Bowler Jonathan W, Bailey R Jayne, North R Alan, Surprenant Annmarie
Institute of Molecular Physiology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK.
Br J Pharmacol. 2003 Oct;140(3):567-75. doi: 10.1038/sj.bjp.0705459. Epub 2003 Aug 26.
ATP receptors present on rat alveolar macrophages (NR8383 cells) were identified by recordings of membrane current, measurements of intracellular calcium, RT-PCR and immunocytochemistry. In whole-cell recordings with a sodium-based internal solution, ATP evoked an inward current at -60 mV. This reversed at 0 mV. The EC50 for ATP was 18 microM in normal external solution (calcium 2 mm, magnesium 1 mm). The currents evoked by 2',3-O-(4-benzoyl)benzoyl-ATP were about five-fold smaller than those observed with ATP. ADP, UTP and alphabeta-methylene-ATP (alphabetameATP) (up to 100 microM) had no effect. ATP-evoked currents were potentiated up to ten-fold by ivermectin and were unaffected by suramin (30-100 microM), pyridoxal-phosphate-6-azophenyl-(2,4-sulphonic acid) (30-100 microM), and brilliant blue G (1 microM). In whole-cell recordings with a potassium-based internal solution and low EGTA (0.01 mm), ATP evoked an inward current at -60 mV that was followed by larger outward current. ADP and UTP (1-100 microM) evoked only outward currents; these reversed polarity at the potassium equilibrium potential and were blocked by apamin (10 nm). Outward currents were also blocked by the phospholipase C inhibitor U73122 (1 microM), and they were not seen with higher intracellular EGTA (10 mm). Suramin (30 microM) blocked the outward currents evoked by ATP and UTP, but not that evoked by ADP. PPADS (10 microM) blocked the ADP-evoked outward current without altering the ATP or UTP currents. RT-PCR showed transcripts for P2X subunits 1, 4 and 7 (not 2, 3, 5, 6) and P2Y receptors 1, 2, 4 and 12 (not 6). Immunocytochemistry showed strong P2X4 receptor expression partly associated with the membrane, weak P2X7 staining that was not associated with the cell membrane, and no P2X1 receptor immunoreactivity. We conclude that rat alveolar macrophages express (probably homomeric) P2X4 receptors, but find no evidence for other functional P2X subtypes. The P2Y receptors are most likely P2Y1 and P2Y2 and these couple through phospholipase C to an increase in intracellular calcium and the opening of SK type potassium channels.
通过膜电流记录、细胞内钙测量、逆转录聚合酶链反应（RT-PCR）和免疫细胞化学方法，鉴定了大鼠肺泡巨噬细胞（NR8383细胞）上的ATP受体。在使用基于钠的细胞内溶液进行的全细胞记录中，ATP在-60 mV时诱发内向电流。该电流在0 mV时反转。在正常细胞外溶液（钙2 mM，镁1 mM）中，ATP的半数有效浓度（EC50）为18 μM。2',3-O-(4-苯甲酰基)苯甲酰基-ATP诱发的电流比ATP诱发的电流小约五倍。ADP、UTP和αβ-亚甲基-ATP（αβmeATP）（高达100 μM）无作用。ATP诱发的电流被伊维菌素增强至十倍，而苏拉明（30 - 100 μM）、磷酸吡哆醛-6-偶氮苯基-(2,4-磺酸)（30 - 100 μM）和亮蓝G（1 μM）对其无影响。在使用基于钾的细胞内溶液和低乙二醇双醚四乙酸（EGTA，0.01 mM）进行的全细胞记录中，ATP在-60 mV时诱发内向电流，随后是更大的外向电流。ADP和UTP（1 - 100 μM）仅诱发外向电流；这些电流在钾平衡电位处反转极性，并被蜂毒明肽（10 nM）阻断。外向电流也被磷脂酶C抑制剂U73122（1 μM）阻断，而在细胞内EGTA浓度较高（10 mM）时未观察到外向电流。苏拉明（30 μM）阻断ATP和UTP诱发的外向电流，但不阻断ADP诱发的外向电流。硫酸吡哆醛-6-苯基-2',4'-二磺酸（PPADS，10 μM）阻断ADP诱发的外向电流，而不改变ATP或UTP诱发的电流。RT-PCR显示有P2X亚基1、4和7（而非2、3、5、6）以及P2Y受体亚型1、2、4和12（而非6）的转录本。免疫细胞化学显示P2X4受体有强表达，部分与细胞膜相关；P2X7染色较弱，与细胞膜无关；未检测到P2X1受体免疫反应性。我们得出结论，大鼠肺泡巨噬细胞表达（可能是同聚体）P2X4受体，但未发现其他功能性P2X亚型的证据。P2Y受体很可能是P2Y1和P2Y2，它们通过磷脂酶C偶联，导致细胞内钙增加和SK型钾通道开放。
