EFFECTS OF OSMOTIC PERTURBATION ON [CA2+](I) AND PH(I) IN RABBIT PROXIMAL TUBULAR CELLS IN PRIMARY CULTURE

The effects of anisosmotic media on intracellular Ca2+ and H+ concentr ations ([Ca2+](i) and pH(i), respectively) were studied to investigate whether these changes play a role in epithelial cell volume regulatio n. [Ca2+](i) and pH(i) 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+ w as extracellular as well as intracellular. Simultaneous with this incr ease in [Ca2+](i), cells slowly acidified from pH(i) of 7.51 +/- 0.03 to 6.86 +/- 0.02. This osmotic swelling-induced acidification could no t be explained by a decrease in the rate of Na+/H+ exchange or increas e in the rate of Cl-/HCO3- exchange. Subsequently increasing medium os molarity from 150 to 500 mosmol decreased the fura 2 ratio below the i nitial level observed in isotonic media, while pH(i) increased from 6. 96 +/- 0.02 to 7.37 +/- 0.03. This decrease in [Ca2+](i) was due to in hibition of Ca2+ influx and to an increase in Ca2+ efflux. The osmotic shrinkage-induced alkalinization was slightly inhibited by ethylisopr opylamiloride, indicative of activation of Na+/H+ exchange. To test wh ether an increase in [Ca2+](i) causes a decrease in pH(i) or vice vers a, pill and [Ca2+](i) were manipulated at isotonic conditions. Surpris ingly, a decrease in [Ca2+](i) was accompanied by a decrease in pH(i) and an increase in pH(i) resulted in an increase in [Ca2+](i), in the absence of osmotic perturbation. In conclusion, changes in [Ca2+](i) a nd pH(i), resulting from osmotic perturbation of proximal tubular cell s in primary culture, appear to be independent phenomena. This study s uggests that both [Ca2+](i) and pH(i) play a role as second messengers in cell volume regulation.