Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling

Abstract

Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor-mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine-induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada -/- and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder.

Figure 1. Ada^–/– mice display spontaneous prolonged penile erection associated with increased CCS relaxation in response to nerve stimulation.

(A) Ada^–/– mice exhibited prolonged penile erections, lasting 8–72 hours. Priapic erections were never observed in control Ada⁺ male mice. (B and C) Ada^–/– mice displayed hypersensitivity to EFS in both dosage-dependent (B) and frequency-dependent (C) manners. Data are means ± SEM. *P < 0.05, Ada^–/– versus Ada⁺. n = 5–10.

Figure 2. Priapic activity seen in Ada^–/– mice is dependent on elevated adenosine in the penis.

(A) Adenosine levels were elevated in the penes of Ada^–/– mice. Inset bar graph shows the average adenosine levels from 3 Ada^–/– mice and 3 wild-type mice. Data are means ± SEM (n = 3). *P < 0.005 versus Ada⁺. (B) The prolonged penile erection in Ada^–/– mice was corrected by intraperitoneal injection of PEG-ADA. n = 3–5. (C) Representative recordings of EFS-induced CCS relaxation (5 V and 30 Hz) using 10 μM phenylephrine–precontracted CCSs of Ada⁺ mice and Ada^–/– mice treated with or without PEG-ADA. (D–F) Average EFS-induced relaxation from the phenylephrine-precontracted CCSs of Ada⁺ mice, Ada^–/– mice, and Ada^–/– mice treated with PEG-ADA. EFS-induced changes in the force of CCS relaxation (D), the duration of relaxation (E), and the combination of force and duration (area under baseline; F). Data are means ± SEM (n = 5–6). *P < 0.05 versus Ada⁺; **P < 0.05 versus untreated Ada⁺; ***P < 0.05 versus untreated Ada^–/–.

Figure 3. Inhibition of ADA activity in wild-type mice induces potent penile CCS relaxation.

(A and B) CCSs of wild-type mice were treated with different concentrations of adenosine in the presence or absence of DCF (5 μM; A) or with 100 μM adenosine in the presence of different concentrations of DCF (0–100 μM; B). The resulting adenosine-induced CCS relaxation was monitored by force transducer. Data are means ± SEM. *P < 0.05 versus adenosine alone; **P < 0.05 versus untreated.

Figure 4. A_2BR signaling is required for adenosine-mediated CCS relaxation.

(A) The extent of adenosine-induced CCS relaxation in wild-type mice with theophylline treatment was measured by a force transducer. Data are means ± SEM (n = 5). *P < 0.05 versus untreated; **P < 0.05 versus adenosine alone. (B) Adenosine-induced CCS relaxation in wild-type, A₁R^–/–, A_2AR^–/–, A_2BR^–/–, and A₃R^–/– mice was measured by a force transducer. Data are means ± SEM (n = 5–10). *P < 0.005 versus wild-type. (C) Adenosine-mediated cAMP production of CCSs from wild-type and A_2BR^–/– mice. Some of the CCSs from wild-type mice were treated with l-NAME or MRS1706. Data are means ± SEM (n = 6–7). *P < 0.05 versus wild-type treated with adenosine alone.

Figure 5. Adenosine stimulates an increase in both cAMP and cGMP in CCSMCs via A_2BR activation.

(A) Adenosine receptor mRNA expression profile in purified primary CCSMCs were determine by quantitative real-time RT-PCR. nd, not determined. (B) cAMP levels of CCSMCs from wild-type mouse penes in the presence of different concentrations of adenosine with or without specific adenosine receptor agonists or antagonists. Data are means ± SEM. *P < 0.05 versus adenosine alone. (C) cAMP levels of CCSMCs from wild-type and A_2BR^–/– mice treated with adenosine, NECA, or forskolin. Data are means ± SEM (n = 4). *P < 0.05 versus untreated wild-type; **P < 0.05 versus untreated A_2BR^–/–. (D) cGMP levels in CCSs of wild-type and A_2BR^–/– mice treated with l-NAME or MRS1706. Data are means ± SEM (n = 6–7). *P < 0.05 versus wild-type treated with adenosine alone. (E) cGMP levels in CCSMCs of wild-type and A_2BR^–/– mice treated with the indicated compounds. Data are means ± SEM. *P < 0.05 versus respective control.

Figure 6. EFS leads to increased cAMP and cGMP production via A_2BR in Ada^–/– mice.

(A and B) EFS-mediated cAMP (A) and cGMP (B) production were measured in phenylephrine-precontracted CCSs of Ada^–/– and Ada⁺ mice with or without MRS1706, ZM241389, and/or l-NAME treatment. Data are means ± SEM (n = 4). *P < 0.05, versus wild-type with EFS; **P < 0.05 versus Ada^–/– with EFS alone; ***P < 0.05 versus wild-type with EFS alone.

Figure 7. Ada^–/– mice develop penile vascular damage and fibrosis subsequent to priapism.

(A–D) Histological examination of the vascular structures in the corpus spongiosum. (A and B) H&E staining. (C and D) Anti–α-SMA immunohistochemical staining. Arrowheads indicate intimal thickening with smooth muscle hypertrophy of the vascular wall in the deep dorsal vein; arrows indicate muscular hypertrophy of the arterial vascular wall. (E–H) Fibrosis in corpus spongiosum (E and F) and corpus cavernosum (G and H) visualized by Masson trichrome staining. Arrowheads denote the extensive fibrosis with extension into the intima of the deep dorsal vein; arrows indicate fibrosis around the lumen of the artery. Original magnification, ×100 (A–D); ×200 (E–H).

Figure 8. Priapic activity in SCD transgenic mice is dependent on elevated adenosine in penis and A_2BR signaling.

(A) Adenosine levels were elevated in the penes of SCD transgenic mice. Data are means ± SEM (n = 5–6). *P < 0.005 versus control. (B) Increased CCS relaxation by EFS in SCD transgenic mice. Data are means ± SEM (n = 5). P < 0.05, SCD transgenic versus control. (C–E) Prolonged penile erection in SCD transgenic mice is due to A_2BR signaling. EFS-induced relaxation in CCSs (C), duration of relaxation (D), and the combination of force and duration (area under baseline; E) were measured by force transducer in mice left untreated or treated with PEG-ADA, MRS1706, or ZM241389. Data are means ± SEM (n = 5). *P < 0.05 versus control; **P < 0.05, versus untreated SCD transgenic; ***P < 0.05 versus untreated control.