The relationship of KCl- and prostaglandin F2 alpha-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 F2 alpha (PGF2 alpha) (2-30 microM) 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 PGF2 alpha, 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 PGF2 alpha-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+]i-tension relationship is dependent upon the type of pharmacological agent that is used to change muscle tension.

Effects of metalloprotease inhibitors on smooth muscle endothelin-converting enzyme activity.

The enzyme responsible for the conversion of exogenous big endothelin-1 to endothelin-1 by porcine coronary arterial smooth muscle has been shown to be a metalloprotease. The potencies of eight metalloprotease inhibitors for this endothelin-converting enzyme were determined. CGS 25015, CGS 26129, and thiorphan inhibited the enzyme activity monophasically with IC50 values of 2.6, 2.4, and 190 microM, respectively. In contrast, the data obtained using phosphoramidon as an inhibitor were best fit by a two-site model. The biphasic concentration-response curve had IC50 values of 4.6 microM and 2.2 mM. Three analogs of phosphoramidon were also tested for enzyme inhibition. Removal of the rhamnose moiety of phosphoramidon reduced the potency (IC50 = 15 microM), whereas substitution of the rhamnose by N-[2-(2-naphthyl)ethyl] improved the potency (IC50 = 2.0 microM). These results identify a thiol and a phosphonyl series of compounds as smooth muscle endothelin-converting enzyme inhibitors. The structure-activity relationships revealed that an aromatic or aliphatic group in the P2' position or an aromatic group in the P1 position of the inhibitor significantly increased the potency.

Purification and characterization of an endothelin degradation enzyme from rat kidney.

A soluble protease which effectively inactivated endothelin-1 was purified 2,700-fold from rat kidney using DE52 anion exchange, concanavalin A-Sepharose, thiopropyl-Sepharose, and Mono P column chromatography. The overall recovery of enzyme activity was 12%. This enzyme appeared to contain two subunits with molecular weights of 34 and 21 kDa. The molecular weight of the active enzyme complex was estimated to be 82 kDa by gel filtration. It was shown to have a pI between 4.8 and 5.2 and a pH optimum of 5.5. Its activity was inhibited by benzyloxycarbonyl-Phe-AlaCHN2 and p-hydroxymercuribenzoic acid, thiol protease inhibitors, and by phenylmethylsulfonyl fluoride, a serine protease inhibitor, but not by metalloprotease inhibitors or other serine protease inhibitors. The purified enzyme degraded endothelin-1 rapidly; KM and Vmax values were 5.9 microM and 0.40 mumol/mg/min, respectively. It removed the carboxyl terminal tryptophan of endothelin-1 by specifically cleaving the Ile20-Trp21 peptide bond, yielding a peptide which was three orders of magnitude less potent than endothelin-1 in causing contraction of porcine coronary arterial rings. In contrast, proendothelin-1 was not degraded by this enzyme. These results are consistent with published findings that show the clearance rate for proendothelin-1 in the pig to be significantly slower than that for endothelin-1. Our study suggests that this enzyme may play a role in the homeostasis of circulating endothelin-1 and contribute to the regulation of vascular tone.

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.

Comparative effects of a selective adenosine A2 receptor agonist, CGS 21680, and nitroprusside in vascular smooth muscle.

CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethyolcarboxamidoa denosine) is an adenosine agonist that has been reported recently to bind selectively to adenosine A2 receptors in rat brain. This adenosine agonist, and the parent compound NECA (5'-N-ethylcarboxamidoadenosine), were found to be potent vasorelaxants of prostaglandin F2 alpha (PGF2 alpha) precontracted porcine coronary smooth muscle with EC50s of 4.5 and 9.7 nM, respectively. Schild analysis of the inhibition of CGS 21680, NECA and 2-chloroadenosine induced relaxation of the porcine coronary artery by CGS 15943 (9-chloro-2-(2-furanyl)[1,2,4]triazolo[1,5-C]quinazolin-5-amine), an A2 receptor antagonist, yielded identical pA2 values for the antagonist (approximately 9.3). This indicates that the same receptor mediates the effects of these three adenosine agonists. NECA and CGS 21680 were equipotent in most vascular preparations except in the canine coronary artery. Porcine coronary arterial rings contracted with PGF2 alpha were relaxed by NECA or CGS 21680 as well as by nitroprusside; those contracted with KCl (40 mM) were relaxed only by nitroprusside. In rabbit aorta, contractions induced by phenylephrine or PGF2 alpha were inhibited by nitroprusside but not by NECA or CGS 21680. Thus, the adenosine A2 receptor agonists, NECA and CGS 21680, are potent vasorelaxants that display regional vascular and species variations that differ from those of nitroprusside.

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 PGF2 alpha-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 Ca(2+)-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 (P(o)) 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.

Two subtypes of the endothelin receptor (ETA and ETB) mediate vasoconstriction in the perfused rat heart.

The effects of endothelin-1 (ET-1) are mediated by two subclasses of the endothelin receptor, ETA and ETB. The Langendorff perfused rat heart was used to determine the endothelin receptor subtype mediating rat coronary vasoconstriction. ET-1 (EC50 = 1.5 x 10(-10) M) and endothelin-3 (ET-3) (10(-11)-3 x 10(-8) M) caused dose-dependent increases in coronary perfusion pressure. BQ-123, a selective antagonist of the ETA-receptor subtype, did not cause a parallel shift in the dose-response curves of ET-1 or ET-3. In the presence of 1-3 x 10(-6) M BQ-123, ET-1 and ET-3 exhibited biphasic dose-response curves, suggesting that vasoconstriction was caused by two receptors. The ETB-selective agonist Suc-[Glu9,Ala11,15]-ET-1(8-21) (IRL 1620) maximally increased perfusion pressure by approximately 50% of the maximal response to ET-1 and was not inhibited by BQ-123. These data suggest that the rat coronary vasoconstrictor effects of ET-1 and ET-3 are mediated by both ETA and ETB receptors.

The relationship between the binding site of [3H]-d-cis-diltiazem and that of other non-dihydropyridine calcium entry blockers in cardiac sarcolemma.

[3H]-d-cis-Diltiazem binds to canine cardiac sarcolemma in a specific, saturable, and reversible manner with a KD = 58.0 +/- 9.5 nM and a receptor site density (maximum binding) of 2.19 +/- 0.24 pmol/mg of protein. Bepridil and verapamil, Ca2+ channel inhibitors, can completely inhibit this binding at nM concentrations. This inhibition was determined from saturation binding data to be due to a change in affinity of the radioligand, suggesting a competitive interaction between the three drugs. However, in dissociation experiments, both bepridil and verapamil increased the dissociation rate of the radioligand. This effect is uncharacteristic of competitive inhibitors and suggests that bepridil and verapamil regulate [3H]-d-cis-diltiazem binding in a negative allosteric fashion through their own distinct binding sites.

Effects of bepridil and diltiazem on [3H]nitrendipine binding to canine cardiac sarcolemma. Potentiation of pharmacological effects of nitrendipine by bepridil.

[3H]Nitrendipine binds to canine cardiac sarcolemma in a specific, saturable and rapid manner. Bepridil, a Ca++ channel inhibitor, stimulates this binding at submicromolar concentrations, but inhibits it noncompetitively at higher concentrations (IC50 = 15.8 microM). The increase in binding was due primarily to a 30% increase in the association rate constant (k+1) of [3H]nitrendipine, causing a decrease in the KD from a control value of 0.40 to 0.28 nM. In contrast to bepridil, diltiazem causes only an increase in [3H] nitrendipine binding without any inhibition. The increase was due primarily to an increase in receptor site density (maximum binding). These results show that the regulatory effects of bepridil and diltiazem on [3H]nitrendipine binding to cardiac tissue are different. The stimulatory effects of bepridil appeared to be pharmacologically relevant as low concentrations of bepridil potentiated the negative inotropic effect of nitrendipine in isolated perfused rat hearts.

Decrease in immunoreactivity and vasoactivity of endothelin-1 after exposure to oxygen.

Endothelin-1 (ET-1) caused a dose-dependent contraction of porcine coronary arterial rings. Preincubation of ET-1 in oxygenated Krebs solution at 37 degrees C resulted in a progressive loss of the contractile activity and immunoreactivity of ET-1. The half-life of ET-1 contractile activity in oxygenated and non-oxygenated buffer was 16.1 and 86.6 min., respectively. The molecular weight of ET-1 exposed to oxygen was identical to that of ET-1. These results indicate that during the contraction of smooth muscle preparations the levels of ET-1 in the tissue bath are steadily decreasing due to exposure to high pO2 levels.

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.

Different affinities and selectivities of endothelin-1 and endothelin-3 binding to various rat tissues.

Inhibition of [125I]endothelin-1 ([125I]ET-1) binding to membrane fractions from various rat tissues by ET-1 and endothelin-3 (ET-3) was investigated. Brain tissue demonstrated a 37-fold higher affinity for ET-1 compared to lung, while a greater than 1000-fold difference in affinity for ET-3 was observed in these two tissues. Furthermore, the ratio of the IC50 value of ET-3 to that of ET-1 in each tissue varied from approximately 2 in brain, kidney and liver to greater than 100 in heart and spleen. These experiments show that the tissues examined have different affinities as well as different selectivities for ET-1 and ET-3.

Effects of adenosine A2 receptor agonists on nucleoside transport.

A series of adenosine A2 receptor agonists were examined for their ability to activate adenosine A2 receptors and inhibit nucleoside transport. A2 receptor activation was measured by the ability of these adenosine agonists to relax porcine coronary smooth muscle, where the compounds varied in their EC50 values from 4.5 nM (CGS 21680A (2-[p-(2-carboxyethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine)] to 3.6 microM (CGS 23321 [2 alpha,3 alpha-dihydroxy-1 beta-hydroxymethyl-4 beta-(2-phenylamino-9- adenyl)-cyclopentane]). Nucleoside transport was measured as the nitrobenzylthioinosine-sensitive cellular accumulation of [3H]uridine into guinea pig erythrocytes at 22 degrees C. The initial velocity of transport was dependent on substrate concentration and a substrate-velocity curve yielded a Km of 78 +/- 16 microM and a Vmax of 0.31 +/- 0.049 mmol/l of cell water per hr (mean +/- S.D., n = 4). Dipyridamole, a known potent inhibitor of nucleoside transport, blocked cellular [3H]uridine accumulation with an EC50 of 29.4 nM. Whereas a number of the adenosine agonists tested showed little or no inhibition of nucleoside transport, CV 1808 (2-phenylaminoadenosine) inhibited transport with an EC50 of 140 nM. In addition, two carbocyclic derivatives of CV 1808, CGS 23321 and CGS 23302 [(-)2S,3R-dihydroxy-4R-hydroxymethyl-1R-[2-(p-ethoxycarbonyl)- phenylamino-9-adenyl]-cyclopentane) inhibited nucleoside transport with respective EC50 values of 366 and 168 nM. The data suggest that these compounds have a different structure-activity relationship for adenosine A2 receptors and for the site mediating nucleoside transport inhibition.

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

IRL 2500 [N-(3,5-dimethylbenzoyl)-N-methyl-(D)-(4-phenylphenyl)-alany l-L- tryptophan] inhibited the binding of [125I]-endothelin-1 (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 sarafotoxin 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.

Effects of metalloprotease inhibitors on the conversion of proendothelin-1 to endothelin-1.

The IC50 values of phosphoramidon, CGS 25015, CGS 26129, thiorphan and benazeprilat for inhibition of endothelin converting enzyme partially purified from porcine aortic endothelial cells were 3.5, 18, 58, > 100 and > 100 microM, respectively. A similar rank order of potency was observed for inhibition of the proendothelin-1 (proET-1) -induced pressor response in the rat where phosphoramidon, CGS 25015, CGS 26129, thiorphan and benazeprilat at 30 mg/kg i.v. produced 65, 57, 27, 12, and 0% inhibition, respectively. A slightly different rank order of potency was obtained in the proET-induced contraction of porcine coronary arteries where IC50 values of < 10, 10-30, 10-30, 30-100 and 30-100 microM were exhibited by CGS 25015, CGS 26129, phosphoramidon, thiorphan and benazeprilat, respectively. These data indicate that the endothelin converting enzymes in the three systems studied are similar, except that phosphoramidon is a slightly more potent inhibitor in the in vitro assay and the in vivo pressor test than in the smooth muscle contraction assay.