Effect of Docosahexaenoic Acid on Voltage-Independent Ca 2+ Entry Pathways in Cultured Vascular Smooth Muscle Cells Stimulated with 5-Hydroxytryptamine

We previously reported that docosahexaenoic acid (DHA) inhibits an increase in intracellular Ca concentration ([Ca ] ) in cultured rat vascular smooth muscle cells (VSMCs) through a mechanism involving mainly voltage-dependent Ca channels; however, the effect of DHA on voltage-independent pathways, such as store-operated and receptor-operated Ca entry, and Ca entry through Na /Ca exchanger (NCX), has not been clarified. In the present study, we investigated the effect of DHA treatment on the expression of transient receptor potential canonical (TRPC) channels, capacitative Ca entry, and Ca entry through NCX in rat cultured VSMCs stimulated with 5-hydroxytryptamine (5-HT). RT-PCR analysis detected TRPC1, TRPC4, and TRPC6 mRNA in cultured VSMCs. DHA treatment for 2 d slightly but significantly decreased TRPC1, but not TRPC4 and TRPC6, mRNA expression. Sarpogrelate, a selective serotonin 5-HT receptor inhibitor, completely inhibited the 5-HT-induced increase in [Ca ] in cultured VSMCs. Ca influx by adding extracellular Ca (1.3 mM) to the Ca -free condition in the presence of 5-HT was partially but significantly inhibited by sarpogrelate. DHA treatment for 2 d had no effect on Ca influx when extracellular Ca was added to the Ca -free condition in the presence of either 5-HT alone or 5-HT with sarpogrelate. KB-R7943, a selective inhibitor of reverse mode NCX, significantly suppressed the 5-HT-induced increase of [Ca ] . Furthermore, DHA treatment for 2 d significantly decreased NCX1 mRNA expression. These results suggest that DHA seems to have little effect on capacitative Ca entry. Through decreasing NCX1 expression, DHA may suppress the 5-HT-induced increase in [Ca ] .