Key role for Kv11.1 (ether‐a‐go‐go related gene) channels in rat bladder contractility

Figures

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  FIGURE 1. Expression of Kcnh2 channels in rat detrusor. (a) Quantitative PCR experiments showing the expression of total Kcnh2, long Kcnh2 isoform (Kcnq2‐L), Kcnh6 and Kcnh7 mRNAs in rat detrusor, normalized to the housekeeping gene Cyc1. The rat brain and heart were used as positive controls. Data are expressed as mean ± standard error of the mean. N = 5. (b) Fluorescent images of smooth muscle cells isolated from rat detrusor and labeled with a mouse monoclonal Kv11.1 antibody (left column) and α‐smooth muscle actin (α‐SMA, middle column). Cells where primary antibodies were omitted (incubated with secondary antibodies only) are also shown (right panel). Each image is representative of five separate dispersals. Scale bar = 10 μm.

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  FIGURE 2. Whole‐cell electrophysiology of isolated detrusor smooth muscle cells. (a, b) Show currents evoked by the protocol shown in (c) in the absence (a) and presence (b) of E4031 (2 μM). The right‐hand panels (a.i, b.i) show a zoom of the initial 200 ms of the test step to highlight the E4031‐sensitive current. (d) Shows a representative set of E4031‐sensitive currents. (e, f) Show the mean current amplitude at different test potentials in the absence and presence of E4031. Data were taken from 8 to 12 cells from four animals (N = 4, *p < 0.05, **p < 0.01, one‐way analysis of variance, error bars represent standard error of the mean).

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  FIGURE 3. Effect of Kv11.1 channel inhibitors on spontaneous contractile activity. (a) Representative trace of basal tone and phasic contraction amplitudes after the addition of 20 μM of the Kv11.1 inhibitors, E4031 (left) and Dofetilide (right). (b) The maximum increase in baseline tone at various concentrations of Kv11.1 inhibitors E4031 (i) and dofetilide (ii), and in the presence of neuronal inhibition with TTX (iii) (N = 5). (c) Percent increase in the amplitude of phasic contractions compared with the control, in the presence of Kv11.1 inhibitors, E4031 (i) and dofetilide (ii), and in the presence of neuronal inhibition (iii) (N = 5) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one‐way analysis of variance, error bars represent standard error of the mean).

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  FIGURE 4. Assessing amplitude of phasic contractions in response to carbachol in the presence and absence of potassium channel inhibitors. (a) Representative traces of contraction amplitudes in response to carbachol before and after the addition of the Kv11.1 inhibitor, E4031 (20 μM) within the same tissue (internal control). (b) Representative traces of contraction amplitudes in response to carbachol before and after the addition of vehicle control (dimethyl sulphoxide). (c) The change in the mean amplitude of contractions taken over a 2‐min period when the effect of Cch reaches a plateau and prior to the addition of the next concentration. The greatest change in amplitude, compared with control was observed in the presence of E4031 with 1 μM CCh (N = 5), *p < 0.05, error bars represent SEM; statistical analysis using two‐way ANOVA, with Bonferroni multiple comparison test. (d) The area under the curve analysis carried out at the maximum change in baseline tone after the addition of each concentration of Cch, in the presence of 20 μM E4031 (N = 5; two‐way ANOVA; error bars represent SEM). ANOVA, analysis of variance; SEM, standard error of the mean.

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  FIGURE 5. Effect of E‐4031 on nerve‐evoked contractions. (a) Electrical field stimulation (EFS) across the frequency range 0.5, 1, 2, 4, 8, 16, and 32 Hz evoked frequency‐dependent contractions. Following treatment with E4031 (20 μM), the stimulation protocol was repeated. Note the enhancement of spontaneous contractions in the presence of E4031. (b.i) Summary data from experimental series (N = 5), normalized to high K+ (60 mM) contraction. (b.ii) Mean data showing the effect of E4031 on EFS (30 Hz, 30 V) with and without prior incubation with TTX (t test). *p < 0.05, ***p < 0.0001; error bars represent standard error of the mean. (Two‐way analysis of variance with Bonferroni post‐hoc test).

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  FIGURE 6. Effect of E‐4031 on contractile activity of a denuded bladder. (a) Electric field stimulation (EFS) of a denuded bladder across a range of frequencies (0.5, 1, 2, 4, 8, 16, 32 Hz). The protocol was conducted before and after incubation with E4031 (20 μM). (b.i) Mean data indicating an increase in baseline tone in the absence (control) and presence of E4031. (b.ii) Average data of increase in phasic contractile amplitude after the addition of E4031 or in the presence of the vehicle (control) (t test). (B.iii) Summary data of EFS‐induced contractions (normalized to K+ (60 mM). *p < 0.05, ***p < 0.0001, N = 5, error bars represent standard error of the mean).

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  FIGURE 7. Effect of the Kv11.1 activator, PD118057 on the amplitude of spontaneous and EFS (electric field‐stimulated) contractions (20 Hz). (a) Representative trace of cumulative concentration of PD118057. (b) Normalized amplitudes of spontaneous and EFS contractions at increasing concentrations of PD118057. (c) Normalized amplitudes of spontaneous and EFS contractions in response to PD118057, relative to the vehicle. N = 5, Error bars represent standard error of the mean, ***p < 0.001. Statistical analysis using two‐way analysis of variance.

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  FIGURE 8. Characterization of baseline tone and phasic contraction amplitude in the presence of BK channel inhibitors in the presence and absence of E4031. (a) Representative trace of basal tone and phasic contraction amplitudes after the addition of 20 μm of the Kv11.1 inhibitor, following preincubation with 300 nM of the BK inhibitor iberiotoxin. (b) Maximum increase in baseline tone in the presence of iberiotoxin with and without E4031 (N = 5). (c) Percent increase in the amplitude of phasic contractions compared with control, in the presence of the Kv11.1 inhibitor, E4031 with and without prior incubation with iberiotoxin (N = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, error bars represent SEM. Statistical testing using one‐way ANOVA (N = 5, one‐way ANOVA, **p < 0.01, error bars represent SEM). ANOVA, analysis of variance; SEM, standard error of the mean.

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  FIGURE 9. The effect of isoprenaline in the presence of potassium channel inhibitors. (a) Decrease in phasic contraction amplitude of detrusor muscle strips upon treatment with Isoprenaline. The relaxation effect of Isoprenaline is significantly counteracted upon pretreatment with E4031 (b) Preincubation with either XE991 or Iberiotoxin did not significantly alter the adrenergic effect of Isoprenaline (N = 5), *p < 0.05; error bars represent standard error of the mean, statistical analysis using two‐way analysis of variance, with Bonferroni multiple comparison test.