Na+/H+ exchange is the major mechanism of pH regulation in cultured sympathetic neurons: measurements in single cell bodies and neurites using a fluorescent pH indicator.
The regulation of intracellular pH in single cell bodies and in neurites of cultured neurons from rat superior cervical ganglion was studied by continuous monitoring of pH transients using the fluorescent indicator bis(carboxyethyl)carboxy-fluorescein. Intracellular pH was 7.03 +/- 0.05 (n = 8) in bicarbonate-free media at pH 7.4 and was not affected by depolarization with high potassium. Brief exposure to NH4Cl caused rapid cytoplasmic acidification followed by an exponential return of intracellular pH to the resting value. The apparent first order rate constant for recovery from an NH4Cl-induced acid load was 0.2 +/- 0.03 min-1 (37 degrees C) and was similar in media at pH 6.5 or 7.8. Recovery from an acid load was blocked by removal of extracellular Na+ or by amiloride but was not dependent on extracellular Cl- or phosphate or blocked by inhibitors of anion transport, in the presence or absence of bicarbonate. Addition of 5-10 mM bicarbonate at pH 7.4 resulted in a slight alkalinization of the cytoplasm and enhanced complete restoration of pHi after an NH4Cl-induced acid load. Nerve growth factor did not affect intracellular pH of either growing cells deprived of nerve growth factor up to 6 days or of newly isolated neurons left at 4 degrees C for a week before exposure to nerve growth factor. Phorbol 12-myristate 13-acetate had no effect on the pH of cell bodies of growing cells and increased pH of cells deprived of nerve growth factor by less than 0.05 pH units. It is concluded that: pH regulation in cultured sympathetic neurons is largely achieved by Na+/H+ exchange; Bicarbonate may also participate in pH regulation, but not by its exchange with Cl-.
Tolkovsky
A M
AM
Department of Biochemistry, University of Cambridge, U.K.
Richards
C D
CD
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003712:Dendrites / Q000378:metabolism; D005452:Fluoresceins; D005728:Ganglia, Sympathetic / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D006859:Hydrogen / Q000493:pharmacokinetics*; D006863:Hydrogen-Ion Concentration; D009414:Nerve Growth Factors / Q000494:pharmacology; D051381:Rats; D012964:Sodium / Q000493:pharmacokinetics*
D005452:Fluoresceins; D009414:Nerve Growth Factors; D000643:Ammonium Chloride; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000584:Amiloride; D006859:Hydrogen; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium