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-trisulp honic acid (HPTS) to reexamine the mechanisms that extrude acid from voltag e-clamped Helix aspersa neurones. Intracellular acid loads were imposed by three different methods: application of weak acid, depolarization and remov al of extracellular sodium. In nominally CO2/HCO3-free Ringer the rate of r ecovery from acid loads was significantly slowed by the potent Na+/H+ excha nge inhibitor 5-[N-ethyl-N-isopropyl]-amiloride (EIPA, 50 mu M). Following depolarization-induced acidifications the rate of intracellular pH (pH(i)) recovery was significantly reduced from 0.41+/-0.13 pH units.h(-1) in contr ols to 0.12+/-0.09 pH units.h(-1) after treatment with EIPA at pH(i) congru ent to 7.3 (n=7). The amiloride analogue also reduced the rate of acid load ing seen during extracellular sodium removal both in the presence and absen ce of the Na+-dependent Cl-/HCO3- exchange inhibitor 4-acetamido-4'-isothio cyanato-stilbene-2,2'-disulphonic acid (SITS, 50 mu M). This is consistent with EIPA inhibiting reverse-mode Na+/H+ exchange. In 2.5% CO2/20 mM HCO3-b uffered Ringer pH(i) recovery was significantly inhibited by SITS, but unaf fected by EIPA. Our results indicate that there are two separate Na+-depend ent mechanisms involved in the maintenance of pH, in Helix neurones: Na+-de pendent Cl-/HCO3- exchange and Na+/H+ exchange. Acid extrusion from Helix n eurones is predominantly dependent upon the activity of Na+-dependent Cl-/H CO3- exchange with a lesser role for Na+/H+ exchange. This adds further wei ght to the belief that the Na+/H+ exchanger is ubiquitous.