title: Phosphodiesterase type 5 is not upregulated by tadalafil in cultures of human penile cells.
abstract: 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.
authors.affiliation: Los Angeles Biomedical Research Institute-Urology, Torrance, CA, USA.
authors.forename: Dolores
authors.initials: D
authors.lastname: Vernet
authors.forename: Thomas
authors.initials: T
authors.lastname: Magee
authors.forename: Ansha
authors.initials: A
authors.lastname: Qian
authors.forename: Gaby
authors.initials: G
authors.lastname: Nolazco
authors.forename: Jacob
authors.initials: J
authors.lastname: Rajfer
authors.forename: Nestor
authors.initials: N
authors.lastname: Gonzalez-Cadavid
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000704:Analysis of Variance; D002243:Carbolines / Q000494:pharmacology*; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D006801:Humans; D008297:Male; D010410:Penile Erection / Q000187:drug effects; D010413:Penis / Q000187:drug effects / Q000201:enzymology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D000068581:Tadalafil
chemical_list: D002243:Carbolines; D010726:Phosphodiesterase Inhibitors; D000068581:Tadalafil; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP