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 re-examine 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 µM). 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 ≅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 µM). 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.