Apple procyanidins induce hyperpolarization of rat aorta endothelial cells via activation of K+ channels.

Apple procyanidins (AP), one of the polyphenol-rich compounds, showed an endothelial-dependent vasorelaxation in rat aorta, but the mechanisms of beneficial effects are still unclear. The present study was designed to clarify the potential role of AP in rat aorta endothelial cells (RAECs). The treatment of RAECs with AP (1-10 µg/ml) resulted in a dose-dependent hyperpolarization with a maximum effect at 10 µg/ml, and for this reason, AP (10 µg/ml) was used in all the following experiments. AP-induced hyperpolarization was significantly inhibited by pretreatment of nonspecific K(+) inhibitor, tetraethyl ammonium chloride or specific K(+) channel inhibitors, iberiotoxin, glibenclamide, 4-aminopyridine and BaCl(2), as well as by high KCl or Ca(2+)-free solution. AP-induced hyperpolarization was also proved using 64-channel multielectrode dish system that can monitor a direct and real-time change of membrane potential. Furthermore, AP treatment caused a significant increase of nitric oxide (NO) production and cyclic guanosine monophosphate levels via endothelial NO synthase messenger RNA expression. The NO production was inhibited by N(G)-monoethyl-l-arginine or Ca(2+)-free solution and was completely abolished by their combination. Also, AP inhibited endothelial proliferation, while the effect was significantly abolished by N(G)-monoethyl-l-arginine or tetraethyl ammonium chloride. These findings suggest that AP induces both hyperpolarization of RAECs via multiple activation of K(+) channels and activation of NO/cyclic guanosine monophosphate pathway via increasing NO production or is responsible for antiangiogenic effect. Diminishment of hyperpolarization as well as NO production of AP in Ca(2+)-free solution implicated that AP would play a crucial role in promoting Ca(2+) influx into endothelial cells so as to promote both actions.

Procyanidins are potent inhibitors of LOX-1: a new player in the French Paradox.

Lectin-like oxidized LDL receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL (oxLDL) and plays multiple roles in the development of cardiovascular diseases. We screened more than 400 foodstuff extracts for identifying materials that inhibit oxLDL binding to LOX-1. Results showed that 52 extracts inhibited LOX-1 by more than 70% in cell-free assays. Subsequent cell-based assays revealed that a variety of foodstuffs known to be rich in procyanidins such as grape seed extracts and apple polyphenols, potently inhibited oxLDL uptake in Chinese hamster ovary (CHO) cells expressing LOX-1. Indeed, purified procyanidins significantly inhibited oxLDL binding to LOX-1 while other ingredients of apple polyphenols did not. Moreover, chronic administration of oligomeric procyanidins suppressed lipid accumulation in vascular wall in hypertensive rats fed with high fat diet. These results suggest that procyanidins are LOX-1 inhibitors and LOX-1 inhibition might be a possible underlying mechanism of the well-known vascular protective effects of red wine, the French Paradox.

Apple procyanidins induced vascular relaxation in isolated rat aorta through NO/cGMP pathway in combination with hyperpolarization by multiple K+ channel activations.

Apple (Malus pumila) procyanidins led to a potent vasorelaxation effect in 1.0 microM phenylephrine-contractive rat thoracic aorta. Relaxation was greatly reduced by 70 mM KCl as well as by removal of the endothelium, suggesting that it was associated with endothelium-dependent hyperpolarization. Neither cAMP synthesis inhibition nor NAD(P)H oxidase inhibition abolished the effect. In contrast, complete abolition by a soluble guanylyl cyclase inhibitor revealed that apple procyanidins were mainly involved in the cGMP production pathways. In the presence of N(G)-monoethyl-L-arginine or tetraethylammonium chloride, the effect was still observed at higher concentrations (>25 microg/ml), while their combination completely diminished the effect. Vasorelaxation was to some extent affected by paxillin, apamin and glybenclamide, and was greatly affected by 4-aminopyridine and by BaCl(2). These results indicate that procyanidin-induced vasorelaxation is associated with NO-cGMP pathway in combination with hyperpolarization due to multiple activation of Ca(2+)-dependent and -independent K(+) channels.