Role of adenosine kinase and AMP deaminase in the regulation of cardiac purine release
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Abstract:
To analyze the relation between cardiac energy status, adenosine formation, and purine release, reliable measurements of the cytosolic concentrations of ATP, ADP, AMP, and adenosine are required. Based on the creatine kinase and myokinase equilibrium, ADP and AMP are determined by 31P nuclear magnetic resonance spectroscopy, whereas free cytosolic adenosine is measured by the S-adenosyl-homocysteine (SAH) technique. Combining these methods with efflux measurements, selective enzyme blockade and a comprehensive model analysis enables a description of both concentrations and flux rates in purine metabolism. In the well-oxygenated heart, adenosine is predominantly formed intracellularly from AMP, but also from S-adenosyl-homocysteine. Net adenosine formation (2.3 nmol/min per g) exceeds coronary venous release (0.07 nmol/min per g) more than 30-fold, because most of the adenosine formed is rephosphorylated by adenosine kinase. This enzyme maintains a low intracellular adenosine and limits both adenosine release and deamination to inosine. In fact, inosine is mainly formed from IMP (1.8 nmol/min per g) the product of AMP deaminase. Inosine, hypoxanthine, xanthine, and uric acid (1.1, 0.4, 0.2, 1.4 nmol/min per g) are the main purine catabolites released. In the oxygen-limited heart, energy status is impaired and AMP increased. Under these conditions, a linear relation between AMP (200–3,000 nmol/liter), net adenosine formation, as well as net inosine formation is observed. It is, thus, the AMP substrate concentration that directly controls adenosine formation by cytosolic 5′-nucleotidase and most likely flux through AMP deaminase. Hypoxia-induced inhibition of adenosine kinase shunts adenosine from the salvage pathway to venous release and causes the amplification of small changes in AMP into a major rise in adenosine. This mechanism plays an important role in the high sensitivity of the cardiac adenosine system to impaired oxygenation. Drug Dev. Res. 45:295–303, 1998. © 1998 Wiley-Liss, Inc.Keywords:
Inosine
Hypoxanthine
AMP deaminase
Adenosine kinase
Adenine nucleotide
Energy charge
Purine metabolism
5'-nucleotidase
Adenosine A1 receptor
In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57+/-22% of whole homogenate 5'-nucleotidase activity and was not significantly different from the other groups. A weak inverse correlation was found between AMP deaminase and cN-I activities (r2=0.18, p<0.01). There were no significant differences between different groups in the activities of cN-I, whole homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities of whole homogenate nucleotidase, total cN and cN-I. The results indicate no changes in the relative expression or catalytic behaviour of cN-I in AMP deaminase-deficient human skeletal muscle, but suggest that increased turnover of AMP by cN-I in working skeletal muscle is due to higher substrate availability of AMP.
AMP deaminase
5'-nucleotidase
Nucleotidase
Adenosine monophosphate
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Abstract— The maximum activities of 5’nucleotidase, adenosine kinase and adenosine deaminase have been measured in several areas of rat and human brain. There is no major difference between the activities of nucleotidase and kinase between rat and human brain, but the activity of deaminase is considerably higher in human brain. The activities of all these enzymes are similar in three areas of rat brain and nine areas of human brain, except for hind brain of the human, which has a low activity of adenosine deaminase. This variation may indicate the existence of different steady‐state concentrations of adenosine in certain areas of the brain. Subcellular fractionation of different areas of rat brain showed that, whereas adenosine kinase and deaminase activities were located mainly in the soluble fractions, 5’nucleotidase was present in all subcellular fractions (i.e. membrane, synaptosomal, mitochondrial and soluble). In particular, there was no major localisation within the synaptosomal fraction. Thus it is unlikely that the regulation of the activities of these enzymes is dependent upon changes within a specific compartment (e.g. synaptosomes) in the brain.
5'-nucleotidase
AMP deaminase
Nucleotidase
Adenosine kinase
Human brain
Adenosine A2B receptor
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1. The maximal activities of 5'-nucleotidase, adenosine kinase and adenosine deaminase together with the Km values for their respective substrates were measured in muscle, nervous tissue and liver from a large range of animals to provide information on the mechanism of control of adenosine concentration in the tissues. 2. Detailed evidence that the methods used were optimal for the extraction and assay of these enzymes has been deposited as Supplementary Publication SUP 50088 (16pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K.,from whom copies can be obtained on the terms indicated in Biochem. J. (1978), 169, 5. This evidence includes the effects of pH and temperature on the activities of the enzymes. 3. In many tissues, the activities of 5'-nucleotidase were considerably higher than the sum of the activities of adenosine kinase and deaminase, which suggests that the activity of the nucleotidase must be markedly inhibited in vivo so that adenosine does not accumulate. In the tissues in which comparison is possible, the Km of the nucleotidase is higher than the AMP content of the tissue, and since some of the latter may be bound within the cell, the low concentration of substrate may, in part, be responsible for a low activity in vivo. 4. In most tissues and animals investigated, the values of the Km of adenosine kinase for adenosine are between one and two orders of magnitude lower than those for the deaminase. It is suggested that 5'-nucleotidase and adenosine kinase are simultaneously active so that a substrate cycle between AMP and adenosine is produced: the difference in Km values between kinase and deaminase indicates that, via the cycle, small changes in activity of kinase or nucleotidase produce large changes in adenosine concentration. 5. The activities of adenosine kinase or deaminase from vertebrate muscles are inversely correlated with the activities of phosphorylase in these muscles. Since the magnitude of the latter activities are indicative of the anaerobic nature of muscles, this negative correlation supports the hypothesis that an important role of adenosine is the regulation of blood flow in the aerobic muscles.
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