A role for Na+/H+ exchange in pH regulation in Helix neurones.
We have used the pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) to reexamine the mechanisms that extrude acid from voltage-clamped Helix aspersa neurones. Intracellular acid loads were imposed by three different methods: application of weak acid, depolarization and removal of extracellular sodium. In nominally CO2/HCO3-free Ringer the rate of recovery from acid loads was significantly slowed by the potent Na+/H+ exchange inhibitor 5-[N-ethyl-N-isopropyl]-amiloride (EIPA, 50 microM). Following depolarization-induced acidifications the rate of intracellular pH (pHi) recovery was significantly reduced from 0.41 +/- 0.13 pH units.h-1 in controls to 0.12 +/- 0.09 pH units.h-1 after treatment with EIPA at pHi approximately equal to 7.3 (n = 7). The amiloride analogue also reduced the rate of acid loading seen during extracellular sodium removal both in the presence and absence of the Na(+)-dependent Cl-/HCO3- exchange inhibitor 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid (SITS, 50 microM). This is consistent with EIPA inhibiting reverse-mode Na+/H+ exchange. In 2.5% CO2/20 mM HCO3-buffered Ringer pHi recovery was significantly inhibited by SITS, but unaffected by EIPA. Our results indicate that there are two separate Na(+)-dependent mechanisms involved in the maintenance of pHi in Helix neurones: Na(+)-dependent Cl-/HCO3- exchange and Na+/H+ exchange. Acid extrusion from Helix neurones is predominantly dependent upon the activity of Na(+)-dependent Cl-/HCO3- exchange with a lesser role for Na+/H+ exchange. This adds further weight to the belief that the Na+/H+ exchanger is ubiquitous.
Willoughby
D
D
Department of Physiology, University of Cambridge, UK. dw212@cam.ac.uk
Thomas
R C
RC
Schwiening
C J
CJ
D000143:Acids / Q000378:metabolism / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000378:metabolism; D027963:Chloride-Bicarbonate Antiporters; D006372:Helix, Snails / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007552:Isotonic Solutions / Q000737:chemistry / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D000077331:Ringer's Solution; D012964:Sodium / Q000502:physiology; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology*
D000143:Acids; D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; D007552:Isotonic Solutions; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D006859:Hydrogen; D000077331:Ringer's Solution; D012964:Sodium; C039614:ethylisopropylamiloride