Adenosine production and its interaction with protection of ischemic and reperfusion injury of the myocardium

Adenosine exerts cardioprotective effects on the ischemic myocardium. A flexibly mounted microdialysis probe was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production) in in vivo rat hearts. The level of adenosine during perfusion of adenosine 5'-adenosine monophosphate (AMP) was given as an index of the activity of ecto-5'-nucleotidase in the tissue. Endogenous norepinephrine (NE) activates both alpha(1)-adrenoceptors and protein kinase C (PKC), which, in turn, activates ecto-5'-nucleotidase via phosphorylation thereby enhancing the production of interstitial adenosine. Histamine-release NE activates PKC, which increased ecto-5'-nucleotidase activity and augmented release of adenosine. Opening of cardiac ATP sensitive K(+) (K(ATP)) channels may cause hydroxyl radical (.OH) generation through NE release. Lysophosphatidylcholine (LPC), an endogenous amphiphiphilic lipid metabolite, also increases the concentration of interstitial adenosine in rat hearts, through the PKC-mediated activation of endogenous ecto-5'-nucleotidase. Nitric oxide (NO) facilitates the production of interstitial adenosine, via guanosine 3',5'-cyclic monophosphate (cGMP)-mediated activation of ecto-5'-nucleotidase as another pathway. These mechanisms play an important role in high sensitivity of the cardiac adenosine system. Adenosine plays an important role as a modulator of ischemic reperfusion injury, and that the production and mechanism of action of adenosine are linked with NE release.     

2',3'-cAMP, 3'-AMP, and 2'-AMP inhibit human aortic and coronary vascular smooth muscle cell proliferation via A2B receptors

Rat vascular smooth muscle cells (VSMCs) from renal microvessels metabolize 2',3'-cAMP to 2'-AMP and 3'-AMP, and these AMPs are converted to adenosine that inhibits microvascular VSMC proliferation via A(2B) receptors. The goal of this study was to test whether this mechanism also exists in VSMCs from conduit arteries and whether it is similarly expressed in human vs. rat VSMCs. Incubation of rat and human aortic VSMCs with 2',3'-cAMP concentration-dependently increased levels of 2'-AMP and 3'-AMP in the medium, with a similar absolute increase in 2'-AMP vs. 3'-AMP. In contrast, in human coronary VSMCs, 2',3'-cAMP increased 2'-AMP levels yet had little effect on 3'-AMP levels. In all cell types, 2',3'-cAMP increased levels of adenosine, but not 5'-AMP, and 2',3'-AMP inhibited cell proliferation. Antagonism of A(2B) receptors (MRS-1754), but not A(1) (1,3-dipropyl-8-cyclopentylxanthine), A(2A) (SCH-58261), or A(3) (VUF-5574) receptors, attenuated the antiproliferative effects of 2',3'-cAMP. In all cell types, 2'-AMP, 3'-AMP, and 5'-AMP increased adenosine levels, and inhibition of ecto-5'-nucleotidase blocked this effect of 5'-AMP but not that of 2'-AMP nor 3'-AMP. Also, 2'-AMP, 3'-AMP, and 5'-AMP, like 2',3'-cAMP, exerted antiproliferative effects that were abolished by antagonism of A(2B) receptors with MRS-1754. In conclusion, VSMCs from conduit arteries metabolize 2',3'-cAMP to AMPs, which are metabolized to adenosine. In rat and human aortic VSMCs, both 2'-AMP and 3'-AMP are involved in this process, whereas, in human coronary VSMCs, 2',3'-cAMP is mainly converted to 2'-AMP. Because adenosine inhibits VSMC proliferation via A(2B) receptors, local vascular production of 2',3'-cAMP may protect conduit arteries from atherosclerosis.     

Biochemical properties of Candida parapsilosis ecto-5'-nucleotidase and the possible role of adenosine in macrophage interaction

Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.     

Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b+Gr1+ cells

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A(2B) adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b(+)Gr1(high) cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b(+)Gr1(high) subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b(+)Gr-1(high) subset had the highest levels of CD73 (ecto-5'-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b(+)Gr-1(int) (ΔMFI of 57.9 ± 6.8) and CD11b(+)Gr-1(-/low) (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b(+)Gr-1(high) cells correlated with high levels of ecto-5'-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5'-nucleotidase substrate 5'-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.     

Cardiac adenosine production in rat genetic models of low and high exercise capacity

We previously demonstrated that Copenhagen (COP) and DA inbred rat strains show a wide difference in a test for aerobic treadmill running that correlated positively with isolated cardiac function. The purpose of this study was to test adenosine production as a candidate intermediate phenotype that may explain part of the difference in running and cardiac performance in these genetic models for low and high aerobic capacity. Adenosine production was measured as the activity of soluble 5'-nucleotidase and membrane-bound ecto-5'-nucleotidase in the membrane pellet and supernatant fractions of left and right ventricular muscle and gracilis muscle taken from 10 DA and 10 COP rats. Ecto-5'-nucleotidase activity in the membrane pellet of hearts from both DA and COP accounted for the vast majority of the total tissue adenosine production (>90% in the left ventricle and >80% in the right ventricle). Ecto-5'-nucleotidase activity in the pellet fraction was significantly higher in the left (22.4%) and right (46.1%) ventricles of DA rats compared with COP rats, with no differences in total protein content. There were no significant differences between the strains for 5'-nucleotidase activity in the cardiac supernatant, the gracilis pellet, or the gracilis supernatant. These data support the hypothesis that an increase in cardiac adenosine production may contribute to the greater aerobic running capacity of the DA rats.     

Ecto-5'-nucleotidase of cultured rat mesangial cells

Because adenosine plays a role in the regulation of glomerular filtration rate and of the release of renin, we examined the possibility of a local source for this mediator. We found that rat cultured glomerular mesangial cells converted 5'-AMP into adenosine. The properties of the enzyme involved in the reaction were those of an ecto-5' nucleotidase: (1) the products of the reaction were generated in the extracellular fluid although no 5'-nucleotidase was released by the cells into the medium; (2) identical activities were found for cultured cells in situ and sonicated cells; (3) the diazonium salt of sulfanilic acid which is a nonpenetrating reagent inhibited up to 75% of the enzyme activity. Ecto-5'-nucleotidase activity of intact cells obeyed Michaelis-Menten kinetics. Apparent Km for 5'-AMP was 0.32 mM. 5'-UMP was a strictly competitive inhibitor. ADP exerted a very powerful inhibitory effect and behaved also as a competitive inhibitor. ATP was inhibitory both by increasing Km and by decreasing Vmax. Ecto-5'-nucleotidase was active in the absence of divalent cations. However, Mg2+, Ca2+, Co2+ and Mn2+ were stimulatory. Zn2+ and Cu2+ suppressed the activity. Concanavalin A, a plant lectin, was markedly inhibitory, suggesting that a glycoprotein moiety was necessary to express enzyme activity. Ecto-5'-nucleotidase activity was not modified during phagocytosis of serum-treated zymosan by mesangial cells. Rat cultured glomerular epithelial cells exhibited a 5'-nucleotidase activity which was 4 times lower than that of the mesangial cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for coordinated induction and repression of ecto-5'-nucleotidase (CD73) and the A2a adenosine receptor in a human B cell line

In the human B cell line P493-6 two mitogenic signals, the Epstein-Barr virus nuclear antigen 2 (EBNA2) and myc, can be independently regulated by means of an estrogen receptor fusion construct or an inducible expression vector, respectively. Shut off of EBNA2, either in the presence or absence of myc, leads to a significant increase in enzymatic activity and surface expression of ecto-5'-nucleotidase (CD73) as well as an increased adenosine receptor response in cyclic AMP formation. Shut off of myc expression has a small additional positive effect on CD73 activity. Among the four different subtypes of adenosine receptors, the A2a receptor exclusively is subject to regulation in this system, which is substantiated by pharmacologic data (specific agonists and inhibitors), as well as on the mRNA level. With up-regulated CD73 and A2a, cells also respond to 5'-AMP with increased cyclic AMP formation. Turn on of EBNA2 has the reverse effect of repression of CD73 and A2a expression. The time course of both induction and repression of CD73 and A2a is rather slow.     

Effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis

Here we report the effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis was 5- to 7-fold higher for the fresh clinical strain, when compared with the ATCC (American Type Culture Collection) strain. ATP hydrolysis was activated in the presence of metronidazole in the ATCC strain, whilst it was inhibited 33% by 50 microM tinidazole in a fresh clinical isolate. The treatment of cells in the presence of metronidazole for 2 h inhibited ATP and ADP hydrolysis, whilst treatment with tinidazole inhibited ATP and ADP hydrolysis only in the fresh clinical isolate. The drugs did not change the ecto-5'-nucleotidase activity for both strains. Our results suggest that the modulation of extracellular ATP and ADP levels during treatment with these drugs could be a parasitic defence strategy as a survival mechanism in an adverse environment.     

Catalytic characteristics of 5'-nucleotidase in the structure of cell membrane rafts in smooth muscles

5'-nucleotidase (EN 3.1.3.5) is widely distributed enzyme occurring in vertebrate, bacterial and plant cells. The main physiological function of 5'-nucleotidase is hydrolysis of 5'-AMP to adenosine and Pi. It was found that the detergent-insoluble membrane domains (rafts) are enriched by proteins possessing high 5'-AMPase activity. This study is aimed to investigate some physical and chemical properties of 5'-nucleotidase, which is present in detergent insoluble membrane domains isolated from pig stomach and lung. It was shown for the first time that catalytic properties of the raft-associated 5'-nucleotidase and of the pure enzyme described in literature differ. Our results demonstrate that the greatest activity of the raft-associated enzyme takes place in the physiological conditions contrary to the pure enzyme. Our data suggest that such changes of 5'-nucleotidase catalytic activity might be due to the disruption of its interaction with membrane rafts.     

Differential macrophage activation alters the expression profile of NTPDase and ecto-5'-nucleotidase

Macrophages are key elements in the inflammatory process, whereas depending on the micro-environmental stimulation they exhibit a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype. Extracellular ATP can act as a danger signal whereas adenosine generally serves as a negative feedback mechanism to limit inflammation. The local increase in nucleotides communication is controlled by ectonucleotidases, such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto-5'-nucleotidase/CD73 (ecto-5'-NT). In the present work we evaluated the presence of these enzymes in resident mice M1 (macrophages stimulated with LPS), and M2 (macrophages stimulated with IL-4) macrophages. Macrophages were collected by a lavage of the mice (6-8 weeks) peritoneal cavity and treated for 24 h with IL-4 (10 ng/mL) or LPS (10 ng/mL). Nitrite concentrations were measured using the Greiss reaction. Supernatants were harvested to determine cytokines and the ATPase, ADPase and AMPase activities were determined by the malachite green method and HPLC analysis. The expression of selected surface proteins was evaluated by flow cytometry. The results reveal that M1 macrophages presented a decreased ATP and AMP hydrolysis in agreement with a decrease in NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M2. In contrast, M2 macrophages showed a higher ATP and AMP hydrolysis and increased NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M1 macrophages. Therefore, macrophages of the M1 phenotype lead to an accumulation of ATP while macrophages of the M2 phenotype may rapidly convert ATP to adenosine. The results also showed that P1 and P2 purinoreceptors present the same mRNA profile in both phenotypes. In addition, M2 macrophages, which have a higher ATPase activity, were less sensitive to cell death. In conclusion, these changes in ectoenzyme activities might allow macrophages to adjust the outcome of the extracellular purinergic cascade in order to fine-tune their functions during the inflammatory set.     

The proposed 5'-nucleotidase inhibitor in human leukemic cells is an artefact

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5'-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577-584) mentioned that this phenomenon resulted from the presence of a 5'-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5'-[3H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [3H]Adenosine derived from 5'-[3H]AMP hydrolysis was further transformed into [3H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [3H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5'-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5'-nucleotidase inhibitor. We did not observe 5'-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.     

Enzymes of adenosine metabolism in the brain: diurnal rhythm and the effect of sleep deprivation

Adenosine plays a role in promoting sleep, an effect that is thought to be mediated in the basal forebrain. Adenosine levels vary in this region with prolonged wakefulness in a unique way. The basis for this is unknown. We examined, in rats, the activity of the major metabolic enzymes for adenosine - adenosine deaminase, adenosine kinase, ecto- and cytosolic 5'-nucleotidase - in sleep/wake regulatory regions as well as cerebral cortex, and how the activity varies across the day and with sleep deprivation. There were robust spatial differences for the activity of adenosine deaminase, adenosine kinase, and cytosolic and ecto-5'-nucleotidase. However, the basal forebrain was not different from other sleep/wake regulatory regions apart from the tuberomammillary nucleus. All adenosine metabolic enzymes exhibited diurnal variations in their activity, albeit not in all brain regions. Activity of adenosine deaminase increased during the active period in the ventrolateral pre-optic area but decreased significantly in the basal forebrain. Enzymatic activity of adenosine kinase and cytosolic-5'-nucleotidase was higher during the active period in all brain regions tested. However, the activity of ecto-5'-nucleotidase was augmented during the active period only in the cerebral cortex. This diurnal variation may play a role in the regulation of adenosine in relationship to sleep and wakefulness across the day. In contrast, we found no changes specifically with sleep deprivation in the activity of any enzyme in any brain region. Thus, changes in adenosine with sleep deprivation are not a consequence of alterations in adenosine enzyme activity.     

Cytochemical localization of 5'-nucleotidase in the frog (Rana pipiens) retina. A histochemical and cytochemical study

Localization of 5'-nucleotidase in the frog retina was investigated using histochemical and cytochemical techniques. Light-microscopic observations revealed the presence of this enzyme in the inner retinal layers (the nerve fiber layer, ganglion cell layer, and inner plexiform layer). Ultrastructural investigations revealed that the enzyme activity is associated with the plasma membranes of the Müller cell processes, whereas the Müller cell processes present in the outer retinal layers did not demonstrate any detectable enzyme activity. This observation would appear to confirm our previous findings, that 5'-nucleotidase is an ectoenzyme, but its distribution in frog retina differs from that in rodents and it is only present in the inner layers of the retina. The prominent localization of 5'-nucleotidase on the glial plasma membrane may be viewed in the context of the widely accepted interaction between neurones and glial cells. Since nucleotides do not penetrate the plasma membrane, a mechanism to produce membrane-permeable adenosine, important for neuronal function, is postulated. It is known that 5'-nucleotidase produces adenosine by hydrolyzing adenosine 5'-monophosphate (5'-AMP). Therefore one would expect that the glial membrane-bound enzyme can accomplish the final step in this mechanism by producing the adenosine in the extracellular spaces.     

Ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio)

Adenosine, a well-known neuromodulator, may be formed intracellularly in the CNS from degradation of AMP and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. This study reports the enzymatic properties of an ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for AMP hydrolysis in a pH range of 7.0-7.5 in the presence of Mg(2+). The enzyme presented a maximal activity for AMP hydrolysis at 37 degrees C. The apparent K(m) and V(max) values for Mg(2+)-AMP were 135.3+/-16 microM and 29+/-4.2 nmol Pi.min(-1).mg(-1) protein, respectively. The enzyme was able to hydrolyze both purine and pyrimidine monophosphate nucleotides, such as UMP, GMP and CMP. Levamisole and tetramisole (1 mM), specific inhibitors of alkaline phosphatases, did not alter the enzymatic activity. However, a significant inhibition of AMP hydrolysis (42%) was observed in the presence of 100 microM alpha,beta-methylene-ADP, a known inhibitor of ecto-5'-nucleotidase. Since 5'-nucleotidase represents the major enzyme responsible for the formation of extracellular adenosine, the enzymatic characterization is important to understand its role in purinergic systems and the involvement of adenosine in the regulation of neurotransmitter release.     

Production of Adenosine from Extracellular ATP at the Striatal Cholinergic Synapse

Abstract: The components of the ectonucleotidase pathway at the immunoaffinity-purified striatal cholinergic synapse have been studied. The ecto-ATPase (EC 3.6.1.15) had a K _m of 131 γ M , whereas the ecto-ADPase (EC 3.6.1.6) had a K _m of 58 γ M , was Ca²⁺-dependent, and was inhibited by the ATP analogue 5'-adenylylimidodiphosphate (AMPPNP). The ecto-5'-nucleotidase (EC 3.1.3.5) had a K _m of 21 γ M , was inhibited by AMPPNP and α,β-methylene ADP, and by a specific antiserum. The V _max values of the ATPase, ADPase, and 5'-nucleotidase enzymes present at this synapse were in a ratio of 30:14:1. Very little ecto-adenylate kinase activity was detected on these purified synapses. The intraterminal 5'-nucleotidase enzyme, which amounted to 40% of the total 5'-nucleotidase activity, was inhibited by AMPPNP, α,β-methylene ADP, and the antiserum, and also had the same kinetic properties as the ectoenzyme. The time course of ATP degradation to adenosine outside the nerve terminals showed a delay, followed by a period of sustained adenosine production. The delay in adenosine production was proportional to the initial ATP concentration, was a consequence of feedforward inhibition of the ADPase and 5'-nucleotidase, and was inversely proportional to the ecto-5'-nucleotidase activity. The function and characteristics of this pathway and the central role of 5'-nucleotidase in the regulation of extraterminal adenosine concentrations are discussed.     

Adenosine kinase and 5'-nucleotidase activity after prolonged wakefulness in the cortex and the basal forebrain of rat

The effect of prolonged wakefulness on adenosine kinase (AK), ecto-5'-nucleotidase and endo-5'-nucleotidase activity was assessed in the present study. Rats were sleep deprived for 3 or 6h, and one group was allowed to sleep 2h of recovery sleep after the 6h deprivation. The cortex and the basal forebrain were dissected, and frozen rapidly on dry ice. The enzyme activity of adenosine kinase was measured by monitoring the conversion of [2-3H]-adenosine into [3H]-adenosine monophosphate (AMP) and the ecto-5'-nucleotidase and endo-5'-nucleotidase activities by monitoring the conversion of [2-3H]-AMP into [3H]-adenosine. The enzyme activities did not change during deprivation or recovery sleep in either cortex or basal forebrain when compared to unhandled controls. Significant diurnal variation in enzyme activities was noted in both brain areas. In the basal forebrain adenosine kinase and both nucleotidases showed their lowest activity in the middle of the rest phase, 6h after lights on, suggesting a low level of adenosine metabolism, both production and degradation at this time point. In the cortex adenosine kinase had a diurnal activity pattern similar to the basal forebrain and the ecto-5'-nucleotidase activity was low already early in the rest phase, 3h after lights on, and remained low until the end part of the rest phase, 8h after lights on. Endo-5'-nucleotidase lacked diurnal variation. These activity patterns may be associated with the lower level of energy metabolism during sleep compared to wakefulness.     

A novel transverse push-pull microprobe: in vitro characterization and in vivo demonstration of the enzymatic production of adenosine in the spinal cord dorsal horn

Adenosine produces analgesia in the spinal cord and can be formed extracellularly through enzymatic conversion of adenine nucleotides. A transverse push-pull microprobe was developed and characterized to sample extracellular adenosine concentrations of the dorsal horn of the rat spinal cord. Samples collected via this sampling technique reveal that AMP is converted to adenosine in the dorsal horn. This conversion is decreased by the ecto-5'-nucleotidase inhibitor, alpha,beta-methylene ADP. Related behavioral studies demonstrate that AMP administered directly to the spinal cord can reverse the secondary mechanical hyperalgesia characteristic of the intradermal capsaicin model of inflammatory pain. The specific adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) inhibits the antihyperalgesia produced by AMP. This research introduces a novel microprobe that can be used as an adjunct sampling technique to microdialysis and push-pull cannulas. Furthermore, we conclude that AMP is converted to adenosine in the dorsal horn of the spinal cord by ecto-5'-nucleotidase and subsequently may be one source of adenosine, acting through adenosine A(1) receptors in the dorsal horn of the spinal cord, which produce antihyperalgesia.     

Oviduct cells express the cyclic AMP-adenosine pathway

The extracellular cAMP-adenosine pathway refers to the local production of adenosine mediated by cAMP egress into the extracellular space, conversion of cAMP to AMP by ectophosphodiesterase (PDE), and the metabolism of AMP to adenosine by ecto-5'-nucleotidase. The goal of this study was to assess whether the cAMP-adenosine pathway is expressed in oviduct cells. Studies were conducted in cultured bovine oviduct cells (mixed cultures of fibroblasts and epithelial cells, 1:1 ratio). Confluent monolayers of oviduct cells were exposed to cAMP (0.01-100 micromol/L) in the presence and absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/L, an inhibitor of both extracellular and intracellular PDE activity), 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX, 100 micromol/L, a xanthine that can inhibit extracellular or ecto-PDE activity at high concentrations), or alpha,beta-methylene-adenosine-5'-diphosphate (AMPCP, 100 micromol/L, an ecto-5'-nucleotidase inhibitor) for 0-60 min. The medium was then sampled and assayed for AMP, adenosine, and inosine. Addition of exogenous cAMP to oviduct cells increased extracellular levels of AMP, adenosine, and inosine in a concentration- and time-dependent manner. This effect was attenuated by blockade of total (extracellular and intracellular) PDE activity (IBMX), ecto-PDE activity (DPSPX), or ecto-5'-nucleotidase (AMPCP). The functional relevance of the cAMP-adenosine pathway is supported by the findings that treatment with adenylyl cyclase stimulants (forskolin plus isoproterenol) resulted in the egress of cAMP (97% extracellular) into the extracellular space and its conversion into adenosine. The extracellular cAMP-adenosine pathway exists in oviduct cells and may play an important role in regulating the biology and physiology of the oviduct. This pathway also may play a critical role in regulating sperm function, fertilization, and early embryo development.     

Anemia induces 5'-nucleotidase in fibroblasts of cortical labyrinth of rat kidney

We recently observed a strong increase of 5'-nucleotidase in renal fibroblasts of rats that were anemic due to an immunity against erythropoietin. In order to test if the change of 5'-nucleotidase was related with anemia, we studied the distribution of the enzyme in irradiated rats treated with phenylhydrazine. The hematocrit of these rats decreased to 15% within 4 days and erythropoietin levels were more than 200 times over controls. After 7 days a histochemical study showed that the enzymatic activity and the immunoreactivity for 5'-nucleotidase was markedly enhanced in the fibroblasts of the cortical labyrinth. There was no modification of 5'-nucleotidase in other cell types of the kidney. The 5'-nucleotidase activity of renal fibroblasts in cell culture increased by 72% upon addition of 160 microM 5'-AMP to the culture medium for 8 days. We propose that anemia provokes an energy deficit in some structure in the cortical labyrinth. This might increase the concentration of 5'-AMP which would induce 5'-nucleotidase. An interesting consequence of these events would be an increased production of adenosine in the direct vicinity of some of its putative targets, the glomerular arterioles and the erythropoietin-producing cells.