Light D B, Dahlstrom P K, Gronau R T, Baumann N L
Department of Biology, Ripon College, 300 Seward St, Ripon, WI 54971, USA.
J Membr Biol. 2001 Aug 1;182(3):193-202. doi: 10.1007/s0023201-0043-z.
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.
我们最近报道，在低渗肿胀过程中，三趾螈红细胞通过一条传导途径释放ATP，且细胞外ATP可增强调节性容积减小（RVD）。本研究旨在确定细胞外ATP是否通过嘌呤受体发挥作用。这是通过三种不同的实验方法完成的：1）溶血研究以检查渗透脆性；2）库尔特计数器以评估RVD；3）全细胞膜片钳技术以测量膜电流。我们发现两种P2激动剂——细胞外ATP和ATPγS，降低了渗透脆性，增强了细胞在低渗休克后的容积恢复，并增加了全细胞电流。此外，2-甲硫基-ATP增强了RVD。相比之下，无论采用何种实验方法，UTP、α,β-亚甲基-ATP、2'-& 3'-O-(4-苯甲酰苯甲酰基)腺苷5'-三磷酸以及P1激动剂腺苷均无作用。此外，P2拮抗剂苏拉明增加了渗透脆性，抑制了RVD，并降低了肿胀细胞的全细胞电导。与之前一项表明细胞肿胀激活钾离子电导的研究一致，在短杆菌肽（一种用于维持高钾离子通透性的阳离子载体）存在的情况下，苏拉明无作用。我们还发现P2拮抗剂磷酸吡哆醛-5-磷酸-6-偶氮苯基-2',4'-二磺酸（PPADS）增加了渗透脆性；然而，反应性蓝2以及P1拮抗剂咖啡因和茶碱均无作用。我们的结果表明，在低渗肿胀过程中，细胞外ATP激活了三趾螈红细胞中的P2受体，进而通过刺激钾离子外流增强了RVD。药理学证据表明存在一种P2X受体亚型。
