Effect of Urocortin and Its Interaction with Adrenocorticotropin (ACTH) Secretagogues on ACTH Release

Peptides, Vol. 19, No. 3, pp. 513–518, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00

PII S0196-9781(97)00464-6

Effect of Urocortin and Its Interaction with Adrenocorticotropin (ACTH) Secretagogues on ACTH Release MEGUMI OZAWA, YUTAKA OKI,1 FUMIE WATANABE, KAZUMI IINO, MASAHIRO MASUZAWA, MASAYASU IWABUCHI AND TERUYA YOSHIMI Second Division, Department of Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu, 431-31, Japan Received 11 September 1997; Accepted 29 October 1997 OZAWA, M., Y. OKI, F. WATANABE, K. IINO, M. MASUZAWA, M. IWABUCHI AND T. YOSHIMI. Effect of urocortin and its interaction with adrenocorticotropin (ACTH) secretagogues on ACTH release. PEPTIDES 19(3) 513–518, 1998.—We examined the effect of urocortin (Ucn) on the adrenocorticotropin (ACTH) release from cultured rat anterior pituitary cells and AtT 20 cells. Synthetic rat (r)Ucn was not soluble in 0.1 N HCl but soluble in alkaline solvents with diminished corticotropin-releasing activity. rUcn dissolved in 0.1 M sodium phosphate buffer as a stock solution maintained its bioactivity and had the equal corticotropinreleasing activity with rat/human corticotropin-releasing factor (r/hCRF). rUcn stimulated the adrenocorticotropin release via CRF-receptors accompanied by the additive effect with r/hCRF, the synergistic effect with arginine vasopressin and the dose-dependent inhibition of a potent CRF-receptor antagonist. © 1998 Elsevier Science Inc. Urocortin Corticotropin-releasing factor CRF receptor CRF-receptor antagonist

Arginine vasopressin

CORTICOTROPIN-releasing factor (CRF), a neuropeptide first isolated from the mammalian hypothalamus, is the major physiological regulator of pituitary adrenocorticotropin (ACTH) release (18). Recently, urocortin (Ucn), and another new neuropeptide of CRF family, was identified from a rat midbrain region (20). Rat (r)Ucn, a 40 aminoacid-mammalian peptide, has 63% sequence identity to urotensin and 45% sequence identity to rat/human (r/h)CRF (20). The previous study demonstrates that Ucn binds and activates CRF receptors with greater potency than r/hCRF, and exhibits neuroanatomical and pharmacological characteristics consistent with a role for this peptide as an endogenous ligand for type 2 CRF receptors (3,20). In this study, we investigated the effect of Ucn on ACTH release from rat pituitary monolayer cells and AtT 20 cells in order to know the details of Ucn interacting 1

Adrenocorticotropin

CRF and arginine vasopressin (AVP) in mediating ACTH release. METHOD Reagents rUcn, r/hCRF, and AVP were purchased from Peptide Institute (Osaka, Japan). Medium 199 and Dulbecco’s Modified Eagle’s Medium (DMEM, 4.5 g/l glucose) were purchased from Sigma Chemical Co. (St. Louis, MO). r/hCRF and AVP were dissolved in 0.1 N HCl at 100 mM as stock solutions and frozen at 280°C. [D-Phe12, Nle21,38, CaMeLeu37] CRF 12– 41 (Human, Rat), a CRF receptor (CRF-R) antagonist, was purchased from Peninsula Laboratories, Inc. (Belmont, CA). Collagenase type 2 and DNase type 1 were purchased from Worthington Biochemical

All correspondence should be addressed to Yutaka Oki, MD. E-mail: [email protected]

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Corp. (Freehold, NJ). Other chemicals were obtained from Katayama Chemicals (Osaka, Japan). Animals Male Sprague–Dawley rats weighing 150 –200 g were obtained from JAPAN SLC Inc. (Hamamatsu, Japan) and housed for one week in a temperature-controlled room (24°C), with lights on from 0600 –1800 h daily and were given free access to standard rat chow and water. They were killed by decapitation on the morning of the pituitary cell preparation.

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Radioimmunoassay (RIA) The ACTH-RIA was performed using rabbit antiserum against ACTH1–24. The characteristics of the assay have been reported previously (16). The minimum detection limit of the assay was 1 pg/tube. Statistics The results are expressed as the mean 6 SEM. Statistical analysis was performed by Student’s two-tailed unpaired t-test, or by one-way analysis of variance (ANOVA), followed by Duncan’s multiple comparison test as appropriate. p , 0.05 was considered significant.

Primary Culture of Rat Anterior Pituitary Cells The procedures used in this study have been described previously (11). In brief, anterior pituitary cells were dispersed with 0.3% collagenase and 80 mg/ml DNase 1. After dispersion, 105 or 2 3 105 cells in 0.5 ml culture medium (Medium 199 supplemented with 10 mM HEPES, 10 mM NaHCO3, 0.1% BSA, 20 U/ml penicillin, 20 U/ml streptomycin, 0.05 mg/ml amphotericin B and 10% FCS) were plated in each well of 48-well tissue culture cluster dishes (Costar, Cambridge, MA). Cells were maintained in static monolayer culture for 4 days in a humidified incubator under a 5% CO2–95% air atmosphere at 37°C. AtT 20 Cell Culture Cells of mouse AtT-20/D16-16v were maintained and subcultured in DMEM culture medium supplemented with 10 mM HEPES, 15 mM NaHCO3, 0.1% BSA, 20 U/ml Penicillin, 20 U/ml Streptomycin, 0.05 mg/ml Amphotericin and 10% FCS at 37°C under 5% CO2–95% air in a humidified chamber. 105 cells in 0.5 ml culture medium were plated in each well of 48-well tissue culture cluster dishes. After passage, AtT20 cells were maintained in static monolayer culture for 2 days. Release Study Day 4 cultured rat anterior pituitary cells were washed three times with 0.5 ml serum-free culture medium (M199), preincubated with 0.5 ml medium for 10 min at 37°C, and then incubated further with 0.5 ml serum free culture medium containing test substances for 4 h to measure ACTH release. Day 2 cultured AtT 20 cells were washed three times and preincubated with serum-free culture medium (DMEM). After preincubation for 10 min, AtT 20 cells were incubated with 0.5 ml serum-free culture medium containing test substances for 24-h to measure ACTH release. After incubation, the medium was transferred to a conical tube and centrifuged at 3 1500 g at 4°C for 5 min, and the supernatant was stored at 220°C until the ACTH determination.

RESULTS Selection of Initial (Stock) Solvent of rUcn and Effects on ACTH Release To prepare the stock solution at 100 mM, we tried to dissolve synthetic rUcn in 0.1 N HCl, 0.1 N NaHCO3, 0.1 N NaOH, H2O, dimethylsulfoxide (DMSO), or 0.1 M sodium phosphate buffer (PB, pH 7.4). rUcn could not be dissolved in 0.1 N HCl. Ucn was able to be dissolved in other solutions, but the corticotropin-releasing activities of them were varied. Figure 1 shows the corticotropin-releasing activities of rUcn dissolved in various initial solvents. Those stock solutions were diluted with serum-free culture medium up to objective concentrations. The most potent activity was observed when rUcn was dissolved in PB. In addition, the activity of rUcn in PB was as potent as that of r/hCRF (8.30 6 0.16 vs. 7.62 6 0.43 ng/well at 1028 M, p . 0.05; Fig. 1). Effects of rUcn and r/hCRF on ACTH Release from AtT20 Cells AtT20 cells were maintained in static monolayer culture and exposed to various concentrations of r/hCRF or rUcn. Both r/hCRF and rUcn increased ACTH release in a dose-dependent manner, and were equipotent to stimulate ACTH release (190.9 6 15.9 vs. 217.2 6 19.7 ng/well at 1028 M, p . 0.05; Fig. 2). Reciprocal Action of Ucn and CRF on ACTH Release When rat anterior pituitary cells were incubated with medium containing r/hCRF (2 3 10212 2 2 3 1028 M), rUcn (2 3 10212 2 2 3 1028 M), or the combination of r/hCRF plus rUcn (each concentration; 10212 2 1028 M), r/hCRF, rUcn and the combination of rUcn plus r/hCRF increased ACTH release in a dose-dependent manner. The combination of rUcn plus r/hCRF stimulated ACTH release as potently as two-fold concentration of either rUcn alone or r/hCRF alone (23.05 6 1.33 ng/well at 1028 M CRF plus 1028 M Ucn vs. 19.85 6 0.72 ng/well at 2 3 1028 M Ucn vs. 20.17 6 1.24 ng/well at 2 3 1028 M CRF, p . 0.05;

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FIG. 1. Comparison of initial solvents of rUcn on ACTH release in rat anterior pituitary cells. rUcn was initially dissolved in DMSO, H2O, 0.1N NaOH, 0.1N NaHCO3, or 0.1 M sodium phosphate buffer (PB, pH 7.4), and diluted with serum-free culture medium. Day 4 cultured rat anterior pituitary cells were incubated with medium containing rUcn for 4 h. Vehicle was incubated with medium containing each initial solvent, of which concentration was the same as each rUcn solution at 1028 M, as vehicle. Each point represents the mean of four determinations. Bars indicate the SEM. Symbols for each solvent are indicated in the figure.

Fig. 3) indicating that the effect of the combination of rUcn plus r/hCRF was additive. Reciprocal Action of rUcn, r/hCRF and AVP on ACTH Release When rat anterior pituitary cells were incubated with culture medium containing r/hCRF (10211 2 1028 M), rUcn (10211

FIG. 2. Effects of rUcn and r/hCRF on ACTH release by AtT20 cells. AtT 20 cells were maintained in monolayer culture, and incubated with various concentrations of r/hCRF or rUcn for 24 h. Each point represents the mean of four determinations. Bars indicate the SEM. *p , 0.05 vs. vehicle.

2 1028 M), AVP (1027 M), or the combination of AVP plus r/hCRF or rUcn, the combination of r/hCRF plus AVP stimulated the ACTH release synergistically, and also the combination of rUcn and AVP potentiated the ACTH release with synergistic increase (Fig. 4). These synergistic

FIG. 3. Reciprocal action of Ucn and CRF on the ACTH release by rat anterior pituitary cells. Day 4 cultured rat anterior pituitary cells were incubated with medium containing r/hCRF (2 3 10212 2 2 3 1028 M), rUcn (2 3 10212 2 2 3 1028 M) or the combination of r/hCRF plus rUcn (each concentration; 10212 2 1028 M). Each point represents the mean of four determinations. Bars indicate the SEM.

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FIG. 4. Reciprocal action of Ucn and AVP on ACTH release by rat anterior pituitary cells. Day 4 cultured rat anterior pituitary cells were incubated with medium containing r/hCRF (10211 2 1028 M), rUcn (10211 2 1028 M), AVP (1027 M), the combination of AVP plus r/hCRF or the combination of AVP plus rUcn. Each point represents the mean of four determinations. Bars indicate the SEM. *, p , 0.05 vs. rUcn alone at each concentration. †p , 0.05 vs. r/hCRF alone at each concentration.

effects were also nearly equal (r/hCRF vs. rUcn at 1028 M, 21.11 6 1.80 vs. 19.74 6 1.82 ng/well, respectively; p . 0.05; Fig. 4). Effect of CRF-R Antagonist under Stimulation of rUcn or r/hCRF Rat anterior pituitary cells were incubated with medium containing CRF-R antagonist (1026 2 1029 M) with 1029 M r/hCRF or 1029 M rUcn. The CRF-R antagonist inhibited ACTH release stimulated by r/hCRF or rUcn with the same magnitude (1029 M r/hCRF alone vs. r/hCRF plus 1026 M CRF-R antagonist; 15.71 6 2.47 vs. 4.92 6 0.66 ng/well, respectively; p , 0.01, 1029 M rUcn alone vs. rUcn plus 1026 M CRF-R antagonist, 13.57 6 1.22 vs. 4.77 6 0.19 ng/well, respectively: p , 0.05; Fig. 5). DISCUSSION rUcn, a 40-amino-acid mammalian peptide, has been cloned as a new member of the CRF family (20). Previous reports show that Ucn stimulates ACTH release by rat anterior pituitary cells more potently than CRF in vivo and in vitro (3,20). Until Ucn was found, CRF had been considered to be the most potent ACTH secretagogue (19). Thus, in this study, we investigated the stability and biological activity of rUcn using cultured rat anterior pituitary cells and mouse pituitary tumor cells in order to know the possible role in regulating the ACTH release. When we prepared the stock solution of rUcn, we tried to dissolve synthetic rUcn in 0.1 N HCl like as CRF. However, rUcn was insoluble in it. The manufacturer’s instruction

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recommends to dissolve rUcn in 1 M acetic acid to prepare the stock solution. However, our preliminary study demonstrated that the solution (medium) containing acetic acid even at 1026 M had the nonspecific stimulation of ACTH release from cultured rat anterior pituitary cells. Therefore, acetic acid was not suitable as the initial solvent. Then, we tested 0.1 N NaHCO3, 0.1 N NaOH, H2O, DMSO, or 0.1 M PB. Although rUcn was soluble in alkaline solutions, distilled water, and DMSO, it has the weaker biological activity than r/hCRF when dissolved in those solvents. Finally, rUcn in PB showed the most potent corticotropin-releasing activity. Thus, we used PB as the initial solvent throughout this study. We examined the corticotropin-releasing activity of rUcn. By pituitary cells of both races, rUcn increased ACTH release in a dose-dependent manner as potently as r/hCRF. Vaughan et al. reported that rUcn is more potent to stimulate ACTH release from rat anterior pituitary in vivo and in vitro (20). The cause of this discrepancy has not been clear, but probably depends on the source of peptide. The actions of CRF-related peptides are mediated by interaction with three types of CRF receptors, which display discrete and fairly exclusive distributions. CRF receptor type 1 mRNA is expressed in the brain and pituitary (13). CRF receptor type 2 has two splice forms; 2a mRNA is found only in the brain, and 2b mRNA is present in both the brain and peripheral tissues, but not pituitary (5,7,8,12,15). rUcn is highly effective in the CRF receptor type 2b mediated action (20). In the pituitary corticotrophs, Ucn is

FIG. 5. Effect of CRF-R antagonist under stimulation of rUcn or r/hCRF. Day 4 cultured rat anterior pituitary cells were incubated with medium containing 1029 M r/hCRF plus CRF-R antagonist (1026 2 1029 M), and 1029 M rUcn plus CRF-R antagonist. Each point represents the mean of four determinations. Bars indicate the SEM. *p , 0.05 vs. rUcn alone (1029 M). p , 0.05 vs. r/hCRF alone (1029 M).

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considered to stimulate the ACTH release via CRF receptor type 1. In our study, rUcn stimulated ACTH release as potently as r/hCRF. If these two peptides share the same receptor, the effect of the combination of rUcn and r/hCRF should be additive. In fact, the combination of rUcn and r/h CRF had the equal potency of corticotropin-releasing activity with the double concentration of either rUcn or r/hCRF. It is known that the combination of CRF and AVP increases ACTH release synergistically (4,11,18). CRF stimulates ACTH release from rat pituitary corticotrophs via adenosine 39, 59-cyclic monophosphate (cAMP-dependent protein kinase-A pathway (2,4,6,9), and AVP activates 1, 4, 5 triphosphate/phospholipid protein kinase-C pathway (1,21). We showed that the combination of rUcn and AVP increased ACTH release synergistically as potently as the combination of CRF and AVP also indicating that Ucn acts on corticotrophs via the same mechanism as CRF. Next to these examinations, we examined the effect of [D-Phe12, Nle21,38, Ca-MeLeu37] CRF 12– 41, which is a more potent CRF receptor antagonist than a-helical CRF9 – 41 (10), on either r/hCRF- or rUcn-induced

ACTH release. It inhibited both the rUcn- and r/hCRFinduced ACTH release from rat anterior pituitary cells in a dose-dependent manner with the same magnitude. This also suggests that Ucn stimulates ACTH release from rat pituitary corticotrophs via the same pathway which CRF activates. Up to the present, several actions of Ucn other than corticotropin-releasing activity have been reported. Ucn potently inhibits the development of edema via interaction with CRF receptors in rat heat-injury model (17). Ucn might be an endogenous CRF-like factor in the brain responsible for the effects of stress on appetite (14). Because Ucn has the highest affinity for type 2 CRF receptor, its important roles on peripheral tissues are expected as autocrine or paracrine. In conclusion, the effect of rUcn on ACTH release is as potent as that of r/hCRF. It is conjectured that Ucn stimulates ACTH release from rat pituitary corticotrophs via the same pathway which CRF activates in vitro. However, the role of endogenous Ucn in mediating ACTH release is still uncertain because the chemical characteristics of Ucn is more unstable than CRF.

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