Effect of the phosphodiesterase 5 inhibitors sildenafil, tadalafil and vardenafil on rat anococcygeus muscle: functional and biochemical aspects

Abstract

1. The anococcygeus muscle is part of the erectile machinery in male rodents. Phosphodiesterase (PDE) 5 inhibitors enhance and prolong the effects of cGMP, which has a key role in penile erection. The aim of the present study was to provide a functional and biochemical comparison of the three PDE5 inhibitors, namely sildenafil, tadalafil and vardenafil, in the rat anococcygeus muscle. 2. Muscle strips were mounted in 4 mL organ baths and isometric force recorded. Levels of cGMP were measured using an enzyme immunoassay kit. Western blots were used to determine PDE5 protein expression. 3. The PDE5 inhibitors concentration-dependently relaxed carbachol-precontracted anococcygeus muscle; however, vardenafil was more potent (pEC(50) = 8.11 +/- 0.05) than sildenafil (7.72 +/- 0.06) or tadalafil (7.69 +/- 0.05). Addition of N(G)-nitro-l-arginine methyl ester (100 micromol/L) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 micromol/L) to the organ baths caused significant rightward shifts in concentration-response curves for all PDE5 inhibitors. 4. Sildenafil, tadalafil and vardenafil (all at 0.1 micromol/L) caused leftward shifts in the glyceryl trinitrate (GTN) concentration-response curves (by 4.0-, 3.7- and 5.5-fold, respectively). In addition, all three PDE5 inhibitors significantly potentiated relaxation responses to both GTN (0.01-10 micromol/L) and electrical field stimulation (EFS; 1-32 Hz), with vardenafil having more pronounced effects. 5. All three PDE5 inhibitors reduced EFS-evoked contractions in a concentration-dependent manner over the concentration range 0.001-1 micromol/L. There were no significant differences between the effects of the three PDE5 inhibitors. 6. Vardenafil (0.01-0.1 micromol/L) was more potent in preventing cGMP degradation in vitro than sildenafil (0.01-0.1 micromol/L) and tadalafil (0.01-0.1 micromol/L). 7. Under control conditions, the expression of PDE5 was higher in the anococcygeus muscle than in the corpus cavernosum. 8. In conclusion, PDE5 inhibitors enhance exogenous and endogenous nitric oxide-mediated relaxation in the rat anococcygeus muscle. The potency of vardenafil was greater than that of either sildenafil or tadalafil.

Fig. 1

Effects of the phosphodiesterase (PDE) 5 inhibitors (a) sildenafil, (b) tadalafil and (c) vardenafil on the relaxations induced by glyceryl trinitrate (GTN) in rat anococcygeus muscle preparations. (●), control; (■), in the presence of 0.1 μmol/L PDE5 inhibitor. Muscles were precontracted with carbachol (10 μmol/L) and experimental values were calculated relative to the maximal changes from the contraction produced by carbachol in each tissue, which was taken as 100%. Data are the mean ± SEM (n = 5). *P < 0.05, **P < 0.01 compared with respective control values (Student’s t-test).

Fig. 2

Effects of the phosphodiesterase (PDE) 5 inhibitors (a) sildenafil, (b) tadalafil and (c) vardenafil on electrical field stimulation (EFS)-induced relaxations of rat anococcygeus muscle strips precontracted with carbachol (10 μmol/L). (●), control; (■), in the presence of 0.1 μmol/L PDE5 inhibitor. Experimental values were calculated relative to the maximal changes from the contraction produced by carbachol in each tissue, which was taken as 100%. Data are the mean±SEM (n = 4–5). *P < 0.05, **P < 0.01 compared with respective control values (Student’s t-test).

Fig. 3

Representative tracings of frequency-dependent relaxations to electrical field stimulation (EFS) in the absence and in the presence of the phosphodiesterase (PDE) 5 inhibitors (a) sildenafil, (b) tadalafil and (c) vardenafil (0.1 μmol/L; n = 4 each). Enhancement of the magnitude and duration of EFS-induced relaxations were observed in the presence of the PDE5 inhibitors. Nitrergic duration was determined as the time elapsed from 50% relaxation to 50% recovery.

Fig. 4

Effect of increasing concentrations of sildenafil (●), tadalafil (■) and vardenafil (○) on the duration of nitrergic relaxations evoked by electrical field stimulation (EFS; 2 Hz, 50 V, 10 s trains every 90 s) in rat anococcygeus muscle strips contracted with carbachol (10 μmol/L). The duration of EFS-induced responses was determined as the time elapsed from 50% relaxation to 50% recovery. Data are the mean±SEM (n = 4). *P < 0.05 compared with sildenafil and tadalafil; ^†P < 0.05 compared with tadalafil (one-way ANOVA followed by Student–Newman–Keuls’ post hoc test).

Fig. 5

Effects of the phosphodiesterase (PDE) 5 inhibitors (a) sildenafil, (b) tadalafil and (c) vardenafil on the contractions induced by electrical field stimulation (EFS) in rat anococcygeus muscle preparations. (□), 0.001 μmol/L; (▨), 0.01 μmol/L; (■), 0.1 μmol/L; (▤), 1 μmol/L. Data were calculated relative to the contraction produced by each frequency of stimulation under control conditions (i.e. in the absence of inhibitor). Data are the mean±SEM.

Fig. 6

Degradation of cGMP in the presence of rat anococcygeus cytosolic extracts in the absence or presence of (a) sildenafil, (b) tadalafil and (c) vardenafil. (●), control; (○), 0.001 μmol/L; (■), 0.01 μmol/L; (□), 0.1 μmol/L. Cyclic GMP (10 μmol/L) was incubated in the presence of cytosolic extract (30 μg protein) at 37°C (see Methods) and cyclic nucleotide content (expressed in μmol/L) was determined after 0, 5, 10 and 30 min of incubation. Data are the mean±SEM (n = 4). The dashed line represents the initial concentration of cGMP (10 μmol/L) for reference. The pIC₅₀ values for sildenafil, tadalafil and vardenafil were 8.33 ± 0.07, 8.24 ± 0.08 and 8.75 ± 0.08, respectively.

Fig. 7

Western blot analysis of phosphodiesterase (PDE) 5 in rat anococcygeus (AcM) and corpus cavernosum (CC). Marks indicate the approximate molecular weight. Tissue extracts were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis, followed by western blotting with anti-phosphodiesterase V antibody, as described in the Methods. Blots were detected with an Advance Chemiluminescence Detection Kit (n = 6). Summarized data of densitometrical analysis are also represented. Each set of determinations was performed in triplicate. H, human recombinant PDE5. **P < 0.01 compared with AcM (Student’s t-test).