Potentiation of mitochondrial Ca2+ sequestration by taurine

Article abstract of DOI:10.1016/S0006-2952(99)00183-5

The effects of taurine (2-aminoethanesulphonic acid) and its analogues, 2-aminoethylarsonic acid, 2-hydroxyethanesulphonic (isethionic) acid, 3-aminopropanesulphonic acid, 2-aminoethylphosphonic acid, and N,N-dimethyltaurine, were studied on the transport of Ca²⁺ by mitochondria isolated from rat liver. Taurine enhanced Ca²⁺ uptake in an apparently saturable process, with a K(m) value of about 2.63 mM. Taurine behaved as an uncompetitive activator of Ca²⁺ uptake, increasing both the apparent K(m) and V(max) values of the process. This effect was not modified in the presence of cyclosporin A (CsA). N,N-Dimethyltaurine also stimulated Ca²⁺ uptake at higher concentrations, but there was no evidence that the process was saturable over the concentration range used (1-10 mM). Aminoethylarsonate was a weak inhibitor of basal Ca²⁺ uptake, but inhibited that stimulated by taurine in an apparently competitive fashion (K(i) = 0.05 mM). The other analogues had no significant effects on this process. Taurine either in the presence or the absence of CsA had no effect on Ca²⁺ release induced by 200 nM ruthenium red. Thus, the mechanism of taurine-enhanced Ca²⁺ accumulation appears to involve stimulation of Ca²⁺ uptake via the uniport system rather than inhibition of Ca²⁺ release via the ion (Na⁺/Ca²⁺ and/or H⁺/Ca²⁺) exchangers or by taurine modulating the permeability transition of the mitochondrial inner membrane. Overall, these findings indicate an interaction of taurine with an as yet unidentified mitochondrial site which might regulate the activity of the uniporter. The unique role of taurine in modulating mitochondrial Ca²⁺ homeostasis might be of particular importance under pathological conditions that are characterised by cell Ca²⁺ overload, such as ischaemia and oxidative stress. Copyright (C) 1999 Elsevier Science Inc.

Full text of DOI:10.1016/S0006-2952(99)00183-5

Biochemical Pharmacology, Vol. 58, pp. 1123–1131, 1999.
© 1999 Elsevier Science Inc. All rights reserved.
ISSN 0006-2952/99/$–see front matter
PII S0006-2952(99)00183-5
Potentiation of Mitochondrial Ca2ϩ Sequestration
by Taurine
Mitri Palmi,*† Gise`le Tchuisseu Youmbi,* Fabio Fusi,* Gian P. Sgaragli,*
Henry B. F. Dixon,‡ Maria Frosini* and Keith F. Tipton§
`
*ISTITUTO DI SCIENZE FARMACOLOGICHE, UNIVERSITA DI SIENA, SIENA, ITALY; ‡DEPARTMENT OF BIOCHEMISTRY,
UNIVERSITY OF CAMBRIDGE, CAMBRIDGE, U.K.; AND §DEPARTMENT OF BIOCHEMISTRY, TRINITY COLLEGE OF
DUBLIN, DUBLIN, IRELAND
ABSTRACT. The effects of taurine (2-aminoethanesulphonic acid) and its analogues, 2-aminoethylarsonic
acid, 2-hydroxyethanesulphonic (isethionic) acid, 3-aminopropanesulphonic acid, 2-aminoethylphosphonic
acid, and N,N-dimethyltaurine, were studied on the transport of Ca2ϩ by mitochondria isolated from rat liver.
Taurine enhanced Ca2ϩ uptake in an apparently saturable process, with a Km value of about 2.63 mM. Taurine
behaved as an uncompetitive activator of Ca2ϩ uptake, increasing both the apparent Km and Vmax values of the
process. This effect was not modified in the presence of cyclosporin A (CsA). N,N-Dimethyltaurine also
stimulated Ca2ϩ uptake at higher concentrations, but there was no evidence that the process was saturable over
the concentration range used (1–10 mM). Aminoethylarsonate was a weak inhibitor of basal Ca2ϩ uptake, but
inhibited that stimulated by taurine in an apparently competitive fashion (Ki ϭ 0.05 mM). The other analogues
had no significant effects on this process. Taurine either in the presence or the absence of CsA had no effect on
Ca2ϩ release induced by 200 nM ruthenium red. Thus, the mechanism of taurine-enhanced Ca2ϩ accumulation
appears to involve stimulation of Ca2ϩ uptake via the uniport system rather than inhibition of Ca2ϩ release via
the ion (Naϩ/Ca2ϩ and/or Hϩ/Ca2ϩ) exchangers or by taurine modulating the permeability transition of the
mitochondrial inner membrane. Overall, these findings indicate an interaction of taurine with an as yet
unidentified mitochondrial site which might regulate the activity of the uniporter. The unique role of taurine
in modulating mitochondrial Ca2ϩ homeostasis might be of particular importance under pathological conditions
that are characterised by cell Ca2ϩ overload, such as ischaemia and oxidative stress. BIOCHEM PHARMACOL 58;7:
1123–1131, 1999. © 1999 Elsevier Science Inc.
KEY WORDS. rat liver mitochondria; taurine; Ca2ϩ transport
Mitochondria have been implicated in the maintenance of
the Ca2ϩ “set-point” in cells [1–3], where control of Ca2ϩ
levels plays a significant role in enzymatic regulation and
energy production [4–8]. Pathological conditions that re-
sult in increased tissue Ca2ϩ concentrations, such as isch-
aemia, oxidative stress, and excito- and neurotoxicity,
depolarisation and further cellular Ca2ϩ influx in a regen-
erative and self-potentiating mechanism. In this context,
removal of Ca2ϩ to the extracellular space and/or uptake
into organelles, including mitochondria, work to restore the
normal cytoplasmic Ca2ϩ concentration, as occurs during
the early stages of recirculation after short-term cerebral
ischaemia [15].
involve impairment of the continued ability of Ca2ϩ
-
regulating organelles, such as sarcolemma, mitochondria,
and sarcoplasmic reticulum, to provide long-term control of
cellular Ca2ϩ levels [9–12]. The subsequent increase in
cytoplasmic Ca2ϩ is widely considered to be a critical
initiating event in the development of damage in cells
destined to die. Although the mechanisms responsible for
cell injury under these conditions are not yet fully clarified,
excessive free cytosolic Ca2ϩ may lead to uncoupling of
mitochondrial oxidative phosphorylation [13] with conse-
quently decreased ATP synthesis [14]. The resulting inac-
tivity of ATP-dependent pumps would lead to membrane
Taurine is a naturally occurring amino acid present in
high (mM) concentrations in mammalian tissues included
liver, where its content is in the range of 3.5–60 ␮mol/g
wet weight (for review see [16]). Despite extensive studies,
there is still controversy about its role(s). One of its possible
functions concerns modulation of Ca2ϩ transport. In many
tissues, taurine has been shown to have marked effects on
the kinetics of Ca2ϩ movement across the membranes of
both cellular and subcellular preparations. A system with a
relatively high affinity (15–20 ␮M) for uptake of Ca2ϩ from
cytosol is stimulated by taurine in several membrane prep-
arations, including cardiac sarcolemma, retinal rod outer
segments, disk membrane, and brain synaptosomes [17–19].
In heart, the normalising action of taurine on both low and
high levels of intracellular Ca2ϩ has been ascribed to direct
modulation of Ca2ϩ channels and/or indirect effects oper-
† Corresponding author: Dr. Mitri Palmi, Istituto di Scienze Farmaco-
logiche, Universita` di Siena, Via Piccolomini 170, 53100 Siena, Italy. Tel.
ϩ39-577-221255; FAX ϩ39-577-281928; E-mail: palmi@unisi.it
Received 25 November 1998; accepted 23 March 1999.

1124
M. Palmi et al.
FIG. 1. Structures of the compounds studied.
ating via Naϩ/Ca2ϩ exchange [20, 21]. In addition to its
ability to modulate cellular Ca2ϩ transport, taurine has also
been reported to influence protein phosphorylation [19],
but no clear relationship between these two processes has
yet been established. In the present study, we used isolated,
functionally intact mitochondria from rat liver as a model
with which to investigate the effects of taurine and some
structurally related analogues on Ca2ϩ transport. An un-
derstanding of the influence of taurine on these processes
might be of importance in pathological and toxicological
conditions characterised by loss of Ca2ϩ homeostasis. A
preliminary account of part of this work has been published
[22].
and N,N-dimethyltaurine was crystallised from 50% aque-
ous ethanol and recrystallised from the same (yield ϭ 6.23 g
[58%]). Elemental analysis gave C, 30.8; H, 7.2; N, 9.0%;
C4H11NO3S requires C, 31.4; H, 7.2; N, 9.1%. A better
yield might well have been obtained if sulphite rather than
bisulphite had been used, i.e. if the pH had been raised, as
sulphite is a better nucleophile. 2-Hydroxyethanesulphonic
(isethionic) acid, 3-aminopropanesulphonic acid, 2-amin-
oethylphosphonic acid, RR*, and all the other reagents
were purchased from Sigma Chimica.
Mitochondrial Preparation
Male Wistar rats (200–250 g) were lightly anaesthetised
with a mixture of Ketavet௡ and Rompun௡ and rapidly
exsanguinated. Livers were rapidly removed and homoge-
nised in a medium containing 250 mM sucrose, 5 mM Tris,
1 mM EGTA, at pH 7.4, and mitochondria were separated
by differential centrifugation [24]. After centrifugation, the
mitochondrial pellet was resuspended in 1–2 mL of a
medium containing 150 mM KCl, 25 mM sucrose, 5 mM
Mg2ϩ, 2.7 mM HEPES (KSH), pH 7.2. The mitochondrial
protein content was determined by the biuret method [25]
using BSA as standard.
MATERIALS AND METHODS
Materials
The structures of taurine and the taurine analogues used in
this work are represented in Fig. 1. 2-Aminoethylarsonic
acid was prepared by periodate oxidation of 2-[(2-hydroxy-
ethyl)amino]ethylarsonic acid, itself made by treating
2-chloroethylarsonic acid with ethanolamine [23]. N,N-
Dimethyltaurine was prepared by adding 2-chloroethyl-
(dimethyl)amine as its hydrochloride (69.4 mmol) to so-
dium bisulphite (70 mmol) in 200 mL of water in a
round-bottomed flask fitted with a water-cooled condenser.
This was boiled under reflux for 2 hr, cooled to room
temperature, and strongly acid ion-exchange resin was
added to it. The solution was rotary evaporated to remove
SO2 and added to a column containing more of the same
resin. The compound was eluted with two column volumes
of 2 M acetic acid and shown to be Naϩ-free by the flame
test. The solution was concentrated by rotary evaporation
Determination of Mitochondrial Ca2؉ Transport
Ca2؉ UPTAKE. Ca2ϩ uptake was determined spectropho-
tometrically in two different media by using antipyrylazo III
* Abbreviations: RR, ruthenium red; CsA, cyclosporin A; TPMP, tri-
phenylmethylphosphonium; K0.5, apparent affinity constant; and ⌬⌿,
membrane potential.

Taurine and Mitochondrial Ca2ϩ Accumulation
1125
difference was only strictly linearly correlated to Ca2ϩ
concentration at concentrations below about 60 ␮M. How-
ever, three successive additions of 10 ␮M CaCl2 at the end
of the experiment gave increases none of which were
significantly different (results not shown, N ϭ 8 in each
case) from one-fifth of the change induced by the 50 ␮M
addition at the start of the experiment. The presence of
ATP and pyrophosphate, both Ca2ϩ-chelating agents, in
medium 2 decreased the free Ca2ϩ concentration, as
measured with antipyrylazo III, by approximately 40%.
However, since the standard 50 ␮M Ca2ϩ concentration
used was greatly in excess of the Km value for the uptake
process, this would have an effect of less than 10% on the
initial velocity (see below). Compounds to be tested were
present in the antipyrylazo-containing buffers.
It has been reported that under de-energised conditions,
the presence of pyrophosphate might sensitise mitochon-
dria to the permeability transition, and increase matrix
volume and swelling; however, when mitochondria are
incubated in the presence of ATP, this phenomenon is
greatly reduced [28]. Although ATP was present in the
pyrophosphate-containing medium (medium 2), experi-
ments to measure mitochondrial swelling were carried out
to assess whether mitochondria underwent any permeabil-
ity transition under these conditions. Mitochondrial swell-
ing was measured by the method of Broekemeier et al. [29]
by monitoring the decrease in light-scattering of a mito-
chondrial suspension. Following preparation in KSH (as
described above), the mitochondria were suspended in the
pyrophosphate-containing medium and placed in a 3-mL
stirred cuvette at 30°. The final suspension, containing 1
mg/mL of mitochondria, was monitored spectrophotometri-
cally at 540 nm in a Shimadzu UV-160 spectrophotometer.
After the attenuance had stabilised, Ca2ϩ was added. The
results showed that under the experimental conditions of
either medium 1 or 2, the addition of 50 ␮M Ca2ϩ did not
cause detectable swelling, either in the absence or presence
of taurine and taurine analogues (data not shown).
FIG. 2. Kinetics of Ca2؉ uptake by rat liver mitochondria in two
different media (medium 1: 195 mM mannitol, 65 mM sucrose,
3 mM HEPES, pH 7.2; medium 2: 92.5 mM KCl, 18.5 mM
K؉MOPS (3-(N-morpholino)propanesulphonic acid, 7.5 mM
Na؉pyrophosphate, pH 7.2). The reaction containing 1 mg/mL
of mitochondrial protein was initiated by adding 50 ␮M CaCl2
(arrow). Two successive calibrating additions of 50 ␮M CaCl2
were made at the end of the experiment. Uptake (in nmol/
min/mg of mitochondrial protein) was determined spectropho-
tometrically from the differential absorbance changes (A710–790
)
using the Ca2؉ indicator antipyrylazo III.
as indicator [26]. In one case, mitochondria (2 mg of
protein) were suspended in a medium (medium 1) consist-
ing of 1.9 mL of buffer (195 mM mannitol, 65 mM sucrose
and 3 mM HEPES, pH 7.2) containing 250 ␮M antipyry-
lazo III to which had been added 13 ␮M rotenone and 10
mM succinate. In the other case, mitochondria (2 mg of
protein) were suspended in 2 mL of experimental medium
(medium 2), consisting of 1.9 mL of buffer (92.5 mM KCl,
18.5 mM KϩMOPS (3-(N-morpholino) propanesulphonic
acid), 7.5 mM Naϩpyrophosphate, pH 7.2) containing 250
␮M antipyrylazo III, to which had been separately added
0.1 mL of 1 mM Mg2ϩ-ATP, creatine phosphokinase (20
␮g/mL), and 5 mM phosphocreatine [27]. When CsA was
present in the experimental media, this was at a concen-
tration of 0.5 ␮M. Both experimental media were equili-
brated at 30°, with stirring, before being placed in a 1-cm
path-length cuvette. Changes in Ca2ϩ concentration were
monitored with time, using a Shimadzu UV-160 spectro-
photometer or a Hewlett Packard diode-array spectropho-
tometer, by measuring the absorbance at 710 nm and
subtracting the absorbance at 790 nm (A710–790). The
experiment was started by adding 50 ␮M CaCl2 to the
cuvette; this caused an initial rapid increase in absorbance
because of the formation of the Ca2ϩ-antipyrylazo III
complex. This was followed by a time-resolvable decrease in
absorbance as a result of the transport of Ca2ϩ into the
mitochondria and its consequent disappearance from the
extramitochondrial solution (see Fig. 2). At the end of the
experiment, after the absorbance decrease had stabilised,
the response of the system was checked by determining the
change in absorbance following the addition of successive
portions of CaCl2. It was found that the absorbance
Ca2؉ RELEASE. Mitochondria were loaded with Ca2ϩ by
incubation with 100 ␮M CaCl2 for a period of 5 min. They
were then treated with 200 nM RR. This concentration of
RR was chosen on the basis of the results of direct titration
of Ca2ϩ uptake by mitochondria (1 mg/mL). The results
(data not shown) indicated that 200 nM RR completely
inhibited the mitochondrial Ca2ϩ uptake stimulated by 100
␮M Ca2ϩ. Addition of RR to inhibit the mitochondrial
Ca2ϩ uniporter caused an increase of Ca2ϩ in the medium,
due to efflux of Ca2ϩ from mitochondria [30]. This was
accompanied by a time-resolvable increase in A710–790 as a
result of Ca2ϩ complexation by antipyrylazo III. Loading
mitochondria with 100 ␮M Ca2ϩ in pyrophosphate-con-
taining medium (medium 2) may induce pore opening,
causing swelling which is prevented by adding CsA. For this
reason, the Ca2ϩ efflux experiments performed in medium
2 were carried out in the presence of CsA (0.5 ␮M).

1126
M. Palmi et al.
RESULTS
Mitochondrial Ca2؉ Influx Rate
Figure 3 shows the effects of 10 mM taurine on the rate of
Ca2ϩ uptake by rat liver mitochondria determined in
medium 1. Taurine also induced a significant and equiva-
lent increase in the rate of Ca2ϩ uptake in medium 2 (data
not shown). The percent changes (mean Ϯ SEM) over
controls were 62.8 Ϯ 2.3 in medium 1 and 64.5 Ϯ 6.5 in
medium 2. When these experiments were repeated in the
presence of 0.5 ␮M CsA, the trends in Ca2ϩ influx were
similar in both media, and the effect of taurine over the
respective (CsA-containing) controls were 78.9 Ϯ 2.94 and
76.5 Ϯ 9.0 in medium 1 and medium 2, respectively. These
values were not significantly different from those observed
in the absence of CsA. When calculated in terms of
cumulative mitochondrial Ca2ϩ uptake over time, the data
from Fig. 3 indicate that in the absence of taurine, about 50
per cent of added Ca2ϩ (100 nmol) was accumulated by 1
mg of mitochondrial protein within 3 min, whereas in the
presence of taurine this quantity increased to 80%. Similar
effects were observed in medium 2 (data not shown). The
effects of taurine were similar either in the presence or
absence of 0.5 ␮M CsA (medium 1: 80.7 Ϯ 1.7 and 81.7 Ϯ
3.7 nmol, respectively; medium 2: 75.9 Ϯ 4.6 and 77.1 Ϯ
8.2 nmol, respectively), indicating that mitochondrial pore
opening did not occur and was not involved in the action
of taurine on Ca2ϩ fluxes.
FIG. 3. Effects of taurine on the rate of Ca2؉ uptake by rat liver
mitochondria. The time–course of the rates of Ca2؉ uptake,
measured in terms of differential absorbance changes (A710–790
)
in antipyrylazo III per min at the times indicated, was deter-
mined in medium 1 (see Methods). Values shown represent the
means ؎ SEM from 3 to 5 separate experiments, each performed
in duplicate. Statistical significance with respect to control
values for **P < 0.01 was determined by Student’s t-test for
unpaired data, followed by Welch’s t-test.
Taurine was added immediately before RR and the rates of
Ca2ϩ release were determined after 2, 4, and 6 min.
Figure 4 shows the effects of different concentrations of
Measurement of Membrane Potential
The mitochondrial ⌬⌿ was determined from the distribu-
tion of the lipophilic cation TPMPϩ across the mitochon-
drial inner membrane [31]. The concentration of TPMPϩ
in the extramitochondrial medium was continuously mon-
itored with a TPMPϩ-selective electrode [32] in an open
thermostatically controlled (30°) and stirred chamber. ⌬⌿
was then calculated from Nernst’s equation, and corrections
due to the binding of TPMPϩ to the mitochondrial
membranes were made as described by Brand et al. [33].
Mitochondria (1 mg/mL of mitochondrial protein) were
suspended in 3 mL of medium containing mannitol 195
mM, sucrose 65 mM, HEPES 3 mM (pH 7.2), and 13.3 ␮M
rotenone, in the absence or presence of taurine (10 mM).
Additions of TPMPϩ were made to calibrate the TPMPϩ
electrode, giving a final concentration of 5 ␮M TPMPϩ.
After the calibration, 10 mM succinate (Kϩ salt) was
added, followed by additions of Ca2ϩ at final concentra-
tions of 10, 20, 30, and 50 ␮M.
FIG. 4. Effects of taurine and analogues on the initial rate of
Ca2؉ uptake by rat liver mitochondria. Initial rates of Ca2؉
uptake, measured in terms of differential absorbance changes
(A710–790) in antipyrylazo III per min following 50 ␮M Ca2؉
addition, were determined in medium 2 (see Methods) in the
absence or presence of taurine, N,N-dimethyltaurine (DMT),
isethionic acid (ISE), aminoethylphosphonic acid (AEP), amin-
opropanesulphonic acid (APS), or aminoethylarsonic acid
(AEA). The rate of Ca2؉ uptake in the absence of taurine or the
analogues was 20.350 ؎ 0.331 nmol/min/mg of mitochondrial
protein (N ؍ 27). Values shown are percentage means ؎ SEM
above control (100%) from 3 to 5 separate experiments, each
determined in duplicate. Statistical significance with respect to
control values for *P < 0.05 and **P < 0.01 was determined by
Student’s t-test for unpaired data, followed by Welch’s t-test.
Statistics
The mean and SEM values from 3 to 5 separate experi-
ments were determined and the percentage differences
between these and controls were then calculated. The
statistical significance of the differences was determined by
Student’s t-test for unpaired data followed by Welch’s
t-test.

Taurine and Mitochondrial Ca2ϩ Accumulation
1127
taurine and some taurine analogues on the initial rates of
Ca2ϩ uptake by rat liver mitochondria determined in
medium 2. Taurine, in the range of concentrations used
(1–10 mM), induced a significant (P Ͻ 0.01) increase in
the rate of Ca2ϩ uptake, which approached a plateau at
about 10 mM taurine. The taurine analogues studied
differed in their effects on Ca2ϩ uptake. In the 1–10 mM
range, isethionic acid, aminopropanesulphonic acid, and
aminoethylphosphonic acid had no statistically significant
effects, whereas N,N-dimethyltaurine had a stimulating
effect only at a high concentration; at 10 mM this effect
was similar to that produced by the same concentration of
taurine. In contrast to the behaviour of taurine, the kinetics
of N,N-dimethyltaurine-induced Ca2ϩ uptake did not ap-
pear to be saturable in the concentration range used.
Among the compounds examined, only aminoethylarsonic
acid inhibited the process: the effect was small, the Ca2ϩ
uptake rate being lowered by 8.0 Ϯ 1.4% of control at a
concentration of 10 mM.
As shown in Fig. 5a, aminoethylarsonate competitively
inhibited the effects of taurine in stimulating the transport
of Ca2ϩ. The apparent Km value for the stimulation of
Ca2ϩ uptake by taurine in the absence of inhibitor, corre-
sponding to the taurine concentration that increased the
velocity of Ca2ϩ uptake by 50% of maximum stimulation,
was approximately 2.63 mM. A Ki for aminoethylarsonate
of approximately 0.05 mM was determined by the method
of Dixon [34], by plotting the dependence of the reciprocal
of the stimulation of the initial velocities of Ca2ϩ uptake
against the aminoethylarsonate concentration at a series of
fixed taurine concentrations (Fig. 5b).
In order to evaluate whether a change in affinity of the
uniporter may account for the effect of taurine on Ca2ϩ
uptake, the dependence of transport on Ca2ϩ concentra-
tion was determined. As shown in Fig. 6, the rates of uptake
increased with increasing Ca2ϩ in a process that was almost
saturated at a Ca2ϩ concentration of 12.5 ␮M. Thus, as
discussed earlier, the reduction in free Ca2ϩ concentration
as a result of chelation in medium 2 would have only a
small effect on the velocity observed in the presence of a 50
␮M Ca2ϩ concentration used in the previous experiments.
By plotting the dependence of the reciprocal of Ca2ϩ
uptake against the reciprocal of Ca2ϩ concentration (see
inset of Fig. 6), the Vmax and K0.5 of the Ca2ϩ uniporter
process could be determined. These data indicated that the
presence of taurine (10 mM) increased both Vmax and K0.5
(from 29.8 Ϯ 4.11 nmol/min/mg and 10.37 ␮M, respec-
tively, in the absence of taurine to 50.9 Ϯ 7.5 nmol/min/mg
and 17.32 ␮M in the presence of 10 mM taurine).
FIG. 5. Antagonism by aminoethylarsonate (AEA) of taurine
(TAU)-stimulated Ca2؉ transport in rat liver mitochondria. (a)
Double-reciprocal plot to indicate the mechanism of inhibition.
(b) Dixon [30] plot of the data to facilitate calculation of the Ki
value. Values shown represent the means ؎ SEM from 3 to 5
separate experiments, each performed in duplicate.
1 and in pyrophosphate-containing medium (medium 2) in
the presence of 0.5 ␮M CsA are shown, the rates of Ca2ϩ
release did not vary significantly in the interval following
RR addition (range 2.2 Ϯ 0.3–3.45 Ϯ 0.5 nmol/min/mg).
Furthermore, in both media, the presence of 10 mM taurine
did not significantly affect the rates of Ca2ϩ efflux.
Effect of Taurine on Membrane Potential
In order to characterise further the mechanism by which
taurine stimulated mitochondrial Ca2ϩ uptake, the effects
of taurine on mitochondrial ⌬⌿ were studied under the
same conditions as those used to determine Ca2ϩ uptake in
medium 1. The distribution of TPMPϩ was measured both
in the steady state before Ca2ϩ and during Ca2ϩ uptake. As
shown in Fig. 8, the addition of 50 ␮M Ca2ϩ induced a
transient drop in ⌬⌿ that slowly recovered to nearly the
initial value as the Ca2ϩ uptake became complete. The
presence of 10 mM taurine did not significantly modify ⌬⌿
Mitochondrial Ca2؉ Efflux
Possible interactions of taurine with the RR-insensitive
Ca2ϩ efflux system of liver mitochondria were determined
using mitochondria that had been loaded with 100 ␮M
Ca2ϩ. The Ca2ϩ uniporter was then inhibited by addition
of 200 nM RR. As shown in Fig. 7, where results in medium

1128
M. Palmi et al.
FIG. 7. Effect of taurine on the rate of Ca2؉ release by rat liver
mitochondria. Ca2؉ efflux stimulated by 200 nM RR was
determined in Ca2؉ (100 ␮M)-preloaded mitochondria in the
presence or absence of 10 mM taurine either in medium 2 in the
presence of 0.5 ␮M CsA or in medium 1. Rates of Ca2؉ efflux,
measured in terms of differential absorbance changes (A710–790
)
in antipyrylazo III per min at the times indicated, represent the
means ؎ SEM from 3 to 5 separate experiments, each performed
in duplicate. None of the values were significantly different
from the respective control values (as determined by Student’s
t-test for unpaired data, followed by Welch’s t-test).
FIG. 6. Ca2؉ concentration dependence of Ca2؉ conductance of
the mitochondrial inner membrane; effect of taurine. Ca2؉
uptake, measured in terms of differential absorbance changes
(A710–790) in antipyrylazo III per min following Ca2؉ addition,
was determined in medium 1. Values represent the means ؎
SEM from 3 to 5 separate experiments, each performed in
duplicate. Statistical significance with respect to control values
for *P < 0.05 and **P < 0.01 was determined by Student’s
t-test for unpaired data, followed by Welch’s t-test. The inset
represents the dependence of the reciprocal of Ca2؉ uptake
against the reciprocal of Ca2؉ concentration.
(isethionate), or elongation of the carbon chain between
these two groups, as in aminopropanesulphonate, abolished
the stimulation of Ca2ϩ uptake. Although N-substituted
N,N-dimethyltaurine did stimulate Ca2ϩ uptake, it did so
only at higher concentrations and the effect was not
saturable over the concentration range used. These results
are at variance with those of Dolara et al. [36], who reported
that both taurine and isethionate stimulate Ca2ϩ accumu-
lation by rat liver mitochondria. However, those effects
were observed only at very high concentrations of Ca2ϩ (10
mM), with no statistically significant effect of either com-
values, either in the steady state or during Ca2ϩ uptake.
Lower external concentrations of Ca2ϩ induced smaller
transient falls in ⌬⌿ which, similarly to those observed
with 50 ␮M Ca2ϩ, were not significantly affected by taurine
(data not shown).
DISCUSSION
Just as taurine has been shown to increase high (15–20
␮M)-affinity Ca2ϩ uptake by different membrane prepara-
tions [14–16], the present work shows that it stimulates
Ca2ϩ uptake by rat liver mitochondria. The effect was
relatively large, since 10 mM taurine increased the rate of
uptake almost twofold. The initial rate of Ca2ϩ uptake by
mitochondria was shown to depend on the taurine concen-
tration in a saturable process. This suggests that taurine acts
by reversibly forming a complex. This cannot be a complex
with Ca2ϩ, since taurine has a very low affinity for this
cation [35]. The effect of taurine was specific, since replace-
ment of the sulphonic group with a phosphonic (aminoeth-
ylphosphonate) or an arsonic (aminoethylarsonate) group,
replacement of its amino group with a hydroxyl group
FIG. 8. Effect of taurine on mitochondrial ⌬⌿ (؊mV). The
measurements were performed both in the steady state before
Ca2؉ addition and during Ca2؉ uptake in medium 1, in the
presence or absence of 10 mM taurine. After calibration, 10 mM
succinate (K؉ salt) was added, followed by addition of 50 ␮M
Ca2؉ (see Methods).

Taurine and Mitochondrial Ca2ϩ Accumulation
1129
pound being found when Ca2ϩ concentrations in the range
1–5 mM were used. Aminoethylarsonate not only failed to
stimulate Ca2ϩ uptake, but antagonised the stimulatory
effects of taurine in an apparently competitive process, with
a Ki that was some two orders of magnitude lower than the
apparent Km for taurine. These data might indicate a
common interaction site for aminoethylarsonate and tau-
rine, thus further substantiating the specificity of the
stimulatory effect of taurine on this process.
were conducted at a physiological resting ⌬⌿ of about 160
mV and Ca2ϩ uptake lowered it to about 126 mV, the
possibility that taurine might have more profound effects at
that lower initial membrane potential remains to be inves-
tigated.
Since mitochondrial influx and efflux of Ca2ϩ may be
occurring simultaneously, as indicated by the relatively low
rates as compared to those seen with Ca2ϩ-depleted mito-
chondria [43], an apparently increased accumulation of this
ion might result from either stimulated uptake or inhibition
of Ca2ϩ extrusion [4]. The observation [22] that taurine
might have an uncoupling effect under the state 4 condi-
tion might also reflect an increased rate of Ca2ϩ cycling
across the mitochondrial membrane. Although a specific
effect of taurine was also observed on state 3 respiration in
the presence of ADP [22], a condition whose rate is known
to be jointly controlled by the electron transport chain and
by the phosphorylating system, there was no direct effect on
state 3 uncoupled respiration, which is largely controlled by
the electron transport chain [44]. Thus, it appears that
taurine may act at the level of the phosphorylating system,
which is mainly limited by the adenine nucleotide translo-
case and the F0–F1 ATP synthase complex.
Analysis of the kinetics of Ca2ϩ uptake indicates that
taurine acts as an “uncompetitive activator” of Ca2ϩ
transport, increasing both the Vmax and K0.5 of the
uniporter process [37]. These opposing effects on K0.5 and
Vmax result in the stimulatory effects of taurine being
dependent on the Ca2ϩ levels in the medium. For Ca2ϩ
concentrations within the range of 3–12.5 ␮M, taurine has
little effect on the rate of Ca2ϩ uptake, whereas the rate is
increased at higher Ca2ϩ concentration. Thus, the effects
of taurine enhance the total buffering capacity of the cell
under conditions of Ca2ϩ overload. Such behaviour might
contribute to the apparently paradoxical effects of taurine
in increasing Ca2ϩ availability under conditions of low
Ca2ϩ, but protecting against Ca2ϩ overload under condi-
tions of high Ca2ϩ availability [16]. In this respect, the
behaviour of taurine resembles that of spermine [38, 39].
However, although spermine and other polyamines in-
crease the extent of Ca2ϩ accumulation, they also decrease
the steady-state velocity of Ca2ϩ uptake [30, 38, 39]. Two
spermine binding sites with dissociation constants of 0.5
and 4.7 mM have been found to modulate Ca2ϩ transport
in liver mitochondria [39]. Although these values are
similar to Km for taurine, the different responses make it
unlikely that one or both of the spermine sites is involved
in the actions of the latter compound. However, the
possibility that other physiologically occurring compounds
may interact with the taurine site, perhaps at lower con-
centrations, cannot be excluded.
As has been shown, for example, in the case of glucagon,
the rate of Ca2ϩ uptake can be affected not only by changes
in the kinetic parameters of the Ca2ϩ uniporter itself but
also by a change in ⌬⌿, which provides the major compo-
nent of the driving force for this uptake process [40].
Indeed, proton translocation by the respiratory chain may
easily become the rate-limiting step for the process of Ca2ϩ
uptake [41, 42]. In the present study, initial rates were
determined which should depend only on the initial mito-
chondrial ⌬⌿. Furthermore, measurement of TPMPϩ dis-
tribution under conditions identical to those used in the
Ca2ϩ uptake experiments showed that taurine did not
modify ⌬⌿, either under resting conditions (before Ca2ϩ
addition) or during Ca2ϩ uptake, indicating that the
stimulation of uptake by taurine primarily reflects a direct
effect on the transport system rather than an indirect effect
operating through alterations in ⌬⌿. The polyamines
spermine and spermidine have also been reported to in-
crease the total Ca2ϩ accumulation, but have little or no
effect on ⌬⌿ [30, 38, 39]. However, as our experiments
Loading mitochondria with Ca2ϩ in the presence of
pyrophosphate might induce opening of the permeability
transition pore in the mitochondrial inner membrane with
release of accumulated Ca2ϩ, a process that is blocked by
low concentrations of CsA [12]. Since 0.5 ␮M CsA did not
modify the effects of taurine on the measured rate of Ca2ϩ
accumulation, such a permeability transition did not occur
under the experimental conditions used. Furthermore, the
stimulatory effects of taurine were also seen in assays that
did not contain pyrophosphate (medium 1). Thus, the
increased Ca2ϩ uptake was not the result of taurine mod-
ulating the pore opening. The possibility that the appar-
ently increased Ca2ϩ uptake in the presence of taurine
might result from effects on Ca2ϩ efflux through an ion
(Naϩ/Ca2ϩ or Hϩ/Ca2ϩ) exchanger may be ruled out on
the basis of the data showing that taurine did not change
the rate of Ca2ϩ efflux stimulated by RR. Finally, since
there were no significant differences between the Ca2ϩ
uptake values determined in medium 1 and medium 2, the
possibility that phosphate ions, derived from pyrophosphate
hydrolysis, were affecting the observed Ca2ϩ accumulation
may be excluded. Thus, under the conditions used in the
present work, pyrophosphate did not affect Ca2ϩ transport
and the mechanism of taurine-enhanced Ca2ϩ uptake was
independent of the presence of either phosphate or pyro-
phosphate. Taken together, these data strongly support the
view that stimulation of Ca2ϩ uptake by taurine resulted
from stimulation of influx rather than inhibition of efflux.
It has recently been reported that taurine decreases the
fluidity of biological membranes, but only when co-admin-
istered with Ca2ϩ, suggesting that an interaction between
taurine and Ca2ϩ affects membrane lipid fluidity [45].
Although the nature of this interaction is not yet clear, a
change in the dynamics of mitochondrial membranes might

1130
M. Palmi et al.
be invoked to explain how taurine modifies Ca2ϩ transport
through them. Thus, taurine might alter the interaction of
the mitochondrial Ca2ϩ uniporter with the lipid bilayer,
thereby affecting its kinetic behaviour.
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calcium ion concentration by rat liver mitochondria. Biochem
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In isolated hepatocytes, taurine was shown to prevent
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icity of different compounds such as hydrazine, carbon
tetrachloride, and 1,4-naphthoquinone [47]. In rats, a
reduction of liver taurine significantly increased the hepa-
totoxicity of carbon tetrachloride [48], whereas treatment
with taurine protected the liver against carbon tetrachlo-
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intracellular Ca2ϩ accumulation [49], suggesting a correla-
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taurine on galactosamine-induced hepatic necrosis [50].
The effects of mitochondrial Ca2ϩ accumulation may be
particularly beneficial for the maintenance of normal cell
function in pathologies characterised by cytoplasmic accu-
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excitotoxicity, and hypoxia/ischaemia–reperfusion. The ob-
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overload in brain tissue that results from hypoxia suggests
that such damage may indeed be improved by taurine
supplementation [51]. Furthermore, taurine has been re-
ported to protect dopaminergic neurones against the neu-
rotoxicity of the mitochondrial inhibitor 1-methyl-4-phe-
nylpyridinium ion (MPPϩ) [52]. Since the mitochondrial
inhibitory action of MPPϩ may, in turn, lead to the
production of oxygen radicals and impaired Ca2ϩ ho-
meostasis [53], the present results suggest that the protec-
tion afforded by taurine may be related to its effects on
mitochondrial function.
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In conclusion, we have shown that taurine potentiates
the initial rate of Ca2ϩ uptake in mitochondria and that
this may be due to its interaction with a specific recognition
site in the mitochondrial membrane. This apparently spe-
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The financial support of the EC BIOMED Programme (BMH1
CT94-1402) and the Ministero degli Affari Esteri is acknowledged.
The authors thank Nicholas Desmond-Smith for help in synthesising
N,N-dimethyltaurine and Dr. M. P. Murphy for stimulating
discussion.
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