Levitra (vardenafil) Info

Rat isolated heart model
Female Wistar rats (180–200g; total of 52) were anaesthetized with pentobarbital sodium (60mgkg−1 i.p.), and hearts were excised, mounted on a Langendorff apparatus and perfused in a constant pressure mode with modified Krebs–Henseleit bicarbonate buffer containing (mM) 118.5 NaCl, 24.8 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2 and 10 glucose. A suture was passed around a major branch of the left coronary artery. After equilibration, hearts were subjected to 30min of regional ischemia by occluding the snared artery followed by 2h of reperfusion as depicted in Figure 1. Control hearts were subjected to only 30min of regional ischemia and then reperfusion. Four groups of hearts were treated with different vardenafil dosages (1, 10, 100 and 1000nM) during the reperfusion period starting 5min before reperfusion. In the next groups, one of three inhibitors was co-infused along with the protective vardenafil dose of 10nM. These inhibitors included the GC inhibitor ODQ (10μM) and the PKG inhibitor KT-5823 (1μM). Finally, three groups of hearts were treated with only ODQ or KT-5823, as noted above, to exclude independent effects of the blockers.

Figure 1
Experimental protocols. All hearts were stabilized for 30min before the experiments. Control hearts received 30min of regional ischemia followed by 2h of reperfusion. Vardenafil (VAR) was administered throughout reperfusion starting from 5min before reperfusion. The blockers 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) and KT-5823 (KT) were given 5min before vardenafil treatment. The blockers alone were given accordingly.

Measurement of infarct size
As previously detailed, the risk zone was delineated with 2–9μm green fluorescent microspheres (Duke Scientific Corp., Palo Alto, CA, USA) and infarction with triphenyltetrazolium chloride staining. The areas of infarct and risk zone were determined by planimetry of each slice and volumes calculated by multiplying each area by slice thickness and summing them for each heart. Infarct size is expressed as a percentage of the risk zone.

HL-1 cardiomyocytes
The murine HL-1 cardiomyocyte cell line was kindly provided by W. Claycomb. HL-1 cells maintain a cardiac-specific phenotype even after repeated passages, and are used as a standard cell line for comparable applications. The cells were maintained in Claycomb medium (JRH Bioscience, Lenexa, KS, USA) supplemented with 10% foetal calf serum, 100Uml−1 penicillin, 100μgml−1 streptomycin and 4mM L-glutamine. The supplemented medium was changed every 24h, and standard incubation conditions of 37°C, 5% CO2 air were used. One day before using, the cells were serum starved.

Assessment of mitochondrial membrane potential
Cells were plated on four-chambered cover slips and loaded with 100nM tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes Inc., Eugene, OR, USA) for 20min, which causes cells to fluoresce in proportion to their mitochondrial membrane potential (ψm). A reduction in TMRE fluorescence served as an indicator of the loss of ψm, which would occur with mPTP formation. If required, canadian vardenafil and blocker were added 5min before TMRE loading, mirroring preconditioning-like effects. Experiments were designed such that four different conditions on one cover slip were always simultaneously evaluated. Afterward, cells were washed twice with TMRE-free medium and then incubated in the selective calcium ionophore calcimycin (100μM), which is known to induce mPTP formation, as seen at reperfusion following myocardial ischemia. Fluorescence intensity was measured at 582nm (FL-2 channel) using a Becton Dickinson (Heidelberg, Germany) FACSCalibur and CellQuest software immediately after the TMRE exposure (baseline value) and after an 80-min calcimycin treatment. Data were gated to exclude debris and the average fluorescence of 8000 cells was computed. Fluorescence measurements provide data in arbitrary units (a.u.), which were expressed as percentage of the baseline values. Each data set contains five groups of TMRE-loaded myocytes studied in parallel: no treatment, addition of calcimycin, addition of calcimycin in the presence of vardenafil, addition of calcimycin in the presence of vardenafil (Levitra) and a given inhibitor and addition of calcimycin in the presence of the inhibitor alone.

Determination of cell death
The integrity of the cell membrane was assessed by flow cytometric analysis of propidium iodide (PI) uptake. Cells were harvested, stressed for 80min with calcimycin (100μM) and treated without TMRE as indicated above, followed by a 5-min incubation in 2μgml−1 PI in PBS at 4°C in the dark. PI uptake was measured by flow cytometry analysis. Ten thousand cells were analysed in each sample; data were gated to exclude debris. Six groups were measured in parallel: untreated control, vardenafil (1nM), vardenafil (Levitra Professional) in the presence of either ODQ (10μM) or DT-2 (125nM), and ODQ and DT-2 alone.

PKG activity measurements
HL-1 myocytes were harvested and four groups were studied in parallel: untreated control, vardenafil, vardenafil plus KT-5823 and KT-5823 alone. After the 80min incubation, cells were homogenized in ice-cold lysis buffer. Fifty micrograms of total protein, as measured with a BCA protein assay (Pierce Biotechnology, Rockfort, IL, USA), were loaded in each lane. After being blocked with milk, the membranes were treated with phospho-specific vasodilator-stimulated phosphoprotein (VASP) Ser239 antibody (1:1000) followed by the secondary antibody (1:5000) conjugated to horseradish peroxidase. Immunoreactive proteins were detected by enhanced chemiluminescence with LumiGLO (Cell Signaling Technology, Beverly, MA, USA). Films were developed and the blots quantified by using a computer scanner. The density of each band was calculated with Sigmagel software. Identical membranes were stripped, reprobed with primary antibody against total, nonphosphorylated VASP and analysed as above. Results were depicted as increase in VASP phosphorylation compared with the total VASP content.

Statistics
Data are presented as mean±s.e.mean. The TMRE fluorescence intensity is presented as a percentage of the respective baseline values. Differences in infarct size, TMRE fluorescence and VASP phosphorylation among groups were compared by one-way ANOVA with Tukey’s post hoc test. A value of P<0.05 was considered significant.

Materials
Canadian Vardenafil was kindly provided by Bayer HealthCare GmbH (Wuppertal, Germany). KT-5823 was obtained from Alexis Pharma and DT-2 and DT-3 were from Biomol (Hamburg, Germany). Phospho-specific VASP Ser239 antibody and antibody against total VASP was from Cell Signaling Technology. All other chemicals were from Sigma-Aldrich Chemie GmbH, Munich, Germany. Wortmannin (WORT), KT-5823 and ODQ were dissolved in DMSO before being diluted in Krebs–Henseleit buffer, resulting in a DMSO concentration of less than 0.01%. Vardenafil (Brand Levitra) was diluted directly in Krebs–Henseleit buffer.