Adenine nucleotide metabolism in pigeon liver and heart: Diurnal changes and correlations between indices

Comp. Biochem. Physiol. Vol. 82B, No. 2, pp. 385 394, 1985 Printed in Great Britain

0305-0491/85 $3.00+0.00 Pergamon Press Ltd

A D E N I N E N U C L E O T I D E M E T A B O L I S M IN P I G E O N LIVER A N D HEART: D I U R N A L C H A N G E S A N D CORRELATIONS BETWEEN INDICES YURY G. KAM1NSKY and ELEN A. KOSENKO Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR (Received 19 February 1985)

A b s t r a c ~ l . The adenine nucleotide and P~ content of pigeon liver and heart were determined, and the energy charge, phosphoryl potential and mass action ratio of adenylate kinase were calculated over the 24 hr period. 2. All the indices of adenine nucleotide metabolism were shown to vary a 2- to 4-fold extent, both in liver and heart. 3. The correlation coefficients for each of the adenine nucleotides and each of the calculated indices were computed and shown to be different for liver and heart. 4. The difference between pigeon liver, pigeon heart and rat liver in the diurnal variation of adenine nucleotide metabolism, in the regulatory mechanism, and in metabolism on the whole is discussed.

INTRODUCTION

Physiological functions in animals vary in a 2 4 h r cycle. These variations can be used to study metabolic interrelationships and to see similarities in or differences between various tissues and animal species under certain conditions. Adenine nucleotides (AN*) play a central role in intermediary metabolism acting as the universal link between energy-yielding and energy-consuming processes in the cell. The energy status of the cell is revealed by the ATP, A D P , A M P and total A N concentrations. A balance between the ATPconsuming and ATP-regenerating reactions is characterized by the phosphorylation potential, P P = [ATP]/[ADP] x [P~], and controlled by the adenylate energy charge, EC = ([ATP] + 1/2[ADP)/([ATP] + [ADP] + [AMP]) (Atkinson and Walton, 1967; Atkinson, 1977). The activity of adenylate kinase, the reversible enzyme, seems to be sufficient to maintain virtual equilibrium between ATP, A D P and A M P throughout the cell. In general, the indices as the sum of AN, the EC and PP, and the mass action ratio of the adenylate kinase reaction (FAK) seem to be constant in the steady state of energy metabolism. Moreover, energy metabolism is regulated by the EC in such a way as to contribute to stabilization of the EC, and /'AK value must be, by the definition of the equilibrium reaction, strongly constant under different conditions. In previous reports, circadian rhythm in A N content of ureotelic rat liver has been described (Robinson et al., 1981; Kaminsky et al., 1984), and A N metabolism has been shown to be in close agreement with a hypothesis on the maintenance of the EC (Atkinson and Walton, 1967; Atkinson, 1977). In uricotelic animals such as birds, the enzyme system of

*Abbreviations: AN, adenine nucleotides; EC, adenylate energy charge; PP, phosphorylation potential; FAK, mass action ratio of the adenylate kinase reaction. 385

the purine ring synthesis is used as a pathway for the excretion of excess nitrogen. The level of the de novo purine synthesis in birds is 15 times that in mammals [Welch and Rudolph (1982) and references within], and pigeons excrete as much as 6-8 g uric acid/day (Krakoff and Karnofsky, 1958), three orders of magnitude more, on the kg body mass basis, than human subjects (0.6 g/day) (Van den Berghe et al., 1977; Van den Berghe, 1981). It implies that the purine nucteotide turnover should be a more dynamic one, and the purine nucleotide level should be subjected to more drastic diurnal variation in birds than in ureotelic animals. The question arises as to how much change in the ATP, A D P , A M P and total A N concentrations would occur and whether the EC and FAK values would be maintained in avian tissues over the 24 hr period. The purpose of this work was to determine the individual and total A N and Pi concentrations, the EC, PP and FAK values in pigeon liver and heart over the natural day and to see some correlations between these indices. MATERIALS AND M E T H O D S Male 16- to 18-month-old pigeons (Columba livia) were entrained to a natural light-dark cycle (lights on 09:00-16:00 hr) in a 5°C dovecote, and had a free access to food and water. The diet consisted of hemp, wheat, oats and sunflower seeds, and vitamins and minerals were given with the water. The birds were allowed to fly out of the dovecote throughout the light period of the day. Animals were sampled randomly at 3 hr intervals to represent 8 time points in the 24 hr cycle, 4 birds per one time point. The pigeons were decapitated, the chest was dissected, and a portion of liver was freeze-clamped within 13 sec, and then the whole heart was freeze-clamped within other 7-12 sec. In some experiments, liver mitochondria were isolated with the conventional method, once washed and suspended in 0.3 M sucrose and 0.01 M Tris-HCl (pH 7.4). Preparation procedures and AN and P, assays were conducted according to the methods described elsewhere (Kosenko, 1981; Kaminsky et al., 1984).

386

YURY G . KAMINSKY a n d ELEN A. KOSENKO

Statistical analysis of the results were carried out according to Gubler and Genkin (1973). RESULTS Adenine nucleotides and Pi in the liver (Table 1)

The ATP content of pigeon liver is between 0.4 and 0.5 #mol/g wet mass during the period from 03:00 to 09:00hr, then increases rapidly up to 1.0pmol/g at 15:00 hr, decreases again to 0.6 0.7/~mol/g at darkness and remains at this level to 24:00 hr. The ADP and AMP levels vary virtually as well, except the additional maxima at 06:00 hr. Thus, the liver content of each individual AN increases 2- to 3-fold (P < 0.001) during the light period of activity, then decreases at darkness, and stays at the daily mean level during the dark period of rest. The total AN pool size varies exactly so, being more than 2-fold higher at 15:00 hr as compared to 09:00 hr (P < 0.001). The Pi concentration is maximum during the period from 18:00 to 24:00 hr and minimum over the 03:00 to 09:00 hr period. The extrema in the Pi level differ 3- to 4-fold (P < 0.001).

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Adenine nucleotide metabolism

387

Table 2. Linear correlation coelticients for diurnal variations in indices of A N metabolism of pigeon liver EC Sum of AN

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The PP value is rather high at 09:00hr, then decreases sharply, attains the minimum during half of the dark period (18:00-24:00hr), and increases sharply at 06:00 hr. The ATP/ADP ratio, like the EC, is maximum at 15:00 and 24:00 hr and minimum at 03:00-06:00 hr, and stays at the daily mean level at other times. T h e / ' A K ratio is maximum during the period from 09:00 to 15:00hr and minimum at 18:00 and 03:00-06:00hr. The extrema differ 2- to 2.5-fold (P < 0.002). Linear correlations between liver indices (Table 2) The sum of AN in pigeon liver correlates with all the ATP, ADP, AMP and Pi concentrations. The EC correlates with ATP, ADP and the sum of AN, while the PP correlates negatively with ADP and Pi. The FAK ratio does not correlate with any index, except the ADP level. Adenine nueleotides and Pi in the heart (Table 3) The concentrations of AN in pigeon heart vary in the other diurnal patterns as compared to those in the liver, but their changes are the same in the magnitude. The ATP content of the heart is maximum at 15:00 and 21:00 hr and minimum at 12:00 and 03:00 hr, the differences between the extrema achieving 2-fold extent (P < 0.005). Both the ADP and AMP contents are maximum at 24:00hr and minimum over the 12: 00 to 21 :00 hr period. The differences between the maximum and minimum values are significant (P < 0.001). The sum of AN varies irregularly over the 24 hr period but in a wide range 2.4-4.2 #mol/g. The Pi concentration in the heart is 2- to 3-times that in the liver, on the average, and varies similarly over the 24 hr period. Calculated indices o f A N metabolism in the heart (Table 3, Fig. 2) The EC increases by 15% with respect to the daily mean value during the period from 09:00 to 21:00 hr, and decreases by 15% during the period from 21:00 to 03:00hr. The difference between the maximum (0.78) and minimum (0.58) levels in the EC is significant at the 0.001 level. The PP value changes slightly over the 24hr period. Only the maximum at 21:00hr and the minimum at 12:00hr differ significantly from the daily mean. Both the ATP/ADP and /'AK ratios are maximum during the period from 15:00 to 21:00 hr and about 2-fold lower at other times of the day-span. Linear correlations between heart indices (Table 4) The sum of AN correlates with ATP, ADP and AMP. The EC correlates positively with ATP, P~ and the PP, and correlates negatively with both ADP and

AMP, and does not correlate with the sum of AN. The PP correlates oppositely with ATP and Pi- The /'AK ratio correlates with ATP, and negatively with ADP and AMP, and quite significantly with the EC. DISCUSSION

Adenine nucleotides play a fundamental role in metabolism. They participate in every metabolic sequence, and each sequence uses or regenerates ATP, the immediate source of chemical energy. Functional relationships between various metabolic sequences depend on the stoichiometry of ATP consumption and production. The intracellular concentrations of AN can alter under different experimental conditions. In controls, their concentrations are believed to be constant, as otherwise it would be impossible to estimate the effect of the environment. The present work, however, showed that the AN concentration in the pigeon tissues varied greatly, 2to 3-fold, under "normal" conditions, over the natural day, without any specific influences on birds. The variation is higher than, for example, that in rat liver under fasting conditions, different dietary and hormonal treatment, in aging, diabetes mellitus, adrenalectomy, over the day-span, and under different perfusion conditions (Veech et al., 1970, 1972; Greenbaum et al., 1971; Faupel et al., 1972; Woods and Krebs, 1973; Seitz et al., 1976; Soboll et al., 1978; Veech, 1978; Berdanier et al., 1979; Robinson et al., 1981; Kaminsky et al., 1983, 1984). Great changes in the AN concentrations were observed in both the heart adopted to quick performance of metabolic and mechanical work, and the liver, not capable to perform a mechanical work and characterized with sluggish, adaptive changes in metabolism. According to Atkinson (1977), measurements of individual concentrations of AN are not important to the study of metabolic function or metabolic regulation in vivo and in vitro. The value of the EC indicates directly the amount of metabolically available energy in the adenylate system, and changes in the EC are directly proportional to the net amount of energy that has been put into or removed from the system. The PP, the sum of AN as the general index of the energy status of the cell, and the/'AK ratio seem to be of particular interest. The sum o f adenine nucleotides Until recently, the ATP content and the total adenylate pool size of the cell were believed to maintain at the constant levels (Lehninger, 1972; Atkinson, 1977; Dynnik and Sel'kov, 1978; Ivanitzky et al., 1978). When comparing the energy system of the cell with an electric battery, Atkinson (1977) postulated that "the adenylate pool has in common with a storage battery the fundamental property

388

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KAMINSKY and

ELEN A. KOSENKO

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that the total amount of material in the system typically remains constant". The comparison is obviously erroneous as the living cell represents an open system and the storage battery is a closed system. Nevertheless the proposed constancy of the sum of AN was the basis of the theories on the regulation in energy metabolism, e.g. on the oscillatory behavior of biochemical systems, and to construct complex mathematical models (Dynnik and Sel'kov, 1978; Ivanitzky et al., 1978). The suggestion that the total AN pool size is constant appeared probable from the numerous data of experiments performed with rat liver preparations in which the sum of AN altered relatively slightly under different physiological and experimental conditions (Veech et al., 1970, 1972, 1979; Greenbaum et al., 1971; Faupel et al., 1972; Woods and Krebs, 1973; Lawson et al., 1976; Seitz et al., 1976; Soboll et al., 1978; Veech, 1978; Berdanier et al., 1979; Robinson et al., 1981; Kaminsky et al., 1983, 1984). In other animal species, however, the sum of AN can be more variable. Thus, in sleeping golden hamster the hepatic AN pool size was reported to be 1.5 times that in wakeful animals (Jones, 1970). In pigeon liver (Kaminsky et al., 1982), chicken liver (Deaciuc and llonca, 1981) and, probably, in tissues of other uricotelic species the sum of AN appeared to be a very dynamical index. As the present work showed, the sum of AN in pigeon liver increased 0.95 2.13 pmol/g during activity (Table 1). For comparison, the sum of AN in rat liver has been shown to be much more stable and varied only in a range 2.63-3.40 (Kaminsky et al., 1984) or 2.95-3.32#mol/g (Robinson et al., 1981) over the 24 hr period. Pigeon liver differs from rat liver as well as from pigeon heart in both quantitative and qualitative aspects of diurnal changes in the sum of AN. It implies that the interpretation of metabolic events in experiments, as well as the construction of mathematical and kinetic models of energy metabolism must be performed with great caution and as dealing with not "the cell in general" but with a specified type of the cell only.

Adenine nucleotide metabolism

389

Table 4. Linear correlation coefficientsfor diurnal variations in indices of AN metabolism of pigeon heart ATP ADP AMP P, EC PP EC 0.698*+ -0.723§ -0.77811 0.448* 0.441" Sum of AN 0.657*+ 0.612t 0.494t 0.000 0.036 FAg 0.698,+ -0.729~ -0.698§ 0.89211 PP 0.349* --0.235 --0.565t *P < 0.05, tP < 0.0025, ;~P < 0.0005, §P < 0.0002, IIP < 00001 A m o n g the mechanisms of the regulation of the total A N pool discussed in the literature, the A M P deaminase mechanism (Atkinson, 1977; Lowenstein, 1972) seems to be preferable by which the increase in the individual levels of A N stimulates and the increase in the Pi level inhibits AMP-deaminase, and vice versa. The administration of glycerol, glucose or fructose either to animals or into a perfused organ was accompanied with the production of abundant phosphorylated metabolites, with the depletion of the cellular Pi and A N pools. This has been explained by the deficiency of Pi for oxidative phosphorylation on the one hand, and simultaneous and enhanced A M P breakdown by AMP-deaminase on the other hand (see Woods and Krebs~ 1973; Van den Berghe et al., 1977; refs in Chapman et al., 1971; Lowenstein, 1972; C h a p m a n and Atkinson, 1973; Atkinson, 1977). Tables 2 and 4 showed that the sum of A N in pigeon liver correlated with Pi that is in coincidence with the AMP-deaminase mechanism of the sum regulation over the 24 hr period, but no correlation was found between the sum of A N and Pi in the heart. Thus, if the AMP-deaminase mechanism of the regulation of the total A N pool functions in the heart, it does not associate with the P~ concentration in the myocardial cells. In any case, either quantitative or even qualitative expression of the mechanisms of the regulation of the total A N pool appears to be distinct in the heart as compared to the liver of the pigeon. The adenylate energy charge

Despite the large variation of the concentrations of individual A N and of their sum in pigeon liver and heart, the EC's were maintained at near-constant levels. It is in accord with Atkinson's principle of EC stabilization by metabolic processes (Atkinson and Walton, 1967; Atkinson, 1977). Therefore, the EC is maintained at a constant level not only in vitro under different metabolic states of the cell (Atkinson and Walton, 1967; Chapman et al., 1971; Chapman and Atkinson, 1973; Atkinson, 1977), but also in vivo under different influences on animals and perfused organs (calculated from data given by Veech et al.,

1970, 1972; Greenbaum et al., 1971; Faupel et al., 1972; Lawson et al., 1976; Seitz et al., 1976; Soboll et al., 1978; Woods and Krebs, 1973) and over the 24 hr period [Tables 1 and 2, Figs 1 and 2; see also Kaminsky et al. (1984) and values calculated from results given by Robinson et al. (1981)]. In pigeon tissues under study and in particular in pigeon liver, the EC appeared to be very small as compared to that in mammals of 0.7~).9 [Chapman et al. (1971) and calculated from foregoing data]. It seems to be of special interest. C h a p m a n and Atkinson (1973) have suggested from data available that the EC below 0.6 is of no physiological significance. In our experiments, the EC in pigeon liver never exceeded 0.63 and attained the minimum value of 0.55 (Table 1). Ridge (1972) believed, and Chapman et al. (1971) have shown using the Eseherichia coli cells that the EC below 0.5 is apparently incompatible with maintenance of the minimal level of homeostasis required for viability, and that any situation that prevents the EC from reaching or exceeding the critical value of 0.5 must be lethal for the cell. Some results selected the lowest EC and mainly from recent literature and enumerated in Table 5 show that it cannot be the case. In the isolated chicken liver perfused with lactate, the EC has been observed to be as small as 0.427 and 0.375 (Deaciuc and Ilonca, 1981). The same has been reported for rat liver perfused with ethanol and avenaciolide (Wimhurst and Harris, 1976) and for chicken hepatocytes incubated with adenosine (Dickson and Langslow, 1978). In partially hepatectomized rabbits, the EC in liver decreased to 0.45 after an injection of octanoyl carnitine into the portal vein (Nakatani et al., 1981). In mice with ligated renal arteries, veins and ureters, the EC in kidney was decreased to 0.3, from the normal value of 0.8, and was recovered after removing ligatures (Warnick and Lazarus, 1981). In the plasmodium of P h y s a r u m polyeephalum, the EC fell to 0.46 in mitosis and rose to 0.74 later (Sachselmaier et al., 1969). In baker's yeast, the EC dropped drastically to 0.1, from nearly 1.0, 10-15min after glucose addi-

Table 5. Some literary data selected by the minimum EC observed (as given by authors or calculated by us) and associated with no death of the organism or cell Species, tissue or preparation Pigeon heart Pigeon liver The plasmodium of Physarum polycephalum Rabbit liver Rat liver Chicken hepatocytes Chicken liver Mouse kidney Rat brain Baker's yeast

The minimum Conditions EC Over the natural 24 hr cycle 0.58 Over the natural 24 hr cycle 0.55 In mitosis 0.46 Octanoyl carnitine administration 0.45 Perfusion with ethanol and avenaciolide 0.44 Incubation with adenosine 0.39 Perfusion with lactate 0.38 Ligation of renal vessels and ureters 0.30 Ligation of carotid arteries 0.10 Incubation with glucose 0.10

Reference This paper This paper Sachselmaieret al. (1969) Nakataniet al. (1981) Wimhurst and Harris (1976) Dicksonand Langslow (1978) Deaciucand Ilonca (1981) Warnikand Lazarus (1981) Kobayashiet al. (1977) Yoshinoand Murakami (1981)

390

YURY G. KAMINSKYand ELENA. KOSENKO

Table 6. Some literary data demonstrating a variability of the FAK ratio (as given by authors or calculated by us) under different physiological and experimental conditions Species, tissue or preparation

Conditions

The range of the FAK ratio

Reference

0.51-0.83

Greenbaum et al. (1971)

0.44-0.88

Veech et al. (1970)

In riro

Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat liver Rat brain Rat brain Hamster liver Gerbil brain

Starvation for 72hr, refeeding with diet high in carbohydrate or fat Starvation for 48 hr, refeeding with diet high in carbohydrate for 3 7 days Starvation for 0-48 hr, adrenalectomy In aging, fasting and administration of salts of succinic acid Control and anesthesia Fasting and ethanol treatment In 50 and 150 days of age Newborn, initial 6 hr of life Days prenatal to days postnatal development Control and hypoxia Control and anoxia Hibernation and awakening Ligation of carotid arteries, restoration

0.34-0.88 0.22 1.94

Seitz et al. (1976) Kaminsky et al. (1983)

0.09 0.42 0.50-2.00 0.30-0.67 1.30-3.87 0.33 1.19 0.35-5.00 5.00-39.0 2.25~9.30 0.50-14.2

Faupel et al. (1972) Veech et al. (1970, 1972) Berdanier et aL (1979) Ballard (1970) Sutton and Pollak (1978) Ridge (1972) Ridge (1972) Jones (1970) Kobayashi et al. (1977)

Perfusion under different conditions Perfusion with glycerol and dihydroxyacetone Perfusion with ethanol and avenaciolide Perfusion with lactate, adenosine and the mixture Perfusion under different conditions Perfasion with Ca 2+, K ÷ and adrenaline Incubation with adenosine Incubation with citrate and ATPase

0.65.-1.40 0.57 1.09 1.53 23.2 0.22-1.19 0.41-2.20 0.87 2.73 1.32 9.00 0.61 12.8

Soboll et al. (1978) Woods and Krebs (1973) Wimhurst and Harris (1976) Deaciuc and Ilonca (1981) Deaciuc and Ilonca (1981) Nishiki et al. (1978) Dickson and Langslow (1978) Wu and Davis (1981)

In vitro

Rat liver Rat liver Rat liver Chicken liver Guinea-pig liver Rat heart Chicken hepatocytes Muscle extract

tion and was normalized again within the next 15 min (Yoshino and Murakami, 1981). These selected data seem to indicate some generality of the EC below 0.5. Such a low EC can be characteristic of some physiological states of the cells of different types. In any case, such a low EC appeared not to be lethal for organisms and organs enumerated in Table 5, and the level of homeostasis is sufficient to maintain the EC following its dramatic fall. Mechanisms of stabilization of the EC can be different in the liver and heart Linear correlations observed between the EC and all ATP, ADP and the sum of AN for the liver agree with the AMP-deaminase mechanism of stabilization of the EC (Chapman and Atkinson, 1973; Atkinson, 1977) by which the decrease in the EC must be compensated by AMP deamination to IMP, therefore by the increase in the mole fractions of ATP and ADP in the total AN pool and by the decrease in the total pool size. In the heart, however, the EC correlated positively with ATP, but negatively with ADP and AMP, while no relationship existed between the EC and total adenylate pool. Such a difference between two tissues can be a result of either (a) lack of the AMP-deaminase mechanism of stabilization of the EC in heart, or (b) essentially a contribution of this mechanism in the heart as compared to the liver, or (c) an interpretation of the AM P-deaminase mechanism of stabilization of the EC to be different. The indices such as the EC, sum of AN and FAR all are expressed arithmetically by the same elements, ATP, ADP and AMP. Therefore, each of these indices must be dependent on one another, and the mutual dependence must be simple. The decrease in the EC seems to be compensated by activation of metabolic pathways leading to its increase (Atkinson and Walton, 1967; Atkinson, 1977),

that is to the ADP and AMP phosphorylation, and vice versa. If so, it is the positive correlation between the EC and ATP and the negative correlation between the EC and both ADP and AMP rather than other correlations that are evidence for the AMPdeaminase mechanism of stabilization of the EC. But, in this case, that mechanism is not exhibited in the liver, and is not accompanied by the decrease in the total AN pool size in the heart. The mechanisms of stabilization of the EC seem to be different in pigeon liver and heart. To elucidate whether this difference is due to quantitative or qualitative aspects a thorough study is necessary. The energy charge concept (Atkinson and Walton, 1967; Atkinson, 1977) was based on virtual equilibrium between concentrations of ATP, ADP and AMP throughout the cell. A foregoing simplicity of mutual relationships between the EC, total AN pool size and FAK ratio requires, in addition, the total AN pool size to be constant. Hence, in an open metabolic system of the cell, such simple interrelationships are impossible. Moreover, if any pair of the indices, e.g. the EC and FAR ratio, were to be stable, then the third related index, the total AN pool size, would also be stable, by the definition of all the indices. In our experiments, the FAK ratio in the liver differed by 2.5-fold at different times of the day and in the heart, by 2-fold. The range of the variation of the FAK ratio seems to be small enough and does not usually receive any attention. Nevertheless, changes in the FAK ratio occur, and their reason is not known. The literature is lacking in the studies demonstrating a constant FAK ratio under different conditions. Some data (obtained by the authors or calculated by us) are given in Table 6. They show that the FAK ratio varies in a wide range of 0.1~40 in different tissues and under different conditions, most deviations of the FAK ratio from the K, the equilibrium constant of adenylate kinase [an apparent constant of 0.8

Adenine nucleotide metabolism (Lehninger, 1972) or the "true" constant of 0.325 (Blair, 1970) or 0.419 (Lawson et al., 1976)] having occurred under physiological conditions. This short list of deviations of the FAK ratio from the K value suggests that adenylate kinase is the near-equilibrium enzyme rather than the equilibrium enzyme under normal physiological conditions. An alternative can be the variation of the distribution of AN as a function of the free cytosolic magnesium concentration. As ATP is hydrolyzed to A D P and AMP, the magnesium ion concentration must rise, as A D P and A M P form much weaker complexes with magnesium than does ATP (Blair, 1970). In addition, the measured A D P and A M P were proposed to be too high due to binding or compartmentation in the tissues that contain mitochondria (Veech et al., 1979). In the temporary ischemic gerbil brain ATP showed a fall of 90% in its initial concentration (Kobayashi et al., 1977) which would argue against quantitatively significant binding of ATP (Veech et al., 1979). The traditional explanation for the FAK ratio variabililty with changing the free magnesium concentration in the cell cytosol was refuted by Ballard (1970) who showed with soluble muscle adenylate kinase that the FA~ ratio was nearly constant (0.70-1.03) at a range of the magnesium concentration of 0.5-6mM and physiological concentrations of AN. This result has been confirmed by Lawson et al. (1976); in the system containing crystalline commercial adenylate kinase, the FAK ratio was maintained at the level of 0.87-0.98 when the magnesium concentration was altered from 0.37 to 1.08 mM at different initial concentrations of ATP (0.04 or 0.94 mM), A D P (0.03 or 1.92 mM) and A M P (0.14 or 1.04mM). Blair (1970) has performed a computer analysis of the dependence of the FAKratio on the free magnesium concentration in a system containing purified adenylate kinase and AN. He has been able to show that, at the constant sum of A N of 5 mM and alternating the free magnesium concentration from 0.166 to 1.68, from 0.51 to 2.3 and from 1.23 to 2.94mM, the FAk ratio varied 0.81-1.22, 0.68-1.22, and 0.58 0.94, respectively. Again, the relative changes in the FAK ratio were small as compared to those given in Table 6, in spite of non-physiologically high concentrations of total magnesium used (4-6mM), capable of forming complexes with almost all AN. Ballard (1970) has suggested that the A M P compartmentation in the cell and accumulation in mitochondria could be responsible for observed variation of the FAK ratio. However, Soboll et al. (1978) have shown with the preparation of isolated perfused rat liver that the A M P concentration in the mitochondrial fraction was decreased by a factor of 2.5 and that A M P in the cytosolic fraction was increased by a factor of 10 on starvation but the FAK ratio as calculated from measured cytosolic A N concentrations was virtually unchanged (1.28-1.33). We measured the A M P content of mitochondria isolated from pigeon liver by a conventional method (not shown) and were able to find it to vary in a range 1.05-1.64nmol/mg protein over the 24hr period. Such a variation in the mitochondrial AMP level seems to be insufficient to alter the tissue A M P

391

concentration and FAK ratio by 2.5-fold. The mitochondrial A M P level correlated with neither the A M P content of the liver (the correlation coefficient r =0.266) nor the FAK ratio (r = 0.110) (it is important that no negative and significant correlation existed). The FAK ratio correlated positively with mitochondrial A M P at 12:00, 15:00, 21:00, 03:00 and 06:00 hr (r = 0.779, P < 0.002 for three mitochondrial preparations per a time point) and correlated negatively and insignificantly with mitochondrial A M P at 09:00, 18:00 and 24:00hr (r = - 0 . 5 6 7 , P ~0.1, N = 9). Thus, the maximum FAK ratio in fed pigeon liver (at 12:00 and 15:00 hr) and the minimum FAK ratio in fasted birds (at 03:00 and 06:00hr) are not due to opposite changes in mitochondrial A M P (otherwise the negative correlation between the FAK ratio and mitochondrial A M P would be expected). Though these results are indirect, they indicate that the compartmentation of A M P is probably not the main cause of the change in the FAK ratio over the 24 hr period. Veech et al. (1979) have discussed the role of intracellular compartmentation of A D P in the deviation of the FAKratio from the K. Their recalculation of free cytosolic A D P based on the assumption of equilibrium between reactants of the combined gtyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction in vivo, yielded the value 20-fold lower than measured cell A D P content, while the FAKratio differed from the K by 2-fold only. The authors concluded that such a low free cytosolic concentration of ADP must be used in calculations of the PP as well as of the FAKratio. However, if so, the FAKratio would be increased by a factor of 400, and for resulting 200-fold deviation of the FAKratio from the K to be compensated, the free cytosolic concentration of A M P lower by a factor of 200, that is as small as 1 #M, would be accepted. It is not the case (see Soboll et al., 1978). We measured also the A D P content of mitochondria isolated from pigeon liver by a conventional method (not shown) and found it varying in parallel with the A M P content and in the range 0.86~2.04 nmol/mg protein over the 24 hr period. As it was mentioned above, it seems to be indirect evidence for the A D P compartmentation not being the main factor responsible for the variation of the FAK ratio over the 24 hr period as well as under conditions enumerated in Table 6. The lack of data on the invariability of the FAK ratio under different physiological and experimental conditions (Tables 1, 2 and 6) suggests that the K (not the FAK ratio) is regulated in vivo and in vitro mainly by the adenylate kinase activity. The fact that adenylate kinase maintains equilibrium between AN was confirmed so far only in vitro (Ballard, 1970; Blair, 1970; Lawson et al., 1976) was accepted a priori for the in vivo conditions. Since the adenylate kinase activity is regulated both in vitro (Pradhan and Criss, 1974; Watanabe and Kubo, 1982) and in vivo (Adelman et al., 1968; Criss, 1970) as well as in the diurnal rhythm (Chemnitz et al., 1979; North et al., 1979), the variation of the enzyme activity, even of the activity of the acknowledged equilibrium enzyme, must be revealed in the distribution of AN. It is the localization of adenylate kinase preferably in the cytosol of muscle and cardiac cells (Walker and Dow, 1982;

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YURY G. KAMINSKYand ELENA. KOSENKO

see Watanabe and Kubo, 1982, for review) and in the mitochondrial intermembrane space of hepatocytes (Lehninger, 1972; Criss, 1970; Watanabe and Kubo, 1982) that is probably the main cause of different relative equilibrium (or different physiological equilibrium) between AN as well as of the different correlations between the EC and AN in pigeon heart and liver. It is of interest that, in pigeon liver, the EC does not correlate with the FAKratio over the 24 hr period (Table 2) but in the heart, a linear correlation exists (Table 4). Similar correlation between the EC and FAK ratio has been found in rat liver during the development from embryonic to 6-hr-old (Ballard, 1970), in trout embryos during gastrulation (Boulekbache, 1981) and in a soluble system with purified adenylate kinase (Blair, 1970). Adenylate kinase is likely to be involved in the regulation of the EC in pigeon heart, as in above systems (Ballard, 1970; Blair, 1970; Boulekbache, 1981; see also Criss, 1970), but the regulation of EC in pigeon liver can be distinct. A possible role for 5'-nucleotidase can be distinguished between cardiac and hepatic AN metabolism. This enzyme activity in pigeon heart was reported to be as much as 40-fold lower than in pigeon liver (Arch and Newsholme, 1978). In this regard, the AMP-deaminase reaction appears to be the exclusive pathway by which AMP can break down in pigeon heart.

EC correlated with the PP significantly over the 24 hr period, the grade correlation coefficient being 0.905 (Kaminsky et al., 1984), 0.857 or 0.905 (as calculated from data given by Robinson et al., 1981). The reason for such a difference between rat liver and pigeon liver is unclear. But these correlations, together with all other results given and discussed in this paper, suggest that pigeon liver, pigeon heart and rat liver are tissues differing on principle with regard to the organization of metabolism, in particular over the 24 hr period. CONCLUSION The ATP, ADP, AMP and Pi content of pigeon liver and heart varies a 2- to 4-fold extent over the 24 hr period. The total AN concentration and the mass action ratio of the adenylate kinase reaction in pigeon liver and heart vary in wide ranges over the 24 hr period. The EC is stabilized in both tissues, but is unusually low compared to the EC in mammalian tissues and is regulated by different mechanisms in pigeon liver as compared to pigeon heart. Adenylate kinase seems to be involved in the regulation of the EC. Pigeon liver, pigeon heart and rat liver represent the tissues differing on principle with respect to the organization of AN metabolism as shown by the diurnal variation in the indices of AN metabolism and by correlations between these indices.

The phosphorylation potential ( P P )

Some investigators believe the EC and PP are synonyms which formulate the phosphorylation state of AN from the same intracellular pool (Ridge, 1972; Atkinson, 1977; Ivanitzky et al., 1978). If it were so, the indices would correlate under different conditions, e.g. over the 24 hr period. In our experiments, the relationship between the EC and PP was absent from pigeon liver (Table 2) but a linear correlation existed in the heart (Table 4). Moreover, the EC correlated with Pi in both the liver and heart, while the PP correlated oppositely with Pi. Such results do not seem unexpected. The PP is a thermodynamical potential (in general, expressed on a logarithm scale) reflecting the capacity of the cell to synthesize ATP from ADP and P~. In liver, the PP is mainly a criterion of mitochondrial performance in energy conservation, and as the function of mitochondria is the consumption of ADP and Pi and the export of ATP, this criterion is exhibited in the cytosol. In muscle tissues, a substantial role in PP generation is played by glycolysis. Unlike the PP, the EC is a phenomenological quotient, not the thermodynamical potential, and reflects simply the relative amount of high energy phosphate bounds of ATP and ADP in the total adenylate pool, and is apparently not dependent on the presence of mitochondria. The difference between pigeon liver and heart in the correlation coefficients reflects not only a different nature of the EC and PP but also a difference between two types of the tissues with respect to metabolism. As shown in previous sections and in the literature (Kaminsky et al., 1982) pigeon liver differs from mammalian liver in various characteristics of phosphorus and carbohydrate metabolism. In rat liver, the

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