ATP-敏感钾通道和内源性腺苷参与刺激蓝斑引起的脊髓抗痛作用

根据蓝斑刺激可以通过脊髓下行性去甲肾上腺素能纤维阻断由背角上传到束旁核神经元的伤害性放电的事实,本实验用脊髓鞘内给予相应工具药的方法,进一步分析了上述下行性抑制作用在脊髓背角中阻止伤害性传入信号向上传递的可能机制,结果发现：（1）鞘内注入ATP-敏感钾通道阻断剂格列苯脲或腺苷受体拮抗剂氨茶碱,均可以阻断或取消刺激蓝斑引起的对束旁核伤害性放电的抑制作用;（2）鞘内注入ATP-钾通道激动剂nic-orandil或腺苷受体激动剂5‘-N-ethylcarboxamido-adenosine(NECA）,都可抑制束旁核神经元的伤害性放电;（3）鞘内注入氨茶碱可阻断鞘内注入nicorandil引起的束旁核痛放电的抑制,再鞘内注入格列苯脲不能阻断鞘内注入NE-CA引致的束旁核痛放电的抑制。这些结果提示：（1）蓝斑刺激在脊髓背角中抑制痛信号的上传,要有ATP-敏感钾通道的激活和内源性腺苷的释放为中介;（2）ATP-敏感钾通道的激活发生在腺苷的释放之前。     

Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.

In fat cells isolated from the parametrial adipose tissue of rats, the addition of purified adenosine deaminase increased lipolysis and cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. Adenosine deaminase markedly potentiated cyclic AMP accumulation due to norepinephrine. The increase in cyclic AMP due to adenosine deaminase was as rapid as that of theophylline with near maximal effects seen after only a 20-sec incubation. The increases in cyclic AMP due to crystalline adenosine deaminase from intestinal mucosa were seen at concentrations as low as 0.05 mug per ml. Further purification of the crystalline enzyme preparation by Sephadex G-100 chromatography increased both adenosine deaminase activity and cyclic AMP accumulation by fat cells. The effects of adenosine deaminase on fat cell metabolism were reversed by the addition of low concentrations of N6-(phenylisopropyl)adenosine, an analog of adenosine which is not deaminated. The effects of adenosine deaminase on cyclic AMP accumulation were blocked by coformycin which is a potent inhibitor of the enzyme. These findings suggest that deamination of adenosine is responsible for the observed effects of adenosine deaminase preparations. Protein kinase activity of fat cell homogenates was unaffected by adenosine or N6-(phenylisopropyl)adenosine. Norepinephrine-activated adenylate cyclase activity of fat cell ghosts was not inhibited by N6-(phenylisopropyl)adenosine. Adenosine deaminase did not alter basal or norepinephrine-activated adenylate cyclase activity. Cyclic AMP phosphodiesterase activity of fat cell ghosts was also unaffected by adenosine deaminase. Basal and insulin-stimulated glucose oxidation were little affected by adenosine deaminase. However, the addition of adenosine deaminase to fat cells incubated with 1.5 muM norepinephrine abolished the antilipolytic action of insulin and markedly reduced the increase in glucose oxidation due to insulin. These effects were reversed by N6-(phenylisopropyl)adenosine. Phenylisopropyl adenosine did not affect insulin action during a 1-hour incubation. If fat cells were incubated for 2 hours with phenylisopropyl adenosine prior to the addition of insulin for 1 hour there was a marked potentiation of insulin action. The potentiation of insulin action by prior incubation with phenylisopropyl adenosine was not unique as prostaglandin E1, and nicotinic acid had similar effects.     

Selective adenosine release from human B but not T lymphoid cell line

Intracellular adenosine formation and release to extracellular space was studied in WI-L2-B and SupT1-T lymphoblasts under conditions which induce or do not induce ATP catabolism. Under induced conditions, B lymphoblasts but not T lymphoblasts, release significant amounts of adenosine, which are markedly elevated by adenosine deaminase inhibitors. In T lymphoblasts, under induced conditions, only simultaneous inhibition of both adenosine deaminase activity and adenosine kinase activities resulted in small amounts of adenosine release. Under noninduced conditions, neither B nor T lymphoblasts release adenosine, even in the presence of both adenosine deaminase or adenosine kinase inhibitors. Comparison of B and T cell's enzyme activities involved in adenosine metabolism showed similar activity of AMP deaminase, but the activities of AMP-5'-nucleotidase, adenosine kinase and adenosine deaminase differ significantly. B lymphoblasts release adenosine because of their combination of enzyme activities which produce or utilize adenosine (high AMP-5'-nucleotidase and relatively low adenosine kinase and adenosine deaminase activities). Accelerated ATP degradation in B lymphoblasts proceeds not only via AMP deamination, but also via AMP dephosphorylation into adenosine but its less efficient intracellular utilization results in the release of adenosine from these cells. In contrast, T lymphoblasts release far less adenosine, because they contain relatively low AMP-5'-nucleotidase and high adenosine kinase and adenosine deaminase activities. In T lymphoblasts, AMP formed during ATP degradation is not readily dephosphorylated to adenosine but mainly deaminated to IMP by AMP deaminase. Any adenosine formed intracellularly in T lymphoblasts is likely to be efficiently salvaged back to AMP by an active adenosine kinase. In general, these results may suggest that adenosine can be produced only by selective cells (adenosine producers) whereas other cells with enzyme combination similar to SupT1-T lymphoblasts can not produce significant amounts of adenosine even in stress conditions.     

Adenosine transport by a variant of C1300 murine neuroblastoma cells deficient in adenosine kinase

The uptake of adenosine by an adenosine kinase deficient variant of C1300 murine neuroblastoma cells has been studied in the absence and in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, a potent adenine deaminase inhibitor. Although 100 micro M inhibitor completely blocks the metabolism of adenosine under the conditions studied, the uptake of adenosine is concentrative, i.e., the intracellular adenosine concentration exceeds the extracellular concentration. This concentrative effect decreases as the concentration of adenosine increases and is hypothesized to be due to the binding of adenosine to an intracellular component. Despite this concentrative effect, we believe that the kinetics of uptake, as determined in experiments with short (10-20 s) uptake periods, reflect the kinetics of adenosine transport by a facilitated diffusion process. This nucleoside transport system appears to be nonspecific in that the transport of adenosine is competitively antagonized by thymidine. It does not appear to be necessary to inhibit adenosine deaminase in order to study transport in these cells as the Km for transport is not affected by the presence of erythro-9-(2-hydroxy-3-nonyl)adenine. However, erythro-9-(2-hydroxy-3-nonyl)adenine does depress the V for transport. This effect of the inhibitor is probably not due to the inhibition of adenosine deaminase as the transport of thymidine is similarly affected.     

Construction and expression of an adenosine deaminase::lacZ fusion gene

A eukaryotic expression vector was constructed in which the coding nucleotide sequences (ADA) of human adenosine deaminase (ADA) were fused in frame with the coding sequences of the bacterial gene lacZ encoding beta-galactosidase (beta Gal). This ADA::lacZ fusion gene was anticipated to encode a hybrid protein that has retained the biological functions of both proteins. Transfection of mammalian cells with the fusion gene resulted in the synthesis of both ADA and beta Gal. Cells expressing the gene could therefore be detected with the histochemical staining procedure that relies on the conversion of the indicator, XGal, by beta Gal. In addition, the transfected cells could be sorted on a fluorescence-activated cell sorter with the use of a vital staining procedure described for the selection of beta Gal-producing cells. Cell lines that harbored the fusion gene were tested for ADA overexpression by exposing them to the cytotoxic adenosine analog 9-beta-D-xylofuranosyl adenine (Xyl-A), in the presence of the ADA inhibitor deoxycoformycin (dCF). Resistance to Xyl-A/dCF was observed in the lines carrying ADA::lacZ and moreover, the fraction of cells that survived a stringent selection for ADA overexpression also exhibited significantly increased levels of beta Gal, which confirmed the direct linkage between ADA and lacZ expression. The use of this and other fusion genes might be useful in the development of gene-therapy protocols where they could help to meet the demand for versatile methods to detect and select cells with newly introduced genes.     

Early postimplantation embryolethality in mice following in utero inhibition of adenosine deaminase with 2'-deoxycoformycin

Adenosine deaminase (ADA) catalyzes the hydrolytic deamination of adenosine (or 2'-deoxyadenosine) to inosine (or 2'-deoxyinosine). Previously, we have shown that ADA activity is subject to strong cell-specific developmental regulation in placental tissues of mice between days 6 and 11 of gestation (Knudsen et al.:Biology of Reproduction 39:937-951, 1988). In the present study, we examined the effects of intrauterine exposure to 2'-deoxycoformycin (dCF; pentostatin), a potent irreversible inhibitor of ADA, on early postimplantation development. Deoxycoformycin was administered to pregnant ICR mice as a single intraperitoneal injection at a dose of 5 mg/kg on one of days 6 through 11 of gestation (plug day 0). A marked increase in the incidence of implantation site resorptions was observed following treatment specifically on days 7 (61% resorbed) or 8 (78% resorbed). No effect was observed following treatment on days 6, 9, 10, or 11. ADA-immunoreactive protein was shown, by ABC-immunoperoxidase staining on days 7 or 8 of gestation, to be present at high levels in decidual cells of the antimesometrial region but at below-detectable levels in the embryo. Treatment of pregnant dams with dCF on day 7 produced a complete (greater than 99%) inhibition of ADA activity in the antimesometrial decidua by 30 min, induced excessive cell death in the prospective neural plate and primary mesenchyme of the trilaminar disc by 6 h, and arrested embryonic development at an early somite stage. These results suggest that the antimesometrial decidua plays a protective role in preventing an inappropriate accumulation of endogenous ADA substrates in the implantation site.     

An analysis of multiple mechanisms of adenosine toxicity in baby hamster kidney cells

Analysis of the response of baby hamster kidney cells to adenosine in the presence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine has revealed two distinct mechanisms of toxicity. The first is apparent at low concentrations of adenosine (less than 5 microM) and is dependent upon the presence of a functional adenosine kinase. The initial toxicity is abolished by uridine, is unrelated to the inhibition of ribonucleotide reductase, and is accompanied by a decrease in the size of the pyrimidine nucleotide pool. Toxicity at higher concentrations of adenosine is adenosine kinase independent and is potentiated by homocysteine thiolactone. An elevation in the intracellular level of S-adenosylhomocysteine, which was observed following treatment with higher concentrations of adenosine (greater than 10 microM), is believed to mediate toxicity at these levels. Interestingly, BHK cells were resistant to intermediate levels of adenosine. The mechanism of resistance is currently unknown, but appears unrelated to a lack of inhibition of adenosine deaminase. It is proposed that substrate inhibition of adenosine kinase may be a determinant of this property.     

Catabolism of adenine nucleotides in adenosine deaminase deficient erythrocytes

$\text{In vitro}$ incubation studies using fluoride and iodoacetate as glycolytic inhibitors have been carried out on red cells of the two subjects with adenosine deaminase deficiency. For comparison, similar studies have also been carried out on red cells from a normal subject and from a child with severe combined immunodeficiency with normal adenosine deaminase activity. The adenosine formed in the adenosine deaminase deficient red cells is a measure of adenosine 5′-phosphate breakdown initiated by 5′-nucleotidase, whereas inosine 5′-phosphate, inosine and hypoxanthine formation is a measure of adenosine 5′-phosphate breakdown initiated by adenylate deaminase. With fluoride as inhibitor, nearly all of the adenosine 5′-phosphate breakdown proceeded by way of adenylate deaminase, while with iodoacetate as inhibitor, 20–30% of the adenosine 5′-phosphate breakdown was initiated by 5′-nucleotidase acting on adenosine 5′-phosphate. In addition, significant amounts of adenine were produced in adenosine deaminase deficient red cells in the presence of the glycolytic inhibitors. Possible explanations for the findings noted in this study are discussed and related to recent studies on the properties of the pertinent purine nucleotide catabolic enzymes.     

Release of adenosine by C1300 neuroblastoma cells in tissue culture

Previous work in our laboratory led us to postulate that N2a cells release adenosine into growth medium, where it acts at the extracellular adenosine receptors to modulate the sensitivity of the cells to the cyclic AMP-elevating effect of adenosine [Green, RD, J Pharmacol Exp Ther 201:610, 1977]. We have now devised a high-performance liquid chromatographic (HPLC) procedure capable of quantitating the concentrations of adenosine in cells and tissue culture media. Growth media of N2a cells and a variant of N2a cells deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT^?) contain 10–20 nM adenosine, while that of a variant deficient in adenosine kinase (AK^?) is elevated severalfold. It appears that the concentration of adenosine in growth media is determined by both the rate at which it is released by cells into the medium and the rate at which it is metabolized by adenosine deaminase present in the serum in the growth medium. Both N2a and AK^? cells release considerable amounts of adenosine into serum-free medium (SFM) over a short period. Adenosine release is greater from AK^? cells and is accelerated by erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), a potent adenosine deaminase inhibitor. This accelerated release is retarded by dipyridamole and homocysteine. Surprisingly, dipyridamole and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20 1724), a potent phosphodiesterase inhibitor, stimulate basal adenosine release from N2a but not from AK^? cells. It remains to be determined if this is due to an effect of these compounds on adenosine kinase. These results give further support for the hypothesis that adenosine in growth medium modulates the sensitivity of the cells to the cyclic AMP-elevating affect of adenosine, and furthermore they suggest that adenosine in growth media may tonically stimulate adenylate cyclase and affect processes controlled by the cyclic AMP:cyclic AMP-dependent protein kinase system.     

Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.     

Activities and some properties of 5''-nucleotidase, adenosine kinase and adenosine deaminase in tissues from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine.

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.     

Enhancement by adenosine of insulin-induced activation of phosphoinositide 3-kinase and protein kinase B in rat adipocytes.

The role of adenosine receptor in regulation of insulin-induced activation of phosphoinositide 3-kinase (PI 3-kinase) and protein kinase B was studied in isolated rat adipocytes. Rat adipocytes are known to spontaneously release adenosine, which in turn binds and stimulates the adenosine A1 receptors on the cells. In the present study, we observed that degradation of this adenosine by adenosine deaminase attenuated markedly the insulin-induced accumulation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), a product of PI 3-kinase. p-Aminophenylacetyl xanthine amine congener (PAPA-XAC), an inhibitor of the adenosine A1 receptor, also inhibited the insulin-induced PtdIns(3,4,5)P3 accumulation. When extracellular adenosine was inactivated by adenosine deaminase, phenylisopropyladenosine, an adenosine A1 receptor agonist, potentiated the insulin-induced accumulation of PtdIns(3,4,5)P3. Insulin-induced activation of protein kinase B, the activity of which is controlled by the lipid products of PI 3-kinase, was also potentiated by adenosine. Prostaglandin E2, another activator of a pertussis toxin-sensitive GTP-binding protein in these cells, potentiated the insulin actions. Thus, the receptors coupling to the GTP-binding protein were found to positively regulate the production of PtdIns(3,4,5)P3, a putative second messenger for insulin actions, in physiological target cells of insulin.     

Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

Isolation of mutant adenosine deaminase by coformycin affinity chromatography

Adenosine deaminase is a purine salvage enzyme that catalyzes the deamination of adenosine and deoxyadenosine. Deficiency of the enzyme activity is associated with T-cell and B-cell dysfunction. Mutant adenosine deaminase has been isolated from heterozygous and homozygous deficient lymphoblast cell lines with the aid of an affinity matrix consisting of coformycin (a potent inhibitor of the enzyme) as the affinity ligand, bound to 3,3'-iminobispropylamine-derivatized Sepharose. Routinely, 80-90% of adenosine deaminase in crude cell homogenates could be bound to the material. Adenosine deaminase was specifically eluted by enzyme inhibitors or less efficiently by high substrate concentrations. Protein preparations isolated from several different deficient cell lines were highly purified and exhibited molecular weights identical to wild-type adenosine deaminase. This method produces a protein that is suitable for structural studies.     

The effects of an induced adenosine deaminase deficiency on T-cell differentiation in the rat

Inherited deficiency of the enzyme adenosine deaminase (ADA) has been found in a significant proportion of patients with severe combined immunodeficiency disease and inherited defect generally characterized by a deficiency of both B and T cells. Two questions are central to understanding the pathophysiology of this disease: (1) at what stage or stages in lymphocyte development are the effects of the enzyme deficiency manifested; (2) what are the biochemical mechanisms responsible for the selective pathogenicity of the lymphoid system. We have examined the stage or stages of rat T-cell development in vivo which are affected by an induced adenosine deaminase deficiency using the ADA inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 2'-deoxycoformycin (DCF). In normal rats given daily administration of an ADA inhibitor, cortical thymocytes were markedly depleted; peripheral lymphocytes and pluripotent hemopoietic stem cells (CFU-S) all were relatively unaffected. Since a deficiency of ADA affects lymphocyte development, the regeneration of cortical and medullary thymocytes and their precursors after sublethal irradiation was used as a model of lymphoid development. By Day 5 after irradiation the thymus was reduced to 0.10-0.5% of its normal size; whereas at Days 9 and 14 the thymus was 20-40% and 60-80% regenerated, respectively. When irradiated rats were given daily parenteral injections of the ADA inhibitor plus adenosine or deoxyadenosine, thymus regeneration at Days 9 and 14 was markedly inhibited, whereas the regeneration of thymocyte precursors was essentially unaffected. Thymus regeneration was at least 40-fold lower than in rats given adenosine or deoxyadenosine alone. Virtually identical results were obtained with both ADA inhibitors, EHNA and DCF. The majority of thymocytes present at Day 9 and at Day 14 in inhibitor-treated rats had the characteristics of subcapsular cortical thymocytes which are probably the most ancestral of the thymocytes. Thus, an induced ADA deficiency blocked the proliferation and differentiation of subcapsular cortical thymocytes which are the precursors of cortical and medullary thymocytes.     

Metabolism of adenosine through adenosine kinase inhibits gluconeogenesis in isolated rat hepatocytes

In isolated hepatocytes from fasted rats, 0.5 mM adenosine inhibited gluconeogenesis from glutamine, lactate and pyruvate. This inhibition was due to adenosine conversion through adenosine kinase. An increase in ketone body release was only observed in the presence of lactate or pyruvate, and the two phenomena (i.e. inhibition of gluconeogenesis and increased ketone-body release) were linked. With alanine, dihydroxyacetone or serine as substrates, adenosine did not change gluconeogenesis; however, its conversion through adenosine kinase also inhibited gluconeogenesis. With asparagine as substrate, 0.5 mM adenosine increased gluconeogenesis; this increase was due to adenosine conversion through adenosine deaminase. However, adenosine conversion through adenosine kinase inhibited gluconeogenesis from asparagine. Thus, whatever the substrate used, adenosine conversion through adenosine kinase inhibited gluconeogenesis. The inhibitory effect of adenosine on gluconeogenesis cannot be related to the decrease in Pi concentration and to the increase in ATP pool. Beside its effect on gluconeogenesis, adenosine inhibited ketogenesis measured without added substrate; adenosine conversion through adenosine kinase was also involved in the inhibition of ketogenesis.     

Role of adenosine deaminase, ecto-(5''-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.     

Development of an antitumor adenosine analog, 3'-ethynyladenosine

3'-ethynyladenosine (EAdo) was an adenosine analog with potent antitumor activity against various human tumor cells in vitro. However, EAdo was enzymatically inactivated by adenosine deaminase (ADA) in vitro and in vivo. Therefore, we synthesized two ADA-resistant EAdo derivatives (2-F-EAdo and EAdo-5'-monophosphate, EAMP) and examined their antitumor activities.     

Chromosome anomalies associated with amplification of the adenosine deaminase gene (ADA) in rat hepatoma cells

Two independently selected series of rat hepatoma cell lines resistant to the drug deoxycoformycin (dCF) were analyzed karyotypically. Several forms of homogeneously staining regions (HSRs) were present on metaphase chromosomes of these cells. In some instances HSRs comprised nearly an entire chromosome, which are among the largest chromosomes in the karyotype. Stable resistance to dCF is acquired in rat cells by overproduction of the enzyme adenosine deaminase (ADA) as a result of amplification of ADA gene sequences. We have localized the amplified ADA gene sequences to HSRs on metaphase chromosomes from both series of dCF-resistant cell lines by in situ hybridization. Based upon the number of ADA gene sequences present and the lengths of the HSRs, we have estimated the size of the amplified unit to range from 450 to 1,000 kb.     

Inhibition of adenosine kinase and adenosine uptake in guinea-pig CNS tissue by halogenated tubercidin analogues

Two halogenated analogues of tubercidin (7-deazaadenosine) viz. 5-iodotubercidin and 5′-deoxy-5-iodotubercidin, previously were shown to be potent inhibitors of guinea-pig brain adenosine kinase activity and adenosine uptake in guinea-pig cerebral cortex slices. A further series of halogenated tubercidin analogues have been investigated; of the 9 compounds tested, 5′-deoxy-5-iodotubericidin was the most potent adenosine kinase inhibitor while 5-iodotubercidin was the most potent in inhibiting the facilitated uptake of adenosine. These compounds may be useful for elucidating the involvement of adenosine kinase in adenosine uptake, the maintenance of intracellular adenosine levels and in the neuromodulatory actions of adenosine in the CNS.