The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism

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Abstract

Apelin is an endogenous ligand of the human orphan receptor APJ. We detected apelin-like immunoreactivity in the adipocytes, gastric mucosa, and Kupffer cells in the liver. We also detected apelin-like immunoreactivity localized within the endothelia of small arteries in various organs. Further, it was found that mean arterial pressure after the administration of apelin-12, apelin-13, and apelin-36 at a dose of 10 nmol/kg in anaesthetized rats was reduced by 26±5, 11±4, and 5±4 mm Hg, 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 to 27.0±1.5 μM. Thus, apelin may lower blood pressure via a nitric oxide-dependent mechanism.

Introduction

Many orphan G-protein-coupled receptors are considered to be specific receptors for unidentified hormones and neuropeptides [1]. In 1993, APJ (putative receptor protein related to AT1) was identified in a human gene by O'Dowd et al. [2]. APJ resembles the angiotensin receptor, but membrane preparations prepared from the APJ-expressing cells do not bind to angiotensin II, suggesting that APJ is an orphan receptor with no known ligand. APJ receptor mRNA is widely expressed in many regions of the central nervous system [3] and in a wide variety of organs including the spleen, thymus, prostate, testis, ovary, small intestine, and colon [4]. Recently, APJ has been found to act as a coreceptor supporting the efficient entry of human immunodeficiency virus (HIV) [4], [5]. These results suggest that APJ may be a receptor for a previously unknown biologically active substance.

In the search for an endogenous ligand of the human orphan receptor APJ, we isolated a novel 36-amino acid peptide ligand for APJ that we named apelin [6]. We isolated the peptide from bovine stomach extracts by monitoring the increase in the extracellular acidification rate induced by receptor–ligand interactions in cells expressing the APJ receptor. The sequences of the 77-amino acid preproproteins were determined from bovine, human [6], rat, and mouse [7], [8] cDNAs. Subsequent studies have found that apelin-13 (preproapelin 65–77) and apelin-17 (preproapelin 61–77), the short C-terminal fragments of apelin-36 (preproapelin 42–77), exhibit much stronger activity than apelin-36 [6], suggesting that the C-terminal portion of apelin-36 may be responsible for its receptor binding and biological activity. Recently, we reported that apelin peptides block the entry of HIV and inhibit HIV infection in cells expressing CD4 and APJ [9].

Apelin mRNA is reportedly distributed in the lung, brain, heart, ovary, testis, adipose tissue, and mammary glands according to reverse-transcription polymerase chain reaction (RT-PCR) [7]. Northern blot analysis detected preproapelin mRNA in the brain, pituitary, adrenal, vas deferens, testis, intestine, heart, spleen, liver, and kidney [8], [10]. A preliminary study suggests that apelin-13 possesses hypotensive properties in rats [8].

In this study, we report that apelin may be present in the endothelia of small arteries and lower blood pressure via a nitric oxide-dependent mechanism.

Section snippets

Chemical synthesis of apelin peptides and production of antiserum against apelin

Apelin peptides were synthesized by a solid-phase synthetic technique using an automatic peptide synthesizer (Model 431, Applied Biosystems) and purified by HPLC after deprotection. Synthetic [Cys0]apelin-17 was conjugated to keyhole limpet hemocyanin (KLH), and was used as an antigen to generate rabbit antiserum against apelin-17. Apelin-17 was chosen as the antigen, because the amino acid sequence of apelin-17 is identical to that of human, rat, mouse, or cow origin. In a radioimmunoassay for

Localization of apelin in rat tissues

Using immunohistochemical techniques, apelin-like immunoreactivity was detected in white adipose tissue, such as the great omentum and mesenterium. Due to the presence of a large amount of fat deposits in the adipocytes, the cytoplasm densely stained by immunoreactive apelin was seen close to the cell membrane (Fig. 1a). The nuclei were devoid of staining. Apelin-like immunoreactivity was also present in the endothelia of the small arteries of the mesenterium (Fig. 1b), omentum, heart, lung,

Discussion

The present study demonstrates that apelin is localized within the endothelia of small arteries in many organs such as the liver, spleen, lung, pancreas, intestine, kidney, and adipose tissues. Apelin peptides were found to significantly lower arterial blood pressure. These results suggest that apelin is involved in the regulation of blood pressure and blood flow. We found that the depressor response of an apelin peptide is greatly influenced by its molecular sizes. The injection of apelin-13

Acknowledgements

We would like to thank Drs. Isao Kobayashi, Hiroyuki Shimizu, and Mitsuhiko Miura for their helpful comments. We appreciate the excellent technical assistance of Ms. Sachiko Sato. The work was supported by the Ministry of Education, Science and Culture of Japan.

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