(-)-epicatechin activation of endothelial cell endothelial nitric oxide synthase, nitric oxide, and related signaling pathways

Abstract

Recent reports indicate that (-)-epicatechin can exert cardioprotective actions, which may involve endothelial nitric oxide synthase (eNOS)-mediated nitric oxide production in endothelial cells. However, the mechanism by which (-)-epicatechin activates eNOS remains unclear. In this study, we proposed to identify the intracellular pathways involved in (-)-epicatechin-induced effects on eNOS, using human coronary artery endothelial cells in culture. Treatment of cells with (-)-epicatechin led to time- and dose-dependent effects that peaked at 10 minutes at 1 mumol/L. (-)-Epicatechin treatment activates eNOS via serine 633 and serine 1177 phosphorylation and threonine 495 dephosphorylation. Using specific inhibitors, we have established the participation of the phosphatidylinositol 3-kinase pathway in eNOS activation. (-)-Epicatechin induces eNOS uncoupling from caveolin-1 and its association with calmodulin-1, suggesting the involvement of intracellular calcium. These results allowed us to propose that (-)-epicatechin effects may be dependent on actions exerted at the cell membrane level. To test this hypothesis, cells were treated with the phospholipase C inhibitor U73122, which blocked (-)-epicatechin-induced eNOS activation. We also demonstrated inositol phosphate accumulation in (-)-epicatechin-treated cells. The inhibitory effects of the preincubation of cells with the calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 indicate that (-)-epicatechin-induced eNOS activation is at least partially mediated via the Ca(2+)/CaMKII pathway. The (-)-epicatechin stereoisomer catechin was only partially able to stimulate nitric oxide production in cells. Together, these results strongly suggest the presence of a cell surface acceptor-effector for the cacao flavanol (-)-epicatechin, which may mediate its cardiovascular effects.

Figure 1. EPI-induced NO synthesis

(A), Time-course of EPI [1 μmol/L]-induced NO synthesis in HCAECs. NO concentration levels show no statistical difference amongst 10, 20 and 30 minutes (ANOVA). (B), EPI dose-response curve on NO synthesis. HCAECs were treated with EPI [0.1 nmol/L – 100 μmol/L] for 10 min. Data are expressed as mean ± SD (n=3).

Figure 2. EPI-induced eNOS activation via Ser-1177 and Ser-633 phosphorylation and Thr-495 dephosphorylation

EPI [1 μmol/L] and BK [1 μmol/L] induced increases in the phosphorylation of residues Ser-1177 and Ser-633 and the dephosphorylation of Thr-495, as compared to control cells. Data are expressed as mean ± SD of n=3, in triplicate. Densitometric mean values of the phosphorylation of eNOS Ser-1177 and Ser-633 in controls were normalized to total eNOS densitometric values, and the results were set arbitrarily to a value equal to 100. EPI- and BK-induced effects were plotted as changes over control. EPI and BK values were statistically different from control. (*p<0.05 EPI vs. BK).

Figure 3. PI3K signaling pathway mediates EPI-induced eNOS activation

HCAECs were treated with WORT or H89 or SH-5 (see text for details) and stimulated with EPI [1 μmol/L] for 10 min. Western blots were probed with specific antibodies against Akt, p-Akt, eNOS, p-eNOS Ser-1177, Ser-633 and Thr-495; NO was measured as stated in the text. (A), EPI-induced AKT phosphorylation; this effect was blocked by WORT and SH5 pre-treatment. Normalized control densitometric levels were arbitrarily set to 100 in all cases. (B), eNOS activity was measured by its phosphorylation on Ser-1177 and Ser-633 and dephosphorylation on Thr-495. Pre-treatment with WORT and H89 leads to a decrease in eNOS phosphorylation on Ser-1177 and Ser-633. SH5 treatment decreased Ser-1177 phosphorylation. (C) The effects of WORT, H89 and SH-5 on EPI-induced NO synthesis. Control values were arbitrarily set to zero. Statistical analysis shows no difference between WORT and H-89. SH-5 effects were significantly different vs. H89. Data are expressed as mean ± SD (n=3). (*p<0.05).

Figure 4. EPI-induced eNOS decoupling from the caveola

HCAECs were treated with EPI [1 μmol/L] for 10 min, lysed and immunoprecipitated with anti-Cav-1 or -CaMI antibodies. Both the immunoprecipitated (IP) and supernatant (SN) phases were analyzed for phosphorylation of Ser-1177, Ser-633 and Thr-495 as well as Cav-1 or CaMI and eNOS by immunoblots. (A) Control HCAECs demonstrate inactive eNOS associated with Cav-1. EPI treated HCAECs show only Cav-1 in the IP and activated eNOS in the SN phase. (B), Control HCAECs demonstrate no association between the active or inactive forms of eNOS and CaMI, and the SN phase demonstrates the inactive form of eNOS. EPI treated HCAECs demonstrate the activated form of eNOS linked to the immunoprecipitated CaMI, whereas the SN phase in EPI-treated HCAECs show only negligible amounts of eNOS. Data are expressed as mean ± SD (n=3).

Figure 5. EPI-induced increase in intracellular IP₃ levels

HCAECs were incubated with EPI [1 μmol/L] for 10 min. Total intracellular [³H]IP₃ accumulation was measured. EPI-treated HCAECs demonstrate greater IP₃ levels than the control. BK [1 μmol/L] was used as a positive control. IP₃ levels decreased with the pre-incubation with U73122. Data are expressed as mean ± SD (n=3). (*p<0.05).

Figure 6. EPI-induced eNOS activation is mediated by the CaM/CaMKII pathway

HCAECs were pretreated with U73122 or KN-93 and stimulated with EPI 1 μmol/L for 10 min. Western blots were probed with specific antibodies for IP3, p-IP3, CaMKII, p-CaMKII, CaMI, p-CaMI, eNOS, p-eNOS Ser-1177, Ser-633 and Thr-495. NO was measured as stated in the text. (A) Phosphorylation of IP₃R, (B) CaMKII and (C) CaMI was observed in EPI-treated HCAECs, and it was diminished in the three proteins with U73122 and KN-93 inhibitors. (D) EPI-induced eNOS Ser-1177 and Ser-633 phosphorylation and Thr-495 dephosphorylation was blocked by U73122 and KN-93. (E) EPI-induced increases in NO synthesis were diminished by U73122 and K-93 pre-treatment. BK [1 μmol/L] was used as a positive control. Data are expressed as mean ± SD (n=3) (*p<0.05).

Figure 7. NO measurements in HCAECs treated with EPI or catechin

NO production was measured in HCAECs treated with EPI or catechin [1 μmol/L]. EPI induces a ~4-fold increase in NO vs. catechin. Data are expressed as mean ± SD (n=3). (*p=<0.05).

Figure 8. EPI-induced eNOS activation in HCAEC

Schematic representation of signaling pathways involved in EPI-induced eNOS activation. BK receptor activated pathways are included (double arrows) to demonstrate similarities.