title: P2X receptors trigger intracellular alkalization in isolated perfused mouse medullary thick ascending limb.
abstract: OBJECTIVE
  Extracellular ATP is an important regulator of renal tubular transport. Recently, we found that basolateral ATP markedly inhibits Na(+) and Cl(-) absorption in mouse medullary thick ascending limb (mTAL) via a P2X receptor. The underlying mechanism that mediates this ATP-dependent transport inhibition in mTAL is, however, unclear. The renal outer medullary K(+) channel (ROMK) is sensitive to intracellular pH where a reduction leads to closing of ROMK. We speculated that P2X receptor stimulation in the TAL could lead to changes in pHi , leading to a reduction in NaCl transport.
  
  
  METHODS
  To test this hypothesis, we measured pHi in single perfused mouse mTALs using the fluorescent ratiometric dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester.
  
  
  RESULTS
  Interestingly, basolateral ATP (100 μm) caused a prominent, reversible intracellular alkalization of mTAL, with an average pHi increase of 0.14 ± 0.02 (n = 14). This was completely abolished by the P2X receptor antagonist periodate-oxidized ATP (50 μm). The P2X receptor-mediated intracellular alkalization required the activity of the apical Na(+) /H(+) exchanger (NHE3). Typically, Gq -coupled receptors cause a significant acidification of tubular epithelial cells, which was confirmed in this study, by P2Y2 and Ca(2+) sensing receptor stimulation.
  
  
  CONCLUSIONS
  This study reports that stimulation of basolateral P2X receptors causes a substantial intracellular alkalization in the isolated perfused mouse mTAL. This intracellular alkalization is mediated through an increased apical NHE3 activity, similar to what we previously observed when tubular transport is inhibited with furosemide. This increased NHE3 activity causes H(+) secretion in the mTAL and provides further support that the TAL is a site of urinary acidification.
authors.affiliation: Department of Biomedicine, Physiology and Biophysics, Aarhus University, Aarhus C, Denmark.
authors.forename: P I A
authors.initials: PI
authors.lastname: de Bruijn
authors.forename: M
authors.initials: M
authors.lastname: Bleich
authors.forename: H A
authors.initials: HA
authors.lastname: Praetorius
authors.forename: J
authors.initials: J
authors.lastname: Leipziger
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000378:metabolism; D017136:Ion Transport / Q000502:physiology; D007679:Kidney Medulla / Q000378:metabolism*; D008138:Loop of Henle / Q000378:metabolism*; D051379:Mice; D011188:Potassium / Q000378:metabolism; D058469:Receptors, Purinergic P2X / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism
chemical_list: D058469:Receptors, Purinergic P2X; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium; D011188:Potassium
keywords: Na+/H+ exchanger 3; P2 receptors; intracellular pH; loop of Henle; purinergic