E2 down-regulates nNOS expression and activity in anterior pituitary cells through ER.

<p>Anterior pituitary cells from 14 days-OVX rats were stabilized for 24 h and incubated with or without 1 nM E2 for 24, 48 or 72 h. (<b>A</b>) nNOS mRNA levels were determined by RT-PCR. (<b>B</b>), NOx concentration was assayed by nitrate reductase-Griess assay. (<b>C</b>), Pituitary cell cultures from OVX rats were incubated with 1 nM E2 and/or with 100 nM ICI 182,780 (an estrogen receptor antagonist), 30 min before E2 treatment. nNOS mRNA levels were determined by RT-PCR. Results represent media ± SE of densitometric values relative to β-actin or nitrite concentration (N = 3). ANOVA followed by Tukey’s test, *p<0.05, **p<0.01 vs. respective control, Δp<0.05 vs. E2.</p

Primers used for semi-quantitative RT-PCR assays.

<p>Primers used for semi-quantitative RT-PCR assays.</p

nNOS-derived pituitary NO production plays an important role in OVX-induced apoptosis.

<p><b>(A)</b> Anterior pituitary primary cell cultures from control and 4, 7 and 14 days-OVX rats were stabilized for 24 h. (<b>B</b>) control and 14 days-OVX pituitary cells were incubated with or without 0.1 mM L-NMMA (a non-selective NOS inhibitor), 0.1 mM L-NAME (a more selective nNOS inhibitor) or 0.1 mM AG (an iNOS selective inhibitor). NOx concentration was determined in the culture media by nitrate reductase-Griess assay after 48 h. (<b>C</b>) control and 14 days-OVX pituitary cells were incubated with or without 0.1 mM L-NAME. Cell viability was determined by MTT assay. Results represent mean ± SE (N = 3) and were expressed as percentage of control. ANOVA followed by Tukey’s test, *p<0.05, **p<0.01 vs. respective control; ΔΔp<0.01 vs. 14 days-OVX rats.</p

Prolactin (PRL) down-regulates nNOS expression and NO production in anterior pituitary cells.

<p><b>(A, B)</b> Anterior pituitary cells from 14 days-OVX rats were stabilized for 24 h and incubated with or without 500 ng/mL PRL for 24, 48 or 72 h. (<b>A</b>) nNOS mRNA levels were determined by RT-PCR. <b>(B)</b>, NOx concentration was assayed by nitrate reductase-Griess assay. (<b>C</b>) Pituitary cell cultures from 14 days-OVX rats were incubated for 48 h with 20 μM Tyrphostin AG490 (Tyr), a Jak2 protein kinase inhibitor, 30 min before 1 nM E2 or 500 ng/mL PRL treatments. nNOS protein levels were determined by western blot (N = 3). Results represent media ± SE of densitometric values relative to β-actin or nitrite concentration (N = 3). ANOVA followed by Tukey’s test, *p<0.05, **p<0.01 vs. respective control; Δp<0.05 vs. Tyr.</p

E2 and NO pathway cross-talk in anterior pituitary cells.

<p>In anterior pituitary cells, nNOS-mediated NO production is primarily associated with cell death by apoptosis. On the other hand, E2 is the main stimulator of PRL secretion, and promotes lactotroph survival and proliferation. NO and E2 pathways reciprocally inhibit each other at multiple points. NO through cGMP inhibits PRL secretion. E2 inhibits nNOS through hypothalamic GnRH inhibition and further down-regulates nNOS either directly or indirectly at pituitary level, through PRL stimulation. In addition, E2 inhibits sGC activity and promotes an imbalance in its subunits’ expression which has been associated with other functions, not related to cGMP production.</p

sGC expression is augmented after OVX and mediates NO-induced pituitary cell death.

<p>Anterior pituitary primary cell cultures from control and 14-days OVX rats were stabilized for 24 h. <b>(A)</b> α1 and β1 protein levels were measured by western blot after 48 h. <b>(B)</b>, α2 and α2i mRNA levels were determined by RT-PCR. (<b>C, D</b>) Cells were incubated with or without 10 μM LY-83583 (a sGC inhibitor) for 48 h. (<b>C</b>) Cell viability was determined by MTT assay. (<b>D</b>) Active caspase-3 protein levels were measured by western blot. Results represent mean ± SE, (N = 3) of relative units corresponding to the protein or mRNA densitometric values relative to β-actin expressed as percentage of control. ANOVA followed by Tukey’s test, *p<0.05, **p<0.01 vs. control, ΔΔp<0.01 vs. 14 days-OVX rats.</p

E2 actions on sGC α1 and β1 mRNAs were mediated by nuclear ER after 6 h of incubation.

<p>Pituitary cells in culture were incubated with vehicle (control) or 1 nM E2 or with 1 nM E2-conjugated to BSA (E2-BSA) unable to trespass cell membrane during 6 h. sGC α1 and β1 mRNAs were evaluated by PCR. <i>Top</i>, a representative PCR. <i>Bottom,</i> average densitometric values. Bars represent mean ± SE of relative units, corresponding to α1 (open bars) and β1 (black bars) densitometric values normalized to β-actin, and are expressed as percent of the control (n = 3). ANOVA followed by Tukey's test, *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. respective controls.</p

Primers used for semi-quantitative RT-PCR assays.

<p>Primers used for semi-quantitative RT-PCR assays.</p

E2 decreased sGC α1 protein expression in a PI3k-dependent pathway involving non-nuclear ER but does not modify sGC β1 after 3 h of incubation.

<p>Pituitary cells in culture were incubated with vehicle (control) or 1 nM estrogen dendrimer conjugate (EDC), unable to trespass cellular membrane for 3 h with or without 50 µM LY294002 (LY), a PI3K inhibitor, 30 min before treatment. Protein expression was evaluated by western blot. (<b>A, B</b>) <i>Top</i>, representative western blots. <i>Bottom</i>, Corresponding average densitometric values. Bars represent mean ± SE of α1 (open bars) and β1 (black bars) protein densitometric values normalized to β-actin, as percent of control (n = 3). ANOVA followed by Tukey's test, *<i>P</i><0.05 vs. respective controls; ΔΔ<i>P</i><0.01 <i>vs</i>. E2; #<i>P</i><0.05 <i>vs</i>. EDC.</p

E2 actions on sGC α1 and β1 mRNAs depended on <i>de novo</i> translation.

<p>Pituitary cells in culture were incubated for 8 h with vehicle (control) or 1 nM E2 with or without 10 µg/mL cycloheximide (CHX), a translation inhibitor. mRNA expression was evaluated by semiquantitative PCR. <i>Top</i>, A representative PCR. <i>Bottom</i>, average densitomentric values. Bars represent mean ± SE of relative units corresponding to densitometric values of α1 (open bars) and β1 (black bars) normalized to β-actin, expressed as percent of control (n = 3). ANOVA followed by Tukey's test, **<i>P</i><0.01 <i>vs</i>. respective controls; Δ<i>P</i><0.05, ΔΔ<i>P</i><0.01 <i>vs</i>. E2.</p