Effects of epoxyeicosatrienoic acids on the cardiac sodium channels in isolated rat ventricular myocytes

Abstract

1. Whole-cell Na+ currents (holding potential, -80 mV; test potential, -30 mV) in rat myocytes were inhibited by 8, 9-epoxyeicosatrienoic acid (8,9-EET) in a dose-dependent manner with 22+/-4% inhibition at 0.5 microM, 48+/-5% at 1 microM, and 73+/-5% at 5 microM (mean +/- S.E.M., n = 10, P<0.05 for each dose vs. control). Similar results were obtained with 5,6-, 11,12-, and 14,15-EETs, while 8,9-dihydroxyeicosatrienoic acid (DHET) was 3-fold less potent and arachidonic acid was 10- to 20-fold less potent. 2. 8,9-EET produced a dose-dependent, hyperpolarized shift in the steady-state membrane potential at half-maximum inactivation (V ), without changing the slope factor. 8,9-EET had no effect on the steady-state activation of Na+ currents. 3. Inhibition of Na+ currents by 8,9-EET was use dependent, and channel recovery was slowed. The effects of 8,9-EET were greater at depolarized potentials. 4. Single channel recordings showed 8,9-EET did not change the conductance or the number of active Na+ channels, but markedly decreased the probability of Na+ channel opening. These results were associated with a decrease in the channel open time and an increase in the channel closed times. 5. Incubation of cultured cardiac myocytes with 1 microM [3H]8,9-EET showed that 25% of the radioactivity was taken up by the cells over a 2 h period, and most of the uptake was incorporated into phospholipids, principally phosphatidylcholine. Analysis of the medium after a 2 h incubation indicated that 86% of the radioactivity remained as [3H]8,9-EET while 13% was converted into [3H]8,9-DHET. After a 30 min incubation, 1-2% of the [3H]8,9-EET uptake by cells remained as unesterified EET. 6. These results demonstrate that cardiac cells have a high capacity to take up and metabolize 8,9-EET. 8,9-EET is a potent use- and voltage-dependent inhibitor of the cardiac Na+ channels through modulation of the channel gating behaviour.

Figure 1. Effect of 8,9-EET on the Na⁺ currents in isolated rat cardiac myocytes

Whole-cell I_Na was elicited from a holding potential of −80 mV to a test potential of −30 mV in the presence of 20 mM [Na⁺]_o at room temperature. A represents raw tracings of Na⁺ currents before (control) and after application of 0.5, 1.0 and 5.0 μM 8,9-EET, followed by the partial reversal of the EET effect upon washout. The capacitive transients are eliminated for cosmetic reasons. B represents the peak whole-cell Na⁺ current in the same cell plotted versus time with pulses elicited at 15 s intervals. The arrows indicate the onsets of the interventions.

Figure 2. Dose-dependent inhibition of the Na⁺ currents by 8,9-EET (A) and arachidonic acid (B)

A shows the magnitude of Na⁺ current inhibition by 0.5, 1.0 and 5.0 μM 8,9-EET as a ratio of Na⁺ currents under control conditions. Na⁺ currents were evoked from a resting potential of −80 mV to a test potential of −30 mV (n = 10, *P < 0.05vs. control). B shows the magnitude of Na⁺ current inhibition by 0.5, 1.0, 5.0 and 10.0 μM arachidonic acid as a ratio of control Na⁺ currents under conditions similar to those shown in A (n = 11, * P < 0.05).

Figure 3. Effect of 8,9-EET on whole-cell (A) Na⁺ current-voltage relations and activation curves (B)

A represents the current-voltage relations in five cells, in the presence of 0 (control, ▪), 0.5 μM (•), 1.0 μM (▴) and 5.0 μM (▾) 8,9-EET. Holding potentials are at −80 mV with pulse durations of 20 ms. Pulses are repeated at 5 mV increments at 5 s intervals. Values are normalized to the peak I_Na amplitude under control conditions and are expressed as means ± s.e.m.B represents the whole-cell Na⁺ current activation curve showing the conductance-voltage relations at baseline (control, ▪) and in the presence of 0.5 μM (•), 1.0 μM (▴) and 5.0 μM (▾) 8,9-EET. Values are normalized to maximal conductance (G_Na,max) and are expressed as means ± s.e.m. The data are curve-fitted using a Boltzmann equation. V_½ is the half-activation value and k is the slope factor. Differences between control values and those with the various doses of 8,9-EET are not statistically significant.

Figure 4. Effect of prepulse potential on the inhibition of Na⁺ current by 8,9-EET

A shows the amplitudes of the Na⁺ currents (test potentials of −20 mV) plotted against the indicated prepulse potentials (500 ms) under control conditions (▪) and after application of 0.5 μM (•), 1.0 μM (▴) and 5.0 μM (▾) 8,9-EET. Each point represents the means ± s.e.m. of nine experiments. B shows the effects of 8,9-EET on the whole-cell Na⁺ current steady-state inactivation curves. The data in A are normalized to maximal Na⁺ current and plotted versus prepulse potentials for control (▪) and after application of 0.5 μM (•), 1.0 μM (▴) and 5.0 μM (▾) 8,9-EET. Data are expressed as means ± s.e.m. The data are fitted with a Boltzmann equation where V_½ represents the half-inactivation potential and k is the slope factor. The V_½ values are significantly different for 1.0 and 5.0 μM 8,9-EET versus control (P < 0.05). The differences in k values are not statistically significant.

Figure 5. Effect of 8,9-EET on the frequency-dependent inhibition of the cardiac Na⁺ currents (A) and the recovery of the Na⁺ currents from inactivation (B)

A shows the peak whole-cell Na⁺ current amplitudes during trains of 20 depolarizing current pulses at a cycle length of 200 ms with holding potentials of −80 mV and test potentials of −30 mV (inset). Results are normalized against the amplitude of the first pulse and are expressed as means ± s.e.m. Responses in the presence of 0 (control, ▪), 0.5 μM (•), 1.0 μM (▴) and 5.0 μM (▾) 8,9-EET are plotted against time (n = 6). B shows the effect of 8,9-EET on the recovery of the Na⁺ current from inactivation using a two-pulse protocol. Conditioning pulses at 0 mV of 500 ms duration are followed by test pulses at −30 mV of 20 ms duration with an intertrain interval of 5 s (inset). Holding potentials are −80 mV with interpulse recovery intervals between 1 and 2000 ms. The results are expressed as means ± s.e.m. for 0 (control, ▪), 0.5 μM (•), 1.0 μM (▴) and 5.0 μM (▾) 8,9-EET (n = 6).

Figure 6. Effect of 8,9-EET on single Na⁺ channel conductance

Currents from single Na⁺ channels in rat ventricular myocytes are recorded in cell-attached configuration from a holding potential of −100 mV to various test potentials as indicated. The raw current tracings under control conditions or after exposure to 5 μM 8,9-EET are shown in A. In B, the unitary Na⁺ current amplitudes are plotted against membrane potentials and the results are fitted with a linear regression equation. The single Na⁺ channel conductance (γ) is 27 pS and 5 μM 8,9-EET does not alter γ (n = 4).

Figure 7. Effect of 8,9-EET on single Na⁺ channel opening probability

Representative single channel recordings in cell-attached membrane patches of rat ventricular myocytes with a holding potential of −100 mV and a test potential of −40 mV before (control) and after application of 5 μM 8,9-EET are shown in A. In the presence of EET, the number of null sweeps is significantly increased. The ensemble-averaged currents from 500 sweeps before (control) and after application of 5 μM 8,9-EET are shown in B. C represents the results from seven experiments measuring single Na⁺ channel opening probability (P_o) under control conditions and in the presence of 5 μM 8,9-EET. P_o is 0.270 ± 0.027 for the controls, and 8,9-EET markedly reduced P_o to 0.048 ± 0.026.

Figure 8. Effect of 8,9-EET on the kinetics of single Na⁺ channels

Single Na⁺ channel currents are recorded in cell-attached patches with holding potentials of −100 mV and test potentials of −40 mV. A shows the Na⁺ channel open duration histograms for 0 (control) and 5 μM 8,9-EET. The open dwell time distributions are fitted with a single exponential equation with time constants (τ) of 163 μs for control and 100 μs for 8,9-EET. B shows the Na⁺ channel closed-duration histograms for control and 5 μM 8,9-EET treatment. The mean closed time of the Na⁺ channel recordings is fitted with a two-exponential equation. Both τ₁ and τ₂ are prolonged by 8,9-EET. τ₁ is 22 μs for control and 108 μs for EET and τ₂ is 1.4 ms for control and 1.95 ms for EET.

Figure 9. Distribution of radioactivity following incubation of rat neonatal myocytes with [³H]8,9-EET

Cells were incubated in medium containing 1 μM [³H]8,9-EET for 30 min to 4 h, after which the cell- and medium-associated lipids were extracted and analysed. The distribution of radioactivity between the cells and medium (quantified by liquid scintillation counting) following a 2 h incubation with [³H]8,9-EET is shown in A. The values are expressed as means ± s.e.m., n = 3. In B, a representative TLC chromatogram (upper panel) shows the distribution of radioactivity in cell lipids following a 30 min incubation with [³H]8,9-EET. PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; GL, glycerides. Similar results were obtained from a duplicate culture. Migration of radiolabelled phospholipid standards (PC and PE) and 8,9-EET are shown in the lower panel.

Figure 10. Metabolites of [³H]8,9-EET found in the medium of cultured rat neonatal myocytes during 2 h (A) and 4 h (B) incubations

Following incubation, lipids were extracted from the medium and separated by reverse-phase HPLC, and the radioactivity was assayed with an on-line flow scintillation detector. In A, after a 2 h incubation, the radiolabelled components co-migrated with 8,9-EET and 8,9-DHET standards. In B, after a 4 h incubation, a major unidentified product formed from [³H]8,9-EET is apparent. Both panels contain a chromatogram from a single culture, but similar results were obtained from a duplicate culture in each case.