Dopamine-Induced Apoptosis of Lactotropes Is Mediated by the Short Isoform of D2 Receptor

Dopamine, through D2 receptor (D2R), is the major regulator of lactotrope function in the anterior pituitary gland. Both D2R isoforms, long (D2L) and short (D2S), are expressed in lactotropes. Although both isoforms can transduce dopamine signal, they differ in the mechanism that leads to cell response. The administration of D2R agonists, such as cabergoline, is the main pharmacological treatment for prolactinomas, but resistance to these drugs exists, which has been associated with alterations in D2R expression. We previously reported that dopamine and cabergoline induce apoptosis of lactotropes in primary culture in an estrogen-dependent manner. In this study we used an in vivo model to confirm the permissive action of estradiol in the apoptosis of anterior pituitary cells induced by D2R agonists. Administration of cabergoline to female rats induced apoptosis, measured by Annexin-V staining, in anterior pituitary gland from estradiol-treated rats but not from ovariectomized rats. To evaluate the participation of D2R isoforms in the apoptosis induced by dopamine we used lactotrope-derived PR1 cells stably transfected with expression vectors encoding D2L or D2S receptors. In the presence of estradiol, dopamine induced apoptosis, determined by ELISA and TUNEL assay, only in PR1-D2S cells. To study the role of p38 MAPK in apoptosis induced by D2R activation, anterior pituitary cells from primary culture or PR1-D2S were incubated with an inhibitor of the p38 MAPK pathway (SB203850). SB203580 blocked the apoptotic effect of D2R activation in lactotropes from primary cultures and PR1-D2S cells. Dopamine also induced p38 MAPK phosphorylation, determined by western blot, in PR1-D2S cells and estradiol enhanced this effect. These data suggest that, in the presence of estradiol, D2R agonists induce apoptosis of lactotropes by their interaction with D2S receptors and that p38 MAPK is involved in this process

DA increases apoptosis in PR1-D2S cells incubated in the presence of E2.

<p>PR1-V, PR1-D2L or PR1-D2S cells were cultured with DMEM-F12-SS in the absence (A) or presence of E2 (1 nM, B) for 48 h. Then the cells were incubated with DA (1 µM-100 µM) in the presence or absence of E2 for 4 h and apoptosis was detected by ELISA. Each column represents the mean ± SE expressed of OD as the percentage of control without DA (n = 5 wells/group). Data were analyzed by ANOVA followed by Dunnett's test. * p<0.05 vs control without DA.</p

A p38 MAPK inhibitor blocks the apoptosis of anterior pituitary cells induced by D2R activation.

<p>Anterior pituitary cells were cultured with E2 (1 nM) for 48 h. Then the cells were preincubated with SB203580 (1 µM), a p38 MAPK inhibitor, for 30 min and then incubated with DA (1 µM, A and B) or CAB (1 µM, C and D) in the presence or absence of SB203580 for 4 h. Each column represents the percentage of TUNEL-positive cells ± CI (>2,000 cells/group, A, C) or the percentage of TUNEL positive lactotropes ± CI (>500 cells/group B, D). Data from at least two separate experiments were analyzed by χ². ** p<0.01 vs respective control without DA or CAB. ∧ p<0.05 vs respective control without SB203589.</p

p38 MAPK is involved in DA-induced apoptosis of PR1-D2S cells.

<p>A: PR1-D2S cells were cultured with E2 (1 nM) for 48 h. Then the cells were preincubated with SB203580 (1 µM), a p38 MAPK inhibitor, for 30 min and then incubated with DA (1 µM) in the presence or absence of SB203580 for 4 h. Each column represents the percentage of TUNEL-positive cells ± CI (>2,000 cells/group). Data from at least two separate experiments were analyzed by χ². ** p<0.01 vs respective control without DA. ∧∧ p<0.01 vs respective control without SB203580. B: PR1-D2S cells were cultured with or without E2 (1 nM) for 48 h. Then the cells were incubated with DA (1 µM) in the presence of E2 or VEH for 15 min, 30 min or 4 h. Proteins were extracted and p38 MAPK and phospho-p38 MAPK were detected by western blot.</p

CAB induces apoptosis of anterior pituitary cells in an E2-dependent manner.

<p>OVX and E2 treated rats were injected with CAB (1 mg/kg, ip) or vehicle (CONTROL) and euthanized 16 h later. Anterior pituitary cells were dispersed and apoptosis was detected by Annexin-V and flow cytometry. Each column represents the mean ± SE of the percentage of apoptotic cells (n = 8 rats/group). Data were analyzed by two-way ANOVA, followed by Tuckey's test. *p<0.05 vs. respective control without CAB, ∧p<0.05 vs respective control without E2.</p

DA increases the percentage of apoptotic PR1-D2S cells incubated in the presence of E2.

<p>PR1-D2S cells were cultured with DMEM-F12-SS in the absence (VEH) or presence of E2 (1 nM) for 48 h. Then the cells were incubated with DA (1 µM) with or without E2 for 4 h. Each column represents the percentage of TUNEL-positive cells ± CI (>2,000 cells/group). Data from at least two separate experiments were analyzed by χ². ** p<0.01 vs respective control without DA. ∧∧ p<0.01 vs respective control without E2.</p