P2X Receptor‐Mediated Inhibition Of NaCl Absorption In The Thick Ascending Limb: No Evidence For Nitric Oxide As Second Messenger

Abstract

BackgroundBasolateral (bl.) P2X receptors acutely and markedly reduce NaCl absorption in mouse medullary thick ascending limb (mTAL). It has been proposed that P2X receptor mediate NO synthesis and subsequent NKCC2 inhibition. P2X receptor stimulation causes an increase in cytosolic Ca2+ and therefore this could be proximal to the generation of NO.ObjectiveHere we tested if blocking NO synthesis, adding an NO donor or hindering Ca2+ influx affects the ATP‐mediated (P2X) transport inhibition.MethodsWe used isolated, perfused mTALs from mice to electrically measure NaCl absorption. By microelectrodes we determined the transepithelial voltage (Vte) and the transepithelial resistance (Rte) and via these the transepithelial Na+ absorption (equivalent short circuit current, I′sc).ResultsWe confirm that bl. ATP (100μM) induced a marked, acute and reversible inhibition of Na+ absorption (28% ± 6%, n=6). In the presence of the NO synthase blocker L‐NAME (100 μM, ~4 min preincubation) the ATP effect remained unaffected (23% ± 9%, n=6). Also higher concentrations and longer incubation with L‐NAME (1 mM, 15min incubation) were without effect. High concentrations of L‐NAME (1 mM) per se reduced transport significantly. Acute addition of the NO donor SNAP (100μM, ~10 min) did not affect tubular transport. Furthermore, the removal of extracellular Ca2+ (100 nM, 2 min preincubation) had no effect on the ATP‐induced transport inhibition ([Ca2+ ]o=100nM: 21% ± 6%, n=5, [Ca2+]o=1μM: 24% ± 4%, n=6).ConclusionWe find no evidence for nitric oxide (NO) as second messenger for P2X receptor‐dependent transport inhibition. Similarly, Ca2+ signaling appears not involved in the ATP effect. It remains undefined how P2X receptors trigger the marked reduction of transport in the TAL.