Immunolocalization, binding, and biological activity of endothelin in rabbit uterus: effect of ovarian steroids

Abstract

Specific immunostaining for endothelin 1 (ET-1) was observed in the endometrium but not myometrium of rabbits. The staining was dramatically affected by subacute treatment with ovarian steroids: epithelial cells were predominantly positive in immature rabbits, whereas, in sex steroid-primed rabbits, ET-1 was mainly localized in the stromal compartment. Binding studies were performed in myometrium of estrogen-treated rabbits using labeled ET-1 and ET-3, the corresponding unlabeled peptides, and sarafotoxin b (SRTX). Mathematical modeling of experimental results indicates that two populations of sites are present in myometrium. One site (R1 = 1 pmol/mg protein) shows approximately the same affinity for ET-1, ET-3, and SRTX [dissociation constant (Kd) 100 pM], whereas the second site (R2 = 10 pmol/mg protein) selectively binds ET-1 (Kd 400 pM). According to binding studies, ET-1 was more potent than SRTX in stimulating uterine contraction "in vitro." The subacute administration of increasing concentrations of 17 beta-estradiol (0.2-200 micrograms/kg for 4 days), but not 17 beta-estradiol (200 micrograms/kg for 4 days) plus progesterone (5 mg/kg for 4 days), stimulates a dose-dependent increase in endothelin receptors in myometrium (half-maximal effective dose = 0.7 micrograms/kg for 4 days). However, estrogen treatment does not affect the concentration of endothelin receptors in myometrial cells in primary culture. Conversely, divalent ions like calcium and magnesium enhance the binding of ET-1 to both uterine membranes and cells. Our results indicate that in rabbit uterus endothelin is present in the endometrium, whereas specific receptors are located in myometrium.