Modulation of cardiac membrane currents by phorbol esters.
Date
1996
Authors
Asai, Tatsuya.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
Protein kinase C (PKC) and protein kinase A (PKA) are important regulators of cardiac cell function. I have used PKC-activated phorbol 12-myristate 13-acetate (PMA) and PKA-activating forskoline (FSK), to investigate the regulation of membrane channels in guinea pig ventricular myocytes.
The whole-cell patch-clamp technique was used to measure currents through Cl$\sp-$ channels $(I\sb{\rm Cl}),$ delayed-rectifying K$\sp+$ channels $(I\sb{\rm K},$ inward-rectifying K$\sp+$ channels $(I\sb{\rm K1}),$ and L-type Ca$\sp{2+}$ channels $(I\sb{\rm Ca,L},I\sb{\rm Na,L}).$ In myocytes dialyzed with standard pCa 9 solutions, the active phorbol ester PMA had no effect on $I\sb{\rm K1},$ moderately increased $I\sb{\rm K}$ and $I\sb{\rm Cl}$ in a concentration-dependent manner (EC$\sb{50}$ 4 and 10 nM, respectively), and frequently depressed $I\sb{Ca,L}.$
$I\sb{\rm Cl}$ activated by PMA was time-independent, displayed marked outward rectification when the dialysate contained 30 mM Cl$\sp-,$ and became almost linear when dialysate Cl$\sp-$ concentration was increased to 140 mM. The current was strongly depressed by the Cl$\sp-$ channel blocker, anthracene-9-carboxylic acid.
Stimulation of $I\sb{\rm Cl}$ and $I\sb{\rm K}$ by PMA was suppressed when myocytes were pretreated with PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or dialyzed with pCa $\sim$11 solution. In addition, stimulation by PMA was not duplicated by the inactive phorbol esters, 4$\alpha$-phorbol 12,13-dedecanoate ($\alpha$PDD) and 4$\alpha$-phorbol ($\alpha$PHR). These results strongly suggest that the stimulatory effects of PMA on Cl$\sp-$ and K$\sp+$ channels are mediated by PKC.
In contrast to the foregoing, the depression of $I\sb{\rm Ca,L}$ induced by PMA (often co-monitored with $I\sb{\rm K}$ in the same cell) occurred $\sim$3 times faster than $I\sb{\rm K}$ stimulation, and was unaffected by the two interventions that inhibited $I\sb{\rm K}$ stimulation (pretreatment with H-7 and pCa $\sim$11 dialysate). Inactive phorbol ester $\alpha$PDD, but not $\alpha$PHR, had PMA-like effects on $I\sb{\rm Ca,L}.$ Similar inhibitory effects of phorbol esters were also observed when Na$\sp+$ was used as a charge-carrier for L-type current. These results suggest that phorbol esters depress Ca$\sp{2+}$ channel currents by a PKC-independent action.
A final objective was to investigate the effects of co-application of maximally-effective concentrations of PMA and FSK on the activation of $I\sb{\rm Cl}$ and stimulation of $I\sb{\rm K}.$ Mutual occlusionary effects of these agents were not detected; rather, there were hints of a synergistic activation of $I\sb{\rm Cl}$ by FSK in PMA-treated myocytes, and mutually additive effects of PMA and FSK on $I\sb{\rm K}.$
Thesis (Ph.D.)--Dalhousie University (Canada), 1996.
The whole-cell patch-clamp technique was used to measure currents through Cl$\sp-$ channels $(I\sb{\rm Cl}),$ delayed-rectifying K$\sp+$ channels $(I\sb{\rm K},$ inward-rectifying K$\sp+$ channels $(I\sb{\rm K1}),$ and L-type Ca$\sp{2+}$ channels $(I\sb{\rm Ca,L},I\sb{\rm Na,L}).$ In myocytes dialyzed with standard pCa 9 solutions, the active phorbol ester PMA had no effect on $I\sb{\rm K1},$ moderately increased $I\sb{\rm K}$ and $I\sb{\rm Cl}$ in a concentration-dependent manner (EC$\sb{50}$ 4 and 10 nM, respectively), and frequently depressed $I\sb{Ca,L}.$
$I\sb{\rm Cl}$ activated by PMA was time-independent, displayed marked outward rectification when the dialysate contained 30 mM Cl$\sp-,$ and became almost linear when dialysate Cl$\sp-$ concentration was increased to 140 mM. The current was strongly depressed by the Cl$\sp-$ channel blocker, anthracene-9-carboxylic acid.
Stimulation of $I\sb{\rm Cl}$ and $I\sb{\rm K}$ by PMA was suppressed when myocytes were pretreated with PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or dialyzed with pCa $\sim$11 solution. In addition, stimulation by PMA was not duplicated by the inactive phorbol esters, 4$\alpha$-phorbol 12,13-dedecanoate ($\alpha$PDD) and 4$\alpha$-phorbol ($\alpha$PHR). These results strongly suggest that the stimulatory effects of PMA on Cl$\sp-$ and K$\sp+$ channels are mediated by PKC.
In contrast to the foregoing, the depression of $I\sb{\rm Ca,L}$ induced by PMA (often co-monitored with $I\sb{\rm K}$ in the same cell) occurred $\sim$3 times faster than $I\sb{\rm K}$ stimulation, and was unaffected by the two interventions that inhibited $I\sb{\rm K}$ stimulation (pretreatment with H-7 and pCa $\sim$11 dialysate). Inactive phorbol ester $\alpha$PDD, but not $\alpha$PHR, had PMA-like effects on $I\sb{\rm Ca,L}.$ Similar inhibitory effects of phorbol esters were also observed when Na$\sp+$ was used as a charge-carrier for L-type current. These results suggest that phorbol esters depress Ca$\sp{2+}$ channel currents by a PKC-independent action.
A final objective was to investigate the effects of co-application of maximally-effective concentrations of PMA and FSK on the activation of $I\sb{\rm Cl}$ and stimulation of $I\sb{\rm K}.$ Mutual occlusionary effects of these agents were not detected; rather, there were hints of a synergistic activation of $I\sb{\rm Cl}$ by FSK in PMA-treated myocytes, and mutually additive effects of PMA and FSK on $I\sb{\rm K}.$
Thesis (Ph.D.)--Dalhousie University (Canada), 1996.
Keywords
Biology, Cell., Biology, Animal Physiology.