Nuttle L C, el-Moatassim C, Dubyak G R
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Mol Pharmacol. 1993 Jul;44(1):93-101.
Extracellular ATP activates two distinct types of P2 purinoreceptor-mediated signaling pathways in macrophages, 1) the rapid formation of nonselective pores/channels in the plasma membrane and 2) a guanine nucleotide-binding protein-dependent stimulation of phosphotidylinositol-specific phospholipase C, with subsequent mobilization of intracellular Ca2+. Several studies have suggested that the pore-forming, or P2z, purinoreceptor may be involved in the cytolytic effects of ATP on macrophages and other cell types. We have identified 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) and UTP as selective agonists for the P2z purinoreceptor and Ca(2+)-mobilizing nucleotide receptor, respectively, in BAC1.2F5 macrophages. In this paper we demonstrate that BzATP and ATP (but not UTP) activate membrane depolarization in BAC1.2F5 cells and also stimulate appropriate electrophysiological responses, consistent with the expression of the P2z purinoreceptor, in Xenopus oocytes injected with poly(A)+ RNA derived from BAC1.2F5 cells. Micromolar concentrations of BzATP or millimolar concentrations of ATP induced a sustained increase in the membrane holding current in these voltage-clamped oocytes. This response was significantly potentiated in the absence of extracellular divalent cations, consistent with the specificity of the P2z purinoreceptor for tetrabasic nucleotides. The sustained currents induced by BzATP or ATP were distinct from the transient and/or oscillating increases in Ca(2+)-dependent Cl- currents that were stimulated by UTP but not BzATP. UTP-stimulated transient currents and nucleotide-dependent increases in aequorin luminescence in poly(A)+ RNA-injected oocytes were independent of extracellular Ca2+ and were correlated with the mobilization of intracellular Ca2+ stores. Sucrose fractionation of the poly(A)+ RNA from BAC1.2F5 cells resulted in the enrichment of mRNA species encoding the components of the P2z purinoreceptor, as well as the Ca(2+)-mobilizing nucleotide receptor, in fractions containing 2.5-4.0-kilobase species.
细胞外ATP可在巨噬细胞中激活两种不同类型的P2嘌呤受体介导的信号通路，1）质膜中快速形成非选择性孔道/通道，2）通过鸟嘌呤核苷酸结合蛋白依赖性刺激磷脂酰肌醇特异性磷脂酶C，随后动员细胞内Ca2+。多项研究表明，形成孔道的P2z嘌呤受体可能参与ATP对巨噬细胞和其他细胞类型的细胞溶解作用。我们已确定3'-O-(4-苯甲酰基)苯甲酰基-ATP（BzATP）和UTP分别是BAC1.2F5巨噬细胞中P2z嘌呤受体和Ca(2+)动员核苷酸受体的选择性激动剂。在本文中，我们证明BzATP和ATP（但不是UTP）可激活BAC1.2F5细胞中的膜去极化，并在注射源自BAC1.2F5细胞的聚(A)+RNA的非洲爪蟾卵母细胞中刺激适当的电生理反应，这与P2z嘌呤受体的表达一致。微摩尔浓度的BzATP或毫摩尔浓度的ATP可诱导这些电压钳制卵母细胞中的膜钳制电流持续增加。在没有细胞外二价阳离子的情况下，这种反应明显增强，这与P2z嘌呤受体对四碱基核苷酸的特异性一致。BzATP或ATP诱导的持续电流不同于UTP刺激的Ca(2+)依赖性Cl-电流的瞬时和/或振荡增加，但BzATP不会刺激这种增加。UTP刺激的瞬时电流和聚(A)+RNA注射卵母细胞中水母发光蛋白发光的核苷酸依赖性增加与细胞外Ca2+无关，并且与细胞内Ca2+储存的动员相关。对BAC1.2F5细胞的聚(A)+RNA进行蔗糖分级分离，导致在含有2.5-4.0千碱基种类的级分中富集了编码P2z嘌呤受体以及Ca(2+)动员核苷酸受体成分的mRNA种类。
