Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic-Dependent Ca2+ Pathways in Rat Pulmonary Artery Smooth Muscle Cells

Abstract

Background/Aims: Adenosine diphosphate ribose (ADPR), a product of β-NAD⁺ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs); however the physiological function of extracellular ADPR is unclear. Methods: Ca²⁺ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y) subtypes were examined in pulmonary arteries. Results: ADPR elicited concentration-dependent increase in [Ca²⁺]_i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca²⁺ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X) antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca²⁺ response, while the P2(XY) inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca²⁺ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca²⁺ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca²⁺ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. Conclusion: ADPR and ATP activate Ca²⁺ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.