EFFECT OF PHOSPHOLIPID HYDROLYSIS BY PHOSPHOLIPASE A(2) ON THE KINETICS OF ANTAGONIST BINDING TO CARDIAC MUSCARINIC RECEPTORS

Activation of phospholipases during prolonged myocardial ischemia coul d contribute to the functional derangement of myocardial cells by alte ring the phospholipid environment of a number of membrane bound protei ns including receptors. The present study examined the kinetics of mus carinic receptor antagonist [H-3]quinuclidinyl benzilate binding ([H-3 ]QNB) to muscarinic receptors of highly purified sarcolemmal membranes under control conditions and after treatment with phospholipase A(2) (PLA(2); EC 3.1.1.4.). Initial binding rates of QNB exhibited saturati on kinetics, when plotted against the ligand concentration in control and PLA(2) treated sarcolemmal membranes. This kinetic behaviour of QN B-binding is consistent with at least a two step binding mechanism. Ac cording to this two step binding hypothesis an unstable intermediate r eceptor-QNB complex(RQNB) forms rapidly, and this form undergoes a sl ow conversion to the high affinity ligand-receptor complex R-QNB. The Michaelis constant K-m of R-QNB formation was 1.8 nM, whereas the diss ociation constant K-d obtained from equilibrium measurements was 0.062 nM. After 5 min exposure of sarcolemmal membranes to PLA(2)QNB bindin g capacity (B-max) was reduced by 62%, and the affinity of the remaini ng receptor sites was decreased by 47% (K-d = 0.116 nM). This PLA(2)-i nduced increase of K-d was accompanied by a corresponding increase of K-m, whereas the rate constants k(2) and k(-2) of the hypothetical slo w conversion step (second reaction step) remained unchanged. These res ults suggest that binding of QNB to cardiac muscarinic receptors induc es a transition in the receptor-ligand configuration, which is necessa ry for the formation of the final high affinity R-QNB complex. PLA(2)- induced changes of the lipid environment result in the inability of a part of the receptor population to undergo this transition, thereby in hibiting high affinity QNB-binding.