Induction of skeletal muscle contracture and calcium release from isolated sarcoplasmic reticulum vesicles by sanguinarine

Abstract

The benzophenanthrine alkaloid, sanguinarine, was studied for its effects on isolated mouse phrenic-nerve diaphragm preparations. Sanguinarine induced direct, dose-dependent effects on muscle contractility. Sanguinarine-induced contracture was partially inhibited when the extracellular Ca(2+) was removed or when the diaphragm was pretreated with nifedipine. Depletion of sarcoplasmic reticulum (SR) internal calcium stores completely blocked the contracture. Sanguinarine induced Ca(2+) release from the actively loaded SR vesicles was blocked by ruthenium red and dithiothreitol (DTT), consistent with the ryanodine receptor (RyR) as the site of sanguinarine action. Sanguinarine altered [(3)H]-ryanodine binding to the RyR of isolated SR vesicles, potentiating [(3)H]-ryanodine binding at lower concentrations and inhibiting binding at higher concentrations. All of these effects were reversed by DTT, suggesting that sanguinarine-induced Ca(2+) release from SR occurs through oxidation of critical SH groups of the RyR SR calcium release channel.

Figure 1

Sanguinarine-induced muscular contracture and paralysis of mouse diaphragm. The effects of 40 μM sanguinarine on nerve-stimulated (trace a and b) or muscle-stimulated (trace c and d) or quiescent (trace e) phrenic nerve-diaphragm isolated from mouse were studied according to the procedure outlined in Methods. In trace b and d, 1×10⁻⁶ g ml⁻¹ α-bungarotoxin (α-BuTx) and 1 μM tetrodotoxin (TTX) were added prior to the addition of sanguinarine, respectively.

Figure 2

Dose-response effects for sanguinarine on muscular contracture in mouse diaphragm. Mouse diaphragm was prepared as described in Methods and the effects of sanguinarine at different doses on contractile force (a) and time to peak tension (b) were measured. Data are expressed as mean of multiple measurements and vertical lines show s.e.mean (n>4).

Figure 3

Reversal of the sanguinarine-induced contracture by sulphydryl reducing agent. The effect of sulphydryl reducing agent, dithiothreitol (DTT), on sanguinarine-induced contracture was examined. In trace b, the DTT was added after removal of the sanguinarine by washing the diaphragm with the fresh Krebs medium when the maximal contractile force was reached. In trace c, 2 mM of DTT was added at the beginning of the contracture without washing. In trace d, 2 mM of DTT was added prior to the addition of sanguinarine.

Figure 4

Induction of Ca²⁺ release by sanguinarine from SR membrane vesicles. SR vesicles (0.6 mg ml⁻¹) were actively loaded with Ca²⁺ and the Ca²⁺ concentration in the medium was monitored by absorbance difference at 710 nm and 790 nm according to the procedures outlined in Methods. Trace 1, Ca²⁺ release induced by 2 μg ml⁻¹ polylysine; traces 2 and 3, Ca²⁺ release induced by 20 μM and 10 μM sanguinarine, respectively; traces 4 and 5, Ca²⁺ release induced by 20 μM sanguinarine with prior addition of 275 μM DTT and 2 μM RR, respectively.

Figure 5

Dose-response effect of sanguinarine on [³H]-ryanodine binding. Sanguinarine, at concentration indicated, were added to the reaction mixture containing 0.5 mg ml⁻¹ SR and 10 nM [³H]-ryanodine. The specific binding of [³H]-ryanodine to the SR were measured according to the procedures outlined in Methods. Control binding was measured with the addition of 0.2% solvent, DMSO. Data are presented as mean±s.e.mean of triplicate measurements from three different preparations.