Sally A. Wiest

Characterization of distinct angiotensin II binding sites in rat adrenal gland and bovine cerebellum using selective nonpeptide antagonists.

We investigated the characteristics of 125I-AII binding to rat adrenal and bovine cerebellar membranes in the presence and absence of new nonpeptide angiotensin II (All) receptor ligands. The imidazole All ligands, DUP753 and WL19, both produced biphasic competition curves to 125I-All binding in rat adrenal glomerulosa and adrenal medulla particles, suggesting the existence of two distinct All binding sites. Antagonist affinity (Ki) and binding capacity (Bmax) for each binding site was determined using nonlinear analysis of competition data fit to a two-site model. The high capacity site (68% of total specific 125I-All bound) in glomerulosa had high affinity for DUP753 (4.6 ± 0.8 nM) and low affinity for WL19 (29 ± 3 μM), and the low capacity site had high affinity for WL 19 (3.3 ± 1.4 nM) and low affinity for DUP753 (51 ± 9 μM). Conversely, in medulla, the high capacity site (77% total binding) had high affinity for WL 19 (19 ± 6 nM) and low affinity for DUP753 (29 ± 8 μM), and the low capacity site had low affinity for WL19 (25 ± 7 μM) but a high affinity for DUP753 (2.8 ± 2.0 nM). In glomerulosa, binding parameters for the nonpeptide ligands at each site derived from monophasic competition curves obtained in the presence of either 0.3 μM DUP753 or WL 19 to selectively block the high or low capacity binding site, respectively, were similar to values determined from the biphasic competition curves. Unlike the nonpeptide inhibitors, unlabeled All yielded monophasic inhibition curves. WL19 and DUP753 also blocked 125I-All binding to bovine cerebellar membranes (Ki = 0.14 ± 0.03 and 170 ± 21 μM, respectively), and both displayed monophasic competition curves. All-induced contraction of rabbit aorta was potently and competitively blocked by DUP753 (K = 3.9 ± 0.4 nM) but not by WL 19. Thus, the Ki values for DUP753 at the DUP753-sensitive binding site in rat adrenal glomerulosa and medulla are similar to the affinity of DUP753 for All receptors in rabbit aorta, suggesting a similarity among these sites. The Ki values for WL19 at the WL19-sensitive site in rat adrenal gland and bovine cerebellum differ from 7–40-fold and represent binding site(s) distinct from the DUP753-sensitive site.

Cellular Electrophysiology of Clofilium, a New Antifibrillatory Agent, in Normal and Ischemic Canine Purkinje Fibers

Summary Intracellular electrophysiological studies were performed on isolated canine cardiac tissues to investigate further the reported ability of clofilium (3 × 10-8- 10-6 M) to selectively increase action potential duration (APD) and refractoriness. In Purkinje fibers from normal dogs, clofilium did not influence (1) the rate of rise of the action potential (Vmax) elicited from normal or depolarized (10 mM potassium) resting potentials, (2) the Vmax of premature potentials elicited during the repolarization phase of a previous action potential or (3) the rate of diastolic depolarization of spontaneously firing Purkinje fibers. The diastolic interval was altered by inserting a single premature impulse during diastole or by varying the basic cycle length. Clofilium (3 × 10-7 M) slightly reduced the time constant for the relation between diastolic interval and APD in concentrations that caused a maximal increase in APD of nonpremature impulses. In dogs subjected to occlusion of the left anterior descending coronary artery 48 hr before study, the APD of surviving Purkinje fibers was longer in the infarcted zone than in the normal zone. Clofilium (3 × 10-8 M) increased APD in both zones but more so in the normal area, thus reducing the disparity of APD between zones. Similarly, clofilium (3 × 10-8 and 3 × 10-7 M) increased the effective refractory period in both zones but more so in the normal area. The increase of APD and refractoriness in normal as well as depolarized or ischemic tissues in the absence of marked changes in Vmax and conduction may decrease the likelihood of reentrant arrhythmias and underlie the antifibrillatory effects in anesthetized dogs.

Binding of [3H]2-(2-benzofuranyl)-2-imidazoline (BFI) to human brain: Potentiation by tranylcypromine

Recently, a new imidazoline (I2) ligand, [3H]2-(2-benzofuranyl)-2-imidazoline (BFI) was shown to be more selective for I2 vs alpha 2 binding in rodent brain. We characterized [3H]BFI binding in human brain cortex and lateral reticular nucleus (NRL). Membranes were incubated with [3H]BFI at 22 degrees C in 50 mM Tris, 1.5 mM EDTA at pH 7.5. Saturation experiments with [3H]BFI (0.5-80 nM) were analyzed using non-linear curve fitting. The NRL had 4X more binding sites than cortex with similar affinity (Bmax = 2085 +/- 732 and 471 +/- 41 fmol/mg protein; KD = 9.3 +/- 3.5 and 11.9 +/- 2.7 nM, respectively). In competition studies, cortical [3H]BFI binding was displaced in order of decreasing potency by clorgyline > BFI > or = cirazoline > idazoxan > or = guanabenz > clonidine > RX821002. The monoamine oxidase (MAO) inhibitor tranylcypromine (TCP) (1 nM-10 microM), markedly enhanced [3H]BFI binding in both NRL and cortex. Enhanced binding was maximal at 300 nM (12 X control) and returned to baseline at 30 microM. Potentiation was not seen with pargyline or clorgyline. TCP did not effect [3H]BFI binding in rat cortex, or [3H]idazoxan binding in human cortex and NRL. In human cortex, inhibition of MAO by preincubation with pargyline (10 micro M) abolished the TCP effect. Upon preincubation with TCP, the stimulation of [3H]BFI binding was dose-dependently related to a simultaneous inhibition of MAO. Thus, [3H]BFI labels a site in human NRL and cortex that appears similar to the previously described I2 site labeled by [3H]idazoxan. However, [3H]BFI binding is dramatically stimulated by TCP in human brain via a mechanism dependent on endogenous MAO activity.

Electrophysiological studies of indecainide hydrochloride, a new antiarrhythmic agent, in canine cardiac tissues

The intracellular electrophysiological properties of a new, orally effective antiarrhythmic agent, indecainide hydrochloride, were studied in isolated canine myocardial preparations stimulated at 1 Hz and superfused with Tyrodes solution. In Purkinje fibers, indecainide (10−6 and 3 x 10−6M) decreased the maximal rate of rise of phase 0 (&OV0312;max), conduction velocity, action potential duration APD, and effective refractory period, and shifted the membrane response curve by 5 mV in a hyperpolarizing direction. In papillary muscle, APD was unchanged, but &OV0312;max was decreased. The effect of the drug on &OV0312;max was rate dependent, but over physiologically relevant cycle lengths (370 to 1,000 ms), &OV0312;max remained relatively constant. In the presence of indecainide (3 × 10−6M) and at a basic cycle length of 333 ms, the rate constant for block onset was 0.06 and 0.1 action potentials−1 in Purkinje fiber and papillary muscle, respectively. The recovery of &OV0312;max from maximum steadystate block was half completed in Purkinje fibers and muscle in 52 and 49 s, respectively. No resting block was apparent in either tissue at normal resting membrane potential. Indecainide had only minimal effects on automaticity arising from normal or depolarized (barium) membrane potentials. Thus, indecainide is a potent class I local anesthetic antiarrhythmic agent that depresses &OV0312;max and conduction in cardiac tissues. The depressant effects of indecainide are completely dependent on prior activation of the tissue, but because of its slow kinetics for recovery from sodium channel block, little additional change in &OV0312;max occurs within physiologically relevant heart rates and prematurity intervais.

Chiral recognition of the angiotensin II (AT1) receptor by a highly potent phenoxyproline octanoamide

Abstract The synthesis and in vitro biological evaluation of a novel series of diastereomeric phenoxyproline octanoamides ( 3–h ) as angiotensin II (AT 1 ) receptor antagonists are reported.

Binding and pharmacologic properties of peptides derived from human and rat angiotensin II (AII) mRNA

We investigated the binding and pharmacologic properties of peptides encoded by complementary mRNA derived from the human and rat angiotensinogen gene (human and rat IIA, respectively). Human IIA (identical with AII in 4 amino acids) inhibited binding of [125I]AII to rat adrenal glomerulosa particles (Ki = 0.62 +/- 0.09 microM) and competitively blocked, with similar potency, the ability of three AII receptor agonists to contract rabbit aorta. Rat IIA affected neither [125I]AII binding to glomerulosa particles nor the contractile response of AII. We conclude that rat IIA does not interact with AII or its receptors and that human IIA acts as a competitive inhibitor of AII at the receptor level.