P2X 4 , P2Y 1 and P2Y 2 receptors on rat alveolar macrophages

• Published in: British journal of pharmacology Vol. 140; no. 3; pp. 567 - 575
• Main Authors: Bowler, Jonathan W, Jayne Bailey, R, Alan North, R, Surprenant, Annmarie
• Format: Journal Article
• Language: English
• Published: 02-02-2009
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0705459

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