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. 2011 Aug;25(8):2823–2830. doi: 10.1096/fj.11-181057

A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation

Jiaming Wen *,†, Almut Grenz ‡, Yujin Zhang *, Yingbo Dai *,†, Rodney E Kellems *, Michael R Blackburn *, Holger K Eltzschig ‡, Yang Xia *,1
PMCID: PMC3136334  PMID: 21566208

Abstract

Normal penile erection is under the control of multiple factors and signaling pathways. Although adenosine signaling is implicated in normal and abnormal penile erection, the exact role and the underlying mechanism for adenosine signaling in penile physiology remain elusive. Here we report that shear stress leads to increased adenosine release from endothelial cells. Subsequently, we determined that ecto-5′-nucleotidase (CD73) is a key enzyme required for the production of elevated adenosine from ATP released by shear-stressed endothelial cells. Mechanistically, we demonstrate that shear stress-mediated elevated adenosine functions through the adenosine A2B receptor (A2BR) to activate the PI3K/AKT signaling cascade and subsequent increased endothelial nitric oxide synthase (eNOS) phosphorylation. These in vitro studies led us to discover further that adenosine was induced during sustained penile erection and contributes to PI3K/AKT activation and subsequent eNOS phosphorylation via A2BR signaling in intact animal. Finally, we demonstrate that lowering adenosine in wild-type mice or genetic deletion of A2BR in mutant mice significantly attenuated PI3K/AKT activation, eNOS phosphorylation, and subsequent impaired penile erection featured with the reduction of ratio of maximal intracavernosal pressure to systemic arterial pressure from 0.49 ± 0.03 to 0.41 ± 0.05 and 0.38 ± 0.04, respectively (both P<0.05). Overall, using biochemical, cellular, genetic, and physiological approaches, our findings reveal that adenosine is a novel molecule signaling via A2BR activation, contributing to penile erection via PI3K/AKT-dependent eNOS activation. These studies suggest that this signaling pathway may be a novel therapeutic target for erectile disorders.—Wen, J., Grenz, A., Zhang, Y., Dai, Y., Kellems, R. E., Blackburn, M. R., Eltzschig, H. K., Xia, Y. A2B adenosine receptor contributes to penile erection via PI3K/AKT signaling cascade-mediated eNOS activation.

Keywords: shear stress, vascular tone, CD73, erectile function

Normal penile erection is under the control of multiple factors and signaling pathways. Nitric oxide (NO) is the best characterized molecule to regulate penile erection. In the penis, NO is produced by neuronal NO synthase (nNOS) and endothelial NO synthase (eNOS) (1, 2). nNOS-induced NO production is well established and mediated by neuronal activation and contributes to initiation of penile erection by inducing cavernosal smooth muscle relaxation (1). Neuron-derived NO is quite transient and is considered to be involved in initiation of penile erection, whereas endothelial cell-derived NO by activation of eNOS, which is abundant in endothelial cells lining penile vessels and the trabecular network, lasts much longer and leads to sustained penile erection. One of the most important stimuli for continuous formation of NO is the viscous drag or shear stress generated by the streaming blood on the endothelial layers (3). However, the specific molecules responsible for shear stress-mediated NO production and the maintenance of penile erection remain unidentified.

One molecule known to be released by shear stress from endothelial cells is ATP (4), which is quickly converted to adenosine by ectonucleotidases. Adenosine has long been implicated in normal and abnormal penile erection (5). Earlier studies in multiple animal species, including humans (6), showed that intracavernous injection of exogenous adenosine resulted in tumescence and penile erection (7–12). Adenosine elicits its effects on target cells by engaging specific G-protein-coupled adenosine receptors, A1R, A2AR, A2BR, and A3R (13). More recent in vitro studies show that impaired A2BR signaling is associated with erectile dysfunction in men (14), and our mouse studies revealed that excess adenosine contributes to priapism via A2BR signaling (15). However, whether shear stress results in increased production of adenosine and whether elevated adenosine is responsible for eNOS phosphorylation and sustained penile erection have not been determined. Here, we sought to investigate whether adenosine is a candidate contributing to shear stress-mediated eNOS activation for normal penile erection and the underlying mechanisms.

MATERIALS AND METHODS

Mice

A2BR-deficient mice were obtained from Michael R. Blackburn (University of Texas Medical School, Houston, TX, USA). These mice were backcrossed ≥10 generations onto the C57BL/6 background and were genotyped according to established protocols (16). For all studies, wild-type (WT) C57BL/6 mice were used as controls. Mice were maintained and housed in accordance with U.S. National Institutes of Health guidelines and with the approval of the Animal Care and Use Committee of the University of Texas Health Science Center at Houston.

Cavernous nerve stimulation and intracavernosal pressure (ICP) measurement

Mice were anesthetized by intraperitoneal injection with 250 mg/kg Avertin. Bilateral cavernous nerve, located posterolateral to the prostate, was identified and isolated. The shaft of the penis was freed of skin and fascia. Small portions of the ischiocavernous muscles were dissected bilaterally to expose each penile crus. Electric stimulation of the cavernous nerve was carried with a bipolar silver electrode, positioned by a micromanipulator and placed around the cavernous nerve. Electrical stimulation was delivered by a Grass stimulator (AD Instruments Inc., Colorado Springs, CO, USA) at 4 voltages with 16 Hz and 5 ms duration for 4 min to induce penile erection (3, 17, 18).

To monitor ICP, the right corpus cavernosum was penetrated by a 25-gauge needle and a heparinized (150 U/ml) mouse jugular catheter (Alzet Osmotic Pumps, Palo Alto, CA, USA) was inserted into the right corpus cavernosum. The cannula inserted into the right corpus cavernosum was connected to a pressure transducer and an amplifier unit. The amplifier was connected to a data acquisition module. The ICP before and after electrical stimulation was recorded on a computer by Chart 5 software (AD Instruments). For intracavernosal drug administration, a separate cannula (30.5-gauge needle, attached to PE-10 tubing and 25-μl Hamilton syringe; Hamilton, Reno, NV, USA) was inserted into the left corpus cavernosum. Polyethylene glycol–adenosine deaminase (PEG-ADA; 2 U) was prepared in a volume of 20 μl of PBS for injection. At 10 min after the injection, ICP was monitored following electrical stimulation.

Systemic mean arterial pressure (MAP) measurement

ICP data were normalized to systemic MAP, which was monitored simultaneously with ICP measurements. The right carotid artery was dissected via a midline cervical incision under the microscope, and a mouse jugular catheter was inserted into the carotid artery. The catheter was connected to a pressure transducer and an amplifier unit. The amplifier was connected to a data acquisition module, and MAP was recorded simultaneously with ICP monitoring on a computer by Chart 5 software (AD Instruments).

PEG-ADA preparation

PEG-ADA was generated by the covalent modification of purified bovine ADA with activated PEG, as described previously (19).

HPLC analysis of tissue extracts and adenine nucleotides

The penes of mice were removed rapidly before and immediately after electrical stimulation and quick-frozen in liquid nitrogen containing a cocktail including 10 μM 2′-deoxycoformycin (DCF; Sigma-Aldrich, St. Louis, MO, USA) to inhibit adenosine deaminase, 10 μM α,β-methylene ADP (APCP) to inhibit ecto-5′-nucleotidase (CD73), and 10 μM dipyridamole to inhibit equilibrium nucleotide transporter activity (20). Nucleotides were extracted from frozen penes using 0.4 N perchloric acid as described previously (15). Tissue extracts and adenine nucleotides were separated and quantified by reverse-phase HPLC (Waters; Millipore Corp., Billerica, MA, USA) analysis on a Partisphere bonded-phase C18 (reverse-phase) cartridge column at a flow rate of 1.5 ml/min (21, 22). To measure adenosine levels in human microvascular endothelial cell (HMEC-1) culture medium, adenosine was fractionated and quantified using reverse-phase HPLC, as described previously (15).

ATP measurement

The determination of ATP levels in HMEC culture medium was assessed by ATP Bioluminescent Assay Kit (Sigma-Aldrich; ref.23).

Total RNA isolation and real-time RT-PCR analysis

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNase-free DNase (Invitrogen) was used to eliminate genomic DNA contamination. Transcript levels were quantified using real-time quantitative RT-PCR. Adenosine receptor transcripts were analyzed using Taqman probes, with primer sequences and conditions as described previously (24–26).

Sheer stress studies using HMEC-1 cells

HMEC-1 cells (a kind gift of Francisco Candal, Centers for Disease Control, Atlanta, GA, USA; ref. 27) were plated on BioFlex collagen type I culture plates (BF-3001C; FlexCell International, Hillsborough, NC, USA). These cells are derived from dermal tissues and were harvested and cultured by a modification of methods described previously (27). HMEC-1 cells were allowed to attach and grow to 90% confluence. The medium was changed to MEM plus 1% FBS. Plates were then placed on a FlexCell FX-4000T Tension Plus System and stretched at 45 dyn/cm2 for 2 h with a sine wave 5 s on and 5s off (4, 28). Some of the cells were treated with APCP, LYS294002, and MRS1706 (each 10 μM) 1 h before inducing sheer stress by adding the compound into the medium. ATP and adenosine levels were measured in the culture medium as described above, while cellular AKT and eNOS phosphorylation were monitored by ELISA kits, as described above.

Phospho-S1177-eNOS and phospho-S473-AKT measurement

The penile tissues or HMEC-1 cells were sonicated in 0.3 ml lysis buffer containing complete protease inhibitors and phosphatase inhibitors (Cell Signaling Technology, Danvers, MA, USA) for 2 min at 25 Hz. The lysate was centrifuged at 20,000 g for 10 min at 4°C. The supernatant was transferred to a new tube for phospho-S1177-eNOS and phospho-S473-AKT detection by PathScan Phospho-eNOS (Ser1177) Sandwich ELISA Kit (Cell Signaling Technology) and PathScan Phospho-Akt1 (Ser473) Chemiluminescent Sandwich ELISA Kit (Cell Signaling Technology), which accurately and sensitively measure phosphorylation of both eNOS and AKT, respectively (29–32).

Statistical analysis

All data are expressed as means ± se. Data were analyzed for statistical significance using GraphPad Prism 4 software (GraphPad Software, San Diego, CA, USA). Student's t tests (paired or unpaired as appropriate) were applied in 2-group analysis. Differences between the means of multiple groups were compared by 1-way analysis of variance (ANOVA), followed by a Tukey's multiple comparisons test. A value of P < 0.05 was considered significant and was the threshold to reject the null hypothesis.

RESULTS

Increased ATP release and elevated CD73 expression are dual factors contributing to increased extracellular adenosine production by shear-stressed endothelial cells

To determine whether shear stress can induce adenosine production by endothelial cells, HMEC-1 cells were exposed to sheer stress at 45 dyn/cm2 for 2 h, as described previously (4, 28), and the concentration of adenosine in the culture medium was determined. We found that shear stress resulted in a significant increase in extracellular adenosine levels (Fig. 1A). Next, to determine whether elevated adenosine produced in response to shear stress is dependent on CD73, HMCE-1 cells were subjected to shear stress in the presence or absence of APCP, an inhibitor of CD73. We found that in the presence of APCP, the shear stress-induced adenosine production was inhibited significantly (Fig. 1A). In contrast, the concentration of extracellular ATP was elevated further (Fig. 1B). In addition, when we extended shear stress from 2 to 24 h, we found that CD73 mRNA expression levels were also elevated significantly (Fig. 1C). Collectively, these findings provide the direct evidence that that sheer-stress mediated ATP release coupled with elevated CD73 gene expression are dual mechanisms underlying the elevated extracellular adenosine under shear stress condition.

Figure 1.

Figure 1.

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CD73 is a key enzyme involved in converting shear stress-mediated released ATP to adenosine. A, B) Adenosine (A) and ATP (B) levels in the supernatants of cultured HMEC-1 cells with or without shear stress in the presence or absence of APCP (CD73 specific inhibitor). C) CD73 gene expression is induced by shear stress in endothelial cells. One group of HMEC-1 cells was subjected to shear stress for 24 h, and a control group was not. CD73 gene expression was determined for each group by quantitative RT-PCR. Data are expressed as means ± se; n = 5–7. *P < 0.05 vs. control; #P < 0.05 vs. shear stress.

Adenosine is responsible for shear stress-mediated eNOS activation via PI3K/AKT signaling cascade in endothelial cells

Because adenosine receptors are hinted to be involved in PI3K cascade (33, 34), it is possible that shear stress-induced adenosine is an unrecognized molecule responsible for shear stress-induced eNOS activation via the PI3K/AKT signaling cascade. To test this possibility, endothelial cells were subjected to shear stress in the presence or absence of APCP or LY294002 (a specific PI3K inhibitor). To measure phosphorylation of eNOS and AKT, we took advantage of ELISA kits which are known to accurately and sensitively monitor phosphorylation of these signaling molecules (29–32). Consistent with early studies, we found that LY294002 inhibited sheer stress-induced eNOS-S1177 and AKT-S473 phosphorylation (Fig. 2A, B). Intriguingly, APCP inhibited shear stress-mediated eNOS-S1177 and AKT-S473 phosphorylation to the same extent as LY294002 (Fig. 2A, B), suggesting that adenosine generated by shear stress is responsible for eNOS activation via PI3K/AKT signaling.

Figure 2.

Figure 2.

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Adenosine activation of A2BR underlies shear stress-induced eNOS activation via PI3K/AKT signaling cascade. A, B) In vitro evidence of shear stress-mediated increased adenosine responsible for eNOS activation via A2BR activation and PI3K/AKT signaling. ELISA measurement of eNOS phosphorylation at S1177 (A) and AKT phosphorylation at S473 (B) in HMEC-1 cells without or with shear stress. Some of the cells with shear stress were treated with LY294002, APCP, or MRS1706. C) Adenosine receptor mRNA expression profiles in HMEC-1 cells were determined by quantitative real-time RT-PCR. Data are expressed as means ± se; n = 5–7. *P < 0.05 vs. control; #P < 0.05 vs. shear stress.

A2BR is responsible for shear stress-mediated eNOS activation via PI3K/AKT signaling cascade in endothelial cells

Adenosine is a signaling molecule functioning through 4 G-protein-coupled adenosine receptors (13, 35, 36). To identify which adenosine receptor is responsible for adenosine-mediated PI3K/AKT eNOS activation with shear stress, first, we used quantitative RT-PCR to determine the expression profile of adenosine receptors in endothelial cells. The results showed that the A2BR was the major adenosine receptor present on these cells (Fig. 2C), suggesting that the A2BR is likely a key receptor sensing elevated adenosine produced by shear stress to induce eNOS activation via the PI3K-AKT signaling cascade. To test this possibility, we employed MRS1706 (an A2BR specific antagonist) to block the A2BR signaling pathway in HMCE-1 cells. We found that MRS1706 significantly inhibited shear stress-induced phosphorylation of eNOS at S1177 and AKT at S473 (Fig. 2A, B), indicating that adenosine A2BR signaling is required for shear stress-induced PI3K/AKT-eNOS activation. Altogether, we determined that adenosine, produced from ATP released by shear stress, functions through the A2BR to induce eNOS activation by regulating the PI3K/AKT signaling cascade in endothelial cells.

Cavernous nerve stimulation induces adenosine production in penile tissue

To test the significance of our in vitro findings, we conducted in vivo experiments to determine whether adenosine levels increase during sustained penile erection. For these studies, we used a well-established method to mimic normal physiological erection by electrical stimulation (ES) of the cavernous nerve to induce an erectile response (3, 17, 18). Flaccid penile tissue was obtained as a control from sham-operated unstimulated mice. At the end of ES, the penile tissues were quickly collected, and adenosine levels were measured. Consistent with our in vitro shear stress experiments, we found that adenosine was elevated significantly in the erected penile tissues compared to the flaccid penile tissues (Fig. 3A). These findings provide the first in vivo evidence that adenosine production increases significantly during penile erection.

Figure 3.

Figure 3.

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Electrical stimulation-mediated elevation of endogenous adenosine in mouse penis contributes to normal penile erection. A) Adenosine was significantly increased in mouse penile tissue following ES; n = 5–7. B) Representative ICP tracing after cavernous nerve stimulation at 4 V for 4 min. C, D) PEG-ADA treatment reduced the maximum ICP (C) and total ICP (D). Data are expressed as means ± se; n = 8–10. *P < 0.05.

Elevated adenosine production contributes to normal penile erection

Next, to determine the physiological role of increased adenosine generated by cavernous nerve stimulation in normal penile erection, ES was performed following intracavernosal injection of either PBS or PEG-ADA (degrades adenosine). We found that intracavernosal injection of PEG-ADA, in contrast to PBS, resulted in a significantly decreased maximal ICP/MAP ratio, from 0.49 ± 0.03 to 0.41 ± 0.05, as well as total ICP (area under the curve), from 5061.13 ± 1261.67 to 3060.89 ± 646.41 mmHg · s, which reflects the maintenance of penile erection, indicating that increased adenosine contributes to maintenance of normal penile erection (Fig. 3B–D). Thus, we have provided the in vivo evidence that cavernous nerve stimulation-induced elevation of endogenous adenosine in penile tissue contributes the maintenance of normal penile erection.

A2BR is required for cavernosal nerve stimulation-mediated penile erection

In vitro studies revealed that the A2BR is critical for shear stress-mediated eNOS activation in endothelial cells (Fig. 2A, B), suggesting that A2BR signaling may contribute to normal penile erection. To determine the in vivo significance of A2BR signaling in normal penile erection, we took advantage of A2BR-deficient mice. Similar to PEG-ADA treatment, we found that genetic deletion of A2BR led to significantly decreased penile erection, with a significant decrease in maximal ICP/MAP ratio, from 0.49 ± 0.03 to 0.38 ± 0.04, as well as total ICP (area under the curve), from 5061.13 ± 1261.67 to 3782.12 ± 1022.44 mmHg · s (Fig. 4), indicating that elevated adenosine-mediated A2BR activation contributes to normal penile erection.

Figure 4.

Figure 4.

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Imparied erectile function in A2BR-deficient mice. A) Representative ICP tracing after cavernous nerve stimulation at 4 V for 4 min. B, C) A2BR-deficient mice had a reduced maximum ICP (B) and total ICP (C). Data are expressed as means ± se; n = 8–10. *P < 0.05.

Genetic deletion of A2BR down-regulates PI3K/AKT-eNOS signaling cascade

Finally, to test the in vivo significance of A2BR-mediated PI3K/AKT signaling in eNOS activation, we measured AKT and eNOS phosphorylation in WT and A2BR-deficient mice with or without cavernous nerve stimulation, as described above. Similar to in vitro findings, we found that eNOS-S1177 and AKT-S473 phosphorylation increased significantly in penile tissues isolated from WT mice in response to cavernous nerve stimulation (Fig. 5A, B), indicating that ES-induced blood influx into the penile tissue generates shear stress, which results in PI3K/AKT-eNOS activation in vivo. In contrast, the level of eNOS-S1177 and AKT-S473 phosphorylation was remarkably reduced in penile tissues isolated from A2BR-deficient mice compared to those of WT mice following cavernous nerve stimulation (Fig. 5A, B). However, no significant differences of eNOS-1177 and AKT-S473 phosphorylation were found in the penile tissues among WT and A2BR-deficient mice in the absence of cavernous nerve stimulation (Fig. 5A, B). Overall, these findings reveal the in vivo essential roles of A2BR-mediated PI3K/AKT signaling cascade in eNOS activation during penile erection.

Figure 5.

Figure 5.

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In vivo significance of A2BR signaling in eNOS activation following cavernous nerve stimulation. A, B) ELISA measurements of eNOS phosphorylation at S1177 (A) and AKT phosphorylation at S473 (B) in penile tissues of WT and A2BR-deficient mice. Data are expressed as means ± se; n = 6–8. *P < 0.05 vs. WT; #P < 0.05 vs. WT + ES. C) Working model of endogenous adenosine generation by shear stress and its function in normal penile erection. Sheer stress-mediated ATP release coupled with increased CD73 gene expression results in increased endogenous adenosine production during penile erection. Elevated adenosine, activating A2BRs on endothelial cells, results in the activation of the PI3K/AKT-eNOS signaling cascade, increased production of NO, and sustained penile erection.

DISCUSSION

In this study, we demonstrate that endogenously produced adenosine contributes to the penile erection via A2BR-mediated eNOS activation in a PI3K/AKT-dependent manner. Significantly reduced erectile function was observed following depletion of endogenous adenosine in WT mice with the intracavernosal injection of PEG-ADA or elimination of A2BR signaling by genetic deletion of the A2BR in mutant mice. Mechanistically, using in vitro study, we demonstrate that increased ATP release and elevated CD73 expression levels are dual mechanisms underlying shear stress-mediated elevation of extracelluar adenosine production by endothelial cells and elevated adenosine signaling via A2BR, responsible for eNOS activation via PI3K/AKT signaling in shear-stress-endothelial cells (Fig. 5C). Thus, our studies reveal a previously unrecognized role for endogenous adenosine in normal penile erection, offer underlying mechanisms for its generation and function in this process, and suggest novel therapeutic strategies for erectile disorder.

Subsequent to initiation of penile erection, blood flow-mediated sheer stress of endothelial cells contributes to the maintenance of penile erection by the activation of PI3K/AKT signaling and the activation of eNOS (37). One molecule known to be released by shear stress from endothelial cells is ATP (4), which is quickly converted to adenosine. However, a role for adenosine as the mediator linking shear stress with eNOS activation was unknown until our studies reported here. We show here that shear stress results in the release of ATP by endothelial cells and the subsequent conversion of ATP to adenosine in an extracellular pathway requiring CD73. In addition, we found that shear stress results in elevation of CD73 gene expression. Thus, both increased ATP release and elevated CD73 gene expression are causative factors contributing to elevation of extracellular adenosine production by shear-stressed endothelial cells. Consistent with in vitro studies, we provide in vivo evidence that adenosine is elevated in erected penile tissue compared to flaccid penile tissue. Thus, we demonstrate for the first time that shear stress-mediated elevation of adenosine production likely contributes to increased adenosine levels during physiological penile erection.

Following neuronal activation-mediated initiation of erection, increased blood flow to the corpus cavernosum generates shear stress on endothelial cells, resulting in the activation of eNOS (3). The activation of eNOS results in a sustained production of NO that promotes cavernosal smooth muscle relaxation and the maintenance of erection. But how shear stress mediates eNOS activation in endothelial cells remained a mystery until our studies revealed a role for adenosine signaling in this process. Consistent with early studies (38), we found that A2BR is a major adenosine receptor expressed in human microvascular cells. Moreover, our findings reveal a novel working model in which endothelial shear stress-mediated release of ATP that is converted rapidly to adenosine plays an important role in a sustained penile erection via A2BR signaling. Specifically, we demonstrate that the increased extracellular adenosine signals through the A2BR to activate the PI3K/AKT signaling cascade resulting in eNOS phosphorylation and activation. Thus, our findings identify shear stress-induced adenosine is a key molecule signaling via A2BR activation to induce eNOS phosphorylation via PI3K/AKT signaling cascade.

Functionally, we demonstrated that elevated adenosine contributes to normal penile erection by inducing eNOS phosphorylation via PI3K/AKT signaling cascade. Specifically, we found that lowering adenosine by PEG-ADA enzyme injection in WT mice significantly reduced normal penile erection induced by cavernous nerve stimulation. Similarly, mice lacking the A2BR also display significantly reduced erectile function. Consistent with our in vitro findings, we found that cavernous nerve stimulation-mediated increased AKT and eNOS phosphorylation in WT mouse penile tissue was decreased significantly in the penile tissues from A2BR-deficient mice. Our study is supported by earlier studies in multiple animal species, including humans (6), showing that intracavernous injection of exogenous adenosine resulted in tumescence and penile erection (7–12). Of significance, our studies are also supported by human studies showing that impaired A2BR signaling is associated with erectile dysfunction in men (14) and mouse studies showing that excess adenosine contributes to priapism via A2BR signaling (15). Taken together, our studies reveal physiological role of adenosine via A2BR signaling in penile erection by induction of eNOS phosphorylation and support a novel concept that impaired adenosine signaling may be a causative factor for erectile disorder.

We have revealed that CD73 is a key ectoenzyme responsible for the production of endogenous adenosine from ATP released from shear-stressed endothelial cells and lower adenosine by PEG-ADA lead to decrease penile erection in mice. Supporting the important role of adenosine in penile erection, an early human in vitro study indicated that impaired A2BR signaling contributes to erectile dysfunction (14), and here we provide mouse in vivo evidence that A2BR contributes to penile erection via eNOS activation and that A2BR-deficient mice display impaired erectile function. However, neither CD73- nor A2BR-deficient mice presented significantly reduced fertility. Supporting the mouse finding, a recent study reported that affected males with inherited CD73 deficiency still reproduce (39). Thus, these studies imply that other factors besides CD73 control adenosine production and/or other signaling pathways besides A2BR signaling may contribute to normal penile erection via eNOS activation. For example, previous study have revealed a role of ADP-mediated feed-forward inhibition of AMPase (CD73) activity, suggesting that other ectonucleotidases besides CD73 may affect nucleotide scavenging (40). Additional factors can affect adenosine production, including equilabrative transporters, which shuttle adenosine in and out of cells, and adenosine deaminase, which converts adenosine to the presumably inactive nucleoside inosine. Thus, extracellular adenosine production is a complex process involved in multiple sequential enzyme reactions and nucleotide transporters. Moreover, Silva et al. (41) have revealed that P2Y1, P2Y2, and possibly P2Y4 are involved as purinergic receptors in eNOS phosphorylation and increased NO generation. Thereby, P2 receptor-mediated eNOS activation may be another signaling pathway contributing to penile erection.

In summary, our work identified a previously unrecognized role of adenosine signaling in penile erection and revealed the underlying mechanisms accounting for adenosine production and signaling pathways involved in this process. It is likely that the significance of our findings is not limited to penile physiology and pathology, but extends to a more general role for adenosine signaling in the regulation of vascular tone. For example, the release of ATP by endothelial cells in response to shear stress, its conversion to adenosine, and the activation of eNOS are also likely to be important signaling pathways in controlling vascular tone. Thus, our studies not only discover a new player contributing to normal penile erection, but also have substantial relevance and implications for the mechanisms by which adenosine regulates vascular tone. Our findings also reveal novel therapeutic possibilities for the treatment of disorders of erectile function and vascular tone.

Acknowledgments

This work was supported by U.S. National Institute of Health grants DK077748 and DK083559 (to Y.X.), HL070952 (to M.R.B.), and HL092188 (to H. E.) and China Scholarship Council grant 2008637068 (to J.W.). The authors declare no conflicts of interest.

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