Role of the Second-Messenger Cyclic-Adenosine 5'-Diphosphate-Ribose on Adrenocorticotropin Secretion from Pituitary Cells
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内分泌学杂志 2005年第5期
Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Eduardo Nunes Chini, Signal Transduction Laboratory, Department of Anesthesiology, Mayo Clinic and Foundation, Rochester, Minnesota 55905. E-mail: chini.eduardo@mayo.edu.
Abstract
We examined the role of the second-messenger cyclic-ADP-ribose (cADPR) on the regulation of ACTH secretion using AtT20 corticotroph tumor cell line. We found that the cADPR antagonist, 8-Br-cADPR, substantially diminished the secretion of ACTH induced by CRH and potassium in these cells, whereas xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, had no effect. In addition, the cADPR agonist, 3-deaza-cADPR, augmented ACTH secretion. The presence of the components of the cADPR system, namely ryanodine receptor, CD38, and cADPR itself, was determined in AtT20 cells. Furthermore, we observed that antagonists of the ryanodine channel and cADPR system can decrease the potassium-induced Ca2+ transients in these cells. These results suggest that cADPR is a second messenger in pituitary cells and regulates ACTH secretion by a mechanism dependent on activation of the ryanodine channel by extracellular Ca2+.
Introduction
THE PRECISE ROLE of intracellular and extracellular Ca2+ pools on intracellular Ca2+ transients and ACTH secretion have not been completely elucidated (1, 2, 3, 4, 5, 6). It is possible that a complex interaction between intracellular and extracellular Ca2+ stores may play a role in ACTH secretion. Indeed, in AtT20 cells, it has been shown that both extracellular Ca2+ influx and Ca2+ release from intracellular Ca2+ stores are involved in regulating ACTH secretion (4, 5, 6). In fact, the ryanodine receptor-channel (RyR) has been implicated as an important component of the signal transduction pathway responsible for the secretion of ACTH (7, 8, 9). The RyR is an intracellular Ca2+ channel that is present on the endoplasmic reticulum (ER) and, on activation, leads to release of Ca2+ from the lumen of the ER to the cytoplasm of the cell (10). Although the RyR is implicated in ACTH release, the regulation of this channel in pituitary cells has not been elucidated to date. In other mammalian cells, the second-messenger cyclic-ADP-ribose (cADPR) has been implicated as an endogenous modulator of the RyR (11, 12, 13). cADPR is metabolized by the protein CD38, a bifunctional enzyme capable of synthesis and hydrolysis of this nucleotide (11, 12, 13, 14). The role of the cADPR-signaling pathway on the neuroendocrine axis has not been completely elucidated. In fact, whether cADPR is implicated in the regulation of ACTH release has not been explored to date. In this study we demonstrate for the first time that cADPR is an endogenous regulator of the secretion of ACTH in pituitary cells. Here we propose that, in pituitary cells, the mechanisms of intracellular calcium transients are mediated by influx of extracellular calcium through voltage-dependent calcium channels (VDCCs), which consequently activate calcium-induced calcium release (CICR) from intracellular stores through the RyRs by a mechanism dependent on the second-messenger cADPR.
Materials and Methods
Culture of cells
The AtT20 corticotroph cell line (American Type Culture Collection, Manassas, VA) was maintained in a T75 flask with DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen). Cultures were maintained in a humidified incubator supplied with 5%CO2-95% air at 37 C. Media were changed twice a week and cells were subcultured as needed by detaching the cells with a Ca2+/Mg2+-free Hanks’ balanced salt solution (HBSS; Invitrogen) containing 0.25% trypsin and 5 mM EDTA.
Fura-2 loading and intracellular free calcium concentration measurements
AtT20 corticotroph cells were plated on 9- x 22-mm coverslips for spectrofluorometer microscopy at a density of 25,000 cells/coverslip and grown until approximately 80% confluent in DMEM with 10% FBS. Cells were made quiescent by changing the medium to DMEM with 1% FBS for 2–3 d and then incubated with 5 μM fura 2-AM for 2 h at 37 C. Cells were then washed in HBSS medium with or without Ca2+, depending on the experiment and imaged alternately with 340/380 nm light (emission 510 nm) using an F-2000 spectrofluorometer (Hitachi, Tokyo, Japan).
KCl-induced Ca2+ transients
The measurement of calcium influx was made after addition of calcium (2 mM) for 1–3 min. To assess calcium release, the cells were washed with HBSS with Ca2+ and cells were incubated for 2–3 min and 1 h with antagonist and agonist of RYR/IP3, inositol 1,4,5-trisphosphate (IP3) receptor; afterward we added 60 mM KCl and the measurement was performed.
Immunoprecipitation and Western blot
Extracts from AtT20 cells were washed with PBS and subjected to sonication (3 x 5 sec) in cell lysis buffer. The homogenates were centrifuged at 10,000 x g for 10 min and the resultant supernatant assayed for protein using the DC protein assay (Bio-Rad Laboratories, Hercules, CA). The lysates (200 μl) were adjusted to contain 200 μg protein and 2 μg mouse monoclonal anti-CD38 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit polyclonal anti-ryanodine receptor (Santa Cruz Biotechnology) and incubated over night at 4 C with gentle rocking. The antibody-protein complex was then immunoprecipitated for 2 h using Protein A/G Plus-Agarose (Santa Cruz Biotechnology), and the pellet was washed two times in cell lysis buffer and resuspended in 30 μl of cell lysis buffer and 30 μl of Laemmli buffer. The supernatants were denatured at 100 C for 3–5 min and 40 μl of sample subject to SDS-PAGE using the Criterion gel system (Bio-Rad) and 4–15% gradient gel. The gels were run at a constant current of 200 V for 70 min followed by transfer to polyvinyl difluoride membranes (Bio-Rad). The membranes were blocked for 1 h in 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 followed by incubation with appropriate primary antibodies and horseradish peroxidase-conjugated secondary or horseradish peroxidase-linked protein A. Blots were visualized by exposing them to BioMax MR film (Eastman Kodak Co., Rochester, NY) using LumiGLO (Cell Signaling Technology, Beverly, MA).
ELISA for ACTH
AtT20 cells were plated at a density of 40,000 per well and grown in DMEM with 10% FBS until approximately 80% confluent. Cells were then made quiescent in DMEM containing 1% FBS for 2–3 d. On the day of the experiment, cells were washed with HBSS once and incubated with different compounds. Afterward the media were collected and ELISA was performed according to the manufacture’s protocol (Research Diagnostics, Inc., Flanders, NJ).
ADPR-cyclase activity
Activity of ADP-ribosyl cyclase was performed using the nicotinamide guanine dinucleotide technique as described previously (15). AtT20 homogenates (1 mg/ml) were incubated in a medium containing 0.2 mM nicotinamide guanine dinucleotide, 0.25 M sucrose, and 40 mM Tris-HCl (pH 7.2) at 37 C. Activity was determined with a fluorometric assay with a 300-nm excitation wavelength and 410 nm emission (15). In key experiments, results were also confirmed with the use of nicotinamide adenine dinucleotide (NAD) as the natural substrate of the enzyme.
cADPR hydrolase activity
Hydrolysis was assayed using cADPR as substrate as described before (16, 17). AtT20 homogenates (1 mg/ml) were incubated in a medium containing 1 μM cADPR, 0.25 M sucrose, and 40 mM Tris-HCl (pH 7.2) at 37 C. Aliquots prior and after the incubation were assayed for remaining cADPR content using the sea urchin egg homogenate bioassay and HPLC as described previously (16, 17). Specific activity was expressed as nanomoles of ADPR produced per milligram protein per minute.
Detection of cADPR levels in AtT20 cells
Nucleotides were extracted from AtT20 cells with 10% trichloroacetic acid (TCA) at 4 C. TCA was removed with water-saturated ether as described previously (18). The aqueous layer containing the cADPR was removed and adjusted to pH 8 with 1 M Tris base. To remove nucleotides other than cADPR, a mixture of hydrolytic enzymes containing (final concentrations) 0.44 U/ml nucleotide pyrophosphatase, 12.5 U/ml alkaline phosphatase, 0.0625 U/ml NADase, and 2.5 mM MgCl2 were added and incubated overnight at 37 C. The enzyme mixture hydrolyzes all nucleotides (including NAD+) in the samples except for cADPR, which is resistant to these enzymes (18, 19). Enzymes were removed with Centricon-3 filters and samples recovered in the filtrate after centrifugation at 4 C and 7500 x g for 90 min (18, 19). The detection of cADPR was performed with some modifications to the cycling method described recently (18, 19). In brief, 0.1 ml cADPR standard or nucleotides extracted from AtT20 cell were incubated with 0.1 ml cycling reagent containing 0.3 μg/ml ADP-ribosyl cyclase, 4 mM nicotinamide, 100 mM sodium phosphate (pH 8), 2% ethanol, 40 μg/ml alcohol dehydrogenase, 8 μM resazurin, 0.04 μg/ml diaphorase, and 4 μM flavin mononucleotide. An increase in the resazurin fluorescence (with excitation at 544 nm and emission at 590 nm) was measured using a Spectramax Gemini XPS fluorescent plate reader (Molecular Devices Corp., Sunnyvale, CA). The recovery rate of exogenous cADPR detected by this method, after TCA extraction of standard concentrations of cADPR, was in the range of 75–80%.
Materials
Fura 2-AM, nifedipine, and xestospongin C were purchased from Calbiochem (San Diego, CA). 3-Deaza-cADPR was purchased from Molecular Probes, Inc. (Eugene, OR). Antibodies were purchased from Santa Cruz Biotechnology. All the others reagents, of the highest purity grade available, were supplied by Sigma Chemical Co. (St. Louis, MO).
Statistics
The reported experiments were repeated three to six times and data expressed as mean ± SE. The unpaired t test was used for statistical analysis. P 0.05 was significant.
Results and Discussion
Presence of the CD38-cADPR-RyR system in AtT20 cells
To determine the role of the second-messenger system modulated by cADPR on the secretion of ACTH, we first determined whether AtT20 pituitary tumor cells expressed the components of this system, including CD38 (a bifunctional enzyme responsible for the synthesis and degradation of cADPR), the RyR (a channel that is activated by cADPR), and cADPR itself. Using Western blot analysis, we determined that AtT20 cells contain CD38 (Fig. 1). Furthermore, we also determined that AtT20 cells are capable of generating cADPR from NAD and degrading cADPR to ADPR (Fig. 1). The synthesis of cADPR was inhibited by known inhibitors of the CD38 cyclase, including nicotinamide, dithiothreitol, and nicotinic acid (Fig. 1). We have previously shown that the expression of CD38 can be increased in smooth muscle cells by incubation with TNF (17, 18, 19) and extend this observation to AtT20 cells (Fig. 1). These data indicate that the enzyme CD38 is present and functional in AtT20 cells. We also observed that AtT20 cells contain RyR (Fig. 1). Furthermore, treatment of cells with the RyR agonist caffeine led to a robust increase in intracellular Ca2+ (Fig. 1). This Ca2+ release was inhibited by the RyR inhibitor ruthenium red. Finally, we observed that cADPR itself is present in AtT20 cells at concentrations of 1.8 ± 0.5 pmol/mg protein. In summary, all the components of the cADPR system are present in AtT20 cells.
FIG. 1. Characterization of the CD38-cADPR-RyR system in AtT20 cells. A, Western blot analysis of AtT20 cells for the enzyme CD38. Cell extracts were obtained from control AtT20 cells and AtT20 cells treated with 50 ng/ml TNF for 24 h. B, The effect of TNF on ADP-ribosyl cyclase activity in AtT20 cells. Cells were treated as indicated and extracts assayed for cyclase activity as described in Materials and Methods. C, The effect of antagonists on ADP-ribosyl cyclase activity. Nict, Nicotinamide; NA, nicotinic acid; DTT, dithiothreitol. D, The measurement of cADPR hydrolase activity in AtT20 cells. Extracts were incubated with 10 μM cADPR and degradation determined as described in Materials and Methods. The specific activity of cADPR hydrolysis was determined to be 0.11 nmol of cADPR/min·mg of protein. E, Western blot analysis for RyR in AtT20 cells. F, The effect of ruthenium red on caffeine-induced intracellular Ca2+ release in fura-2-loaded AtT20 cells. Cells were incubated with 0 or 25 μM ruthenium red 1 min before the addition of caffeine. Results are statistically significant (P 0.05) and expressed as the means of three or four experiments. cGDPR, Cyclic ribose.
Role of the RyR and cADPR on intracellular Ca2+ transients in AtT20 cells
The release of ACTH in AtT20 cells is modulated by intracellular calcium (4, 5, 6). There are several systems of Ca2+ influx and release from intracellular stores (20). Release of Ca2+ from intracellular stores is mediated by several mechanisms including the IP3 and cADPR-ryanodine pathway (20). Conversely, the influx of extracellular Ca2+ is mediated by VDCCs (20). The participation of the ryanodine-mediated pathway in the CRH-stimulated release of ACTH in AtT20 cells has been proposed previously (7, 9). It has been postulated that CRH stimulation of the cell leads to extracellular Ca2+ influx into the cytosol through VDCCs, activating the RyRs, stimulating release of Ca2+ from the ER, and stimulating the secretion of ACTH from the pituitary cells (9). Here we explore this hypothesis using KCl as an activator of the VDCCs. Treatment of AtT20 cells with KCl leads to cell depolarization that in turn activates intracellular Ca2+ transients in the cytosol. We observed that stimulation of AtT20 cells with 60 mM KCl leads to a rapid increase in intracellular free Ca2+ (Fig. 2). This rise in intracellular Ca2+ was nearly abolished by the removal of extracellular Ca2+, indicating that this Ca2+ transient is at least in part mediated by influx of extracellular Ca2+ (Fig. 2). In addition, we observed that the KCl-induced intracellular Ca2+ transient could be inhibited by nifedipine (an inhibitor of the so-called L-type VDCC). In contrast, inhibition of the N-type VDCC with -conotoxin led to a small decrease in the KCl-induced intracellular Ca2+ transient (Fig. 2). It has been previously shown that the intracellular Ca2+ increase induced by extracellular Ca2+ influx through the VDCC can be augmented by activation of release of Ca2+ from intracellular stores (21). This mechanism is referred to as CICR and is mediated by the RyR (21). Furthermore, cADPR as a regulator of the RyR can modulate the CICR properties of this channel (21). In fact, here we observed that the Ca2+ transient induced by KCl can also be inhibited by blocking the RyR with high concentrations of ryanodine, ruthenium red and the inhibition of the cADPR system with 8-Br-cADPR (a cell-permeable cADPR antagonist) (Fig. 2). These data clearly implicate both extracellular Ca2+ influx through L-type VDCCs and Ca2+ release from intracellular cADPR-sensitive Ca2+stores (RyR) as important components of the intracellular Ca2+ transient in AtT20 pituitary cells.
FIG. 2. The role of extracellular Ca2+ and the cADPR-RyR system on KCl-induced intracellular Ca2+ transients. AtT20 cells were loaded with fura-2-AM as described in Materials and Methods. A, Measurement of KCl-induced Ca2+ transients in the presence of 2 mM extracellular CaCl2 or the absence of extracellular CaCl2 (0 Ca2+). B, The effect of VDCC inhibitors on the KCl-induced intracellular Ca2+ transients. AtT20 cells were treated with vehicle (control) or the VDCC inhibitors in the presence of 2 mM extracellular CaCl2. The effect of the VDCC inhibitors was compared with the effect of removing extracellular Ca2+ (0 Ca2+). Cells were stimulated with 60 mM KCl. C, The effect of cADPR-RyR system inhibitors on the KCl-induced intracellular Ca2+ transients. Cells were treated with inhibitors 1 h before the addition of 60 mM KCl. All Ca2+ transients were assayed in the presence of extracellular Ca2+. Results are the means of three or four experiments and are statistically significant (P 0.05).
Role of ryanodine channel on ACTH secretion in AtT20 cells
We compared the levels of ACTH release after incubating the pituitary cells with KCl (60 mM) and CRH (100 nM) in the presence of ryanodine (100 μM). As previously described, the levels of ACTH were increased by incubation of cells with KCl of CRH (Fig. 3). We also observed that ryanodine could decrease the ACTH release induced by both KCl and CRH (Fig. 3). The levels of ACTH were significantly lower in cells with ryanodine and KCl or CRH, compared with control, thus suggesting a role for RyRs in the release of ACTH. (Fig. 3). These data are in agreement with the recent observation by Yamamori et al. (9) that antagonists of the RyR can inhibit ACTH secretion in AtT20 cells.
FIG. 3. Role of the RyR on KCl- and CRH-induced ACTH release. The effect of IP3 or RyR antagonists on KCl- and CRH-induced ACTH release. All experiments were performed in the presence of 2 mM extracellular CaCl2 except where specified ("0" Ca2+). A, AtT20 cells were treated with vehicle (control), 60 mM KCl, or 100 nM CRH and the release of ACTH was determined 3 h after treatment. B and C, AtT20 cells were treated with xestospongin C (Xest. C; 10 μM), ruthenium red (RR; 25 μM), and ryanodine (Rya; 10 μM). NS, Not significant. All other treatments led to significant (P 0.05) difference from control. Results are the means of three or four experiments.
Role of cADPR on ACTH secretion
cADPR appears to be an endogenous regulator of the RyR in vivo (11, 12, 13). Furthermore, it has been previously described that cADPR can activate intracellular Ca2+ release in the pituitary cell (22). Here we explored the role of the cADPR system in ACTH secretion when stimulated by KCl and CRH. We observed a 2.5-fold increase in ACTH levels when cells were stimulated with 60 mM KCl. Conversely, removal of extracellular Ca2+ led to a dramatic decrease in KCl-induced ACTH secretion (Fig. 3), indicating that extracellular Ca2+ plays an important role in KCl-induced ACTH release. Additionally, we observed that 8-Br-cADPR (a cADPR antagonist) could also inhibit KCl and CRH-induced ACTH secretion (Figs. 4 and 5). Also, the cell-permeable cADPR agonist 3-deaza-cADPR led to a 1.5-fold increase in KCl-induced ACTH secretion (Fig. 4A) and prolonged the KCl-induced Ca2+ transients in AtT20 cells (Fig. 4B). These data clearly indicate that cADPR plays an important role on the signal transduction mechanism involved in ACTH secretion in AtT20 cells. In contrast with the role of the cADPR-RyR system, the IP3 system does not have a significant importance on ACTH release from AtT20 cells because IP3 antagonist xestospongin C had no significant effect on KCl- or CRH-induced ACTH secretion (Fig. 3).
FIG. 4. Role of the cADPR system on KCl-induced ACTH release. A, AtT20 cells were pretreated with 100 μM 8-Br-cADPR or 200 nM 3-deaza-cADPR 1 h before the addition of 60 mM KCl. Release of ACTH was determined 3 h after treatment. The results are expressed as percentage of control and are the means of three or four experiments. All the treatments are significantly (P 0.05) different from control. B, AtT20 cells were loaded with fura-2-AM as described in Materials and Methods. Measurement of 60 mM KCl-induced Ca2+ transients in the presence of 2 mM extracellular CaCl2 was determined as in Fig. 2, in cells treated with vehicle (control) or 200 nM 3-deaza-cADPR 1 h before the addition of 60 mM KCl.
FIG. 5. Role of the cADPR system on CRH-induced ACTH release. AtT20 cells were pretreated with 100 μM 8-Br-cADPR 1 h before the addition of 100 nM CRH, and the release of ACTH was determined 3 h after treatment. The results are expressed as percentage of control. Results are the means of three or four experiments. The ACTH release in the presence of 8-Br-cADPR is significantly (P 0.05) different from control.
Conclusion
The results of this study strongly suggest the involvement of cADPR-RyR-mediated calcium mobilization from intracellular stores on the mechanisms of ACTH release in AtT20 corticotroph cell line. Previous studies have shown the involvement of intracellular Ca2+ transients on the mechanism of ACTH release (4, 5, 6). The current hypothesis is that CRH activates the cAMP/protein kinase A pathway, resulting in an influx of extracellular calcium through the VDCCs and stimulating ACTH secretion (1, 2, 3, 4, 5, 6). Presently we know that there are other mechanisms involved as well (7, 8, 9). Some studies previously demonstrated the participation of RyR as one of the mechanisms of ACTH release (7, 8, 9). Therefore, we considered the possible role of an endogenous regulator of RyR, cADPR, as potentially influencing ACTH secretion. Our experiments provide evidence for the presence of cADPR and its regulatory enzyme CD38 in AtT20 cells. These data support a model of ACTH release after depolarization of the cell membrane that includes: 1) activation of VDCCs that allows influx of Ca2+ into the cell; 2) activation of the CICR mechanism through the RyR by a mechanism modulated by cADPR; and 3) elevation in intracellular Ca2, resulting in release of ACTH (Fig. 6).
FIG. 6. Working hypothesis. Our data support a model of ACTH release after depolarization of the cell membrane that includes: 1) activation of VDCCs that allows influx of Ca2+ into the cell; 2) activation of the CICR mechanism through the RyR by a mechanism modulated by cADPR; and 3) elevation of intracellular Ca2+ causing release of ACTH. In the case of CRH, the cAMP-protein kinase A (PKA) system led to the activation of the VDCC. R, ACTH receptor; SR, sarcoplasmic reticulum.
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Address all correspondence and requests for reprints to: Eduardo Nunes Chini, Signal Transduction Laboratory, Department of Anesthesiology, Mayo Clinic and Foundation, Rochester, Minnesota 55905. E-mail: chini.eduardo@mayo.edu.
Abstract
We examined the role of the second-messenger cyclic-ADP-ribose (cADPR) on the regulation of ACTH secretion using AtT20 corticotroph tumor cell line. We found that the cADPR antagonist, 8-Br-cADPR, substantially diminished the secretion of ACTH induced by CRH and potassium in these cells, whereas xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, had no effect. In addition, the cADPR agonist, 3-deaza-cADPR, augmented ACTH secretion. The presence of the components of the cADPR system, namely ryanodine receptor, CD38, and cADPR itself, was determined in AtT20 cells. Furthermore, we observed that antagonists of the ryanodine channel and cADPR system can decrease the potassium-induced Ca2+ transients in these cells. These results suggest that cADPR is a second messenger in pituitary cells and regulates ACTH secretion by a mechanism dependent on activation of the ryanodine channel by extracellular Ca2+.
Introduction
THE PRECISE ROLE of intracellular and extracellular Ca2+ pools on intracellular Ca2+ transients and ACTH secretion have not been completely elucidated (1, 2, 3, 4, 5, 6). It is possible that a complex interaction between intracellular and extracellular Ca2+ stores may play a role in ACTH secretion. Indeed, in AtT20 cells, it has been shown that both extracellular Ca2+ influx and Ca2+ release from intracellular Ca2+ stores are involved in regulating ACTH secretion (4, 5, 6). In fact, the ryanodine receptor-channel (RyR) has been implicated as an important component of the signal transduction pathway responsible for the secretion of ACTH (7, 8, 9). The RyR is an intracellular Ca2+ channel that is present on the endoplasmic reticulum (ER) and, on activation, leads to release of Ca2+ from the lumen of the ER to the cytoplasm of the cell (10). Although the RyR is implicated in ACTH release, the regulation of this channel in pituitary cells has not been elucidated to date. In other mammalian cells, the second-messenger cyclic-ADP-ribose (cADPR) has been implicated as an endogenous modulator of the RyR (11, 12, 13). cADPR is metabolized by the protein CD38, a bifunctional enzyme capable of synthesis and hydrolysis of this nucleotide (11, 12, 13, 14). The role of the cADPR-signaling pathway on the neuroendocrine axis has not been completely elucidated. In fact, whether cADPR is implicated in the regulation of ACTH release has not been explored to date. In this study we demonstrate for the first time that cADPR is an endogenous regulator of the secretion of ACTH in pituitary cells. Here we propose that, in pituitary cells, the mechanisms of intracellular calcium transients are mediated by influx of extracellular calcium through voltage-dependent calcium channels (VDCCs), which consequently activate calcium-induced calcium release (CICR) from intracellular stores through the RyRs by a mechanism dependent on the second-messenger cADPR.
Materials and Methods
Culture of cells
The AtT20 corticotroph cell line (American Type Culture Collection, Manassas, VA) was maintained in a T75 flask with DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen). Cultures were maintained in a humidified incubator supplied with 5%CO2-95% air at 37 C. Media were changed twice a week and cells were subcultured as needed by detaching the cells with a Ca2+/Mg2+-free Hanks’ balanced salt solution (HBSS; Invitrogen) containing 0.25% trypsin and 5 mM EDTA.
Fura-2 loading and intracellular free calcium concentration measurements
AtT20 corticotroph cells were plated on 9- x 22-mm coverslips for spectrofluorometer microscopy at a density of 25,000 cells/coverslip and grown until approximately 80% confluent in DMEM with 10% FBS. Cells were made quiescent by changing the medium to DMEM with 1% FBS for 2–3 d and then incubated with 5 μM fura 2-AM for 2 h at 37 C. Cells were then washed in HBSS medium with or without Ca2+, depending on the experiment and imaged alternately with 340/380 nm light (emission 510 nm) using an F-2000 spectrofluorometer (Hitachi, Tokyo, Japan).
KCl-induced Ca2+ transients
The measurement of calcium influx was made after addition of calcium (2 mM) for 1–3 min. To assess calcium release, the cells were washed with HBSS with Ca2+ and cells were incubated for 2–3 min and 1 h with antagonist and agonist of RYR/IP3, inositol 1,4,5-trisphosphate (IP3) receptor; afterward we added 60 mM KCl and the measurement was performed.
Immunoprecipitation and Western blot
Extracts from AtT20 cells were washed with PBS and subjected to sonication (3 x 5 sec) in cell lysis buffer. The homogenates were centrifuged at 10,000 x g for 10 min and the resultant supernatant assayed for protein using the DC protein assay (Bio-Rad Laboratories, Hercules, CA). The lysates (200 μl) were adjusted to contain 200 μg protein and 2 μg mouse monoclonal anti-CD38 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit polyclonal anti-ryanodine receptor (Santa Cruz Biotechnology) and incubated over night at 4 C with gentle rocking. The antibody-protein complex was then immunoprecipitated for 2 h using Protein A/G Plus-Agarose (Santa Cruz Biotechnology), and the pellet was washed two times in cell lysis buffer and resuspended in 30 μl of cell lysis buffer and 30 μl of Laemmli buffer. The supernatants were denatured at 100 C for 3–5 min and 40 μl of sample subject to SDS-PAGE using the Criterion gel system (Bio-Rad) and 4–15% gradient gel. The gels were run at a constant current of 200 V for 70 min followed by transfer to polyvinyl difluoride membranes (Bio-Rad). The membranes were blocked for 1 h in 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 followed by incubation with appropriate primary antibodies and horseradish peroxidase-conjugated secondary or horseradish peroxidase-linked protein A. Blots were visualized by exposing them to BioMax MR film (Eastman Kodak Co., Rochester, NY) using LumiGLO (Cell Signaling Technology, Beverly, MA).
ELISA for ACTH
AtT20 cells were plated at a density of 40,000 per well and grown in DMEM with 10% FBS until approximately 80% confluent. Cells were then made quiescent in DMEM containing 1% FBS for 2–3 d. On the day of the experiment, cells were washed with HBSS once and incubated with different compounds. Afterward the media were collected and ELISA was performed according to the manufacture’s protocol (Research Diagnostics, Inc., Flanders, NJ).
ADPR-cyclase activity
Activity of ADP-ribosyl cyclase was performed using the nicotinamide guanine dinucleotide technique as described previously (15). AtT20 homogenates (1 mg/ml) were incubated in a medium containing 0.2 mM nicotinamide guanine dinucleotide, 0.25 M sucrose, and 40 mM Tris-HCl (pH 7.2) at 37 C. Activity was determined with a fluorometric assay with a 300-nm excitation wavelength and 410 nm emission (15). In key experiments, results were also confirmed with the use of nicotinamide adenine dinucleotide (NAD) as the natural substrate of the enzyme.
cADPR hydrolase activity
Hydrolysis was assayed using cADPR as substrate as described before (16, 17). AtT20 homogenates (1 mg/ml) were incubated in a medium containing 1 μM cADPR, 0.25 M sucrose, and 40 mM Tris-HCl (pH 7.2) at 37 C. Aliquots prior and after the incubation were assayed for remaining cADPR content using the sea urchin egg homogenate bioassay and HPLC as described previously (16, 17). Specific activity was expressed as nanomoles of ADPR produced per milligram protein per minute.
Detection of cADPR levels in AtT20 cells
Nucleotides were extracted from AtT20 cells with 10% trichloroacetic acid (TCA) at 4 C. TCA was removed with water-saturated ether as described previously (18). The aqueous layer containing the cADPR was removed and adjusted to pH 8 with 1 M Tris base. To remove nucleotides other than cADPR, a mixture of hydrolytic enzymes containing (final concentrations) 0.44 U/ml nucleotide pyrophosphatase, 12.5 U/ml alkaline phosphatase, 0.0625 U/ml NADase, and 2.5 mM MgCl2 were added and incubated overnight at 37 C. The enzyme mixture hydrolyzes all nucleotides (including NAD+) in the samples except for cADPR, which is resistant to these enzymes (18, 19). Enzymes were removed with Centricon-3 filters and samples recovered in the filtrate after centrifugation at 4 C and 7500 x g for 90 min (18, 19). The detection of cADPR was performed with some modifications to the cycling method described recently (18, 19). In brief, 0.1 ml cADPR standard or nucleotides extracted from AtT20 cell were incubated with 0.1 ml cycling reagent containing 0.3 μg/ml ADP-ribosyl cyclase, 4 mM nicotinamide, 100 mM sodium phosphate (pH 8), 2% ethanol, 40 μg/ml alcohol dehydrogenase, 8 μM resazurin, 0.04 μg/ml diaphorase, and 4 μM flavin mononucleotide. An increase in the resazurin fluorescence (with excitation at 544 nm and emission at 590 nm) was measured using a Spectramax Gemini XPS fluorescent plate reader (Molecular Devices Corp., Sunnyvale, CA). The recovery rate of exogenous cADPR detected by this method, after TCA extraction of standard concentrations of cADPR, was in the range of 75–80%.
Materials
Fura 2-AM, nifedipine, and xestospongin C were purchased from Calbiochem (San Diego, CA). 3-Deaza-cADPR was purchased from Molecular Probes, Inc. (Eugene, OR). Antibodies were purchased from Santa Cruz Biotechnology. All the others reagents, of the highest purity grade available, were supplied by Sigma Chemical Co. (St. Louis, MO).
Statistics
The reported experiments were repeated three to six times and data expressed as mean ± SE. The unpaired t test was used for statistical analysis. P 0.05 was significant.
Results and Discussion
Presence of the CD38-cADPR-RyR system in AtT20 cells
To determine the role of the second-messenger system modulated by cADPR on the secretion of ACTH, we first determined whether AtT20 pituitary tumor cells expressed the components of this system, including CD38 (a bifunctional enzyme responsible for the synthesis and degradation of cADPR), the RyR (a channel that is activated by cADPR), and cADPR itself. Using Western blot analysis, we determined that AtT20 cells contain CD38 (Fig. 1). Furthermore, we also determined that AtT20 cells are capable of generating cADPR from NAD and degrading cADPR to ADPR (Fig. 1). The synthesis of cADPR was inhibited by known inhibitors of the CD38 cyclase, including nicotinamide, dithiothreitol, and nicotinic acid (Fig. 1). We have previously shown that the expression of CD38 can be increased in smooth muscle cells by incubation with TNF (17, 18, 19) and extend this observation to AtT20 cells (Fig. 1). These data indicate that the enzyme CD38 is present and functional in AtT20 cells. We also observed that AtT20 cells contain RyR (Fig. 1). Furthermore, treatment of cells with the RyR agonist caffeine led to a robust increase in intracellular Ca2+ (Fig. 1). This Ca2+ release was inhibited by the RyR inhibitor ruthenium red. Finally, we observed that cADPR itself is present in AtT20 cells at concentrations of 1.8 ± 0.5 pmol/mg protein. In summary, all the components of the cADPR system are present in AtT20 cells.
FIG. 1. Characterization of the CD38-cADPR-RyR system in AtT20 cells. A, Western blot analysis of AtT20 cells for the enzyme CD38. Cell extracts were obtained from control AtT20 cells and AtT20 cells treated with 50 ng/ml TNF for 24 h. B, The effect of TNF on ADP-ribosyl cyclase activity in AtT20 cells. Cells were treated as indicated and extracts assayed for cyclase activity as described in Materials and Methods. C, The effect of antagonists on ADP-ribosyl cyclase activity. Nict, Nicotinamide; NA, nicotinic acid; DTT, dithiothreitol. D, The measurement of cADPR hydrolase activity in AtT20 cells. Extracts were incubated with 10 μM cADPR and degradation determined as described in Materials and Methods. The specific activity of cADPR hydrolysis was determined to be 0.11 nmol of cADPR/min·mg of protein. E, Western blot analysis for RyR in AtT20 cells. F, The effect of ruthenium red on caffeine-induced intracellular Ca2+ release in fura-2-loaded AtT20 cells. Cells were incubated with 0 or 25 μM ruthenium red 1 min before the addition of caffeine. Results are statistically significant (P 0.05) and expressed as the means of three or four experiments. cGDPR, Cyclic ribose.
Role of the RyR and cADPR on intracellular Ca2+ transients in AtT20 cells
The release of ACTH in AtT20 cells is modulated by intracellular calcium (4, 5, 6). There are several systems of Ca2+ influx and release from intracellular stores (20). Release of Ca2+ from intracellular stores is mediated by several mechanisms including the IP3 and cADPR-ryanodine pathway (20). Conversely, the influx of extracellular Ca2+ is mediated by VDCCs (20). The participation of the ryanodine-mediated pathway in the CRH-stimulated release of ACTH in AtT20 cells has been proposed previously (7, 9). It has been postulated that CRH stimulation of the cell leads to extracellular Ca2+ influx into the cytosol through VDCCs, activating the RyRs, stimulating release of Ca2+ from the ER, and stimulating the secretion of ACTH from the pituitary cells (9). Here we explore this hypothesis using KCl as an activator of the VDCCs. Treatment of AtT20 cells with KCl leads to cell depolarization that in turn activates intracellular Ca2+ transients in the cytosol. We observed that stimulation of AtT20 cells with 60 mM KCl leads to a rapid increase in intracellular free Ca2+ (Fig. 2). This rise in intracellular Ca2+ was nearly abolished by the removal of extracellular Ca2+, indicating that this Ca2+ transient is at least in part mediated by influx of extracellular Ca2+ (Fig. 2). In addition, we observed that the KCl-induced intracellular Ca2+ transient could be inhibited by nifedipine (an inhibitor of the so-called L-type VDCC). In contrast, inhibition of the N-type VDCC with -conotoxin led to a small decrease in the KCl-induced intracellular Ca2+ transient (Fig. 2). It has been previously shown that the intracellular Ca2+ increase induced by extracellular Ca2+ influx through the VDCC can be augmented by activation of release of Ca2+ from intracellular stores (21). This mechanism is referred to as CICR and is mediated by the RyR (21). Furthermore, cADPR as a regulator of the RyR can modulate the CICR properties of this channel (21). In fact, here we observed that the Ca2+ transient induced by KCl can also be inhibited by blocking the RyR with high concentrations of ryanodine, ruthenium red and the inhibition of the cADPR system with 8-Br-cADPR (a cell-permeable cADPR antagonist) (Fig. 2). These data clearly implicate both extracellular Ca2+ influx through L-type VDCCs and Ca2+ release from intracellular cADPR-sensitive Ca2+stores (RyR) as important components of the intracellular Ca2+ transient in AtT20 pituitary cells.
FIG. 2. The role of extracellular Ca2+ and the cADPR-RyR system on KCl-induced intracellular Ca2+ transients. AtT20 cells were loaded with fura-2-AM as described in Materials and Methods. A, Measurement of KCl-induced Ca2+ transients in the presence of 2 mM extracellular CaCl2 or the absence of extracellular CaCl2 (0 Ca2+). B, The effect of VDCC inhibitors on the KCl-induced intracellular Ca2+ transients. AtT20 cells were treated with vehicle (control) or the VDCC inhibitors in the presence of 2 mM extracellular CaCl2. The effect of the VDCC inhibitors was compared with the effect of removing extracellular Ca2+ (0 Ca2+). Cells were stimulated with 60 mM KCl. C, The effect of cADPR-RyR system inhibitors on the KCl-induced intracellular Ca2+ transients. Cells were treated with inhibitors 1 h before the addition of 60 mM KCl. All Ca2+ transients were assayed in the presence of extracellular Ca2+. Results are the means of three or four experiments and are statistically significant (P 0.05).
Role of ryanodine channel on ACTH secretion in AtT20 cells
We compared the levels of ACTH release after incubating the pituitary cells with KCl (60 mM) and CRH (100 nM) in the presence of ryanodine (100 μM). As previously described, the levels of ACTH were increased by incubation of cells with KCl of CRH (Fig. 3). We also observed that ryanodine could decrease the ACTH release induced by both KCl and CRH (Fig. 3). The levels of ACTH were significantly lower in cells with ryanodine and KCl or CRH, compared with control, thus suggesting a role for RyRs in the release of ACTH. (Fig. 3). These data are in agreement with the recent observation by Yamamori et al. (9) that antagonists of the RyR can inhibit ACTH secretion in AtT20 cells.
FIG. 3. Role of the RyR on KCl- and CRH-induced ACTH release. The effect of IP3 or RyR antagonists on KCl- and CRH-induced ACTH release. All experiments were performed in the presence of 2 mM extracellular CaCl2 except where specified ("0" Ca2+). A, AtT20 cells were treated with vehicle (control), 60 mM KCl, or 100 nM CRH and the release of ACTH was determined 3 h after treatment. B and C, AtT20 cells were treated with xestospongin C (Xest. C; 10 μM), ruthenium red (RR; 25 μM), and ryanodine (Rya; 10 μM). NS, Not significant. All other treatments led to significant (P 0.05) difference from control. Results are the means of three or four experiments.
Role of cADPR on ACTH secretion
cADPR appears to be an endogenous regulator of the RyR in vivo (11, 12, 13). Furthermore, it has been previously described that cADPR can activate intracellular Ca2+ release in the pituitary cell (22). Here we explored the role of the cADPR system in ACTH secretion when stimulated by KCl and CRH. We observed a 2.5-fold increase in ACTH levels when cells were stimulated with 60 mM KCl. Conversely, removal of extracellular Ca2+ led to a dramatic decrease in KCl-induced ACTH secretion (Fig. 3), indicating that extracellular Ca2+ plays an important role in KCl-induced ACTH release. Additionally, we observed that 8-Br-cADPR (a cADPR antagonist) could also inhibit KCl and CRH-induced ACTH secretion (Figs. 4 and 5). Also, the cell-permeable cADPR agonist 3-deaza-cADPR led to a 1.5-fold increase in KCl-induced ACTH secretion (Fig. 4A) and prolonged the KCl-induced Ca2+ transients in AtT20 cells (Fig. 4B). These data clearly indicate that cADPR plays an important role on the signal transduction mechanism involved in ACTH secretion in AtT20 cells. In contrast with the role of the cADPR-RyR system, the IP3 system does not have a significant importance on ACTH release from AtT20 cells because IP3 antagonist xestospongin C had no significant effect on KCl- or CRH-induced ACTH secretion (Fig. 3).
FIG. 4. Role of the cADPR system on KCl-induced ACTH release. A, AtT20 cells were pretreated with 100 μM 8-Br-cADPR or 200 nM 3-deaza-cADPR 1 h before the addition of 60 mM KCl. Release of ACTH was determined 3 h after treatment. The results are expressed as percentage of control and are the means of three or four experiments. All the treatments are significantly (P 0.05) different from control. B, AtT20 cells were loaded with fura-2-AM as described in Materials and Methods. Measurement of 60 mM KCl-induced Ca2+ transients in the presence of 2 mM extracellular CaCl2 was determined as in Fig. 2, in cells treated with vehicle (control) or 200 nM 3-deaza-cADPR 1 h before the addition of 60 mM KCl.
FIG. 5. Role of the cADPR system on CRH-induced ACTH release. AtT20 cells were pretreated with 100 μM 8-Br-cADPR 1 h before the addition of 100 nM CRH, and the release of ACTH was determined 3 h after treatment. The results are expressed as percentage of control. Results are the means of three or four experiments. The ACTH release in the presence of 8-Br-cADPR is significantly (P 0.05) different from control.
Conclusion
The results of this study strongly suggest the involvement of cADPR-RyR-mediated calcium mobilization from intracellular stores on the mechanisms of ACTH release in AtT20 corticotroph cell line. Previous studies have shown the involvement of intracellular Ca2+ transients on the mechanism of ACTH release (4, 5, 6). The current hypothesis is that CRH activates the cAMP/protein kinase A pathway, resulting in an influx of extracellular calcium through the VDCCs and stimulating ACTH secretion (1, 2, 3, 4, 5, 6). Presently we know that there are other mechanisms involved as well (7, 8, 9). Some studies previously demonstrated the participation of RyR as one of the mechanisms of ACTH release (7, 8, 9). Therefore, we considered the possible role of an endogenous regulator of RyR, cADPR, as potentially influencing ACTH secretion. Our experiments provide evidence for the presence of cADPR and its regulatory enzyme CD38 in AtT20 cells. These data support a model of ACTH release after depolarization of the cell membrane that includes: 1) activation of VDCCs that allows influx of Ca2+ into the cell; 2) activation of the CICR mechanism through the RyR by a mechanism modulated by cADPR; and 3) elevation in intracellular Ca2, resulting in release of ACTH (Fig. 6).
FIG. 6. Working hypothesis. Our data support a model of ACTH release after depolarization of the cell membrane that includes: 1) activation of VDCCs that allows influx of Ca2+ into the cell; 2) activation of the CICR mechanism through the RyR by a mechanism modulated by cADPR; and 3) elevation of intracellular Ca2+ causing release of ACTH. In the case of CRH, the cAMP-protein kinase A (PKA) system led to the activation of the VDCC. R, ACTH receptor; SR, sarcoplasmic reticulum.
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