Nickel inhibits β-1 adrenoceptor mediated activation of cardiac CFTR chloride channels

Abstract

Cardiac ventricular myocytes exhibit a protein kinase A-dependent Cl(-) current (ICl.PKA) mediated by the cystic fibrosis transmembrane conductance regulator (CFTR). There is conflicting evidence regarding the ability of the divalent cation nickel (Ni(2+)), which has been used widely in vitro in the study of other cardiac ionic conductances, to inhibit ICl.PKA. Here the action of Ni(2+) on ICl.PKA activated by β-adrenergic stimulation has been elucidated. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. Externally applied Ni(2+) blocked ICl.PKA activated by 1 μM isoprenaline with a log IC50 (M) of -4.107 ± 0.075 (IC50=78.1 μM) at +100 mV and -4.322 ± 0.107 (IC50=47.6 μM) at -100 mV. Thus, the block of ICl.PKA by Ni(2+) was not strongly voltage dependent. Ni(2+) applied internally via the patch-pipette was ineffective at inhibiting isoprenaline-activated ICl,PKA, but in the same experiments the current was suppressed by external Ni(2+) application, indicative of an external site of Ni(2+) action. In the presence of 1 μM atenolol isoprenaline was ineffective at activating ICl.PKA, but in the presence of the β2-adrenoceptor inhibitor ICI 118,551 isoprenaline still activated Ni(2+)-sensitive ICl.PKA. Collectively, these data demonstrate that Ni(2+) ions produce marked inhibition of β1-adrenoceptor activated ventricular ICl.PKA at submillimolar [Ni(2+)]: an action that is likely to involve an interaction between Ni(2+) and β1-adrenoceptors. The concentration-dependence for ICl.PKA inhibition seen here indicates the potential for confounding effects on ICl,PKA to occur even at comparatively low Ni(2+) concentrations, when Ni(2+) is used to study other cardiac ionic currents under conditions of β-adrenergic agonism.

Fig. 1

The effect of extracellular Ni²⁺ on isoprenaline-activated I_Cl.PKA. (A) Panel (Ai) shows the voltage-ramp protocol (holding potential = 0 mV, frequency of application 1/10 s) used for recording Cl⁻ currents. Panel (Aii) shows representative currents, plotted against voltage, obtained during the descending phase of the ramp saw-tooth. Letters indicate traces obtained from the time-points indicated in panel (B). (B) Representative time course of an experiment with currents sampled at +100 mV (open circles) and −100 mV (filled circles) during saw-tooth voltage-ramps; the solid bars at the top indicate application of 1 μM isoprenaline (ISO) and Ni²⁺ at the concentrations indicated. (C) Concentration–response relationship of the effect of Ni²⁺ on I_Cl.PKA. Concentration–responses are shown at +100 mV (open circles) and −100 mV (filled circles). The ‘n’ numbers at each respective concentration are shown in parentheses. Solid and dashed lines represent fits to the data with Eq. (2) at −100 mV and +100 mV respectively. The fitted logIC₅₀ (M) at +100 and −100 mV were respectively −4.107 ± 0.075 and −4.322 ± 0.101; the n_H values for the fits were 1.145 ± 0.187 at +100 mV and 1.019 ± 0.214 at −100 mV.

Fig. 2

Intracellular Ni²⁺ does not inhibit ISO-activated I_Cl.PKA. (Ai) Mean current–voltage relations (mean ± SEM; solid lines shows the mean values and the surrounding dotted lines show SEMs) recorded using pipette solution without intracellular Ni²⁺. Currents were recorded in control extracellular solution (a), in the presence of 1 μM isoprenaline (ISO) (b) and in the presence of 1 μM ISO plus 10 mM Ni²⁺ (c) (n = 6). (Aii) Mean ISO-activated I_Cl.PKA calculated from the data shown in panel (Ai). (Bi) Mean current–voltage relations (mean ± SEM; solid lines show the mean values and the surrounding dotted lines shows SEMs) recorded using pipette solution containing intracellular Ni²⁺ (300 μM). Currents were recorded in control extracellular solution (a), in the presence of 1 μM ISO (b) and in the presence of 1 μM ISO plus 10 mM Ni²⁺ (c) (n = 6). (Bii) Mean ISO-activated I_Cl.PKA calculated from the data shown in panel (Bi).

Fig. 3

Involvement of β-1 adrenoceptors in Ni²⁺-sensitive I_Cl.PKA. (A) Representative time course of an experiment with currents sampled at +100 mV (open circles) and −100 mV (filled circles) during saw-tooth voltage-ramps; the solid bars at the top indicate application of 1 μM isoprenaline (ISO), 10 mM Ni²⁺ and 1 μM atenolol. (B) Representative current–voltage relations obtained during the descending phase of the ramp saw-tooth. Letters indicates traces obtained as indicated in panel (A). (C) Mean current–voltage relations for the Ni²⁺-sensitive (n = 5) and atenolol-sensitive (n = 5) difference currents in the presence of 1 μM ISO. Bars indicate ±SEM. There was no statistically significant difference between the mean Ni²⁺-sensitive and atenolol-sensitive current–voltage relations.

Fig. 4

β-2 adrenoceptors do not activate Ni²⁺-sensitive I_Cl.PKA. (A) Representative time course of an experiment with currents sampled at +100 mV (open circles) and −100 mV (filled circles) during saw-tooth voltage-ramps; the solid bars at the top indicates application of 100 nM ICI 118,551, 1 μM isoprenaline (ISO) and 10 mM Ni²⁺. (B) Representative current–voltage relations obtained during the descending phase of the ramp saw-tooth. Letters indicate traces obtained as indicated in panel (A). (C) Fractional block by 10 mM Ni²⁺ of ISO-activated I_Cl.PKA at +100 mV and −100 mV in the absence (control) and presence of 100 nM ICI 118,551. ICI 118,551 had no statistically significant effect on fractional block at either voltage. Replicate numbers are given in parentheses.