Effects of β-adrenoceptor stimulation on delayed rectifier K(+) currents in canine ventricular cardiomyocytes

Abstract

Background and purpose: While the slow delayed rectifier K(+) current (I(Ks)) is known to be enhanced by the stimulation of β-adrenoceptors in several mammalian species, phosphorylation-dependent regulation of the rapid delayed rectifier K(+) current (I(Kr)) is controversial.

Experimental approach: In the present study, therefore, the effect of isoprenaline (ISO), activators and inhibitors of the protein kinase A (PKA) pathway on I(Kr) and I(Ks) was studied in canine ventricular myocytes using the whole cell patch clamp technique.

Key results: I (Kr) was significantly increased (by 30-50%) following superfusion with ISO, forskolin or intracellular application of PKA activator cAMP analogues (cAMP, 8-Br-cAMP, 6-Bnz-cAMP). Inhibition of PKA by Rp-8-Br-cAMP had no effect on baseline I(Kr). The stimulating effect of ISO on I(Kr) was completely inhibited by selective β₁-adrenoceptor antagonists (metoprolol and CGP-20712A), by the PKA inhibitor Rp-8-Br-cAMP and by the PKA activator cAMP analogues, but not by the EPAC activator 8-pCPT-2'-O-Me-cAMP. In comparison, I(Ks) was increased threefold by the activation of PKA (by ISO or 8-Br-cAMP), and strongly reduced by the PKA inhibitor Rp-8-Br-cAMP. The ISO-induced enhancement of I(Ks) was decreased by Rp-8-Br-cAMP and completely inhibited by 8-Br-cAMP.

Conclusions and implications: The results indicate that the stimulation of β₁-adrenoceptors increases I(Kr), similar to I(Ks), via the activation of PKA in canine ventricular cells.

Figure 1

Effects of isoprenaline (ISO) on rapid delayed rectifier K⁺ (I_Kr) current. (A–C) I_Kr current traces showing tail currents as relaxation of current during repolarization to −40 mV under control conditions (A), following exposure to 100 nM ISO (B) and after washing the cells in ISO-free solution (C). (D) Representative experiment demonstrating the effect of 100 nM ISO on I_Kr and the full suppression of the ISO-induced current by 1 µM E-4041. (E) Voltage-dependence of the activation of I_Kr in control and in the presence of 100 nM ISO. Tail current amplitudes measured at each test potential was normalized to the respective tail current obtained at +20 mV. Symbols and bars are means ± SEM, n= 4.

Figure 2

(A) Cumulative concentration-dependent effect of isoprenaline (ISO) on rapid delayed rectifier K⁺ (I_Kr) tail current amplitude studied in eight myocytes. (B,C) Effects of 100 nM ISO in the presence of 100 nM metoprolol (n= 6) and 300 nM CGP-20712A (n= 4). I_Kr amplitudes were normalized to their respective control values. Columns and bars indicate means ± SEM. *Denotes significant (P < 0.05) differences from control (100%) determined using paired t-test.

Figure 3

Role of the cAMP/PKA pathway in regulation of rapid delayed rectifier K⁺ current (I_Kr). Left-hand column of each pair shows I_Kr tail current amplitudes measured in control (no drug, n= 13), following superfusion with forskolin (3 µM, n= 6), and after internal application of cAMP (250 µM, n= 9), 8-Br-cAMP (250 µM, n= 8), 6-bnz-cAMP (100 µM, n= 8), 8-pCPT-2'-O-Me-cAMP (100 µM, n= 6) and Rp-8-Br-cAMP (100 µM, n= 6). Right-hand column of each pair indicates I_Kr tails obtained following superfusion with 100 nM ISO. Data are means ± SEM. *Indicates significant (P < 0.05) differences from the control (no drug) I_Kr amplitude, determined using unpaired t-test. ⁺Denotes ISO-induced differences from the respective pre-ISO values, determined using paired t-test. ISO, isoprenaline; PKA, protein kinase.

Figure 4

Effects of isoprenaline (ISO) on slow delayed rectifier K⁺ (I_Ks) current. (A–C) I_Ks tail current traces obtained at repolarization to −40 mV under control conditions (A), following exposure to 100 nM ISO (B) and after washing the cells in ISO-free solution (C). (D) Representative experiment demonstrating the effect of 100 nM ISO on I_Ks and the full suppression of the ISO-induced current by 1 µM HMR-1556. (E) Average data obtained in 11 myocytes showing the cumulative effect of 10 nM and 100 nM ISO on I_Ks tail current amplitude. Columns and bars are means ± SEM values. *Indicates significant (P < 0.05) differences from control determined using paired t-test.

Figure 5

Role of the cAMP/PKA pathway in the regulation of slow delayed rectifier K⁺ (I_Ks) current. (A, B) Representative experiments demonstrating the effects of ISO in the presence of Rp-8-Br-cAMP (A) and 8-Br-cAMP (B). In (C), left-hand column of each pair shows I_Ks tail current amplitude measured in control (no drug, n= 11), and after internal application of Rp-8-Br-cAMP (100 µM, n= 5) and 8-Br-cAMP (250 µM, n= 5). Right-hand column of each pair indicates I_Ks tails obtained following superfusion with 100 nM ISO. Data are means ± SEM. *Indicates significant (P < 0.05) differences from the control (no drug)I_Ks amplitude determined using unpaired t-test. ⁺Denotes ISO-induced differences from the respective pre-ISO values determined using paired t-test. ISO, isoprenaline; PKA, protein kinase.