E2 receptors (ERs) mediates cadmium effect on cyclins D1 and D3 and PRL mRNA expression.

<p>Anterior pituitary cells cultures were first incubated with 100 nM ICI 182,780 (ICI) for 20 min and then incubated with vehicle (control) or 10 nM Cd for 72 h. Cyc D1 and D3 (A) and PRL (B) mRNA expression was evaluated by PCR. Bars represent the mean ± SE of densitometric values normalized to GAPDH and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, **p<0.01 vs. control, ##p<0.01 vs. Cd (N=3).</p

Cadmium stimulates anterior pituitary lactotroph proliferation.

<p>Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2 for 96 h. Cell growth was determined by ICC measuring 24 h-BrdU incorporation. Lactotrophs were identified by prolactin-specific antibody and cell nuclei were stained by DAPI. Pictures are representative of three independent experiments performed in triplicate. Bars represent the mean ± SE of BrdU-labeling index expressed as positive BrdU lactrotroph / total lactotroph cell number x100. ANOVA followed by Tukey-Kramer’s test, **p<0.001 vs. control (N=3). </p

Cadmium exposure increases ERα

<p><b>protein expression in anterior pituitary cells</b>. Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2 for 24h. A representative western blot is shown. Bars represent the mean ± SE of densitometric values of full-length ERα (open bars) and ERα46 (black bars) normalized to β-actin expression and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, *p<0.05, **p<0.01, ***p<0.001 vs. respective control; #p<0.05, ###p<0.001 vs. Cd (N=3).</p

Cadmium increases cyclin D1 protein expression in anterior pituitary cells.

<p>Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2 for 72 h. A representative western blot is shown. Bars represent the mean ± SE of densitometric values normalized to β-actin expression and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, *p<0.05 vs. control (N=3).</p

Cadmium increases gene expression of proliferation markers in anterior pituitary cells.

<p>Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2. Gene expression was evaluated by PCR. Bars represent the mean ± SE of densitometric values of cyclins D1 and D3 after 72 h (A) or <i>c-fos</i> after 8-24 h (B) normalized to GAPDH expression and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, *p<0.05, **p<0.001 vs. control (N=3). </p

Additive effect of cadmium and E2 co-treatment on ERα mRNA expression.

<p>Anterior pituitary cell cultures were treated with vehicle (control), 10 nM Cd or 1 nM E2 or 10 nM Cd plus 1 nM E2 for 8 h or 24 h. ERα mRNA expression was evaluated by PCR. Bars represent the mean ± SE of densitometric values normalized to GAPDH. ANOVA followed by Tukey-Kramer’s test, *p<0.05, </p><p>** p<0.01, *** p<0.001 vs. Control; #p<0.05 vs. Cd; <b>^</b>p<0.05 vs. E2 (N=3).</p><p></p

Cadmium increased 23 kDa prolactin (PRL) protein expression in anterior pituitary cells in culture.

<p>Anterior pituitary cells were incubated with 10 nM Cd or vehicle (control) for 8 h. Protein expression was measured by western blot. Bars represent mean ± SEM of PRL densitometric values normalized to β-actin and are expressed as percent of control. **P<0.01, Student’s ‘t’ test (N=3).</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

Primers used for semi-quantitative RT-PCR assays.

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