Effect of ribose supplementation on resynthesis of adenine nucleotides after intense intermittent training in humans.

The effect of oral ribose supplementation on the resynthesis of adenine nucleotides and performance after 1 wk of intense intermittent exercise was examined. Eight subjects performed a random double-blind crossover design. The subjects performed cycle training consisting of 15 x 10 s of all-out sprinting twice per day for 7 days. After training the subjects received either ribose (200 mg/kg body wt; Rib) or placebo (Pla) three times per day for 3 days. An exercise test was performed at 72 h after the last training session. Immediately after the last training session, muscle ATP was lowered (P < 0.05) by 25 +/- 2 and 22 +/- 3% in Pla and Rib, respectively. In both Pla and Rib, muscle ATP levels at 5 and 24 h after the exercise were still lower (P < 0.05) than pretraining. After 72 h, muscle ATP was similar (P > 0.05) to pretraining in Rib (24.6 +/- 0.6 vs. 26.2 +/- 0.2 mmol/kg dry wt) but still lower (P < 0.05) in Pla (21.1 +/- 0.5 vs. 26.0 +/- 0.2 mmol/kg dry wt) and higher (P < 0.05) in Rib than in Pla. Plasma hypoxanthine levels after the test performed at 72 h were higher (P < 0.05) in Rib compared with Pla. Mean and peak power outputs during the test performed at 72 h were similar (P > 0.05) in Pla and Rib. The results support the hypothesis that the availability of ribose in the muscle is a limiting factor for the rate of resynthesis of ATP. Furthermore, the reduction in muscle ATP observed after intense training does not appear to be limiting for high-intensity exercise performance.

Adenine nucleotide synthesis in human erythrocytes depends on the mode of supplementation of cell suspension with adenosine.

In suspensions of washed human erythrocytes, adenosine added in a single dose to concentrations of 0.1-10.0 mmol/l suspension was deaminated at rates ranging from 10 to 50 mmol/l cells h. The sum of adenosine, inosine, and hypoxanthine concentrations in the suspension, as well as the intracellular concentration of ATP, remained constant. In the presence of 25-50 mmol/l orthophosphate, addition of a single dose of adenosine into erythrocyte suspension increased the ATP concentration by up to 280% of the initial level. If the initial adenosine concentrations were greater than 5 mmol/l suspension, ATP increased independently of adenosine concentration to the level determined only by the concentration of orthophosphate. After orthophosphate was returned to its initial level, ATP in erythrocytes began to decrease. In the presence of coformycin, erythrocytes utilised adenosine at a rate of 0.2-0.3 mmol/l cells h. Their adenylate pool increased at a rate of 0.10-0.16 mmol/l cells h for several hours, but intracellular ATP increased only slightly. The energy charge of cells decreased significantly from 0.86 +/- 0.05 (control) to 0.82 +/- 0.06. Adenosine continuously pumped into erythrocyte suspensions at rates of 0.02-5.0 mmol/l cells h for several hours caused the adenylate pool of erythrocytes and intracellular ATP to increase synchronously at a rate of 0.02-0.35 mmol/l cells h. The energy charge of these erythrocytes increased significantly up to 0.91 +/- 0.03. After pumping of adenosine was stopped, the intracellular ATP and the adenylate pool began to decrease, returning sometimes to the initial level in 2-3 h.

Inhibition of adenine nucleotide synthesis: effect on tight junction structure and function of clone 4 MDCK cells.

The formation and maintenance of tight junctions as a barrier to the diffusion of ions and other water-soluble across epithelia is an energy-dependent process. The administration of N-formyl-hydroxyaminoacetic acid (Hadacidin), an analog of aspartate and a competitive inhibitor of adenylosuccinate synthetase, has been shown to inhibit the multiplication of clone 4 MDCK cells and concomitantly reduce the levels of ATP and cAMP (J. Cell. Physiol. 140, 186-194 (1989)). When added to mitotically quiescent confluent cultures of clone 4 MDCK cells, millimolar concentrations of Hadacidin inhibited the generation of transepithelial electrical resistance (TER). In such cultures passive Na+ permeability was similar to controls indicating that the effect of Hadacidin was not on the transcellular pathway. That these cells were viable was demonstrated by their ability to exclude Trypan Blue, and the fact that they remained competent to develop steady state TER upon removal of the inhibitor. Suppression of TER was completely reversed within 48 h of replacing the Hadacidin-supplemented medium with one containing aspartate. Adenosine, but not aspartate, when added simultaneously with the drug, obviated the latter's effect on TER. A mixture of dibutyryl cAMP (db-cAMP) and theophylline was only partially effective in overcoming the effects of Hadacidin on the development of TER and, in fact, markedly delayed its development in control cultures not treated with the drug. When monolayers with established steady state TER were exposed to Hadacidin, no change was noted during the first 24 h. By 48 h, however, TER had decreased to very low values.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine nucleotide synthesis in exercising and endurance-trained skeletal muscle.

Strenuous exercise leads to increased efflux of purine nucleoside and base that should necessitate recovery of adenine nucleotides by either the de novo synthesis or salvage pathway. De novo synthesis of adenine nucleotide was measured in quiescent and contracting muscle of sedentary and exercise-trained rats using an isolated perfused hindquarter preparation. Synthesis rates were assessed by measuring the incorporation of [1-14C]glycine into adenine nucleotide in muscles of both resting and stimulated hindlimbs after 1 h of either low- or high-energy demand isometric contractions. In nonstimulated sedentary and trained muscles, rates of de novo synthesis were similar. The effect of muscle contractions on de novo synthesis varied among muscle fiber types. Contracting, nonfatigued fast-twitch muscle sections showed significant declines in de novo synthesis in both sedentary and trained groups. Rates in slow-twitch red fibers and fatigued fast-twitch white fiber sections were not different from rest. Supplementing the perfusate with 5 mM ribose caused de novo synthesis to rise three- to fourfold in each of the fiber sections. However, the response in synthesis rates due to exercise was similar with or without ribose supplementation. De novo synthesis does not increase during exercise but exhibits an unchanged or reduced rate depending on the expected energy balance within the cell. This would occur if the energy state of muscle exerts significant control over de novo synthesis of adenine nucleotide.

Effect of extracellular potassium on ouabain-sensitive consumption of high-energy phosphate by crayfish giant axons: a study of the energy requirement for transport in the steady state.

Crayfish axons exposed to a high or low extracellular K+ concentration ([K+]o) maintain intracellular Na+ and K+ concentrations constant, for up to 3 h, by adjusting both the Na+/K+ transport "coupling ratio" and turnover rate in compensation for changes in ion fluxes due to altered electrochemical gradients. These findings give rise to the prediction that the steady-state consumption of high-energy phosphate (approximately P) [ATP and phospho-L-arginine (Arg-P)] is inversely proportional to the [K+]o, i.e., directly proportional to the product of membrane conductance and magnitude of the transmembrane electrochemical gradients for Na+ and K+. This investigation was designed to test this hypothesis. The [K+]o did not influence total approximately P consumption (Q approximately P) of the axon. For a [K+]o between 0.5 and 21.6 mM, Q approximately P averaged 52.8 +/- 4.7%/h (n = 44) of the initial [ATP] + [Arg-P]. Unlike total Q approximately P, the ouabain-sensitive portion of Q approximately P was markedly influenced by [K+]o. In 0.5 mM K+o, ouabain poisoning reduced Q approximately P to 8%/h, a result indicating that 85% of the total Q approximately P was ouabain sensitive. For 1.35 mM K+o, the ouabain-sensitive portion was 66%; at 5.4 mM K+o, 45%; and at 13.5 mM K+o, 41%. There was a small but significant increase in the ouabain-sensitive Q approximately P at 21.6 mM K+o, compared with Q approximately P at 5.4 mM K+o. The pattern of effect of [K+]o on Q approximately P was similar to its effect on the electrical power content of the Na+ and K+ electrochemical gradients. In contrast to the generally accepted Na+ flux (JNa)/approximately P stoichiometry of 3, an actual ratio of JNa/approximately P stoichiometry of approximately 33:1 was calculated for the experiments reported here, a result suggesting that cells in a zero-membrane current steady state utilize efficient energy conservation mechanisms that may not operate under non-steady-state conditions.

The neuromuscular and vascular hypotheses of muscular dystrophy: a possible link via adenine nucleotides and phosphate.

A hypothesis is presented that an abnormality of nucleotide synthesis or metabolism might be a primary functional defect underlying Duchenne muscular dystrophy. One aim of this hypothesis is to demonstrate that it is possible to explain a variety of observations in dystrophy, neuromuscular and vascular, on a single hypothesis.

Myocardial lipids and nucleotides of rats fed olive oil or rapeseed oil.

After 1 week, the level of myocardial fatty acids was 4 times greater in young rats fed high erucic rapeseed oil than in those fed oliver oil. The proportion of erucic acid was 5.6% in the mitochondrial fraction, 15.1% in the microsomal fraction, and 34.8% in the floating fat fraction. This incorporation of erucic acid into triglycerides of the floating fat was evidence of esterification. The changes in the mitochondrial lipids did not alter the content of adenine nucleotides of the myocardium nor its apparent capacity to oxidize substrates.

A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17 .

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17beta. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(beta-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.