Background and Purpose: Adenosine is a local mediator that regulates changes in the cardiovascular system via activation of four G protein-coupled receptors (A1, A2A, A2B, A3). Here we have investigated the effect of A2A and A2B-selective agonists on vasodilatation in three distinct vascular beds of the rat cardiovascular system. Experimental Approach: The regional haemodynamic effects of adenosine A2A and A2B selective agonists were investigated in conscious rats. Male Sprague-Dawley rats (350-450g) were chronically implanted with pulsed Doppler flow probes on the renal artery, mesenteric artery and the descending abdominal aorta. Cardiovascular responses were measured following intravenous infusion (3 min for each dose) of CGS 21680 (0.1, 0.3, 1 g.kg-1.min-1) or BAY 60-6583 (4,13.3, 40 g.kg-1.min-1) following pre-dosing with either SCH 58261 (0.1 or 1 mg.kg-1.min-1), PSB 1115 (10 mg.kg-1.min-1) or vehicle. Key Results: The A2A-selective agonist CGS 21680 produced a striking increase in heart rate (HR) and hindquarters vascular conductance (VC) that was accompanied by a significant decrease in mean arterial pressure (MAP) in conscious rats. In marked contrast, the A2B-selective agonist BAY 60-6583 significantly increased HR and VC in the renal and mesenteric vascular beds, but not in the hindquarters. Conclusions and Implications: Taken together, these data indicate that A2A and A2B receptors are regionally selective in their regulation of vascular tone. These results suggest that the development of A2B receptor agonists to induce vasodilatation in the kidney may provide a good therapeutic approach for the treatment of acute kidney injury.

Background: Vascular Endothelial Growth Factor A (VEGF-A) is a key mediator of angiogenesis, primarily signalling via VEGF Receptor 2 (VEGFR2). Endothelial cells also express the co-receptor Neuropilin-1 (NRP1) that potentiates VEGF-A/VEGFR2 signalling. VEGFR2 and NRP1 had distinct real-time ligand binding kinetics when monitored using Bioluminescence Resonance Energy Transfer (BRET). We previously characterised fluorescent VEGF-A isoforms tagged at a single site with tetramethylrhodamine (TMR). Here, we explore differences between VEGF-A isoforms in living cells that co-expressed both receptors. Experimental Approach: Receptor localisation was monitored in HEK293T cells expressing both VEGFR2 and NRP1 using a membrane-impermeant HaloTag and SnapTag technologies. To isolate ligand binding pharmacology at a defined VEGFR2/NRP1 complex, we developed an assay using NanoBiT complementation technology whereby heteromerization is required for luminescence emissions. Binding affinities and kinetics of VEGFR2-selective VEGF165b-TMR and non-selective VEGF165a-TMR were monitored using BRET from this defined complex. Key Results: Cell surface VEGFR2 and NRP1 were co-localised and formed a constitutive heteromeric complex. Despite being selective for VEGFR2, VEGF165b-TMR had a distinct kinetic ligand binding profile at the complex that largely remained elevated in cells over 90 minutes. VEGF165a-TMR bound to the VEGFR2/NRP1 complex with kinetics comparable to those of VEGFR2 alone. Using a binding-dead mutant of NRP1 had no impact on the binding kinetics or affinity of VEGF165a-TMR. Conclusions and Implications: This NanoBiT approach enabled real-time ligand binding to be quantified in living cells at 37°C from a specified complex between a receptor tyrosine kinase and its co-receptor for the first time.