title: Effect of phospholipid hydrolysis by phospholipase A2 on the kinetics of antagonist binding to cardiac muscarinic receptors.
abstract: Activation of phospholipases during prolonged myocardial ischemia could contribute to the functional derangement of myocardial cells by altering the phospholipid environment of a number of membrane bound proteins including receptors. The present study examined the kinetics of muscarinic receptor antagonist [3H]quinuclidinyl benzilate binding ([3H]QNB) to muscarinic receptors of highly purified sarcolemmal membranes under control conditions and after treatment with phospholipase A2 (PLA2; EC 3.1.1.4). Initial binding rates of QNB exhibited saturation kinetics, when plotted against the ligand concentration in control and PLA2 treated sarcolemmal membranes. This kinetic behaviour of QNB-binding is consistent with at least a two step binding mechanism. According to this two step binding hypothesis an unstable intermediate receptor-QNB complex (R*QNB) forms rapidly, and this form undergoes a slow conversion to the high affinity ligand-receptor complex R-QNB. The Michaelis constant Km of R-QNB formation was 1.8 nM, whereas the dissociation constant Kd obtained from equilibrium measurements was 0.062 nM. After 5 min exposure of sarcolemmal membranes to PLA2QNB binding capacity (Bmax) was reduced by 62%, and the affinity of the remaining receptor sites was decreased by 47% (Kd = 0.116 nM). This PLA2-induced increase of Kd was accompanied by a corresponding increase of Km, whereas the rate constants k2 and k-2 of the hypothetical slow conversion step (second reaction step) remained unchanged. These results suggest that binding of QNB to cardiac muscarinic receptors induces a transition in the receptor-ligand configuration, which is necessary for the formation of the final high affinity R-QNB complex. PLA2-induced changes of the lipid environment result in the inability of a part of the receptor population to undergo this transition, thereby inhibiting high affinity QNB-binding.
authors.affiliation: Abteilung Innere Medizin III, Medizinische Klinik, Universität Heidelberg F.R.G.
authors.forename: B
authors.initials: B
authors.lastname: Rauch
authors.forename: F
authors.initials: F
authors.lastname: Niroomand
authors.forename: F C
authors.initials: FC
authors.lastname: Messineo
authors.forename: A
authors.initials: A
authors.lastname: Weis
authors.forename: W
authors.initials: W
authors.lastname: Kübler
authors.forename: W
authors.initials: W
authors.lastname: Hasselbach
mesh_terms: D000818:Animals; D004285:Dogs; D006321:Heart / Q000187:drug effects; D007700:Kinetics; D018727:Muscarinic Antagonists; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D011813:Quinuclidinyl Benzilate / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000737:chemistry; D012508:Sarcolemma / Q000378:metabolism; D014316:Tritium
chemical_list: D018727:Muscarinic Antagonists; D010743:Phospholipids; D011976:Receptors, Muscarinic; D014316:Tritium; D011813:Quinuclidinyl Benzilate; D010741:Phospholipases A; D054467:Phospholipases A2