Phorbol ester activation of chloride current in guinea-pig ventricular myocytes

1. Although earlier studies with phorbol esters indicate that protein kinase C (PKC) may be an important regulator of Cl^- current (I(Cl)) in cardiac cells, there is a need for additional quantitative data and investigation of conflicting findings. Our objectives were to measure the magnitude, time course, and concentration-dependence of I(Cl) activated in guinea-pig ventricular myocytes by phorbol 12-myristate 13-acetate (PMA), evaluate its PKC dependence, and examine its modification by external and internal ions. 2. The whole-cell patch clamp technique was used to apply short depolarizing and hyperpolarizing pulses to myocytes superfused with Na⁺-, K⁺-, Ca²⁺-free solution (36°C) and dialysed with Cs⁺ solution. Stimulation of membrane currents by PMA (threshold ≤ 1 nM, EC₅₀ ≃ 14 nM, maximal 40% increase with ≥ 100 nM) plateaued within 6-10 min. 3. PMA-activated current was time-independent, and suppressed by 1 mM 9-anthracenecarboxylic acid (9-AC). Its reversal potential (E(rev)) was sensitive to changes in the Cl^- gradient, and outward rectification of the current-voltage (I-V) relationship was more pronounced with 30 mM than 140 mM Cl^- dialysate. 4. The relative permeability of PMA-activated channels estimated from E(rev) measurements was I^- > Cl^- >> aspartate. Channel activation was independent of external Na⁺. 5. PMA failed to activate I(Cl) in myocytes pretreated with 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or dialysed with pCa 10.5 solution. Lack of response to 4α-phorbol 12,13-didecanoate (αPDD) was a further indication of mediation by PKC. 6. I(Cl) induced by 2 μM forskolin was far larger than that induced by PMA, suggesting that endogenous protein kinase A is a much stronger Cl^- channel activator than endogenous PKC in these myocytes. 7. The macroscopic properties of PMA-induced I(Cl) appear to be indistinguishable from those of PKA-activated I(Cl). We discount stimulation of PKA by PMA as an explanation, and conclude that endogenous PKC may activate PKA-regulated Cl^- channels in these myocytes.