Involvement of uracil nucleotides in protection of cardiomyocytes from hypoxic stress

Cardiomyocytes express one or more subtypes of P2 receptors for extracellular nucleotides. P2 purinoceptors, which are activated by nucleotides, are classified as P2X or P2Y: P2X receptors are ligand-gated intrinsic ion channels, and P2Y receptors are G protein-coupled receptors. Extracellular pyrimidine and purine nucleotides are released from the heart during hypoxia. Although the cardioprotective effects of purines acting via purinoceptors were studied intensively, the physiological role of uracil nucleotide-responsive P2Y₂, P2Y₄, P2Y₆, and P2Y₁₄ receptors is still unclear, especially in the cardiovascular system. This study revealed that uridine-5′-triphosphate (UTP) protected cultured rat cardiomyocytes during hypoxia and explored the UTP signaling pathway leading to this cardioprotection. We found that UTP, but not UDP or uridine, significantly reduced cardiomyocyte death induced by hypoxia. Incubation with UTP for 1 h, before exposure to hypoxic conditions, protected the cells 24 h later. The cardioprotective effect of UTP was reduced in the presence of the P2 antagonist suramin. In addition, UTP caused a transient increase of [Ca²⁺] _i in cardiomyocytes. Pyridoxal-5′-phosphate-6-azophenyl-2,4- disulfonate (PPADS) or Reactive blue 2 (RB-2), other antagonists of P2 receptors, abolished the [Ca²⁺]_i elevation caused by UTP. We used various inhibitors of the Ca²⁺ signaling pathway to show that UTP elevated levels of [Ca²⁺]_i, originating from intracellular sources, via activation of phospholipase C and the IP₃ receptor. Interestingly, these inhibitors of the Ca²⁺ signaling pathway did not prevent the immediate protective effect caused by UTP. Although mitochondrial K_ATP channels are involved in other preconditioning mediator pathways, the involvement of these channels in the cardioprotective effect induced by UTP was ruled out, because 5-hydroxydecanoic acid (5-HD), a specific inhibitor of these channels, did not prevent the protection.