OBJECTIVE: Tadalafil, a long-acting phosphodiesterase type 5 (PDE5) inhibitor, improves the erectile response by inhibiting cyclic guanosine monophosphate (cGMP) breakdown. Sustained higher levels of cGMP may hypothetically upregulate PDE5 expression and/or activity and lead to tachyphylaxis. We have investigated whether PDE5 upregulation occurs in vitro in cultures of human penile cells subjected to long-term incubation with increasing concentrations of tadalafil in the presence of a nitric oxide (NO) donor. METHODS: Human corpora cavernosa smooth muscle cells (CSMC) and tunica albuginea fibroblasts (TAF) primary cultures were characterized by immunocytochemistry and Western blot, and incubated with graded concentrations of tadalafil for 14 days, adding S-nitroso-N-acetyl penicillamine (SNAP) as an NO donor for the last 24 hours or at time zero, and cGMP levels were measured. Incubations were repeated for 7, 10, and 14 days, in the presence of SNAP, and PDE5 was estimated by Western blot, and at 14 days, by immunocytochemistry combined with quantitative image analysis, and by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Constructs of the human PDE5A promoter expressing luciferase were cloned and transfected into CSMC, and promoter activation by 8-deoxybromo-cGMP (8-Br-cGMP) was measured by luminometry. RESULTS: Incubations of CSMC with SNAP and tadalafil up to 14 days did not upregulate PDE5 mRNA or protein levels. With TAF, PDE5 protein was also not upregulated despite a slight increase in mRNA levels. PDE5 enzyme activity was unaffected by tadalafil in either CSMC or TAF. No upregulation of the PDE5 promoter was observed with up to 2 mM 8-Br-cGMP. CONCLUSIONS: Long-term incubation of human penile cells with tadalafil at concentrations above the in vitro IC50, and around the in vivo Cmax utilized in the clinical setting, did not upregulate PDE5A expression nor decrease cGMP levels. These data suggest that PDE5 upregulation is unlikely to occur in vivo on long-term tadalafil treatment.
OBJECTIVE:Tadalafil, a long-acting phosphodiesterase type 5 (PDE5) inhibitor, improves the erectile response by inhibiting cyclic guanosine monophosphate (cGMP) breakdown. Sustained higher levels of cGMP may hypothetically upregulate PDE5 expression and/or activity and lead to tachyphylaxis. We have investigated whether PDE5 upregulation occurs in vitro in cultures of human penile cells subjected to long-term incubation with increasing concentrations of tadalafil in the presence of a nitric oxide (NO) donor. METHODS:Human corpora cavernosa smooth muscle cells (CSMC) and tunica albuginea fibroblasts (TAF) primary cultures were characterized by immunocytochemistry and Western blot, and incubated with graded concentrations of tadalafil for 14 days, adding S-nitroso-N-acetyl penicillamine (SNAP) as an NO donor for the last 24 hours or at time zero, and cGMP levels were measured. Incubations were repeated for 7, 10, and 14 days, in the presence of SNAP, and PDE5 was estimated by Western blot, and at 14 days, by immunocytochemistry combined with quantitative image analysis, and by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Constructs of the humanPDE5A promoter expressing luciferase were cloned and transfected into CSMC, and promoter activation by 8-deoxybromo-cGMP (8-Br-cGMP) was measured by luminometry. RESULTS: Incubations of CSMC with SNAP and tadalafil up to 14 days did not upregulate PDE5 mRNA or protein levels. With TAF, PDE5 protein was also not upregulated despite a slight increase in mRNA levels. PDE5 enzyme activity was unaffected by tadalafil in either CSMC or TAF. No upregulation of the PDE5 promoter was observed with up to 2 mM 8-Br-cGMP. CONCLUSIONS: Long-term incubation of human penile cells with tadalafil at concentrations above the in vitro IC50, and around the in vivo Cmax utilized in the clinical setting, did not upregulate PDE5A expression nor decrease cGMP levels. These data suggest that PDE5 upregulation is unlikely to occur in vivo on long-term tadalafil treatment.
Authors: Robert A Gelfand; Dolores Vernet; Istvan Kovanecz; Jacob Rajfer; Nestor F Gonzalez-Cadavid Journal: J Sex Med Date: 2014-12-11 Impact factor: 3.802