PubMed · 14506469
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2003-09-29. Vardenafil (levitra) for erectile dysfunction.. https://pubmed.ncbi.nlm.nih.gov/14506469/
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Phosphodiesterase 5 (PDE5) is a major isoform of cGMP phosphodiesterase in a variety of human tumor cell lines and plays a key role in regulating intracellular cGMP concentrations ([cGMP]i). Here, we demonstrate that suppression of PDE5 gene expression by antisense pZeoSV2/ASP5 plasmid transfection results in a sustained increase in [cGMP]i, growth inhibition, and apoptosis in human colon tumor HT29 cells. With stable transfection, antisense transcripts exhibited a specific suppression in PDE5 activity, mRNA levels, and a 93 kDa hPDE5A1 protein. In cloned antisense cells, prolongation of the cell growth doubling times correlate positively with suppressed PDE5 activity and increased [cGMP]i. The growth inhibition in PDE5 antisense clones is due to an increased apoptotic rate and delayed cell-cycle progression. These results corroborate previous findings with the PDE5 inhibitor exisulind and its derivatives showing that sustained [cGMP]i induces apoptosis and growth inhibition in tumor cells. Furthermore, an inducible mitotic inhibitor p21WAF1/CIP1 has been found to account for the delay of cell-cycle progression in PDE5 antisense clones at G2/M phase. A proteolytic cleavage of p21WAF1/CIP1 in the antisense clones is also increased at the later stage of serum stimulation. The protein kinase G (PKG) inhibitor, KT5823, can prevent the cleavage of p21(WAF1/CIP). These data substantiate a pivotal role for PDE5 as a modulator of apoptosis and cell-cycle progression for human carcinoma via a mechanism involving the activation of [cGMP]i/PKG signaling pathways.
It was previously shown that cGMP enhances cAMP-induced Ca2+-influx in Dictyostelium discoideum. This finding is based on experiments done with strains defective in cGMP-hydrolysis, the streamer F cells. In this work, we show that these chemically mutagenized cells display different properties in their cAMP-induced light-scattering response and cAMP-induced Ca2+-influx compared with a cGMP-phosphodiesterase knock-out strain, pdeD KO, generated by homologous recombination. PdeD KO cells possess a reduced Ca2+-influx that is developmentally regulated. This finding contradicts the result of streamer F cells, where cAMP-induced Ca2+-influx is prolonged and elevated. Both mutants, however, showed a three to four-fold delayed response to cAMP at 3-4h of starvation. Thus, the consequence of an elevated cGMP concentration is a delay and an inhibition of Ca2+-influx and not an enhancement. Results obtained with streamer F cells should therefore be interpreted with caution because the mutation(s) responsible for the divergent phenotype to pdeD KO cells has not been identified. We show by the use of membrane-permeant cGMP-analogues in wild type (wt) cells, permeabilized cells and measurements on isolated vesicles that the cause for the reduced Ca2+-influx seems to be due to developmentally regulated Ca2+-channel inhibition by cGMP.
Cyclic GMP (cGMP)-binding cGMP-specific phosphodiesterase (PDE5) activity was found in chick dorsal root ganglia (DRG). PDE5 expression was studied at different stages of development: in embryonic day 10 (E10) and E18 embryos and in 5-day post-hatching chick (P5). The presence of PDE5 was suggested by the ion exchange chromatography elution profile in E18 DRG extracts, where cGMP-specific hydrolytic calmodulin-independent activity was found; in other stages, this activity coeluted with the PDE1 calmodulin-stimulated isoform characterized previously. Inhibition studies supported the hypothesis that the newly identified PDE activity belongs to the PDE5 isoform. Western blot analysis using a PDE5-specific antibody was also carried out and revealed the presence of three specific immunoreactive bands with apparent molecular weights of 98, 93, and 86 kDa, corresponding to the three described splice variants (PDE5A1, PDE5A2, and PDE5A3). The expression in DRG of the three PDE5 isoforms was also confirmed by RT-PCR. Developmental regulation of PDE5 was revealed by the immunoblot analysis at different stages; expression was very low at E10 but an overall substantial increase occurred between E10-18 (about 12-fold, considering the three PDE5 isoforms together). Differences were revealed, however, when a single PDE5 isoform was considered. PDE5A1 and PDE5A3 showed an increase at all stages although more pronounced between E10-18, whereas PDE5A2 underwent a marked increase (about 38-fold) in the first period and remained nearly constant between E18 and P5. This is the first evidence of PDE5 in sensory neurons, and the distinct temporal expression patterns of enzyme isoforms may indicate different physiologic roles in developing and mature chick DRG.