The Purine‐Nucleotide Cycle

Abstract

The contents of citric acid cycle compounds were measured in rat hind limb muscle in situ during rest, exercise, and recovery from exercise. The following changes in citric acid cycle intermediates were observed during exercise for 15 min. The contents of fumarate and malate increased fourfold over resting values. The contents of citrate, isocitrate, and succinate rose 68 'I<, 48 and 87 "' , respectively, but the increase in these intermediates was delayed relative to the increase in fumarate and malate. The content of 2-oxoglutarate did not change significantly. The content of oxaloacetate increased from less than 1 nmol/g dry weight in resting muscle to 8 nmol/g dry weight during exercise. The orthophosphate content doubled. The sum of the contents of citric acid cycle intermediates doubled. The ratios of the contents of malate/fumarate and citrate/isocitrate remained constant. The constancy of the citrate/isocitrate ratio indicates that the concentration of intracellular free Mg2+ is not affected by exercise. All metabolite levels returned to resting values during recovery. Comparison of these data with the rate of operation of the purine nucleotide cycle leads us to conclude that the increase in citric acid cycle intermediates can be accounted for by the operation of the purine nucleotide cycle.The rate of operation of the purine nucleotide cycle in skeletal muscle increases during exercise and recovery following exercise [l, 21. The cycle catalyzes the net reaction : aspartate + GTP + H20 + NH3 + fumarate + G D P + Pi. We now show that the total level of citric acid cycle intermediates in rat skeletal muscle rises by a factor of two during exercise. The changes in citric acid cycle intermediates are compared with the rate of formation of IMP in the absence and presence of hadacidin, an inhibitor of adenylosuccinate synthetase. On the basis of this comparison it is concluded that the operation of the purine nucleotide cycle is sufficient to account for the observed increases in citric acid cycle intermediates in skeletal muscle. Other possible reactions for replenishing or increasing citric acid cycle intermediates are considered in the Discussion.ions participate in the reactions of the citric acid and the purine nucleotide cycles and they exert direct and indirect effects on adenylate deaminase [3]. Determinations of the contents of these substances in skeletal muscle during rest, exercise, and recovery from exercise are also presented. Orthophosphate and Mg2 MATERIALS AND METHODSMale rats of the Sprague-Dawley strain were obtained from Charles River Breeding Laboratories (Wilmington, MA). The animals received food and water ad libitum, and weighed 175-200 g at the time of use.Rats were anaesthetized by intraperitoneal injection of sodium pentobarbital (5 mg/100 g body weight) and the skin from the right hind leg was removed. The sciatic nerve was exposed and a Dastre's electrode (Palmer Co., London, UK) was attached around the nerve in its gluteal course. The leg was fixed to a platform at the ankle with ...

Sites of Superoxide and Hydrogen Peroxide Production by Muscle Mitochondria Assessed ex Vivo under Conditions Mimicking Rest and Exercise

“…Published values were corrected for plasma concentrations where appropriate and for the distribution of intracellular metabolites between cytosol and mitochondria, using the following matrix/cytosol ratios from heart and liver (or the assumed values for metabolites in parentheses): glycerol 3-phosphate, glutamine (dihydroxyacetone phosphate, taurine): 0 (68,69) (83)(84)(85)(86). Targeted free Mg 2ϩ concentrations were 600 M (rest), 600 M (mild aerobic exercise), and 1000 M (intense aerobic exercise) (40,(87)(88)(89). Targeted Na ϩ concentrations were 16 mM for all media (65,89).…”

Regulação metabólica e produção de espécies reativas de oxigênio durante a contração muscular: efeito do glicogênio na manutenção do estado redox intracelular

“…Nesse contexto, diferentes mecanismos anapleróticos controlados por reações de carboxilação e descarboxilação podem controlar o fluxo que regula a entrada e saída de carbonos do ciclo. Entre estes mecanismos, inclui-se as reações do ciclo das purinas (36) e a reação catalisada pela glutamato desidrogenase (37) (Figura 5). Esses mecanismos permitem ajustar as concentrações dos intermediá-rios do ciclo de Krebs em resposta a demanda metabólica.…”

“…Um aumento da atividade anaplerótica pelas vias acima descritas poderia contribuir com o aumento do fluxo de entrada de substratos no ciclo de Krebs (11,20,35,36,38) , favorecendo a atividade da isocitrato desidrogenase e consequentemente a síntese de NADPH + , importante doador de elétrons para a regeneração de glutationa no músculo (32) . Em adição, o fluxo de substratos no ciclo de Krebs pode ser regulado pelo aumento de ADP/ATP, Ca ++ e NAD/NADH + , importantes moduladores do complexo piruvato desidrogenase (15) , e das enzimas reguladoras do ciclo (non-equilibrium, G-), citrato sintase, isocitrato desidrogenase e -cetoglutarato desidrogenase.…”

“…Portanto, qualquer aumento no fluxo dessas vias de formação, independente de NADH, pode favorecer a manutenção da síntese oxidativa de ATP durante a atividade muscular prolongada. Embora as reações acima são descritas como importantes vias anapleróticas, durante o exercício prolongado (11,20,(34)(35)(36)(37)(38) , a redução nos estoques de glicogênio pode comprometer a disponibilidade de substrato na forma de oxalacetato, piruvato e fosfoenolpiruvato para algumas dessas vias, como é o caso das reações catalisadas pela aspartato aminotransferase, alanina aminotransferase, glutamato piruvato transaminase, enzima má-lica e piruvato carboxilase. Dessa forma, aumentando a possibilidade da enzima glutamato desidrogenase e do ciclo das purinas de nucleotídios serem as principais vias anapleróticas durante a atividade muscular prolongada.…”

“…Por outro lado, a depleção dos estoques de glicogênio muscular e/ou redução da glicemia devido ao comprometimento dos estoques de glicogênio hepático reduz substancialmente a performance durante o exercício prolongado (18,49) . A diminuição da concentração de glicogênio resulta em menor disponibilidade de piruvato, um substrato importante para produção de acetil-CoA e para manutenção das vias anapleróticas de produção de intermediários do ciclo de Krebs, incluindo oxalacetato (11,20,33,36,37) . Portanto, um aumento do conteúdo dos intermediários do ciclo de Krebs é fundamental para a manutenção da produção de NADH.…”