Effects of tetracaine on sarcoplasmic calcium release in mammalian skeletal muscle fibres

1.Single muscle fibres were dissociated enzymatically from the extensor dig itorum communis muscle of rats. The fibres were mounted into a double Vasel ine gap experimental chamber and the events in excitation-contraction coupl ing were studied under voltage clamp conditions in the presence and absence of the local anaesthetic tetracaine. 2. Changes in intracellular calcium concentration ([Ca2+](i)) were monitore d using the calcium sensitive dyes antipyrylazo III and fura-2 and the rate of calcium release (R-rel) from the sarcoplasmic reticulum (SR) was calcul ated Tetracaine decreased the maximal attained [Ca2+](i) and suppressed, in a dose-dependent manner, both the early peak and the steady level of R-rel in the voltage range examined. 3. The concentration dependence of the effects on the two kinetic component s of R-rel were almost identical with a half-effective concentration (K-50) of 70 and 71 mu m and a Hill coefficient (n(H)) of 2.7 and 2.3 for the pea k and the steady level, respectively Furthermore, the drug did not alter th e peak to steady level ratio up to a concentration (50 mu M) that caused a 35 +/- 5% reduction in calcium release. Higher concentrations did suppress the ratio but the degree of suppression was voltage independent. 4. Tetracaine (50 mu M) neither influenced the total available intramembran e charge nor altered its membrane potential dependence. It shifted the tran sfer function, the normalized SR permeability versus normalized charge to t he right, indicating that similar charge transfer caused a smaller increase in SR permeability. 5. To explore the site of action of tetracaine further the ryanodine recept or (RyR) calcium release channel of the SR was purified and reconstituted i nto planar lipid bilayers. The reconstituted channel had a conductance of 5 11 +/- 14 pS (n = 8) in symmetric 250 mM KCl that was not affected bq tetra caine. Tetracaine decreased the open probability of the channel in a concen tration-dependent manner with K-50 = 68 mu M and n(H)= 1.5. 6. These experiments show that tetracaine suppresses SR calcium release in enzymatic isolated mammalian skeletal muscle fibres. This effect is due, pr esumably to the decreased open probability of the RyR in the presence of th e drug. Since both the inactivating peak and the steady level of R-rel were equally affected by tetracaine, our observations suggest that there is a t ight coupling between these kinetic components of SR calcium release in mam malian skeletal muscle.