adenosine-5--o-(3-thiotriphosphate) and Hemolysis

We examined the effect of ATP on hypoxia-induced injury in freshly isolated rat renal proximal tubules and compared it with the effects of stable ATP analogues and ATP degradation products. Extracellular ATP significantly reduced hypoxia-induced structural cell damage (lactate dehydrogenase release). P(2)-receptor agonistic ATP analogues, including 2'-methylthio-ATP (2-Me-S-ATP), were also protective. In contrast, the P(1)-agonistic degradation products AMP and adenosine were not protective. Hypoxia-induced functional cell damage (loss of cellular potassium) was not changed by ATP or 2-Me-S-ATP. We therefore conclude that the protective property of ATP is not based on an effect of the degradation products or on a direct effect on cellular energy metabolism. The data indicate that the protective effect of ATP is mediated by P(2) receptors.

Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Energy Metabolism; Hemolysis; Hypotonic Solutions; Hypoxia; Kidney Tubules, Proximal; Kinetics; L-Lactate Dehydrogenase; Male; Potassium; Quinazolines; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Thionucleotides; Triazoles

We recently reported that ATP is released from Necturus erythrocytes via a conductive pathway during hypotonic swelling and that extracellular ATP potentiates regulatory volume decrease (RVD). This study was designed to determine whether extracellular ATP exerts its effect via a purinoceptor. This was accomplished using three different experimental approaches: 1) hemolysis studies to examine osmotic fragility, 2) a Coulter counter to assess RVD, and 3) the whole-cell patch-clamp technique to measure membrane currents. We found extracellular ATP and ATPgammaS, two P2 agonists, decreased osmotic fragility, enhanced cell volume recovery in response to hypotonic shock, and increased whole-cell currents. In addition, 2-methylthio-ATP potentiated RVD. In contrast, UTP, alpha,beta-methylene-ATP, and 2'-& 3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate and the P1 agonist adenosine had no effect regardless of experimental approach. Furthermore, the P2 antagonist suramin increased osmotic fragility, inhibited RVD, and reduced whole-cell conductance in swollen cells. Consistent with a previous study that indicated cell swelling activates a K+ conductance, suramin had no effect in the presence of gramicidin (a cationophore used to maintain a high K+ permeability). We also found the P2 antagonist pyridoxal-5-phosphate-6-azophenyl-2'4-disulfonic acid (PPADS) increased osmotic fragility; however, reactive blue 2 and the P1 antagonists caffeine and theophylline had no effect. Our results show that extracellular ATP activated a P2 receptor in Necturus erythrocytes during hypotonic swelling, which in turn potentiated RVD by stimulating K+ efflux. Pharmacological evidence suggested the presence of a P2X receptor subtype.

Topics: Adenosine; Adenosine Triphosphate; Animals; Cell Size; Erythrocytes; Gramicidin; Hemolysis; Necturus maculosus; Osmotic Fragility; Patch-Clamp Techniques; Potassium; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Suramin; Thionucleotides