Yuting Tang1, Lubing Zhou. 1. Department of Drug Discovery, Johnson and Johnson Pharmaceutical Research and Development, L.L.C, 1000 Rt. 202, Raritan, NJ 08869, USA. ytang@prdus.jnj.com
Abstract
BACKGROUND: Adenosine A1 receptors (A1ARs) modulate various aspects of renal functions, such as hormone release, hemodynamics and tubular absorption. Here we set up to demonstrate the expression of A1ARs in an immortalized cell line (HK-2) derived from normal adult human proximal tubule. We also examined the mechanism whereby A1ARs signal in HK-2 cells and their potential role in renal physiology such as sodium-dependent phosphate transport. METHODS: Ligand binding assay of A1ARs was performed using plasma membranes of HK-2 cells and a selective high-affinity A1AR radioligand [3H]DPCPX. HK-2 cells in 96-well plates were treated with various agents (forskolin, adenosine receptor agonists, and antagonists) to activate or inhibit adenylate cyclase. Intracellular cyclic AMP accumulation was measured using cAMP flashplates. mRNA levels of adenosine receptors in HK-2 cells was determined by real-time PCR technique. Sodium-dependent phosphate transport across cell membrane was measured after 15-minute incubation of phosphorus-33 in transport buffer with HK-2 cells at room temperature. RESULTS: In HK-2 cells, A1ARs were expressed at a density of 211 +/- 74 fmol/mg membrane proteins. [3H]DPCPX bound to A1ARs on HK-2 cell membranes with Kd of 8.3 +/- 2.2 nM. Activation of A1ARs inhibited isoproterenol-stimulated adenylate cyclase activity through pertussis toxin-sensitive Gi protein in HK-2 cells. Coexpression of adenosine A2a receptors at a seemingly lower level than A1ARs was revealed by synergistically activating adenylate cyclase with forskolin. Real-time RT-PCR further demonstrated the expression of both A1AR and A2aAR in HK-2 cells. Sodium-dependent phosphate transport was augmented by activation of A1ARs in HK-2 cells. CONCLUSION: A1ARs are expressed in human proximal tubule epithelial (HK-2) cells and modulate sodium-dependent phosphate transport.
BACKGROUND: Adenosine A1 receptors (A1ARs) modulate various aspects of renal functions, such as hormone release, hemodynamics and tubular absorption. Here we set up to demonstrate the expression of A1ARs in an immortalized cell line (HK-2) derived from normal adult human proximal tubule. We also examined the mechanism whereby A1ARs signal in HK-2 cells and their potential role in renal physiology such as sodium-dependent phosphate transport. METHODS: Ligand binding assay of A1ARs was performed using plasma membranes of HK-2 cells and a selective high-affinity A1AR radioligand [3H]DPCPX. HK-2 cells in 96-well plates were treated with various agents (forskolin, adenosine receptor agonists, and antagonists) to activate or inhibit adenylate cyclase. Intracellular cyclic AMP accumulation was measured using cAMP flashplates. mRNA levels of adenosine receptors in HK-2 cells was determined by real-time PCR technique. Sodium-dependent phosphate transport across cell membrane was measured after 15-minute incubation of phosphorus-33 in transport buffer with HK-2 cells at room temperature. RESULTS: In HK-2 cells, A1ARs were expressed at a density of 211 +/- 74 fmol/mg membrane proteins. [3H]DPCPX bound to A1ARs on HK-2 cell membranes with Kd of 8.3 +/- 2.2 nM. Activation of A1ARs inhibited isoproterenol-stimulated adenylate cyclase activity through pertussis toxin-sensitive Gi protein in HK-2 cells. Coexpression of adenosine A2a receptors at a seemingly lower level than A1ARs was revealed by synergistically activating adenylate cyclase with forskolin. Real-time RT-PCR further demonstrated the expression of both A1AR and A2aAR in HK-2 cells. Sodium-dependent phosphate transport was augmented by activation of A1ARs in HK-2 cells. CONCLUSION: A1ARs are expressed in human proximal tubule epithelial (HK-2) cells and modulate sodium-dependent phosphate transport.
Authors: Robert A Fenton; Søren B Poulsen; Samantha de la Mora Chavez; Manoocher Soleimani; Meinrad Busslinger; Jessica A Dominguez Rieg; Timo Rieg Journal: Am J Physiol Renal Physiol Date: 2015-04-29