Participation of Guanylate cyclase, PKCe; and ERK 1/2 in estradiol effects mediated by membrane estrogen receptor in lactotroph cells

GUTIÉRREZ, S; DE PAUL, AL; PETITI, JP; SOSA, LV; PALMERI, CM; SOAJE, M; MASINI-REPISO A; TORRES, AI

Rio de Janeiro-Brazil

Congreso; 13th International Congress of Endocrinology-ICE 2008; 2008

International Society of Endocrinology and Brazilian Society of Endocrinology and Metabology.

The aim of the present work was to investigate the Guanylate cyclase (GC), protein kinase C (PKC) e and extracellular-signal regulated kinase (ERK) 1/2 participation in estradiol (E2) effects on prolactin secretion and lactotroph proliferation, and to identify membrane estrogen receptors (ER) in lactotroph cells. Primary pituitary cell cultures from female rats were treated with E2, E2 conjugated to bovine serum albumin (E2?BSA) and insulin (In), alone or combined, for 15, 30, 60min and 24h. The cultured cells were processed for PRL secretion by RIA, quantification of lactotroph proliferation by BrDU/PRL and protein detection by western blot (PKCe, ERK1/2 phosphorylated (P) and total, GCa1, b1 and ß-actin). Membrane ERs were immunodetected with specific antibody against classic nuclear ERa and b. Statistical analysis: ANOVA-Tukey. In serum free conditions, E2 and E2-BSA promoted a differential secretory activity on PRL cells but were unable to induce lactotroph cell proliferation. However, both free and conjugated E2 were competent arresting the mitogenic activity promoted by Ins. E2, E2-BSA and Ins induced opposite effects on GCa1 and b1 expression, increasing the GCa1 and inhibing GCb1, while the E2/Ins or E2-BSA/Ins co-incubation induced an augmentation of two GC isoforms. E2, E2-BSA and Ins stimuli increased the PKCe and P-ERK1/2 expression, although combined treatments with E2/Ins or E2-BSA/Ins induced a significant reduction in these levels, in close correlation with the decrease of lactotroph cell proliferation. Pre-treatment with bisindolmaleymide I (PKC inhibitor) significantly inhibited ERK1/2 activation promoted by Ins, without modifying P-ERK1/2 expression levels induced by E2 or E2-BSA. By immuno-electron-microscopy, ERa was localized on the cell-surface of lactotrophs. No immunostaining for ERb was detected. These findings show the presence of ERa in the plasma membrane of lactotroph cells which acts modulating PRL secretion and lactotroph proliferation through GC, PKCe and ERK1/2.