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JEZ 860
THE JOURNAL OF EXPERIMENTAL ZOOLOGY 279:471–475 (1997)
ATP Regulation of a Swelling-Activated Osmolyte
Channel in Skate Hepatocytes
NAZZARENO BALLATORI1* AND JAMES L. BOYER2
Department of Environmental Medicine, University of Rochester School of
Medicine, Rochester, New York 14642
2
Department of Medicine and Liver Center, Yale University School of
Medicine, New Haven, Connecticut 06520
1
ABSTRACT
Hypotonic swelling of isolated skate hepatocytes activates a regulatory volume decrease (RVD) which is achieved in part by the release of taurine and other intracellular organic
osmolytes. Volume-activated taurine efflux appears to be mediated by an anion channel that exhibits
a taurine/chloride permeability ratio of approximately 0.2. Of significance, this channel was shown to
be regulated by intracellular nucleotide. When intracellular ATP was decreased to about 50% of
control levels, channel opening was completely prevented. Many putative ion channel blockers were
found to inhibit the channel indirectly, by depleting intracellular ATP, rather than by directly interacting with the channel. Investigators using these channel blockers in whole cell preparations should
be aware of this alternative mechanism. Cell swelling-activated taurine efflux was also inhibited by
HgCl2, DIDS, and pyridoxal 5-phosphate, at concentrations of these agents that had no effect on
intracellular ATP levels, suggesting additional mechanisms of inhibition and regulation of the volume-sensitive osmolyte channels. J. Exp. Zool. 279:471–475, 1997. © 1997 Wiley-Liss, Inc.
Mechanisms of regulatory volume decrease
(RVD) in animal cells occur through the release
of major intracellular inorganic ions (potassium
and chloride) as well as the release of organic
osmolytes. The relative contribution of the inorganic versus the organic osmolytes to the RVD
differs between species and even among tissues
of a given species. Most marine organisms have
relatively high concentrations of organic osmolytes, and RVD in these animals is associated with
a loss of compounds such as taurine, betaine, sorbitol, inositol, and tetramethylamines. Release of
organic osmolytes avoids the potential perturbing
effects associated with reductions in major intracellular ions.
Our studies characterized the mechanism for
RVD in hepatocytes isolated from the small skate
(Raja erinacea), an osmoconforming marine elasmobranch. Skate hepatocytes are ideal models for
these studies since they contain high concentrations of organic osmolytes, including high concentrations of taurine (65 mM; Ballatori and Boyer,
’92a). Skate hepatocytes are easily isolated and
maintained in cell suspension with high viabilities, and are also easily amenable to patch clamp
analysis. Our studies demonstrated a requirement
for intracellular ATP in the activation of a volume-regulatory organic osmolyte channel.
© 1997 WILEY-LISS, INC.
MECHANISM FOR RVD IN SKATE
HEPATOCYTES
When exposed to an acute hypotonic shock,
skate hepatocytes swell in proportion to the degree of dilution, behaving as nearly perfect osmometers (Ballatori and Boyer, ’92b). However,
volume regulatory mechanisms are rapidly activated after cell swelling, leading to a gradual recovery of cell volume over the next 30–40 minutes
to approach their original size.
To examine whether intracellular K+ is involved
in the RVD response in skate hepatocytes, cellular K+ content was measured by flame photometry, as well as in cells preloaded with 86Rb+ as a
marker for K+. The results demonstrated that cell
swelling had only small effects on K+ content: potassium content was decreased only ~5 and 10%
by 40 and 50% dilution with water, respectively
(Ballatori and Boyer, ’92b).
Because these relatively small changes in K+
efflux in hypotonic medium could not account for
the loss of osmolytes during skate hepatocyte
RVD, additional studies examined the role of another major intracellular osmolyte, taurine, in cell
*Correspondence to: Ned Ballatori, Ph.D., Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY 14642. E-mail: ballatorin@envmed.rochester.edu
472
N. BALLATORI AND J.L. BOYER
volume regulation. In contrast to potassium, there
was a marked stimulation of taurine release after cell swelling: cells diluted with 30, 40, or 50%
H2O released 24, 41, and 65% of intracellular
[14C]taurine over 2 h, respectively (Ballatori and
Boyer, ’92b). Most of the taurine was released during the first 30 min after dilution, with rates of
efflux approaching baseline rates by 60 min. Of
significance, the time course of taurine release
paralleled that of volume recovery, in that most
of the RVD also occurred during the first 30 min
after dilution. Direct amino acid analysis confirmed that there was a net loss of taurine from
the hepatocytes (Ballatori and Boyer, ’92b).
PROPERTIES OF THE SWELLINGACTIVATED TAURINE EFFLUX PATHWAY
IN SKATE HEPATOCYTES
To distinguish whether volume-activated taurine efflux was occurring by a carrier mediated
mechanism or by activation of a channel, additional studies examined [14C]taurine uptake and
efflux during RVD in cells incubated in media containing taurine concentrations from 0.1–100 mM.
Despite increasing concentrations of taurine in the
medium, rate coefficients for [14C]taurine uptake
and efflux were unchanged (Ballatori et al.,’94).
This kinetic pattern is consistent with the activation of a channel rather than a carrier-mediated
process. Studies carried out in collaboration with
Paul Jackson and Kevin Strange (Ballatori et al.,
’95; Jackson et al., ’96) confirmed the presence of
a volume-activated anion channel in skate hepatocytes. When the patch pipette contained taurine at pH 8.2 so that significant amounts of this
amino acid were in anionic form, an inward rectifying taurine current was demonstrated. Permeability and ion conductances for various
anions showed characteristics identical to volume activated anion currents in glia cells previously demonstrated by Jackson and Strange
(’93; Jackson et al., ’94). The relative Ptaurine/PCl
in skate hepatocytes was 0.17.
Studies in the isolated perfused skate liver demonstrated that the channel was localized to the
basolateral membrane of the liver cell (Ballatori
et al.,’94). The channel was immediately inactivated when isotonicity was restored and swollen
hepatocytes rapidly returned to their normal volumes, but was unaffected by the transmembrane
Na+, Cl–, or K+ gradients (Ballatori and Boyer, ’92b;
Ballatori et al., ’94), indicating that neither Na+,
Cl–, nor membrane potential are directly involved
in volume-stimulated taurine transport.
ROLE OF INTRACELLULAR ATP IN
ACTIVATION OF THE TAURINE CHANNEL
Volume-activated taurine efflux from skate
hepatocytes was nearly completely blocked by decreasing the incubation temperature from 15° to
4°C, and by several metabolic inhibitors (Ballatori
and Boyer, ’92b). Cells pretreated for 30 min with
2,4-dinitrophenol, oligomycin, CCCP, antimycin A,
iodoacetate, KCN, sodium azide, or a combination
of iodoacetate plus KCN or iodoacetate plus azide,
released relatively little [14C]taurine following hypotonic cell swelling.
The role of intracellular ATP in volume-activated
taurine transport was studied further by examining the effects of ATP depletion at different times
after the hypotonic stimulus. Administration of
2,4-dinitrophenol, a highly membrane-permeant
metabolic inhibitor nearly completely prevented
any further release of taurine, when added at different times after cell swelling, indicating that
taurine release requires the continual presence of
intracellular ATP (Ballatori et al., ’94). Comparable effects were noted with two other metabolic
inhibitors, antimycin A and the combination of
KCN plus iodoacetate.
The quantitative relation between cellular ATP
content and volume-activated [14C]taurine efflux
was assessed in skate hepatocytes exposed to increasing concentrations of 2,4-dinitrophenol (Fig.
1). 14C-Taurine efflux exhibited a roughly sigmoidal relationship with cellular ATP levels (Ballatori
et al.,’95). Efflux was inhibited by 50% when cellular ATP declined from approximately 7 nmol/
mg protein (~2 mM) to 4 nmol/mg protein, with
essentially complete inhibition when ATP levels
reached 3 nmol/mg protein. When taurine efflux
was plotted against cellular ATP/ADP ratios, a sigmoidal relation was also observed (Fig. 1). There
was a gradual inhibition of taurine efflux until
the ATP/ADP ratio reached ~2, at which point
there was a precipitous decrease in swelling-activated efflux (Ballatori et al., ’95). Dilution alone
produced no effect on ATP levels or ATP/ADP ratios compared to hepatocytes maintained in isotonic medium.
Whole cell patch recording of volume-activated
anion current in skate hepatocytes demonstrated
that these effects of 2,4-dinitrophenol are not directly related to an interaction of this metabolic
poison with the channel (Ballatori et al., ’95). In
contrast, swelling-activated anion current was
critically dependent on the presence of intracellular ATP, as demonstrated in studies where ATP
AN ATP-REGULATED OSMOLYTE CHANNEL
473
Fig. 1. Relation between volume-activated [ 14C]taurine
efflux and ATP content (A), ADP content (B), and ATP/ADP
ratio (C), in isolated skate hepatocytes exposed to increasing
concentrations of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. Hepatocytes preloaded with [14C]taurine
were incubated for 30 min with 2,4-dinitrophenol at concentrations of 0, 5, 10, 25, 50, 100, 200, 500, and 700 µM, and
aliquots of the cell suspension were removed for ATP and
ADP analysis and to assess 14C content of the cells. Hypotonicity was then induced by diluting the remaining cell suspensions 40% with either H2O or Ringer (control), and cellular
14
C content measured 30 min later. Values are means ± SEM
of four to six experiments at each concentration of 2,4-dinitrophenol. Reprinted with permission of Williams & Wilkins
from Ballatori et al. (’95).
was omitted from the patch pipette (Jackson et
al., ’96). When a nonhydrolyzable analog of ATP
was included in the pipette, full activation of
anion current was observed (Jackson et al., ’96),
indicating that channel activation was not dependent on ATP hydrolysis, but rather was related to the binding of ATP to the channel or
some regulatory protein.
Most importantly, when various channel blockers
were examined which inhibited volume-activated
[14C]taurine efflux, no effect on volume-activated
anion current was observed with a number of
these compounds. Only DIDS and pyridoxal 5phosphate significantly inhibited volume-activated
current, whereas compounds such as 2,4-dinitrophenol, ketoconazole and even NPPB produced
little or no inhibition of volume-activated current
(Ballatori et al., ’95).
The mechanism by which these agents were inhibiting [14C]taurine efflux without affecting swelling-activated anion current became apparent
when cellular ATP levels were compared (Fig. 2).
NPPB, glibenclamide, DPC, ketoconazole, gossypol, niflumic acid, quinine, and phenylarsine oxide all decreased cellular ATP concentrations and
ATP/ADP ratios at the same concentrations that
they inhibited taurine efflux (Fig. 2; Ballatori et
al.,’95). Thus, these compounds were inhibiting the
swelling-activated channel indirectly, that is by
depleting cellular ATP, which in turn prevented
channel activation.
In contrast, DIDS and pyridoxal 5-phosphate
inhibited taurine efflux (Fig. 2) and whole cell
anion current (Ballatori et al.,’95), but had no
effect on cellular ATP levels or ATP/ADP ratios.
The DIDS inhibition of whole cell anion current
was rapid (<60 sec) and voltage-dependent, suggesting that DIDS interacts directly with the
channel protein. Pyridoxal 5-phosphate also inhibited whole cell anion current, albeit in a different manner than DIDS. When swollen cells
were exposed directly to the drug, whole cell
anion current dropped very slowly or was
largely unaffected. However, if the cells were
pretreated with the drug for 15–30 min and
then swollen, activation of whole cell current
was dramatically inhibited. Anion current measured 3 min after induction of swelling in cells
pretreated with pyridoxal 5-phosphate was inhibited 92% compared to untreated control cells.
474
N. BALLATORI AND J.L. BOYER
Fig. 2. Effects of ion channel blockers and other transport inhibitors on volume-activated [14C]taurine efflux, cellular ATP content, and ATP/ADP ratios. Skate hepatocytes were
loaded with [14C]taurine, then treated for 30 min with the
indicated concentrations of inhibitors at 15°C. Control cells
received an equal volume of vehicle (dimethylsulfoxide or elasmobranch Ringer). Aliquots of the cell suspensions were then
removed for ATP and ADP analysis and to assess 14C content
of the cells, and hypotonicity was induced by diluting the
remaining cell suspensions 40% with either H2O or Ringer
(control). Cellular 14C content was measured 30 min after
dilution. Inhibitors were not removed prior to dilution, but
remained (40% diluted) throughout the efflux period. Values
are means ± SEM of three to six cell preparations, each performed in duplicate. Reprinted with permission of Williams
& Wilkins from Ballatori et al. (’95).
The slow inhibition of whole cell current by pyridoxal 5-phosphate suggests that the drug may
be acting from an intracellular site. Studies of
ion transporting ATPases have shown that pyridoxal 5-phosphate covalently modifies ATP binding sites (Hinz and Kirley, ’90; Tamura et al., ’86).
Although the mechanism by which pyridoxal 5phosphate inhibits anion current and taurine
efflux is not known, it may be through an interaction with an ATP binding site on the channel
itself or an associated regulatory protein.
MERCURY INHIBITION OF THE
SWELLING-ACTIVATED TAURINE
CHANNEL
A major mechanism by which inorganic mercury
and some organomercurial compounds produce cytotoxicity is by altering ion and nonelectrolyte
transport, and cell volume regulation (Kinter and
Pritchard, ’77). The inability to regulate cell volume disrupts transmembrane ion, solute, and electrical gradients, which in turn disrupt cellular
communication, homeostasis, and metabolic func-
AN ATP-REGULATED OSMOLYTE CHANNEL
tions. Our early studies with skate hepatocytes
confirmed the effects of mercuric chloride on cell
swelling, and further demonstrated that HgCl2
prevents the normal RVD observed after cells are
swollen in hypotonic media (Ballatori et al., ’88a).
RVD in isolated skate hepatocytes was essentially
abolished by pretreatment with micromolar concentrations of mercuric chloride.
To examine the mechanism for inhibition of
RVD, we examined whether mercury and other
sulfhydryl-reactive reagents could also inhibit volume-activated taurine release. All sulfhydryl reagents tested inhibited taurine release during
RVD (Ballatori and Boyer, ’92b). Pretreatment
with 1 mM N-ethylmaleimide or 10 mM diamide
nearly completely prevented [14C]taurine release,
whereas 10 mM t-butyl hydroperoxide and 25 µM
HgCl2 were somewhat less effective.
In contrast to metabolic inhibitors and sulfhydryl reagents, neither ouabain (2 mM) nor furosemide (1 mM) had any effect on [14C]taurine
release during RVD (Ballatori and Boyer, ’92b).
Ouabain at 2 mM effectively blocks 86Rb+ uptake,
and leads to a gradual depolarization of membrane
potential in skate hepatocytes (Ballatori et al.,
’88a). Thus, neither Na+-K+-ATPase nor the cell
membrane potential appear to be directly involved
in volume-stimulated taurine transport.
The effects of mercury were also not explained
by a decrease in cell ATP levels (Ballatori and
Boyer, ’96). Swelling activated [14C]taurine efflux
was progressively inhibited in the presence of increasing concentrations of HgCl2; however, ATP
levels and ATP/ADP ratios remained essentially
unchanged over a range of HgCl2 concentrations
from 0–40 µM. These studies indicate that HgCl2
may be interacting directly with the channel; however, additional studies are needed to test this hypothesis.
ACKNOWLEDGMENTS
Supported in part by National Institutes of
Health Grants DK48823, DK34989, and ES01247,
and by Grant ES03828 to the Center for Membrane Toxicity Studies at the Mount Desert Island Biological Laboratory.
475
LITERATURE CITED
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membrane ion permeability in mercury-induced cell injury:
Studies in hepatocytes of elasmobranch Raja erinacea. Toxicol. Appl. Pharmacol., 95:279–291.
Ballatori, N., R. Wondergem, and J.L. Boyer (1988b) Effects
of L-alanine on membrane potential, potassium (86Rb) permeability and cell volume in hepatocytes from Raja erinacea.
Biochim. Biophys. Acta, 946:261–269.
Ballatori, N., and J.L. Boyer (1992a) Taurine transport in
skate hepatocytes: I. Uptake and efflux. Am. J. Physiol.,
262:G445–G450.
Ballatori, N., and J.L. Boyer (1992b) Taurine transport in
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Ballatori, N., T.W. Simmons, and J.L. Boyer (1994) A volumeactivated taurine channel in skate hepatocytes: Membrane
polarity and role of intracellular ATP. Am. J. Physiol.,
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Ballatori, N., A.T. Truong, P.S. Jackson, K. Strange, and J.L.
Boyer (1995) ATP depletion and inactivation of an ATP-sensitive taurine channel by classic ion channel blockers. Mol.
Pharmacol., 48:472–476.
Ballatori, N., and J.L. Boyer (1996) Disruption of cell volume regulation by mercuric chloride is mediated by an
increase in sodium permeability and inhibition of an
osmolyte channel in skate hepatocytes. Toxicol. Appl.
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Haussinger, D., F. Lang, and W. Gerok (1994) Regulation of
cell function by the cellular hydration state. Am. J. Physiol.,
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Jackson, P.S., and K. Strange (1993) Volume-sensitive anion
channels mediate swelling-activated inositol and taurine
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Jackson, P.S., K. Churchwell, N. Ballatori, J.L. Boyer, and K.
Strange (1996) Swelling-activated anion conductance in
skate hepatocytes: Regulation by cell chloride and ATP. Am.
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of cell membranes. In: Handbook of Physiology — Reactions
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Baltimore, MD. Section 9, Chap. 36, pp 563–576.
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