P2X4, P2Y1 and P2Y2 receptors on rat alveolar macrophages

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Abstract
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.