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Urocortins as cardiovascular peptides
Peptides 25 (2004) 1723–1731
Review
Urocortins as cardiovascular peptides Kazuhiro Takahashia∗ , Kazuhito Totsuneb , Osamu Murakamic , Shigeki Shibaharaa a
Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8575, Japan b Department of Clinical Pharmacology and Therapeutics,Tohoku University, Graduate School of Pharmaceutical Science and Medicine, Aoba-ku, Sendai, Miyagi 980-8578, Japan c Department of Medicine, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8574, Japan Received 30 January 2004; accepted 14 April 2004 Available online 21 September 2004
Abstract Urocortins (Ucn) 1, 2 and 3, human homologues of fish urotensin I, form the corticotropin-releasing factor (CRF) family, together with CRF, urotensin I and sauvagine. Ucn 3 is a novel member of this family and is a specific ligand for CRF type 2 receptor. CRF type 2 receptor is thought to mediate the stress-coping responses, such as anxiolysis, anorexia, vasodilatation, a positive inotropic action on myocardium and dearousal. Endogenous ligands for the CRF type 2 receptor expressed in the cardiovascular tissues, such as the myocardium, have long been unknown. We have shown expression of Ucn 3 as well as Ucn 1 in the human heart. Ucn 3 is also expressed in the kidney, particularly distal tubules. Studies in various rat tissues showed that high concentrations of immunoreactive Ucn 3 were found in the pituitary gland, adrenal gland, gastrointestinal tract, ovary and spleen in addition to the brain, heart and kidney. These observations suggest that Ucn 3 is expressed in various tissues including heart and kidney, and may regulate the circulation in certain aspects of stress and diseases, such as inflammation. Ucn 1 and 3 appear to have important pathophysiological roles in some cardiovascular diseases. © 2004 Elsevier Inc. All rights reserved. Keywords: Urocortin; Stresscopin; Corticotropin-releasing factor; Stress; Heart
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2.
Urocortins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3.
CRF receptors in the heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.
Expression of urocortin 1 in human heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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5.
Expression of urocortin 3/stresscopin in human heart and kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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6.
Distribution of urocortin 3/stresscopin in various rat tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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7.
Biological actions of urocortins in the circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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8.
Fish-derived peptides and human diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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∗
Corresponding author. Tel.: +81 22 717 8116; fax: +81 22 717 8118. E-mail address: [email protected] (K. Takahashi).
0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.04.018
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1. Introduction In the last two decades, several bioactive peptides secreted by the cardiovascular organs were discovered [52]. These include atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) secreted by heart [24,51] and endothelins secreted by vascular endothelium [72]. Adrenomedullin, originally discovered in pheochromocytoma, is also produced by heart and vascular vessels [14,52]. These cardiovascular peptides act as vasoconstrictors (e.g., endothelins) or as vasodilators (e.g., natriuretic peptides and adrenomedullin), and are considered to regulate the cardiovascular function as a circulating hormone, an autocrine factor or a paracrine factor. Urotensins are peptide hormones secreted from fish urophysis, the neuroendocrine organ located in the caudal spinal cord. Urotensin I was considered to be a corticotropinreleasing factor (CRF)-like fish peptide, whereas Ucn II was a somatostatin-like fish peptide. Recent studies have revealed that urotensins or urotensin homologues are present in human, and are highly expressed, particularly in the cardiovascular tissues. Ucn 1, 2 and 3 [17,30,43,69], human homologues of urotensin I, are potent vasodilator peptides, which act on corticotropin-releasing factor receptors (CRF receptors) (Fig. 1), whereas human urotensin II is a potent vasoconstrictor peptide [2]. Thus, these peptides appear to have very important pathophysiological roles in the cardiovascular system. In this review article, we will summarize the expression of Ucns and CRF receptors in the heart, and their biological actions in the cardiovascular system. Moreover, we show our novel results in the regional distribution of Ucn 3 in various rat tissues.
was identified by the molecular cloning technique [13], and shown to be expressed in various tissues and cells, such as brain [18,62], pituitary [19], gastrointestinal tract [33], ovary [34], endometrium [15], placenta [40], synovial tissue [67], lymphocytes [4] and skin [49]. Human Ucn 1 has 63% identity with fish urotensin I and 43% identity with CRF at the amino acid level. Ucn 2 and Ucn 3 are novel members of the CRF family, and are specific agonists for CRF2 receptor [30,43] (Fig. 1). These two peptides were identified by searching the public genome databases. Another group reported stresscopin-related peptide (SRP) and stresscopin (SCP), peptides derived from the same genes as Ucn 2 and Ucn 3, respectively [17]. These two groups interpreted post-translational processing sites differently, and therefore the reported amino acid sequences of the peptides were slightly different between Ucn 2 and SRP, and between Ucn 3 and SCP. Human Ucn 2 is a 38 amino acid peptide that corresponds to the sequence 6–43 of human SRP, a 43 amino acid peptide, and human Ucn 3 is a 38 amino acid peptide that corresponds to the sequence 3–40 of human SCP, a 40 amino acid peptide. Ucn 2 and Ucn 3 have about 20–40% homology with CRF and Ucn 1. The homology between Ucn 2 and Ucn 3 was about 40%. The actions of the CRF-family peptides are mediated by at least two types of G-protein coupled receptors: CRF1 receptor [11] and CRF2 receptor [32]. 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 (Fig. 1). CRF and Ucn 1 bind to both CRF1 receptor and CRF2 receptor, whereas Ucn 2 and Ucn 3 are specific ligands for CRF2 receptor.
2. Urocortins 3. CRF receptors in the heart The CRF family consists of CRF, Ucn 1, Ucn 2 (stresscopin-related peptide; SRP), and Ucn 3 (stresscopin; SCP) as well as fish urotensin I and frog sauvagine. Rat Ucn 1 was discovered as a rat homologue of fish urotensin I and binds to both CRF type 1 receptor (CRF1 receptor) and CRF type 2 receptor (CRF2 receptor) [69] (Fig. 1). Human Ucn 1
Fig. 1. Relationship between corticotropin-relasing factor (CRF) family peptides and CRF receptors, and representative biological actions. Ucn, urocortin; SCP, stresscopin; SRP, stresscopin-related peptide.
CRF2 receptor is composed of at least three different isoforms, CRF2(a) , CRF2(b) and CRF2(c) receptors. In the rat, CRF2(a) receptor is expressed predominantly in the brain, whereas CRF2(b) receptor is expressed in both the brain and peripheral tissues, especially in heart and skeletal muscle tissues [32]. It was reported that treatment with lipopolysaccharide, corticosterone or Ucn 1 decreased expression of CRF2(b) receptor mRNA in the rat heart and in the aorta-derived A7R5 cell line [23], suggesting that CRF2(b) receptor expressed in the cardiovascular organs is related to inflammation and stress. In contrast to rodents, CRF2(a) receptor appears to be the major CRF2 receptor in the brain, heart, and skeletal muscle tissues, whereas CRF2(b) receptor is considered to be the minor isoform in humans [31,68]. In our previous study using RT-PCR, CRF2(a) receptor was expressed both in atria and ventricles obtained at autopsy in all four cases examined [28] (Fig. 2). On the other hand, CRF2(b) receptor was expressed in left atria of all 4 cases and in right atrium of one case. A weak band for CRF1 receptor mRNA was detected in some samples of atria and ventricles. Thus, CRF2(a) receptors
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Fig. 2. Reverse-transcriptase polymerase chain reaction analysis for urocortin 1 (Ucn), CRF, CRF type 1 receptor (CRF-R1), CRF type 2(a) receptor (CRFR2␣), and CRF type 2(b) receptor (CRF-R2) in four human hearts (patients 1–4) in the four constituent chambers (RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle). Total RNA from placenta, pituitary gland, hypothalamus, and left atrium were used as positive controls for urocortin and CRF, CRF-R1, -R2␣, and -R2, respectively. The bottom panel shows GAPDH employed as an internal control. P, Positive control. Reproduced from ref [28] Kimura et al. J Clin Endocrinol Metab 87:340–346; 2002 with kind permission from the Endocrine Society. Copyright 2002, The Endocrine Society.
appear to be the only isoform of CRF receptors detected in both atria and ventricles of the human heart.
4. Expression of urocortin 1 in human heart Endogenous ligands to the CRF2 receptor expressed in the heart have long been unknown. Plasma levels of CRF are very low [50], and CRF expression was low or undetectable in the heart (Fig. 2), suggesting that CRF is unlikely to be an endogenous ligand for the CRF2 receptor expressed in the heart. We and other investigators have recently shown that Ucn 1 is expressed in the heart and cardiomyocytes [9,23,28,35,36,37] and proposed that Ucn 1 is an endogenous physiological ligand, which may act as a paracrine or autocrine factor on the CRF2 receptor in the heart. Ucn 1 mRNA is expressed in all four chambers of human heart [28] (Fig. 2). Immunoreactive (IR)-Ucn 1 is present in the human heart at levels comparable to those found in the brain [28,62] (Fig. 3). Ucn 1 is expressed in rat cardiomyocytes, and its expression levels are increased by heat shock and ischemia [9,37]. Nishikimi et al. [35] have reported that the human myocardium was immunohistochemically positive for Ucn 1, the staining of which was more intense in the failing heart. Furthermore, Ikeda et al. [22] examined Ucn 1 expression in endomyocardial biopsy specimens by immunocytochemistry, and showed that Ucn 1 was expressed more abundantly
in the diseased heart, especially hypertrophic cardiomyopathy and dilated cardiomyopathy, than in the normal heart. Thus, Ucn 1 is upregulated in the diseased heart, and may compensate for cardiac function through its positive inotropic action and vasodilator action. Alternatively, Ucn 1 may promote cardiac hypertrophy because Ucn 1 was reported to stimulate cell proliferation of cardiac non-myocytes [20].
Fig. 3. Urocortin 1-like immunoreactivity in the human heart (mean ± S.E.M., n = 4). RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle. Reproduced from ref [28] Kimura et al. J Clin Endocrinol Metab 87:340–346; 2002 with kind permission from the Endocrine Society. Copyright 2002, The Endocrine Society.
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5. Expression of urocortin 3/stresscopin in human heart and kidney The predicted amino acid sequence of human Ucn 2 (SRP) precursor lacks a consensus proteolytic cleavage site that would allow for C-terminal processing of the peptide [43]. It is therefore unclear whether Ucn 2 is present in human tissues or just an artifact. We therefore firstly studied the expression of Ucn 3/SCP in human tissues. We studied expression of Ucn 3/SCP in the human heart and kidney by radioimmunoassay, immunocytochemistry and reverse transcriptase-polymerase chain reaction (RTPCR) [58]. IR-Ucn 3 was detected by radioimmunoassay in the human heart tissues (0.74–1.15 pmol/g wet weight) and kidney (Fig. 4A). IR-Ucn 3 was present in both ventricles and atria of hearts, and no significant difference was noted among them. The levels in the heart and kidney were comparable with the levels found in the human brain tissues (Fig. 4B). IR-Ucn 1 concentrations in the human heart ranged from 0.92 ± 0.1 pmol/g wet weight in the right atrium to 1.90 ± 0.5 pmol/g wet weight in the left ventricle (mean ± S.E.M.) (Fig. 3). The IR-Ucn 3 levels in the human heart were therefore about 60–80% of IR-Ucn 1 levels. Furthermore, IR-Ucn 3 was present in human plasma (51.8 ± 16.0 pmol/l, n = 5) and urine (266 ± 20 pmol/l, n = 5), which were obtained from healthy male subjects. Reverse phase HPLC showed a broad peak of IR-Ucn 3 eluting in the positions of authentic Ucn 3 and SCP in the heart as well as kidney and hypothalamus, suggesting that the IR-Ucn 3 in these human tissues consists of Ucn 3, SCP and other molecular forms [58]. On the other hand, IR-Ucn 1 in human heart was mainly eluted earlier than authentic human (more than 90% of the IR-Ucn 1), and only a very small amount of the IR-Ucn 1 was eluted in the position of Ucn 1 on HPLC [28]. It is therefore plausible that the actual concentrations of Ucn 3 are higher than those of Ucn 1 in the human heart. Immunocytochemistry showed positive staining of Ucn 3 in the myocardium (Fig. 5A and B), and the proximal and distal tubules of the kidney (Fig. 5C–F). Particularly, strong immunostaining was observed in the distal tubules of the renal cortex (Fig. 4C–E). Renal tubules in the renal medulla were weakly stained for Ucn 3 (Fig. 5F). Negative controls using normal rabbit serum instead of the Ucn 3 antiserum showed no positive immunostaining (Fig. 5G and H). The absorption of the antiserum with synthetic Ucn 3 (10 nmol Ucn 3/ml of diluted antiserum incubated for 16 h at 4 ◦ C) abolished positive immunostaining (data not shown). RT-PCR analysis showed expression of Ucn 3 mRNA in the cerebral cortex, hypothalamus, pituitary, ventricles and atria of heart, and kidney (Fig. 6), indicating that Ucn 3 was produced endogenously in these tissues. 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.
Fig. 4. Immunoreactive urocortin 3 (IR-Ucn III) concentrations in (A) human heart and kidney, and (B) brain and pituitary tissues. RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle. Cerebellum was dissected into hemisphere (h) and vermis (v). Error bars show S.E.M. Reproduced from ref [58] Takahashi et al. J Clin Endocrinol Metab 2004 with kind permission from the Endocrine Society. Copyright 2004, The Endocrine Society.
Thus, we have shown expression of Ucn 3/SCP in the human heart. In previous studies by others, RT-PCR analysis showed that Ucn 3 (SCP) mRNA was expressed in the human heart and kidney as well as other peripheral tissues [17], whereas the RNase protection assay could not detect it in the mouse heart [30]. Furthermore, RT-PCR detected Ucn 1 and Ucn 2 mRNAs in mouse cardiomyocytes, but not Ucn 3 mRNA [6]. There may be a species difference in the expression of Ucn 2 and Ucn 3 between human and mouse.
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Fig. 5. Immunocytochemistry of Ucn 3 in human heart and kidney. (A) and (B): Myocardium (Mc) was positively stained for Ucn 3, whereas endocardium (Endo) and pericardial adipocytes (Adipo) were not. (C), (D) and (E): Renal tubular cells in the renal cortex, particularly, distal tubules were strongly stained for Ucn 3 (shown by arrows in D). (D): A higher magnification of (C). (F): Renal tubular cells in the renal medulla were weakly stained for Ucn 3 (arrows). Typical Ucn 3-positive renal tubules were indicated by arrows (D, E, F). (G) and (H): Negative controls of kidney and heart using normal rabbit serum (1:1000). (G): A serial section of (C) kidney. (H): A serial section of (B) heart. Bars = 100 m. Reproduced from ref [58] Takahashi et al. J Clin Endocrinol Metab 2004 with kind permission from the Endocrine Society. Copyright 2004, The Endocrine Society.
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Fig. 6. Reverse-transcriptase polymerase chain reaction of urocortin 3 (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. The sense primer was 5 -TGATGCCGGTCCACTTCCTG-3 (nucleotide numbers 5/24) and the anti-sense primer was 5 -CCAATTTGCGCCATCAGGTG-3 (complementary to 454/473) (Genbank accession No. AF361943). RT(-) indicate negative controls (samples without reverse-transcriptase treatment). Reproduced from ref [58] Takahashi et al. J Clin Endocrinol Metab 2004 with kind permission from the Endocrine Society. Copyright 2004, The Endocrine Society.
6. Distribution of urocortin 3/stresscopin in various rat tissues
Table 1 Immunoreactive urocortin 3/stresscopin concentrations in rat pituitary glands (mean ± S.E.M., n = 6)
We then studied regional distribution of IR-Ucn 3 in various rat tissues by radioimmunoassay. IR-Ucn 3 is detected in brain, pituitary, heart and kidney (Fig. 7, Table 1), consistent with the findings in human tissues. Furthermore, higher concentrations of IR-Ucn 3 were found in the adrenal glands, ovary, jejunum, spleen and liver (Fig. 7). The highest concentrations of IR-Ucn 3 were found in pituitary glands (Table 1) and adrenal glands (Fig. 7). The wet weight of anterior
Anterior lobe Neurointermediate lobe
Fig. 7. Regional distribution of immunoreactive urocortin 3 (IR-Ucn 3) in rat tissues. Data are shown as mean ± S.E.M. (n = 5). Tissues were obtained from Sprague–Dawley rats (200–300 g) and extracted as previously reported [62,63]. Immunoreactive urocortin 3 levels in tissues were measured by radioimmunoasaay [58].
228.0 ± 21.5 fmol/gland 228.7 ± 28.9 fmol/gland
lobe and neurointermediate lobe was about 5–7 mg/gland and 1–2 mg/gland, respectively. The tissue concentration of IRUcn 3 may therefore be the highest in the neurointermediate lobe among various rat tissues. These findings have raised the possibility that Ucn 3 is related to various biological functions, including endocrine function, stress response, immune response and reproductive function. Adrenal medulla and pheochromocytomas are known to express various peptides and peptide receptors [1,60,61]. It is therefore likely that Ucn 3 is produced in the adrenal medulla. On the other hand, there is accumulating evidence, which shows that adrenal cortex and adrenocortical tumors produce and secrete some peptides, such as adrenomedullin, endothelin-1 and urotensin II [56]. In our preliminary studies, IR-Ucn 3 was detected in the tumor tissue extracts of adrenocortical tumors by radioimmunoassay (K. Takahashi, unpublished observations). It is therefore possible that Ucn 3 is also expressed in the adrenal cortex. Immunocytochemistry of rat heart showed positive immunostaining of Ucn 3 in the myocardium (Fig. 8), consistent with the results in humans. Chanalaris et al. have recently reported that both Ucn 2 (SRP) and Ucn 3 (SCP) mRNA expression levels were increased by hypoxic stress in rat cardiomyocytes [10].
7. Biological actions of urocortins in the circulation Ucn 1 has been demonstrated to have potent coronary vasodilatory and cardiac inotropic effects, and these effects have
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Fig. 8. Immunocytochemistry of urocortin 3 in rat heart. Consistent with the findings in human heart (Fig. 5), rat myocardium (Mc) was positively stained for Ucn 3, whereas endocardium (Endo) was not. Heart tissue was obtained from a Sprague–Dawley rat and fixed in 4% formaldehyde. 4 m paraffinembedded sections were immunostained using the antiserum against human Ucn 3 by the ABC method [45,58]. Bar = 100 m.
been shown to be more potent than CRF [38,39,64]. Ucn 1 has protective effects on cardiac myocytes from ischemic or reperfusion injury [7–9,29,37,46,47]. These protective effects of Ucn 1 were mediated by upregulation of p42/p44 MAPK signaling pathway, activation of protein kinase B/Akt, and induction of K(ATP) channel gene expression. Moreover, Ucn 1 stimulates ANP and BNP secretions from neonatal rat cardiomyocytes [21]. In sheep with experimental heart failure, intravenous infusion of Ucn 1 showed beneficial hemodynamic, endocrine and renal effects, such as increased cardiac output, decreased peripheral resistance, decreased plasma concentrations of renin, aldosterone, and endothelin1, and increased urine volume and sodium excretion [42]. Most of these cardiovascular effects of Ucn 1 are considered to be mediated by CRF2 receptor, which is known to be expressed in cardiomyocytes, and vascular endothelial and smooth muscle cells. It is therefore plausible that Ucn 2 and Ucn 3 have similar cardiovascular effects. It has recently been shown that Ucn 2 and Ucn 3 are cardioprotective against ischemia reperfusion injury in the murine heart [6], and against hypoxia/reoxygenation injury in rat neonatal cardiomyocytes [10]. Ucn 2 and Ucn 3 were more potent in anti-apoptotic effects on cardiomyocytes than Ucn 1. These effects of Ucn 2 and Ucn 3 were shown to be mediated by the p42/44 MARK pathway and the protein kinase B/Akt pathway like Ucn 1. The vasodilator effects of Ucn 1 may be caused, not only by its direct effect on vascular smooth muscle cells, but also by its effect on mast cell degranulation [48]. Ucn 1 and CRF induced rat skin mast cell degranulation and increased vascular permeability [12,48]. This effect appeared to be mediated not by the CRF1 , CRF2 (a) or CRF2 (b) receptor, but perhaps by the CRF2 (c) receptor. Furthermore, Ucn 1 and CRF are synthesized and secreted by human mast cells [27]. The relation of Ucn 2 and Ucn 3 with mast cells have not been reported, however.
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It has been shown that CRF2 receptor is a tonic suppressor of vascularization [3]. CRF2 receptor-deficient mice became hypervascularized postnatally. Activation of CRF2 receptor resulted in reduced vascular endothelial growth factor release from smooth muscle cells and an inhibition of smooth muscle cell proliferation. Although effects of Ucns on vascularization have not been reported, this report has raised the possibility that Ucns modulate (probably suppress) angiogenesis in cancer and ischemic cardiovascular diseases. Ucn 1-deficient mice displayed an impaired acoustic startle response, suggesting that Ucn 1 modulates the acoustic startle response through the Ucn 1-expressing neuron projections from the region of the Edinger–Westphal nucleus [70,71]. On the other hand, there has been no report showing abnormal cardiovascular function in Ucn 1-deficient mice. Ucn 2 and/or Ucn 3 may compensate for the cardiovascular effects of Ucn 1 in Ucn 1-deficient mice. Another possibility is that Ucn 1 (as well as Ucn 2 and Ucn 3) may have important regulatory actions on the cardiovascular system only in certain aspects of stresses, such as myocardial ischemia.
8. Fish-derived peptides and human diseases This review has summarized recent progress in the knowledge of Ucns in the cardiovascular system, and suggested important roles of Ucns in the pathophysiology of some cardiovascular diseases, such as ischemic heart disease. In addition to Ucns, there has been increasing evidence that indicates the importance of other fish-derived peptides in human diseases. For example, melanin-concentrating hormone (MCH) [26] is a potent appetite-stimulating neuropeptide in the mammals [41,52,53], and may be related to the pathophysiology of some types of obesity in human. Urotensin II, a cyclic peptide consisting of 11 amino acids, is one of the most potent vasoconstrictor peptides [2], whereas it acts as a vasodilator on some vessels, probably by the release of nitric oxide and endothelium-derived hyperpolarizing factor [5,25]. Increased plasma levels of urotensin II were reported in patients with renal failure [66], heart failure [44], liver cirrhosis [16], and diabetes mellitus [65], suggesting that urotensin II has important pathophysiological roles in these diseases. Furthermore, urotensin II is expressed in some tumor cells [55,57,59] and stimulates proliferation of tumor cells [57], suggesting that urotensin II is related to the tumor biology. Thus, fish-derived peptides including Ucns appear to be important in human physiology and diseases. The receptors for these peptides are therefore important targets for the discovery of novel drugs for human diseases, such as cardiovascular diseases, obesity, and malignant tumors [54].
Acknowledgments This work was supported in part by Grants-in-aid for Scientific Research (B) (No. 13470030) and (C) (No 13671094)
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from Japan Society for the Promotion of Science; by a Grantin-aid for Scientific Research on Priority Areas (A) (No. 13035005) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; by a Research Grant from the HIROMI Medical Research Foundation (2001); by a Research Grant from the Intelligent Cosmos (2002 & 2003) and by the 21st Century COE program, Medical Science Center for Innovative Therapeutic Development towards the Conquest of Signal Transduction Diseases.
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Review
Urocortins as cardiovascular peptides Kazuhiro Takahashia∗ , Kazuhito Totsuneb , Osamu Murakamic , Shigeki Shibaharaa a
Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8575, Japan b Department of Clinical Pharmacology and Therapeutics,Tohoku University, Graduate School of Pharmaceutical Science and Medicine, Aoba-ku, Sendai, Miyagi 980-8578, Japan c Department of Medicine, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8574, Japan Received 30 January 2004; accepted 14 April 2004 Available online 21 September 2004
Abstract Urocortins (Ucn) 1, 2 and 3, human homologues of fish urotensin I, form the corticotropin-releasing factor (CRF) family, together with CRF, urotensin I and sauvagine. Ucn 3 is a novel member of this family and is a specific ligand for CRF type 2 receptor. CRF type 2 receptor is thought to mediate the stress-coping responses, such as anxiolysis, anorexia, vasodilatation, a positive inotropic action on myocardium and dearousal. Endogenous ligands for the CRF type 2 receptor expressed in the cardiovascular tissues, such as the myocardium, have long been unknown. We have shown expression of Ucn 3 as well as Ucn 1 in the human heart. Ucn 3 is also expressed in the kidney, particularly distal tubules. Studies in various rat tissues showed that high concentrations of immunoreactive Ucn 3 were found in the pituitary gland, adrenal gland, gastrointestinal tract, ovary and spleen in addition to the brain, heart and kidney. These observations suggest that Ucn 3 is expressed in various tissues including heart and kidney, and may regulate the circulation in certain aspects of stress and diseases, such as inflammation. Ucn 1 and 3 appear to have important pathophysiological roles in some cardiovascular diseases. © 2004 Elsevier Inc. All rights reserved. Keywords: Urocortin; Stresscopin; Corticotropin-releasing factor; Stress; Heart
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2.
Urocortins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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CRF receptors in the heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Expression of urocortin 1 in human heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Expression of urocortin 3/stresscopin in human heart and kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Distribution of urocortin 3/stresscopin in various rat tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Biological actions of urocortins in the circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Fish-derived peptides and human diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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∗
Corresponding author. Tel.: +81 22 717 8116; fax: +81 22 717 8118. E-mail address: [email protected] (K. Takahashi).
0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.04.018
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K. Takahashi et al. / Peptides 25 (2004) 1723–1731
1. Introduction In the last two decades, several bioactive peptides secreted by the cardiovascular organs were discovered [52]. These include atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) secreted by heart [24,51] and endothelins secreted by vascular endothelium [72]. Adrenomedullin, originally discovered in pheochromocytoma, is also produced by heart and vascular vessels [14,52]. These cardiovascular peptides act as vasoconstrictors (e.g., endothelins) or as vasodilators (e.g., natriuretic peptides and adrenomedullin), and are considered to regulate the cardiovascular function as a circulating hormone, an autocrine factor or a paracrine factor. Urotensins are peptide hormones secreted from fish urophysis, the neuroendocrine organ located in the caudal spinal cord. Urotensin I was considered to be a corticotropinreleasing factor (CRF)-like fish peptide, whereas Ucn II was a somatostatin-like fish peptide. Recent studies have revealed that urotensins or urotensin homologues are present in human, and are highly expressed, particularly in the cardiovascular tissues. Ucn 1, 2 and 3 [17,30,43,69], human homologues of urotensin I, are potent vasodilator peptides, which act on corticotropin-releasing factor receptors (CRF receptors) (Fig. 1), whereas human urotensin II is a potent vasoconstrictor peptide [2]. Thus, these peptides appear to have very important pathophysiological roles in the cardiovascular system. In this review article, we will summarize the expression of Ucns and CRF receptors in the heart, and their biological actions in the cardiovascular system. Moreover, we show our novel results in the regional distribution of Ucn 3 in various rat tissues.
was identified by the molecular cloning technique [13], and shown to be expressed in various tissues and cells, such as brain [18,62], pituitary [19], gastrointestinal tract [33], ovary [34], endometrium [15], placenta [40], synovial tissue [67], lymphocytes [4] and skin [49]. Human Ucn 1 has 63% identity with fish urotensin I and 43% identity with CRF at the amino acid level. Ucn 2 and Ucn 3 are novel members of the CRF family, and are specific agonists for CRF2 receptor [30,43] (Fig. 1). These two peptides were identified by searching the public genome databases. Another group reported stresscopin-related peptide (SRP) and stresscopin (SCP), peptides derived from the same genes as Ucn 2 and Ucn 3, respectively [17]. These two groups interpreted post-translational processing sites differently, and therefore the reported amino acid sequences of the peptides were slightly different between Ucn 2 and SRP, and between Ucn 3 and SCP. Human Ucn 2 is a 38 amino acid peptide that corresponds to the sequence 6–43 of human SRP, a 43 amino acid peptide, and human Ucn 3 is a 38 amino acid peptide that corresponds to the sequence 3–40 of human SCP, a 40 amino acid peptide. Ucn 2 and Ucn 3 have about 20–40% homology with CRF and Ucn 1. The homology between Ucn 2 and Ucn 3 was about 40%. The actions of the CRF-family peptides are mediated by at least two types of G-protein coupled receptors: CRF1 receptor [11] and CRF2 receptor [32]. 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 (Fig. 1). CRF and Ucn 1 bind to both CRF1 receptor and CRF2 receptor, whereas Ucn 2 and Ucn 3 are specific ligands for CRF2 receptor.
2. Urocortins 3. CRF receptors in the heart The CRF family consists of CRF, Ucn 1, Ucn 2 (stresscopin-related peptide; SRP), and Ucn 3 (stresscopin; SCP) as well as fish urotensin I and frog sauvagine. Rat Ucn 1 was discovered as a rat homologue of fish urotensin I and binds to both CRF type 1 receptor (CRF1 receptor) and CRF type 2 receptor (CRF2 receptor) [69] (Fig. 1). Human Ucn 1
Fig. 1. Relationship between corticotropin-relasing factor (CRF) family peptides and CRF receptors, and representative biological actions. Ucn, urocortin; SCP, stresscopin; SRP, stresscopin-related peptide.
CRF2 receptor is composed of at least three different isoforms, CRF2(a) , CRF2(b) and CRF2(c) receptors. In the rat, CRF2(a) receptor is expressed predominantly in the brain, whereas CRF2(b) receptor is expressed in both the brain and peripheral tissues, especially in heart and skeletal muscle tissues [32]. It was reported that treatment with lipopolysaccharide, corticosterone or Ucn 1 decreased expression of CRF2(b) receptor mRNA in the rat heart and in the aorta-derived A7R5 cell line [23], suggesting that CRF2(b) receptor expressed in the cardiovascular organs is related to inflammation and stress. In contrast to rodents, CRF2(a) receptor appears to be the major CRF2 receptor in the brain, heart, and skeletal muscle tissues, whereas CRF2(b) receptor is considered to be the minor isoform in humans [31,68]. In our previous study using RT-PCR, CRF2(a) receptor was expressed both in atria and ventricles obtained at autopsy in all four cases examined [28] (Fig. 2). On the other hand, CRF2(b) receptor was expressed in left atria of all 4 cases and in right atrium of one case. A weak band for CRF1 receptor mRNA was detected in some samples of atria and ventricles. Thus, CRF2(a) receptors
K. Takahashi et al. / Peptides 25 (2004) 1723–1731
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Fig. 2. Reverse-transcriptase polymerase chain reaction analysis for urocortin 1 (Ucn), CRF, CRF type 1 receptor (CRF-R1), CRF type 2(a) receptor (CRFR2␣), and CRF type 2(b) receptor (CRF-R2) in four human hearts (patients 1–4) in the four constituent chambers (RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle). Total RNA from placenta, pituitary gland, hypothalamus, and left atrium were used as positive controls for urocortin and CRF, CRF-R1, -R2␣, and -R2, respectively. The bottom panel shows GAPDH employed as an internal control. P, Positive control. Reproduced from ref [28] Kimura et al. J Clin Endocrinol Metab 87:340–346; 2002 with kind permission from the Endocrine Society. Copyright 2002, The Endocrine Society.
appear to be the only isoform of CRF receptors detected in both atria and ventricles of the human heart.
4. Expression of urocortin 1 in human heart Endogenous ligands to the CRF2 receptor expressed in the heart have long been unknown. Plasma levels of CRF are very low [50], and CRF expression was low or undetectable in the heart (Fig. 2), suggesting that CRF is unlikely to be an endogenous ligand for the CRF2 receptor expressed in the heart. We and other investigators have recently shown that Ucn 1 is expressed in the heart and cardiomyocytes [9,23,28,35,36,37] and proposed that Ucn 1 is an endogenous physiological ligand, which may act as a paracrine or autocrine factor on the CRF2 receptor in the heart. Ucn 1 mRNA is expressed in all four chambers of human heart [28] (Fig. 2). Immunoreactive (IR)-Ucn 1 is present in the human heart at levels comparable to those found in the brain [28,62] (Fig. 3). Ucn 1 is expressed in rat cardiomyocytes, and its expression levels are increased by heat shock and ischemia [9,37]. Nishikimi et al. [35] have reported that the human myocardium was immunohistochemically positive for Ucn 1, the staining of which was more intense in the failing heart. Furthermore, Ikeda et al. [22] examined Ucn 1 expression in endomyocardial biopsy specimens by immunocytochemistry, and showed that Ucn 1 was expressed more abundantly
in the diseased heart, especially hypertrophic cardiomyopathy and dilated cardiomyopathy, than in the normal heart. Thus, Ucn 1 is upregulated in the diseased heart, and may compensate for cardiac function through its positive inotropic action and vasodilator action. Alternatively, Ucn 1 may promote cardiac hypertrophy because Ucn 1 was reported to stimulate cell proliferation of cardiac non-myocytes [20].
Fig. 3. Urocortin 1-like immunoreactivity in the human heart (mean ± S.E.M., n = 4). RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle. Reproduced from ref [28] Kimura et al. J Clin Endocrinol Metab 87:340–346; 2002 with kind permission from the Endocrine Society. Copyright 2002, The Endocrine Society.
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5. Expression of urocortin 3/stresscopin in human heart and kidney The predicted amino acid sequence of human Ucn 2 (SRP) precursor lacks a consensus proteolytic cleavage site that would allow for C-terminal processing of the peptide [43]. It is therefore unclear whether Ucn 2 is present in human tissues or just an artifact. We therefore firstly studied the expression of Ucn 3/SCP in human tissues. We studied expression of Ucn 3/SCP in the human heart and kidney by radioimmunoassay, immunocytochemistry and reverse transcriptase-polymerase chain reaction (RTPCR) [58]. IR-Ucn 3 was detected by radioimmunoassay in the human heart tissues (0.74–1.15 pmol/g wet weight) and kidney (Fig. 4A). IR-Ucn 3 was present in both ventricles and atria of hearts, and no significant difference was noted among them. The levels in the heart and kidney were comparable with the levels found in the human brain tissues (Fig. 4B). IR-Ucn 1 concentrations in the human heart ranged from 0.92 ± 0.1 pmol/g wet weight in the right atrium to 1.90 ± 0.5 pmol/g wet weight in the left ventricle (mean ± S.E.M.) (Fig. 3). The IR-Ucn 3 levels in the human heart were therefore about 60–80% of IR-Ucn 1 levels. Furthermore, IR-Ucn 3 was present in human plasma (51.8 ± 16.0 pmol/l, n = 5) and urine (266 ± 20 pmol/l, n = 5), which were obtained from healthy male subjects. Reverse phase HPLC showed a broad peak of IR-Ucn 3 eluting in the positions of authentic Ucn 3 and SCP in the heart as well as kidney and hypothalamus, suggesting that the IR-Ucn 3 in these human tissues consists of Ucn 3, SCP and other molecular forms [58]. On the other hand, IR-Ucn 1 in human heart was mainly eluted earlier than authentic human (more than 90% of the IR-Ucn 1), and only a very small amount of the IR-Ucn 1 was eluted in the position of Ucn 1 on HPLC [28]. It is therefore plausible that the actual concentrations of Ucn 3 are higher than those of Ucn 1 in the human heart. Immunocytochemistry showed positive staining of Ucn 3 in the myocardium (Fig. 5A and B), and the proximal and distal tubules of the kidney (Fig. 5C–F). Particularly, strong immunostaining was observed in the distal tubules of the renal cortex (Fig. 4C–E). Renal tubules in the renal medulla were weakly stained for Ucn 3 (Fig. 5F). Negative controls using normal rabbit serum instead of the Ucn 3 antiserum showed no positive immunostaining (Fig. 5G and H). The absorption of the antiserum with synthetic Ucn 3 (10 nmol Ucn 3/ml of diluted antiserum incubated for 16 h at 4 ◦ C) abolished positive immunostaining (data not shown). RT-PCR analysis showed expression of Ucn 3 mRNA in the cerebral cortex, hypothalamus, pituitary, ventricles and atria of heart, and kidney (Fig. 6), indicating that Ucn 3 was produced endogenously in these tissues. 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.
Fig. 4. Immunoreactive urocortin 3 (IR-Ucn III) concentrations in (A) human heart and kidney, and (B) brain and pituitary tissues. RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle. Cerebellum was dissected into hemisphere (h) and vermis (v). Error bars show S.E.M. Reproduced from ref [58] Takahashi et al. J Clin Endocrinol Metab 2004 with kind permission from the Endocrine Society. Copyright 2004, The Endocrine Society.
Thus, we have shown expression of Ucn 3/SCP in the human heart. In previous studies by others, RT-PCR analysis showed that Ucn 3 (SCP) mRNA was expressed in the human heart and kidney as well as other peripheral tissues [17], whereas the RNase protection assay could not detect it in the mouse heart [30]. Furthermore, RT-PCR detected Ucn 1 and Ucn 2 mRNAs in mouse cardiomyocytes, but not Ucn 3 mRNA [6]. There may be a species difference in the expression of Ucn 2 and Ucn 3 between human and mouse.
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Fig. 5. Immunocytochemistry of Ucn 3 in human heart and kidney. (A) and (B): Myocardium (Mc) was positively stained for Ucn 3, whereas endocardium (Endo) and pericardial adipocytes (Adipo) were not. (C), (D) and (E): Renal tubular cells in the renal cortex, particularly, distal tubules were strongly stained for Ucn 3 (shown by arrows in D). (D): A higher magnification of (C). (F): Renal tubular cells in the renal medulla were weakly stained for Ucn 3 (arrows). Typical Ucn 3-positive renal tubules were indicated by arrows (D, E, F). (G) and (H): Negative controls of kidney and heart using normal rabbit serum (1:1000). (G): A serial section of (C) kidney. (H): A serial section of (B) heart. Bars = 100 m. Reproduced from ref [58] Takahashi et al. J Clin Endocrinol Metab 2004 with kind permission from the Endocrine Society. Copyright 2004, The Endocrine Society.
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Fig. 6. Reverse-transcriptase polymerase chain reaction of urocortin 3 (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. The sense primer was 5 -TGATGCCGGTCCACTTCCTG-3 (nucleotide numbers 5/24) and the anti-sense primer was 5 -CCAATTTGCGCCATCAGGTG-3 (complementary to 454/473) (Genbank accession No. AF361943). RT(-) indicate negative controls (samples without reverse-transcriptase treatment). Reproduced from ref [58] Takahashi et al. J Clin Endocrinol Metab 2004 with kind permission from the Endocrine Society. Copyright 2004, The Endocrine Society.
6. Distribution of urocortin 3/stresscopin in various rat tissues
Table 1 Immunoreactive urocortin 3/stresscopin concentrations in rat pituitary glands (mean ± S.E.M., n = 6)
We then studied regional distribution of IR-Ucn 3 in various rat tissues by radioimmunoassay. IR-Ucn 3 is detected in brain, pituitary, heart and kidney (Fig. 7, Table 1), consistent with the findings in human tissues. Furthermore, higher concentrations of IR-Ucn 3 were found in the adrenal glands, ovary, jejunum, spleen and liver (Fig. 7). The highest concentrations of IR-Ucn 3 were found in pituitary glands (Table 1) and adrenal glands (Fig. 7). The wet weight of anterior
Anterior lobe Neurointermediate lobe
Fig. 7. Regional distribution of immunoreactive urocortin 3 (IR-Ucn 3) in rat tissues. Data are shown as mean ± S.E.M. (n = 5). Tissues were obtained from Sprague–Dawley rats (200–300 g) and extracted as previously reported [62,63]. Immunoreactive urocortin 3 levels in tissues were measured by radioimmunoasaay [58].
228.0 ± 21.5 fmol/gland 228.7 ± 28.9 fmol/gland
lobe and neurointermediate lobe was about 5–7 mg/gland and 1–2 mg/gland, respectively. The tissue concentration of IRUcn 3 may therefore be the highest in the neurointermediate lobe among various rat tissues. These findings have raised the possibility that Ucn 3 is related to various biological functions, including endocrine function, stress response, immune response and reproductive function. Adrenal medulla and pheochromocytomas are known to express various peptides and peptide receptors [1,60,61]. It is therefore likely that Ucn 3 is produced in the adrenal medulla. On the other hand, there is accumulating evidence, which shows that adrenal cortex and adrenocortical tumors produce and secrete some peptides, such as adrenomedullin, endothelin-1 and urotensin II [56]. In our preliminary studies, IR-Ucn 3 was detected in the tumor tissue extracts of adrenocortical tumors by radioimmunoassay (K. Takahashi, unpublished observations). It is therefore possible that Ucn 3 is also expressed in the adrenal cortex. Immunocytochemistry of rat heart showed positive immunostaining of Ucn 3 in the myocardium (Fig. 8), consistent with the results in humans. Chanalaris et al. have recently reported that both Ucn 2 (SRP) and Ucn 3 (SCP) mRNA expression levels were increased by hypoxic stress in rat cardiomyocytes [10].
7. Biological actions of urocortins in the circulation Ucn 1 has been demonstrated to have potent coronary vasodilatory and cardiac inotropic effects, and these effects have
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Fig. 8. Immunocytochemistry of urocortin 3 in rat heart. Consistent with the findings in human heart (Fig. 5), rat myocardium (Mc) was positively stained for Ucn 3, whereas endocardium (Endo) was not. Heart tissue was obtained from a Sprague–Dawley rat and fixed in 4% formaldehyde. 4 m paraffinembedded sections were immunostained using the antiserum against human Ucn 3 by the ABC method [45,58]. Bar = 100 m.
been shown to be more potent than CRF [38,39,64]. Ucn 1 has protective effects on cardiac myocytes from ischemic or reperfusion injury [7–9,29,37,46,47]. These protective effects of Ucn 1 were mediated by upregulation of p42/p44 MAPK signaling pathway, activation of protein kinase B/Akt, and induction of K(ATP) channel gene expression. Moreover, Ucn 1 stimulates ANP and BNP secretions from neonatal rat cardiomyocytes [21]. In sheep with experimental heart failure, intravenous infusion of Ucn 1 showed beneficial hemodynamic, endocrine and renal effects, such as increased cardiac output, decreased peripheral resistance, decreased plasma concentrations of renin, aldosterone, and endothelin1, and increased urine volume and sodium excretion [42]. Most of these cardiovascular effects of Ucn 1 are considered to be mediated by CRF2 receptor, which is known to be expressed in cardiomyocytes, and vascular endothelial and smooth muscle cells. It is therefore plausible that Ucn 2 and Ucn 3 have similar cardiovascular effects. It has recently been shown that Ucn 2 and Ucn 3 are cardioprotective against ischemia reperfusion injury in the murine heart [6], and against hypoxia/reoxygenation injury in rat neonatal cardiomyocytes [10]. Ucn 2 and Ucn 3 were more potent in anti-apoptotic effects on cardiomyocytes than Ucn 1. These effects of Ucn 2 and Ucn 3 were shown to be mediated by the p42/44 MARK pathway and the protein kinase B/Akt pathway like Ucn 1. The vasodilator effects of Ucn 1 may be caused, not only by its direct effect on vascular smooth muscle cells, but also by its effect on mast cell degranulation [48]. Ucn 1 and CRF induced rat skin mast cell degranulation and increased vascular permeability [12,48]. This effect appeared to be mediated not by the CRF1 , CRF2 (a) or CRF2 (b) receptor, but perhaps by the CRF2 (c) receptor. Furthermore, Ucn 1 and CRF are synthesized and secreted by human mast cells [27]. The relation of Ucn 2 and Ucn 3 with mast cells have not been reported, however.
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It has been shown that CRF2 receptor is a tonic suppressor of vascularization [3]. CRF2 receptor-deficient mice became hypervascularized postnatally. Activation of CRF2 receptor resulted in reduced vascular endothelial growth factor release from smooth muscle cells and an inhibition of smooth muscle cell proliferation. Although effects of Ucns on vascularization have not been reported, this report has raised the possibility that Ucns modulate (probably suppress) angiogenesis in cancer and ischemic cardiovascular diseases. Ucn 1-deficient mice displayed an impaired acoustic startle response, suggesting that Ucn 1 modulates the acoustic startle response through the Ucn 1-expressing neuron projections from the region of the Edinger–Westphal nucleus [70,71]. On the other hand, there has been no report showing abnormal cardiovascular function in Ucn 1-deficient mice. Ucn 2 and/or Ucn 3 may compensate for the cardiovascular effects of Ucn 1 in Ucn 1-deficient mice. Another possibility is that Ucn 1 (as well as Ucn 2 and Ucn 3) may have important regulatory actions on the cardiovascular system only in certain aspects of stresses, such as myocardial ischemia.
8. Fish-derived peptides and human diseases This review has summarized recent progress in the knowledge of Ucns in the cardiovascular system, and suggested important roles of Ucns in the pathophysiology of some cardiovascular diseases, such as ischemic heart disease. In addition to Ucns, there has been increasing evidence that indicates the importance of other fish-derived peptides in human diseases. For example, melanin-concentrating hormone (MCH) [26] is a potent appetite-stimulating neuropeptide in the mammals [41,52,53], and may be related to the pathophysiology of some types of obesity in human. Urotensin II, a cyclic peptide consisting of 11 amino acids, is one of the most potent vasoconstrictor peptides [2], whereas it acts as a vasodilator on some vessels, probably by the release of nitric oxide and endothelium-derived hyperpolarizing factor [5,25]. Increased plasma levels of urotensin II were reported in patients with renal failure [66], heart failure [44], liver cirrhosis [16], and diabetes mellitus [65], suggesting that urotensin II has important pathophysiological roles in these diseases. Furthermore, urotensin II is expressed in some tumor cells [55,57,59] and stimulates proliferation of tumor cells [57], suggesting that urotensin II is related to the tumor biology. Thus, fish-derived peptides including Ucns appear to be important in human physiology and diseases. The receptors for these peptides are therefore important targets for the discovery of novel drugs for human diseases, such as cardiovascular diseases, obesity, and malignant tumors [54].
Acknowledgments This work was supported in part by Grants-in-aid for Scientific Research (B) (No. 13470030) and (C) (No 13671094)
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from Japan Society for the Promotion of Science; by a Grantin-aid for Scientific Research on Priority Areas (A) (No. 13035005) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; by a Research Grant from the HIROMI Medical Research Foundation (2001); by a Research Grant from the Intelligent Cosmos (2002 & 2003) and by the 21st Century COE program, Medical Science Center for Innovative Therapeutic Development towards the Conquest of Signal Transduction Diseases.
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