Gonadotropin-releasing hormone activates the 12-lipoxygenase pathway in the LβT2 gonadotrope cell line

Previous studies have indicated that arachidonic acid and its lipoxygenase (LO) metabolites play a role in the post-receptor effects of gonadotropin-releasing hormone (GnRH) but the exact role and nature of these putative eicosanoids remain unclear. The potential role of arachidonic acid and LO in GnRH receptor-mediated signaling was investigated in the LβT2 gonadotrope cell line, which expresses gonadotropins (LH and FSH) and GnRH-receptor mRNAs. Western immunobloting of LβT2 cell extracts, performed with a murine leukocyte polyclonal antibody against 12-LO, showed a 70-kD band, suggesting the presence of 12-LO protein in these cells. GnRH nearly doubled the release of 12-hydroeico-satetraenoic acid, a product of the 12-LO enzyme, within 10 min. A specific reverse transcriptase polymerase chain reaction with a set of primers based on the reported sequence of rat brain 12-LO yielded a 170-bp band which showed 100% homology with the expected rat brain 12-LO sequence. Exposure of LβT2 cells to pulsatile GnRH treatment (10 nM, 90-min interpulse, one and three pulses) led to a ∼3-fold increase in 12-LO mRNA levels. In conclusion, we provide evidence for the presence of a 12-LO enzyme in LβT2 cells, the expression and activity of which are increased by short-term/pulsatile exposure to GnRH. LβT2 cells represent a potential model to further study the involvement of 12-LO in GnRH receptor signaling.