17β-oestradiol acts as a negative modulator of insulin-induced lactotroph cell proliferation through oestrogen receptor α, via nitric oxide/guanylyl cyclase/cGMP

Abstract

Objectives: 17β-oestradiol interacts with growth factors to modulate lactotroph cell population. However, contribution of isoforms of the oestrogen receptor in these activities is not fully understood. In the present study, we have established participation of α and β oestrogen receptors in effects of 17β-oestradiol on lactotroph proliferation induced by insulin and shown involvement of the NO/sGC/cGMP pathway.

Materials and methods: Cell cultures were prepared from anterior pituitaries of female rats to evaluate lactotroph cell proliferation using bromodeoxyuridine (BrdUrd) detection, protein expression by western blotting and cGMP by enzyme immunoassay.

Results: In serum-free conditions, 17β-oestradiol and α and β oestrogen receptor agonists (PPT and DPN) failed to increase numbers of lactotroph cells undergoing mitosis. Co-incubation of 17β-oestradiol/insulin and PPT/insulin significantly decreased lactotroph mitogenic activity promoted by insulin alone. Both ICI 182780 and NOS inhibitors (L-NMMA and L-NAME) induced reversal of the anti-proliferative effect promoted by 17β-oestradiol/insulin and PPT/insulin. Moreover, 17β-oestradiol, PPT and insulin increased sGC α1 protein expression and inhibited β1, whereas co-incubation of 17β-oestradiol/insulin or PPT/insulin induced increases of the two isoforms α1 and β1. 17β-oestradiol and insulin reduced cGMP production, while 17β-oestradiol/insulin co-incubation increased this cyclic nucleotide.

Conclusions: Our results suggest that 17β-oestradiol is capable of arresting lactotroph proliferation induced by insulin through ER α with participation of the signalling NO/sGC/cGMP pathway.

Figure 1

 Effects of 17β‐oestradiol (E; 1 nm), PPT and DPN on lactotroph proliferation. None of the concentrations tested increased numbers of BrdUrd‐labelled lactotrophs. The ANOVA‐Tukey test was performed on three independent experiments. Data are expressed as mean ± SEM of triplicate determinations from a representative experiment.

Figure 2

 17β‐oestradiol (E; 1 nm) and all doses tested of α agonist, PPT, reversed mitogenic activity induced by insulin (1000 ng/ml) on lactotrophs. DPN failed to induce any anti‐mitogenic effect. The ANOVA‐Tukey test was performed on three independent experiments. *P < 0.001 versus control, **P < 0.001 versus insulin. Data are expressed as the mean ± SEM of triplicate determinations from a representative experiment.

Figure 3

 Effects of ICI 182780 (100 nm) on lactotroph cell proliferation. This inhibitor reversed anti‐proliferative effects promoted by 17β‐oestradiol (1 nm) or PPT (1 nm) on lactotroph proliferation induced by insulin (1000 ng/ml). The ANOVA‐Tukey test was performed on three independent experiments. *P < 0.001 versus control, **P < 0.001 versus insulin, •P < 0.01 versus 17β‐oestradiol/insulin or PPT/insulin. Data are expressed as mean ± SEM of triplicate determinations from a representative experiment.

Figure 4

 Effects of NOS inhibitors on lactotroph proliferation induced by 17β‐oestradiol (1 nm) and insulin (1000 ng/ml). Inhibition of NOS activity by L‐NMMA (0.1 mm) and L‐NAME (1 mm) reversed anti‐proliferative effects induced by 17β‐oestradiol/insulin. The ANOVA‐Tukey test was performed on three independent experiments. *P < 0.001 versus control, **P < 0.001 versus insulin and •P < 0.01 versus 17β‐oestradiol/insulin. Data are expressed as mean ± SEM of triplicate determinations from a representative experiment.

Figure 5

 Effects of L‐NMMA (0.1 mm) and L‐NAME (1 mm) on lactotroph proliferation induced by 17β‐oestradiol agonists and insulin. Both inhibitors reversed only anti‐proliferative effects exerted by PPT/insulin. The ANOVA‐Tukey test was performed on three independent experiments. *P < 0.001 versus control, **P < 0.01 versus insulin, •P < 0.01 versus PPT/insulin. Data expressed as mean ± SEM of triplicate determinations from a representative experiment.

Figure 6

 sGC α1 and β1 expression in total pituitary extract from cell cultures stimulated with 17β‐oestradiol (E; 1 nm) or insulin (I; 1000 ng/ml) alone or as co‐incubations. Both E or I increased protein expression of sGC α1 and decreased β1. In contrast, E + I induced increases in both isoforms. Bands correspond to representative experiment from a total of three with similar results. The ANOVA‐Tukey test was performed on three independent cell cultures: *P < 0.01 versus control. Data shown as mean ± SEM of triplicate determinations from a representative experiment.

Figure 7

 sGC α1 and β1 expression in total pituitary extract from cell cultures stimulated with PPT (1 nm) or insulin (I; 1000 ng/ml) alone or co‐incubations. Bands correspond to a representative experiment from a total of three which had similar results. The ANOVA‐Tukey test was performed on three independent cell cultures: *P < 0.001 versus control. Data are shown as the mean ± SEM of triplicate determinations from a representative experiment.

Figure 8

 ICI 182780 (100 nm) blocked effects promoted by 17β‐oestradiol (1 nm) or PPT (1 nm) on sGC α1 and β1 expression. Bands correspond to a representative experiment from a total of three which had similar results. The ANOVA‐Tukey test was performed on three independent cell cultures: *P < 0.01 versus control, **P < 0.01 versus 17β‐oestradiol, •P < 0.01 versus PPT. Data are shown as the mean ± SEM of triplicate determinations from a representative experiment.