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Female Reproductive Tract |
Center for Clinical Pharmacology,3 Departments of Medicine and Pharmacology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15261
Clinic for Endocrinology,4 Department of Obstetrics and Gynecology, University Hospital, Zurich, 8091 Zurich, Switzerland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,ß-methylene-adenosine-5'-diphosphate (AMPCP, 100 µmol/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.
female reproductive tract, fertilization, oviduct, phosphodiestrases, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adenosine is another local factor produced within the oviduct that may influence the microenvironment in which fertilization and early embryonic development takes place [7]. The biological effects of adenosine are mediated by several different adenosine receptor subtypes [8], and adenosine receptors are expressed in oviduct cells [9–11], spermatozoa [12–14], and early embryos [15]. Within the oviduct, adenosine regulates rhythmic contractions and the ciliary beat frequency via both A1 and A2A adenosine receptors [9–11]. Modulation of adenylyl cyclase by adenosine involves both stimulatory and inhibitory adenosine receptors and G-proteins [8]. Activation of A1 receptors, which are negatively coupled with adenylyl cyclase, attenuates the sympathetic nervous system [16], an extremely important regulatory system involved in the physiology of reproduction, by inhibiting the release of norepinephrine from sympathetic nerve terminals [16]. Via A1 receptors, adenosine induces rhythmic contraction of the oviduct [10], whereas via A2A receptors, which are positively coupled to the adenylyl cyclase, adenosine regulates ciliary beat frequency of oviduct epithelial cells [11]. Both the rhythmic contraction and ciliary beat frequency are important for oocyte, embryo, and sperm transport. Functional A1 and A2A receptors also exist in human spermatozoa. Via A2A adenosine receptors, adenosine (specific A2A agonists) increases cAMP levels in mouse sperm [14], induces tyrosine phosphorylation in mouse sperm [7], induces sperm capacitation [7, 13, 14], stimulates human sperm motility [12], and increases mouse sperm fertilization ability during the early stages of capacitation [17]. Both A1 and A2A adenosine receptors play different roles in the capacitation process [14]. In this regard, A2A receptors only function in uncapacitated sperm and stimulate adenylyl cyclase/cAMP. In contrast, A1 receptors only function in capacitated sperm and inhibit adenylyl cyclase/cAMP, resulting in inhibition of spontaneous acrosome reactions and maintenance of sperm fertilizing potential. Apart from the effects of adenosine on the oviduct and sperm, the levels of adenosine increase during early embryo development (peri-implantation) and decrease during the postimplantation phase of embryo development [18]. These findings suggest that adenosine may play an important role in the early stages of embryo development. This hypothesis is indirectly supported by the finding that levels of cAMP, a precursor of adenosine, are decreased during pseudopregnancy [19]. The above findings provide evidence that local levels of adenosine within the oviduct may importantly influence the microenvironment for the fertilization process and early embryo development.
Although adenosine may play a significant role in reproductive physiology, the biochemical mechanisms regulating adenosine levels within the female reproductive system are incompletely understood. Recently, we proposed the hypothesis that cAMP may be an important determinant of adenosine production via a biochemical mechanism referred to as the cAMP-adenosine pathway [20]. This pathway involves the conversion of cAMP to AMP and hence to adenosine by the enzymes phosphodiesterase (PDE) and 5'-nucleotidase (5'-NT), respectively. The cAMP-adenosine pathway has both an intracellular and an extracellular arm, i.e., adenosine may be formed within the cell and transported to the extracellular space or may be formed directly in the extracellular space [20] (Fig. 1). The purpose of the present study was to test the hypothesis that the extracellular limb of the putative cAMP-adenosine pathway is a viable metabolic mechanism for the production of adenosine. Oviduct cells were selected for study because autocrine/paracrine factors generated within the oviductal microenvironment play a critical role in regulating fertilization and early embryo development.
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To test this hypothesis, we examined the ability of bovine oviduct cells (mixed cultures of epithelial cells:fibroblasts, 1:1) in culture to convert exogenous cAMP to AMP, adenosine, and inosine. We also assessed the individual capability of epithelial cells and fibroblasts to convert cAMP to adenosine. We also examined whether 3-isobutyl-1-methylxanthine (IBMX; an inhibitor of PDE that penetrates cell membranes [21]), ,ß-methyleneadenosine-5'-diphosphate (AMPCP; an ecto-5'-nucleotidase inhibitor [22]), and 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX; a xanthine that can inhibit PDE but is restricted to the extracellular compartment [23]) alter the conversion of exogenous cAMP to AMP, adenosine, and inosine. To provide physiological and biological evidence for the presence of the extracellular cAMP-adenosine pathway, we examined and compared the formation of intracellular and extracellular cAMP and adenosine in cells stimulated with the adenylyl cyclase activators forskolin and isporoterenol [24]. We also evaluated the relative roles of erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and iodotubericidin, specific inhibitors of adenosine deaminase and adenosine kinase [24], respectively, in regulating adenosine metabolism/catabolism by oviduct cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mixed cultures of epithelial cells and fibroblasts Oviducts of young, cyclic, nonpregnant cows were obtained from the local abattoir, and oviduct cells were cultured in Ham F10 medium (Sigma, Chemie, Buchs, Switzerland) containing 10% fetal calf serum (FCS, steroid-free, one batch; Sigma) according to our previously published method [25]. Confluent monolayers of mixed cell cultures (epithelial cells:fibroblasts, approximately 1:1) after 6–8 days in culture were used. These cell cultures were characterized immunohistochemically as previously described [5, 25]. Monoclonal antibodies to epithelial cell cytokeratin (anti-cytokeratin AE1/AE3; Dako Diagnostiks AG, Zug, Switzerland) and fibroblast vimentin (anti-vimentin VIM 3B4; Dako) were used to identify epithelial cells and fibroblasts in culture, respectively. The peroxidase/anti-peroxidase system (Dako) was used to visualize staining. Because autocrine/paracrine factors generated by both epithelial cells and fibroblasts may be important in regulating the physiology and biology of the oviduct, we used the coculture system to analyze the presence of the cAMP-adenosine pathway in the oviduct. Using the same model, we previously demonstrated that both epithelial cells and fibroblast synthesize autocrine/paracrine factors such as leukemia inhibitory factor [25] and endothelin 1 [26]; similar synthesis was observed in cocultures.
Culture of epithelial cells Epithelial cells were isolated and cultured using our previously described method [25]. Epithelial cells were isolated from bovine oviducts using a nonenzymatic cell isolation procedure and cultured in Ham F10 medium supplemented with FCS and antibiotics. Cell purity of >94% was confirmed by positive immunostaining with anti-cytokeratin AE1/AE3 and negative staining with anti-vimentin.
Culture of fibroblasts Fibroblasts from bovine oviducts were isolated and cultured using our previously described method, using a nonenzymatic cell isolation procedure and selective plating method [25]. Cell purity of >98% was confirmed by positive immunostaining with anti-vimentin antibodies.
cAMP Metabolism Studies
To investigate whether oviduct cells express the cAMP-adenosine pathway, confluent monolayers (epithelial cells:fibroblasts, 1:1) from bovine oviducts were washed twice with Hepes-buffered Hanks balanced salt solution. After washing, monolayers of oviduct cells were treated with 0.5 ml of Dulbecco PBS buffered with Hepes (25 mmol/L) and NaHCO3 (13 mmol/L) in the presence and absence of cAMP (30 µmol/L), IBMX (1 mmol/L; Sigma), IBMX (1 mmol/L) plus cAMP (30 µmol/L), AMPCP (100 µmol/L; Sigma), AMPCP (100 µmol/L) plus cAMP (30 µmol/L), DPSPX (100 µmol/L; Research Biochemicals International, Natick, MA), or DPSPX (100 µmol/L) plus cAMP (30 µmol/L). After 1 h of incubation under standard tissue culture conditions, the supernatant was collected, transferred immediately into ice-cold tubes, and frozen at -70°C until adenosine, inosine, and AMP concentrations were estimated. The remaining cells were solubilized in 0.5 N NaOH, and protein content was measured by bicinchoninic acid (BCA)-protein assay kit (Sochochim, Lausane, Switzerland). Oviduct cells treated in parallel were examined microscopically to ensure that the various treatments caused no toxic effects or cell death.
To evaluate whether the conversion of cAMP to AMP, adenosine, and inosine depends on cAMP concentration, we assayed the formation of these metabolites in the supernatant of cells incubated for 60 min with different concentrations (0.01, 1, 10, 30, and 100 µmol/L) of cAMP. To assess whether the conversion of cAMP to AMP, adenosine, and inosine is time dependent, we quantified the formation of these metabolites in the incubation medium of oviduct cells treated with 30 µmol/L cAMP for 0, 1, 5, 10, 20, 40, and 60 min.
To assess whether the conversion of cAMP to adensoine in mixed cultures is due to both epithelial cells and fibroblasts, we assayed and compared the conversion of exogenous cAMP (30 µM) to adenosine by cultured epithelial cells, fibroblasts, and mixed cultures of epithelial cells:fibroblasts (1:1). Monolayers of the various cell populations were treated with 30 µmol/L cAMP, and after 60 min of incubation under standard tissue culture conditions the supernatant was collected and the extracellular concentrations of adensoine were analyzed by HPLC.
Protocols for Physiological Presence of cAMP-Adenosine Pathway
To assess whether stimulation of adenylyl cyclase causes the egress of cAMP, we stimulated oviduct cells with a combination of adenylyl cyclase activators, forskolin (10 µM), and isoproterenol (0.1 µM) in the presence and absence of IBMX (1 mM). Because the stimulatory effects of forskolin on adenylyl cyclase are greatly enhanced by isoproterenol, which activates adenylyl cyclase via beta-adrenergic receptors [20], we used both activators in oviduct cells to obtain a maximal effect. After 4 h of incubation under standard tissue culture conditions, the supernatant was collected and the cells were treated with 1 ml of ice cold propanediol. The concentrations of cAMP in the supernatant (extracellular) and in the propanediol extracts of cells (intracellular) were analyzed by HPLC with a previously described method [27]. To assess whether egress of cAMP into the extracellular space results in the formation of adenosine, we measured adenosine concentrations in the medium of cells stimulated with forskolin plus isoproterenol. Because, adenosine is rapidly metabolized to inosine by adenosine deaminase (a ubiquitous enzyme) and to AMP by adenosine kinase, their presence in oviduct cells may decrease extracellular adenosine under conditions of physiological stimulation and limit its quantification. Therefore, we conducted experiments to identify the presence of these enzymes in oviduct cells by studying basal formation of adenosine in the presence and absence of adenosine deaminase inhibitor EHNA (10 µM) and the adenosine kinase inhibitor iodotubericidin (IDO; 0.1 µM). We also studied the formation of adenosine following adenylyl cyclase stimulation, in the presence and absence of adenosine catabolism inhibitors.
Adenosine, AMP, and Inosine Analysis
We analyzed adenosine, AMP, and inosine concentrations in the samples with an HPLC system using our previously described method [28]. Samples (supernatant) were thawed and centrifuged at 3000 rpm for 15 min, and 80 µl of each sample was injected into an Isco HPLC system (pump model 2350, gradient programmer model 2360, V4 absorbance detector, 4.6- x 250-mm C18 column with 5-µm particle size; ISCO Chem-Research Data Management System, Lincoln, NE). Mobile phase A was 0.1 mol/L KH2PO4 (pH 6.1), and mobile phase B was 80% 0.01 mol/L KH2PO4 (pH 3.5) and 20% methanol. Mobile phase A was maintained at 100% for 11 min, a 2-min linear gradient to 50% mobile phase A was initiated, 50% mobile phase A was maintained for 21 min, a 2-min linear gradient back to 100% mobile phase A was initiated, and 100% mobile phase A was maintained for at least 24 min before the next sample was injected. The eluant was monitored at a wavelength of 254 nm, and AMP, adenosine, and inosine were measured as the area under the chromatographic peak. The concentration of each substance in the samples is presented as nanomoles per liter per milligram protein.
To analyze cAMP and adenosine concentrations in cells treated with adenylyl cyclase activators, we used our HPLC assay system with fluorescent detection and as described previously [29].
Protein Determination
To determine protein concentrations, cells remaining after removal of cell culture supernatant were solubilized in 0.2% SDS, and the protein was assayed with the BCA-protein assay kit using BSA as a standard. Each experiment was conducted in triplicate and repeated three to four times using cell cultures form different pools of fresh oviducts.
Statistical Analysis
Data are presented as mean ± SEM. Data were analyzed by ANOVA using a Statview program (SAS Institute, Inc., Cary, NC). For individual comparisons for significant differences, both the Fisher least significant difference test and the Student-Newman-Keuls posteriori test were performed. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The metabolism of cAMP to AMP, adenosine, and inosine was concentration dependent (Fig. 2) and time dependent (Fig. 3). Addition of cAMP (30 µmol/L) to oviduct cells caused a time-related increase in the extracellular levels of AMP, adenosine, and inosine (Fig. 3). Compared with oviduct cells not treated with cAMP, the concentrations of AMP, adenosine, and inosine increased significantly in samples incubated for 1–60 min. The maximal increases in AMP and adenosine were observed at approximately 1–40 and 10–60 min of incubation, respectively. The increase in inosine levels did not reach a plateau even after 60 min of incubation, and the formation of total products (AMP plus adenosine plus inosine) between 1 and 60 min remained linear (Fig. 3). Compared with the formation of AMP, the formation of adenosine, inosine, and total products did not attain an equilibrium at the same early time point (1 min) and the formation of adenosine and inosine at the early time points (1–5 min) was much lower. During a reaction, the time for a product to reach a steady state depends on its rate of degradation. The rate of conversion of cAMP to AMP is more rapid than that of AMP to adenosine, and the rate of conversion of adenosine to inosine is more rapid than that of inosine to hypoxanthine, xanthine, and uric acid; these differences in rates may be responsible for the differences in the concentratios of AMP, adenosine, and inosine observed at the early time point.
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The metabolism of cAMP to AMP, adenosine, and inosine was also concentration dependent (Fig. 2). Compared with the untreated controls, AMP and adenosine concentrations were significantly different in oviduct cells incubated for 60 min with cAMP concentrations 
0.1 and 1 µmol/L, respectively. Significant concentrations of inosine were present in oviduct cells incubated with 0.01 µmol/L of cAMP, and at concentrations >1 µmol/L, the concentrations of inosine were greater than those for AMP and adenosine and the concentrations of AMP were greater than those for adenosine. No significant difference in metabolite formation were observed for cells treated with 30 or 60 µmol/L of cAMP, suggesting that a saturation point was reached at 30µmol/L of cAMP. Because cAMP is rapidly metabolized, lower concentrations of cAMP may limit our ability to assess the modulatory effects of inhibitors used to investigate the presence of the cAMP-adenosine pathway. Hence, saturating concentrations (30 µM) of cAMP were used for the experiments. At this concentration, cAMP remained stable over the period of incubation, and during a 60-min incubation period approximately 1% of the total substrate was metabolized.
Figures 4 and 5 illustrate the effects of various inhibitors on the metabolism of cAMP to purines. Compared with concentrations in oviduct cells treated with PBS alone (vehicle), the extracellular (medium) concentrations of AMP, adenosine, and inosine increased significantly in oviduct cells treated with 30 µmol/L of cAMP. In vehicle-treated oviduct cells, the concentrations of AMP and adenosine were near or below the assay detection limit, whereas the concentrations of inosine were 28 ± 6 nmol L-1 mg protein-1. In cAMP-treated cells, the concentrations of AMP, adenosine, and inosine were 149 ± 21, 37 ± 2, and 287 ± 13 nmol L-1 mg protein-1, respectively (p<0.05 vs vehicle-treated oviduct cells).
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Consistent with our hypothesis, metabolism of cAMP into AMP, adenosine, and inosine was significantly inhibited by IBMX (1 mmol/L; Figs. 4 and 5), a PDE inhibitor that crosses cell membranes. Metabolism of cAMP to AMP, adenosine, and inosine was also attenuated by DPSPX (0.1 mmol/L; Figs. 4 and 5), a xanthine that inhibits PDE but is restricted to the extracellular compartment [23]. Compared with concentrations in control oviduct cells treated with cAMP (30 µmol/L) in the absence of DPSPX, the concentrations of AMP, adenosine, and inosine were decreased by >50% in oviduct cells treated with cAMP plus DPSPX (P < 0.05), indicating that DPSPX attenuated the metabolism of cAMP to AMP, adenosine, and inosine. Treatment of oviduct cells with cAMP in the presence of the ecto-5'-NT inhibitor AMPCP (0.1 mmol/L) prevented the metabolism of cAMP to adenosine and inosine (Fig. 5) but not to AMP (Fig. 4).
To confirm that the effects of IBMX, AMPCP, and DPSPX on cAMP metabolism to AMP, adenosine, and inosine were due to their inhibitory effects on specific biochemical pathways and not due to cell toxicity, we conducted microscopic examination of oviduct cells treated similarly and in parallel to the metabolic studies. No morphological changes or membrane damage were observed in oviduct cells incubated with PBS alone versus cells treated with IBMX, AMPCP, or DPSPX in the absence and presence of cAMP.
To confirm that the conversion of cAMP to adenosine by mixed cultures was due to both fibroblasts and epithelial cells, the ability of epithelial cells and fibroblasts to metabolize cAMP to adenosine was evaluated. Both epithelial cells and fibroblasts efficiently metabolized cAMP to adenosine (Fig. 6). The epithelial cells were significantly more efficient than the fibroblasts in converting cAMP to adenosine (Fig. 6). In the mixed cultures, the conversion of cAMP to adenosine was approximately the mean of what was observed in epithelial cells and fibroblasts alone.
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Conversion of Endogenous cAMP
Although these findings support our hypothesis that oviduct cells express the extracellular cAMP-adenosine pathway, whether this pathway is active under physiological conditions cannot be inferred from these findings. To provide evidence that this pathway is physiologically active in oviduct cells, we first conducted experiments to demonstrate that activation of adenylyl cyclase causes egress of cAMP into the extracellular space. We treated the cells with a combination of adenylyl cyclase activators, forskolin and isoproterenol. The intracellular and extracellular cAMP concentrations were dramatically increased in cells treated with adenylyl cyclase stimulators compared with cells treated with PBS (vehicle) alone (Fig. 7). Consistent with our hypothesis, the extracellular concentrations of cAMP were several orders of magnitude higher than the intracellular concentrations (P < 0.05) in both stimulated and nonstimulated cells (Fig. 7). The intracellular and extracellular cAMP concentrations in nonstimulated cells were 4.8 ± 0.2 and 30.98 ± 4.6 pmol ml-1 mg protein-1, respectively, and those in stimulated cells were 113 ± 3.44 and 3858 ± 132 pmol ml-1 mg protein-1, respectively. Of the total cAMP produced, the intracellular and extracellular cAMP accounted for 2.8% and 97.2%, respectively. In cells treated with adenylyl cyclase stimulants in the presence of IBMX (inhibits cAMP metabolism by PDE), the intracellular and extracellular concentrations were significantly enhanced (Fig. 7), and the extracellular concentrations remained several orders of magnitude higher than the intracellular concentrations, at 9270 ± 336 and 293 ± 11 pmol ml-1 mg protein-1, respectively.
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To investigate whether egress of cAMP following adenylyl cyclase activation results in adenosine formation, we assayed adenosine concentrations in the medium of cells treated with adenylyl cyclase stimulants in the presence and absence of IBMX. Similar to concentrations of cAMP, the extracellular concentrations of adenosine were significantly higher than the intracellular concentrations under basal conditions and in cells treated with adenylyl cyclase stimulants (Fig. 8A). However, in contrast to concentrations of cAMP, the extracellular concentrations of adenosine did not increase in cells treated with activators of adenylyl cyclase and were similar to concentrations found in cells treated with vehicle (Fig. 8A). Because adenosine is rapidly metabolized to inosine, the lack of increase in extracellular adenosine concentrations in cells treated with adenylyl cyclase activators may be due to its rapid catabolism. To investigate this possibility, we first evaluated the role of adenosine deaminase and adenosine kinase in metabolizing adenosine in oviduct cells. Treatment of cells with EHNA, an adenosine deaminase inhibitor, but not IDO, an adenosine kinase inhibitor, resulted in a dramatic increase in extracellular adenosine (Fig. 8B), suggesting that adenosine deaminase is responsible for catabolizing extracellular adenosine. Consistent with the extracellular cAMP-adenosine pathway hypothesis, in cells treated with activators of adenylyl cyclase in the presence of adenosine catabolism inhibitors the extracellular concentrations of adenosine were dramatically increased from 3.84 ± 0.71 to 6209 ± 685 pmol ml-1 mg protein-1 (Fig. 9A). Similar to concentrations of adenosine, a marked increase in extracellular cAMP concentrations was also observed in cells treated with adenylyl cyclase stimulants in the presence of adenosine catabolism inhibitors (Fig. 9B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because the intracellular and extracellular ATP pathways of adenosine production require crisis events and the transmethylation pathway is mostly constitutive, the three traditional routes of adenosine biosynthesis are not well suited for physiological modulation. However, we have proposed a fourth pathway, the cAMP-adenosine pathway, for adenosine production that would be more amendable to physiological modulation of adenosine levels by hormones [20, 28, 30, 31]. This pathway may be of particular importance in the extracellular space, because the intracellular formation of adenosine may be diminished by the competition of cytosolic 5'-NT and adenylate kinase for AMP and by the competition of transport mechanisms with adenosine kinase for adenosine (Fig. 1). Therefore, the extracellular limb of the cyclic AMP-adenosine pathway may be quantitatively more important.
Ecto-5'-NT is a ubiquitous enzyme that is tethered to the extracellular face of the plasma membrane via a lipid-sugar linkage [22]. Ecto-5'-NT efficiently metabolizes AMP to adenosine, and oviduct cells [18] and the cells of the blood vessels lining the oviduct (endothelial cells, smooth muscle cells, fibroblasts) are well equipped with ecto-5'-NT [22]. Activation of adenylyl cyclase always causes egress of cAMP into the extracellular space [32, 33]. Therefore, provided that sufficient levels of ecto-PDE exist, activation of adenylyl cyclase would trigger the extracellular metabolism of cAMP to AMP and hence to adenosine. Because these reactions would take place in a highly localized environment, this newly formed adenosine could then act in an autocrine and/or paracrine fashion to amplify, inhibit, and/or expand the local response to hormonal stimulation of adenylyl cyclase. Relatively modest increases in cAMP production could give rise to significant concentrations of adenosine at the cell surface.
Our previous studies in the perfused rat renal vascular bed demonstrate that infusion of cAMP causes a concentration dependent increase in the renal secretion rates of AMP, adenosine, and inosine, and the increases in AMP and adenosine secretion are inhibited by IBMX (PDE inhibitor) and DPSPX (ecto-PDE inhibitor at high concentrations). However, the increase in adenosine but not AMP secretion is blocked by AMPCP (ecto-5'-NT inhibitor) [30]. Our studies in contractile cells (vascular smooth muscle cells and cardiac fibroblasts) provide evidence for a cAMP-adenosine pathway in the cardiovascular system [28].
Because oviduct cells contain both receptor-activated adenylyl cyclase [4, 11] and ecto-5'-NT [34], oviduct cells may also be an important cell type for supporting a cAMP-adenosine pathway. In the present study, we addressed this hypothesis in bovine oviduct cells. In oviduct cells incubated with exogenous cAMP, extracellular concentrations (i.e., those in the medium) of AMP, adenosine, and inosine were increased several fold, and the increases in AMP, adenosine, and inosine were blocked by inhibition of PDE with IBMX and of ecto-PDE with DPSPX. AMPCP blocked the metabolism of exogenous cAMP to adenosine and inosine but not to AMP. These findings are consistent with the hypothesis that the cAMP-adenosine pathway exists in oviduct cells and contributes to the production of adenosine.
The finding that the concentrations of AMP, adenosine, and inosine are increased by addition of cAMP whereas blockade of PDE by IBMX inhibits this process is highly consistent with the existence of the cAMP-adenosine pathway. Several lines of evidence support the suggestion that the metabolism of exogenous cAMP to adenosine occurs mainly in the extracellular space. First, because cAMP is hydrophilic, exogenous cAMP should not penetrate cell membranes, and its conversion to adenosine most likely takes place extracellularly. Second, because AMPCP only inhibits ecto-5'-NT and not endo-5'-NT, the blockade of cAMP metabolism to adenosine by AMPCP is consistent with an extracellular site of metabolism. Third, because DPSPX has a negative charge at physiological pH and is restricted to the extracellular space [32], inhibition by DPSPX of the conversion of exogenous cAMP to AMP and adenosine further supports an extracellular site of metabolism.
The physiological and biological relevance of the extracellular cAMP-adenosine pathway in the oviduct cells is supported by the finding that activation of adenylyl cyclase by forskolin plus isoproterenol resulted in the egress of cAMP into the extracellular space and efficient conversion to adenosine. The fact that conversion of cAMP to adenosine in the extracellular compartment following adenylyl cyclase activation was only detected in the presence of adenosine metabolism inhibitors suggests that adenosine is rapidly metabolized by oviduct cells. More importantly, the finding that inhibition of adenosine deaminase but not adenosine kinase activity resulted in a significant increase in extracellular adenosine levels suggests that adenosine deaminase may play an important role in regulating active extracellular adenosine levels in oviduct cells. Because abnormalities in adenosine deaminase activity have been associated with the pathophysiology of vascular occlusion and abnormal growth [35], it is tempting to speculate that abnormalities in adenosine deaminase activity may influence the physiologic actions of adenosine and induce deleterious effects on the fertilization process within the oviduct. However, further study is required to evaluate the effects and role of adenosine and adenosine deaminase in the pathophysiology of reproduction.
Although the above findings provide evidence that extracellular conversion of cAMP to adenosine, they also indicate that the cells have considerable capacity for converting adenosine to inosine, so that local adenosine concentrations might be quite low. This hypothesis is further supported by our finding that adenosine deaminase, which metabolizes extracellular adenosine to inosine, is very active in oviduct cells. However, in a biological system, when the cAMP-adenosine system is activated concentrations of adenosine at the biophase (unstirred layer) of the membrane would be adequate to activate adenosine receptors, even though adenosine is metabolized to inosine as it diffuses out. Thus, high adenosine deaminase activities provide a rapid off switch for halting adenosine-mediated signaling once cAMP production ceases. The extracellular concentrations of adenosine measured in the culture medium may underestimate the true concentrations within the unstirred biophase of the membrane, which are not measurable.
The finding that oviduct cells can effectively increase extracellular levels of adenosine suggests that generation of adenosine locally at the surface of oviduct cells may be a critical mechanism by which oviduct cells enrich the profertilization microenvironment and regulate ciliary beat frequency, sperm motility and capacitation, fertilization, and early embryo development and transport. Decreased concentrations of cAMP have been associated with ectopic pregnancy and pseudopregnancy [19]. Adenosine concentrations in mouse uterus increase significantly during preimplantation, and these increases are associated with increased 5'-NT expression [22]. These findings suggest that the cAMP-adenosine pathway plays an active role in regulating the biology and physiology of the oviduct and acts as an autocrine/paracrine factor to facilitate fertilization and early embryo development and transport.
Increases in intracellular and extracellular concentrations of cAMP in presence of adenylyl cyclase activators were markedly enhanced in presence of adenosine catabolism inhibitors, suggesting that cAMP-derived adenosine can stimulate adenylyl cyclase and induce intracellular cAMP concentrations via a positive feedback mechanism involving the activation of A2 adenosine receptors (Fig. 1), and this ability may be of physiological/biological relevance. In this context, hormone-stimulated activation of the adenylyl cyclase could trigger a cascade of events that would result in sustained generation of cAMP via the cAMP-adenosine pathway and may play a critical role in regulating the fertilization process. Abnormalities or dysfunction of this mechanism may have deleterious effects and may be associated with infertility.
The results of this study provide the first evidence for the presence of the extracellular cAMP-adenosine pathway in oviduct cells. Hormonal activation of this pathway may play a critical role in regulating oviduct biology and physiology associated with fertilization. Pharmacological augmentation of this pathway could abate infertility associated with tubal dysfunction/abnormalities.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Marinella Rosselli, Department of Obstetrics and Gynecology, Clinic for Endocrinology (NORD1, D-217), University Hospital Zurich, Frauenklinikstrasse 10, 8091 Zurich, Switzerland. FAX: 41 1 255 4439; marinella.rosselli{at}fhk.usz.ch
Received: 10 April 2003.
First decision: 11 April 2003.
Accepted: 14 April 2003.
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P < 0.05 compared with control oviduct cells not treated with camp; 
P < 0.05 compared with control oviduct cells treated with cAMP
