j Mol Cell Cardiol 17, 727 731 (1985)

EDITORIAL REVIEW

T h e C y t o p l a s m i c P h o s p h o r y l a t i o n P o t e n t i a l

Its Possible Role in the Control of Myocardial Respiration and Cardiac Contractility

(Received 2 July 1984, accepted in revised form 29 August 1984)

Introduction Respiratory control of mitochondria was first elucidated by Chance and Williams [3] who characterized five recognizable states and, in particular reference to living muscle cells, focussed on State 3 to State 4 transitions and the role of ADP. Since that time there has always been a recognition of the role of intra- cellular [ADP] and there have been further elaborations of the concept such as the adeny- late energy charge hypothesis of Atkinson [-2]. I t can be shown that in certain physiological circumstances neither of these two criteria satisfactorily predict cellular energy flux and in recent years there has been considerable interest in the role of the cytoplasmic phos- phorylation potential in cellular function [6, 9, 12, 31]. Now the phosphorylation potential is simply equal to [ATP] / [ADP][PI] but its calculation requires the determination of the

free cytoplasmic concentrations of [ADP] and [Pi] as opposed to the analytically measured values. This article looks at the implications of some recent results and concentrates in parti- cular on data in the cardiac literature.

In 1975 Hassinen and Hiltunen [12], using surface fluorometry and spectrophotometry on resting and beating rat hearts, provided evidence that there is a near equilibrium between the phosphorylation state of the adenine nucleotides and the redox state of the respiratory carriers; this was taken as experi- mental evidence confirming the thermodyna- mic equilibrium hypothesis of Klingenberg [16]. This hypothesis has also been supported by the experiments of McGilvery and Murray

[19]. Subsequently, Wilson and colleagues [6, 28, 29] developed, and experimentally tested, a model of mitochondrial respiratory control where respiratory rate is determined by three variables (1) the availability of reducing sub- strates which determine the mitochondrial [ N A D + ] / [ N A D H ] ; (2) the cellular energy supply as expressed in the cytosolic phos- phorylation potential and (3) the rate of oxi- dation ofcytochrome c by cytochrome oxidase and molecular oxygen, which is dependent upon cellular oxygen tension. In 1978 Nishiki et al. [22] applied these concepts to isolated rat heart (Langendorff perfusion) which was made to use energy at different rates. The con- centrations of free cytoplasmic ADP and AMP were calculated from the near equi- librium of the creatine phosphokinase and adenylate kinase reactions [see 13, 31]. The authors found that increasing the respiratory rate by various physiological and pharmacol- ogical manipulations also caused a fall in the cytosolic [ATP] / [ADP][Pi ] ratio.

Further support for the concept's applica- bility to cardiac tissue came from Giesen and Kammermeier [9] who experimented with isolated rat hearts; these authors convincingly showed that respiration depended linearly upon the log of the cytosolic phosphorylation potential. The correlation coefficient was increased from 0.43 to 0.95 by (i) a consider- ation of compartmentat ion of the respective metabolites; (ii) correction for ADP binding (mainly to actin); (iii) consideration of the effects of a change (increase) in the cytosolic pH with increased respiratory activity which

0022-2828/85/080727 + 05 $03.00/0 �9 1985 Academic Press Inc. (London) Limited

728 C. Gibbs

alters the creatine phosphokinase equilibrium (see below)--this is a prediction of Mitchell's chemiosmotic theory of oxidative phos- phorylation [21], and (iv) a consideration of the effect of the respiration induced pH change upon the distribution of Pi between the sarcoplasm and mitochondrial matrix space.

Before leaving this brief consideration of the role of the cytoplasmic phosphorylation potential on respiration rate it should be men- tioned that not all authors accept the concept of maintenance of near thermodynamic equi- librium between the cytosolic adenine nucleo- tide phosphorylation potential and electron transport at the first two phosphorylation steps [27, 30]. Williamson [27] believes that at the present time it is impossible to determine whether respiratory rate is regulated by the proton electrochemical potential via kinetic regulation at the adenine nucleotide trans- locator step or by additional interactions at other sites and in a recent review he focuses attention on the mechanisms regulating the production and utilization ofacetyl-CoA.

Even though the debate is far fi~om settled and although it is evident that there are many feedback pathways controlling the three vari- ables referred to above, the importance of the cytosolic phosphorylation potential cannot be denied. This relatively simple concept has given us considerable insight into the pheno- menon of tissue 'oxygen sensing' over the physiological range of oxygen tension [29]. This problem has been in the physiological literature for decades and has been elegantly discussed by Honig [14] in a recent review.

T h e free e n e r g y c h a n g e for ATP h y d r o l y s i s

The free energy change for a chemical reac- tion is a differential quantity as Wilkie [25, 26] has pointed out so that to give a value for in vivo AG is in a sense misleading as the actual free energy change will depend upon the degree of chemical advancement of the reac- tion (for a clear discussion of this point see, Atkinson, [2]. Wilkie has argued that we should use the term affinity (A) or free energy change (dG/d~) where { (measured in mol) denoted the degree of advancement of the

chemical reaction under consideration. In this article the concept in relation to the in vivo hydrolysis of ATP is considered.

A or dG/d~ = AG~ ..... a

+ RT In ([ADP][PiJ /[ATP]) (I)

where AG~ is the value of A measured under certain standard conditions of molarity, tem- perature and its value will vary with pH and [Mg z+] [1, 18]. At 37~ and a magnesium concentration of 1 mM AG~ = -- 30.5 kJ/mol.

For various reasons it is difficult to measure free ADP (see refs [20, 22, 31] and of recent times the affinity value for ATP hydrolysis is usually calculated from the expression

A = kG~ + R T In

x ([Cr] x [Pi]/[PC][H+]KcK)

where PC is creatine phosphate, Cr is creatine and KeK is the equilibrium constant for the creatine kinase reaction which seems to be in near equilibrium in vivo; its value is taken to be about 109/[moll, unfortunately there is some variation in the literature with values between 0.5 x 109 and 1.8 x 109/[mol] being reported for this reaction in the presence of Mg 2 + ions.

The knowledge that the free energy change of any chemical reaction is linked to the degree of chemical advancement is in all the standard chemical thermodynamic textbooks and biochemists have routinely been employ- ing equation (1) to calculate changes in free energy. There are however special problems in most biological situations because of the complex intracellular milieu within which reactions take place. At physiological pH ionized molecules such as ATP usually exist in several charged forms that complex with cations.

In 1980 Dawson, Gadian and Wilkie [5], using conventional biochemical techniques plus nuclear magnetic resonance in muscle, showed that experimentally induced fatigue caused the free energy change consequent upon ATP hydrolysis to drop from values in the mid 50's (kJ/mol) to a mean value of 41 kJ/mol (a value at which the calcium pump would no longer be able to take calcium up into the sarcoplasmic reticulum against the

Cytoplasmic Phosphorylation Potential 729

concentration gradient (see [11]). Wilkie and colleagues were able to show that the rate constant of the relaxation phase was linearly dependent upon dG/d~. In an earlier paper [4] these same authors had looked at force development using the same fatigue protocol and, although their study did not show any correlation between declining affinity and decreasing force development, subsequent studies in the cardiac literature have sug- gested that this possibility should be examined very seriously.

I t is perhaps worth noting that in most cell types the [ATP] / [ADP] [Pi] ratio ranges from 1 x 10a/M to 5 x 103/M whereas in cardiac muscle the value is considerably higher (,-~ 10S/M) leading to higher affinity values in cardiac muscle than, say, in liver cells. The implication is that much higher levels of ATP are required in a cell type where there is an energy flux that may increase several-fold under certain physiological conditions.

For a long time there has been much specu- lation about the biochemical reasons for the early hypoxic failure of the heart since cardiac failure often occurs when there are consider- able reserves of ATP and PC: indeed under certain experimental conditions the heart will contract quite well with relatively low reserves of ATP present. Problems such as this have led to many theories such as critical cellular compartments [10], tissue acidosis, decreased Ca 2§ influx, inorganic phosphate accumula- tion, [17] and there are several reviews where various postulates have been considered [8, 13, 17]. Recently Hearse [13] discussed some of the various theories and refocussed atten- tion, by his own experiments, on the early fall in myocardial ATP that occurs in a measur- able quantity before the onset of noticeable mechanical failure. This paper plus the work of Wilkie and colleagues discussed above, forms of background to experiments by Kam- mermeier, Schmidt and Jungling [15] who looked at the effect of varying periods of anoxia upon mechanical performance and tissue metabolites (ATP, ADP, PC, Cr and Pi). Making use of published data regarding cellular compartmentat ion and utilizing known equilibrium constants to estimate free ADP they calculated the cytoplasmic phos- phorylation potential and the free energy change of ATP hydrolysis. No metabolic par-

ameter alone accounted for the decline in mechanical performance but there was a steep decline in the calculated affinity of ATP hydrolysis from control values of 61 kJ/mol to values between 50 and 45 kJ/mol and the authors were convinced that changes in the affinity of ATP hydrolysis were much more important than the changes in the actual level of ATP.

Since that paper this type of analysis has been extended to single cardiac cycles. Detect- able changes in ATP, PC and el have been shown to occur over a single cardiac cycle; some of the evidence with gated 31p N M R [7] needs further confirmation as there are prob- lems of interpretation of N M R under such conditions but more classical techniques also indicate cyclic changes. Recently some elegant freeze clamp studies have been made by Wikman-Coffelt and colleagues [23, 24] on rat hearts. Their biochemical data were analyzed in the manner first outlined by Kammermeier and colleagues [15] but they also took into account the changing levels of ADP bound to myosin in diastole as opposed to systole. The authors could show detectable cyclic changes in metabolites in hearts per- fused with a glucose containing medium (particularly if their energy flux was increased by raising either afterload or calcium level and by infusing isoproterenol). No such cyclic changes could be detected in physiological saline containing pyruvate. It was suggested that metabolic changes in these metabolites and in the calculated phosphorylation poten- tial could be detected under these conditions because of rate-limiting steps in glycolysis and the slow transport of NADH into mitochon- dria under this substrate regime. The authors calculated the systolic peak affinity values to be as low as --50 kJ/mol. However the authors believe that the free energy of ATP hydrolysis was probably preserved over the cardiac cycle by the continuous binding and recycling of ADP, see [22], i.e., they argue that it is unlikely that free ADP levels rise as high as one would predict from simply mea- suring ATP hydrolysis. However, in a sub- sequent paper Sievers et al. [23] compared the affinity values measured in failing hearts (Syrian hamsters) with normal hearts (Golden hamsters). I t is quite clear from this elegant study that in spite of the assumptions

730 C. Gibbs

tha t h a v e to be m a d e to ca lcu la te the free [ A D P ] the c a r d i o m y o p a t h i c hear ts had l o w e r dG/d~ATP values in b o t h dias to le and systole t h a n d id the non- fa i l ing hear ts (see thei r T a b l e 3).

I n s u m m a r y , recen t work does suggest tha t u n d e r pa tho log i ca l cond i t ions changes in the aff ini ty of A T P hydrolys is cou ld be a causa l fac to r in ca rd i ac failure. I t also seems as i f there cou ld be a s p e c t r u m o f responses to any c h a n g e in the aff ini ty since the d i f ferent A T P a s e s w i th in a cell will all p r e s u m a b l y be fac ing s o m e w h a t d i f ferent w o r k loads (i.e.

have d i f fe ren t free ene rgy r e q u i r e m e n t s ) . I t wou ld n o t be too surpr is ing to find tha t the free ene rgy n e e d e d to ' p r i m e ' a c rossbr idge or p roduce some c o n f o r m a t i o n a l c h a n g e in a con t rac t i l e p ro t e in (for those w h o ques t ion the crossbr idge hypothes is) m i g h t be d i f fe ren t f rom thef i -ee ene rgy c h a n g e tha t is needed say to p u m p Ca 2+ aga ins t a c o n c e n t r a t i o n gra- d ien t in to the sa rcop lasmic r e t i c u l u m or in to the ex t r ace l l u l a r fluid ( s a r co l emmal Ca 2+ A T P a s e ) or to t r anspor t N a + ions across the cell m e m b r a n e . Th i s concep t seems w o r t h y o f r igorous tes t ing in the c o m i n g years.

C o l i n G i b b s Reader in Physiology, Monash University, Clayton,

Victoria 3168, Australia

KEY WORDS : Cytoplasmic phosphorylation potential ; Respiration of heart

Note: Since this article was written Fiolet et al. J Mol Cell Cardiol 16, 1023-1036 (1984) have produced evidence that the magnitude and direction of transsarcolemmal ion-gradients during anoxia and ischemia are controlled by the free energy of ATP hydrolysis.

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