adenosine-5--(n-ethylcarboxamide) and Neuroblastoma

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Down-Regulation; Glioma; GTP-Binding Proteins; Hybrid Cells; Iloprost; Isoproterenol; Neuroblastoma; Transfection; Tumor Cells, Cultured

The induction of apoptosis by adenosine was studied in the mouse neuroblastoma cell line N1E-115. Apoptosis was characterized by fluorescence and electron microscopy, fluorescence-activated cell sorter (FACS) analysis, and caspase activity assays. A sixteen-hour exposure to 100 microM of adenosine led to chromatin condensation and caspase activation. However, selective agonists for all four adenosine receptors were ineffective. Caspase activation could be blocked partially by an inhibitor of the nucleoside transporter, dipyridamole, and completely by uridine, a competing substrate for adenosine transport. 2'-Deoxycoformycin, an inhibitor of adenosine deaminase, enhanced caspase activation by adenosine but had no effect by itself. Caspase activation could be blocked by 5'-amino-5'-deoxyadenosine, which inhibits the phosphorylation of adenosine by adenosine kinase. These results indicate that adenosine receptors are not involved in adenosine-induced apoptosis in N1E-115 cells, but that uptake of adenosine and its subsequent phosphorylation is required.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Apoptosis; Biological Transport; Carrier Proteins; Deoxyadenosines; Membrane Proteins; Mice; Neuroblastoma; Nucleoside Transport Proteins; Purinergic P1 Receptor Agonists; Receptors, Purinergic P1; Tumor Cells, Cultured

Using receptor-selective agonists and antagonists, the possible presence of both A2a and A2b adenosine receptor subtypes coupled to activation of adenylyl cyclase was investigated in NG108-15 neuroblastoma x glioma hybrid cells. The relatively non-selective adenosine receptor agonist 5'-(N-ethyl carboxamido)-adenosine (NECA; 1 nM-300 microM) produced a biphasic increase in adenylyl cyclase activity in cell homogenates, best fitted to two components with high (EC50 0.7 microM) and low (EC50 16.0 microM) potency, respectively. The selective adenosine A2a receptor agonist CGS-21680 (1 nM-300 microM) also produced a biphasic increase in adenylyl cyclase. The NECA-dependent increase in adenylyl cyclase activity was almost completely inhibited by the non-selective adenosine receptor antagonist xanthine amine congener (XAC; 30 microM), but only partially inhibited by the selective A2a adenosine antagonist 8-(3-chlorostyryl)caffeine (CSC; 1 microM). Experiments were also performed to investigate the time course of NECA-induced desensitization of putative A2a and A2b receptor responses. The A2a-response was quantified using 10 microM CGS-21680, whilst the A2b response was quantified using 100 microM NECA in the presence of 1 microM CSC. The t0.5 for desensitization for each subtype was found to be around 20 min. Neither activation (with dibutyryl cAMP; 1 mM) nor inhibition (with H-89; 10 microM) of cyclic AMP-dependent protein kinase altered the ability of NECA pretreatment to desensitize A2a or A2b receptor-activated adenylyl cyclase. However zinc (200 microM), an inhibitor of G-protein coupled receptor kinase 2 (GRK2), significantly reversed the agonist-induced desensitization of A2a and A2b receptor-activated adenylyl cyclase. These experiments suggest the co-existence of A2a and A2b receptors coupled in a stimulatory fashion to adenylyl cyclase in NG108-15 cells. Furthermore desensitization of A2a and A2b responses occurs at the same rate and may involve a G-protein-coupled receptor kinase.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Enzyme Activation; Glioma; Hybrid Cells; Neuroblastoma; Phenethylamines; Purinergic P1 Receptor Agonists; Receptors, Purinergic P1; Tumor Cells, Cultured

G protein-coupled receptor kinases (GRKs) are thought to be important in mediating the agonist-induced phosphorylation and consequent desensitization of G protein-coupled receptor responses. NG108-15 mouse neuroblastoma X rat glioma cells express a wide range of G protein-coupled receptors and significant levels of GRK2. Therefore, to determine the role of GRK2 in agonist-induced desensitization of various G(s)-coupled receptors in NG108-15 cells, we stably transfected cells with a dominant negative mutant GRK2 construct (Lys220Arg). In homogenates prepared from cells overexpressing the dominant negative mutant GRK2, the acute stimulation of adenylyl cyclase by various receptor and nonreceptor agonists was the same as in control cells stably transfected with plasmid only. NG108-15 cells express both A2a and A2b adenosine receptors, which mediate activation of adenylyl cyclase, with both of these responses being subject to agonist-induced desensitization with a t1/2 of 15-20 min. In dominant negative mutant GRK2 cells, the rates of desensitization of A2a and A2b receptor-stimulated adenylyl cyclase were markedly slower than in plasmid transfected controls, with the latter being similar to wild-type cells. After a 20-min treatment with an adenosine agonist, the desensitization of A2a and A2b receptor-stimulated adenylyl cyclase in dominant negative mutant GRK2 cells was less than half that seen in plasmid transfected control cells. On the other hand, the agonist-induced desensitization of secretin and IP-prostanoid receptor-stimulated adenylyl cyclase was the same in dominant negative mutant GRK2 cells as in plasmid transfected control cells. These results indicate that in intact cells, GRK2 may mediate the desensitization of adenosine A2 receptors. Furthermore, there seems to be selectivity of GRK2 action between G(s)-coupled receptors because the agonist-induced desensitization of secretin and IP-prostanoid receptor-stimulated adenylyl cyclase was not affected by dominant negative mutant GRK2 overexpression.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Animals; beta-Adrenergic Receptor Kinases; Cyclic AMP-Dependent Protein Kinases; G-Protein-Coupled Receptor Kinase 2; G-Protein-Coupled Receptor Kinase 3; Glioma; Hybrid Cells; Mice; Mutation; Neuroblastoma; Purinergic P1 Receptor Agonists; Rats; Receptors, Prostaglandin; Receptors, Purinergic P1; Secretin; Sensitivity and Specificity; Tumor Cells, Cultured

In neuroblastoma x glioma hybrid NG108-15 cells, ATP induced a concentration-dependent increase in the intracellular Ca2+ concentration ([Ca2+]i), accompanied by inositol phosphate formation. Under the same conditions, we found a marked increase in cAMP levels produced by ATP at concentrations similar to those required to increase [Ca2+]i. The Ca2+ ionophore A23187 or bradykinin, which evoked inositol phosphate formation and increases in [Ca2+]i, did not increase, and instead slightly decreased, cAMP content, indicating that ATP-induced cAMP accumulation was not due to activation of Ca(2+)-sensitive adenylyl cyclase. The effect of ATP on cAMP production was not dependent on generation of adenosine caused by ATP hydrolysis. Among several P2 purinoceptor agonists, adenosine-5'-O-(3-thio)triphosphate, 5'-adenylylimidodiphosphate, and adenosine-5'-O-(2-thio)diphosphate evoked both cAMP accumulation and Ca2+ mobilization. In contrast, beta,gamma-methylene-ATP selectively elicited cAMP accumulation, whereas 2-methylthio-ATP and UTP induced only Ca2+ mobilization, without affecting cAMP levels. The potent P2x purinoceptor agonist alpha,beta-methylene-ATP did not induce cAMP accumulation or Ca2+ mobilization. The cAMP accumulation induced by ATP was not affected by the P2 receptor antagonist suramin but was inhibited by P1 receptor antagonists such as 8-(p-sulfophenyl)theophylline, 3-isobutyl-1-methylxanthine, and xanthine amine congener. However, the ATP-induced increase in [Ca2+]i was not affected by suramin or xanthine amine congener. Taken together, these results indicate that ATP activates two distinct purinoceptors that are coupled to different signal transduction systems, one being adenylyl cyclase and the other phospholipase C, in NG108-15 cells. Furthermore, pharmacological profiles of the adenylyl cyclase-coupled receptor were quite different from those of any known purinoceptor subtypes, especially in the unusual sensitivity of the receptor to P1 and P2 receptor agonists and antagonists. It is therefore suggested that ATP-induced cAMP accumulation may be mediated by a novel subtype of purinoceptor in NG108-15 cells.

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Alprostadil; Calcium; Cyclic AMP; Dose-Response Relationship, Drug; Glioma; Hybrid Cells; Inositol Phosphates; Neuroblastoma; Receptors, Purinergic; Tumor Cells, Cultured; Type C Phospholipases

1. Prostacyclin and adenosine A2 receptors activate adenylate cyclase in the neuroblastoma hybrid cell lines NG108-15 and NCB-20. Prolonged exposure of NG108-15 cells to iloprost (a stable analogue of prostacyclin) results in a subsequent reduction in the capacity for adenylate cyclase activation by iloprost, the adenosine analogue 5'-(N-ethyl)-carboxamidoadenosine (NECA) or NaF. In contrast prolonged exposure of NCB-20 cells to iloprost results only in the loss of iloprost responsiveness. 2. Iloprost pretreatment of NG108-15 cells also magnified the morphine-dependent inhibition of iloprost-stimulated adenylate cyclase activity from 36 to 48%. This change was not due to lower iloprost stimulation following desensitization, since the % inhibition of adenylate cyclase activity by morphine in control cells was constant irrespective of enzyme activity. 3. These heterologous effects observed in NG108-15 cells following iloprost pretreatment may involve changes in the GS alpha protein, since there was a reduction of about 30% in the cholera toxin-induced [32P]-ADP-ribosylation of a 45 kDa protein from cell membranes (corresponding to the extent of loss of NECA or NaF responsiveness). A similar reduction was not observed in NCB-20 cells. 4. These results indicate that iloprost pretreatment induces different forms of desensitization in NG108-15 and NCB-20 cell lines. The heterologous desensitization in the former may, like the human platelet, involve a functional loss of GS alpha from the cell membrane. Changes in the activity of GS alpha may also account for the heterologous effects on receptors that mediate inhibition of adenylate cyclase.

Topics: Adenosine; Adenosine Diphosphate; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Cell Line; Epoprostenol; GTP-Binding Proteins; Hybrid Cells; Iloprost; Kinetics; Mice; Morphine; Neuroblastoma; Phosphorus Radioisotopes; Receptors, Epoprostenol; Receptors, Prostaglandin; Sodium Fluoride

1. NaF (10 mM) produced a 2-3 fold increase in adenylate cyclase activity in homogenates of NG108-15 cells incubated in the presence of 1 microM GTP. Higher concentrations of NaF suppressed adenylate cyclase activity. 2. In the presence of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA; 100 microM) or the prostacyclin receptor agonist iloprost (10 nM), NaF produced a much smaller increase in adenylate cyclase activity, whereas in the presence of a saturating concentration of iloprost (1 microM), NaF only inhibited adenylate cyclase activity. 3. Similarly, Gpp(NH)p activated basal adenylate cyclase activity, and inhibited 1 microM iloprost-activated enzyme activity. In the presence of 10 microM forskolin, NaF or Gpp(NH)p increased adenylate cyclase activity synergistically. Analysis of concentration-effect curves indicated that NaF (2 mM) or Gpp(NH)p (100 microM) increased the potency with which forskolin activated adenylate cyclase, whilst reducing the maximum activation of adenylate cyclase by iloprost. 4. Opiate receptors mediate inhibition of adenylate cyclase, and the opiate agonist morphine (100 microM) reduced the capacity of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. Unexpectedly, pertussis toxin treatment enhanced the ability of NaF or Gpp(NH)p to inhibit iloprost-activated adenylate cyclase. 5. In the absence of GTP, NaF and Gpp(NH)p remained able both to activate basal adenylate cyclase and to be synergistic with forskolin in activating the enzyme. In contrast the ability of NaF and Gpp(NH)p to inhibit iloprost-activated adenylate cyclase was substantially lost in the absence of added GTP. These results suggest that NaF modulates adenylate cyclase activity in NG108-15 cell membranes by interacting with the alpha subunits of both G0 and Gi regulatory proteins. The effects of NaF and Gpp(NH)p are critically dependent on the prior mode and extent of activation or inhibition of this transmembrane signalling pathway. This simple system may be of use in assessing alterations in GSO-O interaction following manipulations such as hormone receptor desensitization.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Animals; Cell Membrane; Colforsin; Enzyme Activation; Epoprostenol; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Iloprost; Mice; Morphine; Nervous System Neoplasms; Neuroblastoma; Pertussis Toxin; Sodium Fluoride; Tumor Cells, Cultured; Virulence Factors, Bordetella

We have examined the effects of increasing membrane polyunsaturated fatty acids (PUFAs) on adenosine receptor function in intact N1E-115 neuroblastoma cells. Addition of linoleic acid to the culture medium for 48 h resulted in an approximate threefold increase in the amount of omega 6 fatty acids esterified to membrane phospholipids. Basal cAMP accumulation was significantly higher in the PUFA-enriched cells than in controls, although the differences could be diminished by approximately 75% by treatment of the cells with adenosine deaminase or 8-phenyltheophylline. Exposure of the cultures to the stable adenosine analogue 5'-N-ethylcarboxyamide adenosine (NECA) resulted in concentration-dependent increases in cAMP accumulation. Data from saturation experiments indicated that the maximum amount of cAMP that could be formed in response to NECA in the PUFA-enriched cells was twice that in control cells. Also, the amount of agonist required to elicit half maximal stimulation in the supplemented cells was significantly less than in the control cells (mean values for EC50, 0.85 and 1.43 microM, respectively). The results of this study demonstrate that membrane PUFA have the ability to modify interactions between adenosine receptors and adenylate cyclase in neural cells, a fact that is of potential importance in considering the central role that adenosine plays as a neuromodulator in the nervous system.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cyclic AMP; Fatty Acids, Unsaturated; Linoleic Acids; Membrane Lipids; Neuroblastoma; Neurons; Phospholipids; Receptors, Purinergic; Tumor Cells, Cultured

In IMR32 neuroblastoma cells, the two adenosine receptor agonists N6-R-phenylisopropyladenosine and 5'-N-ethylcarboxamidoadenosine dose-dependently stimulated membrane adenylate cyclase activity with potencies consistent with the presence of adenosine receptors of the A2-subtype. The S enantiomer of N6-R-phenylisopropyladenosine induced a significantly lower stimulation of adenylate cyclase, accordingly to its lower ability to activate adenosine receptors. These effects were selectively counteracted by the adenosine receptor antagonist theophylline and, conversely, were not affected by the A1-adenosine receptor selective blocker 8-cyclopentyl-1,3-dipropylxanthine. No adenosine receptors belonging to the A1-subtype seem, therefore, to be present in this cell line, as also shown by the lack of inhibitory activity of N6-R-phenylisopropyladenosine on both basal and forskolin-stimulated adenylate cyclase activity. Activation of A2-receptors did not modify intracellular basal calcium levels, did not influence calcium influx through voltage-dependent calcium channels and did not modify calcium influx and redistribution induced by muscarinic receptor activation. Prolonged exposure of cells to either N6-R-phenylisopropyladenosine or 5'-N-ethylcarboxamidoadenosine was associated with a small but significant degree of morphological differentiation, comparable to that induced by dibutyryl cAMP, and therefore presumably related to the prolonged increase of intracellular cAMP levels elicited by the two adenosine agonists. After cellular differentiation induced with either dibutyryl cAMP or 5-bromodeoxyuridine, a selective desensitization of A2-receptor stimulated adenylate cyclase activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Calcium; Cell Differentiation; Cell Line; Humans; Neuroblastoma; Phenylisopropyladenosine; Receptors, Purinergic; Theophylline; Tumor Cells, Cultured

The activities of an endogenous nucleoside, 5'-deoxy-5'-methylthioadenosine (MTA), on adenosine sensitive sites such as adenosine A1 and A2 receptors and the P-site, as well as on purine nucleoside transport, have been studied. This nucleoside competitively antagonized the A2 receptor-mediated stimulation of neuroblastoma adenylate cyclase, produced a GTP-dependent and 8-p-sulfophenyltheophylline-sensitive inhibition of adenylate cyclase activity in rat cerebellar membranes, and decreased the spontaneous contractile activity of isolated segments of rabbit jejunum. MTA was neither active at the P-site nor did it diminish the binding of [3H]nitrobenzylthioinosine, a nucleoside transport inhibitor. We conclude that (a) MTA is an agonist at the adenosine A1 receptor but an antagonist at the A2 receptor, and (b) the adenosine receptor which causes relaxation of rabbit jejunum is not a neuroblastoma-type A2 receptor which activates adenylate cyclase.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Animals; Cerebellum; Deoxyadenosines; In Vitro Techniques; Jejunum; Mice; Muscle Contraction; Neuroblastoma; Rabbits; Rats; Receptors, Purinergic; Thioinosine; Thionucleosides

The effect of three stable adenosine analogues, L-phenylisopropyl-adenosine (L-PIA), 2-chloroadenosine, and adenosine 5'-ethylcarboxamide (NECA), on cyclic AMP accumulation was studied in five different cell lines derived from the nervous system. In N18-neuroblastoma cells, with cholinergic properties, all three analogues caused an increased accumulation of cyclic AMP with the following relative order of potency: NECA greater than 2-chloroadenosine greater than L-PIA. The half maximal effect of NECA was obtained at close to 10(-8) M concentration. In the two other neuroblastoma cell lines, 41A3 with cholinergic and NIE115 with adrenergic properties, the two analogues NECA and PIA had similar effects. In glioma C6 and 138 MG cells NECA was also found to be more potent that PIA in elevating cyclic AMP levels. However, the absolute potency of NECA in these cell lines was almost 100 times lower. Phosphodiesterase (PDE) activity in crude homogenates of the five cell lines showed essentially similar Km and Vmax, with the exception that the three neuroblastoma cell lines showed biphasic, the glial cell lines monophasic Eadie-Hofstee plots. Theophylline was equally potent as an inhibitor of PDE in all cell lines, but the non-xanthine, inhibitor rolipram, was more potent against neuroblastoma than glial cell PDE. These results indicate that all five cell lines have adenosine receptors of the A2-subtype. However, the apparent affinity of the adenosine analogues to these receptors was markedly different between the neuroblastoma and glial cell lines. The absolute potency of adenosine analogues may be a poor criterion to classify adenosine receptors, into A1 and A2 subtypes, especially when intact cells are used.

Topics: 2-Chloroadenosine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Cell Line; Cells, Cultured; Cyclic AMP; Glioma; Humans; Kinetics; Neuroblastoma; Phenylisopropyladenosine; Phosphoric Diester Hydrolases; Pyrrolidinones; Rolipram; Theophylline; Vasodilator Agents