sildenafil-citrate and endothall

The beneficial effect of phosphodiesterase 5A inhibition in ischemia/reperfusion injury and cardiac hypertrophy is well established. Inhibition of the cardiac Na(+)/H(+) exchanger (NHE-1) exerts beneficial effects on these same conditions, and a possible link between these therapeutic strategies was suggested. Experiments were performed in isolated cat cardiomyocytes to gain insight into the intracellular pathway involved in the reduction of NHE-1 activity by phosphodiesterase 5A inhibition. NHE-1 activity was assessed by the rate of intracellular pH recovery from a sustained acidic load in the absence of bicarbonate. Phosphodiesterase 5A inhibition with sildenafil (1 μmol/L) did not affect basal intracellular pH; yet, it did decrease proton efflux (J(H); in millimoles per liter per minute) after the acidic load (proton efflux: 6.97±0.43 in control versus 3.31±0.58 with sildenafil; P<0.05). The blockade of both protein phosphatase 1 and 2A with 100 nmol/L of okadaic acid reverted the sildenafil effect (proton efflux: 6.77±0.82). In contrast, selective inhibition of protein phosphatase 2A (1 nmol/L of okadaic acid or 100 μmol/L of endothall) did not (3.86±1.0 and 2.61±1.2), suggesting that only protein phosphatase 1 was involved in sildenafil-induced NHE-1 inhibition. Moreover, sildenafil prevented the acidosis-induced increase in NHE-1 phosphorylation without affecting activation of the extracellular signal-regulated kinase 1/2-p90(RSK) pathway. Our results suggest that phosphodiesterase 5A inhibition decreases NHE-1 activity, during intracellular pH recovery after an acidic load, by a protein phosphatase 1-dependent reduction in NHE-1 phosphorylation.

Topics: Animals; Biological Transport; Cats; Cells, Cultured; Cyclic Nucleotide Phosphodiesterases, Type 5; Dicarboxylic Acids; Enzyme Inhibitors; Hydrogen-Ion Concentration; Immunoblotting; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Okadaic Acid; Phosphodiesterase 5 Inhibitors; Phosphodiesterase Inhibitors; Phosphorylation; Piperazines; Protein Phosphatase 1; Protons; Purines; Ribosomal Protein S6 Kinases, 90-kDa; Sildenafil Citrate; Sodium-Hydrogen Exchangers; Sulfones

This study aimed to identify the signaling pathway for the proposed link between phosphodiesterase-5A (PDE5A) inhibition and decreased cardiac Na(+)/H(+) exchanger (NHE-1) activity.. NHE-1 activity was assessed in rat isolated papillary muscles by the Na(+)-dependent initial pH(i) recovery from a sustained acidosis (ammonium prepulse). ERK1/2, p90RSK and NHE-1 phosphorylation state during acidosis was determined.. PDE5A inhibition (1 μmol/L sildenafil, SIL) did not modify basal pH(i) but significantly blunted pH(i) recovery after sustained acidosis. Although preventing ERK1/2- p90RSK signaling pathway (10 μmol/L U0126) mimicked SIL effect, SIL did not blunt the acidosis-mediated increase in kinases activation. SIL+U0126 did not show additive effect on NHE-1 activity. Then, we hypothesized that SIL could be activating phophasatases (PP1 and/or PP2A) to directly dephosphorylate NHE-1 despite preserved ERK1/2-p90RSK activation. Non-specific phosphatases inhibition (1 μmol/L okadaic acid) canceled SIL effect on pH(i) recovery from acidosis. Same result was observed by inhibiting PP2A either with a lower dose of okadaic acid (1 nmol/L) or, more specifically, with 100 μmol/L endothall. Consistently, NHE-1 phosphorylation at Ser703 increased after acidosis, SIL prevented this effect and PP2A inhibition (endothall) reverted SIL effect.. We suggest that PDE5A inhibitors decrease NHE-1 phosphorylation and activity through a mechanism that involves PP2A activation.

Topics: Acidosis; Animals; Butadienes; Cyclic Nucleotide Phosphodiesterases, Type 5; Dicarboxylic Acids; Hydrogen-Ion Concentration; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Okadaic Acid; Papillary Muscles; Phosphodiesterase Inhibitors; Phosphorylation; Piperazines; Protein Phosphatase 1; Protein Phosphatase 2; Purines; Rats; Ribosomal Protein S6 Kinases, 90-kDa; Sildenafil Citrate; Sodium-Hydrogen Exchangers; Sulfones