Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells
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.
References (19)
- et al.
Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor
Biochem. Biophys. Res. Commun.
(1998) - et al.
Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum
Biochim. Biophys. Acta
(1999) - et al.
Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin
J. Biol. Chem.
(2000) - et al.
Molecular properties of apelin: tissue distribution and receptor binding
Biochim. Biophys. Acta
(2001) - et al.
A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11
Gene
(1993) - et al.
Low stringency hybridization study of the dopamine D4 receptor revealed D4-like mRNA distribution of the orphan seven-transmembrane receptor, APJ, in human brain
Neurosci. Lett.
(1996) - et al.
The effects of centrally administered apelin-13 on food intake, water intake and pituitary hormone release in rats
Biochem. Biophys. Res. Commun.
(2002) - et al.
The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism
Regul. Pept.
(2001) - et al.
Venous dilator effect of apelin, an endogenous peptide ligand for the orphan APJ receptor, in conscious rats
Eur. J. Pharmacol.
(2003)
Cited by (267)
Participation of the nitric oxide pathway in lordosis induced by apelin-13 in female rats
2023, Hormones and BehaviorThe profile of adipokines associated with fibrosis and impaired microcirculation in systemic sclerosis
2023, Advances in Medical SciencesIntravenous administration of apeling-13 induces a depressor response by releasing an unidentified substance
2023, Biochemical and Biophysical Research CommunicationsApelin/APJ system in inflammation
2022, International ImmunopharmacologyAPJ/apelin: A promising target for the treatment of retinopathy of prematurity
2022, Drug Discovery Today