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(Circulation Research. 1997;81:219-228.)
© 1997 American Heart Association, Inc.

Articles

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

Shi-Sheng Zhou, Akira Takai, Makoto Tominaga, , Yasunobu Okada

From the Department of Cellular and Molecular Physiology (S.-S.Z., M.T., Y.O.), National Institute for Physiological Sciences, Okazaki, Japan, and the Department of Physiology (A.T.), School of Medicine, Nagoya (Japan) University.

Correspondence to Y. Okada, MD, PhD, Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan. E-mail okada{at}nips.ac.jp

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 (ED₅₀, 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 Ca²⁺ 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.

Key Words: anion channel • cystic fibrosis transmembrane conductance regulator • Cl^- channel blocker • dephosphorylation • cardiac myocyte