Comparative study of 2',3'-O-isopropylidene adenosine and adenosine transformation in rat liver homogenates

Transformation of synthesized 2',3'-O-isopropylidene adenosine was studied in comparison with adenosine in rat liver homogenates. It is stated that 2',3'-O-isopropylidene adenosine is subjected to deamination similar to adenosine but less intensively. Due to deamination 2',3'-O-isopropylidene inosine is formed from 2',3'-O-isopropylidene adenosine. It is shown that under conditions of the conducted experiments enzymic splitting of the isopropylidene grouping from the preparation does not occur; this substrate contrary to adenosine does not split under the effect of purine nucleoside phosphorylase.     

ADP-ribosyl transferase and NAD glycohydrolase activities in rat liver mitochondria

ADP-ribosyl transferase and NAD glycohydrolase activities have been estimated in mitochondria in mitoplasts as well as in other submitochondrial fractions. A high activity of these two enzymes was present in mitoplasts as compared to the outer membrane preparation or intermembrane compartment. Inhibitor studies provide strong evidence for the involvement of ADP-ribosyl transferase in the process of ADP-ribosylation of mitochondrial proteins. When NAD glycohydrolase was blocked by nicotinamide or 3-aminobenzamide, the incorporation of ADP-ribose into mitochondrial proteins still occurs. ADP-ribosyl transferase activity could also be detected when NAD glycohydrolase was separated by hydroxylapatite chromatography. The protein-linked ADP-ribose moiety appears to be an oligomer in mitochondria.     

Stimulation of glycogenolysis and vasoconstriction by adenosine and adenosine analogues in the perfused rat liver.

Infusion of adenosine into perfused rat livers resulted in transient increases in glucose output, portal-vein pressure, the effluent perfusate [lactate]/[pyruvate] ratio, and O2 consumption. 8-Phenyltheophylline (10 microM) inhibited adenosine responses, whereas dipyridamole (50 microM) potentiated the vasoconstrictive effect of adenosine. The order of potency for adenosine analogues was: 5'-N-ethylcarboxamidoadenosine (NECA) greater than L-phenylisopropyladenosine greater than cyclohexyladenosine greater than D-phenylisopropyladenosine greater than 2-chloroadenosine greater than adenosine, consistent with adenosine actions modulated through P1-purine receptors of the A2-subtype. Hepatic responses exhibited homologous desensitization in response to repeated infusion of adenosine. Adenosine effects on the liver were attenuated at lower perfusate Ca2+ concentrations. Indomethacin decreased hepatic responses to both adenosine and NECA. Whereas adenosine stimulated glycogen phosphorylase activity in isolated hepatocytes, NECA caused no effect in hepatocytes. The response to adenosine in hepatocytes was inhibited by dipyridamole (50 microM), but not 8-phenyltheophylline (10 microM). The present study indicates that, although adenosine has direct effects on parenchymal cells, indirect effects of adenosine, mediated through the A2-purinergic receptors on another hepatic cell type, appear to play a role in the perfused liver.     

The action of extracellular NAD+ on gluconeogenesis in the perfused rat liver

In the rat liver NAD⁺ infusion produces increases in portal perfusion pressure and glycogenolysis and transient inhibition of oxygen consumption. The aim of the present work was to investigate the possible action of this agent on gluconeogenesis using lactate as a gluconeogenic precursor. Hemoglobin-free rat liver perfusion in antegrade and retrograde modes was used with enzymatic determination of glucose production and polarographic assay of oxygen uptake. NAD⁺ infusion into the portal vein (antegrade perfusion) produced a concentration-dependent (25–100 μM) transient inhibition of oxygen uptake and gluconeogenesis. For both parameters inhibition was followed by stimulation. NAD⁺ infusion into the hepatic vein (retrograde perfusion) produced only transient stimulations. During Ca²⁺-free perfusion the action of NAD⁺ was restricted to small transient stimulations. Inhibitors of eicosanoid synthesis with different specificities (indo-methacin, nordihydroguaiaretic acid, bromophenacyl bromide) either inhibited or changed the action of NAD⁺. The action of NAD⁺ on gluconeogenesis is probably mediated by eicosanoids synthesized in non-parenchymal cells. As in the fed state, in the fasted condition extracellular NAD⁺ is also able to exert two opposite effects, inhibition and stimulation. Since inhibition did not manifest significantly in retrograde perfusion it is likely that the generating signal is located in pre-sinusoidal regions.     

Influence of azacytidine on the induction of tyrosine aminotransferase and the NAD metabolism in rat liver

5-Azacytidine was found to inhibit the induction of tyrosine aminotransferase caused by L-tyrosine and hydrocortisone. The inhibitory effect can be overcome by L-methionine. There was only a slight inhibition of the tryptophan induction by 5-azacytidine in normal animals 4 hr after the administration of tryptophan. At 8 and 12 hr, a superinduction could be observed. The NAD content in adrenalectomized animals increased after application of tryptophan and 5-azacytidine. There was a slight inhibition of ADPR transferase in the rat liver.     

Specific adenosine binding proteins from rat liver.

Specific adenosine-binding proteins from homogenates of rat liver have been fractionated on a DEAE-cellulose column. Three major peaks have been identified with respect to histone phosphokinase and cAMP and adenosine-binding activities. Peak I contains only histone phosphokinase activity not stimulated by cAMP. Peak II contains histone phosphokinase slightly stimulated by cAMP. Both cAMP- and adenosine-binding activities are found in this fraction. The major adenosine-binding protein is associated with Peak III. Histone phosphokinase in Peak III which also binds cAMP is stimulated 2-fold by 2.5 muM cAMP whereas adenosine at 2.5 X 10(-4)M inhibits these enzymes equally well in each of three peaks. The specificity of adenosine binding is discussed.     

Subcellular localization and properties of rat liver adenosine diphosphatase.

ADPase (adenosine diphosphatase) was assayed in rat liver homogenates with [beta-32P]ADP as substrate. The activity had a pH optimum of 8.0 and was strongly activated by Mg2+. The intracellular localization was determined by analytical subcellular fractionation with single-step sucrose-density-gradient centrifugation. Selective membrane perturbants were used to enhance the resolution of the various organelles. ADPase was localized to the mitochondria. Mitochondria were isolated by differential centrifugation and subfractionated by selective disruption of the inner and outer membranes. The intramitochondrial localization of ADPase was compared with various marker enzymes and was shown to be concentrated in the outer-membrane fractions. The effects of various inhibitors on the ADPase activity were determined and the possibility that the activity could be due to known enzyme systems was considered. It is concluded that ADP degradation is due to a hitherto unrecognized mitochondrial enzyme.     

5'-Nucleotidase activity and adenosine production in rat liver mitochondria.

The controversial subject of mitochondrial 5'-nucleotidase in the liver was studied employing density gradient fractionation combined with a method for analyzing the distribution profiles of marker enzymes based on multiple regression analysis. Triton WR-1339 was used to improve the separation of mitochondria from lysosomes by the gradient centrifugation technique. Adenosine production was examined further using acetate to increase intramitochondrial AMP, and thus adenosine production, in incubations with gradient centrifugation-purified mitochondria. Distribution analysis of the crude homogenate showed that 5'-nucleotidase activity exists in the mitochondrial fraction. To increase the resolution of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied. In this case the relative mitochondrial activity decreased but 5'-nucleotidase activity was still clearly detectable. The mitochondrial 5'-nucleotidase exhibited a Km of 94 microM and a Vmax of 31 nmol/min per mg protein for AMP. The kinetic data for the Mg2+, ATP, ADP and AOPCP sensitivity of the enzyme showed that it differs from the plasma membrane, lysosome and cytosol 5'-nucleotidases. AOPCP was only a moderate inhibitor, and ATP was a more potent inhibitor than ADP at a 1 mM concentration. The enzyme also showed a requirement of Mg2+. Acetate caused the conversion of intramitochondrial adenylates to AMP and the formation of adenosine. Adenosine concentration increased in the extramitochondrial space in a time-dependent manner, but only trace amounts of nucleotides were detected. The data show that 5'-nucleotidase activity producing adenosine exists in rat liver mitochondria and a concentration-dependent adenosine output from mitochondria by diffusion or facilitated diffusion is also suggested.