The aim of this study was to characterize the sarcoplasmic-endoplasmic reticulum Ca-ATPase (SERCA) isoforms in rabbit masticatory muscles compared with those in fast-twitch muscle. It was hypothesized that combined expression of the SERCA isoforms in fast- and slow-twitch muscles accounts for lower Ca-ATPase activity. SERCA was isolated by differential centrifugation, the isoforms were determined by ELISA, and the activity of each isoform was measured using a colorimetric method. Activity was tested for significance by anova, and the distribution of isoforms was assessed using the chi-square test (P < 0.05) and correlated to SERCA activity using Spearman's rank correlation. SERCA1 was predominant (90.5%) in fast-twitch muscle, whereas a mixture of SERCA isoforms was found in masticatory muscles: 62-78% was SERCA2, 20-37% was SERCA1, and the SERCA3 content was negligible. Depressor muscles showed a significantly higher content (77.8%) of SERCA2, and elevator muscles showed a higher content (35.4%) of SERCA1. Elevator muscles showed higher expression of SERCA2a (58%), and depressor muscles showed higher expression of SERCA2b (20%). The SERCA1 content was mainly SERCA1a and significantly higher for elevator muscles (33%), whereas depressor muscles showed a higher content of SERCA1b (4%). The SERCA1 content of fast-twitch muscle was mainly SERCA1a (88.5%). It is concluded that the mixture of different SERCA isoforms, along with a substantial content of SERCA2b, in masticatory muscles would support lower Ca-ATPase activity and calcium transport.

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.

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