Inhibition of 3H-quinuclidinyl benzylate binding to cardiac muscarinic receptor by long chain fatty acids can be attenuated by ligand occupation of the receptor

Abstract

Phospholipase A 2 (PLA 2) inhibits ligand binding to sarcolemmal muscarinic receptors in heart. To determine whether this effect of PLA 2 is mediated by membrane accumulation of non-esterified fatty acids (FFA), the effect of selected fatty acids on the binding of 3H-quinuclidinyl benzylate ( 3H-QNB) to purified canine sarcolemmal membranes before and after PLA 2 treatment was examined. Equilibrium 3H-QNB binding was inhibited by 5 min exposure of membrane vesicles to oleic, linoleic or arachidonic acid (IC 50 = 6.3 ± 0.9, 9.9 ± 1.1, and 6.8 ± 0.4 μ m, respectively); the saturated fatty acids, stearic and palmitic acid (10 μ m) had no effect. Scatchard analysis of equilibrium binding isotherms showed that the effect of the unsaturated fatty acids to inhibit 3H-QNB binding reflected a decrease of B max and a reduction of the affinity of the remaining receptors. The effect of unsaturated fatty acids was dependent on the mole ratio of fatty acid to membrane phospholipid present ( FFA PL ratio). Washing of fatty acid-treated membranes with bovine serum albumin (BSA) resulted in partial recovery of both maximal binding ( B max) and affinity. The fatty acid-induced reduction of B max was also attenuated if binding was started by simultaneous addition of 3H-QNB and FFA. Similarity of the FFA induced effects on 3H-QNB binding to sarcolemmal muscarinic receptors to those induced by PLA 2 suggest that membrane accumulation of unsaturated fatty acids underlies in part the effect of PLA 2. Furthermore, modification of the receptor-ligand interaction by changes in the membrane lipid composition may be prevented by ligand occupation of the receptor.