Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [14C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2'-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined in cell extracts. The results demonstrate that under physiological conditions, there is a small but significant flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5'-nucleotidase (EC 3.1.3.5), reflecting the markedly higher Vmax/Km ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher Vmax/Km ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

In vitro adenine nucleotide catabolism in African catfish spermatozoa

It has been shown recently that African catfish (Clarias gariepinus) spermatozoa possess relatively low ATP content and low adenylate energy charge (AEC). One of the possible explanations for this phenomenon is that the spermatozoa actively catabolize adenine nucleotides. A relatively high rate of such catabolism could then contribute to the low ATP concentration and low adenylate energy charge observed in the spermatozoa in vitro. To check this hypothesis, we investigated ATP content and adenine nucleotide catabolism in African catfish spermatozoa stored at 4 °C in the presence of glycine as an energetic substrate. Our results indicate that the storage of African catfish sperm at 4 °C in the presence of glycine causes time-dependent ATP depletion. In contrast to ATP, the AMP content increases significantly during the same period of sperm storage, while the ADP increases only slightly. Moreover, a significant increase of inosine and hypoxanthine content was also found. Hypoxanthine was accumulated in the storage medium, but xanthine was found neither in spermatozoa nor in the storage medium. It indicates that hypoxanthine is not converted to xanthine, probably due to lack of xanthine oxidase activity in catfish spermatozoa. Present results suggest that adenine nucleotides may be converted to hypoxanthine according to the following pathway: ATP→ADP→AMP (adenosine/IMP)→inosine→hypoxanthine. Moreover, hypoxanthine seems to be the end product of adenine nucleotide catabolism in African catfish spermatozoa. In conclusion, our results suggest that a relatively high rate of adenine nucleotide catabolism contributes to the low ATP concentration and low adenylate energy charge observed in African catfish spermatozoa in vitro.     

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.     

Adenine nucleotide pool size,adenine nucleotide translocase activity,and respiratory activity in newborn rabbit liver mitochondria

The adenine nucleotide content (ATP+ADP+AMP) of newborn rabbit liver mitochondria was 6.0±0.5 nmol/mg mitochondrial protein at birth, increased rapidly to 14.5±1.7 nmol/mg protein by 2 h postnatal, peaked at 6 h, then decreased gradually to 7.8±0.6 nmol/mg protein by 4 days postnatal. There was a strong positive correlation (r=0.82) between the total adenine nucleotide pool size and adenine nucleotide translocase activity in these mitochondria. In contrast, glutamate + malate-supported State 3 respiratory rates remained constant from birth through the first week of life. State 4 rates also remained constant, as did the respiratory control index and uncoupled respiratory rates. The following conclusions are suggested: (1) The maximum rate of translocase activity is limited by the intramitochondrial adenine nucleotide pool size. (2) In newborn rabbit liver mitochondria, the State 3 respiratory rate is not limited by either the adenine pool size or the maximum capacity for translocase-mediated adenine exchange. (3) In contrast to rat, rabbit liver mitochondria are fully functional at birth with regard to respiratory rates and oxidative phosphorylation. (4) The rapid postnatal accumulation of adenine nucleotides by liver mitochondria, now documented in two species, may be a general characteristic of normal metabolic adjustment in neonatal mammals.     

Compartmentation of high-energy phosphates in resting and working rat skeletal muscle

The subcellular distribution of high-energy phosphates in various types of skeletal muscle of the rat was analysed by subfractionation of tissues in non-aqueous solvents. Different glycolytic and oxidative capacities were calculated from the ratio of phosphoglycerate kinase and citrate synthase activities, ranging from 25 in m. soleus to 130 in m. tensor fasciae latae. In the resting state, the subcellular contents of ATP, creatine phosphate and creatine were similar in m. soleus, m. vastus intermedius, m. gastrocnemius and m. tensor fasciae latae but, significantly, a higher extramitochondrial ADP-content was found in m. soleus. A similar observation was made in isometrically and isotonically working m. gastrocnemius. The extramitochondrial, bound ADP accounted fully for actin-binding sites in resting fast-twitch muscles, but an excess of bound ADP was found in m. soleus and working m. gastrocnemius. The amount of non-actin-bound ADP reached maximal values of approx. 1.2 nmol/mg total protein. It could not be enhanced further by prolonged isotonic stimulation or by increased isometric force development. It is suggested that non-actin-bound ADP is accounted for by actomyosin-ADP complexes generated during the contraction cycle. Binding of extramitochondrial ADP to actomyosin complexes in working muscles thus acts as a buffer for cytosolic ADP in addition to the creatine system, maintaining a high cytosolic phosphorylation potential also at increasing rates of ATP hydrolysis during muscle contraction.     

Alteration of mitochondrial oxidative phosphorylation in aged skeletal muscle involves modification of adenine nucleotide translocator

The process of skeletal muscle aging is characterized by a progressive loss of muscle mass and functionality. The underlying mechanisms are highly complex and remain unclear. This study was designed to further investigate the consequences of aging on mitochondrial oxidative phosphorylation in rat gastrocnemius muscle, by comparing young (6 months) and aged (21 months) rats. Maximal oxidative phosphorylation capacity was clearly reduced in older rats, while mitochondrial efficiency was unaffected. Inner membrane properties were unaffected in aged rats since proton leak kinetics were identical to young rats. Application of top-down control analysis revealed a dysfunction of the phosphorylation module in older rats, responsible for a dysregulation of oxidative phosphorylation under low activities close to in vivo ATP turnover. This dysregulation is responsible for an impaired mitochondrial response toward changes in cellular ATP demand, leading to a decreased membrane potential which may in turn affect ROS production and ion homeostasis. Based on our data, we propose that modification of ANT properties with aging could partly explain these mitochondrial dysfunctions.     

Modification of liver mitochondrial lipids and of adenine nucleotide translocase and oxidative phosphorylation by cold adaptation

The decrease in respiration rate following thyroidectomy is preceded by changes in the lipid composition of the mitochondrial membrane (Hoch, F.L., Subramanian, C., Dhopeshwarkar, G.A. and Mead, J.F. (1981) Lipids 16, 328–334) and in concert, changes in the kinetic parameters of the adenine nucleotide translocase (Mak, I.T., Shrago, E. and Elson, C.E. (1981) Fed. Proc. 40, 398). To demonstrate that physiological adaptation also involves this sequence of events, rats were housed at 8°C for 3–4 weeks. Cold adaptation resulted in a modest (5%) increase in the unsaturation index for the mitochondrial fatty acids comprised of a significant increase in arachidonic acid and a reciprocal decrease in linoleic acid. Phospholipid analysis indicated that cold adaptation increased the mitochondrial phosphatidylethanolamine and reciprocally decreased the phosphatidylcholine content. Concomitantly, cold adaptation resulted in 25–30% increases in rat liver mitochondrial respiratory activities without changing the respiratory control or ADP/O ratios. The kinetic parameters of the adenine nucleotide translocase were determined by the back-exchange method (Pfaff, E. and Klingenberg, M. (1968) Eur. J. Biochem. 6, 66–79). At 0–4 and 10°C, the V_max and K_m of the cold-adapted rat liver adenine nucleotide translocase were not distinguishable from the control values. The K_i values determined by Dixon plot studies for atractylate and palmitoyl-CoA were also comparable between the two groups. However, at 25 and 37°C, cold-adapted rat liver adenine nucleotide translocase exhibited a 20% increase in V_max and a 20% decrease in K_m for external ADP. The results suggest that one adaption to a cold environment involves hormone-mediated changes in the lipid composition in the mitochondrial membranes which in turn modulate the adenine nucleotide translocase and subsequent respiratory activities.     

Quantitative changes in aminoacylation of transfer RNA and in free amino acids during fructose-induced depletion of adenine nucleotides in rat liver

Fructose induces depletion of adenine nucleotides in liver and also strongly inhibits incorporation of radioactive amino acids into protein (Mäenpää, P.H., Raivio, K.O. and Kekomäki, M.P. (1968) Science 161, 1253–1254). In this study we have investigated the effects of fructose on aminoacylation of tRNA and on free amino acids in rat liver. 30 min after d-fructose (30 mmol/kg) was injected intraperitoneally into rats, liver ATP was reduced by 58%, ADP by 42%, AMP by 13%, the ATP/ADP ratio by 30%, and total adenine nucleotides by 48%. Using gas chromatography, the aminoacylation of tRNA was determined by quantifying the endogenous amino acids attached to tRNA in vivo. Aminoacylation was reduced by 31%. With different amino acids, reduction varied from 4% (asparagine plus aspartic acid) to 58% (arginine). On the other hand, the amount of free amino acids in the liver was increased by 24%. The most marked individual change was in alanine, which increased 5.7-times. This may have resulted from a combination of effects involving an increased production of alanine in muscle and liver and decreased hepatic gluconeogenesis from alanine caused by the ATP depletion.     

Inhibition of oxidative phosphorylation by Ca or Sr: A competition with Mg for the formation of adenine nucleotide complexes

Intramitochondrial Sr²⁺, similar to Ca²⁺, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca²⁺ and Sr²⁺ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca²⁺ or 5.0 mM Sr²⁺ when the concentration of Mg²⁺ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg²⁺ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca²⁺ or Sr²⁺ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg²⁺ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr²⁺ it can be concluded that intramitochondrial Ca²⁺ or Sr²⁺ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

Proton permeation, adenine nucleotide translocation and respiratory control in mitochondria cross-linked by dimethylsuberimidate

Rat liver mitochondria or isolated mitoplasts were treated with the cross-linking agent, dimethylsuberimidate, under conditions (pH 7.5; 0°C) which were not detrimental for the coupling quality of the mitochondria and the effect was evaluated on a kinetic basis. When about 25% of the NH₂-groups reacted, the mitochondria or the mitoplasts acquired complete osmotic stability. Succinate oxidation in state 4 was inhibited by about 30–35%. This effect was also observed when the organelles were amidinated by methylacetimidate, a monofunctional imidate which caused no osmotic stabilization. Uncouplers stimulated succinate oxidation in cross-linked mitochondria to the same extent as in the control, in contrast stimulation by ADP was suppressed. Accordingly, the rate of decay of the respiration-dependent cross-membrane proton gradient was only decreased by 25%, whereas the ATPase and adenine nucleotide translocase were strongly inhibited. In the cross-linked mitochondria, the extent of inhibition of the ATPase and of the translocase was found to be the same whether the assays were performed at 30°C (like the respiratory assay) or at 0°C. The effect of methylacetimidate treatment on these activities at the two temperatures was different. At 30°C, the ATPase was not inhibited and the extent of inhibition of ATP translocation was small. At 0°C, the two activities were nearly as much inhibited as in cross-linked mitochondria. Our results suggest that a considerable rigidity can be introduced in the coupling membrane by cross-linking, without a major loss in the initial step of energy conservation. However, the energy conserved in the proton gradient cannot be utilized for ATP synthesis, probably because of the restricted mobility of adenine nucleotide translocase in the cross-linked mitochondria.     

Thymidine nucleotide synthesis and catabolism by CHO cells and their changes during the cell cycle

At 0°C, CHO cells efficiently incorporated [³H]thymidine into the nucleotide fraction, but not into DNA. Upon reincubation of asynchronous cultures at 37°C, 15–25% of the radioactivity contained in the cellular nucleotide fraction was released, in the form of thymidine, into the culture medium. At 0°C, however, radioactivity of the nucleotide fraction was retained within the cells. Similarly, dTMP phosphatase (EC 3.1.3.35) in cell extracts was active at 37°C, but not at 0°C, whereas thymidine kinase (EC 2.7.1.21) was active at both temperatures. If synchronous cultures in Gl phase were prelabeled at 0°C and reincubated at 37°C, almost all radioactivity in the nucleotide fraction was released into the medium, whereas in S-phase cultures nearly all radioactivity of the nucleotide fraction was incorporated into DNA. In synchronous S-phase cultures treated with hydroxyurea, radioactivity in the nucleotide fraction was released into the medium at a rate considerably lower than that observed for Gl-phase cells. Rates of endogenous synthesis of thymidine nucleotides were calculated from changes of cellular thymidine nucleotide content, incorporation of thymidine nucleotides into DNA and release of thymidine into the medium during reincubation of prelabeled cultures in thymidine-free medium. The results obtained (see Table III) reveal marked differences between Gl and S phases with respect to the determinants of thymidine nucleotide metabolism.     

Control of mitochondrial respiration. The contribution of the adenine nucleotide translocator depends on the ATP- and ADP-consuming enzymes

The consequence of the complexity of the metabolic network on the amount of control strength of adenine nucleotide translocator was investigated with isolated rat liver mitochondria. Two experimental systems were compared: (i) mitochondria in the presence of yeast hexokinase (hexokinase system) and (ii) the same system plus additional pyruvate kinase (pyruvate kinase system). In both systems the control strength was analysed for the adenine nucleotide translocator by inhibitor titration studies with carboxyatractyloside and for the hexokinase or pyruvate kinase by changing their relative activities. Experimental results were compared with computer simulation of these systems and that of a third one, where the extramitochondrial ATP / ADP ratio was held constant by perifusion (perifusion system). The results demonstrate quite different flux-dependent control strength of the translocator in the three systems. In the hexokinase system the control strength of the translocator on mitochondrial respiration was zero up to respiration rates of about 60 nmol O₂/mg protein per min. For higher rates, the control strength increased until the maximum value (0.45) was reached in the fully active state. Here, the same value was also found in the pyruvate kinase system. In all other states of respiration the translocator exerts a higher control strength in the pyruvate kinase system than in the hexokinase system. This different behaviour was attributed to the various changes in the adenine nucleotide pattern caused by partial inhibition of the translocator in the hexokinase and pyruvate kinase system. The data clearly show that the sharing of control strength depends not only on the respiration rate but also on the complexity of the metabolic system.     

Evidence for swelling-induced adenosine and adenine nucleotide release in rat cerebral cortex exposed to monocarboxylate-containing or hypotonic artificial cerebrospinal fluids

Recent reports have described a swelling-induced release of adenosine triphosphate (ATP) from a variety of non-nervous system cell types, which may be involved in the regulatory volume decrease (RVD) response. The present study examined the effects of swelling induced by applications of hypotonic or monocarboxylic acid containing artificial cerebrospinal fluid (aCSF) on the release of adenosine nucleotides and adenosine from the in vivo rat cerebral cortex using a cortical cup technique. Hypotonic aCSF (25mM NaCl) elicited a significant increase in adenosine, but not adenine nucleotide, release. Applications of sodium L-lactate, pyruvate, or acetate (all 20mM) evoked increases in adenine nucleotides but not adenosine. D-Lactate (20mM) enhanced adenosine and ATP release. Inhibition of the plasma membrane monocarboxylate transporter with cyano-4-hydroxycinnamate (4-CIN, 2mM) blocked the effects of L-lactate on purine release. These in vivo results demonstrate that osmoregulatory processes in cortical cells evoke an efflux of adenine nucleotides and/or adenosine. In that these purines activate a variety of receptors, it is possible that they may function as autocrine or paracrine signaling agents, facilitating volume regulation and enhancing local blood flow.     

Intracellular distribution of fumarase in rat skeletal muscle

The distribution of fumarase activity between the mitochondrial and cytoplasmic compartments of rat skeletal muscle was studied using the method of Fatania and Dalziel (Biochim. Biophys. Acta 631 (1980) 11–19), fractional extraction technique and a method based on the calculation of mitochondrial protein content in the tissue and on the determination of fumarase activity both in the tissue homogenate and in the isolated mitochondria. We found 10%, 5% and 0% of the total fumarase activity in the cytoplasm using these methods, respectively. The results suggest that no more than 10% of the total fumarase activity is present in the cytosolic fraction of rat skeletal muscle. The metabolic consequences of such distribution of fumarase in skeletal muscle are discussed.     

Increased levels of adenine nucleotides modify the interaction between starch synthesis and respiration when adenine is supplied to discs from growing potato tubers

To investigate the importance of the overall size of the total adenine nucleotide pool for the regulation of primary metabolism in growing potato tubers, freshly cut discs were provided with zero or 2 mM adenine in the presence of 1 or 100 mM [U-¹⁴C]glucose or 100 mM [U-¹⁴C]sucrose in the presence and absence of 20 mM orthophosphate (Pi). Adenine led to a 150–250% increase of the total adenine nucleotide pool, which included an increase of ADP, a larger increase of ATP and an increase of the ATP:ADP ratio. There was a 50–100% increase of ADP-glucose (ADPGlc), and starch synthesis was stimulated. Respiratory oxygen uptake was stimulated, and the levels of glycerate-3-phosphate, phosphoenolpyruvate and α-ketoglutarate decreased. The response to adenine was not modified by Pi. It is proposed that increased ATP stimulates ADPGlc pyrophosphorylase, leading to a higher rate of starch synthesis. The impact on starch synthesis is constrained, however, because increased ADP can lead to a stimulation of respiration and decline of glycerate-3-phosphate, which will inhibit ADPGlc pyrophosphorylase. The quantitative impact depends on the conditions. In the presence of 1 mM glucose, the levels of phosphorylated intermediates and the rate of starch synthesis were low. Adenine led to a relatively large stimulation of respiration, but only a small stimulation of starch synthesis. In the presence of 100 mM glucose, discs contained high levels of phosphorylated intermediates, low ATP:ADP ratios (<3) and low rates of starch synthesis (<20% of the metabolised glucose). Adenine led to marked increase of ATP and 2- to 4-fold stimulation of starch synthesis. Discs incubated with 100 mM sucrose already had high ATP:ADP ratios (>8) and high rates of starch synthesis (>50% of the metabolised sucrose). Adenine led to a further increase, but the stimulation was less marked than in high glucose. These results have implications for the function of nucleotide cofactors in segregating sucrose mobilisation and respiration, and the need for energy conservation during sugar-starch conversions. Received: 9 February 2000 / Accepted: 9 June 2000     

Comparison of AMP deaminase from skeletal muscle of acidotic and normal rats

The deamination of AMP by AMP aminohydrolase (EC 3.5.4.6) serves as the major source of ammonia production in skeletal muscle. It has been suggested that the ammonia may serve either in a buffering capacity to combat acidosis due to the accumulation of lactic acid produced during prolonged muscular activity, or as a substrate for glutamine formation which can ultimately be utilized by the kidney in adapting to metabolic acidosis. In view of this proposal, the properties of the enzyme obtained from skeletal muscle of acidotic rats have been compared with the enzyme from normal muscle. The specific activity of AMP deaminase in crude homogenates of acidotic muscle was not significantly different from normal levels. The enzyme from acidotic muscle was purified to homogeneity and was found to be identical to the enzyme obtained from normal muscle by the criteria of electrophoretic mobility, pH optimum, molecular weight, sedimentation coefficient, subunit composition, amino acid composition, monovalent cation requirement, substrate saturation, and inhibition by ATP, P_i and creatine-P. Thus, if the enzyme functions to prevent acidosis, the ability to respond to changes in the intracellular environment which accompany acidosis must be built into the structure of the enzyme normally found in skeletal muscle. Three lines of evidence strongly support this viewpoint: (a) the rate of deamination is approximately 2-fold higher at pH 6.5 than at pH 7.0, (b) the activity increases linearly with a decrease in the adenylate energy charge, and (c) within the normal physiological range of the adenylate energy charge, the enzyme is operating at only 10–20% of its maximum capacity.     

Calcium-dependent cyclic nucleotide phosphodiesterase inhibition of basal activity by heat-stable factors from rat cerebrum

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca²⁺-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca²⁺-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in K_m for cyclic GMP and a decrease in $\text{V}$. In the presence of calcium ions and purified activator protein, the Ca²⁺-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca²⁺-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was mimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca²⁺-independent activation of Ca²⁺-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca²⁺-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca²⁺-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phosphodiesterase activity.     

Characterization of purine nucleotide metabolism in primary rat muscle cultures

The synthesis and metabolic fate of purine nucleotides were studied, employing labeled precursors, in primary rat muscle cultures. The cultures were found to produce purine nucleotides, by de novo and salvage pathways, both exhibiting dependence on cellular availability of substrate 5-phosphoribosyl-1-pyrophosphate (PPRibP). Depletion of cellular PPRibP decelerated the rate of purine synthesis, whereas increasing PPRibP generation by high Pi concentration in the incubation medium, accelerated purine synthesis. Ribose accelerated purine synthesis, indicating that ribose 5-phosphate availability in the cultured muscle is limiting for PPRibP synthesis. The study in the muscle cultures of the metabolic fate if IMP formed from [14C]formate and that of nucleotides formed from labeled purine bases, revealed that the main flow in the nucleotide interconversions pathways is from AMP to IMP. The flow from IMP to GMP and to AMP appeared to be of a lesser magnitude and virtually no flow could be detected from GMP to IMP. The greatest proportion of radioactivity of purine nucleotides following synthesis by either de novo or salvage pathways, accumulated in IMP, reflecting the relative rates of flows between the various nucleotides and probably also a relatively low, or inhibited activity of the IMP nucleotidase. The results suggest that primary muscle cultures are a plausible model for the study of the role of purine metabolism in muscle work.     

Contracting striated muscle fibres differentiated from primary rat pituitary cultures

Summary Whole pituitaries or adenohypophyses alone of adult female Wistar/ Furth rats were dissociated into single cells by means of two different enzymic disintegration methods. The single-cell suspension was then seeded out and cultured for up to 8 months in tissue culture dishes with untreated and polylysine coated surfaces. The cells were cultured in different sera (horse serum, newborn-calf serum, fetal-calf serum, mixtures of horse and newborn-calf serum, and isogenic rat serum) and also in a serum-free, hormone-supplemented medium. When the cells were cultured in medium containing horse serum (15 %) plus fetal-calf serum (3%) on polylysine-treated surfaces, cell fusion and the development of myotubes could be observed between day 5 and 10 after seeding and, on about day twenty, the formation of multicellular microstructures could be seen. Myotubes in such microstructures differentiate into muscle fibres, and show spontaneous contraction. Striation is visible both light and electron microscopyically. Such a differentiation into striated muscle cells depends on specific culture conditions: the serum used, the formation of microstructures, and the treatment of the culture dishes. There is apparently no previous report of striated muscle cells found in pituitary cultures.     

Inhibition of oxidative phosphorylation by a Ca2+-induced diminution of the adenine nucleotide translocator

The mechanism through which internal Ca²⁺ inhibits oxidative phosphorylation of rat heart mitochondria has been explored. In parallel to a Ca²⁺-induced diminution of the activity of the adenine nucleotide translocator, an efflux of internal adenine nucleotides is observed. The efflux of adenine nucleotides depends on the amount of Ca²⁺ accumulated by the mitochondria and on the time that Ca²⁺ remains in the mitochondria; this efflux is atractyloside insensitive. These results suggest that internal Ca²⁺, by inducing a lowering of the internal concentration of adenine nucleotides, diminishes the rate of exchange of adenine nucleotides via the translocase, and in consequence of oxidative phosphorylation. Under conditions in which the Ca²⁺-induced release of adenine nucleotides takes place, no gross changes of the permeability properties of the membrane are observed. As revealed by studies with arsenate, respiratory activity and the function of the ATPase in the direction of ATP synthesis are not affected by internal Ca²⁺.