two ryanodine receptor subtypes (RyR1 and RyR3) was con- firmed by RT-PCR. We also found that caffeine, a ryanodine receptor agonist, significantly ...
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Endocrinology 145(1):36 –38 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2003-1222
Possible Involvement of Ryanodine Receptor-Mediated Intracellular Calcium Release in the Effect of Corticotropin-Releasing Factor on Adrenocorticotropin Secretion ETSUKO YAMAMORI, YASUMASA IWASAKI, YUTAKA OKI, MASANORI YOSHIDA, MASATO ASAI, MACHIKO KAMBAYASHII, YUTAKA OISO, AND NOBUO NAKASHIMA Departments of Medicine (E.Y., M.Y., M.A., Y.O.) and Clinical Pathophysiology (Y.I., M.K., N.N.), Nagoya University Graduate School of Medicine and Hospital, Nagoya 466-8550, Japan; and Department of Medicine II (Y.O.), Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan We examined the role of intracellular calcium release in the regulation of CRH-induced ACTH secretion using the AtT20 corticotroph cell line. We found that ruthenium red, an inhibitor of ryanodine receptor, substantially diminished the secretory response, whereas Xestospongin C, an inositol 1,4,5triphosphate receptor antagonist, had no effect. Expression of two ryanodine receptor subtypes (RyR1 and RyR3) was con-
firmed by RT-PCR. We also found that caffeine, a ryanodine receptor agonist, significantly stimulated, whereas thapsigargin, which causes depletion of intracellular calcium store, markedly diminished, the ACTH release. These results suggest that ryanodine receptor-mediated calcium-induced calcium release is involved in the regulation of CRH-induced ACTH release. (Endocrinology 145: 36 –38, 2004)
T
Materials and Methods
HE HYPOTHALAMIC NEUROPEPTIDES CRH and arginine vasopressin are known to be two major positive regulators of the hypothalamo-pituitary-adrenal axis (1, 2). CRH released into the portal vein of the pituitary stalk stimulates both synthesis and secretion of ACTH from corticotrope of the anterior pituitary through CRH-R1 receptor (3), which is known to couple with the adenylate cyclase/protein kinase A (PKA) pathway. After CRH stimulation, activated PKA somehow opens the voltage-dependent calcium channel (VDCC), which subsequently increases intracellular calcium and triggers the release of ACTH from secretory granules. Vasopressin, on the other hand, is known to stimulate calcium release from the intracellular calcium pool through the phospholipase C-inositol 1,4,5-triphosphate (IP3) signaling pathway (4). Although the outline of the major signaling pathways of CRH and vasopressin for ACTH release has been largely characterized as mentioned above, the precise molecular mechanism is not completely elucidated. For example, it is not known yet whether extracellular calcium influx alone is totally responsible for the ACTH release by CRH. In this study, using the AtT20 corticotroph cell line and various inhibitors of intracellular signaling events, we further characterized the molecular mechanism of the CRH effect. Our data suggest the involvement of the so-called calcium-induced calcium release through a ryanodine receptor-mediated mechanism in CRH-induced ACTH release.
Reagents Human/rat CRH was purchased from the Peptide Institute (Osaka, Japan). Ruthenium red and caffeine were from Sigma (St. Louis, MO). Thapsigargin and Xestospongin C were from Wako Pure Chemical (Osaka, Japan).
Cell culture AtT20PL cells, a clone of the AtT20 mouse corticotroph cell line in which approximately 0.7 kb of the rat POMC 5⬘-promoter (⫺708 to ⫹64; ⫹1 indicates the transcription start site)-luciferase fusion gene was stably incorporated, were described elsewhere (5, 6). The cells were maintained in a T75 culture flask with DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and antibiotics (50 U/ml penicillin and 50 g/ml streptomycin; Invitrogen) under 5% CO2/95% atmosphere at 37 C. Culture medium was changed twice a week, and the cells were subcultured once a week.
Experimental protocols All experiments were performed twice so as to confirm their reproducibility. For each experiment, the cells were plated in 3.5-cm diameter culture dishes with approximately 50% confluence. The culture medium was changed to DMEM supplemented with 1% FBS on the following day, and the cells were cultured for an additional 3– 4 d, during which the culture medium was changed every other day. On the day of an experiment, culture medium was changed to serumfree medium, and the test substance(s), dissolved in H2O or dimethylsulfoxide in 1000⫻ concentration, were added directly into the culture medium of each dish. After cultivation for the defined time interval, the culture medium was collected for ACTH assay. The inhibitors (ruthenium red, Xestospongin C, and thapsigargin) were added from 1 h before the addition of CRH to the end of experiment.
Abbreviations: FBS, Fetal bovine serum; IP3, inositol 1,4,5-triphosphate; PKA, protein kinase A; VDCC, voltage-dependent calcium channel. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.
RT-PCR procedure RNA was isolated from the AtT20PL or Y1 mouse adrenocortical cell lines using TRIzol reagent (Invitrogen), and 5 g of the total RNA was
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used for the reverse transcriptase reaction with Moloney murine leukemia virus reverse transcriptase (Superscript II; Invitrogen). The cDNA obtained was then amplified by PCR with Taq DNA polymerase (Takara Shuzo, Tokyo, Japan). The sequences of primer sets used were as follows: mouse ryanodine receptor 1 (RyR1), sense 5⬘-GTTCCGGTGCTGGGGACATG-3⬘, antisense 5⬘-TGTGGGCTGTGATCTCCAGA-3⬘; RyR2, sense 5⬘-ACTGCTAAAGTGACCAACAG-3⬘, antisense 5⬘-TTGCATCGCTGAAATCTAGT-3⬘; and RyR3, sense 5⬘-GTTGCAAACCTGTGGAACTC-3⬘, antisense 5⬘-CTACTGGGCTAAAGTCAAGG-3⬘. The PCR condition was as follows: 94 C 5 min for initial denaturation, 35 cycles of amplification (94 C for 30 sec, 60 C for 1 min, 72 C for 2 min), and final extension at 72 C for 10 min.
Measurements and statistics ACTH in culture medium was measured by radioimmunometric assay (ACTH immunoradiometric assay kit, Mitsubishi Chemical, Tokyo, Japan) (5). Samples in each group of the experiments were in triplicate or quadruplicate. All data are expressed as mean ⫾ se. When statistical analyses were performed, data were compared by one-way ANOVA with Duncan’s multiple range test, and P ⬍ 0.05 was considered significant.
Endocrinology, January 2004, 145(1):36 –38 37
FIG. 2. The effects of inhibition of IP3 or ryanodine receptors on basal and CRH-induced ACTH release. AtT20 cells were treated with vehicle, Xestospongin C (XC, 5 M) (A), or ruthenium red (RR, 5 or 10 M) (B) and then treated with vehicle or CRH (100 nM) 1 h later. ACTH secretion during the CRH treatment for 3 h was estimated. *, P ⬍ 0.05 vs. corresponding control (C). #, P ⬍ 0.05 vs. value with CRH alone.
Results RyR1 and RyR3 mRNAs are expressed in AtT20 cells
We first analyzed the expression of three subtypes of ryanodine receptors. In AtT20PL cells, bands corresponded to those of the RyR1 and RyR3, but not RyR2 subtypes of the receptor (Fig. 1). On the other hand, in Y1 cells used as a control, all the subtypes of the receptor were amplified. Inhibition of ryanodine receptor but not IP3 receptor diminishes CRH-induced ACTH release
CRH is believed to activate the voltage-dependent calcium channel, with a resultant influx of extracellular calcium triggering ACTH release (7, 8). Because vasopressin, another ACTH secretagogue, uses calcium from the intracellular calcium pool (9), we tried to see whether this mechanism is also involved in CRH-induced ACTH secretion. Intracellular calcium release is caused by two independent mechanisms, the IP3 receptor- and the ryanodine receptor-mediated pathways. When Xestospongin C, a specific inhibitor of IP3 receptor, was applied, ACTH secretion induced by CRH was not influenced (Fig. 2). On the other hand, ruthenium red, a specific inhibitor of ryanodine receptor, was used, a significant decrease in ACTH release was recognized: CRH-stimulated but not basal ACTH release was diminished at 5 m,
FIG. 3. The effects of caffeine and thapsigardin on basal or CRHinduced ACTH release. A, AtT20 cells were treated with vehicle or caffeine (0.1, 0.5, or 1 mM), and ACTH secretion for 3 h was estimated. *, P ⬍ 0.05 vs. control (C). B, AtT20 cells were treated with vehicle or thapsigargin (1 M) and then treated with vehicle or CRH (100 nM) 1 h later. ACTH secretion during the CRH treatment for 3 h was estimated. *, P ⬍ 0.05 vs. corresponding control (C).
and both basal and stimulated release was decreased at 10 m of ruthenium red. Ryanodine receptor agonist caffeine stimulates ACTH release
If ryanodine receptor-mediated calcium release from the intracellular calcium pool is involved in the CRH-induced ACTH release, stimulation of the receptor will cause the secretion of the hormone. Our results showed that the ryanodine receptor agonist caffeine, at or above 0.5 mm, indeed significantly stimulated ACTH secretion in a dose-dependent manner, although the magnitude was less than that of CRH (Fig. 3A). Depletion of intracellular calcium pool by thapsigargin diminishes basal and CRH-induced ACTH release
FIG. 1. RT-PCR analyses of the expression of ryanodine receptor mRNAs. The DNA fragments with the predicted lengths (479 and 429 bp for mouse RyR1 and RyR3 mRNAs, respectively) were amplified from total RNA derived from AtT20PL cells using specific primer sets for each mRNA. On the other hand, all three subtypes were amplified from Y1 cells using the same primer sets. M, A DNA marker (a 100-bp DNA ladder). Reverse transcriptase-minus control reaction showed no amplification (data not shown).
To further confirm the involvement of intracellular calcium release, we used thapsigargin, an inhibitor of endoplasmic reticulum Ca2⫹-ATPase, which causes depletion of intracellular calcium storage. Under the pretreatment with thapsigargin (1 m), both basal and CRH-induced ACTH secretion was markedly decreased (Fig. 3B). Discussion
The results in this study strongly suggest the involvement of ryanodine receptor-mediated calcium mobilization
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FIG. 4. Schematic representation of the putative intracellular pathways involved in the CRH-mediated ACTH release in AtT20 corticotroph cells. AVP, Arginine vasopressin; CICR, calcium-induced calcium release; CRHR1, CRH receptor type 1; DAG, diacylglycerol; PKC, protein kinase C; V1b, vasopressin receptor type 1b.
through intracellular calcium storage in CRH-induced calcium-mediated ACTH release. The previous notion for CRH-induced ACTH secretion is that CRH stimulates the cAMP/PKA pathway, which in turn activates (possibly through the opening of nonselective cation channels and depolarization) VDCC with a resultant influx of extracellular calcium and subsequent ACTH release (7, 8). This mechanism is in sharp contrast with another ACTH secretagogue, vasopressin, which increases intracellular calcium from intracellular calcium storage through activation of phospholipase C and liberation of IP3 (9). Our data in this study, however, strongly suggest the involvement of intracellular calcium mobilization in the effect of CRH as well. More specifically, the unique aspect of the CRH effect is that ryanodine, not IP3, receptor appears to mediate the release of intracellular calcium, because the effect of CRH was decreased by ryanodine receptor antagonist ruthenium red but not by IP3 receptor antagonist Xestospongin C. Ryanodine receptor agonist caffeine stimulated ACTH, further supporting the mechanism. Ryanodine receptors are shown to be expressed in pituitary cells (10). The mechanism responsible for the activation of ryanodine receptor by CRH has not been made clear in this study. However, ryanodine receptor is usually activated by influx of extracellular calcium (calcium-induced calcium release) (11). It is well known that CRH activates VDCC and causes the increase in intracellular calcium through the channel (8). Furthermore, CRH-induced ACTH release is totally dependent on the influx of extracellular calcium, because CRH fails to stimulate ACTH secretion when mobilization of extracellular calcium is blocked by VDCC antagonists or calciumfree medium (7, 8). Thus, we assume that CRH-stimulated increase in intracellular calcium by VDCC triggers the activation of ryanodine receptor and subsequent calcium mobilization from intracellular calcium storage. In this sense, the calcium-induced calcium release mediated by ryanodine receptor might be serving as a booster effect in CRH-induced ACTH release, because CRH responsiveness under ruthe-
Yamamori et al. • Brief Communications
nium red or thapsigargin treatment was weakened but still remained in this study. The involvement of calcium-induced calcium release in the mechanism of hormone release has recently been shown in other endocrine cells (12, 13). The present and previous findings concerning the CRHACTH signaling are summarized in Fig. 4. CRH stimulates adenylate cyclase through Gs protein and produces cAMP, which activates PKA. PKA elicits the opening of VDCC (probably through the activation of nonselective cation channels and the subsequent membrane depolarization) (14), with a resultant calcium entry and triggering of ACTH secretion. In the latter process, the rise in intracellular calcium causes the ryanodine receptor-mediated calcium-induced calcium release mechanism to unfold, and further enhances the ACTH release. Thus, the effect of CRH on ACTH release may be mediated through multiple mechanisms involving both extracellular and intracellular calcium. Acknowledgments We are indebted to Ms. Tatsuyo Miura for excellent technical assistance. Received September 15, 2003. Accepted October 22, 2003. Address all correspondence and requests for reprints to: Yasumasa Iwasaki, M.D., Ph.D., Department of Clinical Pathophysiology, Nagoya University Graduate School of Medicine and Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: iwasakiy@ med.nagoya-u.ac.jp.
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