17Beta-estradiol (E2) plays an important role in Ca2+ fluxes in several cell types. It has been proposed that some of its effects are of nongenomic origin E2 at vascular smooth muscle level can block calcium entry through L-type calcium channels, this mechanism cannot include vascular endothelial cells (VECs), in which increases in the intracellular calcium concentration ([Ca2+]i) are necessary to NO synthesis. We used male rat aorta ECs in culture loaded with fura-2 and a fluorescence imaging system to evaluate the short-term effects of E2 on [Ca2+]i kinetics. We explored the participation of the intracellular steroid receptor on the effects induced by E2, using tamoxifen (1 microM) and ICI 182,780 (10 microM). Our results showed that E2 (like bradykinin) induced an increase in [Ca2+]i. Such agonist-like effects showed a biphasic curve behavior. The 17beta-estradiol effects were not modified by the presence of the intracellular estradiol-receptor antagonist tamoxifen, but it is blocked in the presence of the ICI 182,780. The 17beta-estradiol effects were obtained even with restriction of steroid-free diffusion into cells (17beta-estradiol-bovine serum albumin). Phospholipase Cbeta activity is involved in these effects, because U-73122, a PLCbeta inhibitor, blocked E2 effects. All E2 effects were of rapid onset (milliseconds), exerted at the membrane level, and of rapid offset. We conclude that estradiol can influence the endothelium physiologic responses through effects of nongenomic origin.

The objective of this work was to evaluate the effects of testosterone (T) and 17beta-estradiol (E(2)) on coronary microvascular endothelial cells (CMECs) of male and female rats. To analyze the short-term effects of such sex steroid hormones on intracellular Ca(2+) concentration ([Ca(2+)](i)) kinetics, we used the chelating agent fura-2 acetoxymethyl ester. We also explored the possibility of testosterone aromatization by using selective inhibitors of the aromatase enzyme cytochrome P-450 aromatase (P450(arom)), aminoglutethimide (4 microM), and 4-hydroxyandrostenedione (4 microM). The presence of P450(arom) was investigated by immunocytochemical and immunoblot assays using peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of rat P450(arom) and by in situ hybridization to locate the aromatase mRNA in such cells. The activity of P450(arom) was demonstrated by the stereospecific loss of the tritium atom of [1beta-(3)H]androstenedione. Our results indicate that both T and E(2) induced a rapid increase in [Ca(2+)](i). The fact that the effects of E(2) and T were carried out within milliseconds suggests that they were exerted at the membrane level and not through intracellular receptors. The possibility of involvement of PLC-beta in these effects is suggested because U-73122 (a PLC inhibitor) blocked the effects of both T and E(2). Immunocytochemical assays indicated the expression of androgenic and estrogenic receptors in these cells. The effects of T were blocked by the selective aromatase inhibitors. We also demonstrated membrane association of P450(arom), expression of the ovary-specific mRNA after in situ hybridization, and E(2) formation resulting from a significant activity of P450(arom) in CMECs. There were no gender-based differences.

We evaluated the impact of concurrent pregnancy and lactation on: nest-building (i.e., digging, straw-carrying, hair-pulling), food intake, milk output, body weight, and the concentration of estradiol and progesterone in blood. Digging was lower in pregnant-lactating (PL) rabbits, compared with pregnant-only (PO) does, on 21-23 d (52±64 vs. 104±86 g, respectively; mean±SD; P<0.05). Straw-carrying was also reduced in PL does on 24-26 d (9±27 vs. 79±94 g; P<0.005), 27-29 (27±56 vs. 99±77 g; P<0.005), and in the total amount of material introduced into the nest box (132±167 vs. 286±217 g; P<0.02). Hair-pulling was expressed by practically all animals. Food intake declined in PO does on the three days preceding parturition (P<0.01) and increased markedly during lactation; this increase was much larger in PL than in lactating-only (LO) rabbits (P<0.01). Milk output was similar between PL and LO does during the fi rst 21 d of lactation but a marked decline in this parameter occurred in PL does from then until 30 d. The differences in nest-building between PL and PO rabbits may be related to the concentrations of estradiol and progesterone on specifi c days of pregnancy. PL does showed signifi cantly higher estradiol levels than PO animals on pregnancy 1 d (33±13 vs. 23±4 pg/mL; P<0.02) and 21 (34±19 vs. 24±6 pg/mL; P<0.05) and also higher levels of progesterone on pregnancy 1 d (4±5 vs. 1±2 ng/mL; P<0.05). However, PL rabbits had lower levels of progesterone on 7 d (6±3 vs. 9±2 ng/mL; P<0.02) and 14 d (8±3 vs. 11±3 ng/mL; P<0.005) than PO does. Our results indicate that the unique endocrine milieu of PL rabbits has a direct bearing on specifi c behavioral and physiological phenomena that impact productivity on the farm.