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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 4 1897-1903
Copyright © 2004 by The Endocrine Society

Expression of Urocortin III/Stresscopin in Human Heart and Kidney

Kazuhiro Takahashi, Kazuhito Totsune, Osamu Murakami, Masayuki Saruta, Masao Nakabayashi, Takashi Suzuki, Hironobu Sasano and Shigeki Shibahara

Departments of Molecular Biology and Applied Physiology (K.Ta., S.S.), Medicine (O.M.), and Pathology (M.S., M.N., T.S., H.S.), Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8575, Japan; and Department of Clinical Pharmacology and Therapeutics (K.To.), Tohoku University Graduate School of Pharmaceutical Science and Medicine, Aoba-ku, Sendai, Miyagi 980-8578, Japan

Address all correspondence and requests for reprints to: Kazuhiro Takahashi, M.D., Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. E-mail: ktaka-md{at}mail.tains.tohoku.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Urocortin III (Ucn III)/stresscopin (SCP) is a novel peptide of the corticotropin-releasing factor (CRF) family and is a specific ligand for the CRF type 2 receptor. We wished to clarify whether Ucn III/SCP is expressed in the human heart and kidney. Immunoreactive Ucn III was detected by RIA in the human heart (0.74–1.15 pmol/g wet weight, mean ± SEM; n = 4) and kidney (1.21 ± 0.30 pmol/g wet weight), which were obtained at autopsy. These levels were comparable to the levels in pituitary (2.72 ± 0.13 pmol/g wet weight; n = 3) and brain tissues (~1–2 pmol/g wet weight). Furthermore, immunoreactive Ucn III was present in human plasma (51.8 ± 16.0 pmol/liter; n = 5) and urine (266 ± 20 pmol/liter; n = 5) obtained from healthy subjects. Reverse-phase HPLC showed a broad peak of immunoreactive Ucn III eluting in the position of synthetic Ucn III in the heart, kidney, and hypothalamus. Material eluting in the position of SCP was also found in the HPLC analysis of these tissue extracts. Immunocytochemistry showed positive staining of Ucn III in the myocardium and the renal tubules, particularly distal tubules. RT-PCR showed expression of Ucn III mRNA in the brain, pituitary, heart, and kidney. The present study has shown expression of Ucn III/SCP in the human heart and kidney as well as brain and pituitary tissues and its presence in plasma and urine. Ucn III/SCP may therefore regulate the cardiac and renal function as a local factor and a circulating hormone.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE CORTICOTROPIN-RELEASING factor (CRF) family consists of CRF, urocortin (Ucn), Ucn II (stresscopin-related peptide), and Ucn III [stresscopin (SCP)] as well as fish urotensin I and frog sauvagine. Ucn III is a novel member of the CRF family and is a specific agonist for CRF type 2 receptor (CRF2 receptor) (1). Ucn III was identified by searching the public human genome databases with significant sequence identity to Ucn II (2). Another group reported SCP, a peptide derived from the same gene as Ucn III (3). Different interpretation of posttranslational processing sites resulted in slightly different amino acid sequences of the peptides; human Ucn III is a 38-amino-acid peptide that corresponds to the sequence 3–40 of human SCP, a 40-amino-acid peptide.

The actions of the CRF-family peptides are mediated by at least two types of G protein-coupled receptors: CRF type 1 receptor (CRF1 receptor) and CRF2 receptor. CRF1 receptor mediates ACTH responses to stress, whereas CRF2 receptor mediates stress-coping responses including anxiolysis, anorexia, vasodilatation, a positive inotropic action on myocardium, and dearousal. CRF and Ucn bind to both CRF1 receptor and CRF2 receptor, whereas Ucn II and Ucn III are specific ligands for CRF2 receptor.

It has been reported that Ucn III is expressed in the brain as well as in the peripheral tissues such as gastrointestinal tract, pancreatic islets, and skin (1, 3, 4, 5). In the rat brain, Ucn III-positive neurons were found predominantly within the hypothalamus and medial amygdala, and the Ucn III fibers were distributed mainly in the hypothalamus and limbic structures (4). CRF2 receptor is expressed in the heart (6, 7). We and other investigators have recently shown that Ucn is expressed in the heart and cardiomyocytes (7, 8, 9) and proposed that Ucn is an endogenous physiological ligand for CRF2 receptor in the heart. RT-PCR analysis showed that Ucn III (SCP) mRNA was expressed in the human heart and kidney as well as other peripheral tissues (3), whereas the RNase protection assay could not detect it in the mouse heart (1). Thus, Ucn III expression in the cardiovascular tissues has not been clarified. Furthermore, Ucn was expressed in human pituitary (10), whereas Ucn III mRNA was not detected in mouse pituitary by the RNase protection assay (3). It has not been determined whether the Ucn III mRNA generates Ucn III or SCP in human tissues. We therefore wished to clarify whether Ucn III/SCP is expressed in human heart and kidney as well as brain and pituitary tissues.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

This study has been approved by the Ethics Committee of Tohoku University School of Medicine. Human heart, kidney, brain, and whole pituitary tissues were obtained at autopsy performed at the Department of Pathology, Tohoku University Hospital, within 4 h postmortem. Human brain tissues and pituitaries were obtained from eight patients (four male and four female, 31–70 yr old). These patients had neither neurological nor endocrinological diseases. Brain tissues (~1 g per sample) were dissected out from frontal lobe, temporal lobe, occipital lobe, thalamus, hypothalamus, medulla oblongata, pons, hippocampus, and cerebellum (hemisphere and vermis), which had no pathological abnormalities macroscopically. Heart and kidney tissues were obtained from four subjects without cardiac or renal diseases (three male and one female, 21–75 yr old). Lung (n = 2), pancreas (n = 2), liver (n = 2), spleen (n = 2), and skeletal muscle (psoas muscle, n = 5) were obtained at autopsy via the same methodology. The tissues were immediately frozen and stored at –80 C before extraction of peptide and RNA. Heart tissues obtained at autopsy (n = 4) and kidney tissues obtained at surgery from patients with renal cell carcinoma (n = 2) were used for immunocytochemistry. The tissues were fixed in 4% formalin and embedded into paraffin. Informed consent was obtained from the family of the subjects in case of autopsy or from the subjects in case of surgery.

Plasma and urine samples were obtained from five male healthy subjects (18–25 yr old). Informed consent was obtained from each subject. Blood samples were obtained from a sc vein in the forearm, collected into tubes containing aprotinin (Trasylol; 500 kallikrein inhibitory U/ml blood; Bayer, Leverkusen, Germany) and EDTA (1 mg/ml of blood), and centrifuged at 4 C. The plasma and urine samples were stored at –30 C until extraction.

Peptide extraction and RIA

Tissues were extracted, as reported previously (11). Briefly, the tissue (~750 mg) was boiled in 2 ml of 1 mol/liter acetic acid for 10 min. Eight milliliters of 50% methanol in 0.5 mol/liter acetic acid was added to each sample, and the tissue was homogenized. The homogenate was centrifuged at 15,000 x g for 30 min. The supernatant was separated, dried by air, reconstituted in assay buffer [0.1 mol/liter phosphate buffer (pH 7.5) containing 0.1% (wt/vol) BSA, 0.2% (vol/vol) Triton X-100, and 0.1% (wt/vol) sodium azide], and assayed.

Plasma and urine samples were extracted with Sep-Pak C18 cartridges (Waters, Milford, MA). Plasma (2 ml) or urine (5 ml) samples were acidified with the same volume of 0.75 mol/liter acetic acid and loaded onto cartridges, which were pretreated with 10 ml of 100% acetonitrile, 10 ml of 100% methanol, and then 10 ml of 0.75 mol/liter acetic acid. After the cartridges were washed by 10 ml of 0.75 mol/liter acetic acid, peptides were eluted with 2 ml of 60% (vol/vol) acetonitrile containing 0.1% (vol/vol) trifluoroacetic acid (TFA). The eluate was dried by air, reconstituted in assay buffer, and assayed. The recovery, which was determined by adding synthetic human Ucn III to plasma or urine before the extraction (100 fmol Ucn III/ml of plasma or urine), yielded 41.7 ± 6.0% (plasma) (mean ± SD; n = 3) and 65.7 ± 3.8% (urine), respectively.

The antiserum against human Ucn III was raised in a rabbit by injecting tyrosyl-Ucn III (Sawady Technology, Tokyo, Japan; custom synthesis) conjugated with BSA (Sigma Chemical Co., St. Louis, MO) by carbodiimide (Peptide Institute, Osaka, Japan). Human Ucn III (Phoenix Pharmaceuticals, Inc., Belmont, CA) was used as a standard. [125I]Tyrosyl-Ucn III prepared by the chloramine T method was used as a radioligand.

The assay was performed in 400 µl assay buffer. The sample or the standard peptide (200 µl) was incubated with antiserum (100 µl) at 4 C for 48 h. The antiserum was used at a final dilution of 1:24,000. [125I]Tyrosyl-Ucn III (~4000 cpm/100 µl) was then added to each sample, followed by an additional 48-h incubation at 4 C. The immune complex was precipitated by adding 100 µl of antirabbit IgG raised in goat (Phoenix) and 500 µl of 10% (wt/vol) polyethylene glycol in water. After a 5-h incubation, the sample was centrifuged at 3000 x g for 30 min and the supernatant was separated. The pellets were counted by the {gamma}-counter.

The assay could detect changes of 4.7 ± 0.3 fmol/tube (mean ± SD; n = 8) from zero at 95% confidence with duplicate tubes. The cross-reactivities were 100% with human SCP (Peptide Institute) but less than 0.001% with other peptides including human CRF, Ucn, human SCP-related peptide (Peptide Institute), neuropeptide Y, endothelin-1, arginine vasopressin, oxytocin, substance P, and vasoactive intestinal polypeptide. Intra- and interassay coefficients of variation were 4.4 and 9.7%, respectively.

Chromatographic characterization of tissue extracts was performed by reverse-phase HPLC using a µBondapak C18 column (3.9 x 300 mm, Waters). The tissue extract was reextracted with a Sep-Pak C18 cartridge (Waters), reconstituted in 0.1% (vol/vol) TFA, and loaded onto the column. The HPLC was performed with a linear gradient of acetonitrile containing 0.1% (vol/vol) TFA from 10–60% at a flow rate of 1 ml/min·fraction over 50 min. Each fraction (1 ml) was collected, dried by air, reconstituted with assay buffer, and assayed.

Immunocytochemistry

Immunocytochemistry was performed by the ABC method using the Vector ABC kit (Vector Laboratories, Burlingame, CA), as previously reported (12). Briefly, 4-µm sections were deparaffinized and incubated in methanol containing 0.3% H2O2 for 30 min and then with normal goat serum (1:20) to block nonspecific staining. Sections were intensely washed in 0.01 mol/liter PBS (pH 7.4) between the procedures. Sections were incubated in antiserum against human Ucn III (1:1000) for 20 h at 4 C. The antiserum against human Ucn III used in RIA was also used in immunocytochemistry. Sections were incubated in biotinylated secondary antibody to rabbit IgG (1:200) for 30 min at room temperature and subsequently incubated with peroxidase-conjugated avidin for 30 min using the Vector ABC kit. These sections were visualized by immersion in 3,3'-diaminobenzidine solution [0.01 mol/liter 3,3'-diaminobenzidine in 0.05 mol/liter Tris-HCl buffer (pH 7.6) and 0.006% H2O2].

In negative controls, Ucn III antiserum preabsorbed with synthetic human Ucn III or normal rabbit serum (at a dilution of 1:1000) was used instead of the Ucn III antiserum. An absorption test for Ucn III immunoreactivity was performed using the antiserum incubated with synthetic human Ucn III (10 nmol peptide/ml diluted antiserum) for 20 h at 4 C before use.

RT-PCR

Total RNA was extracted from tissues by the guanidine thiocyanate-cesium chloride method. Total RNA (6 µg) was treated with 1 U of RNase-free DNase (Promega, Madison, WI) for 15 min at 37 C to eliminate contaminated genomic DNA. It was then denatured at 65 C for 5 min and transcribed at 37 C for 60 min in a reaction mixture (20 µl) containing 0.75 µg oligo-dT, 0.5 mmol/liter dNTP, and 400 U of Moloney murine leukemia virus reverse transcriptase (BRL, Gaithersburg, MD). The reaction was stopped by heating at 95 C for 5 min, diluted with 30 µl water, and stored at –20 C until PCR analysis. One microliter of the reaction mixture was subjected to PCR. The PCR was performed in a total volume of 20 µl containing 0.2 mmol/liter of each dNTP, 0.25 µmol/liter of each primer, and 0.5 U Taq DNA polymerase (Pharmacia, Piscataway, NJ).

The sense primer was 5'-TGATGCCGGTCCACTTCCTG-3' (nucleotide numbers 5–24), and the antisense primer was 5'-CCAATTTGCGCCATCAGGTG-3' (complementary to 454–473) (GenBank accession no. AF361943) (1). After heating at 96 C for 2 min, denaturation, annealing, and elongation were carried out at 96 C for 15 sec, 66 C for 30 sec, and 72 C for 60 sec, respectively, and the reactions were repeated for 35 cycles, followed by 72 C for 5 min. Total RNA samples treated with water instead of reverse transcriptase were used as negative controls. Amplification products were visualized on a 5% polyacrylamide gel stained with ethidium bromide and viewed on an UV box.

Statistics

Data are shown as mean ± SEM unless otherwise stated. Statistical analysis was performed by one-way ANOVA.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Dilution curves of extracts of brain, heart, plasma, and urine were parallel with the standard curve of Ucn III (Fig. 1Go). Immunoreactive Ucn (IR-Ucn) III was detected by RIA in the human heart tissues (0.74–1.15 pmol/g wet weight) and kidney tissues (1.21 ± 0.30 pmol/g wet weight, mean ± SEM; n = 4) (Fig. 2AGo). IR-Ucn III was present in both ventricles and atria of hearts, and no significant difference was noted among them. In the human brain and pituitary, IR-Ucn III was present in every region examined, with the highest levels found in pituitary (2.72 ± 0.13 pmol/g wet weight; n = 3) and hypothalamus (1.79 ± 0.23 pmol/g wet weight; n = 8) (Fig. 2BGo). The levels in the heart and kidney were comparable with the levels found in the human brain tissues. IR-Ucn III was widely distributed also in other tissues, such as lung (1.06 pmol/g wet weight, mean of two cases), pancreas (3.45 pmol/g wet weight, mean of two cases), liver (1.12 pmol/g wet weight, mean of two cases), spleen (0.92 pmol/g wet weight, mean of two cases), and skeletal muscle (0.87 ± 0.09 pmol/g wet weight; n = 5). IR-Ucn III was present in human plasma (51.8 ± 16.0 pmol/liter; n = 5) and urine (266 ± 20 pmol/liter; n = 5).



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  		FIG. 1. A standard curve of human Ucn III and dilution curves of extracts of brain (frontal cortex) ({blacksquare}), heart (left ventricle) ({square}), plasma ({circ}), and urine ({triangleup}). The extracts were serially diluted and assayed. Dilution 1 shows the measurement in extracts derived from brain tissue (24 mg wet weight), heart tissue (40 mg wet weight), plasma (0.4 ml), and urine (0.25 ml), respectively.

 


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  		FIG. 2. IR-Ucn III concentrations in human heart and kidney (A) and brain and pituitary tissues (B). RA, Right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle. Cerebellum was dissected into hemisphere (h) and vermis (v). Error bars show SEM.

 
Reverse-phase HPLC showed a broad peak of IR-Ucn III eluting in the position of authentic Ucn III in the heart and kidney tissues as well as in hypothalamus (Fig. 3Go). Material eluting in the position of authentic SCP was also observed. IR-Ucn III in extracts of plasma and urine was mainly eluted earlier than authentic Ucn III, suggesting that IR-Ucn III in plasma and urine may be modified (Fig. 4Go). Materials eluting in the positions of Ucn III and SCP were also observed in the HPLC analysis of the plasma and urine extracts.



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  		FIG. 3. Reverse-phase HPLC of IR-Ucn III in hypothalamus (A), heart (left ventricle) (B), and kidney (C). The arrows indicate the elution position of Ucn III (UIII) and SCP. Dotted lines show a gradient of acetonitrile.

 


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  		FIG. 4. Reverse-phase HPLC of IR-Ucn III in plasma (A) and urine (B). The arrows indicate the elution position of Ucn III (UIII) and SCP. Dotted lines show a gradient of acetonitrile.

 
Immunocytochemistry showed positive staining of Ucn III in the myocardium (Fig. 5Go, A and B) and the proximal and distal tubules of the kidney (Fig. 5Go, C–F). Particularly, strong immunostaining was observed in the distal tubules of the renal cortex (Fig. 5Go, C–E). Renal tubules in the renal medulla were weakly stained for Ucn III (Fig. 5FGo). Negative controls using normal rabbit serum instead of the Ucn III antiserum showed no positive immunostaining (Fig. 5Go, G and H). The absorption of the antiserum with synthetic Ucn III (10 nmol Ucn III/ml diluted antiserum) abolished positive immunostaining (data not shown).



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  		FIG. 5. Immunocytochemistry of Ucn III in human heart and kidney. A and B, Myocardium (Mc) was positively stained for Ucn III, whereas endocardium (Endo) and pericardial adipocytes (Adipo) were not. C–E, Renal tubular cells in the renal cortex, particularly distal tubules, were strongly stained for Ucn III (shown by arrows in D); D, a higher magnification of C. F, Renal tubular cells in the renal medulla were weakly stained for Ucn III (arrows). Typical Ucn III-positive renal tubules were indicated by arrows (D–F). G and H, Negative controls of kidney and heart using normal rabbit serum (1:1000). G, A serial section of kidney (C); H, a serial section of heart (B). Bars, 100 µm.

 
RT-PCR analysis showed expression of Ucn III mRNA in the cerebral cortex, hypothalamus, pituitary, ventricles and atria of heart, and kidney (Fig. 6Go). RNA samples without reverse-transcriptase treatment gave no band or very weak bands, indicating that effects of genomic RNA contamination into the RNA samples were negligible.



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  		FIG. 6. RT-PCR of Ucn III mRNA in brain, pituitary, heart, and kidneys. Cortex, Cerebral cortex; Hypot, hypothalamus; Pitui, pituitary; RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; Kid 1 and Kid 2, kidneys. RT(–) indicates negative controls (samples without reverse-transcriptase treatment).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We have shown for the first time that IR-Ucn III is present in human heart and kidney tissues in high concentrations comparable to the brain. We have previously reported the presence of IR-Ucn in the human heart (0.92 ± 0.1 pmol/g wet weight in the right atrium and ~1.90 ± 0.5 pmol/g wet weight in the left ventricle) (7). The IR-Ucn III levels in the present study were approximately 60–80% of the previously reported IR-Ucn levels in the heart. The amount of material eluting in the position of Ucn was very small on HPLC of the heart extract, however (7). It is therefore plausible that the actual concentrations of Ucn III are higher than those of Ucn in the human heart. Expression of Ucn III mRNA in heart and kidney indicates endogenous production of Ucn III in these organs. CRF2 receptor is expressed in the heart (6, 7). Physiological ligand(s) for CRF2 receptor expressed in the heart has long been unknown, however, because CRF expression in the heart was very low or undetectable, and plasma levels of IR-CRF were also very low. Recent studies, including ours, have shown that Ucn is a candidate for endogenous ligand(s) to CRF2 receptor in the heart (7, 8, 9). The present study has raised the possibility that Ucn III is another candidate for endogenous ligand(s) to this receptor. Furthermore, the presence of Ucn III in the kidney tissues, particularly in the tubular cells, suggests that Ucn III has some physiological roles in the tubular reabsorption and renal circulation. These findings suggest that Ucn III is a novel autocrine or paracrine regulator in the heart and kidney.

IR-Ucn III is present in human plasma, raising the possibility that Ucn III has a role as a circulating hormone in addition to the role as an autocrine or paracrine factor. On the other hand, the source of Ucn III in plasma remains to be determined. Ucn III is expressed in various tissues and organs, as shown in the present study and in previous studies by others (1, 3, 5). Studies in plasma concentrations of Ucn III in various diseases including heart failure may help to clarify the contribution of heart to plasma IR-Ucn III concentrations. IR-Ucn III is also present in urine. IR-Ucn III concentrations in urine were approximately five times higher than those in plasma. Furthermore, urinary IR-Ucn III levels were much higher than the urinary levels of other vasoactive peptides, such as endothelin-1 (13), B-type natriuretic peptide (14), and neuropeptide Y (15). The positive immunostaining of Ucn III in renal tubular cells, particularly distal renal tubules, suggests that IR-Ucn III in urine is mostly originated from the renal tubular cells.

Chromatographic studies in tissue extracts of hypothalamus, heart, and kidney showed a broad peak eluting around the position of Ucn III. Immunoreactive material was also detected in the position of SCP. Thus, although the main molecular form of IR-Ucn III in the tissues appears to be Ucn III, it is likely that IR-Ucn III in the tissues consists of multiple molecular forms, including SCP. However, we could not deny a possibility that some of these multiple immunoreactive peaks represent an artifact generated during the extraction procedure. On the other hand, IR-Ucn III in the plasma and urine was eluting mainly earlier than authentic Ucn III. These findings suggest that large portions of IR-Ucn III in plasma and urine are modified to more hydrophilic forms than Ucn III and SCP. Peaks eluting in the positions of Ucn III and SCP were also observed in the HPLC of plasma and urine extracts, indicating that Ucn III and SCP are also present in plasma and urine.

Ucn has potent coronary vasodilatory and cardiac inotropic effects, and these effects have been shown to be more potent than CRF (16, 17, 18). In addition to coronary arteries, Ucn produces a marked vasodilatation in arteries and veins derived from various tissues, including renal arteries (19, 20). Ucn has protective effects on cardiac myocytes from ischemic or reperfusion injury (8, 21, 22, 23, 24, 25, 26). These protective effects of Ucn were mediated by up-regulation of p42/p44 MAPK signaling pathway, activation of protein kinase B/Akt, and induction of K(ATP) channel gene expression. Moreover, Ucn stimulates atrial natriuretic peptide and brain natriuretic peptide secretions from neonatal rat cardiomyocytes (27). Ucn III is a specific ligand for CRF2 receptor, whereas Ucn binds to both CRF1 receptor and CRF2 receptor. Because many of these biological actions of Ucn appear to be mediated by CRF2 receptor, it is plausible that Ucn III produces cardiovascular and renal actions similar to those of Ucn. Additional studies are required to clarify whether cardiovascular and renal actions of Ucn III are similar to or different from those of Ucn. After the first submission of this manuscript, it has been reported that Ucn II and Ucn III had protective effects against hypoxia/reoxygenation injury in rat neonatal cardiomyocytes (28) and in the murine heart (29).

Recent studies have shown the importance of some vasoactive peptides in the regulation of the cardiovascular and renal systems. For example, adrenomedullin, a potent vasodilator peptide originally discovered in pheochromocytoma (30), is expressed in many tissues including heart and kidney and may regulate the circulation and renal function, possibly as an autocrine or paracrine factor (31, 32, 33). Urotensin II, a potent vasoconstrictor peptide (34), is also expressed in various kinds of peripheral tissues including heart and kidney as well as the central nervous system (35). The present study has raised the possibility that Ucn III is another novel candidate that plays important regulatory roles in the cardiovascular and renal functions. CRF and Ucn are known to play essential roles in the stress response and inflammation (36). Ucn III, a novel member of the CRF family, may act as an autocrine/paracrine factor or a circulating hormone to maintain circulation through its positive inotropic and vasodilator actions in certain aspects of stress and inflammation.

IR-Ucn III was detected in every region of brain examined, suggesting that Ucn III acts as a neurotransmitter or a neuromodulator, for example in the stress response. IR-Ucn III levels in the human brain were approximately 50% of the IR-Ucn levels in our previous study (37). It is also noteworthy that high concentrations of IR-Ucn III and Ucn III mRNA expression are found in the pituitary gland. Contrary to our findings, Lewis et al. (1) reported that Ucn III mRNA was not detected in mouse pituitary, cerebellum, or cerebral cortex by the RNase protection assay. The discrepant results may be explained by higher sensitivity of the RIA and RT-PCR than the RNase protection assay. Another possible reason for this discrepancy may be a species difference in Ucn III expression between human and mouse. Positive immunostaining of Ucn III was observed in many types of human anterior pituitary cells by immunocytochemistry (Takahashi, K., unpublished data). We have previously reported that Ucn is present in anterior pituitary cells and pituitary adenomas (10). Ucn III may also be an autocrine or paracrine regulator of anterior pituitary hormone secretion like Ucn. On the other hand, physiological actions mediated by CRF2 receptor in the pituitary remain to be determined. Another possible physiological action of Ucn III expressed in the pituitary may be the regulation of the pituitary circulation.

The present study has shown expression of Ucn III in heart and kidney as well as brain and pituitary tissues. The IR-Ucn III in human tissues appears to consist of multiple molecular forms including SCP. These findings suggest that Ucn III/SCP may play important physiological roles in the cardiovascular and renal regulation, possibly in certain aspects of stress and inflammation, in addition to the roles as a neurotransmitter, a neuromodulator, and a neurohormone.


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research (B) (no. 13470030) and (C) (no. 13671094), and a Grant-in-Aid for Scientific Research on Priority Areas (A) (no. 13035005) from the Ministry of Education, Science, Sports, and Culture of Japan, by a research grant from the HIROMI Medical Research Foundation (2001), and by a Research Grant from the Intelligent Cosmos (2002).

Abbreviations: CRF, Corticotropin-releasing factor; IR-Ucn, immunoreactive Ucn; SCP, stresscopin; TFA, trifluoroacetic acid; Ucn, urocortin.

Received September 23, 2003.

Accepted December 22, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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