Nitric Oxide Plays a Key Role in Ovariectomy-Induced Apoptosis in Anterior Pituitary: Interplay between Nitric Oxide Pathway and Estrogen.

Changes in the estrogenic status produce deep changes in pituitary physiology, mainly because estrogens (E2) are one of the main regulators of pituitary cell population. Also, E2 negatively regulate pituitary neuronal nitric oxide synthase (nNOS) activity and expression and may thereby modulate the production of nitric oxide (NO), an important regulator of cell death and survival. Little is known about how ovary ablation affects anterior pituitary cell remodelling and molecular mechanisms that regulate this process have not yet been elucidated. In this work we used freshly dispersed anterior pituitaries as well as cell cultures from ovariectomized female rats in order to study whether E2 deficiency induces apoptosis in the anterior pituitary cells, the role of NO in this process and effects of E2 on the NO pathway. Our results showed that cell activity gradually decreases after ovariectomy (OVX) as a consequence of cell death, which is completely prevented by a pan-caspase inhibitor. Furthermore, there is an increase of fragmented nuclei and DNA cleavage thereby presenting the first direct evidence of the existence of apoptosis in the anterior pituitary gland after OVX. NO production and soluble guanylyl cyclase (sGC) expression in anterior pituitary cells increased concomitantly to the apoptosis. Inhibition of both, NO synthase (NOS) and sGC activities prevented the drop of cell viability after OVX, showing for the first time that increased NO levels and sGC activity observed post-OVX play a key role in the induction of apoptosis. Conversely, E2 and prolactin treatments decreased nNOS expression and activity in pituitary cells from OVX rats in a time- and E2 receptor-dependent manner, thus suggesting interplay between NO and E2 pathways in anterior pituitary

Primers used for semi-quantitative RT-PCR assays.

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

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