The Essential Role Of Adenosine A_2B Receptor Signaling In Priapism And Penile Fibrosis

BackgroundPriapism is defined as abnormally prolonged and persistent penile erection in absence of sexual provocation or desire. This disorder is considered as a urological emergency which need early treatment to avoid the risks of structural damage of the penis and erectile dysfunction (ED), a known complication. Priapism is rarely been seen in general population, but it is associated with a number of different medical conditions, such as sickle cell disease (SCD), ED pharmacotherapeutic complications, neurological disease, and solid malignancies. Due to lack of fundamental understanding of the etiology and pathophysiology of priapism, effective approaches to manage the disorder are limited. Thus, mechanism-based approaches which aim at preventing and curing priapism are needed.Nitric oxide (NO) is the best characterized molecule to regulate penile erection, but that does not exclude a role of other signaling molecules, either released by neruons, and/or endothelial and smooth muscle cells in the regulation of this process. Recently, our lab unexpectedly found that mice deficient in the purine catabolic enzyme adenosine deaminase (ADA) exhibited priapism seen in human, which suggest adenosine may play a key role in priapism and normal penile erection and indicated ADA-deficient mice could be employed as a novel and important model to study priapism like the other well-established animal model of priapism, SCD transgenic (Tg) mice. We further demonstrated that excess adenosine in penile tissue contributed to priapism through adenosine A2B receptor and decreasing adenosine level in penis by ADA enzyme therapy could prevent and reverse the occurrence of priapism. These results all suggested that adenosine signaling pathway plays an important role in the regulation of normal and abnormal penile erection.Adenosine is a well-known potent vasodilator that elicits its physiological effects on target cells by engaging its four specific G-protein-coupled receptors:A1, A2A, A2B, and A3. ADA is the key enzyme in adenosine metabolism, which catalyses irreversible conversion of adenosine to inosine. Based on our previous studies, adenosine A2B receptor is the main adenosine receptor contributing to priapism,which indicate that adenosine A2B receptor signaling pathway may be a novel therapeutic target for erectile disorders, especially priapism and penile fibrosis.In the penis of patients with ischemic priapism, the corpus cavernosum and vascular endothelial cells are exposed to intermittent hypoxic episodes. Hypoxia-inducible factor-1(HIF-1) is a homodimeric transcriptional complex which promotes expression of numerous genes which involve in oxygen and energy-providing substrates supplements under hypoxia condition. And adenosine is also highly induced under ischemic and hypoxia condition, which could regulate HIF-1α expression. It would be quite interesting to determine whether HIF-1α involves in the pathogenesis of priapism.To extend our findings to therapeutic possibility for priapism and penile fibrosis, we choose adenosine A2B receptor specific antagonist PSB1115, which could be used in vivo research, and took the advantages of two independent priapic animal model, ADA-deficient mice and SCD Tg mice to evaluate the efficiency of PSB1115treatment to priapism and penile fibrosis in vivo and to determine the role of adenosine-HIF-1α-eNOS signaling pathway in priapism.MethodOur study is generally consisted of four parts.1. Genotyping of ADA-deficient mice and SCD Tg mice, two well-accepted animal models of priapism.1) We took advantage of ADA-deficient mice which generated from our lab as an animal models of priapism. We routinely treat the ADA-deficient mice with PEG-ADA to make them survive and reproductive system mature. In this study, zymogram analysis was employed to detect the ADA activity at7th days after birth to determine the newborn mice’s genotyping.2) SCD Tg mice express exclusively human sickle hemoglobin and have the major features found in humans with sickle cell disease. For genotyping of these mice, two methods including blood smear test and PCR are used at6weeks after birth.2. Therapeutic effects of adenosine A2B receptor specific antagonist PSB1115on priapism in mice were observed.ADA-deficient mice and SCD Tg mice were treated with PSB1115(10mg.kg-1.d-1) by intraperitoneal injection for2weeks. These mice were divided into6groups (n=5):1) Control:age-matched wild-type (WT) C57BL/6mice+saline2) Control+PSB1115:age-matched wild-type (WT) C57BL/6mice+PSB11153) ADA-/-:ADA-deficient mice+saline4) ADA-/-+PSB1115:ADA-deficient mice+PSB11155) SCD:SCD Tg mice+saline6) SCD+PSB1115SCD Tg mice+PSB1115A well-established technique was used in vivo to mimic normal penile erection by measuring the intracavernosal pressure (ICP) to assess the erectile response to cavernous nerve stimulation. Maximum ICP, total ICP (area under curve) and slope were detected as the main parameters for evaluating the erectile function. HPLC was used to measure the adenosine level in penile tissue.3. To determine the underlying mechanism of adenosine mediated priapism via adenosine A2B receptorStudies of in vivo and in vitro were employed. Penile tissues were collected from6groups. HIF-1α and eNOS gene expressions were measured. Human microvascular endothelial cell-1(HMCE-1) was treated with NECA (adenosine analogy) and/or PSB1115and observed the variation of HIF-1α and eNOS gene expression. HMCE-1with HDF-1α knocked down was used to determine the regulation effect of HIF-1α on eNOS.4. Therapeutic effects of adenosine A2B receptor specific antagonist PSB1115on penile fibrosisMice with PSB1115were treated for6weeks and penile tissues were collected for histological examination (H&E staining, Masson’s Trichrome staining and a-Smooth muscle actin (a-SMA) immuno-histochemistry staining) to evaluate penile fibrosis. Real-time PCR was used to measure fibrotic gene expression profile (α2(Ⅰ) procollagen, TGF-β1, and plasminogen activator inhibitor-1),Results1. ADA-deficient mice homozygote were identified by zymogram analysis and SCD Tg mice homozygote were genotyped by blood smear test and PCR.2. Compared with WT mice, levels of adenosine in penile tissue of ADA-deficient mice and SCD Tg mice were significantly increased. ADA-deficient mice and SCD Tg mice displayed an obvious elevated maximum ICP as well as total ICP (area under curve) and the initial rate of ICP (Slope), compared with control mice. However,2weeks PSB1115treatment for ADA-deficient mice and SCD Tg mice dramatically reduced the maximum ICP, total ICP and slope compared to ADA-deficient mice and SCD Tg mice,which were also similar to control mice.3. Compared with WT mice, ADA-deficient mice and SCD Tg mice had a higher level of HIF-la and eNOS gene expression which were reduced after2weeks PSB1115treatment. In vitro, the results showed that in HMCE-1cells treated with NECA (adenosine analog), the mRNA levels of HIF-1α and eNOS were significantly elevated. However, these elevations were inhibited by PSB1115treatment. In HIF-1a knockdown HMEC-1cells, NECA treatment couldn’t increase the eNOS mRNA level. These results suggested that adenosine mediated eNOS gene expression by modulating HIF-1α via adenosine A2B receptor in endothelial cells.4. We found that PSB1115treatment significantly reduced penile injury and fibrosis measured by H&E staining, immunohistochemistry and Trichrome staining in both mouse models. Consistently, we found that PSB1115significantly reduced fibrotic gene expression including procollagen I, TGF-β1, and plasminogen activator inhibitor-1mRNA. Conclusion1. Elevated levels of adenosine in penile tissue of ADA deficient mice and SCD Tg mice, two well-accepted priapism animal models, contribute to priapism via adenosine A2B receptor.2. Adenosine A2B receptor specific antagonist PSB1115prevents and relieves priapic feature and penile fibrosis both in ADA deficient mice and SCD Tg mice.3. Adenosine-HDF-1α-eNOS signaling pathway contributes to priapism in ADA-deficient mice and SCD Tg mice via adenosine A2B receptor.4. Interfering adenosine A2B receptor signaling is likely an effective therapy to treat and prevent priapism and penile fibrosis and which offers a novel therapy for human with priapism.