Ca-EGTA affects the relationship between [Ca2+] and tension in alpha-toxin permeabilized rat anococcygeus smooth muscle

Abstract

The relationship between calcium concentration ([Ca2+]) and force in smooth muscle can be studied by permeabilizing the sarcolemma and bathing the preparation in a mock intracellular solution. Normally [Ca2+] is set in these solutions using the Ca2+ chelator EGTA in the concentration range of 4-10 mM. This study shows that lowering total EGTA concentration ([EGTA]t) below 10 mM depresses Ca(2+)-activated force generated in 0.1 microM Ca2+. The observed threshold for the effect of EGTAt is 0.2 mM, and the effect is maximal at approximately 10 mM. BAPTA, another Ca2+ chelator, also produces this effect. Tension production in smooth muscle is controlled by acto-myosin interaction. This in turn is mediated by the relative activities of myosin light chain kinase (MLCK) and phosphatase (MLCP). Inhibiting MLCP with Microcystin LR (10 microM), an increase [EGTAt] from 0.2 mM to 10 mM still enhanced force. This suggests that EGTA promotes phosphorylation of myosin by the activation of MLCK and not by inhibition of MLCP.