CHARACTERIZACION OF THE EFFECT OF ETIDOCAINE ON SERCA2a IN MEDIAL PTERYGOID MUSCLE.

Aim: To describe the characteristics of the effect of the local anesthetic etidocaine on the SERCA2a (sarcoendoplasmic reticulum Ca-ATPase isoform 2a) isolated from medial pterygoid muscle. Methods: Membranes sealed vesicles were obtained by differential centrifugation of medial pterygoid muscles from male adult New Zealand rabbits (n=5). SERCA2a was isolated by affinity chromatography. We determined the enzymatic activity (5 experiments performed in duplicate) in the presence of 0-90 mM etidocaine, using the Fiske & Subarrow colorimetric method. To evaluate the effect of etidocaine on the membrane permeability we assayed: 1) enzymatic activity in the presence or absence of calcium ionophore A23187, 2) enzymatic activity of sarcoplasmic reticulum (SR) membranes preincubated in etidocaine, and 3) determination of calcium efflux from actively preloaded vesicles, measuring the levels of the cation in the efflux media using a selective ion microelectrode. Mean values ± SEM of the CI50 for the anesthetic were calculated. We compared determinations of enzymatic activity with or without ionophore, and activity and calcium efflux determinations with and without etidocaine using t test (a=0.05). Results: Etidocaine inhibited the activity (CI50=20.06±1.64 mM) under the enzyme?s optimal conditions (250±10 mmol Pi.mg protein-1.h-1). In the absence of the calcium ionophore the activity was lower (150±7mmol Pi.mg protein-1.h-1, t=8.2 p< 0.0001) at [etidocaine]< 10mM. The length of the incubation time of SR membranes in etidocaine increased the enzymatic activity, reaching an optimal activity value at time=20 min in incubations with 10 mM etidocaine, showing the ionophoric-like effect of the anesthetic. Etidocaine increased the calcium efflux in SR membranes enriched in SERCA2a, (Cain=3.5±1 nmol.mg-1 vs control: Cain=16±0.8 nmol.mg-1, t=9.8 p<0.0001) after 2 min. Conclusions: Etidocaine showed a dual effect on the medial pterygoid SERCA2a: it inhibited the enzymatic activity, but also increased the membrane permeability. Both effects are shown at concentrations lower than those of clinical use (54mM) and could be the cause of miotoxic effects by promoting the calcium efflux from the sarcoplasmic reticulum, favoring the sustained muscle contraction.