Elsevier

Regulatory Peptides

Volume 118, Issue 3, 15 May 2004, Pages 119-125
Regulatory Peptides

Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells

https://doi.org/10.1016/j.regpep.2003.11.002Get rights and content

Abstract

Apelin, the proposed endogenous peptide ligand of the novel G-protein-coupled receptor APJ, has been shown to possess potent vasodilator and positive inotropic effects in rats and humans in vivo. However, in humans, no endogenous source of apelin has been reported. Therefore, based on the presence of APJ and mRNA encoding apelin in human tissues, we investigated the expression of apelin in fresh-frozen human tissue from right atrium, left ventricle, lung, kidney, adrenal and large conduit vessels using immunocytochemistry. Apelin-like immunoreactivity (apelin-LI) was detected in vascular endothelial cells lining blood vessels in the human heart, kidney, adrenal gland and lung and in endothelial cells of large conduit vessels. Apelin-LI was also present in endocardial endothelial cells lining recesses of the right atrium. Apelin-LI was not present or below the level of detection in cardiomyocytes, Purkinje's cells, pulmonary or renal epithelial cells, secretory cells of the adrenal gland, vascular smooth muscle cells, adipocytes, nerves and connective tissue. The restricted presence of apelin-LI in endothelial cells suggests that endothelial apelin may play a role as a locally secreted cardiovascular mediator acting on APJ receptors present on the vascular smooth muscle and on cardiac myocytes to regulate vascular tone and cardiac contractility.

Introduction

APJ was first cloned from a human gene by O'Dowd et al. [7] and defined as an “orphan” G-protein-coupled receptor, since the endogenous ligand was not then known. Expression of the receptor in Chinese hamster ovary cells and reverse pharmacology resulted in the isolation of a putative ligand, a 36 amino acid activator peptide from bovine stomach. Consecutive cloning of human and bovine cDNA encoding the novel peptide, which the authors named apelin, led to its identification as the proposed endogenous ligand for the APJ receptor [1]. Interestingly, although apelin-36 was the first natural gene product discovered, testing of shorter synthetic C-terminal apelin peptides (Fig. 1) for activator potential in the same assay showed that shorter fragments were two orders of magnitude more potent than apelin-36, with the N-terminal pyroglutamyl form, (Pyr1)apelin-13, being most effective [1]. A number of basic amino acids in the sequence of the prepropeptide constitute prerequisite cleaving sites for endopeptidases, and the fact that in bovine colostrum Western blot analysis detected a range of apelin immunoreactive peptides with varying molecular weights suggests the existence of different biologically active apelin peptides [2]. However, the predominant endogenous peptides are (Pyr1)apelin-13 and apelin-36. The levels of apelin-36 exceed those of (Pyr1)apelin-13 in bovine colostrum and rat tissues, but (Pyr1)apelin-13 is more potent in a number of functional reporter assays. Therefore, evidence suggests that (Pyr1)apelin-13 is the final product of post-translational modification and the biologically active endogenous ligand [3], [4], [5], [6].

In rat and human brain, apelin and APJ mRNA is abundantly expressed suggesting a central regulatory role for the receptor system [7], [8], [9], [10]. Apelin has also been proposed to be a regulator of fluid homeostasis as the peptide influences water intake in rats, and in the rat hypothalamus apelin is co-localized with vasopressin in neurones of the supraoptic and paraventricular nuclei [6]. In accordance with these findings, other groups have reported changes in water intake as a result of intraperitoneal [10] or intracerebroventricular [6], [11] administration of apelin in rats.

In the periphery, we have previously localized apelin receptors in rat and human myocardium as well as in the medial layer of human coronary artery, aorta and saphenous vein grafts using [125I]-(Pyr1)Apelin-13 and shown potent constrictor responses to (Pyr1)Apelin-13 in endothelium denuded, isolated human saphenous vein [12]. In agreement with this receptor distribution, intravenous injection of apelin in anaesthetized and conscious Wistar rats leads to a significant decrease in mean arterial blood pressure [6], [10], [13], [14] and has positive inotropic effects in the isolated rat heart [15], suggesting a role for apelin in cardiovascular regulation. Apelin-like immunoreactivity (apelin-LI) has been reported in endothelial cells of rat blood vessels [13] and apelin mRNA is abundantly expressed in cultured human endothelial cells [16]. Therefore we hypothesise that apelin might be an endothelium derived vasoactive mediator, however, the cellular distribution of the peptide in human tissue, has not been examined. We have investigated the presence of apelin-LI in fresh-frozen human tissue using an antiserum raised against the C-terminal dodecapeptide of the apelin sequence.

Section snippets

Materials

Unless stated, all chemicals were obtained from Sigma-Aldrich (Poole, UK). Rabbit-anti-apelin-12 and rabbit-anti-apelin-36 antiserum used in immunocytochemistry was obtained from Phoenix Pharmaceuticals (Belmont, CA, USA). Mouse-anti-human von Willebrand factor monoclonal antibody, secondary antibodies, rabbit-PAP-complex and horseradish-peroxidase-conjugated swine-anti-rabbit antiserum were from DAKO (Glostrup, Denmark). The 96-well microtiter plates were from NUNC (Roskilde, Denmark) and

Specificity control ELISA

In the ELISA, the rabbit-anti-apelin-12 serum used in our immunocytochemistry experiments potently and specifically detected apelin-13 and apelin-36 resulting in a strong signal, even at greater dilutions than those used in the immunocytochemistry protocol. The antiserum bound the apelin-36 fragment with ∼60% higher potency compared to apelin-13 at the concentration range used in the immunocytochemistry protocol. The antiserum did not detect the three endothelial peptides angiotensin II,

Discussion

This is the first report of widespread presence of apelin-LI in vascular endothelial cells of fresh-frozen human tissues. The peptides detected by our immunocytochemistry protocol represent the complete range of known apelin peptides, as the primary antiserum has been raised against a C-terminal dodecapeptide from the preproapelin sequence, a sequence common to all functionally active apelin fragments examined so far. Additionally our enzyme-linked immunosorbent assay demonstrated that the

Acknowledgments

This work was supported by the British Heart foundation and the Cambridge European and Isaac Newton Trusts. We would like to thank Dr. Janet J. Maguire for critical review and discussion of the manuscript.

Cited by (267)

  • Apelin/APJ system in inflammation

    2022, International Immunopharmacology
View all citing articles on Scopus
View full text