The Increasing Adenine Nucleotide Concentration and the Maturation of Rat Liver Mitochondria during...

Differentiation 12, 15-21 (1978) Differentiation C Spnnpcr-Vcrl;ig I U78

The Increasing Adenine Nucleotide Concentration and the Maturation of Rat Liver Mitochondria during Neonatal Development ROSEMARY SUTTON and JOHN K. POLLAK Department of Histology and Embryology, University of Sydney, Sydney 2006 Australia

The levels of adenosine triphosphate, diphosphate, and monophosphate in liver and isolated liver mitochondria were examined in foetal, neonatal, and suckling rats. It was shown that while the total adenine nucleotide level in liver varied only slightly during development, there was a steady increase in the concentration of mitochondrial adenine nucleotides. This increase was most dramatic just after birth. Experiments using pups obtained by caesarean section one day prior to normal birth and kept in a humidicrib for up to two hours, showed that the mitochondrial adenine nucleotide level doubles during this period. This increase is associated with the maturation of the mitochondrial inner membrane as measured by the enhancement of respiratory control.

The results indicate that in addition to the adenine nucleotide translocator - which effects the exchange of adenosine triphosphate and diphosphate - there must be a second transport mechanism present, at least in perinatal mitochondria, which is responsible for the net uptake of adenine nucleotides.

The adenine nucleotides in this study were measured, using a modified luciferin-luciferase assay. In this method the preincubation of sample with appropriate enzymes to convert adenosine monophosphate and diphosphate to adenosine triphosphate, was carried out in the same scintillation vial as the final assay. This eliminated a second sampling step and thereby increased the convenience, speed, and accuracy of this very sensitive method.

Introduction

It has been suggested that the maturation of mitochondria within the hepatocytes of the perinatal rat depends on an increase of adenosine triphosphate (ATP) [l, 21. A rise in the concentration of ATP and total adenine nucleotides in rat liver during the first two hours after birth was first described by Ballard 131. Furthermore the respiratory control index (R.C.I.) of foetal rat liver mitochondria, which is low in comparison to adult values, increases after normal birth or after delivery by caesarean section [l , 41. The R.C.I. of foetal mitochondria can be similarly enhanced in vitro by the addition of ATP or the non-hydrolyzable analogue, adenylyl imidodiphos- phate, to mitochondrial suspensions either prior to or during its measurement [l, 5 , 61.

During normal neonatal development there is a dramatic increase of the ATP and total adenine nucleotide content of hepatic mitochondria which can be correlated with changes in the R.C.I. [6, 71.

As a working hypothesis it has been assumed that at birth changes in either the hormonal status or oxygen availability result in an increase in liver ATP which in turn causes mitochondrial ATP and adenine nucleotides to increase [ l , 21. As a result oxidative phosphorylation becomes more effective.

On examining the literature more closely it became obvious that insufficient information was available on the relationship between the adenine nucleotide levels of liver mitochondria and those of whole liver. Changes in the concentration of adenine nucleotides of the liver have been measured only for the first few hours after

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16 R. Sutton and J. K. Pollak: Adenine Nucleotide Uptake by Mitochondria

birth [31, while for mitochondria only the longer term developmental trends are known [6, 71.

This investigation compares the long-term changes (six days before birth to five days after birth) in hepatocytes and mitochondria, as well as the short-term changes 30-9Omin after caesarean section of 1-day prematurely delivered pups.

This study establishes that in the long term the increase in the mitochondrial adenine nucleotide concentration is in contrast with the relative constancy of the adenine nucleotide concentration of freeze-clamped liver before and after birth.

Short-term experiments show that increases in the R.C.I. of mitochondria, during 30-90 min incubation in a humidicrib, after caesarean section, coincide with the increases in the mitochondrial adenine nucleotide content.

This finding underlines the significance of the increases in the mitochondrial adenine nucleotide concentration, particularly in view of the fact that this is evidence for a transport system that effects a net uptake of adenine nucleotides into mitochondria. No such system has been described so far.

Methods

This study was carried out with rats of a random-bred Wistar strain. Foetal rats of known age were obtained from overnight (16 hour) matings. In maturation studies, foetuses were delivered by caesarean section one day prior to normal birth and kept in a humidicrib at 35-37O C for up to two hours. Unmated 13-15 week old females weighing 150-230g were used as the source of adult rat livers.

Liver Samples. Livers from foetal, neonatal, or suckling rats were freeze-clamped using small clamps chilled in liquid nitrogen. The tissue was broken up in liquid N, to powder, using a glass rod, and transferred to, and weighed in, a centrifuge tube containing ice-cold 0.3 M perchloric acid. After two extractions in acid a portion of the combined supernatants was adjusted with constant mixing to pH 7.0 using 0.6 M KOH with 0.06 M N-2-Hydroxyethy1piperazine-N1-2- ethanesulphonic acid (HEPES) in the presence of Universal Indica- tor. The neutralized supernatant was then stored in liquid N, until analysed. The adenine nucleotides in samples stored in this way were stable for at least six months.

Mitochondria. Rat liver mitochondria were isolated as described pro viously I I I. Protein was determined by the method of Lowry et al. [81 using bovine serum albumin (Fraction V, Sigma St. Louis Mo.) as a standard. Respiratory control was determined using a Rank (Bottisham, Cambs. England) oxygen electrode to measure rates of oxygen uptake 111. For the determination of adenine nucleotides, freshly prepared mitochondria were extracted with ice-cold 0.3 M HCIO, (fmal concentration); the mitochondrial concentration being approximately 10 mg protein/ml. These extracts were neutralized as described above for liver samples.

The Determination of Adenine Nucleotides. The very sensitive luciferase assay of ATP was chosen because of the low levels of adenine

nucleotides found in extracts from single foetal livers and mitochondrial suspensions. Adenosine diphosphate (ADP) and monophosphate (AMP) assays were developed in which total ATP was measured using luciferase after preincubation with the conversion enzymes pyruvate kinase and myokinase. The preincubation was carried out in the same scintillation vial as the fmal assay. The presence of the diluted pyruvate kinase and myokinase enzymes was shown not to interfere with the luciferase assay of ATP. The assay can also be carried out in the presence of high levels of many ‘non-adenine’ nucleotides (Sutton, unpublished results). Conversion of both ADP and AMP is 100% in the range tested (50 to 500 pmol). However, the precautions recommended by Hammerstedt [91 are necessary to ensure low background counts and low variability.

The bioluminescence of the luciferase enzyme reaction was measured using a model 221 l Packard liquid scintillation spectrometer set up in the non-coincidence mode with 100% amplification (101 and a counting period of 0.2 minutes. The first channel was set at 60-80 divisions and the second at 160-180 to allow a channels ratio check for sample quenching. The buffer used - 25 rnM HEPES with 17 mM MgSO, pH 7.5 at 200 C (HEPES/magnesium buffer) gives near maximal light output while also being suitable for the conversion of ADP and AMP.

The Fnefly Lantern Extract (Sigma FLE-50) containing both the enzyme luciferase and the substrate luciferin was made up to 5 ml with distilled H,O, and portions of the supernatant were frozen an diluted on the day, approximately 1 in 5, with HEPES/magnesium buffer so that when 100 p1 of the extract were used a 500 pmol ATP standard gave 100,000-200,OOO counts with a background less than 400. The diluted extract kept on ice gave consistent results for 3-4 hours. Small portions of 1 mM ATP, ADP, and AMP standard solutions were kept frozen at -200 C. These were shown to remain stable for at least one year, as found by other workers [ 111. For each set of analyses a fresh portion was thawed and diluted, 1 in 100, in buffer just prior to use.

Five - 10 pl of each sample were added to 0.5 ml portions of HEPES/magnesium buffer in scintillation vials. Six vials were set up for each sample - duplicates for the ATP, ATP + ADP, and ATP + ADP + AMP assays. To the corresponding vials, 100 pl of the ADP conversion mixture (HEPES/magnesium buffer with 2.5 mM phosphoenolpyruvate, and 10 pg/ml pyruvate kinase) or 100 p1 of the ADP + AMP conversion mixture [HEPES/magnesium buffer with 2.5 mM phosphoenolpyruvate, 10 pg/ml pyruvate kinase + 50pg/ml rnyokinase (Sigma M3003)1, were added and gently mixed. All the vials were then placed in the scintillation counter and further mixed by the movement of the sample changer.

Just prior to counting, the sample volume was made up to 3 ml and mixed. Immediately as the print-out for the previous sample commenced, 100 pl of firefly extract was added. The operation is organized so that there is a constant 18 second delay between the addition of enzyme and the beginning of the 12 second counting period. The ATP + ADP samples were not counted until at kast five minutes after the addition of the conversion mix; ATP + ADP + AMP samples were left for at least 15 minutes to ensure that conversion to ATP was complete.

Results

The levels of liver adenine nucleotides were measured in rats of eight different developmental ages over a range from six days prior to birth to five days after birth.

R. Sutton and J. K. Pollak: Adenine Nucleotide Uptake by Mitochondria

Tabk 1. Adenine nucleotide content of rat liver during development

17

~ ~ ~~

Age days ATP ADP AMP Total Energy adenine charge

pmol/g Liver nucleotides

-6 (2) 1.57 f 0.04 0.75 f 0.06 -4 (4) 1.61 f 0.12 0.86 f 0.22 -3 (3) 1.94 f 0.03 0.57 f 0.07 -2 (10) 1.68 f 0.04 0.77 f 0.07 -1 (25) 1.38 f 0.05 0.57 f 0.04 + I (4) 1.59f 0.16 0.60 f 0.11 +3 (5) 1.68 f 0.08 0.56 f 0.10 +5 (9) 2.06 f 0.06 0.64 f 0.13

0.12 k 0.06 0.11 k 0.04 0.14 f 0.03 0.17 f 0.03 0.18 f 0.02 0.27 f 0.05 0.22 f 0.08 0.16 f 0.06

2.44 f 0.08 2.58 f 0.33 2.65 f 0.02 2.62 k 0.06 2.13 k 0.09 2.46 f 0.06 2.46 k 0.18 2.93 f 0.24

0.80 f 0.03 0.79 f 0.02 0.85 f 0.01 0.80 f 0.01 0.79 f 0.01 0.77 f 0.04 0.81 f 0.04 0.84 0.03

Livers from rats of 8 developmental ages (given in days before (-) or after (+) birth) were freeze- clamped and their adenine nucleotides were assayed as described in the Methods section. The number of experiments are shown in parentheses and the results are presented f SEM.

ATP + 5/,ADP ATP + ADP + AMP

Energy charge = as defined by Atkinson and Walton [201

Table 2. Adenine nucleotide content of mitochondria from rat liver during development

Age ATP ADP AMP Total Energy adenine charge

pmoVg protein nucleotides

-5 (3) 0.74 f 0.02 0.40 f 0.03 0.35 f 0.02 1.49 ? 0.03 0.63 f 0.01 -3 (3) 0.78 f 0.03 0.73 k 0.10 0.61 k 0.05 2.11 f 0.12 0.54 f 0.01 -2 (3) 0.91 f 0.21 0.65 f 0.01 0.70 k 0.02 2.28 f 0.21 0.54 f 0.04 -1 (18) 0.89 k 0.07 0.74 f 0.06 0.99 f 0.10 2.62 f 0.14 0.48 f 0.02 2 ha (8) 1.63 f 0.20 1.34 f 0.13 3.05 f 0.33 6.03 f 0.61 0.38 f 0.01

+ I (1) 1.50 1.72 3.56 6.78 0.35 +3 (5) 1.95 f 0.22 1.82 f 0.06 5.60 f 0.11 9.36 f 0.25 0.30 f 0.15 +5 (5) 2.68 f 0.51 2.88 f 0.49 5.44 f 0.47 11.01 f 1.15 0.37 f 0.03 Adult 3.16 f 0.31 4.09 f 0.26 5.42 f 0.34 12.70 f 0.72 0.41 f 0.01

Mitochondria were isolated from the livers of rats of different developmental ages (gwen in days before (-1 or after (+) birth). The mitochondria were extracted and their adenine nucleotides assayed as described in the Methods section. The number of experiments are shown in parentheses and the results are presented f SEM a These pups were delivered at -1 day and kept in a humidicrib for 2 h

The results shown in Table 1 indicate that the ATP and total adenine nucleotide contents remain fairly constant over this developmental period except for a statistically significant 2096 drop observed in foetuses one day before birth. The significance of the constancy of the adenine nucleotide concentration per gram of liver over a developmental period of 11 days is difficult to assess, as during that period a considerable decrease in the proportion of the haemopoietic tissue contribution of the liver occurs [121. Furthermore during the last days of the foetal life, glycogen accumulates in the liver, making up approximately 8% of the total liver weight [131, while

after birth glycogenolysis depletes the liver of the neonatal rat of glycogen within 12 hours 1141. The level of adenine nucleotides observed in five-day-old suckling rats (2.93 pmol/g, Table 1) is within the range of the values found for normal adults rats (3.06 pmol/g) [151.

The meaningful assay of ATP, ADP, AMP in tis- sues depends on the speed of freeze-clamping the tissue after the first manipulation of the rat commences. Both stress and the anoxia occurring during the interval after killing and before clamping will cause a decrease in ATP and increases in both ADP and particularly AMP [ 161. Strict adherence to, and standardization of, methodo-

18

Table 3. The distribution of ATP and total adenine nucleotides within the mitochondrial compartment of hepatocytes during development


Age ATP Adenine ATP Adenine Mitochondrial volume nucleotide nucleotide as % of hepatocyte

volume pmol in mitochondria isolated from 1 g liver

% in mitochondrial compartment

-4d -3d 6.5 -2d 14.4 -Id 14.1 Od; +4h; +6h; +Id 39.6 + 2d Adult 164.9

14.3% (19). 17.5 0.4 0.7 4.4% (18)’ 36.1 0.9 1.4 13.4% (19). 41.4 1 .o 1.9 13.2% (19)’

11.5%; 12.9%; 18.5%; (14)’

662.9 8.2 26.7 20.0% (19).

137.6 1.9 5.7 23.8% (17). 23.0% (19).

20.7% (18).

The level of ATP and total adenine nucleotides in the mitochondria obtained from 1 g of liver has been calculated using data from Table 2 and reference [l]. This has also been expressed as a percentage of the liver ATP and total adenine nucleotidee, using data from Table 1. For comparison data from serveral morphometric studies on the percentage of the hepatocyte volume occupied by mitochondria is presented. Developmental age is given in days (d) or hours (h) before (-) or af€er (+) birth a These figures represent the references from which the % mitochondrial volumes have been taken

Table 4. Changes in mitochondrial respiratory control after birth ~

Time (min)

0 30 60 90

Control 1.71 f 0.13 2.35 f 0.10 2.70 f 0.16 2.81 f 0.28 +0.5 mM ATP 2.72 f 0.14 2.93 f 0.17 3.07 f 0.13 3.09 f 0.15

Pups were delivered one day prematurely and kept in a humidicrib for up to 90 min. Mitochondria were isolated from the livers and respiratory control was measured using an oxygen electrode. In each case the R.C.I. was measured in the presence and absence 0.5 mM ATP, added to the mitochondria at the beginning of the run. The results are the mean of five experiments; in three of these, mitochondrial adenine nucleotides were also measured; in the other two experiments the liver adenine nucleotidts of littermates were measured (Table 5). SEM’s are given for the R.C.I. data

logy was found to be necessary to reduce the variability of values obtained at different developmental stages. For these determinations the shortest possible time interval was allowed between death of both the maternal and foetal rat and the freeze-clamping of the foetal liver.

In direct contrast to the constancy of the adenine nucleotide concentration in foetal and suckling rat liver is the steady rate of increase in the ATP and total adenine nucleotide concentration of mitochondria over the

same developmental period (Table 2). The greatest rate of change in the mitochondrial adenine nucleotide content occurs during the first 120min of the life of the neonate after caesarean section is carried out one day before natural birth would have occurred (Table 2).

The changes in the adenine nucleotide concentration within the mitochondrial compartment of the cell during development become even more significant when the increase in mitochondrial protein and volume are also


Table 5. Changes in liver and liver mitochondrial adenine nucleotides after birth

19

~ _____

Tirne(min) ATP ADP AMP Adenine Energy nucleotides charge

prnoUg liver

Liver adenine nucleotides

0 (4) 1.23 kO.08 0.54 k 0.04 0.25 f 0.02 2.02 f 0.08 0.74 k 0.02 30 (3) 1.86k0.19 0.26f0.11 0.12f0.15 2.23f0.15 0.89k0.03 60 (4) 2.00f0.10 0.27 fO.05 0.06 fO.03 2.33 fO.16 0.92 kO.02 90 (4) 1.97 f 0.08 0.40 f 0.09 0.10 f 0.05 2.48 f 0.17 0.88 f 0.03

Time(min) ATP ADP AMP Adenine Energy nucleotides charge

prnoVg mitochondrial protein

Mitochondrial adenine nucleotides

0 (3) 0.75 fO.03 0.86 fO.10 0.71 f 0.02 2.32 fO.10 0.51 fO.01 30 (3) 0.98 kO.05 0.81 f 0.12 1.46 f 0.16 3.25 f 0.26 0.43 kO.02 60 (3) 1.14k0.02 1.15f0.15 1.93f0.36 4.23f0.41 0.45k0.03 90 (3) 1.11fO.04 0.97k0.15 1.97f0.24 4.05f0.21 0.40k0.03

Pups were delivered one day prematurely and kept in a humidicrib for up to 90 min. The adenine nucleotides in either freeze-clamped livers or isolated liver mitochondria were measured as described in Methods. The results are expressed f SEM and the number of samples are given in parentheses

taken into consideration. It has been shown by the use of the marker enzyme cytochrome oxidase in whole ho- mogenates and isolated mitochondria, that the amount of mitochondrial protein increases from 8.3 mg/g liver at 3-4 days prenatal to 22.4 mg/g at term or in the 1- day neonate and to 52.2 mg/g liver in the adult rat 111. In Table 3 the results of Table 2 are recalculated in terms of pmol ATP or total adenine nucleotides present in mitochondria derived from 1 gram of liver. In addition the percentage of liver ATP and total adenine nucleotides which are present within the mitochondrial compartment is given (Table 3). For comparison we have included data from morphometric studies [14, 17-191 which indicate the changing size of the mitochondrial compartment, expressed as a percentage of the hepatocyte volume. Clearly there is a very large net uptake of adenine nucleotides by the mitochondrial compartment during development.

As stated previously the in vitro addition of ATP to mitochondrial suspensions increases their R.C.I. [l, 5 , 61. The same effect has been shown to occur in vivo during the maturation of prematurely delivered pups which were left to incubate for up to 9Ominutes in a humidicrib [l, 41 (Table 4). Because of the dramatic increase in the mitochondrial adenine nucleotide content that occurs during the first two hours after birth, the concentrations of the individual adenine nucleotides of perinatal rat liver and rat liver mitochondria were com-

pared after maturing the prematurely born pups for Omin, 30min, 60min, and 9Omin in a humidicrib (Table 5). Such a comparison provides valuable information on the changes that do occur in the adenine nucleotide concentration within the mitochondrial and extramitochondrial compartments of the perinatal liver. In addition, the choice of such short periods to investigate developmental changes minimizes the effects of protein synthesis and any possible contributions due to changes in the non-parenchymal cell population of the liver, though the glycogen content within the parenchymal cell would obviously be expected to drop. The results presented in Table 5 show clearly that during the 30-90 min incubation period in the humidicrib the mitochondrial adenine nucleotides increase to a greater extent than those of the liver. The increase of adenine nucleotides within the mitochondrial compartment suggests that a net uptake of adenine nucleotides from the extra-mitochondrial compartment of the hepatocytes occurs during the incubation period.

Discussion

The increase in the amount and concentration of adenine nucleotides in perinatal rat liver mitochondria during development, raises two points in particular, which warrant discussion. The first one refers specifically to

20 R. Sutton and J. K. Poll&: Adenine Nucleotide Uptake by Mitochondria

the perinatal situation of the rat, while the second one has a more general significance in cell biology.

On the addition of ATP to suspensions of foetal rat liver mitochondria (1.2-2.0 mg protein/2 ml R.C.I. me- dium) an ATP concentration of 0.2 mM is sufficient to increase their R.C.I. [ll . On the other hand, if one as- sumes a specific gravity of 1 for foetal rat liver, then the ATP concentration even in foetal rat liver is about 1.5 mM (Table 1). This then raises the question why these mitochondria have a low R.C.I. It is proposed that it is not the concentration of ATP/cell volume which is critical for effective mitochondrial respiration, but rather the concentration of ATP/mg mitochondrial protein. In the in vitro system, the concentration of added ATP was never less than 200 nmol/mg protein, while within the foetal liver one day before birth it can be calculated to be only 87 nmol/mg mitochondrial protein (total mitochondrial protein was estimated to be 15.8 mg/g liver) [ 11. In addition cellular compartmentation and binding of ATP to other cellular organelles would further decrease the proportion of cellular ATP that is readily available to the mitochondrial compartment. In the prematurely delivered pups kept in a humidicrib there is an increase in the hepatic ATP from 1.23 pmol/g liver to 1.97 pmol/g liver (Table 5) , this can be calculated to amount to an increase from 87 to 125 nmol ATP/mg mitochondrial protein. However, it may not be the mea- surable increase in liver ATP which is responsible for the triggering of the mitochondrial maturation, but rather a transient spike of ATP production, which is the result of a hormone induced, brief burst of glycogenolysis initiated by parturition, as proposed previously 121. Individual mitochondria within the hepatocyte may then respond to localized high concentrations of ATP by hy- percontraction and an increased R.C.I. [ l , 2, 41. These mitochondria due to their more effective phosphorylation of ADP to ATP would be therefore responsible for the maturation of other mitochondria by means of a positive feedback as previously proposed ill, and also for the increase in the overall concentration of ATP within the liver. It should be noted that the hepatic ATP concentration increases by 60%, while the total adenine nucleotide concentration increases by only 23% (Table 5 ) and that part of these increases may be due to a loss of glycogen from the tissue since the nucleotide levels have been expressed in terms of pmol/g liver. Concomi- tant with the increase in the mitochondrial R.C.I. occurring during the first 90 minutes after birth there is a large increase in both ATP (48%) and total adenine nucleotide content (75%) of the mitochondria, which is likely to be an essential part of the maturation pro- cess.

The second interesting point pertains to the enrichment of the adenine nucleotide content of mitochondria as opposed to the exchange-Wusion effected by the adenine nucleotide translocator. As shown in Table 5 , the ATP and the total adenine nucleotide concentration of perinatal liver mitochondria increases by 48% and 75% respectively during the 9Ominute incubation period in the humidicrib. Evidence has been presented, showing that the adenine nucleotide translocators of foetal and adult rat liver mitochondria are essentially similar and therefore an additional mechanism has been proposed to account for the enrichment of mitochondria with adenine nucleotides 11, 71.

Originally it was considered that the low foetal mitochondrial adenine nucleotide values reflected the lower adenine nucleotide content of the foetal hepatocytes as shown by Ballard [31. However, as demonstrated in the present investigation (Table l), the adenine nucleotide concentration of the liver remains fairly constant before and after birth, with the one exception of the 20% decrease which occurs temporarily one day before birth (Table 1). The steadily rising adenine nucleotide concentration of the perinatal rat liver mitochondria during development within hepatocytes with a constant adenine nucleotide content therefore implies an active transport mechanism through an inner mitochondrial membrane which is impermeant to adenine nucleotides, since the percentage distribution of adenine nucleotides between the extramitochondrial and mitochondrial compartments is such as to preclude a membrane freely permea- ble to adenine nucleotides (Table 3).

An alternative explanation for the low adenine nucleotide levels of foetal mitochondria, namely that these mitochondria are more fragile or leaky and lose their adenine nucleotides during isolation, may be discounted, as foetal mitochondria are capable of accumulating adenine nucleotides and retaining these when washed (Sut- ton and Pollak, in preparation). Furthermore it has been repeatedly shown that mitochondrial mass, volume, and protein increases during foetal development [21, hence it is self evident that a mechanism of adenine nucleotide accumulation exists if only to maintain the adenine nucleotide content of the increasing mitochondrial mass.

Without wanting to enter into the arena of the con- troversy of cellular energy control by the energy charge concept [201, it should be pointed out that the perinatal rat liver systems with its changing glycogen metabolism 12,131 and the change in the effectiveness of ADP phosphorylation to ATP in perinatal mitochondria, provides an excellent system which can be manipulated to obtain additional evidence for or against the ‘Energy Charge’

R. Sutton and J. K. Pollak: Adenine Nucleotide Uptake by Mitochondria 21

concept as an ovemding control mechanism in the energy metabolism of the cell.

Acknowledgements: This work was supported by a University of Sydney Research Grant. R.S. was in receipt of an Australian Gov- ernment Post-graduate Scholarship.

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Received May 1978lAccepted July 1978

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