Levitra (vardenafil) Info

Infarct size measurements
No significant differences in coronary flow at baseline and during occlusion among the experimental groups were observed (Table 1). Coronary flow at 5min of reperfusion was significantly increased only in the group treated with 1μM vardenafil (P=0.008). All other groups showed no significant increase in coronary flow at reperfusion, including the protective group with vardenafil at 10nM.

Table 1. Coronary flow for isolated, perfused rat hearts

	Baseline	Occlusion	Reperfusion
Control	12.4±0.7	4.9±0.7	8.6±0.7
VAR (1nM)	13.3±0.3	6.3±0.5	10.7±2.2
VAR (10nM)	12.0±0.5	6.0±1.9	9.2±1.6
VAR (100nM)	12.8±0.6	5.5±0.5	11.1±1.5
VAR (1000nM)	12.5±1.0	4.8±0.4	13.9±1.1*
VAR+ODQ	11.3±1.0	3.9±0.5	7.2±0.6
ODQ	9.4±1.2	4.1±1.4	6.4±0.7
VAR+KT	12.0±0.9	4.8±0.9	7.5±0.5
KT	11.8±1.9	3.7±0.4	6.5±0.6

As shown in Table 2 and Figure 2, treating hearts with 10nM vardenafil starting 5min before reperfusion almost halved infarct size but either lower or higher vardenafil (Levitra Professional) concentrations failed to protect.

Table 2. Infarct size data for isolated, perfused rat hearts

	Risk zone (cm3)	Infarct (cm3)	Infarction (% of risk zone)
Control	0.147±0.013	0.066±0.005	45.8±2.0
VAR (1nM)	0.147±0.024	0.056±0.009	38.5±3.6
VAR (10nM)	0.173±0.015	0.047±0.008	26.2±2.7*
VAR (100nM)	0.186±0.020	0.074±0.013	38.5±3.6
VAR (1000nM)	0.152±0.024	0.068±0.014	43.9±4.0
VAR+ODQ	0.176±0.013	0.077±0.004	44.0±1.8
ODQ	0.198±0.056	0.075±0.016	39.9±3.9
VAR+KT	0.157±0.010	0.067±0.006	42.5±1.9
KT	0.165±0.019	0.075±0.006	45.3±1.9

Figure 2. The effect of different concentrations of vardenafil (Generic Levitra) at reperfusion on infarct size expressed as a percentage of the risk zone. Open symbols represent individual experiments and closed symbols the group means. Infusion of vardenafil during reperfusion was protective only at 10nM, whereas lower and higher concentrations failed to protect.

Figure 3 illustrates that the clear protective effect of vardenafil at 10nM could be abolished by co-treatment with either the GC inhibitor, ODQ, or the PKG blocker, KT-5823 (P<0.05 vs vardenafil), suggesting that activation of both GC and PKG are required for the protective effect of vardenafil. Neither ODQ nor KT-5823 had an effect on infarct size in the absence of vardenafil.

Figure 3. The effect of 10nM vardenafil (VAR) at reperfusion alone and in the presence of kinase blockers on infarct size expressed as a percentage of the risk zone. 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) and KT-5823 blocked vardenafil’s protection, suggesting a role of GC and PKG in the signaling. Neither ODQ nor KT-5823 alone had any effect on infarct size.

Assessment of mitochondrial membrane potential
All groups of HL-1 cardiomyocytes were treated with the selective calcium ionophore calcimycin to induce mPTP formation due to calcium overload. As shown in Figure 4, myocytes that had been exposed to 100μM calcimycin for 80min (control) showed a marked reduction in mean fluorescence, compared with baseline values (100%), indicating a loss of mitochondrial membrane potential (ψm), presumably related to mPTP formation. Treatment with vardenafil (1nM) preserved TMRE fluorescence despite challenge with calcimycin (P<0.01 vs control), indicating a protective role of PDE-5 inhibition on ψm depolarization and, thus, mPTP opening. Higher or lower vardenafil concentrations (0.1 or 100nM) failed to protect (data not shown).

Figure 4. HL-1 cardiomyocytes stained with tetramethylrhodamine ethyl ester and stressed for 80min with the selective calcium ionophore calcimycin (100μM) showed a significant reduction in mean fluorescence expressed as percentage of baseline values. Canadian Vardenafil significantly increased mean fluorescence, indicating that it preserves mitochondrial membrane potential in the face of calcium stress. The vardenafil-induced protection could be blocked by co-treatment with the GC inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), the PKG inhibitor KT-5823 or the PKG inhibitory peptides DT-2 and DT-3. The blockers alone had no effect.

Protection seen after vardenafil treatment at 1nM was abolished when the GC inhibitor, ODQ, was administered simultaneously (P=0.01 vs vardenafil). The same was true for the coadministration of the PKG inhibitor KT-5823 (P=0.03 vs vardenafil). To further confirm a role for PKG and to support the results with KT-5823, the highly specific PKG-blocking peptides DT-2 (125nM) and DT-3 (250nM) were used. As expected, both abolished vardenafil’s protective effect against the calcium load (each P<0.05 vs vardenafil). The inhibitors used here had no effect on TMRE fluorescence when applied alone.

Determination of cell death
In all groups, cells were treated as above, but without TMRE. As shown in Figure 5, myocytes treated with vardenafil showed a significant lower PI uptake than untreated cells, indicating more intact cell membranes and, therefore, less cell death (P=0.03). Protection by vardenafil (Levitra Plus) was abolished with co-treatment of ODQ (10μM) and DT-2 (125nM), further supporting a role for GC and PKG in the protection afforded through PDE-5 inhibition. The inhibitors alone had no effect.

Figure 5. HL-1 cardiomyocytes stressed for 80min with the calcium ionophore calcimycin (100μM) followed by the staining of dead cells with propidium iodide. Treatment with vardenafil (VAR) showed significantly less fluorescence, indicating more viable myocytes. The vardenafil-induced effect was abolished with the GC inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) and the PKG inhibitory peptide DT-2. The blockers alone had no effect.

PKG activity
Phosphorylation of VASP at Ser239 is highly selective for PKG and can therefore serve as a reliable indicator of PKG activity. Although VASP is presumably not present in adult cardiomyocytes, we could nevertheless detect it in the HL-1 myocytes. Treatment of HL-1 cells with generic vardenafil lead to a significant increase of VASP phosphorylation (P=0.03 vs control) over total VASP, whereas control cells showed no significant increase. The vardenafil-induced VASP phosphorylation could totally be abolished after co-treatment with the PKG inhibitor KT-5823 and this inhibitor administered alone showed no effect (Figure 6).

Figure 6. Mean levels of phosphorylated vasodilator-stimulated phosphoprotein (VASP) Ser239 as fold of total VASP in HL-1 myocytes. Total VASP was re-probed from the same membranes. Results represent the mean±s.e.mean of three to four independent experiments per group. There was a significant, PKG-dependent increase in VASP phosphorylation aftorylation after er exposure to vardenafil. Top: representative western blot of phosphorylated VASP and total VASP