Direct Evidence of Gonadotropin-releasing Hormone (GnRH)-stimulated Nitric Oxide Production in the L Beta T-2 Clonal Gonadotropes

An immortal cell line (L beta T-2) with characteristics of gonadotropes, such as LH-containing secretory granules, and LH release responsiveness to GnRH, was used to investigate the effect of GnRH stimulation on nitric oxide (NO) production. RT-PCR analysis showed that mouse nNOS mRNA was expressed in cultured L beta T-2 cells. L beta T-2 cells were treated with the calcium ionophore, A23187, and NO levels were measured as nitrite using the Griess assay. The data clearly showed that NO production was increased dose-dependently by A23187 treatment (0-10(-5) M). Next, changes in the intracellular concentration of ionized calcium ([Ca2+]i) in L beta T-2 cells induced by GnRH were analyzed by quantitative fluorescence microscopy, and [Ca2+]i was found to be increased markedly by GnRH treatment. In fact, exposure of L beta T-2 cells to increasing concentrations of GnRH from 0 to 10(-6) M was found to enhance NO production in a dose dependent manner, with maximal augmentation at 10(-6) M. However, the stimulation of NO production by GnRH at this concentration was significantly attenuated by pretreatment with NG-nitro-L-arginine methyl ester hydrochloride (L-NAME), a NO synthase inhibitor. Taken together, the present results suggest that GnRH treatment results in increased NO production in L beta T-2 clonal gonadotropes, and intracellular calcium augmentation produced by GnRH may be participate in this process. Our findings also indicate that the L beta T-2 cell line is a useful tool for in vitro studies of the autocrine and paracrine roles of NO in the anterior pituitary gland.