Dimethyl sulphoxide relaxes rabbit detrusor muscle by decreasing the Ca2+ sensitivity of the contractile apparatus

Abstract

Background and purpose: The intravesical administration of dimethyl sulphoxide (DMSO) is used to alleviate the symptoms of interstitial cystitis. We investigated the relaxant effect of DMSO and its underlying mechanism in the detrusor muscle.

Experimental approach: The effects of DMSO on contraction, on Ca2+ sensitivity of myofilaments, and on myosin light chain (MLC) phosphorylation were investigated in both intact and alpha-toxin-permeabilized strips of rabbit detrusor muscle.

Key results: In fura-PE3-loaded strips, DMSO (>1%) induced a significant relaxation during sustained contractions induced by 60 mM K+-depolarization or 10 microM carbachol, while having no effect on the [Ca2+](i) level. DMSO decreased the level of MLC phosphorylation during the contractions induced by 60 mM K+ and 10 microM carbachol. DMSO also inhibited both the contraction and MLC phosphorylation induced by calyculin-A in intact strips. In the alpha-toxin-permeabilized preparations, DMSO relaxed the Ca2+-induced contraction and also inhibited the tension development induced by a stepwise increment of Ca2+ concentrations. Such a relaxant effect of DMSO was enhanced in the presence of phosphate.

Conclusions and implications: DMSO relaxes rabbit detrusor muscle by decreasing the Ca2+ sensitivity of myofilaments. Inhibition of the kinase activities involved in myosin phosphorylation may play a major role in DMSO-induced Ca2+ desensitization. Inhibition of the cross-bridge cycling at the step of phosphate release may also contribute to the relaxant effect of DMSO. Such relaxant effects of DMSO could be linked to the therapeutic effect of DMSO in interstitial cystitis.

Figures

Figure 1

The effect of dimethyl sulphoxide (DMSO) on [Ca2+]i and tension during the contractions induced by 60 mM K+ or 10 μM carbachol in intact strips of rabbit detrusor muscle. Representative traces and summaries showing the effect of DMSO on [Ca2+]i and tension during the sustained contractions induced by 60 mM K+ (a) and 10 μM carbachol (b). DMSO was added 10 min after the initiation of the precontraction. After the 10-min exposure, DMSO was then washed out. (c) The concentration–response curves for the DMSO-induced relaxation during the precontractions induced by 60 mM K+ (K) or 10 μM carbachol (CCh). During the sustained phase of the precontraction, the fractional amount of DMSO (DMSO (+)) or distilled water (DMSO (−)) was increased in a stepwise manner at 5-min intervals. The levels of [Ca2+]i and tension obtained at rest and those obtained just before the applications of DMSO or distilled water were assigned values of 0 and 100%, respectively. The data represent the mean±s.e.m. (n=5). **P<0.01; *P<0.05; n.s., not significantly different, in comparison to the values obtained just before the application of DMSO (a, b) and those obtained with the corresponding fractional amount of distilled water (c).

Figure 2

The reversibility of the relaxant effect of dimethyl sulfoxide (DMSO) in intact strips of rabbit detrusor muscle. Representative traces and summaries showing the effect of the preceding treatment with DMSO on the subsequent contractile responses to 60 mM K+ (a) or 10 μM carbachol (b). The strips were first contracted with 60 mM K+ or 10 μM carbachol. DMSO (10%) was applied 10 min later. After 10 min treatment with DMSO, the strips were then incubated in PSS for 15 min and then they were stimulated with the second applications of 60 mM K+ or 10 μM carbachol. The contractile response was evaluated both at the peak of the contraction (peak) and 10 min after initiating the contraction (sustained phase), while the values obtained with the first contraction were considered to be 100%. Data represent the mean±s.e.m. (n=5). n.s., not significantly different.

Figure 3

The effects of dimethyl sulphoxide (DMSO) on the Ca2+-induced contractions in α-toxin-permeabilized strips of rabbit detrusor muscle. (a) Representative traces of tension showing the effect of 10% DMSO on the contraction induced by 3 μM Ca2+. (b–e) Representative traces and summary of the contractile responses to a stepwise increase in the concentrations of Ca2+ in the presence of 10% distilled water (DW) (b), or 5% (c) and 10% (d) DMSO. The concentrations of Ca2+ were increased from 0 to 100 μM (first). After recording the contraction induced by 100 μM Ca2+ in the presence of distilled water or DMSO, the strips were then stimulated with 100 μM Ca2+ in their absence to record 100% level, as indicated by closed triangles. The strips were then completely relaxed in the Ca2+-free CSS for 30 min, and they were again stimulated with 100 μM Ca2+ (second). Data represent the mean±s.e.m. (n=5). *P<0.05 vs the values obtained with the corresponding concentrations of Ca2+ in the presence of 10% distilled water. n.s., not significantly different vs 100% level.

Figure 4

The effects of dimethyl sulphoxide (DMSO) on the phosphorylation of MLC in intact strips of rabbit detrusor muscle. Representative photograph of an immunoblot analysis and a summary of the phosphorylation of MLC obtained in 5.9 mM K+-PSS before the contractile stimulation with 60 mM K+ or 10 μM carbachol (rest), 1–2 min after the stimulation (peak), and 10 min after the stimulation and just before the application of DMSO (sustained) and at 10 min after adding 10% DMSO (DMSO +) or 10% distilled water (DMSO −) during the contractile stimulation. The arrowheads and double arrowheads indicate the unphosphorylated and mono-phosphorylated form of MLC, respectively. Data represent the mean±s.e.m. (n=7). *P<0.05. #P<0.05 vs rest.

Figure 5

The effects of dimethyl sulphoxide (DMSO) and wortmannin (WM) on the contraction and the phosphorylation of MLC induced by calyculin-A (CLA) in the intact strips of rabbit detrusor muscle. (a-e) Representative traces and a summary of the contraction induced by 100 nM CLA, in the absence ((a), (b); CLA in (e)) and presence of 10% DMSO ((c); DMSO → CLA in (e)) or 10 μM WM ((d); WM → CLA in (e)). In (a) and (b), 10% DMSO and 10 μM WM were applied 60 min after initiating the contraction with calyculin-A. In (c) and (d), DMSO and WM were applied 10 min before initiating the contraction. The effects of the post-treatment of DMSO (CLA → DMSO in (e)) and WM (CLA → WM in (e)) on the CLA-induced contraction were evaluated 30 min after the application. The reference response to 60 mM K+ was recorded before starting the experimental protocols. The levels of tension at rest and those obtained 10 min after the stimulation with 60 mM K+ were assigned values of 0 and 100%, respectively. (f) A summary of the levels of the MLC phosphorylation obtained at the time points as indicated on the representative traces in (a-d) (marked with A-H in traces). Data represent the mean±s.e.m. (n=4 for (e); n=7 for (f)).

Figure 6

The synergistic effects of dimethyl sulphoxide (DMSO) and phosphate on the contractions in the α-toxin-permeabilized strips of rabbit detrusor muscle. Representative traces (a–c) and a summary (d) of the effects of 3% DMSO, 10 mM phosphate and their combination on the 3 μM Ca2+-induced contraction in the α-toxin-permeabilized strips. DMSO and phosphate were applied 30 min after initiating the contraction. The level of tension obtained just before the application of DMSO or phosphate and that obtained in the relaxing solution were assigned values of 0 and 100% relaxation, respectively. Data represent the mean±s.e.m. (n=4). *P<0.05 vs the precontraction level (0% relaxation). #P<0.05.

Figure 7

The effect of dimethyl sulphoxide (DMSO) on in vitro MLC phosphorylation. Representative photograph of Coomassie brilliant blue staining and a summary of in vitro MLC phosphorylation by native MLCK or active MLCK in the presence and absence of 10% DMSO. The reaction was terminated at 30 s. The arrowheads and double arrowheads indicate the unphosphorylated and monophosphorylated form of MLC, respectively. Data represent the mean±s.e.m. (n=7). *P<0.05. #P<0.05 vs DMSO(−) and MLCK native (−).