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Department of Biochemistry

Members

Director Mikiya Miyazato
Laboratory chiefs Jun Hino, Takeshi Tokudome, Hiroyuki Kaiya, Kenji Mori, Takashi Nojiri
Research staff Ichiro Okano, Morikatsu Yoshida
Postdoctoral fellows Bae Cho Rong, Yoko Kyomoto, Yasutake Tanaka, Hirohito Nishimura, Koichi Miura, Motofumi Kumazoe, Michiko Fukui
Visiting researchers Yasuhiro Deguchi, Takashi Miyazawa, Takanori Ida, Reiko Hanada, Shinji Katsuragi, Shin Ishikane, Toru Kimura

Research activities

The Department of Biochemistry consists of three laboratories, the Laboratory of Immunochemistry, the Laboratory of Enzyme Chemistry and the Laboratory of Humoral Regulation. We have been investigating bioactive substances, which take important roles for maintaining homeostasis in the cardiovascular system, to elucidate their functions in the signal transduction and regulatory system and their physiological significance. Our final goal is the reconstruction of the signal transduction network in the cardiovascular system on the molecular basis. Cardiovascular function is regulated by many neural and humoral factors, so that the discovery of new signaling molecules help to clarify unknown control mechanisms of the cardiovascular system. Furthermore, we are aiming to introduce these bioactive substances into clinical application.

The study of a novel growth hormone-releasing peptide, ghrelin.

In department of biochemistry, we succeeded to isolate a novel growth hormone (GH)-releasing peptide"ghrelin" and to determine the structure in December 1999. Ghrelin participates not only in the stimulation of GH release but also in the stimulation of feeding centrally and peripherally. In addition, it is revealed that ghrelin is also involved in the regulation of cardiovascular system and energy metabolism, and acts on improvement of cardiac performance and of cachectic patients with chronic heart failure. At present, researches towards medical application of heart failure or eating disorder have started.

(A1) Discovery of ghrelin: its structure, distribution and regulation of the secretion

Growth hormone (GH) is produced and secreted from anterior pituitary gland, and exerts its actions by binding to the GH receptor. GH does not directly stimulate growth, but induces insulin-like growth factor-I (IGF-I) production in the liver. The increased IGF-I stimulates postnatal growth. GH also directly activates several tissues to control metabolism, to regulate water and electrolyte balance, and to control cell growth and differentiation. GH is released from the pituitary gland in a pulsatile manner, and the release is regulated by episodic changes in two hypothalamic hormones, growth hormone-releasing hormone (GHRH) and somatostatin. GH secretion is stimulated by GHRH and inhibited by somatostatin. In 1976, it was revealed that modified opioid peptides had low growth hormone-releasing activity. Since then, many efforts have been made to develop potential synthetic growth hormone secretagogues (GHSs). GHSs are divided into two classes: peptidergic and non-peptidergic. These GHSs act on the pituitary and hypothalamus to release GH, not through the GHRH receptor but through an orphan receptor, the growth hormone secretagogue receptor (GHS-R). These facts indicate that unknown endogenous ligands for the GHS-R may exist. As the endogenous ligand for the GHS-R has not been identified, however, the regulatory mechanism of this growth hormone secretagogue system remains elusive. Recent progress in molecular and cellular biology has made it possible to use orphan receptors as assay systems to purify unknown ligands. This technique uses a cell expressing an orphan receptor to monitor intracellular changes of second messengers, such as cAMP production, calcium concentration and arachidonic acid release. Using this technique, we succeeded in the purification of an endogenous ligand for the GHS-R from stomach extracts and named it"ghrelin"."Ghre" means etymological root for growth, and also represents an abbreviation for"growth-hormone-release" a characteristic effect of ghrelin. Ghrelin is a 28-amino acid peptide and has an unique structure; that is, the third serine residue is modified by an n-octanoic acid. The modification is essential for receptor binding and ghrelin activity such as stimulation of GH secretion from the pituitary. The existence of ghrelin in the stomach showed a new sight into a new mechanism regulating GH secretion not only by hypothalamic peptides but also by another regulatory peptide from digestive tracts. At present, ghrelin has been identified in non-mammalian vertebrates such as fish, amphibian, reptilian and avian, suggesting that ghrelin is a functionally important hormone to maintain homeostasis in at least vertebrate species.

Ghrelin is produced in the stomach fundus in all animals. Ghrelin - producing cells are a closed - type endocrine cells and colocalized with chromogranin A. The cell has been characterized as X/A-like cells that have known since 1960s and the content has been unidentified. In humans and rats, ghrelin also produces in the intestine, heart, pancreas, hypothalamus and placenta other than the stomach. In the pancreas, ghrelin-immunoreactive cells are identified at the periphery of pancreatic islets and colocalized with glucagon, indicating that the cell is pancreatic A cell. In the hypothalamus, ghrelin-immunoreactive neuronal cells can be identified in a very limited region of the hypothalamic arcuate nucleus, where is known to control appetite.

In humans and rats, the secretion of ghrelin is stimulated in starvation, and inhibited after feeding or glucose loading. In addition, insulin, somatostatin and cholecystokinin also inhibit ghrelin secretion. Produce of ghrelin in the stomach is increased by starvation and injection of insulin or leptin. Plasma ghrelin levels are low in obesed human and animal models, and correlated negatively with BMI. On the other hand, plasma ghrelin levels are high in patients with anorexia nervosa or CHF patients with cachexia. Ghrelin is secreted from the stomach, and is involved in many physiological functions such as GH release, feeding, cardiovascular system and energy metabolism as a circulating hormone.

<Recent publications>
  1. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402: 656-660, 1999.
  2. Hosoda H, Kojima M, Matsuo H, Kangawa K: Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun 279: 909-913, 2000.
  3. Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal M S, Suganuma T, Matsukura S, Kangawa K, Nakazato M: Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141: 4255-4261, 2000.
  4. Hosoda H, Kojima M, Matsuo H, Kangawa K: Purification and characterization of rat des-Gln14-Ghrelin, a second endogenous ligand for the growth hormone secretagogue receptor. J Biol Chem 275: 21995-22000, 2000.
  5. Matsuo H, Kojima M, Hayashi Y, Kangawa K: Structure-activity relationship of ghrelin: pharmacological study of ghrelin peptides. Biochem Biophys Res Commun. 287: 142-146, 2001.
  6. Matsumoto M, Kitajima Y, Iwanami T, Hayashi Y, Tanaka S, Minamitake Y, Hosoda H, Kojima M, Matsuo H, Kangawa K: Structural similarity of ghrelin derivatives to peptidyl growth hormone secretagogues. Biochem Biophys Res Commun. 284: 655-659, 2001.
  7. Kaiya H, Kojima M, Hosoda H, Koda A, Yamamoto K, Kitajima Y, Matsumoto M, Minamitake Y, Kikuyama S, Kangawa K: Bullfrog ghrelin is modified by n-octanoic acid at its third threonine residue. J Biol Chem 276: 40441-40448, 2001.
  8. Ariyasu H, Takaya K, Tagami T, Ogawa Y, Hosoda K, Akamizu T, Suda M, Koh T, Natsui K, Toyooka S, Shirakami G, Usui T, Shimatsu A, Doi K, Hosoda H, Kojima M, Kangawa K, Nakao K: Stomach is a major source of circulating ghrelin, and feeding state determines plasma ghrelin-like immunoreactivity levels in humans. J Clin Endocrinol Metab 86: 4753-4758, 2001.
  9. Kaiya H, Van Der Geyten S, Kojima M, Hosoda H, Kitajima Y, Matsumoto M, Geelissen S, Darras VM, Kangawa K: Chicken ghrelin: purification, cDNA cloning, and biological activity. Endocrinology. 143: 3454-3463, 2002.
  10. Shiiya T, Nakazato M, Mizuta M, Date Y, Mondal MS, Tanaka M, Nozoe S, Hosoda H, Kangawa K, Matsukura S. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 87: 240-244, 2002.
  11. Lu S, Guan JL, Wang QP, Uehara K, Yamada S, Goto N, Date Y, Nakazato M, Kojima M, Kangawa K, Shioda S: Immunocytochemical observation of ghrelin-containing neurons in the rat arcuate nucleus. Neurosci Lett. 321: 157-160, 2002.
  12. Date Y, Nakazato M, Hashiguchi S, Dezaki K, Mondal M S, Hosoda H, Kojima M, Kangawa K, Arima T, Matsuo H, Yada T, Matsukura S: Ghrelin is present in pancreatic alpha-cells of humans and rats and stimulates insulin secretion. Diabetes 51: 124-129, 2002.
  13. Kaiya H, Kojima M, Hosoda H, Riley LG, Hirano T, Grau EG, Kangawa K: Amidated fish ghrelin: purification, cDNA cloning in the Japanese eel and its biological activity. J Endocrinol. 176: 415-423, 2003.
  14. Kaiya H, Kojima M, Hosoda H, Riley LG, Hirano T, Grau EG, Kangawa K: Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus. Comp Biochem Physiol B Biochem Mol Biol 135: 421-429, 2003.
  15. Kaiya H, Kojima M, Hosoda H, Moriyama S, Takahashi A, Kawauchi H, Kangawa K: Peptide purification, complementary deoxyribonucleic acid (DNA) and genomic DNA cloning, and functional characterization of ghrelin in rainbow trout. Endocrinology 144: 5215-5226, 2003.
  16. Shimada M, Date Y, Mondal MS, Toshinai K, Shimbara T, Fukunaga K, Murakami N, Miyazato M, Kangawa K, Yoshimatsu H, Matsuo H, Nakazato M: Somatostatin suppresses ghrelin secretion from the rat stomach. Biochem Biophys Res Commun 302: 520-525, 2003.
  17. Hosoda H, Kojima M, Mizushima T, Shimizu S, Kangawa K: Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by post-translational processing. J Biol Chem 278: 64-70, 2003.
  18. Kaiya H, Sakata I, Kojima M, Hosoda H, Sakai T, Kangawa K: Structural determination and histochemical localization of ghrelin in the red-eared slider turtle, Trachemys scripta elegans. Gen Comp Endocrinol 138: 50-57, 2004.

(A2) Growth hormone secretion by ghrelin

Since ghrelin was identified as an endogenous substance instead of artificial synthetic growth hormone secretagogues (GHSs), it shows a strong GH-releasing activity by binding to the GHS receptor. When ghrelin was treated to rat dispersed pituitary cells or injected intravenously, specific GH-releasing activity is observed, and secretion of other pituitary hormones does not stimulate. In humans, a bolus injection of ghrelin increases plasma GH levels in a dose-dependent manner, and the effect was much greater than growth hormone releasing hormone (GHRH). GHRH shows the maximum response of GH release at a dose of 1 microgram/kg, but ghrelin does not show the maximum response even at a dose of 5 microgram/kg. This result suggests a different secretory mechanism for GH release between GHRH and ghrelin. It is also known that a low dose of ghrelin stimulates GH release synergistically, and a high dose of it stimulates GH release additively in combination of GHRH. When a high dose of ghrelin was injected, plasma PRL and ACTH levels also increase. This result is different from the rat data. GH secretion by ghrelin is regulated by two pathways: hypothalamic ghrelin and stomach-derived ghrelin. In fact, icv injection of ghrelin stimulates GH release. On the other hand, plasma GH levels are increased after intravenous injection of ghrelin; cutting of the vagal nerve attenuates the effect. Since the expression of Fos mRNA increases in hypothalamic GHRH neurons after iv injection of ghrelin, transmission of the ghrelin signal through the vagal nerve and GHRH responsive thereafter are involved in a mechanism regulating GH release by peripheral ghrelin.

<Recent publications>
  1. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402: 656-660, 1999.
  2. Date Y, Murakami N, Kojima M, Kuroiwa T, Matsukura S, Kangawa K, Nakazato M. Central effects of a novel acylated peptide, ghrelin, on growth hormone release in rats. Biochem Biophys Res Commun 275: 477-480, 2000.
  3. Takaya K, Ariyasu H, Kanamoto N, Iwakura H, Yoshimoto A, Harada M, Mori K, Komatsu Y, Usui T, Shimatsu A, Ogawa Y, Hosoda K, Akamizu T, Kojima M, Kangawa K, Nakao K: Ghrelin strongly stimulates growth hormone release in humans. J Clin Endocrinol Metab 85: 4908-4911, 2000.
  4. Hataya Y, Akamizu T, Takaya K, Kanamoto N, Ariyasu H, Saijo M, Moriyama K, Shimatsu A, Kojima M, Kangawa K, Nakao K: A low dose of ghrelin stimulates growth hormone (GH) release synergistically with GH-releasing hormone in humans. J Clin Endocrinol Metab 86: 4552, 2001.
  5. Date Y, Murakami N, Toshinai K, Matsukura S, Niijima A, Matsuo H, Kangawa K, Nakazato M: The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology. 123: 1120-1128, 2002.
  6. Yamazaki M, Nakamura K, Kobayashi H, Matsubara M, Hayashi Y, Kangawa K, Sakai T: Regulational effect of ghrelin on growth hormone secretion from perifused rat anterior pituitary cells. J Neuroendocrinol 14: 156-162. 2002.

(A3) Stimulation of feeding and energy metabolism by ghrelin

It has been suggested that ghrelin participates in several physiological effects in the CNS other than the stimulation of GH release because ghrelin produces in the arcuate nuclei of the hypothalamus and GHS-R mRNA expresses in several area of the CNS. In fact, icv injection of ghrelin increases food intake and body weights without changes in plasma insulin, glucose, cholesterol, and triglyceride. GH alone acts to stimulate feeding, but GH-deficient dwarf rats show the stimulation of feeding, indicating the feeding effect is not due to the increase in GH after ghrelin administration. Icv injection of ghrelin antibody inhibits feeding, suggesting central ghrelin is involved in feeding behavior indeed. When ghrelin is injected icv, expression of the Fos mRNA, an index of neural activation, increased in some important neurons to regulate feeding behavior such as neuropeptide Y (NPY), agouti-related protein (AGRP) and orexin neurons. Furthermore, pre-injections of antibodies against these peptides attenuate ghrelin-induced feeding behavior. This indicates that ghrelin-induced feeding is regulated by several neurons regulating feeding. Moreover, ghrelin inhibits leptin-induced decrease in food intake, suggesting ghrelin acts competitively to leptin in these neurons concerning feeding behavior.

Stimulatory effect of feeding by ghrelin secreted from the stomach is induced by the same pathway for GH release described previously: ghrelin binds to the receptor exists at the end of vagus nerve; then an information of empty signal is transmitted to the CNS by inhibiting electrical activity of the vagus nerve afferent. A large amount of subcutaneous ghrelin increases body weight in rodents. The ghrelin injection induces an increase in the respiratory quotient. Thus, it is considered that the increased body weight is due to the fat gain by reducing body fat utilization. In humans and rodents, ghrelin secretion is stimulated in fasting state. Plasma ghrelin levels in humans correlates negatively with BMI, and are low in obesed patients, and are high in anorexia nervosa, cachectic patients with CHF and lung cancer. Thus, ghrelin is activated in a case of negative energy balance, and plays important roles for maintaining homeostasis of the body by the stimulation of feeding, fat deposition and anabolic effect of GH. It is expected that elucidation of the mechanism stimulating food intake by ghrelin would be connected with therapy for eating disorder, obesity and malnutrition.

<Recent publications>
  1. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S: A role for ghrelin in the central regulation of feeding. Nature 409: 194-198, 2001.
  2. Tanaka M, Naruo T, Muranaga T, Yasuhara D, Shiiya T, Nakazato M, Matsukura S, Nozoe S: Increased fasting plasma ghrelin levels in patients with bulimia nervosa. Eur J Endocrinol 146: R1-3, 2002.
  3. Date Y, Murakami N, Toshinai K, Matsukura S, Niijima A, Matsuo H, Kangawa K, Nakazato M. The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology 123: 1120-1128, 2002.
  4. Hosoda H, Kojima M, Kangawa K: Ghrelin and the regulation of food intake and energy balance. Mol Interv 2: 494-503, 2002.
  5. Tanaka M, Naruo T, Nagai N, Kuroki N, Shiiya T, Nakazato M, Matsukura S, Nozoe S: Habitual binge/purge behavior influences circulating ghrelin levels in eating disorders. J Psychiatr Res 37: 17-22, 2003.
  6. Hanada T, Toshinai K, Kajimura N, Nara-Ashizawa N, Tsukada T, Hayashi Y, Osuye K, Kangawa K, Matsukura S, Nakazato M: Anti-cachectic effect of ghrelin in nude mice bearing human melanoma cells. Biochem Biophys Res Commun 301: 275-279, 2003.
  7. Tanaka M, Naruo T, Yasuhara D, Tatebe Y, Nagai N, Shiiya T, Nakazato M, Matsukura S, Nozoe S: Fasting plasma ghrelin levels in subtypes of anorexia nervosa. Psychoneuroendocrinology 28: 829-835, 2003.
  8. Tanaka M, Tatebe Y, Nakahara T, Yasuhara D, Sagiyama K, Muranaga T, Ueno H, Nakazato M, Nozoe S, Naruo T: Eating pattern and the effect of oral glucose on ghrelin and insulin secretion in patients with anorexia nervosa. Clin Endocrinol (Oxf) 59: 574-579, 2003.
  9. Hanada T, Toshinai K, Date Y, Kajimura N, Tsukada T, Hayashi Y, Kangawa K, Nakazato M: Upregulation of ghrelin expression in cachectic nude mice bearing human melanoma cells. Metabolism 53: 84-88, 2004.
  10. Akamizu T, Takaya K, Irako T, Hosoda H, Teramukai S, Matsuyama A, Tada H, Miura K, Shimizu A, Fukushima M, Yokode M, Tanaka K, Kangawa K: Pharmacokinetics, safety, and endocrine and appetite effects of ghrelin administration in young healthy subjects. Eur J Endocrinol 150: 447-455, 2004.

(A4) Cardiovascular effects of ghrelin

Ghrelin has some cardiovascular effects. In rats with chronic heart failure (CHF) by ligation of the left coronary artery, ghrelin was given at a dose of 100 maicrogram/kg subcutaneous (SC) for 3 weeks. CHF rats treated with ghrelin showed a significant greater increase in body weight than that given placebo with concomitant increases in serum GH and IGF-1 levels. They also showed higher cardiac output and left ventricular (LV) dP/dtmax than CHF rats given placebo. Ghrelin increased diastolic thickness of the noninfarcted posterior wall and LV fractional shortening, inhibited LV enlargement in CHF rats. These results indicate that chronic SC administration of ghrelin improved LV dysfunction and attenuated the development of LV remodeling and cardiac cachexia in CHF rats. GHS (ghrelin) receptor mRNA was detected in the rat aortas, left ventricles and atria, suggesting direct effect of ghrelin through GH-independent mechanisms. Ghrelin has beneficial hemodynamic effects through reducing cardiac afterload and increasing cardiac output without an increase in heart rate.

Considering these results of animal model, similar SC treatment was conducted in healthy men. The serum GH levels dose-dependently increased, but no significant changes were observed in corticotropin, cortisol, IGF-1, noradrenaline, adrenaline, heart rate and mean arterial pressure. In contrast, LV ejection fraction increased dose-dependently. These hemodynamic and hormonal changes were still apparent during significant increase in plasma ghrelin levels. These results indicate that SC administration of ghrelin is effective to enhance cardiac performance in humans.

Next, when CHF patients or healthy subjects were given an intravenous infusion or injection of ghrelin, 15-fold increases in serum GH levels was observed. Plasma PRL, ACTH, cortisol epinephrine levels were increased but plasma FSH, LH, TSH, IGF-1and norepinephrine levels did not change. Mean arterial pressure was significantly decreased from -9 to -12 mm Hg without a significant change in heart rate. In addition, ghrelin increased cardiac index and stroke volume index without change in mean pulmonary arterial pressure and pulmonary capillary wedge pressure in CHF patients. These hormonal changes are somewhat different from those in the case of SC administration of ghrelin. Furthermore, these results suggested that ghrelin may inhibit activation of sympathetic nervous system during hypertension, which may be beneficial in treating patients with congestive heart failure.

We also examined plasma ghrelin levels of CHF patients and control healthy subjects. Patients with CHF were divided into two groups, those with cachexia and without cachexia. Plasma ghrelin levels did not differ between all CHF patients and healthy subject (181 +/- 10 vs 140 +/- 14 fmol/mL). However, plasma ghrelin levels were significantly higher in CHF patients with cachexia than in those without cachexia (237 +/- 18 vs 147 +/- 10 fmol/mL). Circulating GH, TNF-alpha, norepinephrine and angiotensin II were also significantly higher in CHF patients with cachexia than in those without cachexia. Plasma ghrelin levels positively correlated with GH and TNF-alpha and negatively correlated with body mass index. Positive energy effects of ghrelin may represent a compensatory mechanism under catabolic-anabolic imbalance in cachectic patients with CHF.

  1. Nagaya N, Kojima M, Uematsu M, Yamagishi M, Hosoda H, Oya H, Hayashi Y, Kangawa K: Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol Regul Integr Comp Physiol 280: R1483-1487, 2001.
  2. Nagaya N, Uematsu M, Kojima M, Ikeda Y, Yoshihara F, Shimizu W, Hosoda H, Hirota Y, Ishida H, Mori H, Kangawa K: Chronic administration of ghrelin improves left ventricular dysfunction and attenuates development of cardiac cachexia in rats with heart failure. Circulation. 104: 1430-1435, 2001.
  3. Nagaya N, Uematsu M, Kojima M, Date Y, Nakazato M, Okumura H, Hosoda H, Shimizu W, Yamagishi M, Oya H, Koh H, Yutani C, Kangawa K: Elevated circulating level of ghrelin in cachexia associated with chronic heart failure: relationships between ghrelin and anabolic/catabolic factors. Circulation 104: 2034-2038, 2001.
  4. Nagaya N, Miyatake K, Uematsu M, Oya H, Shimizu W, Hosoda H, Kojima M, Nakanishi N, Mori H, Kangawa K: Hemodynamic, renal, and hormonal effects of ghrelin infusion in patients with chronic heart failure. J Clin Endocrinol Metab 86: 5854-5859, 2001.
  5. Okumura H, Nagaya N, Enomoto M, Nakagawa E, Oya H, Kangawa K: Vasodilatory effect of ghrelin, an endogenous peptide from the stomach. J Cardiovasc Pharmacol 39: 779-783, 2002.
  6. Nakagawa E, Nagaya N, Okumura H, Enomoto M, Oya H, Ono F, Hosoda H, Kojima M, Kangawa K: Hyperglycaemia suppresses the secretion of ghrelin, a novel growth-hormone-releasing peptide: responses to the intravenous and oral administration of glucose. Clin Sci (Lond) 103: 325-328, 2002.
  7. Shimizu Y, Nagaya N, Isobe T, Imazu M, Okumura H, Hosoda H, Kojima M, Kangawa K, Kohno N. Increased plasma ghrelin level in lung cancer cachexia. Clin Cancer Res 9:774-778, 2003.
  8. Nagaya N, Kangawa K: Ghrelin improves left ventricular dysfunction and cardiac cachexia in heart failure. Curr Opin Pharmacol 3: 146-151, 2003.
  9. Enomoto M, Nagaya N, Uematsu M, Okumura H, Nakagawa E, Ono F, Hosoda H, Oya H, Kojima M, Kanmatsuse K, Kangawa K: Cardiovascular and hormonal effects of subcutaneous administration of ghrelin, a novel growth hormone-releasing peptide, in healthy humans. Clin Sci (Lond) 105: 431-435, 2003.
  10. Nagaya N, Kangawa K: Ghrelin, a novel growth hormone-releasing peptide, in the treatment of chronic heart failure. Regul Pept 114: 71-77, 2003.
  11. Shimizu Y, Nagaya N, Teranishi Y, Imazu M, Yamamoto H, Shokawa T, Kangawa K, Kohno N, Yoshizumi M: Ghrelin improves endothelial dysfunction through growth hormone-independent mechanisms in rats. Biochem Biophys Res Commun 310: 830-835, 2003.
  12. Itoh T, Nagaya N, Yoshikawa M, Fukuoka A, Takenaka H, Shimizu Y, Haruta Y, Oya H, Yamagishi M, Hosoda H, Kangawa K, Kimura H: Elevated Plasma Ghrelin Level in Underweight Patients with Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med 2004 (in press)

The analysis of the new cardiovascular regulation by adrenomedullin and PAMP

Adrenomedullin is a novel cardiovascular regulation peptide which have vasodilative activity that it is discovered from human pheochromocytoma is shown by Dr. Kangawa (Department of Biochemistry, NCVC) and Dr. Kitamura (Miyazaki Med. Col.) in 1993. Adrenomedullin is produced not only in adrenal medulla but also in main organs and cardiovascular system such as lung, heart, kidney and blood vessel, and it is thought to be involved in regulation of cardiovascular system. We have analysed the structure, function, expression and secretion of adrenomedullin, such as association between the pathologies of cardiovascular diseases.

We are now proceeding the analyzing the molecular basis of adrenomedullin signal transduction system in the cardiovascular system, and the use of adrenomedullin in clinical studies about the development of the new method of treatment of the cardiovascular disease, which used adrenomedullin. These research are carried out by the collaboration with the Department of Pharmacology (Dr. Minamino, NCVC) and the Laboratory of Hypertension (NCVC). The following results can get its by the investigations on adrenomedullin until now.

<Recent publications>
  1. Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T: Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192: 553-560, 1993
  2. Kitamura K, Sakata J, Kangawa K, Kojima M, Matsuo H, Eto T: Cloning and characterization of cDNA encoding a precursor for human adrenomedullin. Biochem Biophys Res Commun 194: 720-725, 1993.
  3. Sakata J, Shimokubo T, Kitamura K, Nakamura S, Kangawa K, Matsuo H, Eto T: Molecular cloning and biological activities of rat adrenomedullin, a hypotensive peptide. Biochem Biophys Res Commun 195: 921-927, 1993.
  4. Kitamura K, Kangawa K, Kojima M, Ichiki Y, Matsuo H, Eto T: Complete amino acid sequence of porcine adrenomedullin and cloning of cDNA encoding its precursor. FEBS Lett 338: 306-310, 1994.

(B1) The discovery of the hormone; PAMP that exists in the inside of the adrenomedullin precursor is new cardiovascular regulating peptide.

We have cloned and analyzed the cDNA encoding precursor of adrenomedullin, and discovered that a new hormone existed at the N-terminal portion in the precursor protein of 20 residues. We have named this hypothetical peptide"PAMP" (Proadrenomedullin N-terminal 20 Peptide). Moreover, We have proved the existence of PAMP with purification and determined its structure. PAMP had the transient vasodilative activity that was different from adrenomedullin. In addition, PAMP suppressed the secretion of catecholamine from cultured adrenal medulla and the blood vessel wall sympathetic ganglia. This activity is not with adrenomedullin, and is clarified as the new biologically active peptide that PAMP was concerned with cardiovascular regulation.

<Recent publications>
  1. Kitamura K, Kangawa K, Ishiyama Y, Washimine H, Ichiki Y, Kawamoto M, Minamino N, Matsuo H, Eto T: Identification and hypotensive activity of proadrenomedullin N-terminal 20 peptide (PAMP). FEBS Lett 351: 35-37, 1994.
  2. Shimosawa T, Ito Y, Ando K, Kitamura K, Kangawa K, Fujita T: Proadrenomedullin NH(2)-terminal 20 peptide, a new product of the adrenomedullin gene, inhibits norepinephrine overflow from nerve endings. J Clin Invest 96: 1672-1676, 1995.
  3. Kuwasako K, Kitamura K, Ishiyama Y, Washimine H, Kato J, Kangawa K, Eto T: Purification and characterization of PAMP-12 (PAMP[9-20]) in porcine adrenal medulla as a major endogenous biologically active peptide. FEBS Lett 414: 105-110, 1997.
  4. Kuwasako K, Kitamura K, Kangawa K, Ishiyama Y, Kato J, Eto T: Increased plasma proadrenomedullin N-terminal 20 peptide in patients with essential hypertension. Ann Clin Biochem 36: 622-628, 1999.
  5. Kitamura K, Kangawa K, Eto T.: Adrenomedullin and PAMP: discovery, structures, and cardiovascular functions. Microsc Res Tech 1: 3-13, 2002.

(B2) The genomic structure of human adrenomedullin gene

The genomic structure of human adrenomedullin gene was analyzed for the genetic variation in cardiovascular diseases and the regulation of adrenomedullin expression. As for the gene of the adrenomedullin, it encoded by a gene contained in chromosome 11 and consisting of 4 exons and 3 introns, and it contained TATA box, GC box, cAMP-responsive element (CRE), share stress responsive element (SSRE) and AP-2 binding site in the 5'-promoter region.

<Recent publications>
  1. Ishimitsu T, Kojima M, Kangawa K, Hino J, Matsuoka H, Kitamura K, Eto T, Matsuo H: Genomic structure of human adrenomedullin gene. Biochem Biophys Res Commun 203: 631-639, 1994.
  2. Ishimitsu T, Miyata A, Matsuoka H, Kangawa K: Transcriptional regulation of human adrenomedullin gene in vascular endothelial cells. Biochem Biophys Res Commun 243: 463-470, 1998.
  3. Ishimitsu T, Tsukada K, Minami J, Ono H, Ohrui M, Hino J, Kangawa K, Matsuoka H: Microsatellite DNA polymorphism of human adrenomedullin gene in type 2 diabetic patients with renal failure. Kidney Int 63: 2230-2235. 2003

(B3) The regulation of gene expression level and site of adrenomedullin

We have examined the production site of adrenomedullin, it show that a large amount of adrenomedullin was synthesized and secreted in cultured rat endothelial cell and vascular smooth muscle cell, and the level of expression of adrenomedullin in aorta is comparable to the kidney and ventricle. In addition, the expression of adrenomedullin is regulated with many factors such as IL-1, TNF, lipopolysaccharide, the vasoactive factors and the hormones etc. The secretion amount of adrenomedullin of endothelial cell is comparable to the endothelin, and the specific receptor for adrenomedullin exists in endothelial cell and vascular smooth muscle cell. It is likely that the adrenomedullin produced in the blood vessel wall acts on vascular itself in autocrine and paracrine fashion antagonizing endothelin. Moreover, in the rat endotoxin shock model, administered 5mg/kg of LPS, it indicated that plasma adrenomedullin concentration was sequentially increased after the administration 1 hour after the circa 3 times 3 hours for the circa of 20 times. The gene expression of adrenomedullin increased in all the organ, especially in the blood vessel, the pulmonal, the enteric canal. This result suggests that endotoxin shock account for the plasma adrenomedullin concentration. Adrenomedullin was recommended that it be severely involved in the pathophysiology of the endotoxin shock.

<Recent publications>
  1. Sugo S, Minamino N, Kangawa K, Miyamoto K, Kitamura K, Sakata J, Eto T, Matsuo H: Endothelial cells actively synthesize and secrete adrenomedullin. Biochem Biophys Res Commun 201: 1160-1166, 1994.
  2. Minamino N, Shoji H, Sugo S, Kangawa K, Matsuo H: Adrenocortical steroids, thyroid hormones and retinoic acid augment the production of adrenomedullin in vascular smooth muscle cells. Biochem Biophys Res Commun 211: 686-693, 1995.
  3. Isumi Y, Shoji H, Sugo S, Tochimoto T, Yoshioka M, Kangawa K, Matsuo H, Minamino N: Regulation of adrenomedullin production in rat endothelial cells. Endocrinology 139: 838-846, 1998.
  4. Ono Y, Kojima M, Okada K, Kangawa K: cDNA cloning of canine adrenomedullin and its gene expression in the heart and blood vessels in endotoxin shock. Shock 10: 243-247, 1998.

(B4) Adrenomedullin receptors and signal transudation mechanisms

Adrenomedullin increases the cAMP of platelet and vascular smooth muscle cell through its specific receptor. The receptor that exists in vascular smooth muscle cell can both interact with adrenomedullin and CGRP, and bring about the vasorelaxation with cAMP production. While the receptor that existing in endothelial cell can only bind adrenomedullin. These results suggest that adrenomedullin binds to and activates at least two receptors. These receptors in concert either elevate or decrease intracellular Ca2+ concentration independently of cAMP. And this activation increases the NO production of the NO syntheses, and it explains vasorelaxation.

<Recent publications>
  1. Ono Y, Okano I, Kojima M, Okada K, Kangawa K: Decreased gene expression of adrenomedullin receptor in mouse lungs during sepsis. Biochem Biophys Res Commun 271: 197-202, 2000.
  2. Sata M, Kakoki M, Nagata D, Nishimatsu H, Suzuki E, Aoyagi T, Sugiura S, Kojima H, Nagano T, Kangawa K, Matsuo H, Omata M, Nagai R, Hirata Y: Adrenomedullin and nitric oxide inhibit human endothelial cell apoptosis via a cyclic GMP-independent mechanism. Hypertension 36: 83-88, 2000.

(B5) Plasma adrenomedullin concentration in various diseases

The concentration of adrenomedullin in the each organ was determined, and it showed that adrenomedullin was trapped by the pulmonary circulation and that adrenal body was not the major production site of plasma adrenomedullin. Plasma adrenomedullin concentration is increased in the patients with primary and secondary hypertension and in the patients with renal failure that correlated with disease severity. The improvement of the pathology in the heart failure patient correlates with reduction in plasma adrenomedullin concentration. Plasma adrenomedullin concentration correlated with A-type natriuretic peptide (ANP) and the norepinephrine concentration, it is likely that the elevation of body fluid volume and the activation of the sympathetic nerves influence adrenomedullin secretion. On the other hand, secretion of adrenomedullin is accelerated by endotoxin shock in vivo, we have analyzed that the correlation between the pathology of endotoxin shock in human and the plasma adrenomedullin concentration. Plasma adrenomedullin concentration of the septic shock patients is upregulated with all the patients, and the example that reached 150 times of healthy subjects was detected. Moreover, negative correlation is recognized between in plasma adrenomedullin concentration and peripheral blood pressure, this result suggests that adrenomedullin function as an hypotensor at the time of hypotension of endotoxin shock. Adrenomedullin and PAMP are not only function as circulation hormone but also these functions as autocrine and paracrine local factor like endothelin and CNP in blood vessel. Though adrenomedullin acts of vasodilative factor on the endothelial cell and vascular smooth muscle cell, PAMP acts through the sympathetic nervous in blood vessel and causes vasorelaxation. Adrenomedullin and PAMP in concert regulate the cardiovascular system, but their effects and target cells are different.

<Recent publications>
  1. Ishimitsu T, Nishikimi T, Saito Y, Kitamura K, Eto T, Kangawa K, Matsuo H, Omae T, Matsuoka H: Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J Clin Invest 94: 2158-2161, 1994.
  2. Nishikimi T, Saito Y, Kitamura K, Ishimitsu T, Eto T, Kangawa K, Matsuo H, Omae T, Matsuoka H: Increased plasma levels of adrenomedullin in patients with heart failure. J Am Coll Cardiol 26: 1424-1431, 1995.
  3. Nishio K, Akai Y, Murao Y, Doi N, Ueda S, Tabuse H, Miyamoto S, Dohi K, Minamino N, Shoji H, Kitamura K, Kangawa K, Matsuo H: Increased plasma concentrations of adrenomedullin correlate with relaxation of vascular tone in patients with septic shock. Crit Care Med 25: 953-957, 1997.
  4. Sumimoto T, Nishikimi T, Mukai M, Matsuzaki K, Murakami E, Takishita S, Miyata A, Matsuo H, Kangawa K: Plasma adrenomedullin concentrations and cardiac and arterial hypertrophy in hypertension. Hypertension 30: 741-745, 1997.
  5. Yoshitomi Y, Nishikimi T, Kojima S, Kuramochi M, Takishita S, Matsuoka H, Miyata A, Matsuo H, Kangawa K: Plasma levels of adrenomedullin in patients with acute myocardial infarction. Clin Sci (Colch) 94: 135-139, 1998.
  6. Sugo S, Minamino N, Shoji H, Isumi Y, Nakao K, Kangawa K, Matsuo H: Regulation of endothelin-1 production in cultured rat vascular smooth muscle cells. J Cardiovasc Pharmacol 37: 25-40, 2001.
  7. Ono Y, Okano I, Kojima M, Okada K, Kangawa K.: Decreased gene expression of adrenomedullin receptor in mouse lungs during sepsis. Biochem Biophys Res Commun 271: 197-202, 2000.

(B6) Clinical research to turn to the therapeutic application of adrenomedullin

Analysis of the validity and mechamism of the new treatment of heart failure with adrenomedullin administration.:

Plasma concentration of adrenomedullin were elevated in patients of left heart failure and right heart failure with pulmonary hypertension and correlate with disease severity. And this result shows that, adrenomedullin engages severely in this pathology and it may be a compensatory mechanism of protection. In the animal model, the adrenomedullin administration demonstrated to improve left and right with pulmonary hypertension. So, we have evaluated the effect and mechanism of adrenomedullin administration to heart failure and serious pulmonary hypertension patients.

In consequence, adrenomedullin improves heart failure by acting through circulation, diuresis, neuroendocrine factor (i.e. suppression of aldosterone secretion) in left heart failure. In addition, we have compared adrenomedullin with ANP which has already be employed clinical practice in heart failure. As a result, adrenomedullin have the different action mechanism from ANP which acts through the decrease in the cardiac after-load, the elevation of cardiac output for the fore-loading reduction on cardiac organ using the second messenger which is distinct from adrenomedullin. These results apparently suggest that adrenomedullin administration is effective clinical method for the left heart failure and the serious right heart failure patients.

On the other hand, as a consequence of administration of adrenomedullin to severe pulmonary hypertension patients with right heart failure, adrenomedullin markedly increased cardiac output and decreased pulmonary vascular resistance

The pulmonal vasodilative effect of adrenomedullin (10-8M) is comparable to the acetylcholine (10-4M) and the ATP (10-8M). In addtion, In case of the pulmonary hypertension patients which did not respond to acetylcholin and arrested endothelial cell-dependent vasorelaxation, adrenomedullin administration markedly increased the pulmonary flowrate. Adrenomedullin is effective in right heart failure with pulmonary hypertension, and its potent pulmonal vasodilative effect is acting through the endothelial cell-independet manner as mentioned above.

<Recent publications>
  1. Ishimitsu T, Nishikimi T, Saito Y, Kitamura K, Eto T, Kangawa K, Matsuo H, Omae T, Matsuoka H: Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J Clin Invest 94: 2158-2161, 1994.
  2. Nishikimi T, Saito Y, Kitamura K, Ishimitsu T, Eto T, Kangawa K, Matsuo H, Omae T, Matsuoka H: Increased plasma levels of adrenomedullin in patients with heart failure. J Am Coll Cardiol 26: 1424-1431, 1995.
  3. Nishio K, Akai Y, Murao Y, Doi N, Ueda S, Tabuse H, Miyamoto S, Dohi K, Minamino N, Shoji H, Kitamura K, Kangawa K, Matsuo H: Increased plasma concentrations of adrenomedullin correlate with relaxation of vascular tone in patients with septic shock. Crit Care Med 25: 953-957, 1997.
  4. Sumimoto T, Nishikimi T, Mukai M, Matsuzaki K, Murakami E, Takishita S, Miyata A, Matsuo H, Kangawa K: Plasma adrenomedullin concentrations and cardiac and arterial hypertrophy in hypertension. Hypertension 30: 741-745, 1997.
  5. Yoshitomi Y, Nishikimi T, Kojima S, Kuramochi M, Takishita S, Matsuoka H, Miyata A, Matsuo H, Kangawa K: Plasma levels of adrenomedullin in patients with acute myocardial infarction. Clin Sci (Colch) 94: 135-139, 1998.
  6. Nagaya N, Nishikimi T, Horio T, Yoshihara F, Kanazawa A, Matsuo H, Kangawa K: Cardiovascular and renal effects of adrenomedullin in rats with heart failure. Am J Physiol 276: R213-218, 1999.
  7. Nagaya N, Satoh T, Nishikimi T, Uematsu M, Furuichi S, Sakamaki F, Oya H, Kyotani S, Nakanishi N, Goto Y, Masuda Y, Miyatake K, Kangawa K: Hemodynamic, renal, and hormonal effects of adrenomedullin infusion in patients with congestive heart failure. Circulation 101: 498-503, 2000.
  8. Oya H, Nagaya N, Furuichi S, Nishikimi T, Ueno K, Nakanishi N, Yamagishi M, Kangawa K, Miyatake K: Comparison of intravenous adrenomedullin with atrial natriuretic peptide in patients with congestive heart failure. Am J Cardiol 86: 94-98, 2000.
  9. Nagaya N, Nishikimi T, Uematsu M, Satoh T, Oya H, Kyotani S, Sakamaki F, Ueno K, Nakanishi N, Miyatake K, Kangawa K: Haemodynamic and hormonal effects of adrenomedullin in patients with pulmonary hypertension. Heart 84: 653-658, 2000.

Studies for natriuretic peptide family (ANP, BNP, and CNP).

In 1984, we identified human atrial natriuretic peptide (ANP). We also isolated brain natriuretic peptide from porcine brain in 1988. Since their discovery, extensive research on the field has led these two natriuretic peptides from bench to bedside. ANP (in Japan) and BNP (in US) are now used as clinically useful agents for treatment of congestive heart failure patients. In 1990, we identified the third member of the natriuretic peptide family and termed it as C-type natriuretic peptide. We clarified that CNP, which was originally thought to function only in the central nervous system, also exist in peripheral. Especially in vascular wall, we observed that cultured smooth muscle cells express GC-B, a receptor for CNP, that monocytes, macrophages or endothelial cells produce CNP, and that CNP inhibits proliferation of vascular smooth muscle cells. These results indicate that CNP participates in growth control of smooth muscle cells as a local factor. In fact, it has been proven that CNP have a remarkable effect on suppresing intimal hyperplasia of animals (rats or rabbits) after injury. These results suggest the important role of CNP as a regulator in restenosis after coronary angioplasty and crisis of arteriosclerosis. Recently, we assessed the hypothesis that the in vivo administration of CNP might attenuate cardiac late remodeling after myocardial infarction (MI). When CNP was intravenously infused for 2 weeks in rats with MI, the left ventricular enlargement and dysfunction caused by MI were improved by CNP. CNP significantly attenuated an increase in morphometrical collagen volume fraction and cardiomyocyte area in the non-infarct region. The increases of collagen I, collagen III, ANP, and beta-myosin heavy chain mRNA levels in the non-infarct region were also suppressed by CNP. Therefore, CNP prevents cardiac remodeling after myocardial infarction and might be useful as a novel cardioprotective agent. These data indicate that CNP is a promising agent for treatment of vascular and cardiac diseases.

<Recent publications>
  1. Sudoh T, Minamino N, Kangawa K, Matsuo H: C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain. Biochem Biophys Res Commun 168: 863-870, 1990.
  2. Koller K J, Lowe D G, Bennett G L, Minamino N, Kangawa K, Matsuo H, Goeddel D V: Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science 252: 120-123, 1991.
  3. Furuya M, Aisaka K, Miyazaki T, Honbou N, Kawashima K, Ohno T, Tanaka S, Minamino N, Kangawa K, Matsuo H: C-type natriuretic peptide inhibits intimal thickening after vascular injury. Biochem Biophys Res Commun 193: 248-253, 1993.
  4. Ueno H, Haruno A, Morisaki N, Furuya M, Kangawa K, Takeshita A, Saito Y: Local expression of C-type natriuretic peptide markedly suppresses neointimal formation in rat injured arteries through an autocrine/paracrine loop. Circulation 96: 2272-2279, 1997.

Studies for Guanylin family (Guanylin and Uroguanylin).

There are three guanylate cyclases coupled plasma membrane receptors whose endogenous ligands have been identified. GC-A and GC-B bind natriuretic peptides (ANP, BNP and CNP). Guanylin (15 amino acid long) and uroguanylin (15 to 16 amino acid long) are peptide ligands for GC-C that is located in the intestine, kidney, adrenal gland, pancreas and airway tract. Both peptides stimulate Cl- secretion and possibly inhibit Na+ and H2O absorption in the intestine and renal tubules. We prepared antibodies against synthetic guanylin and uroguanylin, and established respective radioimmunoassays (RIAs) and immunohistochemical methods. By the biochemical and immunohistochemical studies, we clarified their endogenous molecular forms, tissue distributions and cellular sources. Furthermore, we cloned human and rat uroguanylin cDNAs, and determined their precursor structures and the gene expressions. Guanylin and uroguanylin are secreted into the circulation and their plasma levels increased along with the severities of renal failure and heart failure. Further investigations of the guanylin family should furnish new insights into the regulation of water and electrolyte homeostasis.

<Recent publications>
  1. Nakazato M, Yamaguchi H, Kinoshita H, Kangawa K, Matsuo H, Chino N, Matsukura S: Identification of biologically active and inactive human uroguanylins in plasma and urine and their increases in renal insufficiency. Biochem Biophys Res Commun 220: 586-593, 1996.
  2. Miyazato M, Nakazato M, Matsukura S, Kangawa K, Matsuo H: Uroguanylin gene expression in the alimentary tract and extra-gastrointestinal tissues. FEBS Lett 398: 170-174, 1996.
  3. Miyazato M, Nakazato M, Yamaguchi H, Date Y, Kojima M, Kangawa K, Matsuo H, Matsukura S: Cloning and characterization of a cDNA encoding a precursor for human uroguanylin. Biochem Biophys Res Commun 219: 644-648, 1996.
  4. Miyazato M, Nakazato M, Matsukura S, Kangawa K, Matsuo H: Genomic structure and chromosomal localization of human uroguanylin. Genomics 43: 359-365, 1997.
  5. Nakazato M, Yamaguchi H, Date Y, Miyazato M, Kangawa K, Goy M. F, Chino N, Matsukura S: Tissue distribution, cellular source, and structural analysis of rat immunoreactive uroguanylin. Endocrinology 139: 5247-5254, 1998.

Studies for BMP-3b (Bone Morphogenetic Protein-3b), a new factor for osteogenesis and embryogenesis.

We succeeded in cloning of BMP-3b from rat and human bone tissues in 1996. Bone morphogenetic proteins (BMP), members of the TGF-beta superfamily originally were identified as proteins that induce endochondral bone formation in adult animals. Subsequently, many related proteins were identified and found to have a variety of biological functions. BMP-3b is expressed in highly differentiated osteoblasts and has a significant role in them. Moreover, we found that the BMP-3b gene was expressed abundantly in cerebellum and highly in aorta, suggesting that BMP-3b is a growth/differentiation factor which could act in cardiovascular system and neural system. Recently, we demonstrated that BMP-3b is essential factor for head formation in embryogenesis including early neurogenesis, and that regulation of the processing of BMP-3b precursor and assembly plays an important role in embryonic patterning. These results indicate that BMP-3b might be useful for regenerative medicine. We now focus on BMP-3b activity in heart and aorta, therefore we are trying to clarify the function of BMP-3b in cardiovascular system.

<Recent publications>
  1. Takao M, Hino J, Takeshita N, Konno Y, Nishizawa T, Matsuo H, Kangawa K: Identification of rat bone morphogenetic protein-3b (BMP-3b), a new member of BMP-3. Biochem Biophys Res Commun 219: 656-662, 1996.
  2. Hino J, Takao M, Takeshita N, Konno Y, Nishizawa T, Matsuo H, Kangawa K: cDNA cloning and genomic structure of human bone morphogenetic protein-3B (BMP-3b). Biochem Biophys Res Commun 223: 304-310, 1996.
  3. Hino J, Matsuo H, Kangawa K: Bone morphogenetic protein-3b (BMP-3b) gene expression is correlated with differentiation in rat calvarial osteoblasts. Biochem Biophys Res Commun 256: 419-424, 1999.
  4. Hino J, Nishimatsu S, Nagai T, Matsuo H, Kangawa K, Nohno T:Coordination of BMP-3b and cerberus is required for head formation of Xenopus embryos. Dev. Biol. 260: 138-157, 2003.
  5. Hino J, Kangawa K, Matsuo H, Nohno T, Nishimatsu S: Bone morphogenetic protein-3 family members and their biological functions. Front. Biosci. 9: 1520-1529, 2004.

Studies for PACAP (Pituitary Adenylate Cyclase Activating Polypeptide).

PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) was discovered in 1989 by Dr. Miyata, as a hypothalamic neuropeptide showing highest homology with VIP (Vasoactive Intestinal Polypeptide) of secretin/glucagon family. In the cardiovascular system, PACAP has physiological role with vasodepressor activity, vasorelaxant activity and stimulatory activity for catecholamine release from adrenal medulla and positive inotropic action in heart. Further in the central nervous system, PACAP exhibits adenylate cyclase stimulatory activity not only in neuronal cells but also in astrocytes 1,000 times more potently than VIP, and is well-known to function as a neurotransmitter or neuromodulator. In addition, it is worthwhile to notice that PACAP exhibits neurotrophic or neuroprotective action in an in vitro model like glutamate-induced neuronal cell death, or an in vivo model like transient global ischemia. In our laboratory, we focus on the physiological function of PACAP as a neurohumonal factor in cardiovascular system and aim for the clarification of its pathophysiological significance in the cardiovascular disease such as hypertension, myocardial infarction and so on. We investigate the biosynthesis and secretion of PACAP and the relationship with other regulatory factors using the pathophysiological model. In addition, we analyze the molecular mechanism of PACAP function with in vitro system using primary culture of rat cardiomyocytes, aortic endothelial cells, or smooth muscle cells together with characterization of the PACAP receptors to be expressed in these cells. On the other hand, we focus on the functional relationship among neuronal cells, glial cells and vascular cells, and aim for the development of new drug with neuroprotection for the therapy of the ischemic brain damage. In this context, we proceed the clarification of molecular mechanism of direct or indirect neuroprotective activity of PACAP including the analysis of neuron-specific expression of PACAP gene.

<Recent publications>
  1. Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler MD, Coy DH: Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 164: 567-74, 1989.
  2. Ohkubo S, Kimura C, Ogi K, Okazaki K, Hosoya M, Onda H, Miyata A, Arimura A, Fujino M: Primary structure and characterization of the precursor to human pituitary adenylate cyclase activating polypeptide. DNA Cell Biol11: 21-30, 1992.
  3. Miyata A, Sato K, Hino J, Tamakawa H, Matsuo H, Kangawa K: Rat aortic smooth-muscle cell proliferation is bidirectionally regulated in a cell cycle-dependent manner via PACAP/VIP type 2 receptor. Ann N Y Acad Sci 865: 73-81, 1998.
  4. Tamakawa H, Miyata A, Satoh K, Miyake Y, Matsuo H, Arimura A, Kangawa K: The augmentation of pituitary adenylate cyclase-activating polypeptide (PACAP) in streptozotocin-induced diabetic rats. Peptides 19: 1497-1502, 1998.
  5. Miyata A, Sano H, Li M, Matsuda Y, Kaiya H, Satou K, Matsuo H, Kangawa K, Arimura A: Genomic organization and chromosomal localization of the mouse PACAP gene. Ann NY Acad Sci, 921, 344-348, 2000.
  6. Sano H, Miyata A, Horio T, Nishikimi T, Matsuo H, Kangawa K: The effect of pituitary adenylate cyclase activating polypeptide on cultured rat cardiocytes as a cardioprotective factor, Reg. Peptides, 109: 107-113, 2002.
  7. Sugawara H, Inoue K, Iwata S, Shimizu T, Yamada1 K, Mori N, Miyata A: Neural-restrictive silencers in the regulatory mechanism of pituitary adenylate cyclase-activating polypeptide gene expression. Regul Peptides (in press) 2004.

Studies for novel physiological roles of neuromedin U (NMU)

Neuromedin U (NMU), initially isolated from porcine spinal cord by our group, is a brain-gut neuropeptide that has a potent ability to cause uterine smooth muscle contraction. NMU is expressed not only in the central nervous system (CNS) but also in peripheral tissues, and the peripheral activities of NMU have been well characterized. In contrast, the physiological roles of NMU in the CNS have been poorly understood. In 2000, we and other groups identified NMU as an endogenous ligand for two orphan G protein-coupled receptors, FM3 (NMU1R) and FM4 (NMU2R). We also demonstrated that intracerebroventricularly (ICV) administered NMU causes potent inhibition of feeding behavior, and regulates energy homeostasis in rats. These data suggest that NMU is closely involved in the molecular mechanisms of obesity, which is a risk factor for the lifestyle-related illness such as cardiovascular diseases.

On the other hand, we showed that NMU centrally regulates the stress response via corticotropin-releasing hormone (CRH), and that ICV administered NMU elevates arterial blood pressure and heart rate through sympathetic nervous system. Central NMU also inhibits gastric acid secretion via CRH system. Recently, we demonstrated that NMU is expressed in the suprachiasmatic nuclei of the hypothalamus, which is the site of the master pacemaker of circadian rhythm, and NMU is closely involved in the regulation of circadian oscillator system.

<Recent publications>
  1. Minamino N, Kangawa K, Matsuo H: Neuromedin U-8 and U-25: novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochem Biophys Res Commun 130: 1078-1085., 1985.
  2. Minamino N, Kangawa K, Honzawa M, Matsuo H: Isolation and structural determination of rat neuromedin U. Biochem Biophys Res Commun 156: 355-360., 1988.
  3. Kojima M, Haruno R, Nakazato M, Date Y, Murakami N, Hanada R, Matsuo H, Kangawa K: Purification and identification of neuromedin U as an endogenous ligand for an orphan receptor GPR66 (FM3). Biochem Biophys Res Commun 276: 435-438., 2000.
  4. Nakazato M, Hanada R, Murakami N, Date Y, Mondal M S, Kojima M, Yoshimatsu H, Kangawa K, Matsukura S: Central effects of neuromedin U in the regulation of energy homeostasis. Biochem Biophys Res Commun 277: 191-194., 2000.
  5. Hanada R, Nakazato M, Murakami N, Sakihara S, Yoshimatsu H, Toshinai K, Hanada T, Suda T, Kangawa K, Matsukura S, Sakata T: A role for neuromedin U in stress response. Biochem Biophys Res Commun 289: 225-228., 2001.
  6. Chu C, Jin Q, Kunitake T, Kato K, Nabekura T, Nakazato M, Kangawa K, Kannan H: Cardiovascular actions of central neuromedin U in conscious rats. Regul Pept 105: 29-34., 2002.
  7. Mondal M S, Date Y, Murakami N, Toshinai K, Shimbara T, Kangawa K, Nakazato M: Neuromedin U acts in the central nervous system to inhibit gastric acid secretion via CRH system. Am J Physiol Gastrointest Liver Physiol 284: G963-969., 2003.
  8. Hanada T, Date Y, Shimbara T, Sakihara S, Murakami N, Hayashi Y, Kanai Y, Suda T, Kangawa K, Nakazato M: Central actions of neuromedin U via corticotropin-releasing hormone. Biochem Biophys Res Commun 311: 954-958, 2003.
  9. Nakahara K, Hanada R, Murakami N, Teranishi H, Ohgusu H, Fukushima N, Moriyama M, Ida T, Kangawa K, Kojima M: The gut-brain peptide neuromedin U is involved in the mammalian circadian oscillator system. Biochem Biophys Res Commun 318: 156-161. 2004.
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