sildenafil-citrate and 5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine

We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 microM) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Apoptosis; bcl-X Protein; Benzimidazoles; Blotting, Western; Carbocyanines; Caspase 3; Caspases; Cell Survival; Cells, Cultured; Cyclic Nucleotide Phosphodiesterases, Type 5; DNA Primers; DNA, Complementary; Enzyme Activation; Enzyme Inhibitors; Immunohistochemistry; In Situ Nick-End Labeling; L-Lactate Dehydrogenase; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mitochondria; Muscle Cells; Myocytes, Cardiac; Necrosis; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxygen; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperazines; Proto-Oncogene Proteins c-bcl-2; Purines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sildenafil Citrate; Sulfones; Time Factors; Transcription, Genetic; Trypan Blue