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P2X 4 , P2Y 1 and P2Y 2 receptors on rat alveolar macrophages
Author(s) -
Bowler Jonathan W,
Jayne Bailey R,
Alan North R,
Surprenant Annmarie
Publication year - 2003
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0705459
Subject(s) - ppads , suramin , p2y receptor , biophysics , egta , apamin , chemistry , p2 receptor , dids , purinergic receptor , intracellular , membrane potential , phospholipase c , reversal potential , potassium channel , patch clamp , receptor , calcium , biochemistry , biology , organic chemistry , membrane
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 EC 50 for ATP was 18 μ M in normal external solution (calcium 2 m M , magnesium 1 m M ). The currents evoked by 2′,3‐ O ‐(4‐benzoyl)benzoyl‐ATP were about five‐fold smaller than those observed with ATP. ADP, UTP and αβ ‐methylene‐ATP ( αβ meATP) (up to 100 μ M ) had no effect. ATP‐evoked currents were potentiated up to ten‐fold by ivermectin and were unaffected by suramin (30–100 μ M ), pyridoxal‐phosphate‐6‐azophenyl‐(2,4‐sulphonic acid) (30–100 μ M ), and brilliant blue G (1 μ M ). In whole‐cell recordings with a potassium‐based internal solution and low EGTA (0.01 m M ), ATP evoked an inward current at −60 mV that was followed by larger outward current. ADP and UTP (1–100 μ M ) evoked only outward currents; these reversed polarity at the potassium equilibrium potential and were blocked by apamin (10 n M ). Outward currents were also blocked by the phospholipase C inhibitor U73122 (1 μ M ), and they were not seen with higher intracellular EGTA (10 m M ). Suramin (30 μ M ) blocked the outward currents evoked by ATP and UTP, but not that evoked by ADP. PPADS (10 μ M ) 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 P2X 4 receptor expression partly associated with the membrane, weak P2X 7 staining that was not associated with the cell membrane, and no P2X 1 receptor immunoreactivity. We conclude that rat alveolar macrophages express (probably homomeric) P2X 4 receptors, but find no evidence for other functional P2X subtypes. The P2Y receptors are most likely P2Y 1 and P2Y 2 and these couple through phospholipase C to an increase in intracellular calcium and the opening of SK type potassium channels.British Journal of Pharmacology (2003) 140 , 567–575. doi: 10.1038/sj.bjp.0705459