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.

Differential mechanism of the effects of ester-type local anesthetics on sarcoplasmic reticulum Ca-ATPase.

The effect of the local anesthetics procaine and tetracaine on sarcoplasmic reticulum membranes isolated from two masticatory muscles, masseter and medial pterygoid, was tested and compared to fast-twitch muscles. The effects of the anesthetics on Ca-ATPase activity, calcium binding, uptake, and phosphorylation of the enzyme by inorganic phosphate (Pi) were tested with radioisotopic methods. Calcium binding to the Ca-ATPase was non-competitively inhibited, and the enzymatic activity decreased in a concentration-dependent manner. The inhibition of the activity depended on pH, calcium concentration, the presence of the calcium ionophore calcimycin, and the membrane protein concentration. Unlike fast-twitch membranes, the pre-exposure of the masseter and medial pterygoid membranes to the anesthetics enhanced the enzymatic activity in the absence of calcimycin, supporting their permeabilizing effect. Procaine and tetracaine also interfered with the calcium transport capability, decreasing the maximal uptake without modification of the calcium affinity for the ATPase. Besides, the anesthetics inhibited the phosphorylation of the enzyme by Pi in a competitive manner. Tetracaine revealed a higher inhibitory potency on Ca-ATPase compared to procaine, and the inhibitory concentrations were lower than usual clinical doses. It is concluded that procaine and tetracaine not only affect key steps of the Ca-ATPase enzymatic cycle but also exert an indirect effect on membrane permeability to calcium and suggest that the consequent myoplasmic calcium increase induced by the anesthetics might account for myotoxic effects, such as sustained contraction and eventual rigidity of both fast-twitch and masticatory muscles.

Radiation produces irreversible chronic dysfunction in the submandibular glands of the rat.

The exposure to high doses of ionizing radiation during radiotherapy results in severe morphological and functional alterations of the salivary glands, such as xerostomia. In the present study we investigated the chronic effect of a single radiation dose of 15 Gray (Gy) limited to head and neck on rat salivary gland function (salivary secretion and gland mass) and histology. Results indicate that norepinephrine (NE)-induced salivary secretion was reduced significantly at 30, 90, 180 and 365 days after the administration of a single dose of 15 Gy of ionizing radiation compared to non-irradiated animals. The maximal secretory response was reduced by 33% at 30 and 90 days post irradiation. Interestingly, a new fall in the salivary response to NE was observed at 180 days and was maintained at 365 days post irradiation, showing a 75% reduction in the maximal response. The functional fall of the salivary secretion observed at 180 days post irradiation was not only associated with a reduction of gland mass but also to an alteration of the epithelial architecture exhibiting a changed proportion of ducts and acini, loss of eosinophilic secretor granular material, and glandular vacuolization and fibrosis. On the basis of the presented results, we conclude that ionizing radiation produces irreversible and progressive alterations of submandibular gland (SMG) function and morphology that leads to a severe salivary hypo-function.