Effect of exogenous phospholipase A2 treatment on cardiac muscarinic receptors of highly purified canine sarcolemmal vesicles

Effects of phospholipase A2 (PLA2)-catalyzed hydrolysis of sarcolemmal phospholipids on ventricular muscarinic receptors were examined by measuring specific binding of 3H-quinuclidinyl benzilate (3H-QNB) to purified canine sarcolemmal vesicles. Scatchard analysis of 3H-QNB saturation isotherms (25 degrees C, pH 7.4) yielded a dissociation constant (Kd) of 58 +/- 10 pM and maximal binding capacity (Bmax) of 5.7 +/- 1.3 pmol/mg. Pretreatment of the sarcolemmal membranes with PLA2 (1 U/ml) for 5 and 30 mins reduced Bmax to 38% and 7% of control, and increased Kd to 109 +/- 21 and 129 +/- 12 pM, respectively. Washing of PLA2-treated sarcolemmal vesicles with defatted albumin resulted in a partial recovery of Bmax, presumably by removing hydrolysis products. PLA2 also reduced equilibrium binding of 3H-QNB to 43% of control when reactions were started by simultaneous addition of 3H-QNB and 1 U/ml PLA2; however, under these conditions the inhibitory effect of PLA2 could be overcome by increasing 3H-QNB from 30 to 600 pM. PLA2 added at equilibrium (59 mins after reaction start) had no effect on 3H-QNB binding. Lipid hydrolysis by PLA2 was unaffected by the presence of bound 3H-QNB. The ability of ligand occupation and removal of hydrolysis products to attenuate the effects of PLA2-treatment on muscarinic receptor sites may be explained if modification of the membrane lipid bilayer leads to transitions between different states of the receptor.