The dihydropyridine calcium channel blocker benidipine prevents lysophosphatidylcholine-induced endothelial dysfunction in rat aorta

Abstract

Background: Lysophosphatidylcholine (LPC), an atherogenic component of oxidized low-density lipoprotein, has been shown to induce the attenuation of endothelium-dependent vascular relaxation. Although benidipine, a dihydropyridine-calcium channel blocker, is known to have endothelial protective effects, the effects of benidipine on LPC-induced endothelial dysfunction remain unknown. We examined the effects of benidipine on the impairment of endothelium-dependent relaxation induced by LPC.

Methods: Benidipine was administered orally to rats and aortas were then isolated. Aortic rings were treated with LPC and endothelial functions were then evaluated. Additionally, the effects of benidipine on intracellular calcium concentration ([Ca2+]i) and membrane fluidity altered by LPC in primary cultured rat aortic endothelial cells were examined. [Ca2+]i was measured using the fluorescent calcium indicator fura-2. Membrane fluidity was monitored by measuring fluorescence recovery after photobleaching.

Results: Treatment with LPC impaired endothelial function. Benidipine prevents the impairment of relaxation induced by LPC. Acetylcholine elicited an increase in [Ca2+]i in fura-2 loaded endothelial cells. The increase in [Ca2+]i was suppressed after exposure to LPC. Plasma membrane fluidity increased following incubation with LPC. Benidipine inhibited the LPC-induced increase in membrane fluidity and impairment of increase in [Ca2+]i.

Conclusion: These results suggest that benidipine inhibited LPC-induced endothelial dysfunction by maintaining increase in [Ca2+]i. Benidipine possesses membrane stabilization properties in LPC-treated endothelial cells. It is speculated that the preservation of membrane fluidity by benidipine may play a role in the retainment of calcium mobilization. The present findings may provide new insights into the endothelial protective effects of benidipine.

Figure 1

Tracings showing relaxant response to ACh (endothelium-dependent vasodilator) in isolated rat aorta. Benidipine or vehicle was administered orally to rats. Thoracic aortas were isolated at 90 min following benidipine administration. Aortas were cut into rings, and incubated with LPC (5 μmol/L) or ethanol (0.05%) for 60 min. After washing, the rings were pre-contracted using PE (1 μmol/L) and subsequently relaxed using ACh. ACh evoked relaxation in the aortic ring isolated from vehicle-treated rat (A). EDR was markedly attenuated following incubation with LPC (B). LPC-induced attenuation of EDR was inhibited in benidipine-treated rat (C).

Figure 2

Effects of benidipine (A), amlodipine (B) and nifedipine (C) on LPC-induced attenuation of endothelium-dependent relaxation. Drugs were administered orally to rats. Rats were treated as described for Figure 1. Data are expressed as percentage of PE-induced contraction. Each value represents the mean ± S.E. of 6–10 experiments. *P < 0.05 vs. vehicle + LPC group.

Figure 3

Tracings showing [Ca²⁺]_i (indicated by fluorescence ratio of fura-2 at 340 nm and 380 nm) transient in rat aortic endothelial cells. ACh (3 μmol/L) elicited increases in [Ca²⁺]_i in fura-2-loaded endothelial cells. Vehicles of LPC and benidipine had no effect on ACh-induced increases in [Ca²⁺]_i (A). LPC (3 μmol/L) decreased ACh-induced increases in [Ca²⁺]_i (B). Benidipine (10 nmol/L) prevented LPC-induced suppression of increases in [Ca²⁺]_i (C).

Figure 4

Effects of benidipine with or without LPC on ACh-induced increases in [Ca²⁺]_i. Endothelial cells were treated as described for Figure 3. Benidipine (1 or 10 nmol/L) or DMSO was simultaneously added with LPC or ethanol. Changes in ACh-induced increases in calcium are expressed as percentage fluorescence value of ACh-induced increases in calcium prior to LPC or ethanol treatment. Each value represents the mean ± S.E. of 7 cells. *P < 0.05, **P < 0.01, ***P < 0.001 compared between indicated groups.

Figure 5

Effects of PKC activators on ACh-induced increases in [Ca²⁺]_i. Fura-2 loaded endothelial cells were treated with ACh (3 μmol/L) followed by washing. Cells were then incubated with PMA, PDB or DMSO for 30 min. After washing, ACh (3 μmol/L) was added once again. Changes in ACh-induced increases in calcium are expressed as percentage fluorescence value of ACh-induced increases in calcium prior to LPC or ethanol treatment. Each value represents the mean ± S.E. of 7 cells.

Figure 6

Effects of PKC inhibitors with or without LPC on ACh-induced increases in [Ca²⁺]_i. Fura-2 loaded endothelial cells were treated with the protein kinase C inhibitors Ro-31-8220 and calphostin C for 30 min. ACh (3 μmol/L) was added before and after treatment. Data are expressed as percentage value of ACh-induced increases in calcium prior to treatment. Each value represents the mean ± S.E. of 7 cells. **P < 0.01, ***P < 0.001 compared between indicated groups.

Figure 7

Effects of anti-oxidants on ACh-induced increases in [Ca²⁺]_i with LPC. Fura-2 loaded endothelial cells were treated with the anti-oxidants ascorbic acid, PDTC and BHT for 30 min. ACh (3 μmol/L) was added before and after treatment. Data are expressed as percentage value of ACh-induced increases in calcium prior to the treatment. Each value represents the mean ± S.E. of 7 cells. ***, P < 0.001 compared between indicated groups.

Figure 8

Effects of benidipine on fluorescence recovery one second after the end of photobleaching. NBD-PC loaded endothelial cells were treated with LPC (3 μmol/L) or ethanol (0.03%) for 30 min. Benidipine (10 nmol/L) or DMSO was simultaneously added with LPC or ethanol. Each value represents the mean ± S.E. (n = 18–28). *P < 0.05 compared between indicated groups.