Ionic control of intracellular pH in rat cerebellar Purkinje cells maintained in culture.

Abstract

SUMMARY1. Intracellular pH (pHi) was measured in single rat cerebellar Purkinje cells maintained in primary culture using microspectrofluorescence analysis of the intracellularly trapped pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF).2. The ratio of the fluorescence signals measured at 530 nm in response to an alternating excitation at 450 and 490 nm was calibrated using the K+-H+ ionophore nigericin. This calibration gave a steady-state pHi of 7-06 + 0-02 (S.E.M., n = 17) when cells were perfused by a 5 % C02-25 mM-HCO3--buffered solution at an external pH of 7 40 at 37 0C.3. Replacement of external chloride with gluconate in the presence of bicarbonate induced a cytoplasmic alkalinization of about 0 3 pH unit. This alkalinization was independent of external sodium and was greatly reduced by 0 5 mM-DIDS, indicating the presence of a chloride-bicarbonate exchange.4. In bicarbonate-free (HEPES-buffered) solution the steady-state pHi was 7-37 +002 (n = 19), significantly higher than in bicarbonate-buffered solution. Recovery from an intracellular acid load brought about by the ammonium chloride pre-pulse technique was blocked by the removal of external sodium or the addition of 1-5 mM-amiloride, indicating the presence of a sodium-hydrogen exchange.5. In bicarbonate-buffered solution pHi recovery after an acid load was also completely blocked by addition of 1-5 mM-amiloride indicating the absence of a bicarbonate-dependent acid extrusion mechanism.6. Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) induced an amiloride-sensitive alkalinization of about 0 3 pH unit in bicarbonate-buffered solution but had no effect in HEPES-buffered solution. This observation suggests that in cultured Purkinje cells the sodium-hydrogen exchanger could be activated through a protein kinase C pathway only when pHi is maintained at a low physiological value by the activity of the chloride-bicarbonate exchange.

Regulation of Intracellular pH in Single Rat Cortical Neurons in vitro: A Microspectrofluorometric Study

“…Furthermore, the introduc tion of C02/HC03 -following an acidification in the presence of harmaline did not enhance the recovery of pHi' Based on these observations, we conclude that rat cortical neurons did not utilize Na + dependent HC03 -IC1-exchange for regulation of their pHi after an acid load. Our results are thus in accordance with those previously reported for syn aptosomes (Nachsen and Drapeau, 1988) and cere bellar Purkinje cells (Gaillard and Dupont, 1990) (see introductory section).…”

“…We report an average steady-state pHj of 7.00 and 7.09 in cultured rat neurons main tained at 37°C (pHe 7.35) in nominally bicarbonate free and in C02/HC03 --containing media, respec tively. This steady-state pHj compares well with values previously reported for vertebrate neurons maintained under similar conditions (Tolkovsky and Richards, 1987;Nachsen and Drapeau, 1988;Gaillard and Dupont, 1990;Nedergaard et aI., 1991;Raley-Susman et aI., 1991).…”

“…Although different type of neurons have been the subject of subsequent stud ies (Chesler, 1990), there have been comparatively few reports of pH regulation in vertebrate neurons. Some authors have concluded that an Na + IH+ ex changer is solely responsible for acid extrusion in vertebrate neurons (Nachsen and Drapeau, 1988;Gaillard and Dupont, 1990), while others have found evidence for additional HC03 --dependent acid extrusion mechanisms (Chesler, 1986;Pocock and Richards, 1989;Raley-Susman et aI., 1991). There are no previous studies of pH regulation in vertebrate neurons following alkaline transients or concerning the effect of pHe on pHi-regulating mechanisms.…”

A large, sustained Na(+)‐ and voltage‐dependent K+ current in spinal neurons of the frog embryo.

“…To control for the possible effect of removing external Na+ on the Na+-H+ exchanger resulting in the acidification of intracellular pH and subsequent block of 352 Nat-DEPENDENT K+ CURRENT IN SPINAL NEURONS K+ currents (Thomas, 1977;Kaila & Vaughan-Jones, 1987;Deutsch & Lee, 1989;Gaillard & Dupont, 1990), further experiments were done using an intracellular medium containing 100 mm Hepes. When Na+ was removed the outward currents were reduced by 30+ 58% (n = 6, Fig.…”

Intrinsic hydrogen ion buffering in rat CNS neurones maintained in culture

“…Neurones, like other cells, adopt two primary methods to regulate their pHi: immediate chemical buffering of H+ and the transmembrane movement of protons or the acid-base equivalent of other ions. Although there have been several recent studies of pH regulation in mammalian neurones (Tolkovsky & Richards, 1987;Gaillard & Dupont, 1990;Raley-Susman, Cragoe, Sapolsky & Kopito, 1991;Pocock & Richards, 1992;Schweining & Boron, 1994), there has been only one systematic study of proton buffering and its variation with pHi (Katsura, Mellergard, Theander, Ouyang & Siesjo, 1993). This information is required to permit calculation of the quantities of acid equivalents transported during pH regulatory processes, which, in the CNS, is further complicated by the diversity of cell types.…”