Effects of adenosine deaminase on the sensitivity of glucose transport, glycolysis and glycogen synthesis to insulin in muscles of the rat

1. Soleus, extensor digitorum longus (EDL) or hemi-diaphragm muscles of the rat were incubated in the presence of insulin and rates of the processes of glycolysis and glycogen synthesis were measured. 2. The concentrations of insulin required to cause half-maximal stimulation of glycolysis in both soleus and EDL preparations were significantly decreased by the presence of adenosine deaminase in the medium. 3. Adenosine deaminase increased the sensitivity of the process of hexose transport to insulin (in an identical manner to the change in sensitivity of glycolysis) in the EDL preparation. 4. None of the adenosine mediated effects on insulin-stimulated rates of glycolysis were observed in the hemi-diaphragm preparation or on the rates of glycogen synthesis in any of the three muscle preparations. 5. Therefore, changes in the adenosine system in skeletal muscle influence insulin sensitivity regardless of fibre type composition of the muscle.     

31P NMR magnetization-transfer measurements of ATP turnover during steady-state isometric muscle contraction in the rat hind limb in vivo

31P NMR magnetization-transfer measurements have been used to measure the flux between ATP and inorganic phosphate during steady-state isometric muscle contraction in the rat hind limb in vivo. Steady-state contraction was obtained by supramaximal sciatic nerve stimulation. Increasing the stimulation pulse width from 10 to 90 ms, at a pulse frequency of 1 Hz, or increasing the frequency of a 10-ms pulse from 0.5 to 2 Hz resulted in an increase in the flux which was an approximately linear function of the increase in the tension-time integral. The flux showed an approximately linear dependence on the calculated free cytosolic ADP concentration up to an ADP concentration of about 90 microM. The data are consistent with control of mitochondrial ATP synthesis by the cytosolic ADP concentration and indicate that the apparent Km of the mitochondria for ADP is at least 30 microM.     

The structure and evolution of the spider monkey delta-globin gene

We have isolated the delta-globin gene of the New-World spider monkey, Ateles geoffroyi, and compared its nucleotide sequence with those of other primate delta- and beta-globin genes. Among primate delta-globin genes, the rate of nonsynonymous substitutions is much less than the rate of synonymous substitutions. This suggests that primate delta- globin genes may remain under evolutionary conservation, perhaps because hemoglobin A2 has an as yet unknown physiological importance.      

Mass measurements of inositol(1,4,5)trisphosphate in rat cerebral cortex slices using a radioreceptor assay: effects of neurotransmitters and depolarization

The specific binding of [3H] and [32P]Ins(1,4,5)P3 to a particulate preparation of bovine adrenal cortex has been used as a radioreceptor assay to determine the concentration of Ins(1,4,5)P3 in agonist- and depolarization-stimulated rat cerebral cortex slices. The resting concentration of Ins(1,4,5)P3 in slices that had been preincubated in a physiological medium was 18.8 +/- 2.6 pmol/mg prot. Carbachol evoked a rapid and dose-related increase in the concentration of Ins(1,4,5)P3. Maximal stimulation (80%) was already seen at the earliest point (10 sec) examined and was maintained for at least 5 min. The EC50 for carbachol was 75 +/- 17 microM and the response was totally suppressed by the muscarinic antagonist atropine. A direct comparison in the same slices was made between mass determinations and [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 accumulation determined by h.p.l.c. Although an identical time course was observed for cold and radiolabelled Ins(1,4,5)P3, the greater stimulation of [3H]Ins(1,4,5)P3 may indicate changes in specific radioactivity. Of a variety of other receptor agonists studied, only the glutamate receptor agonist quisqualate, and noradrenaline significantly increased the mass of Ins(1,4,5)P3 in cerebral cortical slices. However, depolarizing concentrations of K+ were as effective as carbachol at elevating this second messenger.     

Biochemical adaptation in the skeletal muscle of rats depleted of creatine with the substrate analogue beta-guanidinopropionic acid.

Rats were fed on a diet containing 1% beta-guanidinopropionic acid (GPA), a creatine substrate analogue, for 6-10 weeks to deplete their muscle of creatine. This manipulation was previously shown to give a 90% decrease in [phosphocreatine] in skeletal and cardiac muscle and a 50% decrease in [ATP] in skeletal muscle only. Maximal activities of creatine kinase and of representative enzymes of aerobic and anaerobic energy metabolism were measured in the superficial white, medial and deep red portions of the gastrocnemius muscle, in the soleus and plantaris muscle and in the heart. Fast-twitch muscles were smaller in GPA-fed animals than in controls, but the size of the soleus muscle was unchanged. The activities of aerobic enzymes increased by 30-40% in all fast-twitch muscle regions except the superficial gastrocnemius, but were unchanged in the soleus muscle. The activities of creatine kinase and phosphofructokinase decreased by 20-50% in all skeletal-muscle regions except the deep gastrocnemius, and the activity of glycogen phosphorylase generally paralleled these changes. There were no significant changes in the activities of any of the enzymes measured in the heart. The glycogen content of the gastrocnemius-plantaris complex was increased by 185% in GPA-fed rats. The proportion of Type I fibres in the soleus muscle increased from 81% in control rats to 100% in GPA-fed rats, consistent with a previous report of altered isometric twitch characteristics and a decrease in the maximum velocity of shortening in this muscle [Petrofsky & Fitch (1980) Pflugers Arch. 384, 123-129]. We conclude that fast-twitch muscles adapt by a combination of decreasing diffusion distances, increasing aerobic capacity and decreasing glycolytic potential. Slow-twitch muscles decrease glycolytic potential and become slower, thus decreasing energy demand. These results suggest that persistent changes in the [phosphocreatine] and [ATP] are alone sufficient to alter the expression of enzyme proteins and proteins of the contractile apparatus, and that fibre-type-specific thresholds exist for the transformation response.     

Biochemical pathways in prokaryotes can be traced backward through evolutionary time

For the first time, a credible prokaryotic phylogenetic tree is being assembled by Woese and others using quantitative sequence analysis of oligonucleotides in the highly conservative rRNA. This provides an evolutionary scale against which the evolutionary steps that led to the arrangement and regulation of contemporary biochemical pathways can be measured. This paper presents an emerging evolutionary picture of aromatic amino acid biosynthesis within a large superfamily assemblage of prokaryotes that is sufficiently developed to illustrate a new perspective that will be applicable to many other biochemical pathways.      

An investigation of arterial insufficiency in rat hindlimb. A combined 31P-n.m.r. and bloodflow study.

A small animal model of arterial insufficiency is presented which involves unilateral femoral artery ligation and section. Invoked alterations in metabolism and perfusion of the affected muscle mass have been investigated 12 h, 4, 7 and 14 days post-ligation by 31P-n.m.r. and microsphere infusion, both at rest and during isometric muscle contraction at 1 Hz. At rest, the concentration of phosphocreatine was similar to the mean control value (36.0 +/- 1.0 mM) from 4 days post-ligation, but was significantly lower at 12 h (28.5 +/- 3.6 mM). Inorganic phosphate concentrations were significantly elevated for 7 days post-ligation. No significant differences were noted in intramuscular pH. Upon stimulation of the affected muscle mass, a time-dependent improvement in phosphocreatine utilization was observed such that 14 days post-ligation phosphocreatine utilization was not significantly different from mean control values. A similar amelioration was noted for the contraction-induced fall in intramuscular pH. At rest, no significant differences in bloodflow to the muscles of the ligated limb compared with the unaffected contralateral limb were observed. However, isometric contraction of the affected muscle mass resulted in a markedly reduced hyperaemic response 12 h post-ligation. Thereafter, a time-dependent improvement in tissue perfusion during stimulation was observed which paralleled the improvements in phosphocreatine utilization and intramuscular pH changes. The results presented are discussed with respect to the interrelationship between oxygen delivery, high energy phosphate utilization and force maintenance.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.