PROCYANIDIN B2 STIMULATES THE Na+ /K+ ATPASE IN CACO-2 CELLS BY INDUCING PGE2 RELEASE

Abstract

Procyanidins, are known to exert a wide spectrum of biological effects due to their anti-oxidant and anti-inflammatory properties. They are polyphenolic compounds present in plants and are highly abundant in grapes, apples, and cocoa. Procyanidins are polymers or oligomers of catechin or epicatechin, with varying degrees of polymerization. It was previously reported that procyanidins play a beneficial role in inflammatory bowel disease, a disease characterized by an abnormal immune response leading to inflammation of the intestine, and abnormal water absorption. Water transport in the colon follows ionic movements, mainly those of sodium whose gradient is established and maintained by the activity of the Na+/K+ ATPase or Na+/K+ pump. A decrease in the activity of the Na+/K+ ATPase was found to accompany inflammatory bowel disease and alter water transport across colonic cells. Thus, a correlation between procyanidins and the ATPase was suspected, and was examined in this work using Caco-2 cells as a model. Cells were treated with Procyanidin B2 (17 μM, 2 hrs) and the activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the pump. Procyanidin B2 activated the Na+/K+ ATPase but did not have any effect on its expression. Wortmannin, Calphostin C, and RpcAMP, respective inhibitors of PI3K, PKC, and PKA, abolished the stimulatory effect of Procyanidin B2, indicating that these molecules are along the signaling pathway. The effect of Procyanidin B2 did not appear also in presence of indomethacin, an inhibitor of COX enzymes, but a similar stimulatory effect was observed in presence of exogenous PGE2. This stimulatory effect was still manifested in the simultaneous presence of PGE2, and each of SC-19220, PF-04418948, and L-798106, respective blockers of EP1, EP2, and EP3 receptors, but abolished in presence of BGC-201531, an EP4 blocker. The results suggest that PGE2 acts through EP4. Cells treated with PMA, a PKC activator, in presence of wortmannin, a PI3K inhibitor, still showed a significant increase in the activity of the Na+/K+ ATPase. The stimulatory effect of PMA was however abolished in presence of Indomethacin, indicating that PKC is downstream PI3K and upstream PGE2. PGE2 did not have any effect on the ATPase in presence of RpcAMP, an inhibitor of PKA. SB 202190, a specific inhibitor of p38MAPK, increased the pump’s activity, and an additive stimulatory effect was observed when cells were treated with a combination of PGE2 and SB 202190. The results infer that p38MAPK is not along the signaling pathway and that p38MAPK inhibits the pump at basal levels. It was concluded that Procyanidin B2 activates PI3K leading to PKC activation, and PGE2 synthesis. The latter stimulates PKA through EP4 receptors, leading to the activation of the Na+/K+ ATPase. Polyphenols and some procyanidins were reported to activate the bitter taste receptors (TAS2Rs) which are coupled to G-proteins and expressed in various tissues including the colon. We suspect that Procyanidin B2 acts on the pump through an interaction with TAS2Rs. This hypothesis would need however to be confirmed in the future.