The effects of flavoxate hydrochloride on voltage-dependent L-type Ca2+ currents in human urinary bladder

Abstract

The effects of flavoxate hydrochloride (Bladderon, piperidinoethyl-3-methylflavone-8-carboxylate; hereafter referred as flavoxate) on voltage-dependent nifedipine-sensitive inward Ba(2+) currents in human detrusor myocytes were investigated using a conventional whole-cell patch-clamp. Tension measurement was also performed to study the effects of flavoxate on K(+)-induced contraction in human urinary bladder. Flavoxate caused a concentration-dependent reduction of the K(+)-induced contraction of human urinary bladder. In human detrusor myocytes, flavoxate inhibited the peak amplitude of voltage-dependent nifedipine-sensitive inward Ba(2+) currents in a voltage- and concentration-dependent manner (K(i) = 10 microM), and shifted the steady-state inactivation curve of Ba(2+) currents to the left at a holding potential of -90 mV. Immunohistochemical studies indicated the presence of the alpha(1C) subunit protein, which is a constituent of human L-type Ca(2+) channels (Ca(V)1.2), in the bundles of human detrusor smooth muscle. These results suggest that flavoxate caused muscle relaxation through the inhibition of L-type Ca(2+) channels in human detrusor.

Figure 1

Effects of flavoxate on K⁺-induced contraction (10, 20, 40 and 80 mM) of human detrusor strips. (a) K⁺-induced contraction in the absence (control) and presence of 100 nM flavoxate. (b) K⁺-induced contraction in the absence and presence of 30 μM flavoxate.

Figure 2

Effects of flavoxate (⩾100 nM) on the peak amplitude of K⁺-induced contraction of human detrusor strips, when the peak amplitude of 80 mM K⁺-induced contraction in the absence of flavoxate was normalized as one.

Figure 3

Effects of flavoxate and nifedipine on voltage-dependent Ba²⁺ currents in human detrusor. Whole-cell recording, pipette solution Cs⁺-TEA⁺ solution containing 5 mM EGTA and bath solution 10 mM Ba²⁺ containing 135 mM TEA⁺. (a) Original current traces before (control, (i)) and after application of 10 μM flavoxate (ii), as indicated in (b). (iii) Indicates a current trace just before the application of 10 μM nifedipine. (b) The time course of the effects of application of flavoxate and nifedipine on the peak amplitude of the voltage-dependent Ba²⁺ current evoked by repetitive depolarizing pulses to +10 mV from a holding potential of −60 mV. Time 0 indicates the time when 10 μM flavoxate was applied to the bath.

Figure 4

Concentration–response curves for flavoxate on voltage-dependent Ba²⁺ currents in human detrusor. Relationships between relative inhibition of the peak amplitude of Ba²⁺ current and the concentration of flavoxate at two holding potentials (−60 and −90 mV). The peak amplitude of the Ba²⁺ current elicited by a step pulse to +10 mV from the holding potential just before application of flavoxate was normalized as one. The curves were drawn by fitting the following equation using the least-squares method: Relative amplitude of voltage-dependent Ba²⁺ current=1/{1+(D/K_i) n_H} where K_i, D and n_H are the inhibitory dissociation constant, concentration of flavoxate (μM) and Hill's coefficient, respectively. The following values were used for the curve fitting: −60 mV, K_i=10 μM, n_H=1.1; −90 mV, K_i=56 μM, n_H=1.1. Each symbol indicates the mean of 5–15 observation with±s.d. shown by vertical lines. Some of the s.d. bars are less than the size of the symbol.

Figure 5

Effects of flavoxate on voltage-dependent Ba²⁺ inward currents at a holding membrane potential of −60 mV in human detrusor. The pipette solution was Cs⁺-TEA⁺ solution containing 5 mM EGTA and the bath solution was 10 mM Ba²⁺ containing 135 mM TEA⁺. (a) (i) Original current traces before (control) and after application of 10 μM flavoxate at the indicated pulse potentials. (ii) Inward Ba²⁺ current from (i) scaled to match their peak amplitudes and superimposed. (b) Current–voltage relationships obtained in the absence (control) or presence of 10 μM flavoxate. The current amplitude was measured as the peak amplitude of the Ba²⁺ inward current in each condition. The lines were drawn by eye. (c) Relationship between the test potential and relative value of the Ba²⁺ inward currents inhibited by 10 μM flavoxate, expressed as a fraction of the peak amplitude of the Ba²⁺ inward current evoked by various amplitudes of depolarizing pulse in the absence of flavoxate. Each symbol indicates the mean of five observations with±s.d. shown by vertical lines. The line was drawn by eye.

Figure 6

Effects of flavoxate (30 μM) on the voltage-dependent inactivation of the Ba²⁺ inward currents in human detrusor. Whole-cell recording, pipette solution Cs⁺-TEA⁺ solution containing 5 mM EGTA and bath solution 10 mM Ba²⁺ containing 135 mM TEA⁺. The holding potential was −90 mV. Conditioning pulses of various amplitudes were applied (up to +30 mV, 8 s duration) before application of the test pulse (to +10 mV, 1 s duration). An interval of 20 ms was allowed between these two pulses to estimate possible contamination of the capacitive current. The peak amplitude of Ba²⁺ current evoked by each test pulse was measured before and after application of 30 μM flavoxate. The curves with the solid line; the peak amplitude of Ba²⁺ inward current in the absence and presence of flavoxate without application of any conditioning pulse was normalized as one. The curve with the broken line was normalised to the current at +10 mV upon stepping from −90 mV in 30 μM flavoxate. The lines were draw by fitting the data to the following equation in the least-squares method: I=(I_max–C)/{1+exp [(V–V_half)/k]}+C, where I, I_max, V, V_half, k and C are the relative amplitude of Ba²⁺ inward currents observed at various amplitude of the conditioning pulse (I) and observed with application of the conditioning pulse of −90 mV (I_max), amplitude of the conditioning pulse (V), and that where the amplitude of Ba²⁺ inward current was reduced to half (V_half), slope factor (k) and fraction of the noninactivating component of Ba²⁺ inward current (C). The curves in the absence or presence of flavoxate were drawn using the following values: (control), I_max=1, V_half=−31, k=12 and C=0.09 (flavoxate, 30 μM), I_max=0.68, V_half=−40, k=13 and C=0.06. Each symbol indicates the mean of 5–6 observations with±s.d. shown by vertical lines. Some of the s.d. bars are less than the size of the symbol.

Figure 7

The effects of flavoxate on voltage-dependent Ba²⁺ currents. No pulses were applied for the initial 4 min after application of 30 μM flavoxate. Each symbol shows the size of the mean value of the peak amplitude of the voltage-dependent Ba²⁺ current evoked by the depolarizing pulses after this 4 min from two holding potentials (−90 mV, 0.64±0.07, n=5; −120 mV, 0.66±0.09, n=5). The peak amplitude of the voltage-dependent Ba²⁺ current just before application of flavoxate was normalized as one (control).

Figure 8

Fluorescent images of immunoreactivity for Ca_V1.2 in the human detrusor bundles. (a, b) Ca_V1.2 immunoactivity; clear membranous staining was observed at the tissues of the human urinary bladder smooth muscle layers. (c, d) Negative control: use of Ca_V1.2 antibody preadsorbed with the immunizing antigen never yields any colour reaction. Bar (white line in (d)) represents 200 μm.