Proliferation of vascular smooth muscle cells (VSMC) plays a major role as an initiating event of atherosclerosis. Although estrogen directly inhibits the proliferation of VSMC, the mechanism has not been firmly established. In addition, the effect of raloxifene on VSMC remains unknown. 17Beta-estradiol (E(2)) and raloxifene significantly inhibited the growth of VSMC under growth-stimulated conditions. Since mitogen-activated protein (MAP) kinases have been implicated in VSMC proliferation, the role of MAP kinases in both the E(2)- and raloxifene-induced growth inhibition of VSMC was studied. Both E(2) and raloxifene caused rapid, transient phosphorylation and activation of p38 that was not affected by actinomycin D and was blocked by ICI 182,780. In contrast with p38 phosphorylation, extracellular signal-regulated protein kinase (ERK) phosphorylation was significantly inhibited and c-Jun N-terminal kinase (JNK) phosphorylation was not changed by E(2). Because VSMC expressed both estrogen receptor (ER) alpha and ERbeta, it is not known which of them mediates the E(2)-induced phosphorylation of p38. Although E(2) did not affect the p38 phosphorylation in A10 smooth muscle cells, which express ERbeta but not ERalpha, transfection of ERalpha expression vector into A10 cells rendered them susceptible to induction of p38 phosphorylation by E(2). We then examined whether E(2) and raloxifene induce apoptosis through a p38 cascade. Both E(2) and raloxifene induced apoptosis under growth-stimulated conditions. The p38 inhibitor SB 203580 completely blocked the E(2)-induced apoptosis. Our findings suggest that both E(2)- and raloxifene-induced inhibition of VSMC growth is due to induction of apoptosis through a p38 cascade whose activation is mediated by ERalpha via a nongenomic mechanism.

Neuropeptide W (NPW) is a 30-amino-acid peptide initially isolated from the porcine hypothalamus as an endogenous ligand for the G protein-coupled receptors GPR7 and GPR8. An intracerebroventricular administration of NPW increased serum prolactin and corticosterone concentrations, decreased dark-phase feeding, raised energy expenditure, and lowered body weight. Peripherally, GPR7 receptors are abundantly expressed throughout the gastrointestinal tract; the presence of NPW in the gastrointestinal endocrine system, however, remains unstudied. Using monoclonal and polyclonal antibodies raised against rat NPW, we studied the localization of NPW in the rat, mouse, and human stomach by light and electron microscopy. NPW-immunoreactive cells were identified within the gastric antral glands in all three species. Double immunohistochemistry and electron-microscopic immunohistochemistry studies in rats demonstrated that NPW is present in antral gastrin (G) cells. NPW immunoreactivity localized to round, intermediate-to-high-density granules in G cells. NPW-immunoreactive cells accounted for 90% chromagranin A- and 85% gastrin-immunoreactive endocrine cells in the rat gastric antral glands. Using reversed-phase HPLC coupled with enzyme immunoassays specific for NPW, we detected NPW30 and its C-terminally truncated form, NPW23, in the gastric mucosa. Plasma NPW concentration of the gastric antrum was significantly higher than that of the systemic vein, suggesting that circulating NPW is derived from the stomach. Plasma NPW concentration of the gastric antrum decreased significantly after 15-h fast and increased after refeeding. This is the first report to clarify the presence of NPW peptide in the stomachs of rats, mice, and humans. In conclusion, NPW is produced in gastric antral G cells; our findings will provide clues to additional mechanisms of the regulation of gastric function by this novel brain/gut peptide.