Redox modulation of basal and beta-adrenergically stimulated cardiac L-type Ca(2+) channel activity by phenylarsine oxide

Abstract

1. Phenylarsine oxide (PAO) is commonly used to inhibit tyrosine phosphatase activity. However, PAO can affect a variety of different processes because of its ability to promote sulfhydryl oxidation. In the present study, we investigated the effects that PAO has on basal and beta-adrenergically stimulated L-type Ca(2+) channel activity in isolated cardiac myocytes. 2. Extracellular application of PAO transiently stimulated the basal L-type Ca(2+) channel activity, whereas it irreversibly inhibited protein kinase A (PKA)-dependent regulation of channel activity by isoproterenol, forskolin and 8-CPT-cAMP (8-p-chlorophenylthioadenosine 3',5'-cyclic monophosphate). PAO also inhibited channel activity irreversibly stimulated in the presence of adenosine 5'-(3-thiotriphosphate) tetralithium salt. 3. Neither the stimulatory nor the inhibitory effects of PAO were affected by the tyrosine kinase inhibitor lavendustin A, suggesting that tyrosine phosphorylation is not involved. 4. Extracellular application of the sulfhydryl-reducing agent dithiothreitol (DTT) antagonized both the stimulatory and inhibitory effects of PAO. Yet, following intracellular dialysis with DTT, only the inhibitory effect of PAO was antagonized. 5. The inhibitory effect of PAO was mimicked by intracellular, but not extracellular application of the membrane impermeant thiol oxidant 5,5'-dithio-bis(2-nitrobenzoic acid). 6. These results suggest that the stimulatory effect of PAO results from oxidation of sulfhydryl residues at an extracellular site and the inhibitory effect is due to redox regulation of an intracellular site that affects the response of the channel to PKA-dependent phosphorylation. It is concluded that the redox state of the cell may play a critical role in modulating beta-adrenergic responsiveness of the L-type Ca(2+) channel in cardiac myocytes.

Figure 1

PAO transiently stimulates the basal L-type Ca²⁺ current. (a) Time course of changes in peak Ca²⁺ current recorded during depolarizing voltage-clamp steps to 0 mV, applied every 6 s, following exposure to 100 μM PAO. (b) Individual current traces recorded at time points indicated in panel a. (c) Cumulative responses normalized to the magnitude of the basal Ca²⁺ current recorded under control conditions. ^***The magnitude of the current measured after 1.5–2 min of exposure to PAO (n=30) was significantly larger than that measured under control conditions (P<0.001). NS, the magnitude of the current measured following 3–5 min of exposure to PAO (n=16) was not significantly different than that measured under control conditions.

Figure 2

PAO antagonizes stimulation of the L-type Ca²⁺ current by the β-adrenergic agonist Iso. (a) Time course of changes in peak Ca²⁺ current recorded during depolarizing voltage-clamp steps to 0 mV, applied every 6 s, following exposure to 30 nM Iso alone and Iso plus 100 μM PAO. (b) Time course of changes in peak Ca²⁺ current following exposure to 100 μM PAO alone and PAO plus 30 nM Iso. Insets, individual current traces recorded at time points indicated.

Figure 3

Lavendustin A does not alter the effect of PAO on the basal L-type Ca²⁺ current or the L-type Ca²⁺ current response to Iso. (a) Time course of changes in peak Ca²⁺ current recorded during depolarizing voltage-clamp steps to 0 mV, applied every 6 s, following exposure to 100 μM PAO alone and PAO plus 30 nM Iso in cells that were pre-exposed to 5 μM lavendustin A. Inset, individual current traces recorded at time points indicated. (b) Cumulative responses to PAO in control cells (n=30) and in cells pretreated with lavendustin A (n=12). (c) Cumulative responses to Iso alone (n=6) and PAO plus Iso in control cells (n=6) and in cells pretreated with 5 μM lavendustin A (n=11). NS, the magnitude of the response to PAO or Iso following exposure to PAO was not significantly different in control and lavendustin A-treated cells.

Figure 4

DTT blocks PAO-mediated transient stimulation of the basal Ca²⁺ current. (a) Time course of changes in peak Ca²⁺ current recorded during depolarizing voltage-clamp steps to 0 mV, applied every 6 s, following exposure to 1 mM DTT and DTT plus 100 μM PAO. (b) Individual current traces recorded at time points indicated in panel a. (c) Cumulative responses in cells exposed to 100 μM PAO in the absence (n=30) and presence (n=14) of 1 mM DTT.

Figure 5

DTT reverses PAO-induced inhibition of the L-type Ca²⁺ current response to the β-adrenergic agonist Iso. (a) Time course of changes in peak Ca²⁺ current recorded during depolarizing voltage-clamp steps to 0 mV, applied once every 6 s, following exposure to 100 μM PAO alone, subsequent exposure to 30 nM Iso alone, and Iso plus 1 mM DTT. (b) Individual current traces recorded at time points indicated in panel a. (c) Effect of 100 μM PAO on the response to 30 nM Iso before and after exposure to 1 mM DTT (n=7). ^**Magnitude of the Ca²⁺ current response to Iso was significantly larger following exposure to DTT (P<0.01).

Figure 6

Intracellular but not extracellular application of DTNB attenuates the L-type Ca²⁺ current response to Iso. (a) Time course of changes in peak Ca²⁺ current recorded during depolarizing voltage-clamp steps to 0 mV, applied once every 6 s, following exposure to 30 nM Iso alone, Iso plus 200 μM extracellular DTNB (DTNB_o), and DTNB_o alone. Inset, individual current traces recorded at time points indicated. (b) Time course of changes in peak Ca²⁺ current recorded from a myocyte dialyzed with a pipette solution containing 200 μM DTNB (DTNB_i) following exposure to 30 nM Iso alone. Inset, individual current traces recorded at time points indicated. (c) Response to 30 nM Iso before and following exposure to 200 μM DTNB_o (n=5) and in cells dialyzed with 200 μM DTNB_i (n=10). NS, the magnitude of the response to Iso alone or Iso following exposure to DTNB_o was not significantly different. ^**Magnitude of the current measured in the presence of DTNB_i plus Iso was significantly smaller than the magnitude of the current measured following exposure to Iso in the absence of DTNB_i (P<0.01). Statistical comparisons were performed using the Holm–Sidak method for multiple comparisons.

Figure 7

Intracellular dialysis of myocytes with dithiothreitol (DTT_i) attenuates the ability of PAO to block the L-type Ca²⁺ current response to Iso. (a) Time course of changes in peak Ca²⁺ current recorded from a myocyte dialyzed with a pipette solution containing 1 mM DTT following exposure to 100 μM PAO and subsequent exposure to 30 nM Iso alone. (b) Individual current traces recorded at time points indicated in panel a. (c) Effect of 100 μM PAO on the L-type Ca²⁺ current response to 30 nM Iso in control cells (n=6) and cells dialyzed with 1 mM DTT (n=10). ^**Ability of PAO to inhibit the response to Iso was significantly altered in cells dialyzed with DTT (P<0.01).

Figure 8

PAO irreversibly inhibits the L-type Ca²⁺ current stimulated by Iso, forskolin, 8-CPT-cAMP and Iso in the presence of ATPγS. (a) Time course of changes in peak Ca²⁺ current recorded during depolarizing voltage-clamp steps to 0 mV, applied once every 6 s, following exposure to 200 μM 8–CPT-cAMP alone and 8-CPT-cAMP plus 100 μM PAO. Inset, individual current traces recorded at time points indicated. (b) Time course of changes in peak Ca²⁺ current recorded from a myocyte dialyzed with a pipette solution containing 5 mM ATPγS following exposure to 30 nM Iso alone, washout of Iso and subsequent exposure to 100 μM PAO alone. Inset, individual current traces recorded at time points indicated. (c) Cumulative responses to 30 nM Iso (n=6), 3 μM forskolin (n=4), 200 μM 8-CPT-cAMP (n=6) and 30 nM Iso in the presence of 5 mM ATPγS (n=4) before and after exposure to 100 μM PAO.