|Budget Amount *help
¥11,300,000 (Direct Cost: ¥11,300,000)
Fiscal Year 2002: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2001: ¥7,100,000 (Direct Cost: ¥7,100,000)
The principal aim of this proposal is to study the identification and physiological characterization of bombesin receptor subtype-3 (BRS-3) ligand and to study physiological and biochemical characterization of apelin, a novel gastrointestinal hormone isolated from the stomach.
STUDY 1: Bombesin receptor subtype-3 (BRS-3) is an orphan G protein-coupled receptor that is activated by a receptor agonist, [D-Phe^6, β-Ala^<11>, Phe^<13>, Nle^<14>]Bn(6-14). We found that this agonist stimulated the laminin-mediated adhesion of a human small cell lung cancer cell line, NCI-N417, which expresses native BRS-3. The enhancement of adhesion was accompanied by an increase in vinculin-like immunoreactivity, and diminished in the presence of an anti-Pj integrin antibody, suggesting that the receptor activation stimulates focal adhesion formation. We also suggest that mobilization of intracellular calcium is involved in the mechanism of BRS-3-mediated adhesion of NCI-N417 cells.
STUDY 2: Apelin, a novel
36-amino acid peptide, has been identified to be the endogenous ligand for the APJ receptor. We have examined the presence of apelin mRNA in isolated mouse adipocytes and mouse 3T3-L1 cells, and investigated the effects of various hormones on apelin expression in 3T3-L1 adipocytes. We found that both apelin mRNA and APJ mRNA were expressed in isolated mouse adipocytes, while apelin mRNA, but not APJ mRNA, was detected in 3T3-L1 adipocytes. Furthermore, we found that the level of apelin mRNA increased to 3.9-fold during the differentiation of 3T3-L1 cells to adipocytes. Administration of insulin (1 nM-100 nM) increased 2-to 3-fold the apelin mRNA levels in 3T3-L1 adipocytes, while dexamethasone, (0.1 nM-100 nM) decreased the apelin mRNA levels in a dose-dependent manner. In 3T3-L1 adipocytes, dexamethasone reversed insulin-induced increase in apelin mRNA expression, and insulin reversed dexamethasone-induced decrease in the expression. Thus, insulin and glucocorticoids may act as positive and negative regulators of apelin gene expression in 3T3-L1 adipocytes.
STUDY 3: Apelin, the endogenous peptide ligand of the APJ receptor, is expressed in brain regions implicated in food and water intake. This study investigated for the first time, the effect of apelin-12 oh spontaneous (nocturnal) feeding. Rats received 1, 3, and 10 nmol apelin-12 or saline vehicle in random order by intracerebroventricular injection 10 minutes prior to lights out. Acute effects were observed, with dose-dependent reductions in food intake 2-4 hours after injection. Thus, apelin-12 exerted a delayed inhibitory effect on nocturnal feeding.
STUDY 4: Apelin is an endogenous ligand of the human orphan receptor APJ. We detected apelin-like immunoreactivity localized within the endothelia of Small arteries in various organs, adipocytes, gastric mucosa, and Kupffer cells in the liver. We also found that apelin lowers blood pressure. Mean arterial pressure after intravenous administration of apelin-12, apelin-13, and apelin-36 at a dose of 10 nmol/kg in anesthetized rats was reduced by 26 ± 5 mmHg, 11 ± 4 mmHg, and 5 ± 4 mmHg, respectively. In the presence of a nitric oxide (NO) synthase inhibitor, the effect of apelin-12 on blood pressure was abolished. Furthermore, the administration of apelin-12 (10 nmol/kg) in rats produced a transitory elevation of the plasma nitrite/nitrate concentration from a basal level of 21.4 ±1.6 mM to 27.0 ±1.5 mM. Thus, apelin may play an important role in the regulation of blood pressure. Less