Extracellular Adenine Nucleotide Catabolism in Heart Valves

Enzymes of extracellular nucleotide catabolism, such as ecto-nucleoside triphosphate diphosphohydrolase (eNTPD), ecto-5′-nucleotidase (e5NT) and ecto-adenosine deaminase (eADA), control extracellular concentrations of adenine nucleotides and adenosine, furthermore in that way regulate inflammation, immune response, and platelets aggregation. In valves, disturbances of these processes may lead to their dysfunction and calcification. The aim of this study was to analyze the distribution of enzymes, which are engaged in extracellular nucleotide metabolism on the surface of pig aortic and pulmonary valves in relation to activities in the vessel wall. Activity of e5NT was two times higher on the surface of the aortic valve in comparison to the aorta. The same relation between activity of this enzyme in the pulmonary valve and pulmonary artery can be observed. In contrast, eADA activity on the valve surface is much lower than in the vessel wall. No significant differences were observed between the activity of eNTPD on the valve and the vessel surface. This highlights that pattern of enzymes activities favors the production and retention of adenosine on the valve surface and that its alterations could play a role in valve pathology.     

Effects of 4-Pyridone-3-carboxamide-1β-D-ribonucleoside on adenine nucleotide catabolism in the aortic wall; Implications for atherosclerosis in ApoE-/-LDLR-/- mice

ABSTRACT

4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that had been identified as a substrate for intracellular phosphorylation to form intracellular nucleotides. Previous studies demonstrated that 4PYR adversely affects metabolism of endothelial cells that is known risk factor for atherosclerosis. The purpose of this study was to evaluate effects of 4PYR on the progression of atherosclerosis and changes in extracellular nucleotides degradation on the surface of the vessel wall in the murine model. Methods. Two month old ApoE-/-LDLR-/- mice were subcutaneously injected with 4PYR (4P) twice per day for one month or with saline in controls (C). Then, at the age of eight month hydrolysis rates of ATP, AMP and adenosine were evaluated in the intact aorta sections by HPLC based assays. Oil Red O (ORO) staining that indicates lipid deposition was quantified spectrophotometrically after extraction from the vessel. Serum amyloid A (SAA) content was analyzed with ELISA. Results. Adenosine deamination rate (activity of eADA) increased from 8.7±1.4 nmol/min/cm² in C to 16.0±2.6 nmol/min/cm² in 4P (p<0.05). AMP dephosphorylation rate (activity of e5NT) and ATP hydrolysis rate (activity of eNTPD) were not different between C and 4P. ORO staining in the aorta of 4P mice increased by 75% as compared to C (p<0.01) while SAA content was similar in both groups. Conclusions. This data demonstrated that prolonged exposure to 4PYR of ApoE-/-LDLR-/- mice results in sustained elevation of vascular eADA activity and increased ORO staining indicating endothelial impairment and accelerated atherosclerosis.     

Influence of 4-pyridone-3-carboxamide-1Β-D-ribonucleoside (4PYR) on activities of extracellular enzymes in endothelial human cells

ABSTRACT

Previous studies demonstrated that human endothelial cells were capable to phosphorylate 4-pyridone-3-carboxamide-1β-D-ribonucleoside (4PYR) to monophosphate (4PYMP) and formed another metabolite—an analog of NAD (4PYRAD). Elevated levels of 4PYMP and 4PYRAD had an adverse effect on energy balance—depressed adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) concentration in human endothelial cells. Ecto-enzymes such as ecto-nucleoside triphosphate diphosphohydrolase (eNTPD); ecto-5′-nucleotidase (e5’NT); and ecto-adenosine deaminase (eADA) are involved in controlling of inflammation and platelet aggregation. This study aimed to evaluate influence of 4PYR and its metabolites on activities of extracellular enzymes in human endothelial cells. Endothelial cells (endothelial cell line HMEC-1) were treated with 100 uM 4PYR for 0, 24, 48, or 72 hours. After incubation, intact HMEC-1 cells were incubated with suitable substrate. Simultaneously, in another path of experiments intracellular concentration of 4PYMP and 4PYRAD had been analyzed. Conversion of extracellular nucleotides into their products and intracellular concentration of 4PYMP and 4PYRAD were measured by high performance liquid chromatography (HPLC). We demonstrated that eNTPD and e5’NT activities increase after 72 hours of cell treatment with 4PYR as compared to control (0.40 ± 0.02 versus 0.29 ± 0.02 nmol/min/mg protein; 13.3 ± 0.6 versus 8.30 ± 0.34 nmol/min/mg protein, respectively, mean ± SEM). eADA activity decreases after 24 hours of cells treatment with 4PYR as compared to control (1.55 ± 0.06 versus 1.92 ± 0.13 nmol/min/mg protein, respectively, mean ± SEM). 4PYR and its derivatives have positive effect on ecto-enzymes related with ATP degradation pathway. We conclude that these increases in extracellular enzyme activities are an adaptive response to decreased intracellular ATP and NAD arising from 4PYR uptake. These changes may protect the cells from the inflammatory result of external ATP degradation.     

Changes in cardiac nucleotide metabolism in Huntington's disease

ABSTRACT

Huntington's disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5′NT, ADA, and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5′NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were observed. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling.     

Immunohistochemical and Functional Analysis of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) and Ecto-5′-Nucleotidase (CD73) in Pig Aortic Valves

Extracellular nucleotides control mechanisms such as thrombosis or inflammation that are important in several pathologies, including heart valve disease and calcification. Ectonucleoside triphosphate diphosphohydrolase 1 (eNTPD1, CD39) and ecto-5′-nucleotidase (e5NT, CD73) are ectoenzymes that convert adenosine triphosphate to adenosine diphosphate, adenosine monophosphate and finally to adenosine. Changes in activities of these enzymes influence extracellular nucleotide concentrations and therefore could be involved in valve pathology. This study aimed to analyze type of cells, specific area, level of expression and biochemical function of CD39 and CD73 in pig aortic valves. Samples were collected from aortic valves of domestic pigs. Histological sections were cut from paraffin embedded tissue blocks. Following incubation with primary antibody against CD39 or CD73, washing and secondary goat anti-rabbit secondary antibodies, slides were viewed with NanoZoomer scanner. Substantial expression CD39 and CD73 was observed in two main types of valve cells: endothelial and valve interstitial cells. Subsequently, biochemical function of CD39 and CD73 was evaluated in cells cultured from pig aortic valve. Breakdown of extracellular nucleotides added to cell medium was analyzed with high performance liquid chromatography. In the interstitial cells, the CD73 products formation was much faster than in endothelium, while for the CD39 activity this relation was opposite. Expression and high concentration of CD39 and CD73 products in endothelium are expected, but presence of CD73 in valve interstitial cells is a surprise. We conclude that CD39 and CD73 and their enzymatic activities that convert extracellular nucleotides are highly expressed and could have special function in the valve.     

Activities of purine converting enzymes in heart,liver and kidney mice LDLR−/− and Apo E−/−

Nucleotide metabolism plays a major role in a number of vital cellular processes such as energetics. This, in turn, is important in pathologies such as atherosclerosis.

Three month old atherosclerotic mice with knock outs for LDLR and apolipoprotein E (ApoE) were used for the experiments. Activities of AMP-deaminase (AMPD), ecto5′-nucleotidase (e5NT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) were measured in heart, liver and kidney cortex and medulla by analysing conversion of substrates into products using HPLC.

The activity of ecto5′-nucleotidase differ in hearts of LDLR^?/? and ApoE^?/? mice with no differences in ADA and AMPD activity. We noticed highest activity of e5NT in kidney medulla of the models.

This model of atherosclerosis characterize with an inhibition of enzyme responsible for production of protective adenosine in heart but not in other organs and different metabolism of nucleotides in kidney medulla.     

Effect of Nitrosative Stress on Extracellular Nucleotide Metabolism in Endothelial Cells

Mechanisms of free radical injury involve chemical modification of proteins, lipid derivatives and nucleic acids and consequent loss of its function. However, specific targets and exact sequence of events has not been fully clarified. We determined whether extracellular enzymes that are involved in adenosine formation such as ecto-5′nucleotidase (e5N) and removal such as extracellular form of adenosine deaminase (eADA) could be affected by peroxynitrite. We used intact cell assay system that involves exposure of cultured HMEC-1 cells to substrates followed by HPLC analysis of conversion of substrates into products. We found that e5N and ADA activities decreased by 20–40% after incubation for 20 or 60 minutes with 30 μM peroxynitrite. Decrease of cellular ATP and NAD was also observed. We conclude that besides other cytotoxic effects modification of extracellular enzymes of nucleotide metabolism could be important target for free radical injury.     

Uptake and metabolism of adenosine mediate a meiosis‐arresting action on mouse oocytes

This study was carried out to evaluate the possible role of adenosine uptake and metabolism in mediating the inhibitory actions of this nucleoside on spontaneous mouse oocyte maturation. Uridine blocked ³H‐adenosine uptake by oocyte–cumulus cell complexes (OCCs) and cumulus cell–enclosed oocytes (CEOs) by 82–85%, whereas uptake by denuded oocytes (DOs) was suppressed by 97%. Uridine had no effect on germinal vesicle breakdown (GVB) in CEOs when meiotic arrest was maintained with hypoxanthine or hypoxanthine plus adenosine but reversed the combined inhibitory action of these purines in DOs. Five of six adenosine analogs that bind to purinoceptors demonstrated meiosis‐arresting activity but not in relation to their relative affinities for inhibitory or stimulatory adenosine receptors and only at high concentrations. Moreover, in DOs, uridine reversed the inhibitory effect of 2‐chloroadenosine and 5′‐N‐ethylcarboxamidoadenosine, two receptor agonists that are poor substrates for adenosine‐metabolizing enzymes. Results of experiments with adenosine kinase inhibitors showed that methylmercaptopurine riboside (MMPR) and tubercidin, but not 5′‐amino‐5′‐deoxyadenosine, reversed meiotic arrest maintained by hypoxanthine ± adenosine, but this required an additional inhibitory action on de novo purine synthesis. Inhibition of de novo purine synthesis alone was not sufficient because azaserine failed to reverse meiotic arrest. MMPR was a very potent meiosis‐inducing agent, completely reversing meiotic arrest in CEOs and DOs in the presence of a variety of meiotic inhibitors. The adenosine deaminase inhibitor deoxycoformycin had opposite effects on oocyte maturation depending on the presence or absence of adenosine: the inhibitory action of hypoxanthine alone was bolstered, but the meiosis‐arresting action of adenosine was reversed. These data therefore indicate that at low adenosine concentrations phosphorylation predominates, but at higher adenosine concentrations deaminated products contribute to the meiotic inhibition. This idea was borne out by the ability of inosine to mimic the synergistic interaction of adenosine with hypoxanthine. The action of adenosine is not due to deamination to inosine and conversion to nucleotides through the hypoxanthine salvage pathway because adenosine‐mediated inhibition was not compromised in oocytes from mutant mice unable to salvage hypoxanthine. Mol. Reprod. Dev. 53:208–221, 1999. © 1999 Wiley‐Liss, Inc.     

Stimulation of protocollagen proline hydroxylase activity by nucleoside triphosphates.

Activity of purified protocollagen proline hydroxylase was enhanced several fold by addition of nucleoside triphosphates (3 mM) to the assay medium, but nucleoside mono-and diphosphates were almost inactive. Pyrimidine nucleotides were less effective compared with purine nucleotides, among which GTP was the most effective. dATP and ATP analogues such as adenosine 5′-(β,γ-imino) triphosphate (AMP-PNP), adenosine 5′-(β,γ-methylene) triphosphate (AMP-PCP), etc. were inactive. ATP or GTP showed no additive effect on enzyme activity stimulated by dithiothreitol or bovine serum albumin.     

Polymorphism in exon 6 of the human NT5E gene is associated with aortic valve calcification

ABSTRACT

NT5E encodes ecto-5′-nucleotidase (e5NT, CD73) which hydrolyses extracellular AMP to adenosine. Adenosine has been shown to play a protective role against aortic valve calcification (AVC). We identified two nonsynonymous missense single nucleotide polymorphisms (c.1126A > G, p.T376A and c.1136T > C, p.M379T) in exon 6 of the human NT5E gene. Since both substitutions might affect e5NT activity and consequently alter extracellular adenosine levels, we evaluated the association between NT5E alleles and calcific aortic valve disease in 119 patients (95 patients with AVC and 24 controls). In AVC patients, the frequency of the G allele at c.1126 and the frequency of the GG genotype as well as the frequency of the C allele at c.1136, and the frequencies of CC and TC genotypes tended to be higher as compared to controls. The allele and genotype frequencies in AVC patients and controls were also compared to those calculated from the 1000 Genomes Project data for control individuals of European ancestry (n = 503). We found that the frequency of the C allele at c.1136 is significantly higher in patients with AVC than in the European controls (0.111 vs. 0.054, P = 0.0052). Moreover, e5NT activity in aortic valves showed a trend toward lower levels in AVC patients with CC and TC genotypes than in those with the TT genotype. Our findings indicate that the genetic polymorphism of NT5E may contribute to the pathogenesis of calcific aortic valve disease and that the C allele of SNP c.1136 is associated with an increased risk of AVC.     

Functional Analysis of Expression of Human Ecto-Nucleoside Triphosphate Diphosphohydrolase-1 and/or Ecto-5′-Nucleotidase in Pig Endothelial Cells

Adenine nucleosides and nucleotides are important signaling molecules involved in control of key mechanisms of xenotransplant rejection. Extracellular pathway that converts ATP and ADP to AMP, and AMP to adenosine mainly mediated by ecto-nucleoside triphosphate diphosphohydrolase 1, (ENTPD1 or CD39) and ecto-5′-nucleotidase (E5NT or CD73) respectively, is considered as important target for xenograft protection. To clarify feasibility of combined expression of human ENTPD1 and E5NT and to study its functional effect we transfected pig endothelial cell line (PIEC) with both genes together. To do this we have produced a dicistronic construct bearing F2A sequence in frame between human E5NT and human ENTPD1 coding sequences. PIEC cells were mock-transfected as transfection control or transfected with plasmids encoding human ENTPD1 or human E5NT. PIEC cells were exposed to 50 μM ATP or 50 μM ADP or 50 μM AMP. Conversion of extracellular substrates into products (ATP/ADP/AMP/adenosine) was measured by HPLC in the media collected at specific time intervals. Following addition of AMP, production of adenosine in the medium of E5NT/ENTPD1- and E5NT- transfected cells increased to 14.2 ± 1.1 and 24.5 ± 3.4 μM respectively while it remained below 1 μM in controls and in ENTPD1-transfected cells. A marked increase of adenosine formation from ADP or ATP was observed only in E5NT/ENTPD1-transfected cells (11.7 ± 0.1 and 5.7 ± 2.2 μM respectively) but not in any other condition studied. This study indicates feasibility and functionality of combined expression of human E5NT and ENTPD1 in pig endothelial cells using F2A sequence bearing construct.     

Effect of extracellular adenosine 5′-triphosphate on Ca2+ efflux from freshly isolated adult rat cardiomyocytes

The effect of extracellular adenosine 5′-triphosphate (ATP) on Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes was examined. ATP stimulated the efflux of ⁴⁵Ca²⁺ from the cells in a concentration-dependent manner (0.01–1 mM). The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by adenosine-5′-O-(3-thiotriphosphate) (ATP-γs) and α,β-methylene-ATP and adenosine 5′-diphosphate, but not by adenosine 5′-monophosphate and adenosine. The ATP-stimulated ⁴⁵Ca²⁺ efflux was not affected by deprivation of the extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. These results indicate that ATP stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane P₂ purinoceptors which may couple to Na⁺/Ca²⁺ exchange.     

The role of the CD39–CD73–adenosine pathway in liver disease

Extracellular adenosine triphosphate (ATP) is a danger signal released by dying and damaged cells, and it functions as an immunostimulatory signal that promotes inflammation. The ectonucleotidases CD39/ectonucleoside triphosphate diphosphohydrolase‐1 and CD73/ecto‐5′‐nucleotidase are cell‐surface enzymes that breakdown extracellular ATP into adenosine. This drives a shift from an ATP‐driven proinflammatory environment to an anti‐inflammatory milieu induced by adenosine. The CD39–CD73–adenosine pathway changes dynamically with the pathophysiological context in which it is embedded. Accumulating evidence suggests that CD39 and CD73 play important roles in liver disease as critical components of the extracellular adenosinergic pathway. Recent studies have shown that the modification of the CD39–CD73–adenosine pathway alters the liver's response to injury. Moreover, adenosine exerts different effects on the pathophysiology of the liver through different receptors. In this review, we aim to describe the role of the CD39–CD73–adenosine pathway and adenosine receptors in liver disease, highlighting potential therapeutic targets in this pathway, which will facilitate the development of therapeutic strategies for the treatment of liver disease.     

Altered activities of anti-atherogenic enzymes LCAT, paraoxonase, and platelet-activating factor acetylhydrolase in atherosclerosis-susceptible mice

We examined whether the putative anti-atherogenic enzymes LCAT, paraoxonase (PON), and platelet-activating factor acetylhydrolase (PAF-AH) are impaired in 8 week old atherosclerosis susceptible apolipoprotein E (apoE)(-/-) and LDL receptor (LDLr)(-/-) mice and whether plasma concentrations of bioactive oxidized phospholipids accumulate in plasma. ApoE(-/-) mice had reduced (28%) LCAT activity and elevated lysophosphatidylcholine and bioactive oxidized phospholipids (1-palmitoyl-2-oxovaleryl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine) compared with controls on the chow diet. Elevated oxidized phospholipids and reduced LCAT activity may, in part, contribute to spontaneous lesions in these mice on a chow diet. A Western diet decreased LCAT activity further (50% of controls) and PON activity was decreased 38%. The LDLr(-/-) mice showed normal LCAT activity on chow diet and little accumulation of oxidized phospholipids. On a Western diet, LDLr(-/-) mice had reduced LCAT activity (21%), but no change in PON activity. All genotypes had reduced PAF-AH activity on the Western diet. ApoE(-/-) and LDLr(-/-) mice, but not controls, had elevated plasma bioactive oxidized phospholipids on the Western diet.We conclude that impairment of LCAT activity and accumulation of oxidized phospholipids are part of an early atherogenic phenotype in these models.     

Differential thermographic features of some crystalline biopolymers

Crystalline proteins, nucleic acids, nucleotides, and nucleosides have been examined by differential thermal analysis. Characteristic thermograms are illustrated for globular, serum, and blood plasma proteins, calf thymus DNA, sodium triticonucleate, sodium thymonucleate, sperm DNA, yeast RNA, adenosine-3′-phosphate, adenosine-5′-phosphate, disodium adenosine triphosphate, adenosine, and deoxyadenosine. A pronounced effect of moisture on the differential thermal properties of DNA has been observed. It is suggested that solid-state denaturation is one of the prominent thermal effects recorded by the differential thermal analysis of proteins and nucleic acids.     

Extracellular Cyclic AMP—Adenosine Pathway in Isolated Adipocytes and Adipose Tissue

Objective: Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)–adenosine pathway in adipose tissue. Research Methods and Procedures: Sixteen miniature Yucatan swine (Sus scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 μL/min with Ringer's solution containing no addition, varying levels of cyclic AMP, 10 μM isoproterenol, or 10 μM isoproterenol plus 1 mM α,β‐methylene adenosine 5′‐diphosphate (AMPCP), a 5′‐nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 μM isoproterenol, or 1 μM isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. Results: Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP‐provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. Discussion: These data suggest the existence of a cyclic AMP—adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified.     

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi apparatus vesicles

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3′-phosphate 5′-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.     

Elevation of guanosine 3′,5′-monophosphate level by adenosine in cerebellar slices of guinea pig

Effect of adenosine on the level of guanosine 3′,5′-monophosphate in guinea pig cerebellar slices was investigated. Adenosine increased the concentration of guanosine 3′,5′-monophosphate in the slices 3–4-fold. Upon removal of adenosine from the medium, the concentration of guanosine 3′,5′-monophosphate returned to the initial level. AMP, ADP or ATP also increased the guanosine 3′,5′-monophosphate level to the same extent as adenosine, while adenine or other nucleotides were not effective. In the absence of Ca²⁺ in the incubation medium, adenosine did not increase the concentration of guanosine 3′,5′-monophosphate in cerebellar slices although level of adenosine 3′,5′-monophosphate was elevated by adenosine.Anticholinergic agents, adrenergic blocking agents or antihistaminics did not prevent the increase of guanosine 3′,5′-monophosphate by adenosine indicating that the effect of adenosine was not mediated by the release of neurotransmitters.The combination of adenosine with depolarizing agents showed an additive effect on the level of guanosine 3′,5′-monophosphate indicating that adenosine increased the level of guanosine 3′,5′-monophosphate by a different mechanism from the depolarization.     

Partial purification and characterization of S-adenosylhomocysteine hydrolase isolated from Saccharomyces cerevisiae

S-Adenosylhomocysteine (SAH) hydrolase was purified 25-fold from bakers' yeast by chemical methods and column chromatography. The purified enzyme could readily synthesize SAH from adenosine and homocysteine, but could hydrolyze only negligible amounts of SAH. The purified enzyme showed no activity towards S-adenosylmethionine, methylthioadenosine, or adenosine. Several nucleotides, sulfhydryl compounds, and ribose could not replace adenosine or homocysteine in the reaction mixture. SAH could be hydrolyzed by SAH hydrolase if commercial adenosine deaminase was included in the reaction mixture. Under these conditions l-homocysteine could act as a product inhibitor. A number of compounds structurally similar to adenosine and homocysteine were found to inhibit synthesis of SAH from adenosine and homocysteine. The strongest inhibitors were adenine, adenosine-3'-monophosphate, adenosine-2'-monophosphate, adenosine diphosphate, adenosine triphosphate, and adenosine-5'-monophosphate. The biosynthetic and hydrolytic activity of SAH hydrolase in yeast cell ghosts was similar to the activity of the enzyme in vitro.