Phosphatase-Mediated Enhancement of Cardiac cAMP-Activated Cl − Conductance by a Cl − Channel Blocker, Anthracene-9-Carboxylate

Abstract

Abstract An aromatic carboxylate, anthracene-9-carboxylic acid (9-AC), is known as a Cl − channel blocker. However, variable 9-AC effects have hitherto been reported on the cardiac cAMP-activated Cl − conductance, when applied extracellularly. We have reexamined the 9-AC effect on the Cl − conductance activated by isoproterenol or forskolin in guinea pig ventricular myocytes under whole-cell patch-clamp conditions. The inward current was blocked by 9-AC at ≥0.5 mmol/L, but in contrast, the outward current was enhanced at much lower concentrations (ED 50 , ≈13 μmol/L). 9-AC applied by the intracellular perfusion technique increased both the inward and outward currents. In the presence of intracellular 9-AC, deactivation of the conductance after washout of isoproterenol or forskolin was largely prevented. 9-AC produced an enhancing effect, even after inhibiting the deactivation process by okadaic acid (OA), whereas it failed to produce additional effects in the presence of orthovanadate. Intracellular application of 9-AC together with OA virtually abolished the current deactivation. The 9-AC effects on the Cl − conductance were not dependent on intracellular Ca 2+ or pH. Putative inhibitors of alkaline (bromotetramisole) and acid phosphatases (tartrate) were without effect. 9-AC failed to inhibit the activities of purified protein phosphatase (PP)-1, -2A, and -2C. In the extract of guinea pig ventricle, 9-AC (≥10 μmol/L for full action) significantly inhibited a fraction of endogenous phosphatase activity that was sensitive to orthovanadate but not to OA, bromotetramisole, and tartrate. It is concluded that 9-AC blocks cardiac cAMP-activated (cystic fibrosis transmembrane conductance regulator) Cl − conductance from the extracellular side but enhances the conductance from the intracellular side by inhibiting an orthovanadate-sensitive phosphatase distinct from PP-1, -2A, -2B, or -2C and alkaline or acid phosphatase.