RESUMO
Uma série de estudos tem sido realizada para compreensão do metabolismo de glicogênio muscular durante o exercício. Estudos clássicos apontaram uma associação entre as reservas iniciais de glicogênio muscular e o tempo de sustentação do esforço. O glicogênio muscular diminui de forma semi-logarítmica em função do tempo, mas a concentração desse substrato não chega a zero, o que sugere a participação de outros mecanismos de fadiga na interrupção do exercício prolongado. Nesse tipo de atividade, a depleção de glicogênio, primeiro, ocorre nas fibras de contração lenta, seguida pela depleção nas de contração rápida. A diminuição na taxa de utilização de glicogênio muscular está sincronicamente ligada ao aumento no metabolismo de gordura, mas o mecanismo fisiológico é pouco compreendido. Estudos recentes sugerem que uma diminuição da insulina durante o exercício limitaria o transporte de glicose pela membrana plasmática, causando um aumento no consumo de ácidos graxos. Alguns estudos têm demonstrado, também, que a própria estrutura do glicogênio muscular pode controlar a entrada de ácidos graxos livres na célula, via proteína quinase. Fisicamente, a molécula de glicogênio se apresenta de duas formas, uma com estrutura molecular menor (aproximadamente, 4,10(5) Da, Proglicogênio) e outra maior (aproximadamente, 10(7) Da, Macroglicogênio). Aparentemente, a forma Proglicogênio é metabolicamente mais ativa no exercício e a Macroglicogênio mais suscetível a aumentar com dietas de supercompensação. Maior concentração de hipoxantinas e amônia no exercício com depleção de glicogênio muscular também foi relatada, mas estudos com melhor controle da intensidade do esforço podem ajudar a elucidar essa questão.
A large number of studies have been conducted to understand muscle glycogen metabolism during exercise. Classical studies demonstrated a relationship between the pre-exercise muscle glycogen content and duration of exercise. Muscle glycogen declines in a semilogarithmic manner in function of time, but glycogen concentration does not reach zero, which suggests that other fatigue mechanisms participate in the interruption of prolonged exercise. In this type of activity, glycogen depletion occurs first in slow twitch fibers followed by fast twitch fibers. The decrease in the rate of muscle glycogen utilization is synchronized with an increased rate of fat uptake, but the physiological mechanism is not well understood. Recent studies suggest that the decline of insulin during exercise could be a limiting factor of glucose transport through the plasma membrane, which increases the uptake of fatty acids. Others studies have also demonstrated that the structure of muscle glycogen itself can regulate the cellular uptake of free fatty acids via protein kinase. Physically, the glycogen molecule has two forms, one with a smaller molecular structure (approximately 4.10(5) Da, proglycogen) and another one with a larger molecular structure (approximately 10(7) Da, macroglycogen). Apparently, the proglycogen form is more metabolically active during exercise and the macroglycogen form is more susceptible to increase with supercompensation diets. Higher concentrations of hypoxanthines and ammonia during exercise with muscle glycogen depletion have been reported, but studies that control exercise intensity better are necessary to help shed light on this issue.
Assuntos
Esforço Físico/fisiologia , Glicogênio/metabolismo , Hipoxantinas/metabolismo , Insulina/metabolismo , Músculos/metabolismoRESUMO
The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-D-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent K(m) of 320 +/- 10 microM and a Vmax of 5.6 +/- 0.9 pmol/10(6) cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41 +/- 6, 240 +/- 27 and 59 +/- 8 microM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5 +/- 0.3, 11 +/- 3 and 0.16 +/- 0.006 microM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290 +/- 14 microM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells.
Assuntos
Proteínas de Transporte/efeitos dos fármacos , Endotélio Vascular/metabolismo , Hipoxantinas/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Tioinosina/análogos & derivados , Adenosina/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Linhagem Celular , Endotélio Vascular/enzimologia , Humanos , Hipoxantina , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Nucleosídeos , Tioinosina/farmacologiaRESUMO
Luminol-enhanced chemiluminescence was used to determine the effects of diethyldithiocarbamate, dipyridamole, catechin and verapamil on the generation of reactive oxygen species in human leukocytes, and on superoxide generated by chemiluminescence of the hypoxanthine xanthine-oxidase reaction. These agents reduced the luminol enhanced chemiluminescence response of activated leukocytes, most probably by inhibiting the superoxide generation reaction. On the other hand, citrate and diethylcarbamazine, produced a slight increase of the luminol enhanced chemiluminescence of leukocytes.
Assuntos
Leucócitos/metabolismo , Medições Luminescentes , Superóxidos/metabolismo , Catequina/farmacologia , Dipiridamol/farmacologia , Ditiocarb/farmacologia , Humanos , Hipoxantina , Hipoxantinas/metabolismo , Leucócitos/efeitos dos fármacos , Luminol/farmacologia , Proteínas Opsonizantes , Saccharomyces cerevisiae , Verapamil/farmacologia , Xantina Oxidase/metabolismoRESUMO
El dano provocado por la pancreatitis experimental en perros, determina un aumento significativo de la actividad de la Guanasa. Este aumento se produce a las 2 hs de la isquemia en tejidos y a las 24 hs en suero. Asi mismo se determina que la actividad es similar en cabeza y cola y está ausentela actividad en el cuerpo del páncreas. Finalmente se observa que mientras las oxipurinas séricas, aumentan en forma similar a la Guanasa sérica, la Guanina sé-rica, disminuye significativamente recién a las 24 hs. Estos resultados indicanque la actividad de guanasa es un excelente "marcador" del dano pancreático en perros
Assuntos
Animais , Masculino , Cães , Guanina Desaminase/metabolismo , Isquemia , Pâncreas/irrigação sanguínea , Pâncreas/metabolismo , Pancreatite/enzimologia , Hipoxantinas/metabolismo , Isquemia , Xantinas/metabolismoRESUMO
Luminol chemiluminescence induced by the xanthine or hypoxanthine-O2-xanthine oxidase system is analyzed and compared. Characteristics of the light emission curves were examined considering the conventional reaction scheme for the oxidation of both substrates in the presence of xanthine oxidase. The ratio of the areas of the rate of superoxide production during substrate oxidation to uric acid. The O2-. to uric acid ratio for each substrate can account for differences in xanthine and hypoxanthine-supported light emission, since uric acid is a strong inhibitor of O2-.-dependent luminol chemiluminescence. These results are consistent with a free radical scavenging role for uric acid. A similar but weaker scavenging effect of xanthine may also contribute to the observed differences in chemiluminescent yields between both substrates.
Assuntos
Hipoxantinas/metabolismo , Luminol , Piridazinas , Xantina Oxidase/metabolismo , Xantinas/metabolismo , Animais , Bovinos , Ditionita/farmacologia , Hipoxantina , Cinética , Medições Luminescentes , Leite/enzimologia , Espectrofotometria Ultravioleta , Superóxidos/metabolismo , Ácido Úrico/metabolismo , XantinaRESUMO
Superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) (SOD) and ferricytochrome c are used to check the effects on luminol chemiluminescence induced by a xanthine or hypoxanthine/xanthine oxidase/oxygen system. Luminol chemiluminescence has been attributed to superoxide anion radical (O2.-) in this system. From kinetic studies on the light intensity vs. time curves it is demonstrated that addition of SOD into the system does not affect the mechanism of O2.- generation, whilst ferricytochrome c dramatically alters the time-course of the reaction. This is interpreted as the effect of cytochrome c redox cycling by reaction with H2O2, modifying oxy-radical generation in the reaction medium. Also, an alternative mechanism for luminol chemiexcitation is proposed under certain experimental conditions.
Assuntos
Grupo dos Citocromos c/farmacologia , Medições Luminescentes , Luminol/metabolismo , Piridazinas/metabolismo , Superóxido Dismutase/farmacologia , Xantina Oxidase/metabolismo , Catálise , Hipoxantina , Hipoxantinas/metabolismo , Cinética , Oxigênio/metabolismo , Espectrofotometria Ultravioleta , Superóxidos/metabolismo , Xantina , Xantinas/metabolismoRESUMO
To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a "supersuppressor" selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HATr) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT- mutants - selected by their resistance to 6-thioguanine (TGr) - and TK- mutants - selected by their resistance to 5-bromodeoxyuridine (BrdUrdr) - are HATs. Therefore, from HPRT- TK- double nonsense mutants, whose phenotype would be TGr BrdUrdr (HATs), simultaneous HPRT+ TK+ double phenotypic revertants could be obtained by selecting HATr (TGs BrdUrds) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TGr variants, 53 of which were HATs and the rest HATr. Among 36 TGr (HATs) variants tested, 23 did not revert to HATr, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT- nonsense mutants because they were very stringent (had less than 2% of wild-type [3H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrdr) in 7 of these strains allowed us to isolate 29 TGr BrdUrdr (HATs) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HATr (TGs BrdUrds) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.
Assuntos
Linhagem Celular , Mutação , Animais , Fusão Celular , Células Clonais , Técnicas de Cultura/métodos , Congelamento , Teste de Complementação Genética , Variação Genética , Hipoxantina , Hipoxantinas/metabolismo , Preservação de TecidoRESUMO
1. The pattern of purine base uptake in culture epimastigotes of Trypanosoma cruzi can be predicted from cell growth rate and cell concentration, with the late log phase showing the greatest variability. 2. Uptake rates are unexpectedly low in the reproductive tissue amastigotes and high in the non-reproductive blood trypomastigotes. It is suggested that blood trypomastigotes metabolize and accumulate reserves of purine metabolites, whereas amastigotes depend on degradation of host cell RNA and nucleotides as purine sources. 3. All parasite forms salvage hypoxanthine and guanine in preference to adenine. Nifurtimox, dipyridamole and cytochalasin have no effect on uptake, whereas amphotericin B, allopurinol, xanthine and urate inhibit it. The alterations caused by urate are complex, apparently involving inhibition of the monooxypurine phosphoribosyltransferase and induction of permeation of purines into the cells.
Assuntos
Purinas/farmacocinética , Trypanosoma cruzi/metabolismo , Adenina/metabolismo , Animais , Membrana Celular/metabolismo , Guanina/metabolismo , Hipoxantinas/metabolismo , Pentosiltransferases/farmacocinéticaRESUMO
The uptake of adenine, guanine and hypoxanthine by Trypanosoma cruzi culture epimastigotes was studied over short time periods at 22 degrees C. The uptake process is concentrative, driven by the purine phosphoribosyltransferases and saturable at about 10 microM hypoxanthine and 1 microM adenine or guanine. Each purine base is apparently taken up by a separate route and the oxypurines are regulated in parallel. Adenine inhibits oxypurine uptake. When the material was incubated with guanine or hypoxanthine for more than 2 min, a decrease in the nucleotide/free base ratio was observed, indicating inhibition of the guanine and hypoxanthine phosphoribosyltransferases. The uptake rates during the ascending phase of uptake and of culture growth rates were of the order of 50 for hypoxanthine and 20 for guanine, relative to that of adenine. These rates were within the physiological range for cell growth in these cultures. During the phase of descending growth and decreased purine uptake activity rates, uptake was depressed to 20% of the rates required for the growth observed in the cultures. It is proposed that the decline in growth rate leads to an increase of the nucleotide pools in the cells which inhibit uptake. This depression may be a cause of the mitotic blockade which occurs during the stationary phase.
Assuntos
Purinas/metabolismo , Trypanosoma cruzi/metabolismo , Adenina/metabolismo , Animais , Meios de Cultura , Guanina/metabolismo , Hipoxantinas/metabolismo , Pentosiltransferases/metabolismoRESUMO
Leishmania mexicana mexicana promastigotes, axenic amastigotes, and amastigotes derived from Vero cells were examined for de novo purine synthesis and mechanisms of purine salvage. Both promastigotes and axenic amastigotes were incapable of de novo purine synthesis, as shown by the lack of [14C]formate and [14C]glycine incorporation into purine nucleotide pools. However, the ready incorporation of [14C]hypoxanthine, [14C]adenine, and [14C]guanine suggested that purine salvage pathways were operating. In addition, a significant percentage (greater than or equal to 60%) of the total label from these purine precursors was associated with adenylate nucleotides. Nucleotide pool levels of axenic amastigotes were consistently greater but the specific activities were less than those of promastigotes, suggesting a slower rate of purine metabolism in the axenic amastigote form. Similar results were obtained from amastigotes isolated from infected Vero cells.
Assuntos
Adenina/metabolismo , Guanina/metabolismo , Hipoxantinas/metabolismo , Leishmania/metabolismo , Nucleotídeos de Adenina/biossíntese , Animais , Linhagem Celular , Chlorocebus aethiops , Nucleotídeos de Guanina/biossíntese , Hipoxantina , Leishmania/crescimento & desenvolvimento , Nucleotídeos de Purina/biossínteseRESUMO
Promastigotes of Leishmania braziliensis panamensis absorbed the purines adenine, hypoxanthine, adenosine and inosine by a combination of diffusion and mediated components. When the uptake rates for these substrates were corrected for diffusion and compared, the purine bases adenine and hypoxanthine were transported at a significantly slower rate than the purine nucleosides adenosine and inosine. Competitive interactions among those purines tested confirmed the presence of mediated and diffusion components and suggested that three transport loci may be operating (Fig. 6). The first transport locus, designated Locus 1, transported inosine, Locus 2, the purine bases hypoxanthine and adenine and Locus 3, adenosine. In addition, adenine and hypoxanthine inhibited the uptake of one another competitively. A comparison of Ki values derived from double reciprocal plots of labelled hypoxanthine and adenine uptake in the presence of the unlabelled substrates as inhibitors suggested that adenine has a greater affinity for the transport locus.