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 (Icl) 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 Icl 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(+)-, Ca(2+)-free solution (36 degrees C) and dialysed with Cs+ solution. Stimulation of membrane currents by PMA (threshold < or = 1nM, EC50 approximately equal to 14 nM, maximal 40% increase with > or = 100 nM) plateaued within 6-10 min. 3. PMA-activated current was time-independent, and suppressed by l mM 9-anthracenecarboxylic acid (9-AC). Its reversal potential (Erev) 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 Erev measurements was I- > Cl- > > aspartate. Channel activation was independent of external Na+. 5. PMA failed to activate Icl in myocytes pretreated with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or dialysed with pCa 10.5 solution. Lack of response to 4 alpha-phorbol 12, 13-didecanoate (alpha PDD) was a further indication of mediation by PKC. 6. Icl induced by 2 microM 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 Icl appear to be indistinguishable from those of PKA-activated Icl. We discount stimulation of PKA by PMA as an explanation, and conclude that endogenous PKC may activate PKA-regulated Cl- channels in these myocytes.
Shuba
L M
LM
Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
Asai
T
T
McDonald
T F
TF
D000818:Animals; D000838:Anions / Q000378:metabolism; D000873:Anthracenes / Q000494:pharmacology; D018118:Chloride Channels / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D006168:Guinea Pigs; D006352:Heart Ventricles; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000838:Anions; D000873:Anthracenes; D018118:Chloride Channels; D005576:Colforsin; C010030:9-anthroic acid; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate