Control of Cell pH in the T84 Colon Cell Line
M.A. Ramirez
1
, R. Toriano
2
, M. Parisi
2
, G. Malnic
1
1
Depto. de Fisiologia e Biofı ´sica, Instituto de Cie ˆncias Biome ´dicas, Univ. Sa ˜o Paulo, Brazil
2
Lab. de Biomembranas, Depto. de Fisiologia, Facultad de Medicina, Univ. Buenos Aires, Argentina
Received: 3 April 2000/Revised: 22 June 2000
Abstract. Cell pH regulation was investigated in the T84
cell line derived from epithelial colon cancer. Cell pH
was measured by ratiometric fluorescence microscopy
using the fluorescent probe BCECF. Basal pH was 7.17
± 0.023 (n 48) in HEPES Ringer. After acidification
by an ammonium pulse, cell pH recovered toward nor-
mal at a rate of 0.13 ± 0.011 pH units/min in the presence
of Na
+
, but in the absence of this ion or after treatment
with 0.1 mM hexamethylene amiloride (HMA) no sig-
nificant recovery was observed, indicating absence of
Na
+
independent H
+
transport mechanisms in HEPES
Ringer. In CO
2
/HCO
-
3
Ringer, basal cell pH was 7.21 ±
0.020 (n 35). Changing to HEPES Ringer, a marked
alkalinization was observed due to loss of CO
2
, followed
by return to the initial pH at a rate of -0.14 ± 0.012 (n
8) pH/min; this return was retarded or abolished in the
absence of Cl
-
or after addition of 0.2 mM DIDS, sug-
gesting extrusion of bicarbonate by Cl
-
/HCO
-
3
exchange.
This exchange was not Na
+
dependent. When Na
+
was
added to cells incubated in 0 Na
+
Ringer while blocking
Na
+
/H
+
exchange by HMA, cell alkalinization by 0.19 ±
0.04 (n 11) pH units was observed, suggesting the
presence of Na
+
/HCO
-
3
cotransport carrying HCO
-
3
into
these cells, which was abolished by DIDS. These ex-
periments, thus, show that Na
+
/H
+
and Cl
-
/HCO
-
3
ex-
change and Na
+
/HCO
-
3
cotransport participate in cell pH
regulation in T84 cells.
Key words: T84 colon cells — Cell pH — Na
+
/H
+
ex-
change — Cl
-
/HCO
-
3
exchange — Na
+
/HCO
-
3
cotrans-
port
Introduction
The mammalian colon is an important site of water and
electrolyte absorption. The mechanisms of this transport
have been detailed in a number of investigations (Rajen-
dran, Geibel & Binder, 1995; Singh et al., 1995a; Greger
et al., 1997). Several colon cell lines, such as Caco2 and
T84, have been used to study such mechanisms “in
vitro.” It has been shown that these cells present several
properties of epithelial tissue such as polarity and inter-
cellular connections (Dharmsathaphorn et al., 1984;
Osypiw et al., 1994; Tai et al., 1996; Chanson, White &
Garber, 1996). Studies on several ion channels present
in these cells, as well as on aspects of volume regulation
and action of peptide hormones and neurotransmitters on
their ion transport properties, are available (Worrel et al.,
1989; Santos & Reenstra, 1994; Dagher et al., 1994;
Devor & Frizzell, 1998).
These findings show that the cell lines discussed
above present several properties common to cells studied
“in vivo,” indicating that they may be used as cell models
for intestinal epithelium. However, cells in culture fre-
quently lose some of the properties seen in the tissues
from which they originate. Thus, some cultured fibro-
blast lines lack the Na
+
/H
+
exchanger that is found in the
“in vivo” tissue, a property that was found to be valuable
for the expression of exogenous cloned exchangers,
which has contributed much to the study of the molecular
biology of these transporters (Wakabayashi, Shigekawa
& Pouyssegur, 1997). On the other hand, MDCK cells,
originated from dog renal tissue, present most acid-base
transporters found in intercalated cells from the mamma-
lian collecting duct, such as Na
+
/H
+
exchanger isoforms,
vacuolar H
+
-ATPase and H
+
-K
+
ATPase (Vilella et al.,
1992; Ferna ´ndez & Malnic, 1998).
A number of studies on transport of components of
the acid-base system in Caco-2 cells have been reported.
These cells contain Na
+
/H
+
and HCO
-
3
transporters and a
H
+
-K
+
ATPase in their cell membrane (Abrahamse, Bin-
dels & Van Os, 1992; Osypiw et al., 1994). However,
for T84 cells little is known about the mechanisms regu-
lating cell pH and transcellular H
+
and HCO
-
3
transport
(Arreola, Melvin & Begenisich, 1995). Correspondence to: G. Malnic
J. Membrane Biol. 177, 149–157 (2000)
DOI: 10.1007/s002320001108
The Journal of
Membrane
Biology
© Springer-Verlag New York Inc. 2000