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Endocrinology Vol. 141, No. 10 3603-3610
Copyright © 2000 by The Endocrine Society

ARTICLES

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

Lan Chen, Daisuke Maruyama, Masahide Sugiyama, Takafumi Sakai, Chihiro Mogi, Masakatsu Kato, Reiko Kurotani, Nobuyuki Shirasawa, Atsushi Takaki, Ulrich Renner, Yukio Kato and Kinji Inoue

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS NOW well established that anterior pituitary function is controlled not only by hypothalamic factors but also by local regulatory systems. Cell to cell communication in the anterior pituitary gland through autocrine or paracrine factors is of fundamental importance for the systematic regulation of this organ (1). It has become evident that folliculo-stellate (FS) cells may contribute to intrapituitary communication and modulate the response of endocrine cells to hypophysiotropic factors (2, 3).

FS cells are agranular cells characterized by a star-shaped structure and envelop endocrine cells with their long cellular processes (4). This cell type is known to synthesize other peptides, such as interleukin-6 (IL-6) (5), vascular endothelial cell growth factor (6), basic fibroblast growth factor (bFGF) (7), glutamine synthase (GS) (8), and neuronal nitric oxide synthase (nNOS) (9), and to express the receptors for pituitary adenylate cyclase-activating peptide (PACAP) (10). Additionally, it was recently demonstrated that pituitary FS cells possess metabotropic ATP receptors (P2Y2), which act through G protein-mediated activation of phospholipase C (PLC), inositol trisphosphate production, and Ca2+ release from intracellular Ca2+ stores (11).

In contrast to hormone-producing cells, FS cells seem to be multifunctional (4). To reveal the functions of FS cells, studies involving pure FS cells are needed. The TtT/GF cell line established in our laboratory is a good model for FS cells (12); however, some of its functions, such as nNOS production, differ from those of normal pituitary FS cells. This difference may be caused by the diversity of FS cells in the anterior pituitary gland or by the fact that TtT/GF cells lose these characteristics during prolonged cultivation. To clarify all functions of FS cells, especially NO production and its biological significance in the anterior pituitary gland, we attempted to establish another type of FS cell line that expresses nNOS messenger RNA (mRNA).

On the other hand, we recently established a new immortal cell line, named Tpit/F1, which was derived from anterior pituitary cells of a temperature-sensitive large T antigen transgenic mouse. Tpit/F1 cells were originally separated from cells that were closely associated with endothelial cells. Our preliminary experiment indicated that Tpit/F1 cells are a nonhormone-producing cell type and have long cell processes. RT-PCR experiments also showed that this cell type is positive for the mRNAs of nNOS, S-100 protein, and bFGF, so we speculated that this cell line may be a novel FS cell line. For the characterization of Tpit/F1 cells, we here performed a cytophysiological study on Tpit/F1 cells, cytologically characterizing them as a new FS cell line. The possible role of ATP in NO secretion via an ATP-induced increase in intracellular Ca2+ ([Ca2+]i) in FS cells is also discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of Tpit/F1 cells
The Tpit/F1 cell line was derived from an anterior pituitary gland of a temperature-sensitive large T antigen transgenic mouse. The methods employed here were described previously (13). Briefly, tissue was obtained from a T antigen transgenic mouse and cultivated for about 1 yr at 33 C. After prolonged cultivation at 33 C, three independent cell clones were obtained. One of them, called Tpit/E, was characterized as an endothelial cell line. The other two cell lines were derived from cells closely associated with endothelial cells with a characteristic star-shaped morphology and expressed messages for bFGF and S-100 protein. These two cell lines were designated Tpit/F1 and Tpit/F2. Because these two cell lines showed similar characteristics, we only used Tpit/F1 cells for further studies.

Cell culture
Tpit/F1 cells were cultured in medium comprising a mixture of half DMEM (Life Technologies, Inc., Grand Island, NY) and half Ham’s F-12 (Life Technologies, Inc.), which was supplemented with 10% (vol/vol) normal horse serum (Nichimen American, Los Angeles, CA) and 2.5% (vol/vol) FBS (BioWhittaker, Inc., Walkersville, ML). The cells were cultured at 33 C under a humidified atmosphere of 5% CO2 in air.

Electron microscopic observation
The method for electron microscopic observation was described previously (12). Briefly, after removing the medium, the cultured cells were prefixed with 2.5% glutaraldehyde and 1% OsO4.

The cells were then dehydrated with ethanol and embedded in a resin according to a routine method. After heat polymerization of the resin, ultrathin sections were prepared and observed under an electron microscope.

Measurement of IL-6 production by Tpit/F1cells
When the culture was nearly confluent, the initial culture medium was removed, and the cells were washed twice with PBS. Then medium supplemented with 1% FCS was added to the cells together with different concentrations of PACAP-38 (Bachem, Bubendorf, Switzerland). After 24 h, supernatants were collected, centrifuged, and assayed for IL-6. To study the effect of glucocorticoids on the PACAP-mediated IL-6 production by Tpit/F1 cells, 10 nM dexamethasone (Sigma, St. Louis, MO) and 100 nM glucocorticoid receptor antagonist RU 486 (Roussel, Romainville, France) were used.

The amount of IL-6 in the culture medium was determined with a highly specific and sensitive ELISA kit (R&D Systems, Minneapolis, MN). The detection limit of the assay was 3 pg/ml IL-6.

GS assay
Tpit/F1 cells were incubated at 35 C for 18 h with or without 100 nM dexamethasone, and then after washing with PBS three times, the medium was replaced with 10 mM sodium phosphate buffer, pH 7.2, containing 0.1% Nonidet P-40, and the cells were stored at -80 C until GS activity was assayed. GS activity in the supernatants was measured spectrophotometrically by means of the glutamine-r-glutamyl transfer assay described by Miller et al. (14) with minor modifications. The final GS assay solution comprised 100 mM L-glutamine, 50 mM imidazole-HCl (pH 6.8), 0.5 mM manganase chloride, 50 mM hydroxylamine-HCl, 25 mM potassium arsenate, and 0.2 mM disodium ADP. Sixty-minute incubations were performed at 37 C. Each reaction was terminated by the addition of 1.0 ml 0.37 M FeCl3, 0.3 M trichloroacetic acid, and 0.6 M HCl at 4 C for 30 min. The supernatants were harvested after centrifugation at 10,000 rpm for 15 min, and r-glutamyl hydroxamate was measured at 505 nM. One unit of GS activity was defined as the formation of 1 mmol r-glutamyl hydroxamate in 15 min under the assay conditions used.

[Ca2+]i measurement
The method employed here was described in detail previously (15). Dispersed Tpit/F1 cells plated on poly-D-lysine (Sigma)-coated coverslips were incubated with 2 µM fura-PE3/AM (Wako Pure Chemicals, Osaka, Japan) at 37 C for 90 min in a perifusion medium (composed of 137.5 mM NaCl, 5.0 mMKCl, 2.5 mMCaCl2, 0.8 mM MgCl2, 10.0 mM glucose, 20.0 mM HEPES, 0.6 mM NaHCO3, and 0.1% BSA, pH 7.4) and then placed in a flow-through chamber mounted on the stage of a microscope. Recording was performed at room temperature (22–25 C). A Quanticell 700 system (Applied Imagine, Sunderland, UK) was employed for all dynamic video imaging and image processing. Excitation wavelengths (340 and 380 nm) were produced by means of a computer-controlled rotating filter wheel between a xenon lamp and the microscope. The emission light at 510 nm was passed to an image-intensifying charge-coupled device camera (Photonics Science, Tunbridge Wells, UK). The resulting image at each wavelength was averaged, digitized, captured, and stored. The time resolution was set at 6 sec between ratio frames. The ratio of emitted fluorescence at the two excitation wavelengths (340 and 380 nm) was converted to the Ca2+ concentration according to the method of Grynkiewicz et al. (16).

Drug applications
ATP and UTP were obtained from Sigma. The PLC inhibitor 1-(6-[17ß-3-methoxyestra-1,3,5-(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5,dione (U-73122), its inactive analog 1-(6-[17ß-3-methoxyestra-1,3,5-(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73343), and a potent and selective inhibitor of sarcoplasmic and endoplasmic reticulum Ca2+-ATPases, thapsigargin, were purchased from Wako Pure Chemicals (Osaka, Japan). The calcium channel blocker gadolinium (Gd3+) was obtained from Nacalai Tesque (Kyoto, Japan).

RT-PCR method
The following primers were designed for the detection of mouse nNOS complementary DNAs (cDNAs): sense primer, 5'-AGCAACCTACCAGCTCAAGGA-3'; and antisense primer, 5'-AATAGTGATGGCCGACCTGAG-3'. The fragment size was 209 bp. Pituitary-restricted transcription factor (Ptx1) primers were designed based on the sequences of the mouse Ptx1 cDNAs: sense primer, 5'-CCGTGAACTGAATGTAGGGAA-3'; and antisense primer, 5'-AGAGCTGAGCCCTTCTCCTC-3'. The PCR product of the Ptx1 primers was 298 bp.

The initial template denaturation was conducted for 10 min at 94 C. The cycle profile was as follows: 1 min at 94 C (denaturation) and 1 min at 62 C (nNOS) or 55 C (Ptx1; annealing and extension). Forty (nNOS) and 45 (Ptx1) cycles of the profile were run, and the final extension step was increased to 10 min. To examine the specificity, the 298-bp Ptx1 RT-PCR product was digested at 37 C for 2 h with restriction enzyme MspI. Analysis of the RT-PCR products and restriction fragments was performed by agarose gel electrophoresis. The specificity of the amplified cDNA fragments of nNOS was also verified by digestion with a restriction enzyme (SacI). Templates obtained from the pituitary gland and L929 mouse fibroblastic cell line were used as positive and negative controls, respectively.

Measurement of nitrite production
NO was measured as nitrite according to the method described by Yamada et al. (17). In brief, NO3- in the Tpit/F1 cell culture medium was reduced to NO2-, and then the NO2- was mixed with Griess reagent. The absorbance of the color of the product dye at 540 nm was measured with a flow-through spectrophotometer (NOD-10, Eicom, Kyoto, Japan). The Griess reagent comprised 1.25% HCl containing 5 g/L sulfanilamide and 0.25 g/liter N-naphthylethylenediamine.

Statistics
Each of the experiments was repeated at least three times. The data for different groups were compared using Fisher’s protected least significant difference test. P < 0.05 was considered statistically significant. The data are expressed as the mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
General morphology of Tpit/F1 cells
The Tpit/F1 cells had a flat, star-like appearance, with many cytoplasmic processes (Fig. 1AGo). As shown in Fig. 1BGo, electron microscopic observation revealed that the cytoplasmic organelles of Tpit/F1 cells are poorly developed. This morphological appearance is similar to that of FS cells in the pituitary gland.



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  		Figure 1. Light (A) and electron (B) microscopic appearances of Tpit/F1 cells. The Tpit/F1 cells have long cytoplasmic process (arrow). The cytoplasmic organelles are poorly developed (B). N, Nucleus; arrowhead, endoplasmic reticulum. Magnification: A, x280; B, x20K.

 
Ptx1 gene expression by Tpit/F1 cells
The RT-PCR experiment results clearly showed that Tpit/F1 cells expressed Ptx1 mRNA (Fig. 2Go). The specificity of the amplified cDNA fragments of Ptx1 was successfully verified by restriction digestion with MspI. Three expected restriction fragments (49, 110, and 139 bp) were detected. The Tpit/F1 cells also expressed S-100 protein and bFGF; however, they did not express any known anterior pituitary hormones (data not shown).



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  		Figure 2. Ptx1 gene expression by Tpit/F1 cells, as determined by RT-PCR. A, The expected PCR product of 298 bp and the expected MspI-generated restriction fragment (49, 110, and 139 bp) were detected. The ß-actin PCR product was detected at 105 bp. B, Positive and negative controls for RT-PCR are shown. Both the pituitary gland (P) and Tpit/F1 cells (F) are positive, but L929, a mouse fibroblast cell line (L), is negative.

 
Effects of dexamethasone on GS activity and of PACAP-38 on IL-6 production by Tpit/F1 cells
It is known that the GS activity of FS cells in the anterior pituitary increases with glucocorticoid stimulation. The similarity of Tpit/F1 cells to FS cells was demonstrated in the present study. Namely, the GS activity of Tpit/F1 cells markedly increased in the presence of dexamethasone (100 nM; Fig. 3Go).



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  		Figure 3. Effect of dexamethasone on the GS activity of Tpit/F1 cells. Dexamethasone administration significantly increased the GS activity of Tpit/F1 cells. Bars represent the mean ± SE (n = 4) for both the dexamethasone-treated and untreated groups. *, P < 0.01 vs. the control group.

 
PACAP-38 clearly induced IL-6 production by Tpit/F1 cells in a dose-dependent manner at concentrations ranging from 1–1000 nM. Dexamethasone blocked PACAP-stimulated (100 nM) IL-6 production, and the glucocorticoid receptor antagonist RU 486 reversed the inhibitory effect of dexamethasone (Fig. 4Go).



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  		Figure 4. Dose-dependent stimulation of IL-6 production by PACAP-38 and blockade of the PACAP-38 effect by dexamethasone in Tpit/F1 cells. The IL-6 production response is dose related. Dexamethasone blocked PACAP-stimulated (100 nM) IL-6 production, and a glucocorticoid receptor antagonist (RU486) reversed this inhibitory effect (n = 3 for each concentration of PACAP-38). Bars, Mean ± SE. *, P < 0.05 vs. control group; **, P < 0.01 vs. control group; #, P < 0.01 vs. PACAP-38 (100 nM); ##, P < 0.05 vs. PACAP-38 (100 nM)/dexamethasone (Dex).

 
ATP increases [Ca2+]i in Tpit/F1 cells
ATP (0–100 µM) was applied to cells for 2–3 min. In each group approximately 50% of the cells showed an increase in [Ca2+]i when challenged with 1 µM ATP. The proportion of cells responding to ATP increased to 79–94% with higher ATP concentrations (10 or 100 µM), but none of the cells responded to 0.1 µM ATP. The responses to ATP are summarized in Fig. 5Go. The effect of ATP on [Ca2+]i was concentration dependent.



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  		Figure 5. ATP-evoked increase in [Ca2+]i of Tpit/F1 cells. Data are expressed as percentages of those in the case of cells exhibiting a measurable increase in [Ca2+]i. The data shown are the mean ± SE of independent experiments with different ATP concentrations, as follows: 0 µM ATP, mean of three experiments, n = 4 cells/experiment (results for three independent experiments, four single cells were examined in each independent experiment); 0.1 µM ATP, three experiments, n = 4 cells/experiment; 1 µM ATP, five experiments, n = 7–34 cells/experiment; 10 µM ATP, 22 experiments, n = 3–34 cells/experiment; 100 µM ATP, 4 experiments, n = 5–15 cells/experiment. *, P < 0.05; **, P < 0.001 (vs. 0 µM ATP group).

 
ATP evoked Ca2+ release from intracellular stores in Tpit/F1 cells
To investigate the relative contributions of Ca2+ entry and Ca2+ release from intracellular stores to the ATP-induced [Ca2+]i increase, Ca2+-free medium was used. Ca2+-free medium was introduced 2 min before the application of 10 µM ATP and was continuously perifused until the end of the recording. The initial transient increase in [Ca2+]i caused by ATP was not affected by the Ca2+-free medium, whereas the late sustained phase was completely suppressed (Fig. 6Go, A and B). A Ca2+ channel blocker, Gd3+ (100 µM), exerted a similar effect on the response to ATP (Fig. 6CGo). These data show that extracellular ATP evoked a rapid increase in [Ca2+]i in Tpit/F1 cells, and that this effect did not require the presence of extracellular Ca2+ and was not abolished by pretreatment with gadolinium, indicating that it represents the mobilization of intracellularly stored Ca2+. The percentage of sensitive cells in each condition is presented in Fig. 8Go. Furthermore, when the intracellular Ca2+ stores had been depleted after 50 min perfusion of thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, the [Ca2+]i increase induced by subsequent application of 10 µM ATP was not observed (Fig. 7Go). No increase in [Ca2+]i in Tpit/F1 cells caused by ATP was detected in any experiment involving pretreatment with thapsigargin (Fig. 8Go).



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  		Figure 6. A, A typical response of Tpit/F1 cells to 10 µM ATP. [Ca2+]i abruptly increased, remained at a high concentration, and then gradually decreased to the basal level. B, A typical response of Tpit/F1 cells to 10 µM ATP in Ca2+-free perifusion medium. [Ca2+]i abruptly increased followed by a fast decrease to the basal level. C, A typical response of Tpit/F1 cells to 10 µM ATP after Gd3+ pretreatment. A similar calcium response as that in B was observed. The ATP (10 µM) treatment time is indicated by the stippled bar.

 


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  		Figure 8. Comparison of the [Ca2+]i increases in Tpit/F1 cells treated with ATP and UTP under different conditions. The bar chart in Fig. 4Go shows the percentages of cells exhibiting a measurable increase in [Ca2+]i under different conditions (percentage of responding cells). The data shown are the mean ± SE of independent experiments under each set of conditions, respectively. 10 µM ATP, 22 experiments, n = 3–34 cells/experiment (results for 22 independent experiments, 3–34 single cells were examined in each independent experiment); zero Ca2+, six experiments, n = 4–12 cells/experiment; 100 µM Gd3+, four experiments, n = 6–16 cells/experiment; 1 µM thapsigargin, three experiments, n = 6–7 cells/experiment; 10 µM UTP, four experiments, n = 3–9 cells/experiment; 10 µM U-73122, five experiments, n = 9–23 cells/experiment; 10 µM U-73343, three experiments, n = 10–13 cells/experiment. *, P < 0.05; **, P < 0.001 (vs. 10 µM ATP group).

 


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  		Figure 7. Effect of thapsigargin on the ATP-induced [Ca2+]i increase. Five-minute treatment with 1 µM thapsigargin, a Ca2+-ATPase inhibitor known to deplete inositol 1,4,5-triphosphate-sensitive Ca2+ stores, was sufficient for complete depletion of intracellular Ca2+ stores. The action of subsequently applied 10 µM ATP was completely abolished. The shaded bar indicates the 50-min period of thapsigargin application (1 µM). The black bar indicates 10 µM ATP application after 2-min replacement with normal medium (open bar).

 
Specific P2Y2-purinoceptor-mediated effect of ATP
To characterize the ATP receptor subtypes involved in these effects, we used 10 µM UTP in Tpit/F1 cells. UTP caused a similar increase in [Ca2+]i to ATP, with an initial peak followed by a small sustained plateau (data not shown). The percentage of UTP-evoked Ca2+-responsive cells is presented in Fig. 8Go. UTP and ATP stimulate calcium transients of the same order of magnitude and with similar temporal characteristics, which suggests the presence of the P2Y2-purinoceptor subtype (18, 19) in Tpit/F1 cells.

In common with other P2Y receptors, the P2Y2 receptors from different species are all coupled to PLC, the activation of which increases inositol 1,4,5-trisphosphate production and elevates [Ca2+]i (19, 20). To determine whether PLC activity is required for the ATP-evoked Ca2+ response, the PLC inhibitor U-73122 was applied to the bath solution. As illustrated in Fig. 8Go, U-73122 (10 µM) decreased the percentage of cells showing a measurable increase in [Ca2+]i, whereas the proportion of responsive cells in the presence of the inactive structural analog U-73343 (10 µM) was within the normal range of variability. An explanation for these observations is that P2Y2 receptors cause the release of Ca2+ from inositol 1,4,5-trisphosphate-sensitive intracellular stores through activation of PLC.

Effect of ATP on nNOS mRNA and nitrite production by Tpit/F1 cells
RT-PCR was performed to determine the expression of nNOS mRNA in Tpit/F1cells treated with or without ATP (0–100 µM). PCR amplification of nNOS resulted in the expected single band corresponding to 209 bp for both Tpit/F1 cells and mouse pituitary. The PCR products of Tpit/F1 cells cultured for 3 h in the control medium and in medium containing ATP (0.01 µM) each only gave a faint band. In contrast, high doses of ATP (0.1–100 µM) significantly stimulated nNOS mRNA expression in Tpit/F1 cells (Fig. 9Go). The role of ATP in the control of NO production by Tpit/F1 cells was also determined. Three hours of incubation with ATP (10 and 100 µM) induced a high level of NO production by Tpit/F1 cells (Fig. 10Go).



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  		Figure 9. RT-PCR for nNOS. A, ATP-stimulated nNOS gene expression by Tpit F1 cells, as determined by RT-PCR. Upper panel, RT-PCR was performed to detect nNOS mRNA in Tpit/F1 cells stimulated with 0–100 µM ATP. PCR amplification resulted in a single band with the expected product size of 209 bp for both Tpit/F1 cells and mouse pituitary. Total RNA extracted from a mouse pituitary was used as a positive control. A nNOS reaction mixture without an added template was used as a negative control. Lower panel, RT-PCR with primers specific for ß-actin. The relative levels of nNOS mRNA were estimated by using the ß-actin mRNA expression levels in the cells. B, Positive and negative RT-PCR controls for nNOS are shown. Both the pituitary gland (P) and Tpit/F1(F) are positive, whereas L929, mouse fibroblast cell line (L), is negative.

 


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  		Figure 10. Effect of ATP on NO secretion by Tpit/F1 cells. ATP (10 and 100 µM) significantly increased NO secretion into the medium compared with the control. n = 4 for each concentration of ATP. Bars, Mean ± SE. *, P < 0.01; **, P < 0.001 (vs. control group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, many cell lines have been established from temperature-sensitive T antigen transgenic (Ttg) mice (21, 22). The Ttg mouse produces the T antigen, which is unstable at a high temperature such as 39 C, and the mice are born and grow almost normally at body temperature (37 C). When some cells obtained from Ttg mice were cultured at a low temperature (33 C), the T antigen became stable, and the cells started to grow. On continuous cell cultivation at a low temperature, some cells become immortal cell lines. To obtain cell lines derived from the anterior pituitary gland, we cultured an anterior pituitary gland from a Ttg mouse and finally established three kinds of cell lines, Tpit/E, Tpit/F1, and Tpit/F2. Tpit/E cells were characterized as an endothelial cell line. Tpit/F1 cells originate from cells closely associated with endothelial cells; however, their cytological characteristics have not been determined. We therefore performed morphological and cytological studies on Tpit/F1 cells.

Our results showed that Tpit/F1 cells produce IL-6 and that its production is stimulated by PACAP, similar to that in pituitary FS cells (10, 23). Very recently, it was reported that GS is only localized in FS cells in the anterior pituitary and that GS synthesis is increased by glucocorticoids, which suggests that glucocorticoid receptors are present in FS cells (8). This similarity to FS cells was demonstrated by the finding that the GS activity of Tpit/F1 cells increased in the presence of dexamethasone, an agonist of glucocorticoids. Tpit/F1 cells also expressed the mRNAs of S-100 protein, bFGF, and Ptx1. In particular, the expression of Ptx1, which is known to be expressed in the common ancestral cells of hormone-producing pituitary cells and FS cells (24, 25), indicated that Tpit/F1 cells are derived from a similar lineage as anterior pituitary cells. Most interestingly, nNOS, which is expressed in gonadotropes and FS cells in the anterior pituitary gland (9), was also detected in Tpit/F1 cells by RT-PCR. These characteristics of Tpit/F1 cells together with their morphological features are similar to those of FS cells in the anterior pituitary gland and suggest that Tpit/F1 cells are usable as a model cell line of FS cells.

On the other hand, it is becoming clear that nucleotides such as ATP play important roles as extracellular messengers in addition to their well established role in cell metabolism (26, 27). These roles are now known to be mediated by a family of ATP receptors designated P2 purinoceptors (28). The idea of extracellular ATP regulation of anterior pituitary hormone secretion is also beginning to gain acceptance (20, 29). Using a primary pituitary culture system, very recently, it was demonstrated that pituitary FS cells possess metabotropic ATP receptors (P2Y2) (11). The P2Y2 receptor is a member of the P2Y G protein-coupled receptor family and is sensitive to both ATP and UTP (18, 19). P2Y2 receptors from different species are all coupled to the enzyme PLC, thereby increasing inositol phosphate production and elevating [Ca2+]i (19, 20). If Tpit/F1 cells are similar to authentic FS cells, ATP must stimulate Tpit/F1 cells through P2Y2 receptors. We therefore designed a series of experiments.

The data obtained in the present experiments were entirely consistent with the results of a primary pituitary cell culture (11). As UTP was found to act like ATP in stimulating the [Ca2+]i increase in Tpit/F1 cells, and the Ca2+ response is prevented by the PLC inhibitor U-73122, but not by its inactive analog U-73343, it appears that the P2Y2 subtype of ATP receptor, a G protein-linked, Ca2+-mobilizing membrane receptor, is responsible for the increase in [Ca2+]i in Tpit/F1 cells (18, 19, 20). However, the possible involvement of another pathway, such as the ryanodine receptor, could not be excluded, because the suppression by U-731223 was not complete in Tpit/F1 cells.

It is clear that the ATP-stimulated Ca2+ increase in Tpit/F1 cells did not require the presence of extracellular Ca2+ and was blocked by the Ca2+-ATPase inhibitor thapsigargin (30), which indicates that the augmentation of [Ca2+]i in Tpit/F1 cells is not a consequence of increased Ca2+ entry, but represents the release of Ca2+ from intracellular stores. Moreover, the ATP-induced [Ca2+]i increase in Tpit/F1 was not abolished by treatment with gadolinium, a Ca2+ channel blocker, which is known to inhibit ATP stimulation of channel activity (31). As a matter of fact, the results of the present experiments suggest that ATP increases [Ca2+]i through mobilization of internally stored Ca2+ mediated by inositol trisphosphate production, followed by an influx of Ca2+ from extracellular sources.

Meanwhile, growing evidence indicates that NO is an important intracellular and intercellular messenger involved in the control of a wide range of physiological events (9, 32). For example, Kato et al. (15) demonstrated that NO inhibited GH-releasing hormone-stimulated GH secretion by using an isolated pituitary cell perifusion system (33). NO synthesis by nNOS is a Ca2+-regulated process. Ca2+ interacts with calmodulin to activate nNOS, which then converts arginine into NO and citruline (32). Both in vivo and in vitro experiments have demonstrated that gonadotropes and FS cells in the anterior pituitary gland express nNOS (9, 34). In the present study, experiments were performed to examine the involvement of ATP-induced [Ca2+]i augmentation in the control of NO secretion by Tpit/F1 cells. Our results clearly showed that Tpit/F1 cells expressed nNOS mRNA and that this expression was increased by ATP administration. NO secretion into the medium was also increased by ATP treatment.

It has been reported that ATP is stored together with a hormone in the secretory granules of endocrine cells, as in the case of adrenal chromaffin cells (35, 36), and is coreleased with them on exocytosis. This implies a possible paracrine mechanism by which ATP is released from secretory granules in hormone-secreting cells in the anterior pituitary gland (27, 37). This coreleased ATP may then stimulate NO production via an ATP-induced increase in [Ca2+]i in FS cells, which are known to envelop neighboring endocrine cells with their long cytoplasmic processes (24). Finally, NO released from FS cells may act on endocrine cells and regulate hormone secretion, as schematically illustrated in Fig. 11Go. Therefore, we hypothesize that FS cells do not produce any known hormones themselves, but indirectly influence anterior pituitary hormone secretion in part through NO.



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  		Figure 11. Schematic illustration of the biological significance of ATP-mediated NO secretion to the intercellular communication between FS cells and endocrine cells.

 
In summary, on the basis of the above observations, it is suggested that Tpit/F1 cells have characteristics similar to those of FS cells in the anterior pituitary gland. Thus, this cell line is capable of exhibiting responses consistent with those of normal pituitary FS cells and represents a potentially valuable tool with which to study the cellular mechanisms underlying pituitary FS cell functions.


   Footnotes
 
1 We will deposit the Tpit/F1 cells in the RIKEN Cell Bank soon. This new cell line will be supplied at RIKEN Cell Bank (e-mail: cellbank@ rtc.riken.go.jp). Back

Received April 4, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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