In this study, we investigated the effects of pelargonidin, an anthocyanidin found in many fruits and vegetables, on endothelium-independent vascular contractility to determine the underlying mechanism of relaxation. Isometric contractions of denuded aortic muscles from male rats were recorded, and the data were combined with those obtained in western blot analysis. Pelargonidin significantly inhibited fluoride-, thromboxane A2-, and phorbol ester-induced vascular contractions, regardless of the presence or absence of endothelium, suggesting a direct effect of the compound on vascular smooth muscles via a different pathway. Pelargonidin significantly inhibited the fluoride-dependent increase in the level of myosin phosphatase target subunit 1 (MYPT1) phosphorylation at Thr-855 and the phorbol 12,13-dibutyrate-dependent increase in the level of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation at Thr202/Tyr204, suggesting the inhibition of Rho-kinase and mitogen-activated protein kinase kinase (MEK) activities and subsequent phosphorylation of MYPT1 and ERK1/2. These results suggest that the relaxation effect of pelargonidin on agonist-dependent vascular contractions includes inhibition of Rho-kinase and MEK activities, independent of the endothelial function.

A comprehensive collection of proteins senses local changes in intracellular Ca2+ concentrations ([Ca2+]i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca2+ concentrations in cat esophageal smooth muscle cells. To measure [Ca2+]i levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca2+]i in the cells. Pretreatment with EGTA, an extracellular Ca2+ chelator, decreased the S1P-induced increase in [Ca2+]i, and an L-type Ca2+-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca2+ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP3 receptor blocker, the S1P-evoked increase in [Ca2+]i was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of Gi-protein, suppressed the increase in [Ca2+]i evoked by S1P. These results suggest that the S1P-induced increase in [Ca2+]i in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca2+ from the InsP3-sensitive Ca2+ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca2+ via the L type Ca2+ channel, which was dependent on activation of the S1P4 receptor coupled to PTX-sensitive Gi protein, via phospholipase C-mediated Ca2+ release from the InsP3-sensitive Ca2+ pool in cat esophageal smooth muscle cells.