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Direct Evidence of Gonadotropin-Releasing Hormone (GnRH)-Stimulated Nitric Oxide Production in the LβT-2 Clonal Gonadotropes

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Abstract

An immortal cell line (LβT-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 LβT-2 cells. LβT-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 LβT-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 LβT-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 LβT-2 clonal gonadotropes, and intracellular calcium augmentation produced by GnRH may be participate in this process. Our findings also indicate that the LβT-2 cell line is a useful tool for in vitro studies of the autocrine and paracrine roles of NO in the anterior pituitary gland.

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Authors and Affiliations

  1. Department of Cell Biology, Faculty of Science, Saitama University, Urawa, Japan

    Lan Chen, Takafumi Sakai, Shinya Sakamoto & Kinji Inoue

  2. Department of Physiology I, Nippon Medical School, Tokyo, Japan

    Masakatsu Kato

Authors
  1. Lan Chen
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  2. Takafumi Sakai
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  3. Shinya Sakamoto
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  4. Masakatsu Kato
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  5. Kinji Inoue
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Chen, L., Sakai, T., Sakamoto, S. et al. Direct Evidence of Gonadotropin-Releasing Hormone (GnRH)-Stimulated Nitric Oxide Production in the LβT-2 Clonal Gonadotropes. Pituitary 2, 191–196 (1999). https://doi.org/10.1023/A:1009988808338

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