Vol. 274, Issue 6, R1677-R1686, June 1998
Hypotonic-stimulated taurine efflux in skate erythrocytes:
regulation by tyrosine phosphatase activity
Mark W.
Musch1,2,
Erin M.
Davis-Amaral2,3,
Karen L.
Leibowitz3, and
Leon
Goldstein2,3
1 Department of Medicine,
Inflammatory Bowel Disease Center, University of Chicago, Chicago,
Illinois 60637; 2 Mount Desert
Island Biological Laboratory, Salsbury Cove, Maine 04672; and
3 Division of Biology and
Medicine, Brown University, Providence, Rhode Island 02912
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ABSTRACT |
Treatment of skate erythrocytes with
FCCP, dinitrophenol, or sodium azide lowers ATP levels and inhibits
Na+-independent taurine uptake
after hypotonic volume expansion. Inside-out vesicles isolated from
hypotonic volume-expanded cells demonstrate greater
Na+-independent taurine uptake,
and pretreatment of cells with FCCP abolishes this stimulation.
Addition of ATP to the vesicles does not restore stimulated taurine
uptake, suggesting that ATP does not act as a ligand modulator on the
transporter. Therefore the role of protein phosphorylation was
investigated. Because known protein kinase inhibitors have previously
been found to have little effect on taurine fluxes in skate
erythrocytes, we focused on the effects of protein phosphatase
inhibition. When volume-expanded cells were returned to isotonic
medium, taurine flux returned to basal values more slowly after
treatment with the tyrosine phosphatase inhibitor pervanadate,
suggesting that dephosphorylation may regulate inactivation. A similar
effect of phosphatase inhibitors was observed in the inside-out
vesicles from volume-expanded cells: the reversal of stimulated taurine
uptake takes place more slowly in vesicles prepared from cells that had
been incubated with pervanadate. Band 3, a major protein involved in
the taurine transport pathway, shows increased tyrosine phosphorylation
after hypotonic volume expansion. Pervanadate treatment of the cells
potentiates and prolongs the increased phosphorylation. Therefore
tyrosine phosphorylation of band 3 may play an important role in the
activation of taurine fluxes after volume expansion.
volume regulation; elasmobranchs; phosphatase
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INTRODUCTION |
HYPOTONIC VOLUME EXPANSION of cells results in
compensatory mechanisms to reduce cell volume back to normal, the
regulatory volume decrease (RVD). An important mechanism in this
process is the stimulated efflux of organic solutes (osmolytes). The
-amino acid taurine is an osmolyte commonly used by cells in RVD. In a number of diverse cell systems (29) including erythrocytes (9),
hypotonic conditions stimulate the efflux of taurine severalfold.
A number of transport systems have been hypothesized to mediate the
hypotonic-stimulated taurine efflux, including a swelling-activated Cl
channel (17). However,
this channel has not been found in erythrocytes from a number of
species, although these cells demonstrate hypotonic-stimulated taurine
efflux (5). Volume-expanded taurine efflux in erythrocytes is blocked
by inhibitors of the anion exchanger, band 3, such as stilbenes and
pyridoxal 5-phosphate (PLP) but is not inhibited by
Cl channel blockers (e.g.,
diphenylcarboxylic acid) (5, 8-10). Cloning and expression of
trout band 3 in Xenopus oocytes
demonstrated that band 3 could mediate
Na+-independent taurine efflux (7,
10), suggesting that band 3 functions either as an osmolyte channel or
regulates an osmolyte channel in fish erythrocytes.
How band 3 might modulate taurine efflux is not understood. A number of
biochemical alterations to band 3 have been demonstrated to occur
during volume expansion. These alterations include formation of band 3 tetramers (24), higher affinity of ankyrin for band 3 (23), and
increase in DIDS binding and band 3 phosphorylation (25). This latter
result suggests that phosphorylation may play a role in the formation
of a taurine-transporting complex involving band 3.
The swelling-activated Cl
channels of C6 glioma cells and skate hepatocytes, which also allow
taurine to permeate under volume-expanded conditions, have been shown
to be modulated directly by ATP (16, 17). Therefore the present studies
were undertaken to determine whether a similar effect of ATP occurs on
the volume-activated taurine flux in skate erythrocytes. The use of
inside-out vesicles (IOVs) was developed to test the effects of ATP and
additional modulators that can be added directly to transporters on the
vesicles. These studies show that ATP does not act at a ligand
modulator on the taurine transporter in IOVs. The term ligand modulator is used here to mean a molecule that binds and alters the activity of
an effector. In the case of the present studies, this would apply to
ATP binding directly to the volume-activated taurine transporter. In
the present studies, we present data that ATP is not a ligand modulator
of taurine transport but, rather, that protein phosphorylation
and/or dephosphorylation may play an important role in
regulation of taurine transport.
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METHODS |
Isolation of cells.
Little skates (Raja erinacea) were
caught off Frenchman's Bay, ME, or Woods Hole, MA, and kept in running
seawater. Blood was removed from a tail vessel into a heparinized
syringe. Cells were pelleted (400 g
for 2 min at room temperature), and the plasma and buffy coat were
removed. Erythrocytes were resuspended in 5 vol of isotonic (940 mosM)
elasmobranch incubation medium [940 EIM; composition in mM: 300 NaCl, 5.2 KCl, 2.7 MgSO4, 5 CaCl2, 370 urea, and 15 Tris (pH
7.4)] and washed twice.
Cell volume response.
A 45% erythrocyte suspension prepared as above was added to 3.5 ml of
940 EIM with or without pervanadate (vanadate was oxidized to
pervanadate by mixing 0.5 mM sodium orthovanadate with 1.5 mM hydrogen
peroxide and was assumed to be 0.5 mM pervanadate) or 3.3 ml of
hypotonic (460 mosM) elasmobranch incubation medium (460 EIM) with or
without pervandate. A concentrated solution (0.2 ml) of 200 mM NaCl and
170 mM urea was added to the hypotonic flasks to revert them to an
isotonic medium after 10 min. The controls contained 1.5 mM hydrogen
peroxide. Experiments were performed at 15°C in a shaking water
bath. At 0, 10, and 20 min, samples were collected in microhematocrit
tubes and were centrifuged for 3 min in a hematocrit centrifuge.
Hematocrit was measured with the aid of a millimeter ruler and
expressed as a ratio of packed cells to cells plus supernatant.
Flux measurements.
Uptake of taurine was measured when cells were treated with 10 µM
FCCP, 100 µM dinitrophenol (DNP), or 10 mM sodium azide to deplete
ATP. Cells were suspended to 20% hematocrit and pretreated with FCCP,
DNP, or azide in 940 EIM for 2 h, after which an aliquot was removed
and the cell pellet was weighed before and after drying. ATP was
measured in the cells by extracting the dry cell pellet with 7%
perchloric acid and measuring the ATP by emitted luminescence using
luciferase. The luciferase was obtained from bacteria expressing the
enzyme, and the assay was utilized only over the range at which ATP
concentration was linearly proportional to light.
To initiate uptakes, cells (200 µl) were added to 2 ml of 940 or 460 Li+-containing EIM (Li-EIM) with
0.4 µCi/ml
[3H]taurine.
Li+ is used to replace all
Na+ salts in the 940 and 460 EIM
used in the uptakes so that the Na+-dependent taurine uptake was
not measured. Previous experiments have shown that the
Na+-independent uptake closely
resembles the hypotonic-stimulated efflux (9): the efflux occurs
through a pathway that also allows taurine to enter after hypotonic
stimulation. Uptakes were terminated by removing aliquots of cells (2 ml) at 0, 15, and 30 min and rapidly pelleting. Cells were washed
rapidly three times with 2 ml of ice-cold 940 Li-EIM and were extracted
with 7% perchloric acid (1 ml), protein was precipitated by
centrifugation (12,000 g for 2 min),
400 µl of the supernatant were removed, and taurine taken up by the
cell was counted. An aliquot of the uptake medium was removed to
determine specific activity of
[3H]taurine. This
value is then used along with the cell
[3H]taurine to
determine the uptake by the cells.
Efflux of taurine was measured as previously described (9). Briefly,
cells were loaded with 2 µCi/ml
[3H]taurine for 3 h at
15°C in 940 EIM. The cells were washed successively with 10, 1, and
then 0.1 mM taurine, all in 940 EIM, to remove labeled taurine not
taken up by the cells. The cells were then resuspended at 20%
hematocrit in 940 EIM; an aliquot was removed to determine the specific
activity of
[3H]taurine taken up
by the cells and was then diluted ~1:10 (300 µl cells into 3.5 ml
of medium) into either 940 or 460 (as above except 100 mM NaCl and 250 mM urea) EIM with or without pervanadate to initiate efflux. Before the
flux measurements, to inhibit tyrosine phosphatases,
[3H]taurine-loaded
cells at 20% hematocrit were incubated for 20 min with 0.5 mM
pervanadate in 940 EIM after having been washed to remove taurine not
taken up. In all experiments, vanadate was oxidized to pervanadate by
mixing 0.5 mM sodium orthovanadate with 1.5 mM hydrogen peroxide and
was assumed to be 0.5 mM pervanadate. Addition of hydrogen peroxide
alone (1.5 mM) did not affect hypotonic-stimulated taurine flux (data
not shown). In some cases, both with and without pervanadate treatment,
cells were volume expanded for 10 min, and NaCl was then added to
return to isotonic conditions. Aliquots (500 µl) were removed at 0, 10, and 20 min and rapidly pelleted in a microcentrifuge (30 s at
12,000 g). An aliquot of the
supernatant (400 µl) was removed and
[3H]taurine was
quantified by liquid scintillation spectroscopy. Fluxes were calculated
from the [3H]taurine
released and the specific activity of
[3H]taurine taken up
by the cells in each experiment.
Isolation of vesicles.
Cells were obtained, washed, and resuspended at 50% hematocrit. Cells
were diluted to 10% hematocrit in regular
(Na+-containing) 940 or 460 EIM.
When appropriate, cells were treated with phosphatase inhibitors or
other agents including transport inhibitors before dilution for times
specified in RESULTS. IOVs were
prepared by a modification of the procedures used in sheep erythrocytes
(18, 19). Cells were rapidly diluted 1:10 into lysis buffer [10
mM Tris (pH 7.4), 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride
(PMSF) with 10 µg/ml leupeptin, benzamidine, and aprotinin]. Ghosts were pelleted (12,000 g for 30 s at 4°C), and the pellet was vigorously resuspended in 5 ml of
lysis buffer and pelleted again. Generally, two cycles of resuspension
in lysis buffer were required to make ghosts white. The ghosts were
resuspended in spectrin-extraction buffer (0.2 mM EDTA with protease
inhibitors as above) and sheared by passing through a 27-gauge needle.
Samples were spun at 12,500 g for 5 min at 4°C to remove unbroken cells, nuclei, and mitochondria. The
supernatant was applied to a Dextran T-70 cushion (5% wt/vol) in 5 mM
Tris (pH 7.4) with 0.2 mM EDTA and centrifuged for 45 min at 30,000 g at 4°C. Vesicles were collected at the interface and were pelleted (40,000 g for 30 min at 4°C). Vesicles
were resuspended in 940 or 460 Li-EIM. To achieve rapid equilibration
with vesicle uptake buffers of the same ionic composition and
osmolarity as the EIMs used for cell experiments, nystatin (1 µg/ml)
was added, and when appropriate, agents such as arachidonic acid,
quinine, DIDS, and PLP were added during this time. Although cells had
been treated with inhibitors before vesicle preparation, addition of
inhibitors during the vesicle equilibration period ensured replacement
of any inhibitors lost during vesicle preparation. After 15 min,
nystatin was removed by diluting vesicles 10-fold with uptake buffers
(940 or 460 Li-EIM) plus BSA (500 µg/ml) and centrifuged again
(40,000 g for 30 min at 4°C).
Vesicles were resuspended in small volumes of uptake buffer and used
immediately for assay. Uptake was initiated by adding vesicles to
uptake buffer with 10 µCi/ml
[3H]taurine. Samples
were stopped by dilution of vesicles with 2 ml of ice-cold uptake
buffer and immediate filtration through 0.45-µm filters. Samples were
washed with an additional 4 ml of ice-cold uptake buffer and filters
were solubilized and counted.
Sidedness of the vesicles was determined by measuring
acetylcholinesterase in the presence and absence of Triton X-100 (28). Acetylcholinesterase activity has been shown to be directed only toward
the outside of erythrocytes.
Immunoprecipitation of band 3 and tyrosine phosphorylation.
Cells were volume expanded as described either with or without
pretreatment with pervanadate. Cells were pelleted and brought up in
immunoprecipitation buffer [composition in mM: 150 NaCl, 10 Tris (pH 7.4), 1 EDTA, 1 PMSF, and 10 µg/ml leupeptin and
aprotinin]. The use of 10 mM pervanadate in the
immunoprecipitation buffer was required, even if the cells had not been
treated with pervanadate, since, in preliminary experiments, the
phosphotyrosine signal on band 3 was detectable but very low when
pervanadate was omitted from the buffer. The lysis buffer contained
EDTA, which dramatically lowers the effectiveness of pervanadate as a
phosphatase inhibitor (15). Because the erythrocyte has abundant
phosphatases of a number of types, many of which are inhibited by
pervanadate, we used an excess of pervanadate (10 mM) to overcome the
effect of EDTA. Band 3 was immunoprecipitated using a polyclonal
antiserum against the NH2-terminal
cytoplasmic domain of human band 3 (a gift of Dr. P. Low, Purdue
University, West Lafayette, IN). Complexes were brought down using
protein A-agarose, and samples were resolved on 10% SDS-PAGE.
Phosphotyrosine was detected with a monoclonal anti-phosphotyrosine
4G10 antibody and developed by use of a chemiluminescene kit.
Materials.
[3H]taurine was
purchased from NEN (Boston, MA); Dextran T-70 and protein A-agarose
were from Pharmacia (Piscataway, NJ); HAWP filters were from Millipore
(Medford, MA); SuperSignal chemiluminescent kit was from Pierce
(Rockford, IL); antibody 4G10 was from Upstate Biotechnology (Lake
Placid, NY); and nystatin was from Life Technologies (Grand Island,
NY). Luciferase kit and components were from Promega (Madison, WI). All
chemicals were of the highest grade available and purchased from Fisher
Chemical (Itasca, IL) or Sigma Chemical (St. Louis, MO).
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RESULTS |
Effect of ATP depletion on taurine flux.
To determine whether ATP is required for volume-expanded taurine
efflux, cells were pretreated with the mitochondrial uncouplers FCCP
(10 µM) or DNP (100 µM) or the metabolic poison azide (10 mM) for
120 min. ATP was measured in perchloric acid extracts from the cells.
FCCP treatment reduced cell ATP to <10% (3.11 ± 0.67 vs. 0.26 ± 0.12 µmol ATP/g dry cell wt), DNP reduced cell ATP somewhat
less at 2 h (2.89 ± 0.69 vs. 0.62 ± 0.09 µmol ATP/g dry cell
wt), and azide also lowered ATP levels (3.38 ± 0.56 vs. 0.85 ± 0.21 µmol ATP/g dry cell wt). When FCCP-, DNP-, or azide-treated cells are volume expanded, taurine flux is greatly inhibited (Fig. 1), suggesting that ATP is involved in the
opening of the transport path for taurine. Uptake measurements were
performed rather than efflux, since the necessary preloading of the
cells with [3H]taurine
could be minimal in FCCP-, DNP-, or azide-treated cells. We have
previously shown that volume-stimulated,
Na+-independent taurine uptake
into skate erythrocytes represents the same transport pathway as efflux
of taurine that is activated after volume expansion (9). Data are
presented as the ratio of uptake at 460 to 940 mosM in untreated (open
bars) and FCCP-, DNP-, or azide-treated cells (hatched bars). In the
case of DNP- and azide-treated cells, uptakes were determined at 30 min, and for FCCP-treated cells, the uptakes were measured over 15 min.
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Fig. 1.
Effect of ATP depletion on volume-stimulated taurine uptake in intact
erythrocytes. Cells were ATP depleted using FCCP (10 µM),
dinitriphenol (DNP, 100 µM), or sodium azide (10 mM) for 2 h, and
then [3H]taurine
uptake was performed in assay medium osmolarities of 940 and 460 mosM
Li+-containing elasmobranch
incubation medium (940 and 460 Li-EIM) with (hatched bars) or without
(open bars) inhibitor. Results are expressed as ratio of taurine uptake
in 460 or 460 + inhibitor divided by uptake in 940 with or without
ATP-lowering agents. Values shown are means ± SE;
n = 3-4.
* P < 0.05 compared with
untreated cells by Student's
t-test.
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Effects of ATP depletion and pharmacological inhibitors of taurine
efflux in vesicles.
To investigate how ATP and other agents affect vesicle taurine efflux,
preparation of isolated IOVs, which demonstrate volume-stimulated taurine fluxes (uptakes, since vesicles are IOVs), was developed. In
this way, agents such as ATP or inhibitors, which might interact directly with the transport protein, could be tested. Taurine uptake
into IOVs was performed in
Li+-containing buffers rather than
Na+, since a small percentage of
the vesicles were right side out and express a
Na+-dependent taurine transporter
that is active under all conditions. The percentage of vesicles that
were right side out was determined by measuring acetylcholinesterase,
an enzyme which faces solely to the outside of erythrocytes. When the
activity of the enzyme was measured in the absence of detergent (Triton
X-100), only activity from right-side-out vesicles was determined,
whereas with the addition of detergent, activity from inside the
vesicles is measured as well. In four separate IOV preparations tested, the percentage right side out was 11 ± 4; thus most of the vesicles were inside out. Also, all vesicles were treated with nystatin to alter
their intravesicular salt concentrations and the osmolarity after the
isolation procedure. Thus the treatment of all vesicles with nystatin
was constant, and effects observed in vesicle taurine uptake reflect
differences in the regulated transport activities of the vesicles and
are not an artifact of vesicle isolation or treatment.
Taurine transport into the IOVs occurred relatively slowly at 15°C
and was linear for at least 5 min (Fig. 2).
Thus most vesicle fluxes were measured at 5 min. IOVs derived from
cells that were hypotonically stressed (460 EIM) for 10 min before
vesicle preparation demonstrated a greater initial rate of uptake than
IOVs from cells incubated under isotonic conditions (940 EIM for 10 min; Fig. 2A). In Fig. 2, 460/460
indicates vesicles made from cells previously incubated in 460 EIM for
10 min, and vesicle uptakes were subsequently measured in
Li+-containing uptake medium of
460 mosM for varying lengths of time as indicated on the
x-axis. 460/940 indicates the exact
same vesicles (from cells incubated in hypotonic medium for 10 min),
but vesicle uptake medium was
Li+-containing 940 mosM for
varying lengths of time. Similarly, 940/940 indicates vesicles made
from cells preincubated in isotonic medium and then fluxed in
Li+-containing uptake medium of
940 for varying lengths of time; 940/460 indicates these same vesicles
but fluxed in Li+-containing
hypotonic uptake medium of 460 for the various times. The uptake into
IOVs from isotonic cells is stimulated by incubating the vesicles in
hypotonic medium (940/460), but not during the initial phase. This
increase in taurine uptake during the later phases may be due to
expansion of the intravesicular volume induced by hypotonic medium. It
is also possible that the increased uptake observed in the 940 vesicles
placed into the 460 buffer may be due to activation of the
volume-stimulated transport pathway that remains intact, but this is a
less likely explanation, since the incubation medium lacks ATP.
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Fig. 2.
Time course of taurine uptake by inside-out vesicles. Erythrocytes were
incubated in isotonic medium (940) for 10 min, and vesicle taurine
uptake was assayed for varying lengths of time in either isotonic
(940/940) or hypotonic (940/460) media, or erythrocytes were incubated
for 10 min in hypotonic medium (460), and vesicle taurine uptake was
assayed in either hypotonic (460/460) or isotonic (460/940) medium for
time points indicated. Vesicles were prepared and uptake measured as
described in METHODS. Values are means ± SE; n = 3. A and
B are from same experiment with
different time scales.
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To confirm that the taurine transport in the vesicles was by the same
pathway as in the intact cells, a variety of agents known to inhibit
cellular taurine transport were tested. These inhibitors were incubated
with the cells before making the vesicles and were also included in the
nystatin buffer, since many may work at an extracellular site now
inside the IOV. The anion-exchange inhibitors, DIDS and PLP, both
potently blocked the uptake of taurine in IOVs from volume-expanded
cells (Fig. 3). The
Cl channel inhibitor
diphenylcarboxylic acid (DPC), which does not block the efflux in whole
cells, did not affect the uptake in IOVs. Quinine, a taurine channel
blocker, and the fatty acid, arachidonic acid, modestly inhibited the
taurine uptake (Fig. 3). The rank order of potency (at concentrations
used) in IOVs was DIDS 
PLP > quinine > arachidonate >>>
DPC and in intact cells PLP DIDS > quinine arachidonate. Thus those inhibitors that inhibit
hypotonic-stimulated efflux with the greatest potency in intact cells
(8-10) also inhibit the hypotonic-stimulated vesicle uptake,
suggesting that the same transport pathway is responsible for both.
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Fig. 3.
Effect of inhibitors DIDS, pyridoxal 5-phosphate (PLP), quinine,
arachidonic acid (Arach), and diphenylcarboxylic acid (DPC) on taurine
uptake in vesicles from volume-expanded cells. Taurine uptake in
vesicles from volume-expanded cells (for 10 min) was assayed for 5 min
in hypotonic uptake buffer with and without inhibitors. Inhibitors were
both sealed in cells while contents were clamped and were also included
in uptake buffer. 940/940 represents vesicles from nonexpanded cells
assayed in isotonic uptake buffer. Data are means ± SE;
n = 3. * P < 0.05; + P < 0.01;
++ P < 0.001 compared with
460/460 by ANOVA.
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ATP depletion of the intact cells (FCCP treatment for 120 min before a
5-min incubation of vesicles in 940 or 460 EIM) greatly reduced the
hypotonic stimulation of taurine uptake in the IOV (Fig.
4). ATP depletion had little effect on the
5-min vesicle uptake under isotonic conditions (940/940). Addition of
ATP (5 mM) to vesicles did not activate vesicle taurine uptake at 5 min in the IOV from FCCP-treated volume-expanded cells or in the IOV from
FCCP-treated isotonic cells. To eliminate the possibility that the lack
of effect of ATP was due to hydrolysis (erythroyctes are a very rich
source of phosphatases), a nonhydrolyzable analog [adenosine
5'-O-(thiotriphosphate), 5 mM] was used. This analog also did not stimulate uptake in the
vesicles from ATP-depleted cells, suggesting that the effect of ATP is
not a ligand modulator on the transporter but that a cellular metabolic
event requiring ATP may be involved.
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Fig. 4.
Effect of cellular ATP depletion on vesicle taurine uptake and ATP
repletion in assay buffer. Taurine uptake in vesicles from isotonic
(940/940) and volume-expanded (460/460) cells (for 10 min) either
untreated or pretreated with FCCP (10 µM for 120 min in isotonic
buffer at 15°C) was measured for 5 min in hypotonic (460/460)
uptake buffer. ATP or adenosine
5'-O-(thiotriphosphate)
(ATP S) was added to uptake buffer at 5 mM. Data shown are
means ± SE; n = 3. ++ P < 0.001 compared with
control cells by ANOVA.
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Effect of vanadate on taurine efflux in intact cells.
The potential involvement of tyrosine phosphorylation and
dephosphorylation in hypotonic-stimulated taurine efflux was
investigated by treatment of intact cells with the tyrosine phosphatase
inhibitor vanadate. Because some oxidized states of vanadate are more
potent inhibitors of phosphatases (14, 15), vanadate was always
oxidized to pervanadate with hydrogen peroxide immediately before use. Hydrogen peroxide treatment by itself did not produce any of the effects of oxidized vanadate (data not shown). After hypotonic stress,
taurine efflux from erythrocytes increases rapidly (10 min), and the
rate of taurine efflux begins to decrease within 20 min (Fig.
5). In cells treated with pervanadate, the
larger increase in taurine efflux that is observed persists over a
longer period of time (Fig. 5), suggesting that dephosphorylation may be an important step in inactivation of the transporter. Pervanadate treatment of cells in isotonic medium stimulates a small, but significant increase in taurine efflux, suggesting that cycles of
phosphorylation and dephosphorylation may be important in the regulation of taurine transport even under isotonic conditions. By its
ability to inhibit cellular phosphatases (and some kinases), pervanadate may change the phosphorylation state and promote the formation of an active state of transport. When volume-expanded cells
are returned to isotonic conditions, taurine efflux returns toward
basal levels over a period of time (Fig.
6). To determine whether pervanadate alters
the rate of return of taurine efflux in intact cells, erythrocytes were
volume expanded, and NaCl was then added to return cell volume to
normal by increasing osmolarity back to 940. In these experiments, 15 min were allowed for cells to return to basal volumes before effluxes
were measured. Return of medium osmolarity from 460 to 940 rapidly
reverses the stimulation of taurine efflux (Fig. 6). Hypotonically
stimulated cells treated with pervanadate and then returned to isotonic
medium show much less of a decrease in taurine efflux, suggesting that
pervanadate may promote the formation (or retention) of the active
state of taurine transport.
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Fig. 5.
Effect of pervanadate on cellular taurine efflux. Erythrocytes were
labeled with
[3H]taurine, and
efflux was performed under isotonic conditions with (940 + P) or
without (940) pervanadate or under volume-expanded conditions with (460 + P) or without (460) pervanadate. Data shown are means ± SE;
n = 4. RBC, red blood cells.
* P < 0.05 compared with
nonpervanadate-treated cells at same osmolarity.
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Fig. 6.
Effect of pervanadate on osmotic reversal of hypotonic-stimulated
cellular taurine efflux. Efflux from
[3H]taurine-labeled
erythrocytes was assayed at isotonic (940) or volume-expanded (460)
conditions. NaCl was added to aliquots of volume-expanded cells either
untreated (460-940) or treated with pervanadate (460P-940P).
Data shown are means ± SE; n = 3. * P < 0.05, 460P-940P vs.
460-940.
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To test whether the effects of pervandate on taurine efflux might have
been due to an alteration of the cell volume responses to osmotic
changes by the inhibitor, we measured hematocrits of cells suspended in
different media with and without pervanadate. Incubation conditions
were identical to those used to measure taurine effluxes. Hematocrits
of cells in isotonic medium rose from 0.110 ± 0.004 to 0.135 ± 0.004 (n = 3) in the presence of pervanadate. This increase in cell volume may have contributed to the
increase in taurine flux observed in the presence of pervanadate. Hematocrits of cell suspensions incubated in 460 medium rose from 0.109 ± 0.005 in 940 to 0.179 ± 0.002 without pervanadate and from
0.108 ± 0.002 to 0.185 ± 0.003 with pervanadate
(n = 3). Hematocrits of cell
suspensions incubated in 460 medium and then returned to 940 medium
fell to 0.084 ± 0.001 without pervanadate and to 0.097 ± 0.002 with pervanadate (n = 3). Thus, in
hypotonic-stimulated cells, the effects of pervanadate on cell volume
responses (hematocrit changes) were small compared with the effects of
the inhibitor on taurine effluxes (see Fig. 5). In addition vanadate
causes cell swelling by inhibiting
Na+-K+-ATPase
(4) and expanding the pool of intracellular electrolytes and obligated
water. This method of cell swelling is known to be a poor stimulus for
the efflux of intracellular osmolytes such as taurine (22).
Effect of pervanadate on taurine uptake by vesicles.
Next, we tested whether the pervanadate effect observed with intact
cells was also observed in vesicles made from the volume-expanded cells
in hypotonic medium. Cells were pretreated with and without 0.5 mM
pervanadate for 10 min and then volume-expanded in 460 Li-EIM with and
without 0.5 mM pervanadate (to correct for any loss during
pretreatment) for varying periods of time. At different time points of
incubation, cells were removed, and vesicles were prepared. Therefore
the times indicated on the x-axis are
times after exposure of cells to hypotonic medium. Taurine uptake was then measured for 5 min in all of the vesicles. In these experiments, pervanadate was included in the lysis, vesicle preparation, and uptake
buffer for only the pervanadate group. Pervanadate prolonged the effect
of volume expansion on vesicle taurine uptake (Fig. 7). The maximal change was slightly larger
(not significantly), but the return back to basal flux was
significantly slowed. Therefore, just as observed in intact cells,
dephosphorylation appears to be an important step in the inactivation
of the taurine transporter in vesicle preparations as well.
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Fig. 7.
Effect of tyrosine phosphatase inhibition on vesicle taurine uptake.
Vesicles were made from cells that were incubated for varying times in
hypotonic (460) medium with either no addition (control) or pervanadate
pretreatment (0.5 mM for 10 min in 940 EIM before dilution and volume
expansion in 460 EIM). Pervanadate was also included in hypotonic
exposure buffer, lysis buffer, and uptake buffer for pervanadate group.
As indicated by time on x-axis, cells
were removed at varying lengths of time after hypotonic exposure for
both groups, vesicles were isolated, and a 5-min vesicle uptake was
measured. + P < 0.05;
++ P < 0.01 compared with
control cells by Student's t-test.
Values are means ± SE; n = 3.
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Effect of volume expansion on phosphotyrosine level of band 3.
To determine whether volume expansion altered the level of
phosphotyrosine in band 3 and whether pervanadate potentiated this effect, band 3 was immunoprecipitated from cells incubated in isotonic
and hypotonic media for varying times after stimulation. Under basal
conditions, little or no phosphotyrosine was detected on band 3 by
using a specific antiphosphotyrosine antibody. With hypotonic
stimulation, the level of band 3 phosphotyrosine increased but returned
to near basal levels by 60 min (Fig. 8).
Treatment with pervanadate under isotonic conditions (for 10 min before 0 time indicated in Fig. 8) increased the basal level of
phosphotyrosine dramatically, and this level could be increased further
by hypotonicity (Fig. 8). The increase in band 3 phosphotyrosine
decreased only slowly with prevanadate present. By use of NIH Image
software and maximal tyrosine phosphorylation at 5 min set to 100%,
the values for the time points in Fig. 8 were as follows: in nontreated cells, isotonic (Iso) at 0 min 1.2 ± 1.2, Iso at 60 min 2.1 ± 2.1, hypotonic (Hypo) at 2 min 16.9 ± 1.5, 5 min 100, Hypo at 10 min 93.0 ± 3.8, Hypo at 30 min 68.0 ± 9.9, and Hypo at 60 min 3.6 ± 2.9; in pervanadate-treated cells using its 5 min as 100%: Iso at 0 min 40.2 ± 2.4, Iso at 60 min 41.2 ± 4.5, Hypo at 2 min 92.7 ± 2.9, 5 min 100, Hypo at 10 min 102.0 ± 1.5, Hypo at
30 min 101.8 ± 0.9, and Hypo at 60 min 91.4 ± 2.3 (n = 4). This increased phosphotyrosine was found to be on the
NH2-terminal cytoplasmic domain of
band 3, since similar patterns of phosphotyrosine can be detected in a
41-kDa tryptic fragment, which has been characterized to be the
cytoplasmic domain of the protein (data not shown).
View larger version (55K):
[in this window]
[in a new window]
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Fig. 8.
Levels of band 3 phosphotyrosine in control and volume-expanded cells
incubated with and without pervanadate. Band 3 was immunoprecipitated
from cells incubated in isotonic medium (Iso; 0 and 60 min) or
hypotonic (460) medium (Hypo; 2, 5, 10, 30, and 60 min) in absence of
pervanadate or after 10 min of treatment with pervanadate. Pervanadate
was included in incubation and all lysis and immunoprecipitation
buffers when appropriate. Image shown is representative of those of 4 separate experiments.
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The reversibility of band 3 tyrosine phosphorylation was also
investigated in cells in which the cell volume was rapidly returned to
basal levels by addition of salt and urea to cells in hypotonic media
(NaCl and urea were added in small volumes to return osomolarity to 940 from 460). This maneuver inhibited the previously activated taurine
flux (see above) and the cellular volume (presented in Fig. 6 legend).
Reversal of volume markedly reversed the level of tyrosine
phosphorylation of band 3 in cells not treated with pervanadate.
However, with pervanadate present the reversal was less evident (Fig.
9). The maximal tyrosine phosphorylation
was set to 100% (for all 4 experiments, this was hypotonic point in pervandate-treated cells). The levels of band 3 phosphotyrosine determined by densitometry were as follows: in cells without
pervanadate, 940 control 1.2 ± 1.2%, 460 at 10 min 28.9 ± 10.4%, and reversal to isosmotic 6.7 ± 3.4%. In
pervanadate-treated cells, these values were 940 control 38.7 ± 6.9%, 460 at 10 min 100%, and reversal to isosmotic 90.9 ± 16.6%, (n = 4). Thus the
reversibility of the phosphorylation state of band 3 on returning cells
from hypotonic to isotonic medium follows a pattern similar to that of
taurine efflux.
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 9.
Reversal of band 3 tyrosine phosphorylation by rapidly reducing cell
volume. Cells were analyzed for band 3 tyrosine phosphorylation under
isosmotic conditions (940), 10 min after hypotonic medium (460), and
then 10 min after adding solutes (NaCl and urea) to return osmolarity
to 940 (460/940). Band 3 was immunoprecipitated from lysates and
analyzed for tyrosine phosphorylation as previously described. Image
shown is representative of 4 experiments; means ± SE are presented
in text.
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| |
DISCUSSION |
Stimulation of solute efflux is a rapid way for a cell to recover from
volume expansion. Many different solutes are used to accomplish the
RVD, and in the case of the skate erythrocyte, taurine is the
predominant solute lost during RVD (9). We (9) and others (8) have
presented evidence for the involvement of the anion exchanger, band 3, in volume-activated taurine efflux. In addition to pharmacological
evidence, the most convincing argument for band 3 as a participant in
taurine efflux during RVD is the data that trout band 3 expressed in
Xenopus oocytes can mediate a taurine
efflux (7, 10). How band 3 is altered to accomplish this function is as
yet not understood. We have demonstrated that band 3 undergoes
oligomerization to a preferred tetramer state in volume-expanded
conditions (24) and also that the affinity of ankyrin, the cytoskeletal
protein for band 3, is increased at the same time (23). The relation of
these two occurrences to each other is unknown but may involve
phosphorylation and/or dephosphorylation events.
In the present study, we found that cellular ATP is required for a
volume-stimulated taurine efflux. This effect is carried over during
the isolation of plasma membrane vesicles made from ATP-depleted cells.
Addition of ATP to the vesicles does not reverse the effect of ATP
depletion. Therefore a cellular metabolic process requiring ATP may be
involved. It is also possible that ATP may act on an accessory protein
which is lost during preparation of the IOV. However, we present
evidence to link a cellular ATP-dependent event, tyrosine
phosphorylation, with activation of taurine transport. Using the
tyrosine phosphatase inhibitor pervanadate, we found an effect of the
inhibitor on both tyrosine phosphorylation and taurine efflux at the
cellular level as well as in the isolated IOV. After volume expansion,
the amount of phosphotyrosine increases in band 3, a major target for
erythrocyte tyrosine kinases. When cells are treated with pervanadate,
a basal level of band 3 phosphotyrosine can be readily observed, and
this level increases after hypotonic stress. Treatment with pervanadate
under isotonic conditions stimulates a small, but significant taurine
efflux in the erythrocytes, suggesting that cycling of phosphorylation
and/or dephosphorylation regulates taurine efflux even in
nonstimulated cells. In hypotonically stressed cells, pervanadate also
enhances the stimulation of taurine efflux. It should be noted that the
skate erythrocyte, like most other cells, has a number of proteins that
contain phosphotyrosine under basal conditions. In 4 of 12 experiments,
we found a low level of band 3 basal phosphotyrosine under isotonic
conditions when the immunoblots were also allowed to develop for longer
periods of time. The phosphotyrosine levels of other proteins were
increased, notably proteins at 105, 87, 72, 65, 42, and 27 kDa (data
not shown). The identities of these proteins are unknown. However, they
may include kinases or phosphatases involved in the regulation of
taurine transport, or they could be other proteins that complex with
band 3 and mediate taurine efflux.
Vanadate is a potent protein phosphotyrosine phosphatase inhibitor, and
the erythrocyte has a number of phosphotyrosyl protein phosphatases (1,
3, 33). One of these phosphatases, either type 1B or a closely related
phosphatase, has been demonstrated to have a close association with
band 3 by immunoprecipitation studies (33). Although vanadate and
pervanadate are used with the expectation that they are specific and
potent inhibitors of phosphotyrosyl phosphatases, they may have other
effects. Vanadate, however, also has additional effects on certain
kinases (11), some of which are present in erythrocytes (2).
Furthermore, due to the structural similarity of vandate and its
oxidized forms to phosphate, a number of enzymes and transporters have
been shown to be affected by vanadate, including
Na+- and
H+-ATPases, some
Ca2+-ATPases, and the multidrug
resistance transporter P-glycoprotein (4, 26, 27, 30). In addition, we
observed that pervanadate caused cell swelling in both control and
hypotonically treated erythrocytes. Hematocrits of cells incubated in
940 EIM increased 23% in the presence of pervanadate and hematocrits
of cells incubated in 460 EIM with pervanadate also increased, though
slightly (~3%). Thus the small increase in cell taurine efflux seen
in 940 EIM with pervanadate (Fig. 5) could have been due to the cell
swelling after pervanadate treatment. How much cell swelling
contributed to the pervanadate stimulation of taurine efflux at 460 is
difficult to judge. Although the stimulation of efflux (3-fold) was
much greater than the increase in cell volume (3%), the cells in 460 EIM are quite swollen (~65% compared with cells in 940 EIM), and a
little additional swelling could conceivably have had a significant effect on passive taurine release. Thus the effects of pervanadate on
cell taurine efflux should be interpreted with caution.
If the above cautions are kept in mind, it is still possible to make
the point that pervanadate is having both biochemical (band 3 phosphorylation) as well as functional effects on the skate
erythrocyte. Pervandate not only modulates the taurine transport by the
cells but also causes increased phosphotyrosine in band 3. It is
difficult to directly compare phosphorylation level with taurine
transport activity, and it is not a 1:1 correlation. This suggests that
other steps may be involved, such as interaction with other erythrocyte
proteins, which could regulate activity of band 3. Hypotonic stimulated
taurine efflux may result from the interaction of band 3 with one or
more modulators, and it is possible that ATP could be a regulator of
one of the modulatory proteins. We have previously shown that one
cytoskeletal protein, ankyrin, alters its association with band 3 under
volume-expanded conditions (23), and different associations with band 3 may be the case for other proteins.
How the altered phosphotyrosine of band 3 may regulate the interaction
of band 3 with itself (i.e., to form a tetramer) or with other proteins
is not understood. Tyrosine 8 is possibly the major site of
phosphorylation of band 3. However, tyrosine 21 (also in the
cytoplasmic NH2-terminal) as well
as additional tyrosines in other domains of the protein, such as 359 and 904, may also be phosphorylated (6, 11, 31, 32). Tyrosine phosphorylation alters the interaction of band 3 with certain glycolytic enzymes (21, 32); disrupting the interaction allows the
glycolytic enzymes to be active. When band 3 is required in RVD, the
lack of interaction with these cellular proteins may unmask different
interactions and additional functions for band 3. These may include
interaction of band 3 with itself to form an oligomer (perhaps a
tetramer, which we have shown), and this oligomeric complex may be
stabilized by interaction with ankyrin and, perhaps as we suggested
(24), may lead to an increased efflux of taurine.
The tyrosine phosphorylation of band 3 that occurs after volume
expansion must be catalyzed by a tyrosine kinase in the cell. We have
previously tried genistein to inhibit the volume-expanded taurine
efflux with modest success. Even at quite high concentrations only a
20% inhibition was observed (25). Band 3 is an excellent substrate for
p72syk, and in preliminary experiments, we have observed syk activity
in the erythrocytes. The in vitro p72syk activity is poorly inhibited
by genistein. Thus genistein may have failed to inhibit taurine flux
significantly because of its lack of action on skate erythrocyte
tyrosine kinases.
Perspectives
The relationship between cell metabolism and volume regulation has
attracted attention in recent years (for review, see Ref. 12). For
example, during transport of metabolites across absorptive epithelia,
glycolysis and proteolysis cell solute concentrations rise and volume
increases. These conditions necessitate an RVD to return cells back to
basal volume. Similarly, after a hypotonic stress, cells swell and
regulate their volume by releasing solutes. The solutes released may be
electrolytes and/or nonelectrolytes (osmolytes) dependent on
the cell type. Although some of the solutes released are not involved
in cellular metabolism (e.g., taurine), other solutes are intermediary
metabolites (e.g., myoinositol, glycerol) and cannot be lost in
unlimited amounts without impairing cellular metabolism. Jackson et al.
(16, 17) have suggested that ATP may play a pivotal role in regulating
the loss of essential metabolites during cell volume regulation. For
example, ATP regulates the volume-stimulated organic anion channel
(VSOAC); activation of the channel by hypotonic stress requires the
presence of a critical level of ATP in the cell. This ensures that loss
of metabolites involved in ATP synthesis will be limited, since fall in
metabolite levels below a certain point will cause a drop in cellular
ATP concentration and close the swelling-activated channel.
In the case of VSOAC, ATP appears to regulate channel activity by
acting as a ligand modulator, since both ATP and its nonmetabolizable analogs facilitate channel activation. However, in the present study,
depletion of cellular ATP caused inhibition of the swelling-activated osmolyte channel, an inhibition that could not be relieved, in the
vesicle preparation at least, by addition of ATP or its analogs to the
assay medium. It is therefore likely that the requirement for ATP may
involve a protein phosphorylation step requiring a cytoplasmic kinase
for channel opening. Nevertheless, the result is the same as in the
case of ATP and VSOAC: fall in ATP below a critical level closes the
osmolyte channel and prevents excessive loss of essential metabolites
that are lost while the channel is open.
| |
ACKNOWLEDGEMENTS |
L. Goldstein thanks Prof. E. A. Newsholme, Dept. of Biochemistry,
University of Oxford, UK, for helpful discussions concerning ATP and
cell volume regulation during L. Goldstein's visit to Oxford sponsored
by a Wellcome Research Travel Grant (APP 0850).
| |
FOOTNOTES |
This work was supported by National Science Foundation Grant
IBN-9505567 (to L. Goldstein) and National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-38510, DK-42086, and DK-47722
(to M. W. Musch).
Address for reprint requests: L. Goldstein, Div. of Biology and
Medicine, Brown University, Box G B-311, Providence, RI 02912.
Received 30 July 1997; accepted in final form 23 February 1998.
| |
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Abstract
Introduction
