Effect of estrogen on ACh-induced contraction of GSMCs in male versus female rats. GSMCs from male and female rats were stimulated with ACh in the presence or absence of E2, an activator of the majority of ERs, and observed under a microscope. Images of treated and non-treated single cells were acquired and the extent of cell contraction was measured. ACh-induced contraction was significantly reduced in female cells compared with that in male cells. E2 significantly reduced ACh-induced contraction in cells from both sexes, but to a greater extent in GSMCs from females compared with those from males. Values are expressed as the mean ± standard error of the mean (n=50 cells from 10 male or 10 female rats). *P<0.05 vs. M/ACh; #P<0.05 vs. F/ACh; €P<0.05 vs. M/ACh+E2. ACh, acetylcholine; GSMCs, gastric smooth muscle cells; E2, 17β-estradiol; M, male group; F, female group.