Role of intracellular pH in secretion from adrenal medulla chromaffin cells.
The role of intracellular pH in stimulus-secretion coupling was investigated in cultured bovine adrenal medullary chromaffin cells. NH4Cl (1-25 mM) did not affect basal catecholamine or ATP release but markedly inhibited nicotine- or high K+-induced release by up to 60%. The inhibition had a rapid onset (less than 1 min) and was maximal at about 5 mM NH4Cl. The effect of NH4Cl was largely sustained over 20 min and was reversed upon NH4Cl removal. Sodium propionate did not affect secretion but partially reversed the inhibition by NH4Cl in a concentration-dependent manner. Methylamine (10 mM) produced a similar, but slower, inhibition than NH4Cl. Monensin (1-10 microM) inhibited catecholamine secretion by 30-60%, and its effect was reduced in the presence of NH4Cl. Using the fluorescent Ca2+ probe Fura-2, we found that the increase of [Ca2+]i following stimulation was not altered by concentrations of NH4Cl which inhibited secretion maximally. Measurement of cytosolic pH (pHi) with the fluorescent probe 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) revealed an alkalinization by NH4Cl (2.5-25 mM) of 0.1-0.23 pH units and acidification by sodium propionate (10-20 mM) of 0.2-0.25 pH units, with intermediate combined effects. Monensin (1 microM) caused a cytosolic acidification of 0.26 pH units. All pHi changes were partly recovered in 15 min. Fluorescence quenching measurements using the weakly basic fluorescent probe acridine orange indicated the accumulation of the probe into acidic compartments, presumably the chromaffin granules, which was strongly reduced by both NH4Cl and monensin. From these findings we conclude that the pH of the chromaffin granule modulates secretion by affecting some step in the secretory process unrelated to the rise in [Ca2+]i.
Kuijpers
G A
GA
Laboratory of Cell Biology and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
Rosario
L M
LM
Ornberg
R L
RL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000313:Adrenal Medulla / Q000378:metabolism*; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D002837:Chromaffin Granules / Q000187:drug effects / Q000378:metabolism*; D002838:Chromaffin System / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D008744:Methylamines / Q000494:pharmacology; D009538:Nicotine / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D014701:Veratridine / Q000494:pharmacology
D002395:Catecholamines; D008744:Methylamines; D000643:Ammonium Chloride; D001464:Barium; D009538:Nicotine; D014701:Veratridine; D000255:Adenosine Triphosphate; C027451:methylamine; D011188:Potassium; D002118:Calcium