Joseph L. Balwierczak

Benzofused Macrocyclic Lactams as Triple Inhibitors of Endothelin-converting Enzyme, Neutral Endopeptidase 24.11, and Angiotensin-converting Enzyme

The purpose of this study was to identify endothelin-converting enzyme (ECE) inhibitors that also possess inhibitory activity for neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE). The ortho-substituted benzofused macrocyclic lactams, such as CGS 26670, are generally potent NEP inhibitors but poor ACE inhibitors. CGS 26670 inhibited ECE activity with an IC50 of 600 nM, whereas it inhibited NEP and ACE activities with IC50 values of 0.9 and > 10,000 nM, respectively. This compound also prevented the conversion of big endothelin-1 (big ET-1) to ET-1 by denuded porcine coronary arterial smooth muscle with an IC50 of 200 nM. The ACE inhibitory activity is greatly is greatly improved in metasubstituted benzofused macrocyclic lactams. For example, CGS 26582 inhibited ECE, NEP, and ACE activities with IC50 values of 620, 4, and 175 nM, respectively. When injected at 30 mg/kg i.v. in conscious rats, followed by a challenge with big ET-1 at 1 nmol/kg i.v., this compound suppressed by 44% the increase in mean arterial blood pressure owing to the generation of ET-1 by ECE. Because ECE, NEP, and ACE play regulatory roles in cardiovascular and renal function, triple inhibitors of these enzymes may represent a novel class of agents for treatment of cardiovascular and renal diseases.

Evidence that BKCa channel activation contributes to K+ channel opener induced relaxation of the porcine coronary artery

The rank order of potency of a series of benzopyran and cyanoguanidine K+ channel openers (KCOs) for causing relaxation of the PGF2α-precontracted porcine coronary artery was determined. Glyburide, an inhibitor of KATP channels, showed an apparent competitive inhibition of the vasorelaxant activity of the KCOs. The pA2 values of glyburide when cromakalim and CGP 14877 (P1060) were used as vasorelaxants were 7.66 and 7.83, respectively. Charybdotoxin (40 nM), an inhibitor of BKCa channels, also caused a significant inhibition of the cromakalim mediated relaxation of the porcine coronary artery. In order to clarify the site of action of these KCOs, we identified a K+ channel current in single porcine coronary arterial cells and measured channel activity in the presence of these compounds. The prominent K+ ion current in these cells had characteristics typical of the conventional large Ca2+-activated K+ channel BKCa present in other smooth muscle cells. Using symmetrical K+ concentrations, the channel had a conductance of 214 pS and was found to be sensitive to [Ca2+]i and membrane potential. The KCOs were found to reversibly increase the open probability (Po) of the channel without changing channel conductance. The potency of the KCOs to increase K+ channel opening was similar to the potency of these compounds to cause coronary artery relaxation. These results indicate that the porcine coronary artery contains the BKCa channel and that this channel, along with other types of K+ channels (KATP), mediate the vasorelaxant effects of K+ channel openers.

Characterization of a potent and selective endothelin-B receptor antagonist, IRL 2500

Summary: IRL 2500 [N-(3,5-dimethylbenzoyl)-N-methyl-(D)-(4-phenylphenyl)-alanyl-L-tryptophan] inhibited the binding of [125I]-endothelin-l (ET-1) to human ETB (IC50 1.3 ± 0.2 nM) and ETA (IC50 94 ± 3 nM) receptors expressed in transfected Chinese hamster ovary (CHO) cells. In in vitro studies, IRL 2500 inhibited the sarafo-toxin S6c (STX6c)-mediated contraction of the dog saphenous vein (pKb 7.77) and the STX6c-induced relaxation of the preconstricted rabbit mesenteric artery (pKb 6.92). In the anesthetized rat, IRL 2500 (10 mg/kg, i.v.) inhibited the initial transient decrease in mean arterial pressure (MAP) induced by the ETB-selective agonist IRL 1620 (0.5 nmol/kg, i.v.). IRL 2500 also attenuated the IRL 1620-mediated increase in renal vascular resistance (RVR) in the anesthetized rat. Therefore, IRL 2500 is a potent and selective ETB receptor antagonist that can be used to delineate ET responses mediated by the ETB receptor.

The relationship of KCl‐ and prostaglandin F2α‐mediated increases in tension of the porcine coronary artery with changes in intracellular Ca2+measured with fura‐2

1 Changes in intracellular free Ca2+concentration ([Ca2+]i) were measured simultaneously with changes in muscle tension by use of the fluorescent Ca2+indicator, fura‐2, in coronary arterial rings of the Pig. 2 Changes in [Ca2+]i were measured by monitoring the ratio of fluorescence due to excitation at 340 nm (F340) to that at 380 nm (F380). 3 Increases in tension of the porcine coronary artery induced by prostaglandin F2α (PGF2α) (2–30 μm) and KCl (25–70 mm) were accompanied by increases in the F340/F380 fluorescence ratio of fura‐2. 4 KCl‐induced increases in muscle tension, equivalent to those produced by PGF2α, were observed to occur in the presence of a higher [Ca2+]i. 5 Nearly complete relaxation of KCl‐induced contractions by sodium nitroprusside (SNP) was accompanied by only a partial reversal of the increase in [Ca2+]i that occurred during contraction. 6 Complete relaxation of the PGF2α‐contracted coronary artery by cromakalim (BRL 34915) was accompanied by a nearly complete reversal of the increase in [Ca2+]i caused by the contractile agent. 7 The contractile state of smooth muscle is not an indicator of [Ca2+]i. The [Ca2+]itension relationship is dependent upon the type of pharmacological agent that is used to change muscle tension.

A simple assay for the measurement of conversion of big endothelin-1 to endothelin-1 by smooth muscle

The conversion of exogenous big endothelin-1 to endothelin-1 by the smooth muscle of denuded porcine coronary arterial strips was measured by radioimmunoassay. Repeated incubations of a porcine coronary arterial strip with the same concentration of big endothelin-1 yielded similar rates of conversion over a 4 h period. This property allowed the determination of a control rate of endothelin-1 conversion by a coronary arterial strip followed by measurement of rates under various conditions. The assay described in this report can be used to investigate the biochemical properties and inhibitor profiles of the extracellular enzyme mediating the conversion of big endothelin-1 to endothelin-1. The Km and Vmax of the enzyme were 34 +/- 2 microM and 88 +/- 8 fmol/min, respectively. Conversion of big endothelin-1 to endothelin-1 was optimal at pH 7.0 and was inhibited by classic metalloprotease inhibitors.

Differential Cleavage of Urodilatin and Atrial Natriuretic Factor by Thrombin and Protease 3.4.24.11

AbstractHuman urodilatin (residues 95–126) and atrial natriuretic factor (residues 99–126, based on ANF prohor-mone sequence) were incubated separately with three proteases, thrombin, angiotensin converting enzyme (ACE), and neutral endopeptidase 3.4.24.11 (NEP). Thrombin cleaved urodilatin on the carboxyl side of arginine98 to yield ANF but under the same conditions did not cleave h-ANF. Neither urodilatin nor ANF was cleaved by ACE. ANF was rapidly degraded by NEP resulting in a major product cleaved between amino acid residues Cysl05 and Phe106. Urodilatin was also cleaved by NEP and the amino acid sequencing of the cleaved product revealed the site of cleavage to be the same Cys105-Phe106 site as for ANF with a second cleavage site at Gly118-Leu119. However, cleavage of urodilatin by NEP proceeded much more slowly when compared to ANF. A comparison of the affinities of ANF and urodilatin for purified NEP from rabbit kidney revealed Km values of 11.7 and 3.1 μM, respectively. The turnover rates (kcat/K...