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.     

Characterization of a cell culture model for the study of adenosine deaminase- and purine nucleoside phosphorylase-deficient immunologic disease.

The absence of erythrocytic adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) has been associated with severe immunodeficiency disease in children. We have developed a cell culture model to study the possible relationships between purine salvage enzymes and immunologic function using an established T cell lymphosarcoma (S49) and a potent inhibitor of ADA, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA). Wild-type S49 cells are killed by dexamethasone or dbc AMP, and adenosine (5 muM) in the presence of an ADA inhibitor (6 muM EHNA) also prevents the growth of and kills these S49 cells. It has been proposed that adenosine is toxic to lymphoid cells by virtue of its ability to increase the intracellular concentrations of cyclic AMP. We examined the sensitivity of three mutants of S49 cells, with distinctive defects in some component of cyclic AMP metabolism or action, to killing by adenosine and EHNA. All three mutants are resistant to killing by isoproterenol or cholera toxin and two are resistant to dbc AMP itself, but all are sensitive to killing by adenosine and EHNA. Similarly, two dexamethasone-resistant S49 mutants are as sensitive to adenosine and EHNA as are the wildtype cells. We have also simulated the purine nucleoside phosphorylase deficiency in S49 cells by adding inosine and adenosine to the growth medium. In the presence of EHNA or inosine, the toxic effects of adenosine can be partially reversed by addition of (10-20 muM) uridine, an observation suggesting that adenosine is toxic as the result of its inducing pyrimidine starvation.     

Regional Difference in Responsiveness of Adenosine-Sensitive Cyclic AMP-Generating Systems in Chronic Epileptic Cerebral Cortex of the Rat

Cyclic AMP accumulation in brain slices incubated with adenosine or the adenosine analogue 2-chloroadenosine was examined in different areas of rat cerebral cortex following a unilateral injection of FeCl2 solution into the sensorimotor cortex to induce chronic epileptic activity. In the epileptic cortex, cyclic AMP accumulation in cortical slices was elicited three- to 11-fold by adenosine. The elicitation by adenosine of cyclic AMP accumulation was markedly inhibited by the adenosine antagonist 8-phenyltheophylline. In anterior cortical areas of rats in which the appearance of electrographic isolated spikes was dominant either ipsilateral or contralateral to the injection site 8 days or more after the injection, the adenosine-elicited accumulation of cyclic AMP was greater on the side of dominant spike activity than on the other. In anterior cortical areas of rats showing nearly equal spike activity on the two sides 19 days or more after the injection, the cyclic AMP accumulation was greater on the side ipsilateral to the injection site than on the other. In anterior and posterior cortical areas of rats showing spike-and-wave complexes and isolated spikes 1 month or more after the injection, the cyclic AMP accumulation was greater on the ipsilateral side than on the other. Similar regional differences in the adenosine-elicited accumulation of cyclic AMP were detected in the presence of the adenosine uptake inhibitor dipyridamole or the phosphodiesterase inhibitor DL-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724). The cyclic AMP accumulation was elicited five- to 17-fold by 2-chloroadenosine, in which case the elicitation was markedly inhibited by 8-phenyltheophylline. Regional differences in the 2-chloroadenosine-elicited accumulation of cyclic AMP were similar to those with adenosine and were detected in the presence of Ro 20-1724 or adenosine deaminase. The regional differences which correlated with the electrographic discharge patterns were due mainly to persistent changes in cyclic AMP accumulation on the primary epileptic side. These results suggest that alterations in adenosine-sensitive cyclic AMP generation in the cortex are associated with the neurochemical process leading to chronic iron-induced epilepsy.     

Alpha adrenergic receptor mediated formation of cyclic AMP in rat spinal cord

Methoxamine and phenylephrine (PE), postsynaptic alpha adrenergic agonists stimulated the accumulation of cyclic AMP in spinal cord tissue slices. Naphazoline, oxymetazoline and clonidine, previously shown to have greater efficacy at presynaptic alpha receptors did not alter accumulation and, in fact, blocked the PE response. The PE-stimulation was completely inhibited by postsynaptic alpha antagonists, incompletely by agents which bl ock presynaptic alpha receptors, and slightly by the beta blocker propranolol. Pe-stimulated accumulation was potentiated by phosphodiesterase inhibition (RO 20-1724). In contrast to previous reports on the requirement of the copresence of adenosine for alpha receptor stimulated accumulation of cyclic AMP in neuronal tissue, the PE-stimulation in spinal cord slices was unchanged by adenosine receptor blockade (theophylline), hydrolysis of endogenous adenosine (adenosine deaminase), inhibition of adenosine deaminase (EHNA) or blockade of adenosine uptake (dipyridamole). Added adenosine increased basal accumulation and produced a marked potentiation of the PE response. From this data it is evident that, in spinal cord tissue slices, there occurs a postsynaptic alpha adrenergic receptor linked to cyclic AMP accumulation which does not require the presence of other neurohumoral agents for activation.     

Adenosine Regulates the Survival of Avian Retinal Neurons and Photoreceptors in Culture

Adenosine modulates the survival of chick embryo retinal neurons in culture. When cultures were incubated for 3 days and refed with fresh medium, a large proportion of neurons died in the subsequent 3 days of culture. This cell death was prevented by preincubation of cultures for at least 24h with adenosine plus the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an adenosine uptake blocker nitrobenzylthioinosine (NBI), the adenosine A_2A receptor agonist 2-[4-(2-carboxyethyl) phenethylamino]-5-N-ethylcarboxamidoadenosine (CGS21680), or the permeant cyclic AMP analog 8-bromo cyclic AMP, but not the A₁ receptor agonist cyclohexyladenosine (CHA). Adenosine deaminase induced cell death when added to culture medium, and this effect was prevented by EHNA. Cell death was not observed when the medium was replaced by a conditioned medium from sister cultures. The data strongly suggest that adenosine regulates the survival of developing retinal neurons by a long-term activation of A_2A receptors and the increase of cyclic AMP levels.     

Inhibition of 3',5'-nucleotide phosphodiesterase in guinea pig cerebral cortical slices

Several compounds have been tested for their activity as inhibitors of 3′,5′-nucleotide phosphodiesterase in brain cortical slices from guinea pig. SQ 20,009 (1-ethyl-4-isopropylidenehydrazino)-1H-pyrazolo (3,4-b)pyridine-5-carboxylate, ethylester, hydrochloride), a very potent inhibitor of 3′,5′-nucleotide phosphodiesterase from rat and rabbit brain shows only moderate activity as 3′,5′-nucleotide phosphodiesterase inhibitor when tested in brain slices. It enhances cyclic AMP accumulation only when slices are stimulated by histamine. It does not affect cyclic AMP levels when histamine/norepinephrine are used as stimuli of cyclic AMP formation and decreases the activity of adenosine as stimulant slightly. Ro 20–1724 (4-(3-butoxy-4-methoxy)-2-imidazolidinone) a potent inhibitor of canine cerebral cortex PDE activity effectively augments the increase in cyclic AMP under all stimulating conditions mentioned, as does to a somewhat smaller extent the more water soluble Ro 20–2926 (4-(3-ethoxy-ethoxy-4-methoxy)-2-imidazolidinone). Dose-response curves for Ro 20–1724 under three stimulating conditions of increased cyclic AMP formation (0.1 mm histamine, 0.1 mm histamine/0.1 mm norepinephrine, 0.1 mm adenosine) yield an ED₅₀ of about 20 μm in all instances. A significant increase over respective controls is seen even at 1 μm Ro 20–1724 (histamine/norepinephrine). The drugs may be useful as tools for studying the regulation of cyclic AMP levels in the central nervous system.     

Adenosine and Cyclic AMP in Rat Cerebral Cortical Slices: Effects of Adenosine Uptake Inhibitors and Adenosine Deaminase Inhibitors

The endogenous levels of adenosine functionally linked to cyclic AMP systems in rat cerebral cortical slices are regulated by both adenosine deaminase and adenosine uptake systems. 2'-Deoxycoformycin (2'-DCF), an adenosine deaminase inhibitor, slightly increased basal, adenosine, and norepinephrine-elicited accumulations of cyclic AMP, whereas dipyridamole, an uptake inhibitor, had an even greater effect on cyclic AMP accumulations under the same conditions. Combinations of 2'-DCF and dipyridamole elicited a greater effect than either compound alone. Neither 2'-DCF nor dipyridamole significantly augmented accumulations of cyclic AP elicited by a depolarizing agent, veratridine, suggesting that the adenosine "released" during neuronal depolarization of brain slices is not as subject to inactivation by uptake or deamination as endogenous adenosine in control brain slices. The accumulation of cyclic AMP elicited by a combination of norepinephrine and veratridine was greater than additive. The response to a pure beta-adrenergic agonist, isoproterenol, was not potentiated by 2'-DCF, dipyridamole, or veratridine, consonant with minimal interaction of endogenous adenosine with beta-adrenergic systems.     

Adenosine and deoxyadenosine induces apoptosis in oestrogen receptor-positive and -negative human breast cancer cells via the intrinsic pathway

In this study we have examined the cytotoxic effects of different concentrations of adenosine (Ado) and deoxyadenosine (dAdo) on human breast cancer cell lines. Ado and dAdo alone had little effect on cell cytotoxicity. However, in the presence of adenosine deaminase (ADA) inhibitor, EHNA, adenosine and deoxyadenosine led to significant growth inhibition of cells of the lines tested. Ado/EHNA and dAdo/EHNA-induced cell death was significantly inhibited by NBTI, an inhibitor of nucleoside transport, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effects were not affected by 8-phenyltheophylline, a broad inhibitor of adenosine receptors. The Ado/EHNA combination brought about morphological changes consistent with apoptosis. Caspase-9 activation was observed in MCF-7 and MDA-MB468 human breast cancer cell lines on treatment with Ado/EHNA or dAdo/EHNA, but, as expected, caspase-3 activation was only observed in MDA-MB468 cells. The results of the study, thus, suggest that extracellular adenosine and deoxyadenosine induce apoptosis in both oestrogen receptor-positive (MCF-7) and also oestrogen receptor-negative (MDA-MB468) human breast cancer cells by its uptake into the cells and conversion to AMP (dAMP) followed by activation of nucleoside kinase, and finally by the activation of the mitochondrial/intrinsic apoptotic pathway.     

Forskolin and phosphodiesterase inhibitors release adenosine but inhibit morphine-evoked release of adenosine from spinal cord synaptosomes.

The effects of forskolin, Ro 20-1724, rolipram, and 3-isobutyl-1-methylxanthine (IBMX) on morphine-evoked release of adenosine from dorsal spinal cord synaptosomes were evaluated to examine the potential involvement of cyclic AMP in this action of morphine. Ro 20-1724 (1-100 microM), rolipram (1-100 microM), and forskolin (1-10 microM) increased basal release of adenosine, and at 1 microM inhibited morphine-evoked release of adenosine. Release of adenosine by Ro 20-1724, rolipram, and forskolin was reduced 42-77% in the presence of alpha,beta-methylene ADP and GMP, which inhibits ecto-5'-nucleotidase activity by 81%, indicating that this adenosine originated predominantly as nucleotide(s). Significant amounts of adenosine also were released from the ventral spinal cord by these agents. Ro 20-1724 and rolipram did not significantly alter the uptake of adenosine into synaptosomes. Although Ro 20-1724 and rolipram had only limited effects on the extrasynaptosomal conversion of added cyclic AMP to adenosine, IBMX, a phosphodiesterase inhibitor with a broader spectrum of inhibitory activity for phosphodiesterase isoenzymes, significantly inhibited the conversion of cyclic AMP to adenosine and resulted in recovery of a substantial amount of cyclic AMP. As with the non-xanthine phosphodiesterase inhibitors, IBMX increased basal release of adenosine and reduced morphine-evoked release of adenosine. Adenosine released by IBMX was reduced 70% in the presence of alpha,beta-methylene ADP and GMP, and release from the ventral spinal cord was 61% of that from the dorsal spinal cord. Collectively, these results indicate that forskolin and phosphodiesterase inhibitors release nucleotide(s) which is (are) converted extrasynaptosomally to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine-elicited accumulation of cyclic AMP in brain slices: potentiation by agents which inhibit uptake of adenosine

The uptake and incorporation of low concentrations of radioactive adenosine into guinea pig cerebral cortical slices is effectively inhibited by dipyridamole, hexobendine, papaverine, 6-($\text{p}$-nitrobenzylthio) guanosine, 5′-deoxy-adenosine and N⁶-phenyladenosine and ineffectively inhibited by other adenosine analogs such as 2-chloroadenosine, 3′-deoxyadenosine and tubercidin or by phosphodiesterase inhibitors such as theophylline, isobutylmethylxanthine, and N, 0-dibutyrylcyclic AMP. When uptake of 10–20

adenosine is inhibited 50–70% by dipyridamole, hexobendine, papaverine or 6-($\text{p}$-nitrobenzylthio)-guanosine, the adenosine-elicited accumulation of cyclic AMP is potentiated 2–3 fold. Potentiation of the effects of low concentrations of adenosine by various agents parallels more closely their efficacy as inhibitors of adenosine uptake rather than their potency as phosphodiesterase inhibitors. Amine-elicited accumulations of cyclic AMP are enhanced by hexobendine, dipyridamole, papaverine and 6-($\text{p}$-nitrobenzylthio) guanosine and this enhancement is blocked by an adenosine antagonist, theophylline. The stimulatory effects of the adenosine analogs, 5′-deoxyadenosine, 2-chloroadenosine and N⁶-phenyladenosine are blocked by theophylline and potentiated by hexobendine. The results are compatible with the hypothesis that the specific inhibition of uptake of adenosine potentiates adenosine or amine-elicited accumulations of cyclic AMP by increasing the effective extracellular concentration of adenosine within the slice. The inhibition or stimulation of cyclic AMP accumulation by adenosine analogs is consonant with differential activities as agonist or antagonist at an extracellular adenosine receptor.     

Further studies of the mechanism(s) of polyunsaturated-fatty-acid-mediated increases in intracellular cAMP formation in N1E-115 neuroblastoma cells

Following earlier observations that increasing the polyunsaturated fatty-acid (PUFA) content of N1E-115 neuroblastoma cells elevated basal and adenosine (Ado)-stimulated intracellular cyclic AMP (cAMP) formation, we carried out studies to determine the mechanism(s) by which PUFA exerted their modulatory effects. Basal increases in cAMP in the PUFA-enriched (PUFA⁺) cells were evident with short (60 sec) exposure to a phosphodiesterase inhibitor (Ro 20-1724), and increased to a maximum at 20 min; they were not observed in the absence of Ro 20-1724. Forskolinstimulated cAMP formation in the presence of the Ro compound was 2- to 3-fold higher in the PUFA⁺ cells. Basal elevations in cAMP were reduced by 70% by exposing the PUFA⁺ cells to Ado deaminase (ADA) or to an Ado antagonist, and were further increased by inhibiting ADA, which suggested that they could be producing endogenous Ado that activated stimulatory Ado receptors. However, this did not appear to involve PUFA-mediated stimulation of 5-nucleotidase activity or inhibition of [³H]Ado uptake. Overall, the results of this study indicated that multiple mechanisms are involved in PUFA modulation of cAMP formation.Abbreviations used PUFA polyunsaturated fatty acid(s) - ACase adenylate cyclase - PDE phosphodiesterase - ADA adenosine deaminase - 2-DCF 2-deoxycoformycin - 8-PT 8-phenyltheophylline - DPR dipyridamole - CPA cyclopentyladenosine - DMEM Dulbecco's modified Eagle's medium - Ro 20-1724 4-(3-butoxy-4-methoxybenzyl) imiazolidin-2-one - BSA bovine serum albumin     

Differential cardioprotection with selective inhibitors of adenosine metabolism and transport: Role of purine release in ischemic and reperfusion injury

In a previous report, we have demonstrated that simultaneous inhibition of nucleoside transport and adenosine deaminase accumulates endogenous adenosine and protects the myocardium against stunning. The differential cardioprotective effects of erythro-9(2-hydroxy-3-nonyl)-adenine (EHNA), a potent inhibitor of adenosine deamination but not transport, and p-nitrobenzylthioinosine (NBMPR), a selective blocker of adenosine and inosine transport, are not known.Thirty-seven anaesthetized adult dogs were instrumented to monitor left ventricular performance using sonomicrometery. Dogs were randomly assigned into four groups. The control group (n = 8) received only the vehicle solution. Treated groups received saline containing 100 M EHNA (EHNA-group, n = 7), 25 M NBMPR (NBMPR-group, n = 7), or a combination of 100 M EHNA and 25 M NBMPR (EHNA/NBMPR-group, n = 10). Hearts were subjected to 30 min of normothermic global ischaemia and 60 min of reperfusion while on bypass. Adenine nucleotides, nucleosides, oxypurines and NAD⁺ were determined in extracts of transmural myocardial biopsies using HPLC. TTC staining revealed the absence of necrosis in this model.Drug administration did not affect myocardial ATP metabolism and cardiac function in the normal myocardium. Ischemia caused about 50% ATP depletion and accumulation of nucleosides. The ratio between adenosine/inosine at the end of ischemia was 1:10, 1:1, 1:1 and 10:1 in the control, EHNA-, NBMPR- and EHNA/NBMPR-group, respectively. Upon reperfusion, both nucleosides washed out from the myocardium in the control and EHNA-group while retained in the myocardium in the NBMPR and EHNA/NBMPR groups. Ventricular dysfunction 'stunning' persisted in the control group (52%) and in the EHNA-treated group (32%) after 30 min of reperfusion. Significant improvement of function was observed in the EHNA group only after 60 min of reperfusion. LV function recovered in the NBMPR- and EHNA/NBMPR-treated groups during reperfusion. ATP recovery occurred only when animals were pretreated with the combination of EHNA/NBMPR and remained depressed in the control group and EHNA and NBMPR-treated groups. At post mortem, TTC staining revealed the absence of myocardial necrosis.Superior myocardial protection was observed with inhibition of nucleoside transport by NBMPR alone or in combination with inhibition of adenosine deaminase by EHNA. Selective blockade of nucleoside transport by NBMPR is more cardioprotective than inhibition of adenosine deaminase alone in attenuating myocardial stunning. It is not known why EHNA partially inhibit adenosine deaminase, in vivo.     

Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex.

Adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) metabolism in rat renal cortex was examined. Athough the cyclic AMP and cyclic GMP phosphodiesterases are similarly distributed between the soluble and particulate fractions following differential centrifugation, their susceptibility to inhibition by theophylline, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 1-methyl-3-isobutylxanthine (MIX) are quite different. Ro 20-1724 selectively inhibited both renal cortical-soluble and particulate cyclic AMP degradation, but had little effect on cyclic GMP hydrolysis. Theophylline and MIX effectively inhibited degradation of both cyclic nucleotides, with MIX the more potent inhibitor. Effects of these agents on the cyclic AMP and cyclic GMP content of cortical slices corresponded to their relative potency in broken cell preparations. Thus, in cortical slices, Ro 20-1724 (2 mm) had the least effect on basal (without agonist), carbamylcholine, and NaN₃-stimulated cyclic GMP accumulation, but markedly increased basal and (parathyroid hormone) PTH-mediated cyclic AMP accumulation, MIX (2 mm) which was as effective as Ro 20-1724 in potentiating basal and PTH-stimulated increases in cyclic AMP also mediated the greatest augmentation of basal, carbamylcholine, and NaN₃-stimulated accumulation of cyclic GMP. By contrast, theophylline (10 mm) which was only 12% as effective as Ro 20-1724 in increasing the total slice cyclic AMP content in the presence of PTH was much more effective than Ro 20-1724 in potentiating carbamylcholine and NaN₃-mediated increases in cyclic GMP. These results demonstrate selective inhibition of cyclic nucleotide phosphodiesterase activities in the rat renal cortex and support the possibility of multiple cyclic nucleotide phosphodiesterases in this tissue. Furthermore, both cyclic nucleotides appear to be rapidly degraded in the renal cortex.     

Role of nucleoside transport and purine release in a rabbit model of myocardial stunning

Previously, we have demonstrated the role of nucleoside transport and purine release in post-ischemic reperfusion injury (myocardial stunning) in several canine models of ischemia. Since rabbits are deficient of xanthine oxidase, it is not known whether selective blockade of purine release is beneficial in a rabbit model of coronary artery occlusion and reperfusion (stunning). Therefore, we determined the hemodynamic and metabolic correlates in response to myocardial stunning in the presence or absence of selective nucleoside transport blocker (p-nitrobenzylthioinosine, NBMPR) and adenosine deaminase inhibitor (erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA).Sixty adult anaesthetized rabbits were surgically prepared for hemodynamic measurements. After stabilization period, the left anterior descending coronary artery was occluded for 15 min and reperfused for 30 min. Transmural myocardial biopsies were obtained from the ischemic LAD area and from the non-ischemic posterior (circumflex, CFX) segment of the myocardium.Rabbits (n = 60) were randomly assigned to either the control or the EHNA/NBMPR-treated group (n = 30 each). Each group was further divided to either functional or metabolic groups (n = 15 each subgroup). Each animal received intravenously 30 ml of either a vehicle solution or 100 M EHNA and 25 M NBMPR 10 min before ischemia.Although administration of EHNA/NBMPR did not affect the heart rate, it did cause mild hypotension (about 20-30%). Fifteen minutes of LAD occlusion resulted in significant ATP depletion and concomitant accumulation of nucleosides in both groups (p < 0.05 vs. baseline and non-ischemic CFX segment). AMP was higher in the LAD compared to the CFX segment. Significant accumulation of adenosine was observed in the treated group compared to the control group.It is concluded that EHNA/NBMPR induced site specific entrapment of adenosine of nucleoside transport in the rabbit heart, in vivo.     

Activation of cyclic AMP-dependent protein kinase and stimulation of protein phosphorylation in response to adenosine in C-1300 murine neuroblastoma

DEAE-cellulose chromatography of the 20,000g supernatant fraction of homogenates of C-1300 murine neuroblastoma (clone N2a) yields one major and two minor peaks of cyclic AMP-dependent protein kinase activity. Assessment of the endogenous activation state of the enzyme(s) reveals that the enzyme is fully activated by the treatment of whole cells with adenosine (10 μM) in the presence of the phosphodiesterase inhibitor Ro 20 1724 (0.7 mM). This treatment produces a large elevation in the cyclic AMP content of the cells. The treatment of whole cells with adenosine alone (1–100 μM) or Ro 20 1724 alone (0.1–0.7 mM) produces minimal elevations in cyclic AMP but nevertheless causes significant activations of cyclic AMP-dependent protein kinase. The autophosphorylation of whole homogenates of treated and untreated cells was studied using [γ-³²P] ATP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Treatments which activate cyclic AMP-dependent protein kinase selectively stimulate the incorporation of ³²P into several proteins. This stimulation is most prominent in the 15,000-dalton protein band. The addition of cyclic AMP to phosphorylation reactions containing homogenate of untreated cells stimulates the phosphorylation of the same protein bands. These results indicate that adenosine may have regulatory functions through its effect on the cyclic AMP: cyclic AMP-dependent protein kinase system.     

Adenosine-Elicited Accumulation of Adenosine 3'', 5''-Cyclic Monophosphate in the Chick Embryo Retina

The cyclic AMP level of 17-day-old chick embryo retina increased from 20 to 331 pmol/mg protein when the tissue was incubated for 20 min in the presence of 4-(3-butoxy-4-methoxybenzyl-2-imidozolinone) (RO 20-1724). The addition of 0.5 mM-3-isobutyl-1-methylxanthine (IBMX) or 0.5 units/ml of adenosine deaminase (EC 3.5.4.4) to the medium reduced the increase of cyclic AMP content from 20 to 100 pmol/mg protein. Dipyridamole did not interfere with the rise of the retinal cyclic AMP level observed with RO 20-1724. The EC50 of 6-amino-2-chloropurine riboside (2-chloroadenosine)-elicited accumulation of cyclic AMP of retinas incubated in the presence of RO 20-1724 plus adenosine deaminase was approximately 1 microM. When retina incubation was carried out in the presence of 0.5 mM-IBMX, the 2-chloroadenosine dose-response curve was shifted to the right two orders of magnitude. Maximal stimulation of the cyclic AMP level of 17-day-old chick embryo retina incubated in the presence of 0.5 mM-IBMX was observed at 1 mM-adenosine concentration. This effect was not blocked by dopamine antagonists. Guanosine and adenine did not affect the retinal cyclic AMP level. AMP and ATP had a slight stimulatory effect. Adenosine response of embryonic retina increased sharply from the 14th to the 17th embryonic day. A similar, but not identical adenosine effect was observed in cultured retina cells.     

Change of Cyclic AMP Level in Synaptosomes from Cerebral Cortex; Increase by Adenosine Derivatives

The endogenous level of cyclic AMP in incubated synaptosomes from cerebral cortex of guinea pigs was investigated after the addition of various agents to the incubation medium. It appeared that the synaptosomal suspension already contained exogenous adenosine. Preincubation with theophylline or with adenosine deaminase (ADase) decreased both the exogenous level of adenosine and the intrasynaptosomal level of cyclic AMP. The level of cyclic AMP was reincreased by the addition of adenosine agonists, especially 2-chloroadenosine. This increase was antagonized by deoxyadenosine and was not inhibited by dipyridamole. These results suggest that the adenosine derivatives in the synaptic cleft regulate the level of cyclic AMP in nerve terminals through adenosine receptor on the presynaptic membrane. ADP, ATP, dopamine, and histamine also stimulate the formation of cyclic AMP in the ADase-treated synaptosomes.     

Chemotactic peptide induces cAMP elevation in human neutrophils by amplification of the adenylate cyclase response to endogenously produced adenosine

The transient increase in human neutrophil cAMP levels induced by the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) is shown to be caused by amplification of adenylate cyclase response to endogenously produced adenosine. The FMLP-stimulated increase in neutrophil cAMP was potentiated markedly by a nonmethylxanthine cAMP phosphodiesterase inhibitor (Ro 20-1724). By inhibiting the degradation of newly formed cAMP, Ro 20-1724 rendered the FMLP-induced cAMP elevation persistent rather than transient. The role of endogenously produced adenosine in this phenomenon is demonstrated by the ability of either adenosine deaminase or theophylline, an adenosine receptor antagonist, to prevent FMLP-stimulated cAMP elevation. The general nature of the FMLP-potentiated cAMP response is indicated by the finding that FMLP-treated neutrophils, in the presence of exogenously supplied adenosine deaminase, exhibited augmented cAMP generation in response to three different types of receptor agonists: 2-chloroadenosine, prostaglandin E1, and L-isoproterenol. Moreover, like the neutrophil cAMP increase caused by FMLP alone, the ability of FMLP to augment cAMP response to 2-chloroadenosine in adenosine deaminase-treated cells was short-lived and declined after 1.0 min of exposure to FMLP. Preincubation of neutrophil suspensions with the adenylate cyclase inhibitor SQ 22,536 completely prevented FMLP-induced cAMP generation. Furthermore, when neutrophil suspensions were preincubated with concentrations of Ro 20-1724, which apparently maximally inhibit cAMP phosphodiesterase, a 30-s incubation with FMLP still resulted in substantially elevated cAMP levels. It therefore appears that FMLP raises cAMP by activating adenylate cyclase rather than inhibiting cAMP phosphodiesterase.     

Adenosine deaminase 1 and concentrative nucleoside transporters 2 and 3 regulate adenosine on the apical surface of human airway epithelia: implications for inflammatory lung diseases

Adenosine is a multifaceted signaling molecule mediating key aspects of innate and immune lung defenses. However, abnormally high airway adenosine levels exacerbate inflammatory lung diseases. This study identifies the mechanisms regulating adenosine elimination from the apical surface of human airway epithelia. Experiments conducted on polarized primary cultures of nasal and bronchial epithelial cells showed that extracellular adenosine is eliminated by surface metabolism and cellular uptake. The conversion of adenosine to inosine was completely inhibited by the adenosine deaminase 1 (ADA1) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The reaction exhibited Km and Vmax values of 24 microM and 0.14 nmol x min(-1) x cm(-2). ADA1 (not ADA2) mRNA was detected in human airway epithelia. The adenosine/mannitol permeability coefficient ratio (18/1) indicated a minor contribution of paracellular absorption. Adenosine uptake was Na+-dependent and was inhibited by the concentrative nucleoside transporter (CNT) blocker phloridzin but not by the equilibrative nucleoside transporter (ENT) blocker dipyridamole. Apparent Km and Vmax values were 17 microM and 7.2 nmol x min(-1) x cm(-2), and transport selectivity was adenosine = inosine = uridine > guanosine = cytidine > thymidine. CNT3 mRNA was detected throughout the airways, while CNT2 was restricted to nasal epithelia. Inhibition of adenosine elimination by EHNA or phloridzin raised apical adenosine levels by >3-fold and stimulated IL-13 and MCP-1 secretion by 6-fold. These responses were reproduced by the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and blocked by the adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline (8-SPT). This study shows that adenosine elimination on human airway epithelia is mediated by ADA1, CNT2, and CNT3, which constitute important regulators of adenosine-mediated inflammation.     

Characterization of Inhibitor-Sensitive and -Resistant Adenosine Transporters in Cultured Human Fetal Astrocytes

Abstract: The kinetic characteristics of [³H]adenosine uptake, the extent to which accumulated [³H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis-Menten kinetic values of K_T and V_max, and the sensitivities with which nucleoside transport inhibitors blocked [³H]adenosine accumulations were determined in cultured human fetal astrocytes. K_T and V_max values for accumulations of [³H]-labeled purines using 15-s incubations in the absence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and the adenosine kinase inhibitor 5′-iodotubercidin (ITU) were 6.2 µM and 0.15 nmol/min/mg of protein for the high-affinity and 2.6 mM and 21 nmol/min/mg of protein for the low-affinity components respectively. In the presence of EHNA and ITU, where <4% of accumulated [³H]adenosine was metabolized, transport per se was measured, and kinetic values for K_T and V_max were 179 µM and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [³H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of “concentrative” uptake in that the intracellular levels of [³H]-labeled purines (adenosine plus its metabolites) were 1.4-fold higher than in the medium. No apparent concentrative accumulations of [³H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [³H]adenosine transport; for the inhibitor-sensitive components the IC₅₀ values were 0.7 nM for NBI, 1.3 nM for DPR, and 3.3 nM for dilazep, and for the inhibitor-resistant component the IC₅₀ values were 2.5 µM for NBI, 5.1 µM for dilazep, and 39.0 µM for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor-sensitive and -resistant adenosine transporters in nontransformed human cells.