Molecular Pharmacology, Vol 20, 363-370, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Antagonism of Calmodulin by Local Anesthetics

Inhibition of Calmodulin-Stimulated Calcium Transport of Erythrocyte Inside-Out Membrane Vesicles

¹ Department of Physiology and Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032

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(-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 V_max of 49.4 ± 3.9 nmoles/mg of IO vesicle protein per minute. The K_0.5 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 K_0.5 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 K_0.5 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 K_0.5 for calmodulin and reduced V_max for Ca²⁺ uptake. The reduction of V_max by local anesthetics was dose-dependent: e.g., 35% reduction at 0.25 mM dibucaine and 65% reduction at 0.5 mM dibucaine. V_max 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.