Extracellular cAMP-adenosine Pathway Research Articles

Impact of the cAMP efflux and extracellular cAMP-adenosine pathway on airway smooth muscle relaxation induced by formoterol and phosphodiesterase inhibitors

β2-adrenoceptors agonists and phosphodiesterase (PDE) inhibitors are effective bronchodilators, due to their ability to increase intracellular cyclic AMP (cAMP) levels and induce airway smooth muscle (ASM) relaxation. We have shown that increment of intracellular cAMP induced by β2-adrenoceptors agonist fenoterol is followed by efflux of cAMP, which is converted by ecto-PDE and ecto-5′-nucleotidases (ecto-5′NT) to adenosine, leading to ASM contraction. Here we evaluate whether other classical bronchodilators used to treat asthma and chronic obstructive pulmonary disease (COPD) could induce cAMP efflux and, as consequence, influence the ASM contractility. Our results showed that β2-adrenoceptor agonists formoterol and PDE inhibitors IBMX, aminophylline and roflumilast induced cAMP efflux and a concentration-dependent relaxation of rat trachea precontracted with carbachol. Pretreatment of tracheas with MK-571 (MRP transporter inhibitor), AMP-CP (ecto-5′NT inhibitor) or CGS-15943 (nonselective adenosine receptor antagonist) potentiated the relaxation induced by β2-adrenoceptor agonists but did not change the relaxation induced by PDE inhibitors. These data showed that all bronchodilators tested were able to induce cAMP efflux. However, only β2-adrenoceptor-induced relaxation of tracheal smooth muscle was affected by cAMP efflux and extracellular cAMP-adenosine pathway.

Extracellular cAMP-Adenosine Pathway Signaling: A Potential Therapeutic Target in Chronic Inflammatory Airway Diseases.

Adenosine is a purine nucleoside that, via activation of distinct G protein-coupled receptors, modulates inflammation and immune responses. Under pathological conditions and in response to inflammatory stimuli, extracellular ATP is released from damaged cells and is metabolized to extracellular adenosine. However, studies over the past 30 years provide strong evidence for another source of extracellular adenosine, namely the “cAMP-adenosine pathway.” The cAMP-adenosine pathway is a biochemical mechanism mediated by ATP-binding cassette transporters that facilitate cAMP efflux and by specific ectoenzymes that convert cAMP to AMP (ecto-PDEs) and AMP to adenosine (ecto-nucleotidases such as CD73). Importantly, the cAMP-adenosine pathway is operative in many cell types, including those of the airways. In airways, β2-adrenoceptor agonists, which are used as bronchodilators for treatment of asthma and chronic respiratory diseases, stimulate cAMP efflux and thus trigger the extracellular cAMP-adenosine pathway leading to increased concentrations of extracellular adenosine in airways. In the airways, extracellular adenosine exerts pro-inflammatory effects and induces bronchoconstriction in patients with asthma and chronic obstructive pulmonary diseases. These considerations lead to the hypothesis that the cAMP-adenosine pathway attenuates the efficacy of β2-adrenoceptor agonists. Indeed, our recent findings support this view. In this mini-review, we will highlight the potential role of the extracellular cAMP-adenosine pathway in chronic respiratory inflammatory disorders, and we will explore how extracellular cAMP could interfere with the regulatory effects of intracellular cAMP on airway smooth muscle and innate immune cell function. Finally, we will discuss therapeutic possibilities targeting the extracellular cAMP-adenosine pathway for treatment of these respiratory diseases.

The cAMP-Adenosine Feedback Loop Maintains the Suppressive Function of Regulatory T Cells.

Therapeutic manipulation of regulatory T cells (Tregs) has been regarded as a promising approach for the treatment of immune disorders. However, a better understanding of the immunomodulatory mechanisms of Tregs and new safe and effective methods to improve the therapeutic effects of Tregs are highly desired. In this study, we have identified the key roles of a cAMP-adenosine positive feedback loop in the immunomodulatory function of Tregs. Adult male C57BL/6J mice were used for an experimental autoimmune uveitis (EAU) model, Tregs, and uveitogenic T cells (UTs). In established EAU, induced Tregs (iTregs) administration alleviated the inflammatory response. In vitro, iTregs inhibited UTs proliferation and inflammatory cytokine production. Mechanistically, cAMP is partially responsible for iTreg-mediated inhibition on UTs. Importantly, intracellular cAMP regulates CD39 expression and CD39-dependent adenosine production in iTregs, and cAMP directly participates in iTreg-derived adenosine production by a CD39 signaling-independent extracellular cAMP-adenosine pathway. Moreover, extracellular adenosine increases the intracellular cAMP level in Tregs. More importantly, increasing the cAMP level in iTregs before transfer improves their therapeutic efficacy in established EAU. Notably, the cAMP-adenosine loop exists in both iTregs and naturally occurring Tregs. These findings provide new insights into the immunosuppressive mechanisms of Tregs and suggest a new strategy for improving the therapeutic efficacy of Tregs in established autoimmune disease.

Global expression profiling and pathway analysis in two different population groups in relation to high altitude.

High altitude (HA) is associated with number of stresses. Response of these stresses may vary in different populations depending upon altitude, duration of residency, ancestry, geographical variation, lifestyle, and ethnicities. For understanding population variability in transcriptome, array-based global gene expression profiling was performed on extracted RNA of male volunteers of two different lowland population groups, i.e., Indians and Kyrgyz, at baseline and day 7 of HA exposure (3200m). A total of 97 genes were differentially expressed at basal in Kyrgyz as compared to Indians (82 downregulated and 15 upregulated), and 196 were differentially expressed on day 7 of HA (118 downregulated and 78 upregulated). Ingenuity Pathway Analysis and gene ontology highlighted eIF2 signaling with most significant negative activation z score at basal in Kyrgyz compared to Indians with downregulation of various L- and S-ribosomal proteins indicating marked translational repression. On day 7, cAMP-mediated signaling is most enriched with positive activation z score in Kyrgyz compared to Indians. Plasma cAMP levels were higher in Kyrgyz on day 7 compared to Indians. Extracellular adenosine levels were elevated in both the groups upon HA, but higher in Kyrgyz compared to Indians. Valedictory qRT-PCR showed upregulation of ADORA2B and CD73 along with downregulation of ENTs in Kyrgyz compared to Indians indicating elevated levels of extracellular nucleotides mainly adenosine and activation of extracellular cAMP-adenosine pathway which as per literature triggers endogenous protective mechanisms under stress conditions like hypoxia. Thus, transcriptome changes at HA are population-specific, and it may be necessary to take care while interposing similar results in different populations.

The Extracellular cAMP-Adenosine Pathway in Airway Smooth Muscle.

In the respiratory tract, intracellular cAMP has a key role in the smooth muscle relaxation induced by the β2-adrenoceptor/Gs protein/adenylyl cyclase axis. In other tissues, cAMP also works as an extracellular messenger, after its efflux and interstitial conversion to adenosine by ectoenzymes. The aim of this study was to identify cAMP efflux and the "extracellular cAMP-adenosine pathway" in the airway smooth muscle. First, we tested the ability of β2-adrenoceptor agonists formoterol or fenoterol to increase the extracellular cAMP in isolated tracheal rings from adult male Wistar rats. The effects of adenosine, cAMP, 8-Br-cAMP, fenoterol, or formoterol were also evaluated in the isometric contraction of control or carbachol (CCh) precontracted tracheas, normalized as the percentage of CCh-induced response. Fenoterol and formoterol induced 70%-80% relaxation and increased extracellular cAMP levels by up to 280%-450%. Although exogenous cAMP or adenosine evoked phasic contractions, the membrane-permeable cAMP analog 8-Br-cAMP induced relaxation of CCh-precontracted tracheas. The simultaneous inhibition of adenosine degradation/uptake with EHNA [erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride] plus uridine increased by 3-fold the maximum cAMP-induced contraction, whereas it was significantly reduced by AMPCP [adenosine 5'-(α,β-methylene)diphosphate; an ecto-5'-nucleotidase inhibitor], and by adenosine receptor antagonists CGS-15943 (nonselective) or DPCPX (8-cyclopentyl-1,3-dipropylxanthine) (A1 selective). Finally, CGS-15943 shifted to the left the concentration-relaxation curve for fenoterol. In conclusion, our results show that airway smooth muscle expresses the extracellular cAMP-adenosine pathway associated with contracting effects mediated by A1 receptors. The cAMP efflux triggered by fenoterol/formoterol indicates that the extracellular cAMP-adenosine pathway may play a role in balancing the relaxant effects of β2-adrenoceptor agonists in airways, which may impact their bronchodilation effects.

Characterization of cAMP-phosphodiesterase activity in bovine seminal plasma.

The second messenger cyclic adenosine monophosphate (cAMP) has a central role in sperm physiology. Extracellular cAMP can be sequentially degraded into 5'AMP and adenosine by ecto-phosphodiesterases (ecto-PDE) and ecto-nucleotidases, a phenomenon called extracellular cAMP-adenosine pathway. As cAMP-adenosine pathway is involved in sperm capacitation, we hypothesize that extracellular PDEs are functionally present in seminal plasma. Exclusively measuring cAMP-PDE activity, total activity in bovine seminal plasma was 10.1±1.5 fmoles/min/μg. Using different family-specific PDE inhibitors, we showed that in seminal plasma, the major cAMP-PDE activity was papaverine sensitive (47.5%). These data support the presence of PDE10 in bovine seminal plasma and was further confirmed by western blot. In epididymal fluid, total cAMP-PDE activity was 48.2±14.8 fmoles/min/μg and we showed that the major cAMP-PDE activity was 3-isobutyl-methylxanthine insensitive and thus ascribed to PDE8 family. PDE10A mRNAs were found in the testis, epididymis, and seminal vesicles. cAMP-PDE activity is present in bovine seminal plasma and epididymal fluid. The results suggest a role for ecto-PDEs present in those fluids in the signaling pathways involved in sperm functions.

New perspectives in signaling mediated by receptors coupled to stimulatory G protein: the emerging significance of cAMP efflux and extracellular cAMP-adenosine pathway.

G protein-coupled receptors (GPCRs) linked to stimulatory G (Gs) proteins (GsPCRs) mediate increases in intracellular cyclic AMP as consequence of activation of nine adenylyl cyclases , which differ considerably in their cellular distribution and activation mechanisms. Once produced, cyclic AMP may act via distinct intracellular signaling effectors such as protein kinase A and the exchange proteins activated by cAMP (Epacs). More recently, attention has been focused on the efflux of cAMP through a specific transport system named multidrug resistance proteins that belongs to the ATP-binding cassette transporter superfamily. Outside the cell, cAMP is metabolized into adenosine, which is able to activate four distinct subtypes of adenosine receptors, members of the GPCR family: A1, A2A, A2B, and A3. Taking into account that this phenomenon occurs in numerous cell types, as consequence of GsPCR activation and increment in intracellular cAMP levels, in this review, we will discuss the impact of cAMP efflux and the extracellular cAMP-adenosine pathway on the regulation of GsPCR-induced cell response.

Human monocytes respond to extracellular cAMP through A2A and A2B adenosine receptors

In this study, we test the hypothesis that cAMP, acting as an extracellular mediator, affects the physiology and function of human myeloid cells. The cAMP is a second messenger recognized as a universal regulator of several cellular functions in different organisms. Many studies have shown that extracellular cAMP exerts regulatory functions, acting as first mediator in multiple tissues. However, the impact of extracellular cAMP on cells of the immune system has not been fully investigated. We found that human monocytes exposed to extracellular cAMP exhibit higher expression of CD14 and lower amount of MHC class I and class II molecules. When cAMP-treated monocytes are exposed to proinflammatory stimuli, they exhibit an increased production of IL-6 and IL-10 and a lower amount of TNF-α and IL-12 compared with control cells, resembling the features of the alternative-activated macrophages or M2 macrophages. In addition, we show that extracellular cAMP affects monocyte differentiation into DCs, promoting the induction of cells displaying an activated, macrophage-like phenotype with reduced capacity of polarized, naive CD4(+) T cells into IFN-γ-producing lymphocytes compared with control cells. The effects of extracellular cAMP on monocytes are mediated by CD73 ecto-5'-nucleotidase and A2A and A2B adenosine receptors, as selective antagonists could reverse its effects. Of note, the expression of CD73 molecules has been found on the membrane of a small population of CD14(+)CD16(+) monocytes. These findings suggest that an extracellular cAMP-adenosine pathway is active in cells of the immune systems.

Contribution of the Extracellular cAMP-Adenosine Pathway to Dual Coupling of β2-Adrenoceptors to Gsand GiProteins in Mouse Skeletal Muscle

β(2)-Adrenoceptor (β(2)-AR) agonists increase skeletal muscle contractile force via activation of G(s) protein/adenylyl cyclases (AC) and increased generation of cAMP. Herein, we evaluated the possible dual coupling of β(2)-AR to G(s) and G(i) proteins and the influence of the β(2)-AR/G(s)-G(i)/cAMP signaling cascade on skeletal muscle contraction. Assuming that the increment of intracellular cAMP is followed by cAMP efflux and extracellular generation of adenosine, the contribution of the extracellular cAMP-adenosine pathway on the β(2)-AR inotropic response was also addressed. The effects of clenbuterol/fenoterol (β(2)-AR agonists), forskolin (AC activator), cAMP/8-bromo-cAMP, and adenosine were evaluated on isometric contractility of mouse diaphragm muscle induced by supramaximal direct electrical stimulation (0.1 Hz, 2 ms duration). Clenbuterol/fenoterol (10-1000 μM), 1 μM forskolin, and 20 μM rolipram induced transient positive inotropic effects that peaked 30 min after stimulation onset, declining to 10 to 20% of peak levels in 30 min. The late descending phase of the β(2)-AR agonist inotropic effect was mimicked by either cAMP or adenosine and abolished by preincubation of diaphragm with pertussis toxin (PTX) (G(i) signaling inhibitor) or the organic anion transporter inhibitor probenecid, indicating a delayed coupling of β(2)-AR to G(i) protein which depends on cAMP efflux. Remarkably, the PTX-sensitive β(2)-AR inotropic effect was inhibited by the A(1) adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine and ecto-5'-phosphodiesterase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt, indicating that β(2)-AR coupling to G(i) is indirect and dependent on A(1) receptor activation. The involvement of the extracellular cAMP-adenosine pathway in β(2)-AR signaling would provide a negative feedback loop that may limit stimulatory G protein-coupled receptor positive inotropism and potential deleterious effects of excessive contractile response.

The Extracellular cAMP-Adenosine Pathway Regulates Expression of Renal D1 Dopamine Receptors in Diabetic Rats

Activation of D1 dopamine receptors expressed in the kidneys promotes the excretion of sodium and regulates sodium levels during increases in dietary sodium intake. A decrease in the expression or function of D1 receptors results in increased sodium retention which can potentially lead to the development of hypertension. Studies have shown that in the absence of functional D1 receptors, in null mice, the systolic, diastolic, and mean arterial pressures are higher. Previous studies have shown that the expression and function of D1 receptors in the kidneys are decreased in animal models of diabetes. The mechanisms that down-regulate the expression of renal D1 receptor gene in diabetes are not well understood. Using primary renal cells and acutely isolated kidneys from the streptozotocin-induced rat diabetic model, we demonstrate that the renal D1 receptor expression is down-regulated by the extracellular cAMP-adenosine pathway in vitro and in vivo. In cultures of primary renal cells, a 3 mm, 60-h cAMP treatment down-regulated the expression of D1 receptors. In vivo, we determined that the plasma and urine cAMP levels as well as the expression of 5'-ectonucleotidase, tissue-nonspecific alkaline phosphatase, and adenosine A2a receptors are significantly increased in diabetic rats. Inhibitors of 5'-ectonucleotidase and tissue-nonspecific alkaline phosphatase, α,β-methyleneadenosine 5'-diphosphate, and levamisole, respectively, blocked the down-regulation of D1 receptors in the primary renal cells and in the kidney of diabetic animals. The results suggest that inhibitors of the extracellular cAMP-adenosine pathway reverse the down-regulation of renal D1 receptor in diabetes.

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5'-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5'-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine.

Cyclic AMP in Rat Ileum: Evidence for the Presence of an Extracellular Cyclic AMP-Adenosine Pathway

Extracellular adenosine plays a relevant role in regulating intestinal motility and preventing inflammatory processes. Adenosine 3',5'-cyclic monophosphate (cAMP) extruded from cells may be converted to adenosine monophosphate and then to adenosine by ecto-phosphodiesterase and CD73/ecto-5'nucleotidase, respectively, thus representing a source of adenosine. Our purpose was to assess the existence of a functional extracellular cAMP-adenosine pathway in intestinal tissue, obtaining evidence for CD73 expression and evaluating the effect of cAMP on ileum motility. The formation of cAMP metabolites in rat ileum strips incubated with exogenous cAMP or [(3)H]cAMP was monitored by high-performance liquid chromatography. CD73 was detected by immunoprecipitation and immunofluorescence. The functional activity of exogenous cAMP on ileum strips was recorded by measuring tension changes. In ileum strips, the generation of cAMP-derived adenosine monophosphate, adenosine, and inosine was time and concentration dependent and was blocked by phosphodiesterase or CD73 inhibitors in a manner consistent with exogenous cAMP being processed through the extracellular cAMP-adenosine pathway. Accordingly, [(3)H]cAMP uptake in ileum strips was negligible. Immunofluorescence revealed CD73 surface expression on intestinal smooth muscle cells and intact smooth muscle. Exogenous cAMP concentration-dependently increased ileum muscle tension partially inhibited by adenosine inactivation or receptor blockade. Forskolin-stimulated endogenous cAMP induced concentration-dependent ileum relaxations. A functioning extracellular cAMP-adenosine pathway featuring CD73 expression is present in rat ileum and affects intestinal motility. Extracellular cAMP may therefore act on intestinal muscle both directly by binding to specific smooth muscle cell membrane sites and indirectly through its degradation products.

The Extracellular cAMP-Adenosine Pathway Significantly Contributes to the in Vivo Production of Adenosine

The extracellular cAMP-adenosine pathway is the cellular egress of cAMP followed by extracellular conversion of cAMP to adenosine by the sequential actions of ecto-phosphodiesterase and ecto-5'-nucleotidase. Although detailed studies in isolated organs, tissues, and cells provide evidence for an extracellular cAMP-adenosine pathway, whether this mechanism contributes significantly to adenosine production in vivo is unclear. 1,3-Dipropyl-8-p-sulfophenylxanthine is restricted to the extracellular compartment due to a negative charge at physiological pH and, at high concentrations (> or =0.1 mM), blocks ecto-phosphodiesterase. Here, we show that administration of 1,3-dipropyl-8-p-sulfophenylxanthine at a dose that provided concentrations in plasma and urine of approximately 0.3 and 6 mM, respectively, inhibited urinary adenosine excretion. In Sprague-Dawley rats i.v., 1,3-dipropyl-8-p-sulfophenylxanthine (10 mg + 0.15 mg/min) significantly decreased by 48 and 39% the urinary excretion of adenosine (from 3.57 +/- 0.38 to 1.87 +/- 0.14 nmol/30 min; p = 0.0003) and the ratio of urinary adenosine to cAMP (from 0.93 +/- 0.08 to 0.57 +/- 0.06; p = 0.0044), respectively, without altering blood pressure, renal blood flow, or glomerular filtration rate. Although 1,3-dipropyl-8-p-sulfophenylxanthine transiently increased urine volume and sodium excretion, these effects subsided, yet adenosine excretion remained reduced. Thus, changes in systemic and renal hemodynamics and excretory function could not account for the effects of 1,3-dipropyl-8-p-sulfophenylxanthine on adenosine excretion. Additional experiments showed that 1,3-dipropyl-8-p-sulfophenylxanthine, as in Sprague-Dawley rats, significantly attenuated adenosine excretion and the ratio of urinary adenosine to cAMP in both Wistar-Kyoto rats and spontaneously hypertensive rats. We conclude that the extracellular cAMP-adenosine pathway significantly contributes to the in vivo production of adenosine.

CAMP-Adenosine Pathway in the Proximal Tubule

The "extracellular cAMP-adenosine pathway" refers to the conversion of cAMP to AMP by ecto-phosphodiesterase, followed by metabolism of AMP to adenosine by ecto-5'-nucleotidase, with all the steps occurring in the extracellular compartment. This study investigated whether the extracellular cAMP-adenosine pathway exists in proximal tubules. Freshly isolated proximal tubules rapidly converted basolaterally administered cAMP to AMP and adenosine. Proximal tubular cells in culture (first passage) rapidly converted apically administered cAMP to AMP and adenosine. In both freshly isolated proximal tubules and cultured proximal tubular cells, conversion of cAMP to AMP and adenosine was affected by a broad-spectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), an ecto-phosphodiesterase inhibitor (1,3-dipropyl-8-p-sulfophenylxanthine), and a blocker of ecto-5'-nucleotidase (alpha,beta-methyleneadenosine-5'-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In cultured proximal tubular cells, but not freshly isolated proximal tubules, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine plus inosine, and these changes were also modulated by the inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. Conversion of renal interstitial (basolateral) cAMP and AMP to adenosine in vivo was shown by microdialysis coupled with ion trap mass spectrometry. Western blot analysis showed A1, A2A, and A3 receptors on both apical and basolateral proximal tubular membranes, with A1 and A2A receptors more highly expressed on basolateral compared with apical membranes. We conclude that cAMP that reaches either the apical or basolateral membranes of proximal tubular cells is converted in part to adenosine that has ready access to adenosine receptors.

The extracellular cyclic AMP-adenosine pathway in renal physiology.

Many cell types in the kidney express adenosine receptors, and adenosine has multiple effects on renal function. Although adenosine is produced within the kidney by several biochemical reactions, recent studies support a novel mechanism for renal adenosine production, the extracellular cAMP-adenosine pathway. This extracellular cAMP-adenosine pathway is initiated by efflux of cAMP from cells following activation of adenylyl cyclase. Extracellular cAMP is then converted to adenosine by the serial actions of ecto-phosphodiesterase and ecto-5'-nucleotidase. When extracellular cAMP is converted to adenosine near the biophase of cAMP production and efflux, this local extracellular cAMP-adenosine pathway permits tight coupling of the site of adenosine production to the site of adenosine receptors. cAMP in renal compartments may also be formed by tissues/organs remote from the kidney. For example, stimulation of hepatic adenylyl cyclase by the pancreatic hormone glucagon increases circulating cAMP, which is filtered at the glomerulus and concentrated in the tubular lumen as water is extracted from the ultrafiltrate. Conversion of hepatic-derived cAMP to adenosine in the kidney completes a pancreatohepatorenal cAMP-adenosine pathway that may serve as an endocrine link between the pancreas, liver, and kidney.

Adenosine biosynthesis in the collecting duct.

Adenosine regulates tubular transport in collecting ducts (CDs); however, the sources of adenosine that modulate ion transport in CDs are unknown. The extracellular cAMP-adenosine pathway refers to the conversion of cAMP to AMP by ectophosphodiesterase, followed by metabolism of AMP to adenosine by ecto-5'-nucleotidase, with all steps occurring in the extracellular compartment. The goal of this study was to assess whether the extracellular cAMP-adenosine pathway exists in CDs. Studies were conducted in both freshly isolated CDs and in CD cells in culture (first passage) that were derived from isolated CDs. Purity of CDs was confirmed by microscopy, by Western blotting for aquaporin-1, aquaporin-2, bumetanide-sensitive cotransporter type 1, and thiazide-sensitive cotransporter; and by reverse transcription-polymerase chain reaction for adenosine receptors. Both freshly isolated CDs and CD cells in culture converted exogenous cAMP to AMP and adenosine. In both freshly isolated CDs and CD cells in culture, conversion of cAMP to AMP and adenosine was affected by a broad-spectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), an ectophosphodiesterase inhibitor (1,3-dipropyl-8-p-sulfophenylxanthine), and a blocker of ecto-5'-nucleotidase (alpha,beta-methylene-adenosine-5'-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In CD cells in culture, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine, and these changes were also modulated by the aforementioned inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. In conclusion, the extracellular cAMP-adenosine pathway is an important source of adenosine in CDs.

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.

Role of the extracellular cAMP-adenosine pathway in renal physiology.

Adenosine exerts physiologically significant receptor-mediated effects on renal function. For example, adenosine participates in the regulation of preglomerular and postglomerular vascular resistances, glomerular filtration rate, renin release, epithelial transport, intrarenal inflammation, and growth of mesangial and vascular smooth muscle cells. It is important, therefore, to understand the mechanisms that generate extracellular adenosine within the kidney. In addition to three "classic" pathways of adenosine biosynthesis, contemporary studies are revealing a novel mechanism for renal adenosine production termed the "extracellular cAMP-adenosine pathway." The extracellular cAMP-adenosine pathway is defined as the egress of cAMP from cells during activation of adenylyl cyclase, followed by the extracellular conversion of cAMP to adenosine by the serial actions of ecto-phosphodiesterase and ecto-5'-nucleotidase. This mechanism of extracellular adenosine production may provide hormonal control of adenosine levels in the cell-surface biophase in which adenosine receptors reside. Tight coupling of the site of adenosine production to the site of adenosine receptors would permit a low-capacity mechanism of adenosine biosynthesis to have a large impact on adenosine receptor activation. The purposes of this review are to summarize the physiological roles of adenosine in the kidney; to describe the classic pathways of renal adenosine biosynthesis; to review the evidence for the existence of the extracellular cAMP-adenosine pathway; and to describe possible physiological roles of the extracellular cAMP-adenosine pathway, with particular emphasis on the kidney.

Cardiac fibroblasts express the cAMP-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, and the metabolism of AMP to adenosine by ecto-5'-nucleotidase. The goal of this study was to assess whether the cAMP-adenosine pathway limits cardiac fibroblast growth. Studies were conducted in ventricular cardiac fibroblasts maintained in 3-dimensional cultures. Addition of exogenous cAMP to cardiac fibroblasts increased extracellular levels of AMP, adenosine, and inosine in a concentration-dependent and time-dependent manner. This effect was attenuated by blockade of total phosphodiesterase activity (3-isobutyl-1-methylxanthine), ectophosphodiesterase activity (high concentration of 1, 3-dipropyl-8-p-sulfophenylxanthine), or ecto-5'-nucleotidase (alpha, beta-methylene-adenosine-5'-diphosphate). Treatment with exogenous cAMP inhibited cell growth as assessed by DNA synthesis ((3)H-thymidine incorporation), cell proliferation (cell counts), and protein synthesis ((3)H-leucine incorporation). Antagonism of A(2) (KF17837) or A(1)/A(2) (low concentration of 1, 3-dipropyl-8-p-sulfophenylxanthine), but not A(1) (8-cyclopentyl-1, 3-dipropylxanthine), adenosine receptors blocked the growth-inhibitory effects of exogenous cAMP, but not the growth inhibitory effects of 8-bromo-cAMP (stable cAMP analogue). The growth-inhibitory effects of exogenous cAMP were enhanced by the combined inhibition of adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl) adenine] and adenosine kinase (iodotubercidin). In conclusion, the extracellular cAMP-adenosine pathway exists in cardiac fibroblasts and attenuates cell growth. Pharmacological augmentation of this pathway could abate pathological cardiac remodeling in heart disease.