Evolving mechanisms of action of alverine citrate on phasic smooth muscles

Abstract

Background and purpose: We have investigated the mechanisms underlying the paradoxical ability of the antispasmodic, alverine, to enhance spontaneous activity in smooth muscles while suppressing evoked activity.

Experimental approach: The effects of alverine on spontaneous and induced contractile activity were examined in preliminary experiments with various smooth muscles. More detailed effects were also investigated by recording membrane potential, intracellular Ca2+ concentration ([Ca2+]i) and tension from single-bundle detrusor smooth muscle (DSM) of the guinea-pig urinary bladder.

Key results: Alverine (10 microM) increased the frequency and amplitude of spontaneous action potentials, transient increases in [Ca2+]i and associated contractions. Alverine also decreased action potential rate of decay, suggesting inhibition of L-type Ca channel inactivation. Charybdotoxin (50 nM) but neither cyclopiazonic acid (10 microM) nor Bay K 8644 (10 microM) attenuated alverine-induced enhancement of spontaneous contractions. Alverine suppressed contractions produced by high K (40 mM) or ACh (10 microM), without affecting electrical responses and with little suppression of increases in [Ca2+]i. This feature was very similar to that of the effects of the Rho kinase inhibitor Y-27632 (10 microM).

Conclusions and implications: Alverine may increase Ca influx during action potentials due to inhibition of the inactivation of L-type Ca channels, but may also suppress evoked activity by inhibiting the sensitivity of contractile proteins to Ca2+. The proportional contribution of Ca-dependent and Ca-independent contractions in DSM may differ between spontaneous and evoked activity, necessitating further investigations into the interactions between these pathways for assessing the therapeutic potential of alverine to treat DSM dysfunction.

Figures

Figure 1

Effects of 10 μM alverine on spontaneous activity. (a) Alverine-induced spontaneous activity in a previously inactive strip of detrusor, (b) alverine increased phasic activity in a strip of taenia caecum previously showing regular slow contractions. The drug was applied in the superfusing solution at the arrow. Scale bars (a) ordinate 2 mN, abscissa 1 min, (b) ordinate 20 mN, abscissa 1 min.

Figure 2

Effects of alverine on the spontaneous contractile activity of strips dissected from the guinea-pig detrusor. The drug was applied continuously at the line at the concentration indicated. The lower traces are the same as the upper traces but on a faster time scale.

Figure 3

Effects of alverine on the spontaneous contractile activity of smooth muscle strips dissected from the taenia of the guinea-pig caecum (a and b) and the portal vein (c). Scale bars are 10 mN for (a) and (b), 1 mN for (c). The whole trace lasts for 3.6 h.

Figure 4

Paradoxical effects of alverine on the guinea-pig detrusor. Electrical field stimulation was applied every 5 min (50 V, 20 Hz, 1 s, 0.05 ms pulse width). Alverine was added at the first arrow and washed out at the second arrow.

Figure 5

Effects of alverine on spontaneous action potentials, contractions and Ca transients recorded from DSMs of the guinea-pig bladder. Alverine (10 μM) facilitated spontaneous action potentials (Aa) and corresponding contractions (Ab) in a single-bundle DSM. It also increased the amplitude and reduced the rate of decay (Ac). Overlaid action potentials (solid line in control, dotted line in alverine) are averaged traces of 30 action potentials each. In another DSM preparation, alverine (10 μM) enhanced spontaneous Ca transients (Ba) and corresponding contractions (Bb). It also increased the basal Ca level (Ba) and caused a sustained contraction (Bb). With a fast time scale (C and D), alverine (10 μM) increased the amplitude of individual Ca transients (Ca) and contractions (Cb). Scale bars on the right in (Da) and (Db) refer to all Ca and tension traces, respectively.

Figure 6

Effects of cyclopiazonic acid (CPA), charybdotoxin (CTX) and Bay K 8644 on alverine-induced facilitations of spontaneous contractions recorded from DSMs of the guinea-pig bladder. In a single-bundle DSM, CTX (50 nM) caused a threefold increase in the amplitude of spontaneous contractions (A). Subsequent addition of alverine (10 μM) increased the amplitude of ‘single' contractions by only 20%. The overlaid traces (Ab) showed averaged ‘single' contractions in control (dash), in CTX (solid) and in CTX plus alverine (dotted) preparations. CPA (10 μM) increased the frequency and amplitude of spontaneous contractions (B). Subsequent application of alverine (10 μM) increased the amplitude of spontaneous contractions by about 100%. Bay K (10 μM) initiated bursting contractions (C), and subsequent application of alverine (10 μM) caused a further twofold increase in their amplitude. Scale bar in (C) also refers to (Aa) and (B).

Figure 7

Effects of alverine on changes in Ca and contractile responses induced by high K solution and acetylcholine (Ach) recorded from DSMs of the guinea-pig bladder. In a DSM bundle, increasing [K+]o caused a phasic increase in [Ca2+]i, which was followed by a sustained increase in [Ca2+]i (Aa) and an associated contraction (Ab). In the same preparation that had been treated with alverine (10 μM), a subsequent increase in [K+]o caused a similar increase in [Ca2+]i (Ba) but caused a much smaller contraction (Bb). In another DSM, ACh (10 μM) caused a biphasic increase in [Ca2+]i (Ca), and an associated contraction (Cb). In the same preparation, alverine (10 μM) suppressed ACh-induced contractions (Db) but had little effect on the ACh-induced increase in [Ca2+]i (Da). Scale bars in (Da) and (Db) refer to all Ca and tension traces, respectively.

Figure 8

Effects of alverine on high K solution and ACh-induced depolarizations recorded from DSMs of the guinea-pig urinary bladder. In a DSM preparation, increasing [K+]o depolarized the membrane by about 20 mV and increased the frequency of action potentials (Aa). In the same preparation that had been treated with alverine (10 μM), an increase in [K+]o caused a similar depolarization (Ab). In another DSM, ACh (10 μM) depolarized the membrane by about 25 mV, and also initially increased the frequency of action potentials and then their generation ceased (Ba). In the same preparation, alverine (10 μM) did not prevent either ACh-induced depolarization or its effects on action potentials (Bb). Scale bars in (Bb) refer to all traces.

Figure 9

Effects of Y-26763 on changes in Ca and contractile responses induced by high K solution and acetylcholine (Ach) recorded from DSMs of the guinea-pig bladder. In a DSM bundle, increasing [K+]o caused a phasic increase in [Ca2+]i, which was followed by a sustained increase in [Ca2+]i (Aa) and an associated contraction (Ab). In the same preparation that had been treated with Y-26763 (10 μM), a subsequent increase in [K+]o caused a similar increase in [Ca2+]i (Ba) but caused a much smaller contraction (Bb). In another DSM, ACh (10 μM) caused a biphasic increase in [Ca2+]i (Ca), and an associated contraction (Cb). In the same preparation, Y-26763 (10 μM) suppressed ACh-induced contractions (Db), while having little effect on the ACh-induced increase in [Ca2+]i (Da). Scale bars in (Da) and (Db) refer to all Ca and tension traces, respectively.