Involvement of uracil nucleotides in protection of cardiomyocytes from hypoxic stress

Abstract

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 P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 14 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 [Ca 2+ ] 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 [Ca 2+ ] i elevation caused by UTP. We used various inhibitors of the Ca 2+ signaling pathway to show that UTP elevated levels of [Ca 2+ ] i , originating from intracellular sources, via activation of phospholipase C and the IP 3 receptor. Interestingly, these inhibitors of the Ca 2+ 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.

Uridine-5′-triphosphate (UTP) reduces infarct size and improves rat heart function after myocardial infarct

“…We have previously shown that activation of UTP receptors protects newborn rat cardiomyocytes against hypoxic damage and significantly reduces the cell death caused by hypoxia via activation of P2Y 2 receptors [26]. In the present study, we extend this observation and show that prior treatment with UTP reduces the ischemic damage to the rat heart using an in vivo model.…”

“…We have previously shown that activation of a UTP receptor prevented intracellular calcium elevation as a result of hypoxia and maintained an increased level of ATP in cultured cardiomyocytes. Suramin, a P2Y 2 receptor antagonist, impeded these protective effects [26]. In the present study, we further established the intracellular protective effects, as evidenced by preventing mitochondrial Ca 2+ overload following hypoxia in cardiomyocyte cultures.…”

“…Whereas the effects of purine nucleosides and nucleotides in myocardial infarction and ischemia have been intensively studied [2,20], the role of pyrimidine nucleotides under hypoxic conditions has not been well explored [18], although the level of both ATP and UTP were found to be elevated as a result of ischemia [7]. Using rat cell culture we found that UTP protected cardiomyocytes from hypoxic damage through the activation of P2Y 2 receptors [26].…”

“…The hypoxic damage was characterized at the end of the hypoxic period by morphological and biochemical evaluation. Continuous monitoring of [Ca 2+ ] i during hypoxia was realized in a special barrier well, where cells were protected from oxygen by a laminar counterflowing layer of argon gas as previously described [26]. The coverglasses with cultured cells were placed at the bottom of the well.…”

Agonists and Antagonists for P2 Receptors

“…P2Y receptors have been linked to either induction/suppression of apoptosis or hypoxic stress and proliferation (Kim et al 2003a, Yitzhaki et al 2005, Coutinho-Silva et al 2005.…”