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.
Chen
L
L
Department of Cell Biology, Faculty of Science, Saitama University, Urawa, Japan.
Sakai
T
T
Sakamoto
S
S
Kato
M
M
Inoue
K
K
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics; D052248:Nitric Oxide Synthase Type I; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D007476:Ionophores; D012333:RNA, Messenger; D009569:Nitric Oxide; D007987:Gonadotropin-Releasing Hormone; D000001:Calcimycin; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester