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Cytological Characterization of a Pituitary Folliculo-Stellate-Like Cell Line, Tpit/F1, with Special Reference to Adenosine Triphosphate-Mediated Neuronal Nitric Oxide Synthase Expression and Nitric Oxide Secretion¹

Department of Regulation Biology, Faculty of Science, Saitama University (L.C., D.M., M.S., T.S., C.M., K.I.), Urawa 338-8570, Japan; Department of Physiology, Nippon Medical School (M.K.), Tokyo 113-8602, Japan; Department of Pathology, Tokai University School of Medicine (R.K.), Tokai 259-1193, Japan; Department of Anatomy, Wakayama Medical College (N.S.), Wakayama 640-8155, Japan; Department of Integrative Physiology, Graduate School of Medical Sciences, Kyushu University (A.T.), Fukuoka 812-8582, Japan; Department of Endocrinology, Max Planck Institute of Psychiatry (U.R.), Munich D-80804, Germany; and Department of Life Science, Meiji University (Y.K.), Kawasaki 214-8571, Japan

Address all correspondence and requests for reprints to: Kinji Inoue, Ph.D., Laboratory of Cell Biology, Department of Regulation Biology, Faculty of Science, Saitama University, 255 Shimo-ohkubo, Urawa, Saitama 338-8570, Japan. E-mail: mailto:kininoue{at}seitai.saitama-u.ac.jp

An immortal nonhormone-producing cell line with a characteristic star-shaped morphology, named Tpit/F1, was derived from an anterior pituitary gland of a temperature-sensitive large T antigen transgenic mouse. To characterize Tpit/F1 cells, we performed cytological studies, which revealed that Tpit/F1 cells express the messenger RNAs of neruonal nitric oxide (NO) synthase, S-100 protein, basic fibroblast growth factor, and pituitary-restricted transcription factor. The Tpit/F1 cells response to pituitary adenylate cyclase-activating peptide comprised the stimulated secretion of interleukin-6. Furthermore, glucocorticoids stimulate glutamine synthase production by Tpit/F1 cells. Considering these cytological characteristics together with their morphology, we deduced that Tpit/F1 cells are derived from pituitary folliculo-stellate (FS) cells.

Our cytophysiological analyses of Tpit/F1 cells revealed that intracellular Ca²⁺ increased dose dependently on ATP administration (0–100 µM), and that this effect did not require the presence of extracellular Ca²⁺ and was not abolished by treatment with gadolinium, a Ca²⁺ channel blocker. The ATP-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i) was completely abolished by treatment with the Ca²⁺-adenosine triphosphatase (Ca²⁺-ATPase) inhibitor thapsigargin, which suggests that ATP increases [Ca²⁺]_i by mobilizing internally stored Ca²⁺ followed by an influx of Ca²⁺. Moreover, UTP was equipotent with ATP in causing the [Ca²⁺]_i increase in Tpit/F1 cells. Also, the Ca²⁺ response was prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. From these results we therefore concluded that ATP acts on Tpit/F1 cells via P2Y₂-purinoceptors. Interestingly, both neuronal nitric oxide synthase messenger RNA and NO secretion were increased by ATP administration (10 and 100 µM). These results suggest the biological significance of the topological colocalization of FS cells and endocrine cells. Namely, ATP is cosecreted with hormones from endocrine cells and stimulates NO production by FS cells, and the released NO may regulate neighboring endocrine cell and blood vessels.

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