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CARDIOVASCULAR

Characterization of the Relaxant Response to N,N'-Dipropyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine in Porcine Coronary Arteries

Institute of Pharmacy, Free University of Berlin, Berlin, Germany

Received January 22, 2007; accepted February 20, 2007.

	Abstract

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N,N'-Dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines show structural analogy with estrogens and selective estrogen receptor modulators. Because the vasodilator properties of these compounds are unknown, we investigated their potential to relax porcine coronary arteries and determined the mechanism(s) of relaxation. Isolated porcine coronary arterial rings were suspended in organ chambers, precontracted with KCl (30 mM), and the relaxant response was determined by measurement of changes in isometric force. Dependent on the chemical structure, the drugs induced concentration-dependent relaxation in rings with and without endothelium. N,N'-Dipropyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine (8) was most potent and showed a 12- to 15-fold higher vasodilatory effect than 17-estradiol (E2). The vasorelaxation was independent of endothelium. Calcium concentration-dependent contractions in high-potassium depolarizing medium were insurmountably inhibited by 8. The effect of the L-type Ca²⁺ channel activator (S)-(–)-Bay K 8644 [(S)-(–)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine-carboxylic acid methyl ester], which induced a leftward shift of Ca²⁺ contraction, was blocked by 8. The relaxant response to 8 was unaffected by the estrogen receptor antagonist ICI 182,780 (7-[9-[(4,4,5,5,5-pentafluoropentyl]-sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17-diol) and K⁺ channel blockers, i.e., TEA, glibenclamide, and 4-aminopyridine. Furthermore, the vasodilatory effect of 8 was unaffected by the adenylyl cyclase inhibitor SQ 22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine], the guanylyl cyclase inhibitor ODQ [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one], the protein kinase A inhibitor KT 5720 [(9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg: 3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester], the protein kinase G inhibitor KT 5823 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester], and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Western blot analysis demonstrated that 8, unlike E2, raloxifene, and tamoxifen, failed to stimulate p38 MAPK. It is concluded that N,N'-dipropyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine induces endothelium-independent relaxation of coronary arteries; the mechanism apparently involves inhibition of L-type Ca²⁺ channels. The drug may be protective against cardiovascular diseases.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Chemical structures of N,N'-dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines, diethylstilbestrol, hexestrol, tamoxifen, and resveratrol. For R, see Table 1.

Smooth muscle relaxations to estrogen [17-estradiol (E2)], synthetic estrogens (DES, HEX), and SERMs (tamoxifen, raloxifene, resveratrol) have been demonstrated in blood vessels, papillary muscle, and ileum of different species (Himori, 1977; Figtree et al., 1999, 2000; Hutchison et al., 2001; Martinez et al., 2003; Orshal and Khalil, 2004; Li et al., 2006; Novakovic et al., 2006). Interestingly, a common mechanism has been attributed to the relaxant effects of E2, DES, tamoxifen, raloxifene, and resveratrol, implying an inhibition of L-type Ca²⁺ channels (Song et al., 1996; Figtree et al., 1999, 2000; Orshal and Khalil, 2004; Zakharov et al., 2004; Liew et al., 2005). Moreover, an activation of K⁺ channels may additionally play a role in estrogen and SERM-induced relaxation of smooth muscles (White et al., 1995; Diaz et al., 2004; Novakovic et al., 2006; Leung et al., 2007). Furthermore, E2-induced relaxation has been shown to be mediated by activation of adenylyl cyclase and protein kinase G (Keung et al., 2005).

It is unknown whether N,N'-dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines (compounds 1–10, see Table 1) share the coronary artery vasodilator properties of estrogens and SERMs. Due to their structural similarity to synthetic estrogens and SERMs, we hypothesized that these drugs may elicit comparable vasorelaxant responses and a similar mode of action. We used the isolated porcine coronary artery as an in vitro assay to characterize the vascular responses to these drugs because pigs are the predominant model for coronary ischemia (Ngai et al., 1983; Nikol et al., 2001). In our study, N,N'-dipropyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine (8) emerged as the most potent vasorelaxant compound. Therefore, we selected this drug to further examine the potential involvement of estrogen receptors and the roles of the endothelium, K⁺ and L-type Ca²⁺ channels, adenylyl cyclase, guanylyl cyclase, protein kinase A, and protein kinase G in isolated porcine coronary arteries. Since E2, raloxifene, and resveratrol have recently been shown to activate the p38 mitogen-activated protein kinase (MAPK) pathway (Das et al., 2006; Moritz et al., 2006; Seval et al., 2006), we also examined whether this signaling pathway coupling might be involved in the vasorelaxant response to compound 8.

	Materials and Methods

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Isolated Tissue Bath Protocol. Porcine hearts were obtained from the local slaughterhouse and placed in ice-cold oxygenated modified Krebs-Henseleit solution (KHS) of the following composition: 118 mM NaCl, 4.7 mM KCl, 1.6 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, and 11 mM D-glucose, pH 7.4. The left anterior descending coronary artery (ramus interventricularis anterior) and the right coronary artery (A. coronaria dextra) were dissected from the hearts, cleaned of fat and adhering tissue, and cut into rings (approximately 2–3-mm outer diameter and 3–4 mm in length). In experiments where rings without endothelium were used, the intimal surface of the rings was gently rolled with a pair of tweezers to destroy the endothelium. Rings were mounted between two stainless steel hooks, placed in 20-ml water-jacketed organ chambers, and constantly exposed to oxygenated modified KHS (95:5% O₂/CO₂, pH 7.4, 37°C). Tissues were equilibrated for 60 min under a resting tension of 2.0 g with buffer replacement after 30 min. Isometric force was measured with FMI TIM-1020 isometric force transducers connected to a TSE 4711 transducer coupler and a Siemens C 1016 compensograph (Siemens, Munich, Germany). During a period of 200 min, tissues were stimulated three times with KCl (30 mM), with 5- and 3-min washing periods between each challenge. Alternatively, when the involvement of p38 MAPK was studied, tissues were stimulated once with KCl (30 mM) and twice with prostaglandin F₂ (PGF₂)(3 µM). The presence or absence of endothelium was assessed functionally by measuring the extent of endothelium-dependent relaxation following application of substance P (10 nM) after the third KCl or PGF₂ challenge. To inhibit vascular eicosanoid production by cyclooxygenase, experiments were performed in the continuous presence of indomethacin (6 µM).

Comparative Relaxant Responses to N,N'-Dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines (Compounds 1–10) on Precontracted Porcine Coronary Arteries. Coronary arterial rings with or without endothelium were contracted with KCl (30 mM). When the contractile response had reached a plateau (usually after 10 min), N,N'-dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines (10 µM) were added. The relaxant responses were continuously measured and evaluated after 120 min.

Cumulative Concentration-Response Curves to Selected Ethylenediamines (Compounds 4–6 and 8). Coronary arterial rings with or without endothelium were contracted with KCl (30 mM). When the contractile response had reached a plateau, cumulative concentration-response curves to E2, compounds 4–6 and 8 were established from 10 nM to 100 µM in 0.5-log increments. In separate experiments, relaxation to 8 was examined in the absence and presence of the specific estrogen receptor antagonist ICI 182,780 (10 µM).

Reversibility of the Relaxant Response to 8. To examine whether compound 8-induced relaxation was reversible, KCl (30 mM)-precontracted arterial rings were relaxed by 8 (10 µM) for 120 min. The rings were then washed one, three, five, seven, or nine times for 10 min. Each washing was followed by a 5-min rest. After the final washing, the tissues in each group were allowed to reequilibrate for 120, 90, 60, 30, and 0 min. After a further period of 45 min, a final contraction to KCl (30 mM) was established. The same procedure was applied to check the reversibility of the relaxant response to E2 (10 µM).

Effect of 8 on Potassium Channels. To examine the involvement of K⁺ channels in the vasorelaxant response to 8, arterial rings with endothelium were incubated with the nonselective large-conductance Ca²⁺-activated and voltage-sensitive K⁺ channel inhibitor TEA (1 mM), the ATP-sensitive K⁺ channel inhibitor glibenclamide (100 µM), or the voltage-sensitive K⁺ channel inhibitor 4-aminopyridine (4-AP) (1 mM). Inhibitor concentrations were chosen according to Yildiz et al. (2005). After an incubation period of 30 min, KCl (30 mM) was added. When the contractile response had reached a plateau, a cumulative concentration-response curve to 8 (10 nM–30 µM) was established. It should be mentioned that TEA and 4-AP did not affect the KCl-induced contraction. Glibenclamide, however, reduced the KCl-induced contraction from 114 ± 5% (vehicle) to 45 ± 4% (n = 7) relative to the third KCl contraction.

Effect of 8 on Calcium Channels. Arterial rings without endothelium were incubated in Ca²⁺-free high-K⁺ (60 mM) depolarizing KHS (Ebeigbe et al., 1988). The tissues were then stimulated with CaCl₂ (3 mM). A cumulative concentration-response curve to CaCl₂ was established in the absence or presence of 8 (0.3–10 µM) and 8 plus ICI 182,780 (1 µM), respectively. In additional experiments, a CaCl₂ dose-dependent curve was established in the absence or presence of the L-type Ca²⁺ channel activator (S)-(–)-Bay K 8644 (0.1 µM; Jiang et al., 1991) and (S)-(–)-Bay K 8644 (0.1 µM) plus 8 (0.3–10 µM), respectively. In these experiments, no precipitation of calcium at the concentrations used was observed.

Relaxation to 8 and Signaling Pathway Coupling. To examine the involvement of adenylyl cyclase, guanylyl cyclase, protein kinase A, and protein kinase G in the relaxant response to 8, arterial rings with endothelium were incubated for 30 min with the adenylyl cyclase inhibitor SQ 22536 (100 µM), the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (30 µM), the protein kinase A inhibitor KT 5720 (300 nM), and the protein kinase G inhibitor KT 5823 (1 µM) before contracting the rings with KCl (30 mM). Inhibitor concentrations were chosen according to Keung et al. (2005). A concentration-response curve to 8 (10 nM–30 µM) was established on each ring when the KCl contraction had reached a plateau.

To examine a possible role of p38 MAPK in the vasorelaxant response to 8, arterial rings with or without endothelium were precontracted with PGF₂ (3 µM) following a 30-min incubation with SB 203580 (10 µM), an inhibitor of p38 MAPK (Teng et al., 2005). Compound 8 (10 µM) was administered when the contractile response to PGF₂ had reached a plateau. The relaxant response to 8 in the absence or presence of antagonist was continuously measured and evaluated after 120 min.

Western Blotting. Western blotting for nonphosphorylated or phosphorylated p38 MAPK was carried out as described recently (Moritz et al., 2006). Rings of porcine coronary artery with or without endothelium were set up in organ chambers as above. Tissues were exposed to PGF₂ (3 µM), 8 (10 µM), raloxifene (3 µM), tamoxifen (10 µM), and E2 (10 µM) or precontracted with PGF₂ (3 µM) and treated with 8 (10 µM), raloxifene (3 µM), tamoxifen (10 µM), and E2 (10 µM), respectively. Control tissues were not exposed to any compound (basal conditions). After 120 min, the rings were quickly removed from the organ chambers and immediately frozen in liquid nitrogen. Frozen segments were then homogenized in ice-cold lysis buffer [80 mM sodium -glycerophosphate, 20 mM HEPES, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 10 µg/ml aprotinin, 1 µM leupeptin, 500 µM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 1 mM EDTA] and kept on ice for 30 min. The homogenate was centrifugated at 10,000g at 4°C for 15 min. Protein concentrations were determined as described previously (Bradford, 1976). Samples were diluted in sample buffer. Equal amounts of protein from each sample were separated on 4% stacking gel and 12% resolving gel for SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto nitrocellulose membranes by Western blotting. Nonspecific binding of proteins was blocked with 3% bovine serum albumin. Membranes were subsequently incubated with rabbit anti-nonphosphorylated or anti-phosphorylated p38 MAPK primary antibody (1:1000). After washout of antibody showing nonspecific binding, bands were detected by probing with a horseradish peroxidase-conjugated secondary antibody. Bands were visualized using an enhanced chemiluminescence detection system (Amersham Life Sciences, Little Chalfont, Buckinghamshire, UK). Both nonphosphorylated and phosphorylated p38 MAPK bands were analyzed by densitometry using an image analyzer (Bio-Rad, Hercules, CA).

Drugs. Dinoprost tromethamine (PGF₂) was obtained as gift from Upjohn (Kalamazoo, MI). N,N'-Dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines (compounds 1–10) were synthesized according to previously published studies (von Angerer, 1982; Karl et al., 1988; Gust et al., 1995). The following drugs were purchased: 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 17-estradiol, indomethacin, raloxifene hydrochloride, TEA, ODQ, SQ 22536, KT 5720, KT 5823, and SB 203580 from Sigma-Aldrich (Taufkirchen, Germany). 4-AP, (S)-(–)-Bay K 8644, glibenclamide, ICI 182,780, and substance P were from Tocris (Bristol, UK).

All drugs were dissolved in deionized water to a 1 to 100 mM stock solution with the exceptions of indomethacin and raloxifene, which were made soluble in ethanol (50% v/v) and (S)-(–)-Bay K 8644, which was made soluble in ethanol (96% v/v). E2, glibenclamide, KT 5823, KT 5720, and ODQ were dissolved in dimethyl sulfoxide (DMSO). N,N'-Dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines were dissolved in DMSO (50% v/v) and an equimolar amount of 1 N-HCl. Stock solutions were diluted in deionized water. Final organ bath concentrations of DMSO were less than 0.2% (1% glibenclamide, 0.3% ODQ), and those of ethanol did not exceed 0.05%.

Data Analysis. Data are presented as a mean ± S.E.M. for the number of animals indicated by n. Agonist potencies were expressed as pEC₅₀ values (negative logarithm to base 10 of the molar concentration of the agonist producing 50% of the maximum response). Maximal responses were expressed as E_max values (percentage of the maximum contractile response to KCl, PGF₂, or CaCl₂). Multiple comparisons between treatment groups were performed using analysis of variance followed by a Tukey's test. All other statistical evaluations were carried out using Student's t test (unpaired for comparison of means between independent experiments, paired for comparison of means between experiment and control) after checking the homogeneity of the variances by F test. P values < 0.05 were considered to be significant.

	Results

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Comparative Relaxant Responses to N,N'-Dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines on Precontracted Porcine Coronary Arteries (Compounds 1–10). Among the compounds tested (1–10; for numbering, see Table 1), only 4 to 6, 8, and 10 (10 µM each) significantly relaxed precontracted arterial rings with or without endothelium (Fig. 2). Vasorelaxation started immediately after administration of the compounds. This was in agreement with the vasorelaxant effect of E2. There was no difference between the relaxant response to 4 to 6, 8, and 10 in rings with intact endothelium compared with rings without endothelium. d,l-Compounds were more potent than meso-compounds (Fig. 2).

View larger version (15K): [in this window] [in a new window]   Fig. 2. Comparative relaxant response to meso-N,N'-dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines, 1, 3, 5, 7, and 9 (10 µM each; top) and d,l-N,N'-dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines, 2, 4, 6, 8, and 10 (10 µM each; bottom) after 120 min in porcine coronary arterial rings with (+E) and without (–E) endothelium. Data are expressed as percentage of contraction induced by 30 mM KCl (mean ± S.E.M. for four to nine animals). *, P < 0.05.

Cumulative Concentration-Response Curves to Selected Compounds (4–6 and 8). Because compounds 4 to 6 and 8 were most potent in relaxing porcine coronary arteries, we established cumulative concentration-response curves to these drugs. Agonist potencies and maximal responses are summarized in Table 2. The effects were not different in tissues with endothelium compared with those without endothelium. The most potent vasodilator was compound 8. This drug showed a 12- to 15-fold higher vasodilatory effect than E2 (Fig. 3). Vasorelaxation to 8 was not inhibited by ICI 182,780 (10 µM) in endothelium-intact or -denuded arterial rings (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Relaxant response to E2 and compound 8 in porcine coronary arterial rings with (+E) and without (–E) endothelium. Data are expressed as percentage of contraction induced by 30 mM KCl (mean ± S.E.M. for five to seven animals).

Reversibility of 8-Induced Relaxation. To examine the reversibility of the response to 8 in arterial rings with endothelium, KCl (30 mM)-constrictory responses were compared at the start of the experiment with KCl responses at the end of the experiment following repeated washings over a total period of 130 min. Exposure to 8 (10 µM; 2-h incubation) resulted in an inhibition of the final KCl response. Responses to KCl were 95 ± 2% in control experiments and were reduced to 27 ± 1, 32 ± 5, 29 ± 2, 25 ± 4, and 26 ± 2% after 10, 30, 50, 70, or 90 min of washout after exposure to 10 µM8 (n = 4). This was in contrast to the full reversibility of the response to E2. Responses to KCl were 107 ± 3% in control experiments and 111 ± 6, 112 ± 6, 106 ± 3, 101 ± 4, and 108 ± 3% after 10, 30, 50, 70, or 90 min of washout after exposure to 10 µME2(n = 4).

Effect of 8 on Potassium Channels. In arterial rings with endothelium, cumulative concentration-response curves to 8 were constructed in the absence (vehicle, KHS; pEC₅₀, 5.89 ± 0.08; n = 7) and presence of different K⁺ channel blockers. The relaxation to 8 was not inhibited by TEA (1 mM; pEC₅₀, 5.89 ± 0.08; n = 7), glibenclamide (100 µM; 5.82 ± 0.07; n = 7), and 4-AP (1 mM; pEC₅₀, 5.93 ± 0.06; n = 7; Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Relaxant response to compound 8 in the absence or presence of K+ channel inhibitors in porcine coronary arterial rings with endothelium. Data are expressed as percentage of contraction induced by 30 mM KCl (mean ± S.E.M. for seven animals).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Inhibition of the contractile response to CaCl2 by compound 8 in the absence or presence of ICI 182,780 in porcine coronary arterial rings without endothelium. Data are expressed as percentage of contraction induced by 3 mM CaCl2 (mean ± S.E.M. for two to six animals).

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effects of (S)-(–)-Bay K 8644 on CaCl2-induced contractions in the absence and presence of compound 8 in porcine coronary arterial rings without endothelium. Data are expressed as percentage of contraction induced by 3 mM CaCl2 (mean ± S.E.M. for two to six animals).

The Relaxant Response to 8 and Signaling Pathway Coupling. The purpose of these experiments was to elucidate the signaling pathways involved in the vasorelaxant response to 8. Relaxation to 8 in arterial rings with endothelium was not inhibited by SQ 22536 (100 µM), ODQ (30 µM), KT 5720 (300 nM), and KT 5823 (1 µM), respectively (Fig. 7). Furthermore, SB 203580 (10 µM) failed to inhibit the relaxant response to 8 (10 µM) in PGF₂ precontracted arterial rings. In rings with endothelium, relaxation amounted to 69 ± 7% in the absence of SB 203580 and 61 ± 15% in the presence of SB 203580 (n = 4; P > 0.05). In rings without endothelium, relaxation was 63 ± 6% in the absence of SB 203580 and 69 ± 9% in the presence of SB 203580 (n = 4; P > 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 7. Relaxant response to compound 8 in the absence or presence of the adenylyl cyclase inhibitor SQ 22536 (100 µM), the guanylyl cyclase inhibitor ODQ (30 µM), the protein kinase A inhibitor KT 5720 (300 nM), and the protein kinase G inhibitor KT 5823 (1 µM) in porcine coronary arterial rings with endothelium. Data are expressed as percentage of contraction induced by 30 mM KCl (mean ± S.E.M. for four animals).

Because 8 showed numerous pharmacological similarities to estrogens or SERMs regarding its vasorelaxant effect and E2, raloxifene, and tamoxifen elicited an activation of p38 MAPK, we also measured levels of phosphorylated and nonphosphorylated p38 MAPK by Western blotting in tissues exposed to 8. In contrast to tissues exposed to raloxifene (3 µM), tamoxifen (10 µM), or E2 (10 µM), 8 (10 µM) failed to induce an increase in levels of phosphorylated p38 MAPK over control levels both in arterial rings with endothelium and without endothelium irrespective of whether the vessels were precontracted with PGF₂ (3 µM) or not (Figs. 8 and 9). The effects of the drugs on p38 MAPK phosphorylation were evaluated after 120 min, a period of time after which relaxation induced by the drugs reached a plateau. There was no increase in phosphorylated p38 MAPK after 120 min in tissues exposed to PGF₂ (3 µM) alone in intact and endothelium-denuded vessel rings (Figs. 8 and 9).

View larger version (32K): [in this window] [in a new window]   Fig. 8. Immunoblot of porcine coronary artery proteins separated by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with a primary antibody against nonphosphorylated (p38) and phosphorylated p38 MAPK (phospho-p38). Rings of porcine coronary artery with (+E; A) or without (–E; B) endothelium were set up in organ chambers and exposed to different treatments: no drug (basal conditions), compound 8 (10 µM), PGF2 (3 µM), raloxifene (3 µM), PGF2 (3 µM) + compound 8 (10 µM), and PGF2 (3 µM) + raloxifene (3 µM). After 120 min, rings were rapidly frozen, homogenized, and then subjected to SDS-PAGE. Raloxifene data are from Moritz et al. (2006). The blot is representative of four different experiments, with comparable results.

View larger version (24K): [in this window] [in a new window]   Fig. 9. Immunoblot of porcine coronary artery proteins separated by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with a primary antibody against nonphosphorylated (p38) and phosphorylated p38 MAPK (phospho-p38). Rings of porcine coronary artery with (+E; A) or without (–E; B) endothelium were set up in organ chambers and exposed to different treatments: no drug (basal conditions), PGF2 (3 µM), E2 (10 µM), tamoxifen (10 µM), PGF2 (3 µM) + E2 (10 µM), and PGF2 (3 µM) + tamoxifen (10 µM). After 120 min, rings were rapidly frozen, homogenized and then subjected to SDS-PAGE. The blot is representative of four different experiments, with comparable results.

	Discussion

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N,N'-Dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines represent a class of compounds that are structurally related to estrogens and SERMs. Structural similarity is particularly high compared with HEX due to the lack of the styrene double bond and tamoxifen due to the presence of a basic nitrogen. Among the compounds tested, d,l-compounds were more potent than the corresponding meso-compounds in relaxing porcine coronary arterial rings. Thus, relaxation to N,N'-dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines was dependent on the stereochemistry of the compounds. Interestingly, the observation that the d,l-stereoisomers showed a higher agonist potency than the corresponding meso-compounds in relaxing blood vessels is exactly opposite to their estrogen receptor affinity (von Angerer, 1982) and argues against an involvement of classic estrogen receptors in the vasorelaxant response to these drugs (see below). Furthermore, within the series of d,l-compounds, only those drugs showed significant relaxation in which the basic nitrogens were further substituted with methyl, ethyl, propyl, or butyl. There was no difference in the relaxant response to the compounds in porcine coronary arterial rings with or without endothelium. This is in agreement with relaxations to E2, tamoxifen, and raloxifene in this tissue (Salas et al., 1994; Hutchison et al., 2001; Moritz et al., 2006; Leung et al., 2007). Compound 8 with a propyl group at each basic nitrogen atom was most potent. Agonist potency of 8 was 12- to 15-fold higher than that of E2. The effect of 8 was unaffected by ICI 182,780, indicating that classic ICI 182,780-sensitive estrogen receptors were not involved in the vasorelaxant response to this compound. Incubation with 8 shifted the calcium concentration-dependent contraction curve to the right and reduced the maximal response. The inhibition of the calcium contraction was not affected by ICI 182,780. This is in line with observations on the vasorelaxant response to E2 and raloxifene in porcine coronary arteries (Teoh et al., 1999; Moritz et al., 2006; Leung et al., 2007). As in the case of E2, an acute or nongenomic signaling mechanism seems to be involved in the relaxant response to 8.

The observation that 8 insurmountably inhibited Ca²⁺-induced contractions in high-K⁺ depolarization medium suggests that this drug is acting via a calcium antagonist mechanism to induce relaxation in porcine coronary arteries. Further evidence for a calcium antagonist mode of action has been provided by experiments using the L-type Ca²⁺ channel activator, (S)-(–)-Bay K 8644. The leftward shift of concentration-response curves to Ca²⁺ caused by (S)-(–)-Bay K 8644 was concentration-dependently inhibited by 8. A calcium antagonist effect also has been demonstrated in the vasorelaxant response to E2 and raloxifene in rabbit and porcine coronary arteries (Jiang et al., 1991; Figtree et al., 1999; Moritz et al., 2006; Leung et al., 2007). Further studies using electrophysiological recordings of voltage-gated Ca²⁺ currents in porcine coronary artery smooth muscle cells are needed to substantiate the calcium antagonist effect of 8.

A compound that relaxes an artery constricted by modest elevation in extracellular K⁺ (e.g., 30 mM) could be considered a possible K⁺ channel opener (Nelson and Quayle, 1995). K⁺ channels have been shown to be involved in the vasorelaxant response to estrogens in porcine coronary arteries (White et al., 1995). K⁺ channels in arterial smooth muscle include voltage-dependent, Ca²⁺-activated (BK_Ca) and ATP-sensitive K⁺ (K_ATP) channels (Hirst and Edwards, 1989; Nelson and Quayle, 1995). The present study shows that vasorelaxation to 8 seems not to be mediated by: 1) increasing potassium efflux through large-conductance Ca²⁺-activated K⁺ BK_Ca channels, 2) ATP-sensitive K⁺ (K_ATP), and 3) voltage-sensitive K⁺ (K_V) channels. Indeed, the BK_Ca and K_V channel inhibitor TEA, the selective K_V channel inhibitor 4-AP, and the ATP-sensitive K⁺ (K_ATP) channel inhibitor glibenclamide failed to inhibit relaxation to 8. Our observation that glibenclamide reduced the KCl-induced precontraction may be explained by the ability of this drug to induce vasorelaxation via an inhibitory effect on Ca²⁺ influx through Ca²⁺ channels (Chan et al., 2000).

In porcine coronary artery, the time course of relaxation to N,N'-dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines was comparable with E2. In the majority of cases, relaxations to single concentrations of N,N'-dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines showed a plateau within 120 min. However, in contrast to estradiol but in accordance with raloxifene (Moritz et al., 2006) and tamoxifen (A. Moritz and H. H. Pertz, unpublished data), relaxations to N,N'-dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines were not reversed after repeated washings in porcine coronary artery.

Experiments elucidating the signaling pathway that leads to vasorelaxation induced by 8 indicate that adenylyl cyclase, guanylyl cyclase, protein kinase A, and protein kinase G are not involved. This is consistent with the vasorelaxant effect of E2, which was not affected by inhibitors of guanylyl cyclase and protein kinase A in porcine coronary arteries (Keung et al., 2005). However, in contrast to our results with 8, adenylyl cyclase and protein kinase G seem to be involved in vasorelaxation induced by E2 in this tissue (Keung et al., 2005).

It has been shown recently by our group that the relaxant response to raloxifene in porcine coronary arteries is at least in part dependent upon activation of the p38 MAPK pathway (Moritz et al., 2006). Therefore, we used raloxifene as a reference to elucidate whether the p38 MAPK pathway may be involved in the relaxant response to compound 8 in this tissue. E2, raloxifene, tamoxifen, and resveratrol have also been shown to activate p38 MAPK in isolated human endometrial cells and in the isolated perfused rat heart, respectively (Das et al., 2006; Seval et al., 2006). We also checked E2 and tamoxifen for their ability to stimulate p38 MAPK because we hypothesized that the action of these drugs might underlie the same cellular mechanism in porcine coronary arteries. In contrast to raloxifene, tamoxifen, and E2, compound 8 failed to trigger the p38 MAPK pathway in this tissue. This is consistent with our observation in functional experiments that SB 203580, an inhibitor of p38 MAPK, failed to affect compound 8-induced relaxation. Accordingly, our Western blot experiments showed no increase in phosphorylated p38 MAPK 120 min after the addition of 8 alone or 8 following precontraction with PGF₂ in porcine coronary arterial rings with or without endothelium. In contrast, raloxifene, tamoxifen, and E2 alone or raloxifene, tamoxifen, and E2 following precontraction with PGF₂ induced phosphorylation of p38 MAPK. Under the same experimental conditions, precontraction with PGF₂ on its own had no effect on p38 MAPK phosphorylation levels. We studied the effect on p38 MAPK after 120 min because we intended to estimate the degree of p38 MAPK phosphorylation at the time of maximal relaxation induced by compound 8, raloxifene, tamoxifen, and E2, respectively. It should be mentioned that recent studies on porcine coronary arterial smooth muscle cells have demonstrated an activation of p38 MAPK by PGF₂. PGF₂-induced p38 MAPK phosphorylation occurred rapidly (within minutes), reached a peak at 10 min, and decreased within 60 min (Teng et al., 2005). The ineffectiveness of PGF₂ to show an activation of p38 MAPK after 120 min in the present study is consistent with this observation.

In conclusion, the present study shows that 8 relaxes porcine coronary arteries with an agonist potency that was 12- to 15-fold higher than the potency of E2. The drug acts on vascular smooth muscle of coronary arteries apparently by inhibiting Ca²⁺ entry via L-type Ca²⁺ channels. This action is acute, nongenomic, and independent of the endothelium or ICI 182,780-sensitive estrogen receptors. K⁺ channels do not seem to be involved in the relaxant response to the drug. In contrast to E2, raloxifene, and tamoxifen, p38 MAPK activation seems not to play a role in the relaxant response to 8. Further studies are required to elucidate whether 8 may be protective against cardiovascular diseases.

	Acknowledgements

 
We thank Dr. Th. Paulke and M. Uwarow of the Lehr- und Versuchsanstalt für Tierzucht und Tierhaltung (Teltow-Ruhlsdorf, Germany) for providing pig hearts for the studies.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.120337.

ABBREVIATIONS: DES, diethylstilbestrol; HEX, hexestrol; SERM, selective estrogen receptor modulator; E2, 17-estradiol; MAPK, mitogen-activated protein kinase; KHS, Krebs-Henseleit solution; PGF₂, prostaglandin F₂; ICI 182,780, 7-[9-[(4,4,5,5,5-pentafluoropentyl]sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17-diol; 4-AP, 4-aminopyridine; (S)-(–)-Bay K 8644, (S)-(–)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine-carboxylic acid methyl ester; SQ 22536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; KT 5720, (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester; KT 5823, (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester; SB 203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; PAGE, polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide.

Address correspondence to: Heinz H. Pertz, Institute of Pharmacy, Free University of Berlin, Königin-Luise-Strasse 2 + 4, 14195 Berlin, Germany. E-mail: hpertz{at}zedat.fu-berlin.de

	References

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