Adenosine formation and release from neonatal-rat heart cells in culture.

The incorporation of [3H]adenosine (10 microM) into neonatal-rat heart cell nucleotides was inhibited in a concentration-dependent manner, such that 50% inhibition was obtained with 0.75 microM-dipyridamole, 0.26 microM-hexobendine or 0.22 microM-dilazep. Adenosine formation was accelerated 2.5-fold to 2.1 +/- 0.3 nmol/10(7) cells in 10 min when cells were incubated with a combination of 30 mM-2-deoxyglucose and 2 micrograms of oligomycin/ml. Of the newly formed adenosine, 6 +/- 2% was in the cells. Dipyridamole, hexobendine or dilazep (10 microM) increased the amount of adenosine in the cells and decreased that in the medium such that 45-50% of the newly formed adenosine was in the cells. Antibodies which inhibited ecto-5'-nucleotidase by 98.7 +/- 0.3% did not alter the rate of adenosine formation or its distribution between cells and medium. We conclude that adenosine was formed in the cytoplasm during catabolism of cellular ATP and was released via the dipyridamole-sensitive symmetric nucleoside transporter.     

Adenosine Formation and Release by Embryonic Chick Neurons and Glia in Cell Culture

Adenosine formation and release were studied in 48-h-old cultured ciliary ganglia and confluent peripheral and CNS glial cultures from embryonic chicks. Metabolic poisoning induced by 30 mM 2-deoxyglucose and 2 micrograms/ml oligomycin reduced ATP concentration by 90%. An increase in adenosine accounted for 15-40% of the fall in ATP. Dilazep (3 X 10(-6) M), a nucleoside transport inhibitor, decreased both incorporation of adenosine (an index of nucleoside transport) and release of adenosine by 80-90%. Dilazep trapped the newly formed adenosine intracellularly. A concentration of alpha, beta-methylene ADP that inhibited ecto-5'-nucleotidase by 80-90% did not alter the concentration of adenosine or AMP in neurons, glia, or medium. The results demonstrate that adenosine is formed intracellularly and exported out of the cell via the nucleoside transporter. The participation of ecto-5'-nucleotidase was excluded.     

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.     

Stimulus- and cell-type-specific release of purines in cultured rat forebrain astrocytes and neurons

Adenosine is formed during conditions that deplete ATP, such as ischemia. Adenosine deaminase converts adenosine into inosine, and both adenosine and inosine can be beneficial for postischemic recovery. This study investigated adenosine and inosine release from astrocytes and neurons during chemical hypoxia or oxygen-glucose deprivation. In both cell types, 2-deoxyglucose was the most effective stimulus for depleting cellular ATP and for evoking inosine release; in contrast, oxygen-glucose deprivation evoked the greatest adenosine release. alpha,beta-Methylene ADP, an inhibitor of ecto-5'nucleotidase, significantly reduced adenosine release from astrocytes but not neurons. Dipyridamole, an inhibitor of equilibrative nucleoside transporters, inhibited both adenosine and inosine release from neurons. Erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase, reduced neuronal inosine release evoked by oxygen-glucose deprivation but not by 2-deoxyglucose treatment. These data indicate that (1). astrocytes release adenine nucleotides that are hydrolyzed extracellularly to adenosine, whereas neurons release adenosine per se, (2). inosine is formed intracellularly and released via nucleoside transporters, and (3). inosine is formed by an adenosine deaminase-dependent pathway during oxygen-glucose deprivation but not during 2-deoxyglucose treatment. In summary, the metabolic pathways for adenosine formation and release were cell-type dependent whereas the pathways for inosine formation were stimulus dependent.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

Uptake of Adenosine by Isolated Rat Brain Capillaries

Abstract: Adenosine uptake by isolated rat brain capillaries is a carrier-mediated, temperature- and pH-sensitive process. The K _m value for adenosine uptake is 4.74 μ m and the V _max is 21.7 picomol/mg protein/10 min. This is a high-affinity uptake system that can be cross-inhibited by several nucleosides and by the adenosine analogs tubercidin and 5'-deoxyadenosine. The uptake is very sensitive to inhibition by papaverine, hexobendine, and dipyridamole. These results confirm the existence of a nucleoside transport system associated with the blood-brain barrier observed during in vivo studies.     

Adenosine Transport by Primary Cultures of Neurons from Chick Embryo Brain

Abstract: The transport of adenosine was studied in pure cultures of neurons from chick embryo brain. In order to avoid complications due to adenosine metabolism, the cells were depleted of ATP by treatment with cyanide and iodoacetate prior to incubation with [³H]adenosine. During the 5-25-s periods used for transport assays, no significant adenosine metabolism was detectable. ATP depletion reduced the initial rate of adenosine entry by less than 10%, but blocked over 90% of the radioactivity accumulated by untreated cells after 15 min. Elimination of sodium or chloride from the uptake medium had no effect on adenosine transport activity. The kinetics of adenosine entry into ATP depleted neurons obeyed the Michaelis-Menten relationship and yielded a K_m of 13 μM and V_max of 0.15 nmol/min/mg protein. The neuronal transport system has apparent selectivity for adenosine, since thymidine, inosine, or guanosine gave significant inhibition only at levels 10-100-fold higher than [³H]adenosine. Adenosine derivatives ( N ⁶-cyclohexyl-, N⁶-benzyl-, N⁶-methyl-, and 2-chloroadenosine) were more effective inhibitors; p -nitrobenzylthioinosine and dipyridamole were the most potent compounds found. These results describe a high-affinity, facilitated diffusion system for adenosine in cerebral neurons, which could participate in terminating regulatory actions of this compound in the nervous system.     

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [³H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with K_m = 370 μM and V_max = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na⁺ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (K_m = 12 μM; V_max = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N⁶-benzyladenosine. In decreasing order of potency, N⁶-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.     

Metabolic and functional consequences of cytosolic 5'-nucleotidase-IA overexpression in neonatal rat cardiomyocytes

Adenosine exerts a spectrum of energy-preserving actions on the heart negative chronotropic effects. The pathways leading to adenosine formation have remained controversial. In particular, although cytosolic 5'-nucleotidases can catalyze adenosine formation in cardiomyocytes, their contribution to the actions of adenosine has not been documented previously. We recently cloned two closely related AMP-preferring cytosolic 5'-nucleotidases (cN-IA and -IB); the A form predominates in the heart. In this study, we overexpressed pigeon cN-IA in neonatal rat cardiomyocytes using an adenovirus. cN-IA overexpression increased adenosine formation and release into the medium caused by simulated hypoxia and by isoproterenol in the absence and presence of inhibitors of adenosine metabolism. Adenosine release was not affected by an ecto-5'-nucleotidase inhibitor, alpha,beta-methylene-ADP, but was affected by a nucleoside transporter, dipyridamole. The positive chronotropic effect of isoproterenol (130 +/-3 vs. 100 +/-4 beats/min) was inhibited (107 +/-3 vs. 94 +/-3 beats/min) in cells overexpressing cN-IA, and this was reversed by the addition of the adenosine receptor antagonist 8-(p-sulfophenyl)theophilline (120 +/- 3 vs. 90 +/- 4 beats/min). Our results demonstrate that overexpressed cN-IA can be sufficiently active in cardiomyocytes to generate physiologically effective concentrations of adenosine at its receptors.     

Synthesis of adenine nucleotides from exogenous adenosine in the perfused rat heart under normoxic conditions and after ischemia

The rate of synthesis of myocardial adenine nucleotides from exogenous adenosine was studied in the isolated rat heart perfused under normoxic conditions and following ischaemia. The rate of incorporation of adenosine depended on the extracellular concentration of the precursor, following Michaelis-Menten kinetics with a apparent Km of 51.3 microM and a maximal rate of incorporation of about 1 100 nmol g-1 (wet wt.) 30 min-1. The adenosine uptake induced an increase in ATP concentration (+ 20%) when the exogenous concentration of precursor exceeded 10 microM. Following low-flow ischaemia (0.5 ml/min, 30 min), the rate of incorporation of 5 microM adenosine was diminished (-23%), but adenine nucleotide level restoration was favoured by the nucleoside administration. After total ischaemia (24 min), the extent of the decrease in adenosine incorporation was the same as in the case of moderate ischaemia but adenine nucleotide content was not restored.     

An examination of the efficiency of glucose and glutamine as energy sources for cultured chick pigment epithelial cells

Embryonic chick pigment epithelial cells in culture require glucose as their major energy source for long-term growth, pigment formation, and colony organization. Cell number increases with glucose concentration at least up to 5.0 mM. Cells can be grown with glutamine as the major energy source but produce comparable cell numbers for only the first 3 days in culture, after which they cease growing. However, they are able to metabolize glutamine at a two to sixfoid higher rate than cells grown in the presence of glucose as measured by CO₂ release and by incorporation into protein. In cells grown in the presence of both glucose and glutamine, basal ATP levels were 31.1 nmoles/mg protein; P-creatine averaged 15.2 nmoles/mg protein and showed marked variability between experimental groups. During starvation, P-creatine levels fell while ATP levels remained relatively constant. Glucose was required for the recovery of P-creatine to prestarvation levels when measured 5 min after refeeding. Because of these marked changes in P-creatine concentration as a function of nutritional status, the ATP/P-creatine ratio becomes a useful measure of the energy state of the cell.     

Measurement of rat plasma adenosine levels during normoxia and hypoxia.

A stop solution containing EDTA, EGTA, dipyridamole, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and d,l-alpha-glycerophosphate has been used to prevent adenosine formation and loss from rat femoral arterial blood samples prepared for measurement of plasma adenosine levels. The femoral arterial plasma adenosine concentration in normoxic rats was 79.2 +/- 12.7 nM. During a 5 min period of hypoxia (8% oxygen) plasma adenosine increased to 190.2 +/- 32.2 nM. A resting plasma adenosine level of circa 80 nM, which is 10X lower than most previous estimates, approximates the threshold levels of adenosine required for arterial dilation.     

Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture.

1. Adenosine, a potent vasodilator, is transported very efficiently by pig aortic endothelium in monolayer culture (approx. 50pmol/min per 10(6) cells at 2 micrometer). Uptake proceeds by diffusion at high (millimolar) substrate concentrations, and by two discrete transport processes (Km approx. 3 micrometer and 250 micrometer) at lower concentrations. Over 90% of the adenosine taken up at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides (mainly ATP). 2. The high-affinity process is selectively inhibited by dipyridamole and by nitrobenzylthioinosine. Adenine preferentially inhibits the lower-affinity process, papapaverine inhibits both transport processes, and inosine has no significant effect. 3. Pig aortic smooth-muscle cells in culture show no high-affinity transport system for adenosine; uptake is much slower at low concentrations than that by endothelium (approx. 5pmol/min per 10(6) cells at 2 micrometer). Over 80% of the incorporated adenosine at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides. 4. The uptake of adenosine by smooth-muscle cells is powerfully inhibited by adenine, but dipyridamole is much less potent than in endothelium. 5. We conclude that endothelial cells are mainly responsible for the removal of circulating adenosine.     

Effects of nucleoside transport inhibitors and adenine/ribose supply on ATP concentration and adenosine production in cardiac myocytes

Adenosine plays an important role in protection of the heart before, during and after ischemia. Nucleoside transport inhibitors (NTI) increase adenosine concentration without inducing ischemia by preventing its uptake and metabolism in cardiac cells. However, prolonged effects of nucleoside transport inhibitors on adenosine and nucleotide metabolism and its combined effect with nucleotide precursors has not been established in cardiomyocytes. The aim of this study was to investigate the effect of two nucleoside transport inhibitors, dipyridamole (DIPY) and nitrobenzylthioinosine (NBTI) alone or combined with adenine and ribose on adenosine production and ATP content in cardiomyocytes.Rat cardiomyocytes were isolated using collagenase perfusion technique. Isolated cell suspensions were incubated for up to 480 min with different substrates and inhibitors as follows: (1) control; (2) 100 M adenine and 2.5 mM ribose; (3) 10 M DIPY; (4) 1 M NBTI; (5) DIPY, adenine and ribose and (6) NBTI, adenine and ribose. Five M EHNA (erythro-9(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase) was added to all incubations. After incubation, extracts of myocyte suspension were analysed by HPLC for adenine nucleotides and metabolite concentrations.ATP content decreased in cardiomyocytes after 8 h of incubation with DIPY, while no change was observed with NBTI or without inhibitors. Adenosine concentration increased with both DIPY and NBTI. In the presence of adenine and ribose an elevation in ATP concentration was observed, but no significant change in adenosine content. In the presence of DIPY or NBTI together with adenine and ribose, an enhancement in cardiomyocyte ATP concentration was observed together with an increase in adenosine content. This increase in adenosine production was especially prominent with DIPY.In conclusion, dipyridamole causes a decrease in ATP concentration in isolated cardiomyocytes by mechanisms other than nucleoside transport inhibition. Addition of adenine/ribose with dipyridamole prevents the depletion of ATP. Combination of adenine/ribose with nucleoside transport inhibitors may also further enhance adenosine concentration and thus, could be more effective as pharmacological agents for treatment.     

Role of adenosine in hypoxic ventilatory depression

The role of adenosine in the ventilatory depression induced by hypoxia was studied in 82 spontaneously breathing urethan-anesthetized 4-day-old rabbit pups. Respiration was monitored with a pneumotachograph. The animals were exposed to hypoxia (6% O2 in N2) for 30 min or until the occurrence of terminal apnea. In all animals hypoxia produced an initial increase in ventilation followed by a decrease. In the control group 52% of the animals became apneic after 7 min of hypoxic exposure. By contrast, pretreatment with dipyridamole (10 or 20 mg/kg), an adenosine uptake blocker, significantly shortened the time needed to reach apnea. Thus at 7 min of hypoxia 93% of the animals that received dipyridamole became apneic. On the other hand, administration of adenosine antagonists 8-p-sulfophenyltheophylline (5 or 8 mg/kg) and aminophylline (10 or 25 mg/kg) significantly prolonged the time required to produce apnea. Only 20% of the animals that received these antagonists became apneic at 7 min of hypoxia. These results suggest that adenosine is potentially involved in the ventilatory depression produced by hypoxia in neonatal rabbit pups.     

Mycoplasma contamination greatly enhances the apparent transport and concentrative accumulation of formycin B by mammalian cell culture

S49 mouse leukemia cells exhibit both equilibrative and Na(+)-dependent, concentrative formycin B transport. The latter represents only a minor nucleoside transport component and is detectable only when equilibrative nucleoside transport is inhibited by dipyridamole or another transport inhibitor. Thus in uncontaminated S49 cells formycin B accumulated only to slightly above the intracellular-extracellular equilibrium level. In contrast, in suspensions of S49 cells contaminated with mycoplasma, formycin B accumulated in the intracellular water space in unmodified form to 40-50-times the extracellular concentration in a dipyridamole-independent manner during 90 min of incubation at 37 degrees C. The mycoplasma active formycin B transport system was inhibited by all nucleosides tested, including thymidine and deoxycytidine, which are not substrates for the concentrative nucleoside transporter of S49 cells. Mycoplasma contamination was detected by the presence of cell-associated adenosine phosphorylase activity.     

Hypoxia induces adenosine release from the rat carotid body

The effect of hypoxia on the release of adenosine was studied in vitro in the rat whole carotid body (CB) and compared with the effect of hypoxia (2%, 5% and 10% O(2)) on adenosine concentrations in superior cervical ganglia (SCG) and carotid arteries. Moderate hypoxia (10% O(2)) increased adenosine concentrations released from the CBs by 44%, but was not a strong enough stimulus to evoke adenosine release from SCG and arterial tissue. The extracellular pathways of adenosine production in rat CBs in normoxia and hypoxia were also investigated. S-(p-nitrobenzyl)-6-thioinosine (NBTI) and dipyridamole were used as pharmacological tools to inhibit adenosine equilibrative transporters (ENT) and alpha,beta-methylene ADP (AOPCP) to inhibit ecto-5'-nucleotidase. Approximately 40% of extracellular adenosine in the CB came from the extracellular catabolism of ATP, under both normoxic and hypoxic conditions. Low pO(2) triggers adenosine efflux through activation of NBTI-sensitive ENT. This effect was only apparent in hypoxia and when adenosine extracellular concentrations were reduced by the blockade of ecto-5'-nucleotidase. We concluded that CB chemoreceptor sensitivity could be related to its low threshold for the release of adenosine in response to hypoxia here quantified for the first time.     

Insulin stimulation of glucose entry in cultured human fibroblasts.

The effect of insulin on glucose entry has been studied in monolayer cultures of human diploid fibroblastic cells. Influence of insulin on total cell glucose incorporation was evaluated using [14C] glucose. Glucose incorporation was increased up to two-fold in the presence of insulin. Insulin action occurred within 30 minutes and could be observed with insulin concentrations as low as 10(-10) M (10 microU)ml). The action of insulin was enhanced by preincubation in glucose-free medium. After glucose starvation the cells converted glucose primarily to glycogen and nucleotides, and the stimulation by insulin was observed equally in both fractions. Influence of insulin on the kinetics of hexose transport was studied using 2-deoxyglucose and 3-0-methyl glucose. A large diffusion component was corrected using rho-chloromercuribenzoic acid or phloridzin. Km for facilitated diffusion averaged 1.9 mM for 2-deoxyglucose and 5.3 mM for 3-O-methyl glucose, and Vmax ranged from 10-24 nmoles/min/mg cell protein. Insulin resulted in a 150% increase in Vmax with no significant change in Km. The data suggest that human diploid fibroblasts can be a useful system for the study of insulin's glucoregulatory action.     

Inhibition of hippocampal synaptic activity by ATP, hypoxia or oxygen-glucose deprivation does not require CD73

Adenosine, through activation of its A(1) receptors, has neuroprotective effects during hypoxia and ischemia. Recently, using transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1), we reported that nucleoside transporter-mediated release of adenosine from neurons was not a key mechanism facilitating the actions of adenosine at A(1) receptors during hypoxia/ischemia. The present study was performed to test the importance of CD73 (ecto-5'-nucleotidase) for basal and hypoxic/ischemic adenosine production. Hippocampal slice electrophysiology was performed with CD73(+/+) and CD73(-/-) mice. Adenosine and ATP had similar inhibitory effects in both genotypes, with IC(50) values of approximately 25 μM. In contrast, ATP was a less potent inhibitor (IC(50) = 100 μM) in slices from mice expressing hENT1 in neurons. The inhibitory effects of ATP in CD73(+/+) and CD73(-/-) slices were blocked by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and were enhanced by the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI), consistent with effects that are mediated by adenosine after metabolism of ATP. AMP showed a similar inhibitory effect to ATP and adenosine, indicating that the response to ATP was not mediated by P2 receptors. In comparing CD73(-/-) and CD73(+/+) slices, hypoxia and oxygen-glucose deprivation produced similar depression of synaptic transmission in both genotypes. An inhibitor of tissue non-specific alkaline phosphatase (TNAP) was found to attenuate the inhibitory effects of AMP and ATP, increase basal synaptic activity and reduce responses to oxygen-glucose deprivation selectively in slices from CD73(-/-) mice. These results do not support an important role for CD73 in the formation of adenosine in the CA1 area of the hippocampus during basal, hypoxic or ischemic conditions, but instead point to TNAP as a potential source of extracellular adenosine when CD73 is absent.