Effect of estrogen on muscarinic acetylcholine receptor expression in rat myometrium.

We report the effect of acute estrogen treatment in the expression of muscarinic acetylcholine receptors (mAChRs) in myometrium. Strips were obtained from rats in estrus (control) and treated with estrogen, 24h before the experiments. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed and m2, m3 and m5 mAChR mRNA subtypes were detected in myometrium from both groups. [(3)H]Quinuclidinyl benzilate [(3)HQNB] binding studies indicated that estrogen treatment did not change the affinity and density of mAChRs in myometrial membranes. Displacement curves of [(3)HQNB] with different mAChRs antagonists indicated a one-site fit for all antagonists tested. Comparison of pK(i) values indicated a significant correlation to M(2)-mAChR subtype. Functional studies, however, showed that estrogen treatment increased myometrium sensitivity to carbachol and the calculated apparent affinity values were significantly correlated to M(3)-mAChR. Furthermore, the pharmacological profile of the two populations of mAChR was not affected by estrogen. In conclusion, these results provide evidence for the presence of M(2)- and M(3)-mAChR, at the mRNA and protein level, in the rat myometrium and indicate that estrogen induces an increase in myometrial responsiveness to mAChR agonists.

Estrogen receptor ESR1 regulates the phospholipase C-inositol phosphate signaling in the hippocampus from rats in proestrous and estrous phases.

The aim of the present study was to investigate the involvement of estrogen receptors in the activation of phospholipase C (PLC)-phosphoinositide hydrolysis in the hippocampus from rats in estrous and proestrous phases. 17ß-Estradiol (E2) and ESR1-selective agonist PPT, but not ESR2-selective agonist DPN, induced a rapid increase on total [³H]-inositol phosphate accumulation in the hippocampus from both rats. These effects are mediated by PLC activation, since the inhibition of this protein decreased the total [³H]-inositol phosphate accumulation. The pretreatment with ESR1 and ESR2 antagonist ICI 182,780, but not with GPER antagonist G-15, blocked the total [³H]-inositol phosphate accumulation induced by E2 and PPT, confirming that ESR1 is upstream component regulating this rapid effect. SRC family of protein tyrosine kinases inhibitor PP2 blocked the total [³H]-inositol phosphate accumulation induced by E2 and PPT in hippocampus, suggesting that ESR1 undergoes translocation from the nuclei to the plasma membrane region via SRC to activate rapid signaling pathways. Furthermore, the magnitude of the response to E2 and PPT was higher in hippocampus from rats in proestrous than in estrous. On the other hand, the expression of the ESR1 is higher in hippocampus from rats in estrous than in proestrous, indicating that the regulation of this receptor by estrous cycle does not play a role in the magnitude of the response to E2 and PPT in hippocampus. In conclusion, our results indicate that E2 activates SRC-mediated translocation of ESR1 to the plasma membrane, which results in the activation of PLC-inositol phosphate signaling pathway in rat hippocampus. Thus, these rapid estrogen actions in hippocampus might be a key step mediating cellular events important for learning and memory.

Estrogen receptors mediate rapid activation of phospholipase C pathway in the rat endometrium.

The aim of the present study was to investigate the activation of rapid signaling events by 17ß-estradiol in the rat uterus. 17ß-Estradiol induced a rapid increase of total [3H]-inositol phosphate accumulation in the whole uterus and endometrium, but not in the myometrium. The effect of 17ß-estradiol in the endometrium was blocked by phospholipase C (PLC) inhibitor (U73122), estrogen receptors antagonist (ICI 182,780), exportin CRM1 inhibitor (leptomycin B) and selective inhibitor of the SRC family of protein tyrosine kinases (PP2). Furthermore, a selective agonist of ESR1 (PPT) and a selective agonist of GPER (G-1) also induced a rapid increase of total [(3)H]-inositol phosphate accumulation in the endometrium. The G-1 effects were blocked by GPER antagonist (G-15). 17ß-Estradiol and G-1 promoted an additive effect on total [3H]-inositol phosphate accumulation. In conclusion, the present results indicate that a rapid activation of the PLC-mediated phosphoinositide hydrolysis occurred in the rat endometrium after 17ß-estradiol stimulation, and this effect was mediated by ESR1 that underwent nuclear export after hormone stimulation, and that GPER activation may play an additive role for this response. These rapid actions might be one of the key steps that mediate the estrogen-dependent activation of cellular events in the endometrium.

Effects of estrogen on muscarinic acetylcholine receptors in the rat hippocampus.

The aim of the present study was to investigate whether different estrogen manipulations have effects on the expression of muscarinic acetylcholine receptors (mAChRs) in the adult female rat hippocampus. Hippocampus was obtained from rats in proestrus (control), ovariectomized for 2, 10 and 15 days, ovariectomized for 15 days and treated with 17beta-estradiol for 7 days, and treated with 17beta-estradiol immediately after ovariectomy for 21 days. Rats' estrogen status was monitored by measuring estradiol plasma levels and uterus relative weight. [3H]quinuclidinyl benzilate ([3H]QNB) binding studies indicated that ovariectomy time-dependently increases the number of mAChRs in hippocampus when compared to those obtained from control rats. Estradiol treatments for 21 days avoid the effect of ovariectomy. However, the estradiol treatments for 7 days after 15 days of ovariectomy slightly change the number of mAChRs. In conclusion, these results showed that ovariectomy time-dependently increases mAChRs number in the rat hippocampus. In addition, these data suggest that treatment with estradiol initiated within a specific period of time after the loss of ovarian function may be effective at preventing specific effects of hormone deprivation on hippocampus.