Abstract 3927: Decreased Activity of NHE-1 by PDE5A Inhibition is Due to an Increased Activity of Protein Phosphatase 1 (PP1)
The beneficial effect of PDE5A inhibition in myocardial infarction is well established as it is the protection brought about by NHE1 inhibition after ischemia/reperfusion. Interestingly, PDE5A inhibition was reported to be accompanied by decreased NHE1 activity by an unknown mechanism. Experiments were performed in isolated cat cardiomyocytes to get insight into the intracellular pathway involved in the reduction of NHE1 activity induced by PDE5A inhibition with sildenafil (SIL, 1 μmol/L). NHE1 activity was assessed by the initial rate of intracellular pH (pHi) recovery from an acute acidic load in the absence of bicarbonate. Proton efflux (JH), expressed in mmol/L/min, was estimated by multiplying dpHi/dt by the buffer capacity (β). JH comparison among groups was done at a common pHi of 6.8. Whereas SIL did not affect basal pHi (7.18±0.03, n=10 vs 7.17±0.03, n=10), it decreased JH after the acidic load from 2.06±0.22 (n=7) to 1.24±0.17 (n=10, P<0.05). This effect was mimicked by another PDE5A inhibitor, EMD3605227/5 (0.1 μmol/L; JH: 1.31±0.16, n=6, P<0.05) and reverted by the PKG inhibitor KT5823 (1 μmol/L) (JH: 2.13±0.27, n=12). Okadaic acid at a concentration (100 nmol/L) that blocks protein phosphatases PP1 and PP2A, reverted Sil effect (JH: 2.97±0.39, n=10). In contrast, 1 nmol/L okadaic acid (concentration that uniquely inhibits PP2A) did not (1.34±0.21, n=12, P<0.05); suggesting that only PP1 was involved in SIL-induced NHE1 inhibition. On line with these data, endothall, a PP2A specific inhibitor, failed to prevent SIL effect. Acidosis increased NHE1 phosphorylation estimated by anti P-Ser 14 –3–3 binding motif antibody in immunoprecipitated NHE1 (128±9.5 % compared to basal pHi), effect that was prevented by SIL (82±11.7%). When upstream kinases involved in NHE1 activation (ERK1/2 and p90RSK) were examined no changes in the acidosis-promoted increased phosphorylation were detected in the presence of SIL (as percentage of basal pHi: P-ERK1/2 = 189±23, n=11 vs. 187±31, n=7 and P-p90RSK=187±17, n=11 vs. 201±30, n=8 without and with SIL respectively). In conclusion, we provide data supporting that PDE5A inhibition decreases NHE1 activity during pHi recovery after an acidic load by decreasing NHE1 phosphorylation probably through PP1 activation.