Urinary bladder instability induced by selective suppression of the murine small conductance calcium-activated potassium (SK3) channel

Abstract

Small conductance, calcium-activated potassium (SK) channels have an important role in determining the excitability and contractility of urinary bladder smooth muscle. Here, the role of the SK isoform SK3 was examined by altering expression levels of the SK3 gene using a mouse model that conditionally overexpresses SK3 channels (SK3T/T). Prominent SK3 immunostaining was found in both the smooth muscle (detrusor) and urothelium layers of the urinary bladder. SK currents were elevated 2.4-fold in isolated myocytes from SK3T/T mice. Selective suppression of SK3 expression by dietary doxycycline (DOX) decreased SK current density in isolated myocytes, increased phasic contractions of isolated urinary bladder smooth muscle strips and exposed high affinity effects of the blocker apamin of the SK isoforms (SK1-3), suggesting an additional participation from SK2 channels. The role of SK3 channels in urinary bladder function was assessed using cystometry in conscious, freely moving mice. The urinary bladders of SK3T/T had significantly greater bladder capacity, and urine output exceeded the infused saline volume. Suppression of SK3 channel expression did not alter filling pressure, threshold pressure or bladder capacity, but micturition pressure was elevated compared to control mice. However, SK3 suppression did eliminate excess urine production and caused a marked increase in non-voiding contractions. The ability to examine bladder function in mice in which SK3 channel expression is selectively altered reveals that these channels have a significant role in the control of non-voiding contractions in vivo. Activation of these channels may be a therapeutic approach for management of non-voiding contractions, a condition which characterizes many types of urinary bladder dysfunctions including urinary incontinence.

Figure 1. Distribution of SK3 immunoreactivity in the urinary bladder wall

A, SK3 immunoreactivity in a bladder cross-section from a control (C57BL6) mouse. Positive SK3 immunoreactivity (red) was seen in smooth muscle and urothelium. The inset shows a negative control, in which the tissue slice was pretreated with excess antigenic peptide. B, whole mount urothelial preparations stained for SK3 channels. Immunoreactivity is increased relative to control in SK3^T/T overexpressor urothelium, while DOX treatment suppresses SK3 immunostaining. C, whole mount smooth muscle preparations stained for SK3 channels obtained from control, SK3^T/T-overexpressing and SK3^T/T-suppressed mice.

Figure 2. Whole-cell SK currents recorded from mouse urinary bladder smooth muscle cells

A, family of currents elicited by 100 ms depolarization pulses from −70 to +10 mV in bladder myocytes from control (C57BL6), SK3^T/T-overexpressing (SK3^T/T) and SK3-suppressed (SK3^T/T+ DOX) mice in the presence of the BK channel inhibitor iberiotoxin (100 nM) and in the presence of the SK channel inhibitor apamin (1 μM, continued presence of iberiotoxin). B, apamin-sensitive current (I_SK) obtained by subtracting the current in the presence of apamin (and iberiotoxin) from the current in the presence of iberiotoxin alone. C, mean outward current density recorded in the presence of iberiotoxin. D, mean outward current density recorded in the presence of apamin (and iberiotoxin). E, I_SK density. *P < 0.05 vs. C57BL6, †P < 0.05 vs. SK3^T/T (one-way ANOVA).

Figure 3. Regulation of mouse urinary bladder smooth muscle phasic contractions by SK3 channels

A, recordings of spontaneous phasic contractions in urinary bladder smooth muscle strips isolated from control, SK3^T/T-overexpressing and SK3^T/T-suppressed mice. B, spontaneous phasic contraction frequency in C57BL6, SK3^T/T and SK3^T/T+ DOX bladder strips. *P < 0.05 vs. C57BL6; †P < 0.05 vs. SK3^T/T (one-way ANOVA). C, apamin-induced increase in phasic contraction amplitude, expressed as a percentage of the maximum apamin-induced effect in each strip. *P < 0.05 vs. C57BL6; †P < 0.05 vs. SK3^T/T (two-way ANOVA).

Figure 4. Urodynamic function in control, SK3^T/T-overexpressing and SK3^T/T-suppressed mice

A, cystometrograms (CMGs) from control (C57BL6), wild-type (SK3^+/+), SK3^T/T-overexpressing (SK3^T/T) and SK3^T/T-suppressed (SK3^T/T+ DOX) mice. The records from the SK3^T/T-overexpressing and the SK3^T/T-suppressed animal are from the same mouse studied prior to DOX treatment and then again 2 weeks after continuous DOX administration via the drinking water. The red trace shows the infused volume during continuous filling cystometry and the green trace shows the output of urine recorded on an analytical balance beneath the animal cage. Micturition was associated with a sharp rise in intravesical pressure (v). Note the substantial increase in bladder capacity in the SK3^T/T animal and the increase in non-voiding contractions (*) after DOX treatment. B, summary showing infused and voided volumes. C, residual volume measured by subtracting the voided volume from the infused volume. A positive value indicates urine remains in the bladder after voiding, whereas a negative value indicates that endogenous urine production was high enough to increase the amount of voided urine to a level greater than the amount of saline infused. D, non-voiding contractions per CMG were defined as increases in intravesical pressure of at least 5 mmHg that were not associated with measurable voiding. *P < 0.05 vs. C57BL6; †P < 0.05 vs. SK3^+/+; ‡P < 0.05 vs. SK3^T/T (one-way ANOVA).