Effects of osmotic perturbation on intracellular ca2+ and h+
concentration in rabbit proximal tubular cells in primary
culture.
Raat, N. J. H., C. H. Van Os, and R. J. M. Bindels.
Department of Cell Physiology, University of Nijmegen, P.O. Box
9101, 6500 HB Nijmegen, The Netherlands, TEL.: ..-31-80-617191 or
614207, FAX.: ..-31-80-540525
APStracts 2:0063F, 1995.
The effects of anisosmotic media on intracellular Ca2+ and H+
concentrations, [Ca2+]i and pHi, 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 BCECF.
Changing medium osmolarity from 300 to 150 mosM 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 mosM 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 ethyl-isopropyl
amiloride, 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. In conclusion, changes in [Ca2+]i
and pHi resulting from osmotic perturbation of proximal tubular cells
in primary culture appear to be independent phenomena. This study
suggests that both [Ca2+]i and pHi play a role as second messengers
in cell volume regulation.
Received 28 November 1994; accepted in final form 4 April 1995.
APS Manuscript Number F418-4.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 25 April 1995.