Phosphatase-mediated enhancement of cardiac cAMP-activated Cl-conductance by a Cl- channel blocker, anthracene-9-carboxylate.
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 > or = 0.5 mmol/L, but in contrast, the outward current was enhanced at much lower concentrations (ED50, approximately 13 mumol/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 (> or = 10 mumol/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.
Zhou
S S
SS
Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Japan.
Takai
A
A
Tominaga
M
M
Okada
Y
Y
D000249:Adenosine Monophosphate / Q000502:physiology*; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology*; D002478:Cells, Cultured; D002712:Chlorides / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000502:physiology*; D005260:Female; D006168:Guinea Pigs; D018408:Patch-Clamp Techniques; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D016276:Ventricular Function
D000873:Anthracenes; D002712:Chlorides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000249:Adenosine Monophosphate; C010030:9-anthroic acid; D010749:Phosphoprotein Phosphatases