[(125)I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man

Abstract

1. We have determined the binding characteristics of [(125)I]-(Pyr(1))Apelin-13, a putative ligand for the APJ orphan receptor in human cardiovascular and rat tissue and investigated the functional properties of (Pyr(1))Apelin-13 in human saphenous vein. 2. The binding of [(125)I]-(Pyr(1))Apelin-13 to sections of human heart tissue was time dependent and rapid at 23 degrees C. Data were fitted to a single site model with an association rate constant (k(obs)) of 0.115 min(-1). [(125)I]-(Pyr(1))Apelin-13 also dissociated from a single site with a dissociation rate constant of 0.0105 min(-1). 3. In saturation binding experiments [(125)I]-(Pyr(1))Apelin-13 bound to human left ventricle with a K(D) value of 0.35+/-0.08 nM, B(max) of 4.3+/-0.9 fmol mg(-1) protein with a Hill slope of 0.97+/-0.04 and to the right atria with a K(D) of 0.33+/-0.09 nM, B(max) of 3.1+/-0.6 fmol mg(-1) protein and a Hill slope of 0.93+/-0.05. 4. [(125)I]-(Pyr(1))Apelin-13 binding sites were localized using autoradiography to human cardiovascular tissue, including coronary artery, aorta and saphenous vein grafts. In rat tissue a high density of receptors were localized to the molecular layer of the rat cerebellum, rat lung, rat heart and low levels in the rat kidney cortex. 2. (Pyr(1))Apelin-13 potently contracted human saphenous vein with a pD(2) value of 8.4+/-0.2 (n=8). The maximum response elicited by the peptide was 22.6+/-6% of 100 mM KCl. 6. We provide the first evidence of APJ receptor expression, relative densities and functional properties of (Pyr(1))Apelin-13 in human cardiovascular tissue.

Figure 1

Structure of [¹²⁵I]-(Pyr¹)Apelin-13. [¹²⁵I]-(Pyr¹)Apelin-13 was prepared from the unlabelled material (Pyr¹)Apelin-13 by conjugation with monoiodinated (¹²⁵I) Bolton & Hunter reagent, which labelled the Lys⁸ residue.

Figure 2

Time-dependent association of [¹²⁵I]-(Pyr¹)Apelin-13 binding to sections of human left ventricle. Tissue sections were incubated for 150 min at room temperature. Data represents a single experiment, with an association rate constant (k_obs) of 0.109 min⁻¹ for this particular experiment.

Figure 3

Time course for dissociation of 0.15 nM [¹²⁵I]-(Pyr¹)Apelin-13 from sections of human left ventricle. Tissue sections were incubated for 30 min and washed in excess of Tris buffer for differing time points up to 4 h. A representative curve is shown, with a dissociation rate constant of 0.016 min⁻¹ for this particular experiment.

Figure 4

Saturation binding curve for [¹²⁵I]-(Pyr¹)Apelin-13. Increasing concentrations of radioligand (0.01 – 1.25 nM) were incubated with sections of human left ventricle for 30 min at room temperature. Curve represents a typical result, with a dissociation constant (K_D) of 0.3 nM and maximal receptor density (B_max) of 4.2 fmol mg⁻¹ protein.

Figure 5

(A) Autoradiographical localization of APJ receptor to rat brain with high density in the molecular layer (m) of the rat cerebellum. (B) Section stained with heamatoxylin and eosin. Scale bar=2 mm.

Figure 6

Autoradiographical localization of APJ receptors to (A) human left ventricle section. (B) Transverse section of a human non-diseased coronary artery. (C) Longitudinal section of a pulmonary artery. (D) Transverse section of a saphenous vein graft with binding in both the media (m) as well as the intimal (i) smooth muscle layers. No image was detected in the presence of 1 μM cold ligand. Scale bars=5 mm (A and C), 2 mm (B and D).

Figure 7

Cumulative concentration response curves to (Pyr¹)Apelin-13 (0.1 – 300 nM) in human saphenous vein. This trace is representative of eight experiments.

Figure 8

Concentration response curve to (Pyr¹)Apelin-13 in human saphenous vein. Data points are mean±s.e.mean, n=8.