & COOPER, S. Evaluation of oxidative phosphorlyation in hearts from euthroid, hypothroid and hyperthroid rats. American Journal of Physiology 235, 212-219 (1978). OGAWA, S., ROTTENBERG, H., BROWN, T. R., SHULMAN, R. G., CASTILLO, C. L. & 31P nuclear magnetic resonance study of rat liver mitoGLYNN, P. High-resolution chondria. Proceedings of the National Academy of Sciences 75, 1796-1800 (1978). OWEN, C. S. & WILSON, D. F. Control of respiration by the mitochondrial phosphorylation state. Archives of Biochemistry and Biophysics 161, 581-591 (1974). PAGE, E., MCCALLISTER, L. P. & POWER, B. Stereological measurements of cardiac ultrastructures implicated in excitation-contraction coupling. Proceedings of the National Academy of Sciences 68, 1465-1466 (1971). PFAFF, E., KLINGENBERG, M., RITT, E. & VOGELL, W. Korrelation des unspezifisch permeablen mitochondrialen Raumes mit dem “Intermembran-Raum”. European Journal of Biochemistry 5, 222-232 (1968). POOLE-WILSON, P. A. Measurement of myocardial intracellular pH- in pathological states. Journal of Molecular and Cellular Cardiology 10, 51 l-526 (1978). REYNAFARJE, B. & LEHNINGER, A. L. An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function. Proceedings of the National Academy of Sciences 75, 47884792 (1978). ROSE, I. A. The state of magnesium in cells as estimated from the adenylate kinase equilibrium. Proceedings of the National Academy of Sciences 61, 1079-1086 (1968). KRAUSE,

PHOSPHORYLATION 40.

41.

42. 43.

44.

45.

46.

47.

POTENTIAL

905

AND OXYGEN CONSUMPTION

SERAYDARIAN, K., MOMMAERTS, W. F. H. M. & WALLNER, A. The amount and compartmentalization of adenosine diphosphate in muscle. Biochimica et biophysics acta 65, 443-460 (1962). SIESS, E. h. & WIELAND, 0. H. Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells. Biochemical Journal 156, 91-102 (1976). SLATER, E. C., ROSING, J. & MOL, A. The phosphorylation potential generated by respiring mitochondria. Biochimica et biophysics acta 292, 534-553 (1973). THAYER, W. S., Tu, Y.-S. L. & HINKLE, P. C. Thermodynamics of oxidative phosphorylation in bovine heart sub-mitochondrial particles. Journal of Biological Chemistry 252, 8455-8458 ( 1977). WILSON, D. F. & DUTTON, P. L. The oxidation-reduction potentials of cytochromes M and cc3 in intact rat liver mitochondria. Archives of Biochemistry and Biophysics 136, 583-584 (1970). WILSON, D. F., OWEN, C., MELA, L. & WEINER, L. Control of mitochondrial respiration by the phosphat potential. Biochemistry and Biophysics Research Communication 53, 326-333 (1973). WILSON, D. F., OWEN, C. S. & HOLIAN, A. Control of mitochondrial respiration: A quantitative evaluation of the roles of cytochrome and oxygen. Archives of Biochemistry and Biophysics 182, 749-762 (1977). GEVERS, W. Generation of protons by metabolic processes in heart cells. Journal of Molecular and Cellular Cardiolog 9, 867-873 (1977).

Appendix The

calculation

was carried

of the cytosolic

out starting

from

log W-1, -

[Pi-lc

and

according

concentration the following

= pH,

-

of inorganic

pH,

[4, 13, 191

= ApH

PH,

= pKa,

pH,

= pK,,

pH

[Pit,t],

(1)

+ log

[Pi”-], ,pi~lc

=

+ log E m

(5) inserted

pH,

[Pi”-1,

+

(3)

(4)

[Pi-],

= [Pi”-lc + [Pi-lc

[PitOtlc (4) and

(2)

we have [PbiJm

Equations

=

to Henderson-Hasselbalch

1 at physiological

phosphate

equations:

in Equations

= p&

(5)

(3) and

+ log rpitot1;.,

(2) yields

cpi-lm 1 In

(6)

906

J.

GIESEN

AND

H.

KAMMERMEIER

and

pHc = p& Equations

+ log [pitotl&

(6) and (7) solved for [Pi-lm

Equations

rpi-lc 1 c

or [Pi-lC respectively

(7) result in

Lpi-lm = [Pit& . [H+lm J&Z+ [H’lm

(8)

Lpi-l = F%& . EH+lc c Kr2 + [H+lc

(9)

(8) and (9) inserted

in Equation

(1) yields

[Pitdm * W+l, --. [H+lc F’itdc . W+lc Ka2 + [H+lm [H+l m L2 + [H’lc Assuming

an extracellular

space volume

0.75

volume.

we have :

V, . Pitotl~ + Vm . [Ph3tlm

Pila -zzz V, = cytosolic

of 25% of wet weight

(10)

vc + VI0

Vm = mitochondial

matrix

volume.

Where [Pi,] is equal to the total cellular free inorganic phosphate obtained by substraction of extracellular inorganic phosphate (0.4 PM g-l) and 8% bound phosphate (c.f. Table 2) according to the following equation

[P& = [Piltiss- ([Wtiss . 0.08+ [Wext,,,.) [Pi]tiSs = total analyzed [Pilextrac

inorganic

= extracellular = 0.4

since volume

PM

9-l

VC

v

In

[Wa -

0.75 (12) inserted 5 -

[P& 0.75

-

of tissue.

= 4

(11) Equation 5

Equation

content

concentration

W.W.

relationship

follows from Equation

phosphate

phosphate

in Equation

4 [Pitotlc

. Kc32

(12) 4

[Pit0tlO

=

Pitotlm

(10) yields

IX+1m =-. W+lc Wtotlc . W+lc + W+lm v+1 m &2 + [H+lc ’

(13)

PHOSPHORYLATION

POTENTIAL

AND

OXYGEN

CONSUMPTION

Equation (13) solved for [PirOtlC results in the real cytosolic concentration 5 [PitOtla 0.75 Pitt0tl,=

cH+lc -.

[H+l m

[PimlC = cystolic [Pi-],

= matrix

[Pi2-]C = cytosolic

K,2

CH+l m * K,z + [H+lm

4 [Hi], W+l c + W’lc + Ku + [H+lm

[H,PO,-] [H,PO,-] [HP0,2-]

[Pi”-],

= matrix

[HPOd2-]

[Pi&J,

= cytosolic

total inorganic

[Pi,,,],

= matrix

K,,

inorganic

total inorganic

= second dissociation

phosphate phosphate

constant

concentration. concentration.

of inorganic

phosphate.

907 phosphate