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 secretion

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.