Vitamin K2 (menatetrenone) induces iNOS in bovine vascular smooth muscle cells: no relationship between nitric oxide production and gamma-carboxylation.

It has been recently reported that vitamin K2 (menaquinone-4: menatetrenone, VK2) has an anti-atherogenic effect as well as the ability to produce clotting factors and improve osteoporosis. However, the mechanism by which VK2 acts on atherosclerosis is still unclear. In this paper, we investigated the effects of vitamin K and its side chain on NO production as an anti-atherogenic substance in a cultured vascular system. Treatment of bovine vascular smooth muscle cells (SMC) with VK2 (30 microM) caused a time-dependent (24-72 h) increase in the nitrite (NO2) level in the conditioned medium, but not in bovine vascular endothelial cells. Classical NOS inhibitor (L-nitro arginine) and iNOS-specific inhibitors completely blocked the increased nitrite level induced by VK2 treatment, but D-nitro arginine could not it. Immunostaining and Western blotting analysis showed that VK2 induced iNOS protein in the SMC. VK2 has a naphtoquinone nucleus, which is identical in menadione (VK3), and an unsaturated side chain, which is called geranylgeraniol (GGO). To determine whether the structure of VK2 was related to an increasing nitrite level, we investigated the nitrite level in conditioned medium treated with VK3 or GGO. Neither VK3 nor GGO treatment of SMC increased the nitrite level. In addition, warfarin, an inhibitor of VK2-dependent gamma-carboxylation, did not affect the increased nitrite level induced by VK2 in SMC. In conclusion, VK2 caused NO production through iNOS induction in bovine SMC, that was not related to the structure of VK2, naphtoquinone nucleus or its side chain, independently of gamma-carboxylation.