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Corticotropin-Releasing Factor (CRF) and the Urocortins Differentially Regulate Catecholamine Secretion in Human and Rat Adrenals, in a CRF Receptor Type-Specific Manner

Departments of Clinical Chemistry-Biochemistry (E.D., C.T., M.V., A.A., A.N.M.), Pharmacology (V.M., I.C., A.G.), and Forensic Sciences (E.M.), School of Medicine, University of Crete, Heraklion GR-710 03, Crete, Greece; Departments of Pharmacology (E.C.) and Histology-Embryology (M.L.), School of Medicine, Democritus University of Thrace, Alexandroupolis, GR-68 100, Greece; Department of Neuroscience (J.S.), J. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96822

Address all correspondence and requests for reprints to: Andrew N. Margioris, Department of Clinical Chemistry-Biochemistry, School of Medicine, University of Crete, Heraklion GR-710 03, Crete, Greece. E-mail: andym{at}med.uoc.gr.

	Abstract

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Corticotropin-releasing factor (CRF) affects catecholamine production both centrally and peripherally. The aim of the present work was to examine the presence of CRF, its related peptides, and their receptors in the medulla of human and rat adrenals and their direct effect on catecholamine synthesis and secretion. CRF, urocortin I (UCN1), urocortin II (UCN2), and CRF receptor type 1 (CRF₁) and 2 (CRF₂) were present in human and rat adrenal medulla as well as the PC12 pheochromocytoma cells by immunocytochemistry, immunofluorescence, and RT-PCR. Exposure of dispersed human and rat adrenal chromaffin cells to CRF₁ receptor agonists induced catecholamine secretion in a dose-dependent manner, an effect peaking at 30 min, whereas CRF₂ receptor agonists suppressed catecholamine secretion. The respective effects were blocked by CRF₁ and CRF₂ antagonists. CRF peptides affected catecholamine secretion via changes of subplasmaliminal actin filament polymerization. CRF peptides also affected catecholamine synthesis. In rat chromaffin and PC12 cells, CRF₁ and CRF₂ agonists induced catecholamine synthesis via tyrosine hydroxylase. However, in human chromaffin cells, activation of CRF₁ receptors induced tyrosine hydroxylase, whereas activation of CRF₂ suppressed it. In conclusion, it appears that a complex intraadrenal CRF-UCN/CRF-receptor system exists in both human and rat adrenals controlling catecholamine secretion and synthesis.

	Introduction

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THE CATECHOLAMINERGIC SYSTEM and the hypothalamus-pituitary-adrenal axis comprise the two major adaptation mechanisms to stress. The two axes interact at several levels to coordinate their response to stressful stimuli. Over the years, multiple reports have provided evidence that a complex corticotropin-releasing factor (CRF)-based system exists within the adrenal glands coordinating the two stress axes at a peripheral, intraadrenal level. Indeed, CRF, urocortin I (UCN1), urocortin II (UCN2) (in humans stresscopin-related peptide), urocortin III (in humans stresscopin), their receptors CRF receptor type 1 (CRF₁), CRF receptor type 2 (CRF₂), and the decoy receptor CRF-binding protein have been reported to be present in whole human, rodent, bovine, and canine adrenals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). However, there is no work studying the CRF system in the adrenal medulla, as a whole, vis-à-vis the distribution of each particular CRF peptide and their receptors as well as their effects on catecholamine secretion and synthesis. The information available so far is fragmented and involves either rodent or human adrenals (12, 13).

We have previously shown that CRF stimulates catecholamine production in adrenal chromaffin cells (14, 15, 16). CRF elevates adenylate cyclase activity in rat adrenal membranes and cAMP in bovine chromaffin cells (3), whereas UCN1 elevates cAMP in PC12 cells (17). The significance of CRF peptides on adrenal medulla has been demonstrated in glucocorticoid-supplemented CRF null mice in which tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, and phenylethanolamine N-methyltransferase (PNMT), the enzyme catalyzing the conversion of norepinephrine (NE) to epinephrine (E), are lower, compared with CRF (+/+) mice (18). Furthermore, after immobilization stress, the expression of PNMT does not appear to increase in CRF null mice to the same extent as in CRF(+/+) mice (19). Similar data have been reported in CRF₁ null mice in which the levels of E are almost half that of wild-type mice of the same strain, whereas PNMT mRNA are even lower at almost 25% (20). ACTH treatment of CRF₁ null mice improves but does not completely restore E and PNMT mRNA levels. Interestingly, the morphology of chromaffin cells in CRF₁ null mice appears to be dramatically different, compared with that of the wild-type, exhibiting depletion of epinephrine-storing secretory granules, a finding not completely restored by ACTH (20). Finally, UCN1 has also been shown to induce TH expression in PC12 cells via the cAMP/protein kinase A pathway (17).

The aim of the present work was first to map the distribution of CRF peptides and their respective receptors in sections of human and rat adrenals and subsequently examine the role of each component of the system in catecholamine synthesis and secretion, in vitro. For this purpose we used human cadaveric adrenals from traffic accident victims, fresh rat adrenals, and the PC12 cell line. Selective and nonselective CRF₁ and CRF₂ receptor agonists and antagonists were used to examine their effects on catecholamine secretion, actin filament dynamics, and TH expression (21).

	Materials and Methods

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Reagents and antibodies
Rat/human recombinant CRF was purchased from Tocris (Ellisville, MO). Cortagine (nonpeptide CRF₁ antagonist), antisauvagine-30 (synthetic CRF₂ antagonist), and mouse UCN2 (CRF₂ agonist) were provided by Dr. J. Spiess (J. Burns School of Medicine, University of Hawaii, Honolulu, HI), whereas antalarmin (nonpeptide CRF₁ antagonist) was provided by Dr. G. P. Chrousos (National Institute of Child Health and Human Development, National Institutes of Health). The pharmacological tyrosine hydroxylase inhibitor, L-2-methyl-3-(-4hydroxyphenyl)-alanine (AMPT) and L-aromatic amino acid decarboxylase inhibitor, 3-(hydrazinomethyl)-phenol hydrochloride (NSD-1015) were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Mouse monoclonal antibodies against TH and actin were obtained from Chemicon (Temecula, CA). The antisera used for CRF, UCN1, and UCN2 protein detection were purchased from Phoenix Pharmaceuticals Inc. (Belmont, CA). The antisera used for CRF receptor protein detection in human sections, the anti-CRF₁ (4467a-CRF₁) and anti-CRF₂ (2064a-CRF₂), were kindly provided by Dr. D. Grigoriadis (Neurocrine Bioscience Inc. San Diego, CA) (22, 23), whereas the antisera used for CRF receptors protein detection (sc-12381 and sc-20550) in both human and rat sections were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies used were antimouse fluorescein isothiocyanate-labeled conjugated, antimouse tetramethylrhodamine isothiocyanate conjugated, and antimouse horseradish peroxidase conjugated and were all purchased from Chemicon. Bradford Coomassie Brilliant Blue G-250 was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA), and nitrocellulose membranes for Western blotting were purchased from Millipore (Bedford, MA). Immunoreactive bands were visualized with an enhanced chemiluminescence kit from PerkinElmer Life Sciences (Norwalk, CT). Collagenase A, DNase I, collagen, poly-L-lysine, phallacidin, and trypsin were all obtained from Sigma. All sterile tissue apparatus were obtained from Corning (Corning, NY). RPMI 1640, DMEM-F12, L-glutamine, fetal calf serum (FCS), penicillin/streptomycin, and horse serum (HS) were purchased from Invitrogen (Carlsbad, CA). All other chemicals and reagents were obtained from Sigma, if not stated otherwise.

Human and rat primary adrenal chromaffin cell cultures
Cadaver human adrenals were obtained from traffic accident victims from our Forensic Medicine Department. The glands were taken according to the standard protocols and processed within 12 h of death. The University of Crete Ethics Committee for human studies approved the procedure and use of specimen. Rat adrenals were obtained from adult female Sprague-Dawley rats. The experimental protocols were approved by the Laboratory Animal Care Committee and the Public Veterinary Department of the Heraklion prefecture, Crete, Greece. In brief, human or rat adrenal medulla was cut in small pieces and incubated in freshly prepared dispersion medium containing 1 mg/ml collagenase A and 0.1 mg/ml DNase I (human adrenals) or 2 mg/ml collagenase A, 0.1 mg/ml DNase I, and 0.125% trypsin (rat adrenals) for a maximum of 1 h in water bath. Then human or rat cell suspensions were incubated in 0.146 M NH₄Cl for 1–2 min on ice for the elimination of erythrocytes. Human chromaffin cells were cultured in DMEM-F12 supplemented with 10 mM L-glutamine, 4.5 mM glucose, 15 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% HS, and 2.5% FCS, whereas rat chromaffin cells were cultured in DMEM-F12 supplemented with 10 mM L-glutamine, 3.7 g/liter sodium bicarbonate, 4.5 mM glucose, 15 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 20% FCS in collagen-coated plates for 2 d. One day before each experiment, the initial culture media were changed with serum-free media supplemented with 0.1% BSA. Cells were then stimulated with CRF, UCN1, UCN2, or cortagine at various time intervals.

PC12 rat pheochromocytoma cell culture
PC12 cells were obtained from two sources: Dr. M. Greenberg (Children’s Hospital, Boston, MA) and the American Type Culture Collection (Manassas, VA). Cells were grown in RPMI 1640 containing 10 mM L-glutamine, 15 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10% HS, and 5% FCS at 5% CO₂ and 37 C. One day before each experiment, the initial culture media were changed with serum-free media supplemented with 0.1% BSA.

Immunocytoshemistry
Immunohistochemistry was performed on paraffin-embedded tissue sections (4–6 µm), which had been routinely fixed in 4% buffered formalin. Paraffin sections were deparaffinized in xylene and rehydrated in graded alcohol to TBS [50 mM Tris, 150 mM NaCl (pH 7.4)]. The slides were micro waved for 3 x 4 min in 10 mM citrate, washed in TBS, incubated for 10 min with 3% H₂O₂, and blocked for 10 min with Power Block (BioGenex, San Ramon, CA). Slides were then incubated for 75 min with anti-CRF₁ (4467a; 1:1500) or anti-CRF₂ (2064a; 1:1500) diluted in 10% normal rabbit serum in PBS. Alternatively, slides were incubated with anti-CRF₁ (sc-12381; 1:50) or anti-CRF₂ (sc-20550; 1:50) diluted in 2% BSA in TBS overnight at 4 C. For the detection of CRF peptides or TH, slides were incubated for 60 min with anti-CRF (H-019–06; 1:100), anti-UCN1 (H-019–14; 1:100), anti-UCN2 (H-019–24; 1:50), or anti-TH (MAB318; 1:100), all diluted in TBS containing 2% BSA. For preabsorption studies, primary antibodies were incubated with or without the respective blocking peptides overnight at 4 C. Subsequently all the antibodies used were then washed with TBS, incubated with biotinylated secondary antigoat antibody (BioGenex) for 20 min at room temperature, and finally streptavidin-conjugated horseradish peroxidase for 20 min at room temperature. Peroxidase-staining reaction was performed with 3,3'-diaminobenzidine tetrahydrochloride (3 min) and stopped in tap water. Sections from all tissues were counterstained in Mayer’s hematoxylin (1 min) and covered with coverslips. In negative controls, primary antibodies were omitted. Tissues were photographed with a digital camera (Nikon, Tokyo, Japan) under a microscope (Olympus Tokyo, Japan).

Confocal laser-scanning microscopy
Cells were seeded into 8-well chamber slides coated with poly-L-lysine. At the end of each treatment period, cells were fixed by incubation in 3.7% formaldehyde and permeabilized by 0.2% Triton X-100 before blocking. Subsequently cells were incubated with rhodamine conjugated to phalloidin (Molecular Probe, Inc., Eugene, OR). The coverslips were analyzed using a confocal laser-scanning module (Leica Corp., Lasertechnik, Heidelberg, Germany) attached to an inverted microscope (IM35; Carl Zeiss, Oberkochen, Germany) equipped with an argon-krypton ion laser. Confocal images were acquired using a 63/1.25 oil immersion objective and CLSM software (Leica).

Immunofluorescence
PC12 cells were grown in chamber slides. The cells were fixed and permeabilized by acetone followed by 0.01% Triton X-100 before blocking. For the detection of CRF receptors in the membrane section, cells were not permeabilized before blocking. Subsequently cells were incubated with primary antibodies for 1 h. For preabsorption studies, primary antibodies were incubated with or without the respective blocking peptides overnight at 4 C. Finally, cells were incubated with secondary antibodies and mounted with antifade reagent containing 4',6-diamidino-2-phenylindole (DAPI) for nuclei visualization and covered with coverslips. The slides were photographed under an Olympus microscope with a Leica camera.

Measurement of catecholamines
Cells were grown in six-well plates, coated with poly-L-lysine (PC12 cells) or collagen (human and rat chromaffin cells). After stimulation, the concentration of endogenous catecholamines in the culture media was measured by HPLC; catecholamines were extracted by alumina columns as per the manufacturer’s protocol (Chromsystems, Munich, Germany). Subsequently the extracts were injected into HPLC (Agilent, Palo Alto, CA) equipped with an electrochemical CLC 100 detector (Chromsystems). The signals were recorded in an analog digital converter (HP-35900 C; Hewlett-Packard, Böblingen, Germany) connected to a computer. The concentration of endogenous catecholamines in the culture media was also measured by a highly sensitive RIA (TriCat RIA, RE29395; IBL Immuno Biological Laboratories, Hamburg, Germany) used as previously described (24). Cells were harvested and sonicated for quantification of total cellular proteins as previously described (21). Data are presented as nanograms catecholamines per milligram of protein. No significant difference was found between the mean concentrations of catecholamines measured by the HPLC method and RIA.

RT-PCR analysis for CRF receptors
Total RNA from cells was extracted and processed for the PCR as described previously (24). Each cycle consisted of 30 sec at 94 C, 60 sec at 55 C, and 60 sec at 72 C for 35 cycles to detect the product at the exponential phase of the amplification. The primers for rat CRF₁ were 5'-AGGCGGGATCCAGGCAGTAGAGA-3' (sense) and 5'-TCCCGGTAGCCATTGTTTGTCGTG-3' (antisense), whereas the primers for rat CRF₂ were 5'-CTGGTGGCTGCTTTCCTGGTTTTC-3' (sense) and 5'-ATGGGGCCCTGGTAGATGTAGTCC-3' (antisense). Oligonucleotides were synthesized by MWG-Biotech (Ebersberg, Germany). Ten microliters of the amplified product were separated on a 2% agarose gel and visualized by ethidium bromide staining.

Real-time PCR for TH
For quantitation of rat TH mRNA, cDNA was synthesized and processed as described previously (24). For quantitation of human TH mRNA, cDNA was synthesized and processed as described for rat TH mRNA with some modifications: each cycle consisted of 40 sec at 94 C, 40 sec at 60 C, and 40 sec at 72 C for a maximum of 40 cycles. No by-products were present in the reaction as indicated by the dissociation pattern provided at the end of the reaction and agarose gel electrophoresis (data not shown). The primers for human TH were 5'-CCCTGGTTCCCAAGAAAAGT-3' (sense) and 5'-GCGTGGTGTAGACCTCCTTC-3' (antisense).

Western blot analysis
PC12 cell lysates were electrophoresed through a 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose membranes as described previously (25). Membranes were processed according to standard Western blotting procedures. To detect protein levels, membranes were incubated with anti-TH antibody and then exposed to X-Omat AR films (Kodak, Rochester, NY). A PC-based Image Analysis program was used to quantify the intensity of each band (Image Analysis, Inc., Ontario, Canada). To normalize for protein content, the blots were stripped and stained with antiactin antibody; the concentration of TH was normalized vs. actin.

Statistical analysis
For the statistical evaluation of our data, we used ANOVA, post hoc comparison of means followed by two multiple comparison tests: Fisher’s least significance difference and the Newman-Keuls test. For data expressed as percent changes, compared with control values, we used the nonparametric Kruskal-Wallis test for several independent samples.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mapping of CRF peptides and their receptors in human and rat adrenal medulla.
Immunohistochemical mapping was performed in sections of six nonpathological human and eight nonpathological rat adrenals using antisera against TH, CRF, UCN1, UCN2, CRF₁, and CRF₂ as described in Materials and Methods. Figure 1A (TH panel) depicts chromaffin cells in human sections stained with TH antibody. The adrenal cortex served as control and did not stain with the TH antibody as expected. UCN2 and the CRF₂ receptor were mainly expressed in the adrenal cortex (Fig. 1A, UCN2 and CRF₂ panels), CRF was abundantly expressed in both the adrenal medulla and cortex (Fig. 1A, CRF panel), whereas UCN1 and the CRF₁ receptor were mainly expressed in the adrenal medulla (Fig. 1A, UCN1 and CRF₁ panels). It should be noted that the specificity of the antibodies used was tested by preincubation with the respective peptides as described in Materials and Methods. Preabsorbed antibodies did not stain (Fig. 1A, last row).

View larger version (237K): [in this window] [in a new window]   FIG. 1. Distribution of CRF-related peptides and their receptors CRF1 and CRF2 in human and rat adrenal medulla. Chromaffin cells were identified by anti-TH staining. A, Human adrenal sections were prepared and then stained with anti-TH, UCN2, CRF, UCN1, CRF1, or CRF2 as described in Materials and Methods; TH-positive cells were exclusively localized in the medulla (first line; TH). CRF2 and its endogenous agonist UCN2 were mainly expressed in the cortex (second and sixth lines; UCN2 and CRF2), whereas CRF was abundantly expressed throughout the adrenals (third line; CRF). CRF1 and its endogenous agonist UCN1 were mainly expressed in the medulla (fourth and fifth lines; UCN1 and CRF1). Sections exposed to antibodies preabsorbed with their corresponding peptides revealed no specific staining (seventh line). B, Rat adrenal sections. TH-positive cells were exclusively localized in the medulla (first line; TH). Sections not exposed to primary antibodies (first line; NEG). UCN2, CRF, UCN1, CRF1, and CRF2 were strongly expressed in the medulla (second to sixth lines; UCN2, CRF, UCN1, CRF1, and CRF2). Sections exposed to antibodies preabsorbed with their corresponding peptides revealed no specific staining (seventh line). Photos in A (column a), A (seventh line), and B (column a) were magnified x40, whereas photos in A (columns b–d), B (columns b–d), and B (seventh line) were magnified x250. Med, Medulla; retic, reticularis; fasc, fasciculate; glom, glomerulosa; caps, capsule.

View larger version (31K): [in this window] [in a new window]   FIG. 2. CRF peptides and their receptors CRF1 and CRF2 in PC12 cells. A, PC12 cells were fixed, permeabilized, and then stained with antibodies against UCN2, CRF, and UCN1 as described in Materials and Methods. DAPI was used for the visualization of cells nuclei. Cells not exposed to first antibody (NEG). B, PC12 cells were fixed, permeabilized, and then stained with antibodies against CRF1 (first line; green dye), CRF2 (second line; green dye), and TH (second column; red dye) or stained with DAPI (third column; blue dye). Fourth column depicts cells triple stained with CRF1/TH/DAPI (first line) or CRF2/TH/DAPI (second line). C, PC12 cells expressed CRF1 and CRF2 in the membrane section. Cells were fixed and then stained with antibodies against CRF1 or CRF2. Cells not exposed to first antibody (NEG). D, RT-PCR analysis of the expression of CRF1 and CRF2 receptors in PC12 cells. Rat brain was used as positive control. No PCR product was detected in the negative control samples. The results were representatives of two independent experiments.

To determine the optimal time point for the acute effect of CRF peptides, rat chromaffin cells were stimulated with the CRF₁ and CRF₂ receptor agonists CRF or UCN1 at 10^–7 M and NE release was measured (Fig. 3). More specifically, exposure of rat chromaffin cells to CRF or UCN1 at 10^–7 M for 30 min induced NE secretion to 36.8 ± 3.1 ng NE per milligram of protein or 37.3 ± 4.4 ng NE per milligram of protein, compared with 20.9 ± 0.5 ng NE per milligram of protein of control cells, respectively. Time-response experiments were also performed for CRF and UCN1 on E (human and rat chromaffin cells) or dopamine (DA) (PC12 cells) secretion. The resulting curves were similar to those observed for NE (rat chromaffin cells), and they are not shown here.

View larger version (8K): [in this window] [in a new window]   FIG. 3. Time-dependent effects of UCN1 and CRF on NE secretion from dispersed rat chromaffin cells. Dispersed rat chromaffin cells were incubated with UCN1 or CRF at several time points and NE secretion was measured. UCN1 or CRF at 10–7 M induced NE secretion at 30 min. Data are expressed as nanogram NE per milligram of protein (mean ± SE, n = 11 of four independent experiments for CRF, n = 6 of three independent experiments for UCN1). *, P < 0.05 denotes significant statistical difference, compared with parallel controls.

CRF₂ agonist.
Figure 4, A and B, depict the inhibitory effect of UCN2 on NE and E secretion in rat chromaffin cells. Exposure of rat chromaffin cells to UCN2 at 10^–10 M for 30 min suppressed NE secretion by 44 ± 9% and E secretion by 38 ± 7%, compared with parallel controls. Moreover, exposure of human chromaffin cells to UCN2 in doses ranging from 10^–8 to 10^–7 M for 30 min significantly suppressed E secretion below control levels (Fig. 4C). Furthermore, exposure of PC12 cells to UCN2 in doses ranging from 10^–10 to 10^–8 M for 30 min significantly suppressed DA secretion below control levels (Fig. 4D).

View larger version (16K): [in this window] [in a new window]   FIG. 4. UCN2 suppressed whereas cortagine stimulated acute catecholamine secretion from dispersed rat and human chromaffin and PC12 cells. Rat and human chromaffin and PC12 cells were incubated with UCN2 or cortagine at 10–10 to 10–7 M for 30 min, and NE (rat chromaffin cells), E (rat chromaffin cells), E (human chromaffin cells), or DA (PC12 cells) were measured. UCN2 suppressed catecholamine secretion below control levels in both rat and human chromaffin as well as PC12 cells (A–D). In contrast, cortagine induced catecholamine secretion above control levels in both rat and human chromaffin and in PC12 cells (E–H). Data are expressed as percentage change, compared with control values (mean ± SE, n = 3 of two independent experiments in rat and human primary cell cultures and n = 4 of two independent experiments in PC12 cell cultures). *, P < 0.05 denotes significant statistical difference, compared with parallel controls.

Effects of nonselective CRF receptor ligands on catecholamine secretion
Exposure of rat chromaffin cells to UCN1 at 10^–7 M significantly induced NE secretion at 30 min by 78 ± 3%, compared with parallel controls (Fig. 5A). Furthermore, exposure of rat chromaffin cells to UCN1 at 10^–10 M significantly suppressed E secretion by 24 ± 8%, compared with parallel controls, whereas at 10^–7 M, it induced E secretion by 78 ± 4%, compared with parallel controls (Fig. 5B). In human chromaffin cells, UCN1 at all doses tested significantly reduced E secretion (Fig. 5C). In PC12 cells, UCN1 at 10^–8 M significantly reduced DA secretion by 32 ± 8%, compared with parallel control cells (Fig. 5D).

View larger version (17K): [in this window] [in a new window]   FIG. 5. Dose-dependent effect of UCN1 and CRF on catecholamine secretion from dispersed rat and human chromaffin and PC12 cells. Rat and human chromaffin and PC12 cells were incubated with UCN1 or CRF at 10–10 to 10–7 M for 30 min, and NE (rat chromaffin cells), E (rat chromaffin cells), E (human chromaffin cells), or DA (PC12 cells) were measured. UCN1 and CRF affected catecholamine secretion in a dose-dependent and tissue-specific manner. Data are expressed as percentage change, compared with control values (mean ± SE, n = 6 of three independent experiments in rat primary cell cultures, n = 3 of two independent experiments in human primary cell cultures, and n = 4 of two independent experiments in PC12 cell cultures). *, P < 0.05 denotes significant statistical difference, compared with parallel controls.

Effect of CRF receptor antagonists
To confirm the specificity of each receptor signal, we exposed PC12 cells to CRF receptor agonists in the absence or presence of antalarmin (a CRF₁ antagonist) or antisauvagine-30 (a CRF₂ antagonist) for 30 min, and DA secretion was measured (Fig. 6A). Indeed, the inhibitory effect of UCN2 at 10^–10 M on DA secretion was blocked by antisauvagine-30 at 10^–8 M and not antalarmin at 10^–8 M. The stimulatory effect of cortagine at 10^–8 M on DA secretion was blocked by antalarmin at 10^–6 M and not antisauvagine-30 at 10^–6 M. The effect of UCN1 at 10^–8 M on DA secretion was blocked by antisauvagine-30 at 10^–6 M and not antalarmin at 10^–6 M. The stimulatory effect of CRF at 10^–8 M on DA secretion was blocked by both antalarmin at 10^–6 M and antisauvagine-30 at 10^–6 M. Similar results were obtained in primary rat and human chromaffin cells (data not shown).

View larger version (19K): [in this window] [in a new window]   FIG. 6. Differential effects of CRF1 and CRF2 ligands on catecholamine secretion and actin polymerization in rat chromaffin and PC12 cells. A, Cells were exposed to UCN2 (10–10 M), cortagine (Cort; 10–8 M), UCN1 (10–8 M), and CRF (10–8 M) with or without antalarmin (antal) or antisauvagine-30 (a-sav) for 30 min, and DA was measured. CRF1 and CRF2 antagonists blocked the effects of CRF receptor ligands on DA secretion in PC12 cells. B, Cells were exposed to CRF (10–7 M) with or without phallacidin (Phal) for 30 min, and E secretion was measured. The actin filament stabilizer phallacidin blocked the stimulatory effect of CRF on E secretion at 30 min in rat chromaffin cells. Data are expressed as a percentage of parallel controls exposed only to vehicles (mean ± SE, n = 3 of two independent experiments). *, P < 0.05 or **, P < 0.01 denote significant statistical difference, compared with parallel controls, whereas #, P < 0.05 denotes significant statistical difference, compared with cells exposed to CRF peptides alone. C, PC12 cells were exposed to CRF, cortagine (Cort), UCN1, and UCN2 at 10–8 M for 30 min and subsequently were fixed, permeabilized, and stained with rhodamine-phalloidin as described in Materials and Methods. con, Control; neg, cells were fixed, permeabilized without rhodhamine-phalloidin staining. The photos are representative of two independent experiments.

CRF receptor ligands affect catecholamine synthesis and the TH transcript and protein levels
Long-term effects of selective CRF receptor agonists
CRF₂ agonist.
Exposure of rat chromaffin cells to the predominantly CRF₂ agonist UCN2 at 10^–9 M for 48 h stimulated NE by 187 ± 19% and E by 461 ± 120%, compared with parallel controls (Fig. 7, A and B). Exposure of human chromaffin cells to UCN2 at 10^–9 M reduced E by 55 ± 4%, compared with controls (Fig. 7C). Exposure of PC12 cells to UCN2 for 48 h induced DA in doses ranging from 10^–10 to 10^–9 M (Fig. 7D).

View larger version (17K): [in this window] [in a new window]   FIG. 7. Effects of UCN2 and cortagine on catecholamine synthesis by dispersed rat, human chromaffin, and PC12 cells. Cells were exposed to UCN2 (A–D) or cortagine (E–H) at 10–10 to 10–7 M at 48 h and NE (rat chromaffin cells), E (rat chromaffin cells), E (human chromaffin cells), or DA (PC12 cells) were measured. UCN2 and cortagine induced catecholamine synthesis in rat chromaffin and PC12 cells, in a dose-dependent manner. Exposure of dispersed human chromaffin cells to cortagine induced catecholamine synthesis, whereas exposure to UCN2 suppressed catecholamine synthesis. Data are expressed as percentage of parallel controls exposed only to vehicles (mean ± SE, n = 3 of two independent experiments). *, P < 0.05 denotes significant statistical difference, compared with parallel controls.

Long-term effects of nonselective CRF receptor agonists
UCN1 at 10^–10 to 10^–7 M significantly induced NE and E production at 48 h. UCN1 at 10^–10 M induced NE and E by 226 ± 43 and by 564 ± 14%, respectively, compared with parallel controls (Fig. 8, A and B). Exposure of human chromaffin cells to UCN1 at 10^–9 M significantly induced E by 22 ± 2%, compared with parallel controls (Fig. 8C), whereas at 10^–8 M, it reduced E production by 66 ± 1%, compared with parallel controls. In PC12 cells, UCN1 at 10^–7 M significantly induced DA by 76 ± 21%, compared with parallel controls (Fig. 8D).

View larger version (17K): [in this window] [in a new window]   FIG. 8. Dose-dependent effects of UCN1 and CRF on catecholamine production from dispersed rat and human chromaffin and PC12 cells. Rat and human chromaffin and PC12 cells were exposed to UCN1 (A–D) or CRF (E–H) at 10–10 to 10–7 M for 48 h, and NE (rat chromaffin cells), E (rat chromaffin cells), E (human chromaffin cells), or DA (PC12 cells) were measured. Data are expressed as percentage change, compared with parallel controls (mean ± S E, n = 6 of three independent experiments in rat primary cell cultures, n = 3 of two independent experiments in human primary cell cultures, and n = 4 of two independent experiments in PC12 cell cultures). *, P < 0.05 denotes significant statistical difference, compared with parallel controls.

Effect of CRF, UCN1, UCN2, and cortagine on TH expression
The rate-limiting step in the biosynthesis of catecholamines is TH. Figure 9A depicts the quantification of TH transcript by real-time PCR analysis of dispersed human chromaffin cells exposed to UCN2 or cortagine for 24 h. Data are presented as fold difference, compared with controls. The presence of UCN2 at 10^–9 M for 24 h in the media reduced TH mRNA levels in a statistically significant manner by 0.5 ± 0.1-fold, compared with parallel controls, whereas the presence of cortagine at 10^–9 M elevated TH mRNA levels by 2.0 ± 0.77-fold, compared with the parallel controls. Similar results were observed at the protein level, as determined by Western blot analysis (data not shown). Exposure of PC12 cells to CRF peptides affected TH mRNA levels as measured by real-time PCR analysis (Fig. 9B). Exposure of UCN2 at 10^–10 M, UCN1 at 10^–7 M, or CRF at 10^–10 M for 6 h induced TH mRNA levels by 0.8 ± 0.3-, 1.1 ± 0.3-, or 2.6 ± 1.4-fold, compared with parallel controls. Similar results were observed at the protein level, as determined by Western blot analysis. Thus, exposure of PC12 cells for 24 and 48 h to UCN2, UCN1, or CRF resulted in increased TH protein levels (Fig. 9C).

View larger version (39K): [in this window] [in a new window]   FIG. 9. Differential effects of UCN1, UCN2, CRF, cortagine, and antagonists on TH transcript and protein levels. A, Human chromaffin cells were incubated for 24 h in the presence of 10–9 M UCN2 or cortagine (Cort) , and the mRNA levels of TH were measured in cell extracts with real-time PCR. Data are presented as fold change, compared with controls (con). B, PC12 cells were incubated for 3–6 h in the presence of 10–10 M UCN2, 10–7 M UCN1, or 10–10 M CRF, and the mRNA levels of TH were measured in cell extracts with real-time PCR. Data are presented as fold change, compared with controls. C, PC12 cells were incubated for 6–48 h in the presence of 10–10 M UCN2, 10–7 M, UCN1 or 10–10 M CRF and protein levels of TH were measured in cells extracts by Western blot analysis. Levels of the TH protein were normalized against actin presented as TH to actin ratio. PC12 cells were exposed for 48 h to UCN1 with or without antisauvagine-30 (a-sav) or antalarmin (antal), and the DA was measured by HPLC (D) and the protein levels of TH by Western blotting (E). Data are expressed as percentage change, compared with parallel controls (con), mean ± SE, n = 4 of two independent experiments in human primary cell cultures, n = 6 of three independent experiments in TH mRNA and protein levels in PC12 cell cultures, and n = 3 of two independent experiments in DA measurements in PC12 cell cultures). **, P < 0.01 denotes significant statistical differences compared with parallel controls, whereas #, P < 0.05 denotes significant statistical difference, compared with cells exposed to UCN1 alone.

We further confirmed our results by measuring the effect of UCN1 on TH production in PC12 cells in the absence or presence of CRF₁ and CRF₂ antagonists. Cells were incubated for 48 h with 10^–7 M UCN1 in the presence or absence of antisauvagine-30 or antalarmin, and protein levels of TH were measured in cell extracts by Western blot analysis (Fig. 9E). The stimulatory effects of UCN1 in TH production were blocked by the CRF₁ and CRF₂ antagonists.

To confirm the data suggesting that CRF-related peptides affected the biosynthetic process of catecholamines, we exposed PC12 cells to CRF-related peptides in the presence or absence of AMPT (a tyrosine hydroxylase inhibitor) or NSD-1015 (an L-aromatic amino acid decarboxylase inhibitor). Cells were exposed to 10^–10 M UCN2, 10^–8 M cortagine, 10^–7 M UCN1, or 10^–10 M CRF for 48 h with or without preincubation with 10^–6 M AMPT or NSD-1015 for 1 h. The results showed that either AMPT or NSD-1015 blocked the stimulatory effect of CRF-related peptides on DA secretion (Fig. 10). Indeed, the stimulatory effect of UCN2 at 10^–10 M on DA was blocked by AMPT or by NSD-1015. Similarly, the effect of cortagine at 10^–8 M on DA was also blocked by AMPT or NSD-1015. Moreover, the stimulatory effect of UCN1 at 10^–7 M on DA was blocked by AMPT or NSD-1015. Finally, the stimulatory effect of CRF at 10^–10 M on DA was blocked by AMPT or NSD-1015.

View larger version (13K): [in this window] [in a new window]   FIG. 10. Effect of CRF-related peptides on the pathway of catecholamine biosynthesis. Cells were exposed to 10–10 M UCN2, 10–8 M cortagine (Cort), 10–7 M UCN1, or 10–10 M CRF with or without AMPT at 10–6 M or NSD-1015 (NSD) at 10–6 M for 48 h. The effects of CRF-related peptides were completely blocked by either AMPT or NSD-1015. Data are expressed as a percentage change, compared with parallel controls exposed only to vehicles (mean ± SE, n = 6 of three independent experiments). **, P < 0.01 denotes significant statistical difference, compared with parallel controls, whereas #, P < 0.05 denotes significant statistical difference, compared with cells exposed to CRF peptides alone.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our functional studies on dispersed rat and human adrenal chromaffin cells supports the above mentioned hypothesis, i.e. that the UCN1/CRF/CRF₁ receptor system plays a different physiological role, compared with that of UCN2/CRF₂ receptor system. Indeed, activation of the CRF₁ receptor resulted in induction of catecholamine secretion, an effect peaking at 30 min, whereas activation of the CRF₂ receptor suppressed catecholamine secretion. It should be mentioned here that human and rat chromaffin and PC12 cells exhibited the same responses, indicating that this hypothesis may be valid across species. This hypothesis was further supported by additional experiments using selective CRF receptor antagonists in which specific blockade of CRF₁ or CRF₂ receptors confirmed the receptor-type specificity of the effect on catecholamine secretion. Thus, the inhibitory effect of UCN2 was blocked by antisauvagine-30 (a specific CRF₂ antagonist) but not by antalarmin (a nonpeptide CRF₁ antagonist), whereas the stimulatory effect of cortagine on catecholamine secretion was blocked by antalarmin but not by antisauvagine-30.

The acute effects of CRF peptides on catecholamine secretion appear to be mediated by changes of actin filament polymerization, facilitating the exocytosis of the catecholamine secretory vesicles. PC12 cells exposed to CRF₁ ligands showed a broken subplasmalemmal ring of actin filaments with areas of filamentous actin disassembly, whereas cells exposed to CRF₂ ligands showed an enhanced filamentous actin assembly in either the subplasmalemmal area beneath the cell membrane or the cytoplasmic section. In rat chromaffin cells, phallacidin, an actin filament stabilizer, blocked CRF₁-induced catecholamine secretion. These data taken together suggest that the effect of CRF₁ or CRF₂ ligands on catecholamine secretion involved changes of actin filament polymerization. It should be noted that induction of the severing protein 14–3-3 or sciderin results in an acute depolymerization of acting and to a parallel elevation of catecholamine secretion from chromaffin cells (27, 28).

The effects of CRF₁ and CRF₂ receptor agonists were dose and receptor affinity specific. More specifically, the peak suppressive effect of UCN2 on NE/E secretion from rat chromaffin cells occurred at 10^–10 M. At higher concentrations (10^–8 to 10^–7 M) UCN2 binds also to the CRF₁ receptor (IC₅₀ of 350 nM, compared with 0.25 nM of CRF₂), thus attenuating its suppressive effect due to the antagonistic effect of CRF₁ activation. The same holds true for the synthetic CRF₁ receptor agonist cortagine. Indeed, its peak effect occurred between 10^–10 to 10^–9 M, at a concentration at which is a pure CRF₁ receptor agonist, whereas at higher concentration (10^–8 to 10^–7 M), it starts binding the CRF₂ receptor (IC₅₀ 2.6 nM for CRF₁ receptor and 540 nM for CRF₂ receptor (29)], thus attenuating its stimulatory effect. It should be noted that the binding affinity of UCN1 to CRF₁ is 0.17 nM and to CRF₂ 0.86 nM; the binding affinity of CRF to CRF₁ is around 1.6 nM and to CRF₂ 42 nM (30). CRF and UCN1 decreased E levels in human chromaffin cells, whereas they induce NE and DA in rat chromaffin and PC12 cells. The observed discrepancy between human and rat adrenal chromaffin cells in their response to CRF or UCN1 may be due to different stoichiometry of the two receptors present in human and rat chromaffin cells at the time of the experiment or difference in desensitization mechanisms between the two species. Accordingly, the net effect of UCN1 or CRF on catecholamine secretion depends on the local availability of the receptors at the time of their exposure to these peptides as well as their local concentration.

CRF peptides also affected catecholamine synthesis, an effect mediated by TH in both human and rodent adrenals. Indeed, in human chromaffin cells, the inhibitory effect of UCN2 on TH mRNA levels at 24 h resulted in inhibition of catecholamine production at 48 h, whereas cortagine induced both the TH mRNA levels and catecholamine production. These results indicate that the effect of CRF-related peptides on catecholamine production in humans appears to involve activation of catecholamine biosynthetic pathway. In dispersed rat adrenal chromaffin and PC12 cells, all tested CRF agonists, irrespective of their affinity to the type of CRF receptor, elevated the TH mRNA levels at 6 h and TH protein levels at 24–48 h and finally catecholamines at 48 h. The effect of UCN1 on TH protein levels and catecholamines was abolished by both CRF₁ and CRF₂ antagonists. Interestingly, antisauvagine-30 suppressed the effect of UCN1 on TH protein levels below control values in PC12 cells. These results suggest the presence and possible secretion of endogenous CRF receptor agonists. Indeed, UCN1, CRF, and UCN2 were present in our PC12 cells as revealed by the immunofluorescence studies. While we were working on this part of the work, a published report corroborated our data showing that UCN1 induced the expression of TH in rat pheochromocytoma PC12 cells, without altering catecholamine production, an effect blocked by -helical CRF-(9–41) (17). However, our data are not directly comparable with that of Nanmoku et al. (17) because they use a slightly different cell line, the PC12 subclone RCB009, which does not express the CRF₁ and CRF₂ receptors but expresses only the CRF_2ß receptor. Our PC12 cells express both CRF₁ and CRF₂ receptors as per our immunocytochemistry and RT-PCR data. In addition, Nanmoku et al. report that 8 h incubation with UCN1 induced TH mRNA levels in PC12 cells, but they do not indicate the time points of their measurement of catecholamines. It is thus possible that they have missed the window during which catecholamine production is induced. Indeed, our experiments showed that there is a time-dependent fluctuation in catecholamines’ production. Furthermore, Nanmoku et al. measured catecholamines levels using an HPLC system equipped with a spectrofluorometer, a detector that is less sensitive, compared with electrochemical detection that we have in this study.

To confirm our data, we also used the tyrosine hydroxylase inhibitor, AMPT, and the L-aromatic amino acid decarboxylase inhibitor, NSD-1015. They both inhibited the effect of CRF agonists on catecholamine synthesis, further strengthening our hypothesis that the effect of CRF₁ and CRF₂ agonists on catecholamines at 48 h involved TH activation, in agreement with data obtained from CRF null mice in which the expression of TH and PNMT in their adrenal medulla was lower, compared with CRF(+/+) mice (18). Similar data have been reported for CRF₁ null mice in which the levels of E and PNMT are lower, compared with their wild-type counterparts (20). Thus, our study corroborates published reports confirming the close association between CRF peptides and induction of TH. It is possible that prolonged exposure to stress peptides during chronic stress results in induction of TH synthesis and thus elevation of stored E in the secretory vesicles of adrenal medullary cells augmenting or suppressing the magnitude of catecholaminergic response toward prolonged threats to the internal milieu.

Finally, regarding the discrepancy between the histochemical data of the UCN2/CRF₂ system in human adrenal medulla in which its staining was very low, compared with the CRF/UCN1/CRF₁ system in both human and rat adrenal medulla and the intensity of their biological effects, we propose two possible explanations. It is quite possible that the concentration of CRF₂ receptors in the human chromaffin cells is sufficient enough for them to have a measurable biological effect despite the lower histochemical staining or that the process of dispersion and in vitro culture of human chromaffin cells somehow induces their expression of CRF₂ receptors. It should be mentioned here that such changes in the proportion of receptor subtype expression with culture have been shown for a number of receptors including, adrenergic and angiotensin receptors. Indeed, primary cultures of adult rat hepatocytes showed that the ß-adrenergic response and its receptor number increased markedly during short-term culture (31). It seems that this heterogeneity in the number of receptor is influenced by specific cellular contacts in vivo (32).

In conclusion, our data suggest that a complex intramedullary CRF-UCN/CRF receptor system exists in both human and rat adrenals, controlling catecholamine synthesis and secretion. Activation of either the CRF₁ or CRF₂ receptors results in opposing effects on catecholamine secretion. Thus, the local concentration of CRF peptides in the adrenals, and perhaps also in the brain, may affect catecholamine secretion and synthesis under a complex mechanism that depends on the ad hoc concentration of ligands and receptor availability. Our data, validated in primary human and rat chromaffin cells, highlight a potential difference between primates and rodents in the local regulation of catecholamine secretion.

	Footnotes

 
This work was supported by the Hellenic Secretariat for Research and Technology (ENE 01E386) and a grant from Medicon Hellas Co.

Disclosure Statement: E.D., C.T., V.M., E.C., I.C., M.V., A.A., M.L., J.S., E.M., A.G., and A.N.M. have nothing to declare.

First Published Online December 28, 2006

Abbreviations: AMPT, L-2-Methyl-3-(-4hydroxyphenyl)-alanine; CRF, corticotropin-releasing factor; CRF₁, CRF receptor type 1; CRF₂, CRF receptor type 2; DA, dopamine; DAPI, 4',6-diamidino-2-phenylindole; E, epinephrine; FCS, fetal calf serum; HS, horse serum; NE, norepinephrine; NSD-1015, 3-(hydrazinomethyl)-phenol hydrochloride; PNMT, phenylethanolamine N-methyltransferase; TBS, Tris and NaCl; TH, tyrosine hydroxylase; UCN1, urocortin I; UCN2, urocortin II.

Received July 19, 2006.

Accepted for publication December 20, 2006.

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