Porcine Cardiac Membranes Research Articles

Evidence for a multiple binding mode of bispyridinium-type allosteric modulators of muscarinic receptors

The ligand binding properties of muscarinic receptors can be modulated by allosterically acting compounds. Here, a set of novel bispyridinium-type compounds was investigated which were designed to study structure-activity relationships and to provide more insight into the molecular events underlying the allosteric delay of the dissociation of [ 3H] N-methylscopolamine from muscarinic M 2 receptors in porcine cardiac membranes. The parent compound, a non-substituted bispyridinium oxime, displayed a weak allosteric potency and was unable to prevent radioligand dissociation at maximum concentrations. Introduction of either a phthalimidomethyl-moiety or a dichlorobenzyl-moiety at one end of the parent compound led to a considerable increase of the allosteric activity with regard to both the potency and the maximum effect. In these unilaterally ring-substituted bispyridiniums, homologous contralateral non-aromatic modifications were accompanied by divergent potency shifts depending on whether the unilateral ring was phthalimidomethyl or dichlorobenzyl. The findings point to a multiple binding mode of bispyridinium compounds at M 2 receptors in the [ 3H] N-methylscopolamine-occupied state, i.e., different orientations of the compounds at the allosteric binding area or even an interaction with distinct allosteric recognition sites.

Allosteric effect on muscarinic M 2-receptors of derivatives of the alkane-bis-ammonium compound W84. Comparison with bispyridinium-type allosteric modulators

The symmetrically shaped W84 = N,N,N′,N′-tetramethyl- N,N′-bis-(3-phthalimido-propyl)- N,N′-hexane-1,6-diyl-bis-ammonium dibromide is a potent allosteric stabilizer of antagonist-binding to cardiac muscarinic M 2-receptors. The ability of unilaterally shortened W84 derivatives to allosterically retard the dissociation of [ 3H] N-methylscopolamine ([ 3H]NMS) from M 2-receptors was determined in porcine cardiac membranes (3 mM MgHPO 4, 50 mM TrisHCl, pH 7.3, 37°C). Shortening was accompanied by a reduction of the allosteric activity. For instance, W84 prolonged the half-life of [ 3H]NMS dissociation (control t 1 2 = 2.0 min) by a factor of 2 at a concentration of EC 50 = 1.3 μM, whereas a derivative unilaterally lacking phthalimidopropyldimethylammonium was 40-fold less potent. It is concluded that the whole W84 molecule interacts with the allosteric site of the receptor. The structure-activity relationships found with this series of agents did not parallel findings made previously with similarly modified derivatives of the bispyridinium compound DUO 3, ( E,E)-1,1′- (1,3-propanediyl)-bis[4-[[(2,6-dichlorobenzoxyl)imino]-methyl]pyridinium] dibromide, despite considerable similarity with respect to molecular shape and charge distribution.

Pharmacologic Profile of EndothelinA/B Antagonist, [Thr18,γMethylLeu19]Endothelin-1

The pharmacologic profile of [Thr18,gamma methyl Leu19]endothelin-1 (TM ET-1) was investigated in several in vitro and in vivo studies. We found that TM ET-1 inhibited 125I-ET-1 binding in porcine cardiac membrane (ETA receptor) and in bovine brain membrane (ETB receptor), with IC50 values of 0.7 and 0.25 nM, respectively. These values were almost comparable to those for ET-1. TM ET-1 had no effect on intracellular Ca2+ concentration ([Ca]i) in A10 cells mediated by ETA receptors, even at 10(-5) M, or in mouse peritoneal macrophages (MPMs) mediated by ETB receptors, even at 10(-6) M. Increases in [CA]i in A10 cells by ET-1 and in MPMs by ET-3 were completely blocked by pretreatment with 10(-7) M of TM ET-1. In porcine right coronary arteries (PCAs) and in great cardiac veins (PCVs), TM ET-1 caused no contraction at concentrations < or = 10(-6) and 10(-7) M, respectively, although it competitively inhibited ET-1-induced contraction of PCAs and Ala1,3,11,15-ET-1-induced constriction of PCVs with pA2 values of 7.0 and 9.2, respectively. Furthermore, TM ET-1 was more potent than BQ123, an ETA-specific antagonist, in inhibiting the rise in perfusion pressure in a hind-limb preparation in vitro and the increase in blood pressure in rats. These results suggest that TM ET-1 is a potent ETA and ETB antagonist without agonistic effects and can be used in future studies to help clarify the physiological role of ET.