Na+/H+ exchange in granulosa cells of the three largest preovulatory follicles of the domestic hen.
Sodium-dependent intracellular pH (pHi) regulation was compared in granulosa cells from the three largest avian ovarian follicles by monitoring the pHi with biscarboxyethylcarboxyfluorescein, a dye whose fluorescence increases with alkalinity. Collagenase-dispersed granulosa cells obtained from the largest (F1), second largest (F2), and third largest (F3) preovulatory follicles about 2-3 hr prior to expected ovulation of F1 were used in the present study. The resting pHi measured in nominally bicarbonate free buffer with extra-cellular Na+ (Nao+ = 144 mM) and external pH (pHo) of 7.3 was about 6.8 in cells from F1, F2, and F3. There was no correlation between the stage of follicular development and the pHi whether the follicles were removed in the early or late preovulatory period. After acute cytoplasmic acidification by exposure of cells to nigericin in choline+ buffer, or by the abrupt removal of ammonium chloride, complete recovery of pHi occurred in 4-5 min. The rate and magnitude of the recovery were dependent upon the concentration of Nao+ and were abolished when Nao+ was replaced completely by choline+. Recovery in the presence of Nao+ was inhibited dose-dependently by amiloride (sodium-hydrogen exchange inhibitor). There was no difference between the rate and the extent of pHi recovery in acid-loaded cells obtained from F1, F2, and F3. Furthermore, by varying the concentration of Nao+ between 0 and 144 mM both young and matured granulosa cells extruded acid at the same rate. In addition, amiloride inhibited the Nao+ dependency of pHi recovery to a similar degree in F1, F2, and F3 cells. Our observations demonstrate in avian granulosa cells the existence of a Nao+-dependent, amiloride-sensitive pHi regulatory system that is equally effective in cells obtained from the three largest yolk-filled follicles.
Asem
E K
EK
Department of Obstetrics and Gynecology, University of Ottawa, Ontario, Canada.
Tsang
B K
BK
D000584:Amiloride; D000643:Ammonium Chloride; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis; D002645:Chickens / Q000378:metabolism*; D005260:Female; D005498:Follicular Phase; D006107:Granulosa Cells / Q000032:analysis / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D000584:Amiloride; D006859:Hydrogen; D012964:Sodium