Simultaneous imaging of intracellular [Ca2+] and pH in single MDCK and glomerular epithelial cells.
The interrelationships between changes in intracellular calcium concentration ([Ca2+]i) and intracellular pH in Madin-Darby canine kidney cells and kidney glomerular epithelial cells exposed to various stimuli were analyzed simultaneously using a new design of a fluorescence video microscope. Cells were double labeled with indo 1 and SNARF 1 dyes and were excited simultaneously at 350 and 540 nm. Images at four emission wavelengths were captured simultaneously at 405, 475, 575, and 640 nm at 30 frames/s for the two ratio dyes. SNARF sensitivity to pH between 6.5 and 8.0 was unchanged by [Ca2+]i. The SNARF ratio maps were used to correct the pH-dependent changes in the calculation of local cell calcium. NH4Cl loading produced the expected alkalinization and a concurrent rise in [Ca2+]i. When the NH4Cl was removed and the cells became acidic, a second rise in [Ca2+]i was recorded. Both changes in [Ca2+]i were from intracellular stores since they persisted in the absence of extracellular calcium. The findings demonstrate the need for pH correction of indo 1 recordings.
Wiegmann
T B
TB
Renal Section, Veterans Affairs Medical Center, Kansas City 64128.
Welling
L W
LW
Beatty
D M
DM
Howard
D E
DE
Vamos
S
S
Morris
S J
SJ
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007211:Indoles; D007425:Intracellular Membranes / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration
D005456:Fluorescent Dyes; D007211:Indoles; D000643:Ammonium Chloride; C048960:indo-1; D002118:Calcium