SUMMARY1. Double-barrelled, neutral-carrier pH-sensitive micro-electrodes were used to measure the intracellular pH (pHi) and the pHi regulation of neuropile glial (n.g.) cells and of identified neurones of the leech Hirudo medicinalis.2. The distribution of H+ in the n.g. cells and in the neurones was found not to be in electrical equilibrium. The mean pHi ofthe n.g. cells was 6-87 + 0 13 (mean + S.D.of mean here and for all following data n = 27) in HEPES-buffered leech saline (pHo = 7 4) and 7418 + 0-19 (n = 13) in 2 % C02-11 mM-HC03--saline. The mean pHi was 7'28+0-1 (n = 20) in Retzius neurones and 7-32+0415 (n = 12) in noxious neurones in HEPES-buffered leech saline, and 7-20+0415 (n = 10) and 7-27 +016 (n = 6) in 2 % C02-1 1 mM-HC03--buffered saline in these two types of neurones, respectively.3. The cytoplasmic buffering power, as calculated by the change in pHi following the change from 2 % C02-11 mM-HC03-to 5 % C02-22 mM-HCO.-in the leech saline, was 20-30 mM/pH unit in the n.g. cells and between 12 and 33 mM/pH unit in the neurones.4. The recovery of pHi in n.g. cells from an experimentally induced acidification (addition and removal of 20 mm-NH4Cl) was dependent on the presence of external Na+. Independent of the buffer system used, pHi recovery was inhibited when external Na+ was exchanged by N-methyl-D-glucamine. Amiloride (2-3 mM) reduced the rate of pHi recovery by about 50 % in these n.g. cells.5. In CO2-HCO3--free saline, or in the presence of the anion exchange blocker 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonic acid (SITS, 0 5 mM), pHi recovery from an acid load was often slowed by up to 50 % in n.g. cells. This suggests that there is a significant contribution of a HC03--dependent membrane transport to pHi regulation in n.g. cells.6. When a HEPES-buffered saline was exchanged by a 2 % C02-11 mM-HCO3-buffered saline, the pHi of n.g. cells increased by 0-31 pH units. This alkaline shift was reversible upon removal of the CO2-HC03-and was absent in the Na+-free saline. It was not inhibited by 1 mM-furosemide or by 0 5 mM-SITS. Successive addition of first Na+ and then CO2-HCO3-to an Na+-free HEPES-buffered saline produced two separate phases of intracellular alkalization, suggesting two different pHi regulation transport systems.J. W. DEITMER AND W.-R. SCHLUE 7. In sensory and Retzius neurones pHi recovery from acidification was also dependent on external Na+, both in the presence and in the absence of CO2-HC03-.The rate of pHi recovery was reversibly reduced by 2 mM-amiloride by up to 90 % in these neurones.8. In conclusion, bicarbonate buffer affects n.g. cell pHi differently from the way in which it affects neuronal pHi. The regulation of pHi depends on the presence of external Na+ in both n.g. cells and neurones. The experiments suggest the presence of an Na+-H+ exchange and a SITS-sensitive HCO3--and Na+-dependent acid extrusion mechanism across n.g. (and nerve cell) membranes and another, SITSinsensitive HC03-and Na+-dependent transport system only in n.g. cells.

We used the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to monitor intracellular pH (pHi) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally CO2/HCO3(-)-free medium buffered to pH 7.40 with HEPES at 37 degrees C, the cells had a mean pHi of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO2 and 25 mM HCO3(-) caused the steady-state pHi to increase by an average of 0.35, suggesting the presence of a HCO3(-) -dependent acid-extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO3(-)-free (HEPES-buffered) solutions, the application and withdrawal of 20 mM extracellular NH4+ caused pHi to fall to a value substantially below the initial one. pHi spontaneously recovered from this acid load, stabilizing at a value approximately 0.1 higher than the one prevailing before the application of NH4+. In other experiments conducted on cell bathed in HEPES-buffered solutions, removing extracellular Na+ caused pHi to decrease rapidly by 0.5. Returning the Na+ caused pHi to increase rapidly, indicating the presence of an Na(+)-dependent/HCO3(-)-dependent acid-extrusion mechanism; the final pHi after returning Na+ was approximately 0.08 higher than the initial value. This pHi recovery elicited by returning Na+ was not substantially affected by 50 microM ethylisopropylamiloride (EIPA), but was speeded up by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS).(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARY1. Intracellular pH (pHi) and the mechanism of pHi regulation have been investigated in cultured oligodendrocytes from mouse spinal cord using doublebarrelled neutral-carrier H+-selective microelectrodes. The distribution of H+ was not in electrochemical equilibrium. The pHi was more alkaline than the pH of the bathing medium (pHO), namely 7-5 at pHo 7-2 and 7-6 at pHo 7-4. 2. Removal of HCO3-from the bathing medium reduced the steady-state pHi by 0 4 units. An increase in extracellular K+ caused, with a delay, an increase in pHi. A decrease in pHO to 6-2 caused an acidification of pHi by 0 5 units.3. The pHi regulation was studied by applying and subsequently removing NH4, which resulted in an acidification of the cell. The subsequent recovery of pHi could then be analysed. The recovery from an acidification by 1 pH unit lasted 3-10 min.In HCO3--free solution pHi recovery was slowed. 4. In HCO3--free solution pHi recovery was completely blocked when either Na+ was removed or when amiloride was applied indicating an exclusive activation of the Na+-H+ exchanger.5. In the presence of HCO3 , removal of Na+ also completely blocked pH1 recovery. When Na+ was readded, pHi recovered. In HCO3--containing solution amiloride slightly slowed, but did not block pHi recovery.6. Removal of Cl-or application of SITS, DIDS or furosemide, blockers of Cl--coupled transport mechanisms, did not affect the pHi recovery in the presence of HCO3-.7. In conclusion, oligodendrocytes possess two mechanisms regulating pHi, a Na+-H+ exchanger and a Na+-HCO3-co-transporter while the latter is clearly more potent. It follows that pHi regulation of oligodendrocytes is dependent on the transmembrane Na+ gradient and is strictly separated from regulation of internal Cl.

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