Drug action of benzocaine on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle.

The effect of the local anesthetic benzocaine on sarcoplasmic reticulum membranes isolated from fast-twitch muscles was tested. The effects on Ca-ATPase activity, calcium binding and uptake, phosphoenzyme accumulation and decomposition were assessed using radioisotopic methods. The calcium binding to the Ca-ATPase was noncompetitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner (IC50 47.1 mM). The inhibition of the activity depended on the presence of the calcium ionophore calcimycin and the membrane protein concentration. The pre-exposure of the membranes to benzocaine enhanced the enzymatic activity in the absence of calcimycin, supporting the benzocaine permeabilizing effect, which was prevented by calcium. Benzocaine also interfered with the calcium transport capability by decreasing the maximal uptake (IC50 40.3 mM) without modification of the calcium affinity for the ATPase. It inhibited the phosphorylation of the enzyme, and at high benzocaine concentration, the dephosphorylation step became rate-limiting as suggested by the biphasic profile of phosphoenzyme accumulation at different benzocaine concentrations. The data reported in this paper revealed a complex pattern of inhibition involving two sites for interaction with low and high benzocaine concentrations. It is concluded that benzocaine not only exerts an indirect action on the membrane permeability to calcium but also affects key steps of the Ca-ATPase enzymatic cycle.

Amide-type local anesthetics action on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle.

Myotoxic effects related to intracellular Ca(2+) disturbances have been reported for local anesthetics. Such effects might derive from Ca-ATPase dysfunction. The aim of this work was to describe the effect of lidocaine and bupivacaine on the sarcoplasmic reticulum (SR) Ca-ATPase from fast-twitch skeletal muscle and to identify the affected steps of the enzyme's cycle. SR sealed vesicles were isolated from rabbit fast-twitch muscles by ultracentrifugation. The effect of the anesthetics on Ca-ATPase activity was assessed with a colorimetric method and Ca(2+) binding, uptake, phosphorylation of the enzyme by ATP, Ca(2+) dissociation kinetics and phosphoenzyme formation and decomposition levels were tested with radioisotopic methods. Lidocaine and bupivacaine inhibited Ca-ATPase activity with half-maximal inhibitory concentrations (Ki) of 25.3 and 31.4 mM, respectively, and the steady-state Ca(2+) transport ability with Ki values of 33.6 and 46.5 mM, decreasing the maximal transport rate without modification of the Ca(2+) or ATP affinity for the enzyme. This is consistent with an absence of competition for the transport and catalytic sites. The anesthetics did not inhibit Ca(2+) binding but inhibited the phosphorylation partial reactions. Ca(2+) dissociation kinetics was not affected, but the phosphoenzyme levels were decreased, and the decomposition rate of the phosphoenzyme became faster in the presence of the anesthetics. It is concluded that lidocaine and bupivacaine at concentrations available in pharmaceutical formulations for clinical medical and dental uses inhibit the SR Ca-ATPase through inhibition of key phosphorylation steps of the enzymatic cycle.