Effects of osmotic perturbation on [Ca2+]i and pHi in rabbit proximal tubular cells in primary culture.
The effects of anisosmotic media on intracellular Ca2+ and H+ concentrations ([Ca2+]i and pHi, respectively) were studied to investigate whether these changes play a role in epithelial cell volume regulation. [Ca2+]i and pHi were measured in rabbit proximal tubular cells in primary culture using the fluorescent ratio probes fura 2 and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Changing medium osmolarity from 300 to 150 mosmol resulted in a rapid transient increase in fura 2 ratio from 0.89 +/- 0.02 to 1.15 +/- 0.03, which lasted for several minutes and returned to base line within 10 min. The source of Ca2+ was extracellular as well as intracellular. Simultaneous with this increase in [Ca2+]i, cells slowly acidified from pHi of 7.51 +/- 0.03 to 6.86 +/- 0.02. This osmotic swelling-induced acidification could not be explained by a decrease in the rate of Na+/H+ exchange or increase in the rate of Cl-/HCO3- exchange. Subsequently increasing medium osmolarity from 150 to 500 mosmol decreased the fura 2 ratio below the initial level observed in isotonic media, while pHi increased from 6.96 +/- 0.02 to 7.37 +/- 0.03. This decrease in [Ca2+]i was due to inhibition of Ca2+ influx and to an increase in Ca2+ efflux. The osmotic shrinkage-induced alkalinization was slightly inhibited by ethylisopropylamiloride, indicative of activation of Na+/H+ exchange. To test whether an increase in [Ca2+]i causes a decrease in pHi or vice versa, pHi and [Ca2+]i were manipulated at isotonic conditions. Surprisingly, a decrease in [Ca2+]i was accompanied by a decrease in pHi, and an increase in pHi resulted in an increase in [Ca2+]i, in the absence of osmotic perturbation.(ABSTRACT TRUNCATED AT 250 WORDS)
Raat
N J
NJ
Department of Cell Physiology, University of Nijmegen, The Netherlands.
van Os
C H
CH
Bindels
R J
RJ
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D011817:Rabbits
D002118:Calcium