Calcium ionophores increase intracellular pH in chicken granulosa cells.
Several hormone agonists exert their physiological actions by triggering an inositol phospholipid-Ca2+ signalling cascade and cytosolic alkalinization. Although calcium ionophores have been used extensively to probe the role of Ca2+ in the regulation of steroidogenesis in granulosa cells, the precise relationship between changes in intracellular Ca2+ (Ca2+i) and pH (pHi) is unclear. In the present study we have used a fluorescent pH indicator, 2'7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein, to examine the influence of two Ca2+ ionophores, ionomycin and 4-Bromo-A23187 (4-Br-A23187), on pHi in chicken granulosa cells. Chicken granulosa cells from the largest preovulatory follicle were incubated with Ca2+ ionophores (0-2 microM) and/or inhibitors of Na+/H+ antiport (amiloride, dimethylamiloride and ethylisopropyl amiloride; 0.5, 5 and 50 microM respectively) in the presence of Na+ (or choline+; 0-144 mM) and/or Ca2+ (0-10 mM). Ionomycin or 4-Br-A23187 elicited a rapid and sustained cytosolic alkalinization. The magnitude of increase in pHi was dependent on the concentration of the Ca2+ ionophore and the presence of extracellular Ca2+ but independent of extracellular Na+. Pretreatment of the cells with amiloride or its analogues failed to affect the increase in pHi induced by the Ca2+ ionophores significantly. These findings demonstrate that, in addition to their widely reported effects on Ca2+i redistribution in granulosa cells, 4-Br-A23187 and ionomycin cause Ca(2+)-dependent cytosolic alkalinization. This action of the Ca2+ ionophores is independent of the Na+/H+ antiport. Caution must be exercised in using Ca2+ ionophores as probes to define the role of Ca2+ in the regulation of granulosa cell function.
Asem
E K
EK
Department of Physiology and Pharmacology, School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907.
Li
M
M
Tsang
B K
BK
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000001:Calcimycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism; D002413:Cations, Divalent; D002645:Chickens; D004347:Drug Interactions; D005260:Female; D005452:Fluoresceins; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin / Q000494:pharmacology*; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D002413:Cations, Divalent; D005452:Fluoresceins; D017923:Sodium-Hydrogen Exchangers; D000001:Calcimycin; D015759:Ionomycin; C033170:4-bromo-A-23187; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D002118:Calcium