Antihypertensive Effect of a Novel Angiotensin II Receptor Blocker Fluorophenyl Benzimidazole: Contribution of cGMP, Voltage-dependent Calcium Channels, and BKCa Channels to Vasorelaxant Mechanisms

Figures

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  FIGURE 1. Effect of minoxidil (10 mg kg−1) and FPD (50 mg kg−1 and 100 mg kg−1) once daily for 14 days on SBP, DBP, MAP, and HR in spontaneously hypertensive rats (A–D) (mean ± S.E.M., n = 6; p < 0.05, * significance compared to SHR).

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  FIGURE 2. Traces showing angiotensin II (1 µM)-induced contraction in isolated rat superior mesenteric arterial rings (A) and in a pre-incubated arterial ring with FPD (10 µM) (B). Contraction is expressed as the mean ± S.E.M. (n = 6); *p < 0.05 compared to control (C).

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  FIGURE 3. Traces showing acetylcholine (10 µM)-induced relaxation in endothelium-intact (A) and endothelium-denuded (B) isolated rat superior mesenteric arterial rings pre-constricted with U46619 (100 nM). Traces showing FPD-induced concentration dependent relaxation in endothelium-intact (C) and endothelium-denuded (D) arterial rings pre-constricted with U46619 (100 nM). E shows the sigmoidal concentration response curve obtained in (C, D). Relaxation is expressed as the mean ± S.E.M. (n = 8) percentage reversal of U46619-induced contraction.

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  FIGURE 4. Traces showing FPD-induced concentration dependent relaxation in rat superior mesenteric arterial rings pre-constricted with 80 mM of high K+ MKHS (A) and 25 mM of high K+ MKHS (B). Traces showing FPD-induced concentration dependent relaxation in arterial rings pre-incubated with potassium channel blockers; tetraethylammonium (TEA, 1 mM), iberiotoxin (10 nM), 4-aminopyridine (4-AP, 300 µM), glibenclamide (10 µM), and barium chloride (BaCl2, 30 µM) for 20 min and pre-constricted with U46619 (100 nM)/NE (10 µM) (C–G). Relaxation is expressed as the mean ± S.E.M. (n = 6; *p < 0.05 compared to control and 25 mM of high K+ MKHS) percentage reversal of contraction (H). (I) shows the sigmoidal concentration response curve obtained in (A−D).

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  FIGURE 5. Traces showing FPD-induced concentration dependent relaxation in rat superior mesenteric arterial rings pre-incubated with ODQ (10 µM) for 20 min and pre-constricted with U46619 (100 nM) (A). Concentration response curve of FPD in the presence of ODQ (10 µM) (B). Relaxation is expressed as the mean ± S.E.M. (n = 6) percentage reversal of contraction. The bar diagram (C) depicts the mean intracellular cGMP level in treated rat superior mesenteric arterial tissues. Vertical bar represents mean ± S.E.M. (n = 6; *p < 0.05 compared to control).

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  FIGURE 6. Representative pictures of cells derived from rat aorta stained with markers for VSMCs. Mesenchymal marker alpha-smooth muscle actin (α-SMA) (green) and F-actin (red) were stained positive in VSMCs. The nucleus of VSMC was stained by DAPI (blue) (40X).

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  FIGURE 7. Representative traces of intracellular K+ concentration stimulated/inhibited by FPD (10 µM), FPD (30 µM), TEA (10 mM), and KCl (80 mM) in rat aortic VSMC loaded with FluxOR II Green (A). (B) depicts the area under the curve (AUC; * significantly increased compared to control; # significantly decreased compared to control; n = 10). C shows the representative images of the cells.

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  FIGURE 8. Tracings of concentration dependent Bay K8644-induced contraction alone and in the presence of FPD (10 µM) and nifedipine (1 µM) in isolated rat superior mesenteric artery (A). Concentration response curve of Bay K8644 alone, in combination with FPD (10 µM for 20 min) and nifedipine (1 µM) (B) (mean ± S.E.M., n = 8; *p < 0.05 compared to control).

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  FIGURE 9. Representative traces of cytosolic Ca2+ concentration ([Ca2+]i) stimulated by NE (10 µM), Bay K8644 (1 µM) alone, and in the presence of FPD (10 and 30 µM) in rat aortic VSMC loaded with Fluo-4 (A, D); area under the curve (B, E) (AUC, * significantly decreased compared to NE/Bay K8644 treated cells; n = 10); representative images of the cells (C, F).

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  FIGURE 10. Molecular insight, interactive amino acid residues, and binding site conformation of FPD (green) with control compound (element color) docked on sGC subunit β (homology model_1). (A), BKCa channels (PDB ID: 3NAF). (B) VDCC (PDB ID: 1T0H). (C) angiotensin II receptor 1 (PDB ID: 4YAY). (D).

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  FIGURE 11. Effect of FPD as a single acute oral dose at 5, 50, 300, and 1,000 mg kg−1 on absolute organ weight, relative organ weight, and differential leukocytes count (DLC) (A–C) in Swiss albino mice (mean ± SEM, n = 6). Effect of FPD as sub-acute oral dose at 0.1, 1, 10, and 100 mg kg−1 once orally for 28 days on absolute organ weight, relative organ weight, and DLC (D–F) in Swiss albino mice (mean ± S.E.M., n = 6).