TY - JOUR T1 - Antagonism of Calmodulin by Local Anesthetics JF - Molecular Pharmacology JO - Mol Pharmacol SP - 363 LP - 370 VL - 20 IS - 2 AU - M. VOLPI AU - R. I. SHA’AFI AU - M. B. FEINSTEIN Y1 - 1981/09/01 UR - http://molpharm.aspetjournals.org/content/20/2/363.abstract N2 - Transport of calcium across the erythrocyte membrane is regulated by a Ca2+-activated, Mg2+-dependent ATPase which is stimulated by calmodulin. The regulation of Ca2+ transport by calmodulin was studied in inside-out membrane vesicles (IO vesicles) prepared from erythrocytes treated with protease inhibitors and lysed in media containing ethylene glycol bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid and EDTA. Ca2+ uptake by IO vesicles was absolutely dependent on ATP, and the basal rate of uptake was 14.3 ± 1.1 nmoles/mg of IO vesicle protein per minute at a saturating concentration of free Ca2+ in the medium. Calmodulin stimulated Ca2+ uptake by an average of 3.5-fold to a Vmax of 49.4 ± 3.9 nmoles/mg of IO vesicle protein per minute. The K0.5 for calmodulin was 4.9 ± 0.9 nM and the maximal initial rate of Ca2+ uptake was attained at about 64 nM calmodulin. In a medium containing 80 mM Na+, calmodulin reduced the K0.5 for Ca2+ from 8.9 ± 1.8 µM (n = 3) to 5.0 ± 0.3 µM, but in a medium in which all Na+ was replaced by K+, calmodulin decreased K0.5 Ca2+ to 1.4 µM free Ca2+. Certain local anesthetics and drugs with local anesthetic-like properties (e.g., dibucaine, tetracaine, QX572, proadifen, mepacrine, quinine, propranolol, phenacaine, monocaine, and procaine), as well as trifluoperazine, inhibited calmodulin-stimulated Ca2+ uptake into IO vesicles. Calmodulin-stimulated (Ca2+ + Mg2+)-ATPase activity was inhibited to a degree equivalent to the effect on calcium uptake. Basal Ca2+ uptake (minus calmodulin) was only weakly inhibited; e.g., dibucaine at 1.0 mM inhibited stimulation by calmodulin 96.9%, but reduced basal Ca2+ uptake by only 11.4%. The local anesthetics increased the K0.5 for calmodulin and reduced Vmax for Ca2+ uptake. The reduction of Vmax by local anesthetics was dose-dependent: e.g., 35% reduction at 0.25 mM dibucaine and 65% reduction at 0.5 mM dibucaine. Vmax could not be restored to control levels by the addition of an excess of either calmodulin or Ca2+. The antagonism of calmodulin by local anesthetics appeared to have elements of both competitive and noncompetitive inhibition. The antagonism versus Ca2+ was uncompetitive. These results are in agreement with our previous findings that certain local anesthetics are antagonists of calmodulin-stimulated (Ca2+ + Mg2+)-ATPase and calmodulin-stimulated cyclic nucleotide phosphodiesterase. It is suggested that certain of the well-known inhibitory effects of local anesthetics on Ca2+-dependent cellular functions may be due to inhibition of the role played by calmodulin in facilitating these Ca2+-dependent processes. ER -