From the Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom and the § Department of Biomedical Sciences and CNR Centre for Study of Biological Membranes, University of Padova, Via Trieste 75, 35121 Padua 17, Italy
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ABSTRACT |
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Increases in the concentration of free ATP within
the islet Increases in extracellular glucose concentration stimulate the
exocytosis of insulin from islet Increases in the total intracellular concentration of ATP have been
measured in isolated islets (6) and cell lines (7) exposed to increases
in extracellular glucose concentration. However, the measured changes
are generally small and difficult to interpret because of the large
depot of intragranular ATP, and the presence of non- The role of changes in free Ca2+ ion concentration
([Ca2+]) in regulating The use of firefly luciferase, targeted to discrete intracellular
domains, should provide an extremely sensitive method of monitoring
free [ATP] dynamically and at the subcellular level. In previous
studies, we have shown that photon counting imaging of total luciferase
activity in single cells provides a convenient means to measure changes
in gene expression in single cells (15, 16). Luciferase has previously
been employed to measure intracellular ATP concentration in single
cardiac myocytes (17) and hepatocytes (18), but only after
microinjection of the purified protein. In recent reports, Maechler
et al. (19, 20) have shown that expression of recombinant
luciferase provides a means of monitoring cytosolic ATP concentration
in large populations (>10,000) of INS-1 Plasmid Construction--
Cytosolic (untargeted) firefly
luciferase in plasmid pGL3 basic (Promega) was placed under
cytomegalovirus immediate gene control by subcloning the
cytomegalovirus promoter element from plasmid pcDNA3 (Invitrogen)
as an 876-nucleotide BglII-HindIII fragment into
the upstream multiple cloning site of pGL3, generating plasmid cLuc.
Plasma-membrane targeted luciferase was prepared by polymerase chain
reaction amplification of the 617-nucleotide translated region (minus
stop codon) of synaptosome-associated protein of 25 kDa cDNA, with
primers 5' T.TTT.GAC.GAG.ACC.ATG.GCC.GAG.GAC.GCA and
5'TT.TTC.CAT.GGT.ACC.ACT.TCC.CAG.CAT.CTT (NcoI
sites underlined) and the 629-nucleotide polymerase chain reaction
fragment digested and subcloned into the plasmid pGL3-control (Promega)
under SV40 immediate early gene promoter control, generating plasmid
pmLuc. Correct orientation of the insert was verified by restriction mapping with SmaI and SalI, and confirmed by
automated DNA sequencing. To allow higher levels of expression of this
construct under cytomegalovirus promoter control, the
HindIII-BamHI fragment of pmLuc was subcloned into plasmid pcDNA3; essentially identical data were obtained with
either plasmid. For the preparation of mLuc, DNA sequences encoding a
mitochondrial presequence and the hemagglutinin HA1 tag were added to
the luciferase cDNA as follows. A fragment of wild-type luciferase
cDNA was first amplified from plasmid pGL2 (Promega) using the
following primer:
5'AAAG.CTT.AAT.GGA.AGA.CGC.CAA.AAA.CAT.AAA.GAA.A (corresponding to the sequence encoding amino acids 1-9 of luciferase; HindIII site underlined) and
GAA.GAT.GTT.GGG.GTG.TTG.TAA.CAA.T (downstream of the endogenous
ClaI site of the luciferase cDNA and encoding amino
acids 456-465). The polymerase chain reaction product was digested
with the enzymes HindIII and ClaI and fused in
frame to the ClaI/HindIII fragment encoding the
HA1 tag (22). A ClaI fragment was thus generated that, in an
appropriately prepared pBSK+ plasmid, could be fused in frame with the
EcoRI/HindIII fragment encoding the
amino-terminal 33 amino acids of cytochrome oxidase subunit 8 (COX8)
(25 amino acids of the cleavable presequence plus 8 amino acids of the
mature polypeptide) (23) and the ClaI/SalI fragment encoding the carboxyl-terminal portion of luciferase (amino
acids 457-556). The whole final construct (shown schematically in Fig.
1) was excised via PstI/SalI digestion and cloned
into the expression vectors VR1012 (Vical Research Inc., San Diego, CA)
under modified cytomegalovirus promoter control.
Cell Culture, Microinjection, and Imaging--
Primary rat islet
Measurements of Tetramethylrhodamine Ethyl Ester (TMREE)
Fluorescence--
Cells were maintained at 37 °C for 2 h in
serum-free Dulbecco's modified Eagle's medium containing 3 mM glucose, and then loaded with TMREE (10 nM)
for 60 min. Confocal imaging was performed in KREBS, initially
containing 3 mM glucose, using a Leica TCS-NT inverted
confocal microscope, fitted with a × 40 oil immersion objective,
with illumination at 568 nm from a krypton/argon laser. Fluorescence
from groups of 4-7 single cells was analyzed off-line; this
compensated for the considerable movement of individual mitochondria in
and out of the confocal plane.
Immunocytochemistry--
Cells were fixed and permeabilized
24 h after microinjection using 4% (v/v) paraformaldehyde plus
0.2% Triton X-100. Primary polyclonal rabbit anti-luciferase antibody
(Promega) was revealed with tetra-methyl-rhodamine-conjugated
anti-rabbit immunoglobulin G (Sigma). Confocal images were obtained
using a Leica TCS 4D/DM IRBE laser scanning confocal microscope
equipped with a krypton/argon laser (568 nm excitation line) and
analyzed off-line using a Silicon Graphics workstation.
Kinetic Analysis of Luciferase in Vitro--
MIN6 cells were
transiently transfected with the lipoamine Tfx-50TM
(Promega) as per the manufacturer's instructions. Cells were extracted
into buffer comprising 20 mM Hepes, 0.1% Triton X-100 (pH
7.2) and assayed in intracellular medium (see above) using an LB-9501
luminometer (EG & Berthold, Bad Wildbad, Germany).
Statistical Analysis and Other Methods--
Data are presented
as means ± S.E. for the number of observations given. For
Km measurement of the different luciferases, statistical significance was determined by Fischer test for the improvement in fitting individual Km values compared with fitting a common value (28). ATP content of MIN6 cell populations was determined in extracts using firefly lantern extracts (29). Intracellular pH was measured by monitoring fluorescence changes at the
single cell level using 2'7'-bis(carboxyethyl) 5(6)carboxyfluorescein as an intracellular pH indicator. Briefly, cells grown on glass coverslips and preloaded with 2'7'-bis(carboxyethyl)
5(6)carboxyfluorescein for 20 min were perifused continuously with
Krebs-Ringer bicarbonate medium at a flow rate of 2 ml
min Expression and Calibration of Targeted Firefly
Luciferases--
Luminescence imaging of firefly luciferase provides
adequate resolution at the single cell level but is not readily
amenable to confocal or deconvolution methods necessary to achieve
subcellular resolution (24). We therefore used the molecular approach
of targeting luciferase to distinct subcellular domains by fusion with
specific peptide sequences. Fig. 1 shows
the constructs used in this study. Mitochondrially targeted luciferase
(mLuc) was based on wild-type Photinus pyralis firefly
luciferase (Mr 65 kDa), extended at the amino
terminus via the 26-amino acid amino-terminal signal peptide of
cytochrome c oxidase subunit VIII (23). This presequence is
cleaved soon after mitochondrial import of the parental protein (30).
pmLuc was generated by fusion with synaptosome-associated protein of 25 kDa, a neuoronal v-SNARE (11) targeted to the plasma membrane and
neurite extensions after palmitoylation at two amino-terminal cysteine
residues (31). These constructs targeted luciferase to the
mitochondrial matrix and the plasma membrane respectively (Fig. 1), as
predicted from the behavior of aequorin and green fluorescent protein
targeted by identical strategies (32, 33). Limited localization (< 10% of total) to a vesicular, intracellular compartment was also
observed after expression of pmLuc and may represent association with
secretory vesicles (34).
The sensitivity of recombinant expressed luciferase to [ATP] was
first determined in cell extracts. These assays were performed in the
presence of likely intracellular concentrations of CoA (0.01 mM) (35), a known regulator of enzyme activity (36). The
presence of CoA greatly reduces the complex "flash" kinetics of the
enzyme, believed to result either from the formation of the
luciferyl·AMP intermediate, or the accumulation of the reaction product, oxyluciferin (37), probably by enhancing the breakdown of the
less stable enzyme·luciferyl·CoA intermediate (36, 38). Calibration
of the responses of each expressed and extracted luciferase to [ATP]
indicated a similar Km for [ATP] of each
construct, close to 1 mM (Fig.
2).
We next demonstrated that luminescence from the expressed recombinant
chimeras could be imaged in single living cells over the likely time
frame of changes in intracellular [ATP]. Photon production was imaged
in single luciferase-expressing MIN6 cells using an intensified and
cooled charge-coupled device camera (15, 39) attached to an inverted
optics microscope equipped with a × 10 objective lens. In the
presence of 1 mM luciferin, this technique allowed 2-5-s
resolution in MIN6 cells, in which high levels of luciferase expression
could be achieved, and 5-s resolution in primary
To determine the value of [ATP] in each subcompartment, we monitored
the luminescence of single cells before and after permeabilization in
the presence of ATP (Fig. 2). Single MIN6 cells were permeabilized with
digitonin at the likely subdomain pH (7.2 for the cytosol and
sub-plasma membrane region, or pH 7.8 for the mitochondrial matrix),
intracellular free CoA (0.01 mM) (35) and Mg2+
(0.5 mM) (41, 42). Permeabilization caused a
time-dependent decrease in luminescence from each
construct, presumably reflecting loss of ATP from the cell cytosol or
conversion of intramitochondrial ATP to ADP (for mLuc). Re-addition of
ATP caused the re-appearance of luminescence after an initial small
burst, as observed in extracts (Fig. 2). Comparison of the steady-state
luminescence level before and after permeabilization with the obtained
standard curves indicated resting [ATP] values in the low
mM range in each compartment (Fig. 2).
Role of Factors Other Than Intracellular [ATP] in Photon
Production by Recombinant Luciferases in MIN6 Cells--
To confirm
that any stimulus-induced luminescence change was due, principally, to
changes in intracellular [ATP] and was unlikely to be the result of
changes in the concentration of other luciferase substrates or
effectors, we monitored the effects of changes in these parameters on
firefly luciferase activity in vitro and in living cells.
Because luciferase activity was increased in vitro by
increases in pH (activity at 1 mM ATP: 1.0, 1.2, and 1.4 arbitrary units at pH 6.8, 7.2, and 7.6, respectively), we first
investigated whether pH changes may contribute to any observed
luminescence change in response to cell stimulation. Transient
intracellular alkalinization of cells with 10 mM
NH4Cl increased the luminescence output from cells
expressing each construct by 15-20%, whereas acidification with 10 mM Na+ acetate caused a decrease in light
output by about the same amount. Under the conditions used in these
studies, exposure of MIN6 cells to 30 mM glucose caused
little or no change in intracellular pH (26). However, 70 mM K+ caused a small (3-5%) decrease in
2'7'-bis(carboxyethyl) 5(6)carboxyfluorescein fluorescence ratio, which
could be mimicked by treatment with 4 mM
Na+-acetate (data not shown), likely to correspond to a pH
decrease of < 0.05 pH units (43). We next tested the effects of
increasing CoA concentration on extracted luciferase. In islets, Liang
and Matschinsky (35) have reported that increasing extracellular glucose from 2.5 to 25 mM raised islet CoA content by about
6% during a 30-min perfusion, from 6.8 pmol/µg DNA (equivalent to 6.8 µmol/liter, assuming 10 ngDNA·islet Control of Mitochondrial [ATP] by Glucose and Intracellular
Ca2+--
As a functional assay of the correct targeting
of mitochondrial luciferase in living cells, we determined whether
[ATP)c and [ATP]m could be altered
independently in the presence of atractyloside, a potent inhibitor of
the mitochondrial adenine nucleotide translocase (10). As shown in Fig.
3, exposure to atractyloside caused a time-dependent decrease in luminescence from cells
microinjected with cytosolic luciferase (Fig. 3). By contrast, in cells
microinjected with cDNA encoding mitochondrial luciferase, an
anti-parallel increase in the luminescence of mitochondrial luciferase
was apparent.
Exposure to 30 mM glucose of MIN6 cells expressing mLuc
provoked a rapid, stable (for at least 10 min) increase in luminescence (Fig. 4, a and b).
Blockade of Ca2+ influx slowed the apparent glucose-induced
[ATP]m increase but had little or no effect on the final
extent of the [ATP]m change (Fig. 4b; Table
I). Furthermore, the effect of glucose
could be mimicked, in part, by an increase in intracellular
[Ca2+], provoked by exposure to high [K+]
(Fig. 5, a and b).
Unlike the stable luminescence increase observed in response to
elevated glucose, this K+-induced [ATP]m
increase was transient (Fig. 5a; Table I). Simultaneous addition of glucose and high [K+] caused a substantial
decreased the half-time for the glucose-induced [ATP]m
increase and a small increase in the maximum extent of the luminescence
change, compared with glucose alone (Table I). Confirming that changes
in [Ca2+]c or
[Ca2+]m were central to the effects of
K+, addition of K+ in the absence of external
Ca2+ resulted in a marked decrease in [ATP]m
(Fig. 5b, Table I).
To determine directly whether increases in intracellular
[Ca2+] activated mitochondrial oxidation, and the
contribution of this activation to the effect of glucose on
[ATP]m, we monitored on-line the mitochondrial membrane
potential,
Changes in pH gradient across the mitochondrial inner membrane were
assayed as the increase in fluorescence upon addition of nigericin,
which allows exchange of K+ for H+ across the
inner mitochondrial membrane (46). When added to naïve cells,
nigericin caused an increase in TMREE fluorescence of 10 ± 2%
(n = 7 cells, two separate experiments). Stimulation by
glucose and K+ appeared to diminish the size of the
fluorescence change caused by the subsequent addition of nigericin to
about half that observed in naïve cells. However, the relative
sizes of the fluorescence changes suggest that stimulation of
In the absence of extracellular Ca2+, addition of
K+ caused a decrease in TMREE fluorescence (Fig.
5d) that mirrored the decrease in [ATP]m
provoked by K+ in the absence of external Ca2+
(Fig. 5b). By contrast, stimulation with 30 mM
glucose in Ca2+-free medium gave an increase in
fluorescence, of 30 ± 3% (n = 13 cells, three
separate experiments) after 6 min, but the response was smaller and
slower than that in the presence of external Ca2+ (Fig.
4d). This pattern of dependence on extracellular
Ca2+ ions closely resembled that of [ATP]m
(see above, Figs. 4 and 5). Together, these data indicate that the
effect of glucose on [ATP]m may be mediated both by an
increase in substrate supply and through the activation by
Ca2+ of mitochondrial dehydrogenases and the respiratory chain.
In order to explore whether increases in intramitochondrial free
[Ca2+] could be directly responsible for the measured
elevations in intracellular free [ATP] in response to exposure to
high K+, we used the Ca2+-sensitive
photoprotein, recombinant aequorin, targeted to the mitochondrial
matrix (24, 27, 47, 48). Challenge of cells with K+
provoked a transient increase in mitochondrial [Ca2+], as
indicated by a rapid burst in aequorin-derived luminescence (data not
shown), which peaked 6 s after stimulation with K+.
Whether K+ depolarization led to any further, sustained,
increase in [Ca2+]m could not be resolved
conclusively with this approach, due to the limited sensitivity of
aequorin in single cells (24). However, studies on MIN6 populations
expressing mitochondrial aequorin (49) suggest that this may be the case.
Control of [ATP]c and [ATP]pm by
Glucose and Ca2+ Ions--
Exposure to elevated glucose
concentrations (30 mM) caused a marked increase in both
[ATP]c and [ATP]pm (Fig.
6, Table I). However, the kinetics of the
subsequent changes were distinct in these two cytosolic compartments
(Fig. 6; Table I). In particular, under conditions in which transient
increases in [ATP]c were elicited by the elevation of
glucose (Fig., 6), [ATP]pm was increased stably (Fig.
6d; Table I). In further contrast to the
[ATP]m changes, removal of extracellular Ca2+
completely blocked detectable glucose-induced increases in both compartments (Fig. 6, b and e; Table I).
Similarly, K+-addition provoked an increase in
[ATP]c, which recovered more quickly than
[ATP]m (compare Figs. 7 and
5 and see Table I). Depolarization of cells with high external
[K+] also caused an increase in [ATP]pm in
the sub-plasma membrane domain, which was closely similar in magnitude
to the change in [ATP]c, but of slightly longer duration
(Fig. 7; Table I). Simultaneous addition of K+ with glucose
had no significant effect on the maximum extent of the glucose-induced
increase in [ATP]c but instead slowed its relaxation to
prestimulatory values (Table I). By contrast, co-addition of
K+ with glucose enhanced the initial increase in
[ATP]pm but prompted a more rapid return to basal levels.
K+ addition at 3 mM glucose and in the absence
of extracellular Ca2+ caused a marked decrease in both
[ATP]c and [ATP]pm (Table I).
These clear changes in [ATP]c and [ATP]pm
were not associated with any statistically significant change in total
cellular ATP content after exposure to 70 mM
K+. Thus, the ATP content of unstimulated MIN6 cells was
1641 ± 415 pmol·100,000 cells Response of Primary Islet
As in MIN6 cells, increasing the concentration of glucose in the
medium from 3 to 30 mM caused a time-dependent
increase in luminescence, but here the increase was smaller and
more stable, reaching a statistically highly significant plateau
(p < 0.001 for the difference between 30 data points
acquired before and 4 min after increasing the glucose concentration to
30 mM). Thus, in one islet preparation in which five cells
were analyzed simultaneously, the maximum luminescence increase,
observed approximately 200 s after stimulation, was between 3.5 and 23.5% (Fig. 8a), indicative of
Exposure to high external K+ increased [ATP]c
in We describe here, for the first time, rapid dynamic imaging of
free intracellular ATP concentration in subcompartments of single
living cells. We show this to be feasible because the measured Km values for ATP of each of the expressed
recombinant luciferases were similar and in the low mM
range. These values are much higher than those reported for purified
firefly luciferase (e.g. 63 µM) (37, 51) and
although we have not analyzed in detail the reason(s) for this, altered
posttranslational modification of the enzyme seems possible. However,
it might also be noted that our assay conditions, unlike those of the
early in vitro studies, were designed to mimic the
intracellular ionic environment and involved appropriate pH values, the
presence of CoA, physiological salt concentrations, and high (1 mM) luciferin concentrations. However, our values are still
lower than those obtained recently by Maechler et al. (19)
who used an in vivo calibration approach. This involved the
exposure of Staphylococcus Resting [ATP] in Regulation of Mitochondrial ATP Synthesis by Glucose and
Ca2+ Ions--
In this work, we investigated first the
regulation by glucose of [ATP)m, because in this cell
type, mitochondrial metabolism is likely to contribute to > 90%
of all ATP synthesis (44, 55). Because resting [ATP]m was
close to the observed Km for ATP of firefly
luciferase, glucose-induced increases in luminescence reported somewhat
larger increases in [ATP]m (e.g. a 15%
increase in luminescence would correspond to a 35% increase in
[ATP]m). We considered first how provision of the
substrate, glucose, interacted with increases in intramitochondrial
[Ca2+], to enhance respiratory chain activity and hence
ATP synthesis. This is a complex problem because, according to the
accepted view of metabolism-secretion coupling in the
In apparent contrast to the present measurements of free [ATP] in
discrete subcellular domains, it has been reported that in the intact
islet, increases in total ATP content provoked by glucose were
potentiated by pharmacological blockade of Ca2+ influx (14,
66, 67). However, careful inspection of other studies (68) suggests
that elevation of intracellular [Ca2+] by K+
plus diazoxide has no effect on, or causes a small increase in, total
islet ATP content at high glucose concentrations. The differences between these earlier studies and the present work are likely to have
several causes. First, it should be stressed that there is no a
priori reason why total islet ATP content should directly represent free [ATP] in subdomains of the living
In the absence of external Ca2+, K+ stimulation
resulted in a parallel decrease in Regulation of [ATP]c--
In the Regulation of [ATP]pm--
In contrast to
[ATP]c, [ATP]pm remained elevated for up to
10 min after the beginning of incubation with 30 mM
glucose. Thus, glucose appears to provoke the formation of a
microdomain of [ATP] beneath the plasma membrane, which is regulated
differently from [ATP] in the rest of the cytosol. How might this be
achieved? As well as the activation of metabolism, increases in
[Ca2+]c are likely in the Oscillations in Intracellular [ATP] and Activated Insulin
Secretion--
Oscillatory release of insulin (82) may be essential to
ensure normal glycaemia in vivo (83), although the
mechanism(s) by which Conclusion--
We describe a new method for monitoring free ATP
concentrations dynamically and in subdomains of single living cells.
This technology should be applicable to the investigation of a wide variety of cell biological phenomena involving changes in intracellular [ATP] (84). With regard to the -cell may couple elevations in blood glucose to insulin
release by closing ATP-sensitive K+
(KATP) channels and activating Ca2+ influx.
Here, we use recombinant targeted luciferases and photon counting
imaging to monitor changes in free [ATP] in subdomains of single
living MIN6 and primary
-cells. Resting [ATP] in the cytosol
([ATP]c), in the mitochondrial matrix
([ATP]m), and beneath the plasma membrane
([ATP]pm) were similar (~1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased
free [ATP] in each domain with distinct kinetics. Thus, sustained
increases in [ATP]m and [ATP]pm were
observed, but only a transient increase in [ATP]c.
However, detectable increases in [ATP]c and
[ATP]pm, but not [ATP]m, required
extracellular Ca2+. Enhancement of glucose-induced
Ca2+ influx with high [K+] had little effect
on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these
changes, glucose increased the mitochondrial proton motive force, an
effect mimicked by high [K+]. These data support a model
in which glucose increases [ATP]m both through enhanced
substrate supply and by progressive
Ca2+-dependent activation of mitochondrial
enzymes. This may then lead to a privileged elevation of
[ATP]pm, which may be essential for the sustained closure
of KATP channels. Luciferase imaging would appear to be a
useful new tool for dynamic in vivo imaging of free ATP concentration.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells. This is probably achieved
by an increase in glycolysis and flux through the citrate cycle (1),
leading to elevated intracellular levels of likely coupling factors
(2), including ATP. Closure of ATP-sensitive K+ channels
(3-5) then leads to plasma membrane depolarization and the influx of
Ca2+ through voltage gated Ca2+ channels.
-cells (8).
Furthermore, such measurements give no indication of the concentration
of unbound ATP. Unfortunately, measurements of free [ATP] in living
cells, for example by 31P NMR (9), cannot easily be
extended to the islet micro-organ and do not provide sufficient
sensitivity to detect changes at the cellular or subcellular level.
This is an important question because differences in [ATP] at
different intracellular sites have been predicted. In particular,
locally high ATP consumption by the plasma membrane
Na+-K+ and Ca2+-ATPase, may mean
that [ATP] is lower in this domain than in the bulk of the cell
cytosol (1). Similarly, the electrogenic nature of the mitochondrial
ATP/ADP translocase (10) is predicted to create differences in ATP/ADP
ratio across the inner mitochondrial membrane (cytosolic high).
-cell metabolism and ATP
concentration is controversial. Increases in [Ca2+]
following plasma membrane depolarization act both to stimulate ATP
requiring processes (i.e. secretory granule movement and
exocytosis) (11) and possibly to enhance mitochondrial oxidative
metabolism (12, 13). Recent measurements of total ATP content of whole islets have suggested that the former may dominate and that
Ca2+ influx may diminish glucose-induced increases in
ATP/ADP ratio (14).
-cells. Unfortunately, such
measurements fail to take account of the likely heterogeneity in the
behavior of individual
-cells (21). Here, we use photon counting to
image ATP concentrations dynamically and at the subcellular level in
single living primary
-cells and derived MIN6 cells. We further
extend this technique to allow the imaging of [ATP] in two cellular
subdomains, the mitochondrial matrix and the subplasmallemal region, by
the molecular targeting of luciferase. We demonstrate that exposure to
elevated glucose concentrations causes increases in [ATP] in each of
the compartments analyzed, coincident with an increase in mitochondrial membrane potential. Comparison of the kinetics of [ATP] changes, and
dependence on increases in mitochondrial free [Ca2+]
([Ca2+]m),1
also suggests that activation of strategically located mitochondria may
preferentially enhance [ATP] immediately beneath the plasma membrane.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells were isolated by collagenase (PanPlus, Serva) digestion and
purified on a discontinuous bovine serum albumin gradient (16). Cells
were dissociated with trypsin before 24 h culture on
Cell-TakTM-treated glass coverslips. Plasmids (0.2-0.4
mg·ml
1 in 10 mM Tris, 0.2 mM
EDTA) were microinjected using glass borosilicate capillaries and an
Eppendorf 5171 transjector/micromanipulator, as described (16, 24).
MIN6 cells were cultured on poly-L-lysine-treated coverslips (25). Injected primary
-cells cells were cultured 16-24
h in an atmosphere 5% CO2, and at 3 mM
glucose. For all experiments in which the effects of elevated glucose
or K+ concentrations were tested, MIN6 cells were cultured
at 3 mM glucose for 24 h before imaging. Such cells
responded robustly to elevated glucose concentrations with enhanced
insulin secretion (4-10-fold above basal levels) (26). Cells were
imaged in modified Krebs-Ringer bicarbonate medium (0.2 ml) comprising
125 mM NaCl; 3.5 mM KCl; 1.5 mM
CaCl2; 0.5 mM MgSO4; 0.5 mM KH2PO4; 2.5 mM NaHCO3, 10 mM Hepes-Na+, pH 7.4, containing the indicated glucose concentration and equilibrated with
95:5 O2:CO2. Cells were maintained on the
temperature-controlled (37 °C) stage of an Olympus IX-70 microscope
(UPlanApo × 10, 0.4 numerical aperture air objective), located in
a sealed dark housing. Medium was rapidly (<2 s) changed by the
addition of an equal volume through a remotely located syringe. For
calibration of signals, cells were lysed in "intracellular medium"
comprising 20 mM Hepes, 140 mM KCl, 5 mM NaCl, 10.2 mM EGTA, 6.67 mM
CaCl2, 1 mM luciferin, 20 µg·ml
1 digitonin plus additions of ATP,
MgSO4, and CoA as indicated. Data were captured with an
intensified charge-coupled device camera comprising a low-noise S-20
multi-alkali photocathode and three in-series microchannel plates
(Photek ICCD216; Photek Ltd., St. Leonards-on-Sea, East Sussex, United
Kingdom), maintained at 4 °C. Single photon events were captured at
25-ms intervals by time-resolved imaging, which allowed the spatial and
temporal coordinates of each photon event to be held in matrix format.
In this way, luminescence changes of any individual cell or group of
cells could be analyzed for the entire time course of an individual
experiment. When required, images corresponding to the selected
area of interest within the image field were generated
retrospectively over the desired integration period. Aequorin
imaging was performed in cells expressing mitochondrially targeted
aequorin (27) as described previously (24).
1 on the stage of a Nikon Diaphot microscope equipped
with a × 40 oil immersion objective. The ratio of the emitted
light at two excitation wave lengths (440/490 nm) was used to monitor
intracellular pH, using commercially available software (Cairn
Instruments, Faversham, Kent, United Kingdom) for data acquisition.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Targeting strategies for firefly
luciferase. a, cLuc; b, mLuc; c,
pmLuc. Cytosolic luciferase (lacking the carboxyl-terminal Ser-Lys-Leu
peroxisomal targeting motif (38) and engineered for optimal codon usage
in mammalian cells) provided the basis for cLuc and pmLuc, and
wild-type luciferase provided the basis for mLuc. MIN6 cells were
microinjected and cultured 24 h before fixation, permeabilization,
and immunocytochemical analysis (see under "Materials and
Methods"). Individual optical slices (0.5 µm) are shown.
Black bar represents 100 nucleotides.
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Fig. 2.
Dependence of luciferase activity on [ATP]
in vivo. a-c, photon production by 6-30 single
living MIN6 cells was measured by photon counting digital imaging (see
under "Materials and Methods") in the Krebs-Ringer bicarbonate
medium containing 1 mM luciferin. Cells were imaged 24 h after microinjection with cLuc (a), mLuc (b),
or pmLuc (c). After establishment of a steady state of
photon production, the camera was switched off (resulting in the
observed decrease in photon detection rate), and cells were
permeabilized by the replacement of the medium with intracellular
medium containing 20 µg·ml 1 digitonin plus 10 mM ATP and 9-10.5 mM MgCl2.
Broken traces correspond to data integration over 2 s,
and solid lines correspond to a rolling average of between 5 (a and b) and 15 (c) individual
points. Permeabilization in the absence of added ATP caused a
time-dependent decrease in single cell luminescence to <30
photon·s
1 over the whole field, close to the observed
dark count (in the absence of cells) for this camera. These data were
essentially unaffected by the addition of ADP over the likely range of
free intracellular concentration, 0.1-1.0 mM. At all ATP
concentrations tested, luminescence was absolutely dependent upon the
presence of Mg2+ ions consistent with MgATP as the true
substrate of the luciferase reaction. At the free [Mg2+]
used in these studies (0.5 mM), approximately 90% of ATP
is in the MgATP2
form, such that these
Km values closely represent the
Km for MgATP2
. Horizontal broken
lines indicate the pre- and post-ATP addition steady state levels
from which the intracellular ATP concentrations were obtained.
Bars indicate 120 s. Kinetic analyses were performed
using soluble extracts of cells transfected with cLuc (a),
mLuc (b), or pmLuc (c). Light output was measured
using a photon counting luminometer in intracellular medium (see under
"Materials and Methods") supplemented with 0.01 mM CoA
at pH 7.2 (cLuc and pmLuc) or pH 7.8 (mLuc). MgCl2 was
added in the range 1.5-10 mM to provide a constant free
Mg2+ concentration of 0.5 mM, calculated using
METLIG software (85). Light output was integrated over 30 s.
Calculated Km values (mM) for ATP were
obtained by nonlinear regression analysis (FigP, Cambridge Biosoft,
Cambridge United Kingdom) assuming Michaelis-Menten kinetics, using ATP
concentrations in the range 0.1-10 mM. Combining data from
three entirely separate luciferase preparations gave
Km values (in mM) of: cLuc, 1.18 ± 0.45; mLuc, 1.67 ± 0.61; and pmLuc, 0.65 ± 0.13. No
statistically significant difference (see under "Materials and
Methods") was revealed between the Km values for
luciferase expressed in different compartments. Assuming these
constants, the above experiment gave values for [ATP]c,
[ATP]m, and [ATP]pm of 1.0, 1.2, and 0.9 mM, respectively.
-cells (see below,
Fig. 8). As observed previously (40) this concentration of luciferin
was close to saturating for photon production by single cells, with
little further increase in luminescence observed in the presence of 2 mM luciferin or above (data not shown). Ouabain (100 µM) caused a small increase in luminescence from cells
expressing cLuc (5 ± 1%, n = 16 cells) but a
more substantial increase in luminescence from cells expressing pmLuc
(14 ± 1.5%, n = 15 cells) and mLuc (11.5 ± 1.5%, n = 16), as expected by the relief of ATP
consumption by the plasma membrane Na+/K+-ATPase. It should be noted that these
and smaller changes could readily be detected. Indeed, changes in
luminescence in populations of 8-12 cells of as little as 3% were
found to be statistically significant when integrated over a 40-s
interval (results not shown).
1, and a cell
volume of 2 pl) to 7.2 pmol/µg DNA. In our hands, a 500% increase in
[CoA], from 10 to 50 µM, was required to increase luciferase luminescence in cell extracts by 28%. It should be noted
that an increase in intracellular concentration of O2,
another key luciferase substrate, is unlikely in response to elevated extracellular glucose concentrations, because these usually provoke increases in O2 consumption (44) and an increased
intracellular NAD(P)H/NAD(P)+ ratio (see below). Finally,
no changes in the luminescence were apparent in cells transfected with
the non-ATP-utilizing luciferase from the sea pansy, Renilla
reniformis (16) (data not shown).
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Fig. 3.
Effect of atractyloside on luminescence from
populations of single MIN6 cells expressing mLuc (a)
or cLuc (b). Populations of cells expressing
luciferase in the mitochondrial matrix (a) (15 cells) or
cytosol (b) (18 cells) were imaged in the presence of 3 mM glucose. Atractyloside (50 µM) was added
at the upward arrow. Panels show the luminescence of single
cells during a 20-s integration beginning at the position of the
downward arrowheads. Bars beneath the panels
correspond to 15 µm, and the time bars correspond to
120 s. Pseudocoloring is as follows: black, 0;
blue, 1-5; green, 6-7; yellow, 8-9;
red, 10-12; and white, 13
photon·s
1·pixel
1.
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Fig. 4.
Control of [ATP]m and
by glucose. In a,
luminescence was measured from a single cell (left) or a
population of 51 single cells (right) expressing mLuc,
whereas TMREE fluorescence (c and d) was
monitored as described under "Materials and Methods." Dashed traces
in c and d correspond to changes observed in the
absence of additions. Where indicated, [glucose] was increased to 30 mM (glc), whereas nigericin (2 µM)
(nig) was added as indicated to TMREE-loaded cells. Data
shown in traces b and d were from populations of
16 and 13 single cells, respectively, maintained in the absence of
extracellular Ca2+ (medium supplemented with 1 mM EGTA). Images of single cells (inserts in
a and b) correspond to 30-s integrations of
luminescence at the downward arrowheads shown. Scales and
pseudocoloring are as in Fig. 3. Dashed lines in
c and d show fluorescence changes observed in
control, unstimulated cells.
Mean changes in [ATP]-dependent luminescence in the
cytosol, mitochondrial matrix, and sub-plasma membrane region of
single - and MIN6 cells
) were
calculated by direct examination of rolling average data
(e.g. see Fig. 3). The data shown correspond to means ± S.E. for the number of single cells and separate cultures given in
parentheses. N.A., not applicable. Changes in luminescence represent
increases, except for those marked (
).
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Fig. 5.
Control of [ATP]m by external
K+. Measurements were performed essentially as
described in Fig. 4, but with additions of 70 mM KCl
(K+) in place of additional glucose. Cell populations
comprised 25 (a), 15 (b), 21 (c), or
15 (d) single cells.
, after loading the cells with a dye, TMREE (Figs. 4,
c and d, and 5, c and d),
which rapidly equilibrates across the inner mitochondrial membrane
according to
(45). Both elevated glucose and K+
concentrations provoked time-dependent increases in
mitochondrial fluorescence of cells loaded with TMREE. Thus,
K+ stimulation provoked a rapid, 33 ± 3% increase
(n = 31 cells, 6 separate experiments) in
, that
peaked after ~90 s (Fig. 5). By contrast, stimulation by glucose
alone gave a slower, but more substantial, increase in fluorescence
equal to 39 ± 6% (n = 16 cells, three separate
experiments) after 6 min (Fig. 4). This glucose-induced increase in
thus occurred more slowly than [ATP]m (Fig. 4,
c versus a). Similar to the effect on
[ATP]m, simultaneous addition of high [K+]
with glucose provoked a peak response similar to that observed with
K+ alone, but then led to a time-dependent
decrease in
, which returned to prestimulatory values at extended
time periods (> 300 s).
by
K+ and glucose outweigh any change in pH gradient, so that
both stimuli caused a net increase in mitochondrial proton motive force (Figs. 4 and 5).
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Fig. 6.
Control of [ATP]c and
[ATP]pm by glucose. Measurements of luminescence
were performed as described in Fig. 4, using cells expressing cLuc
(a-c) or pmLuc (d-f) as shown. c and
f show the effects of control additions of medium (without
30 glucose). Pseudocolors and scale bar are as in Fig. 3.
Experiments involved populations of 51 (a), 18 (b), 11 (c), and 8 (d) cells.
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Fig. 7.
Control of [ATP]c and
[ATP]pm by K+. Measurements of
luminescence were performed as described in Fig. 5, using cells
expressing cLuc (a and b) or pmLuc (c
and d) as shown. Pseudocolors and scale bar are
as in Fig. 3. Experiments involved populations of 21 (a), 16 (b), 28 (c), and 6 (d) cells.
1, compared with
1514 ± 372, 60 s after exposure to 70 mM
K+ (p = 0.08 with respect to unstimulated
cells by paired Student's t test), and 1644 ± 371 pmol·100,000 cells
1 120 s after K+
stimulation (p = 0.49; n = 9 separate
experiments, each comprising two MIN6 cultures).
-cells to Increases in Extracellular
Glucose Concentration and Ca2+ Influx--
We next
determined whether the above observations, performed on a derived
-cell line, could be extended to primary living
-cells from adult
animals. In primary, as opposed to clonal cells, it has been suggested
that responses to increasing glucose concentration occur on an
"all-or-nothing" basis, with increasing recruitment of individual
cells with elevated [glucose] (50). Fig.
8 shows the changes in luminescence of
-cells, microinjected with plasmid cLuc and exposed 24 h later
to elevated glucose concentrations or high (70 mM)
K+. Typical increases of the luminescence of single cells
are represented in the pseudocolor images presented in Fig. 8.
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Fig. 8.
Glucose- and K+-induced changes
in [ATP]c in primary living
-cells. Single primary
-cells or cell
populations of 5-14 single
-cells were injected with plasmid cLuc
and incubated for 10 min in 3 mM glucose before stimulation
with 30 mM glucose (a), 70 mM
K+ (b), of 30 mM glucose plus 70 mM K+ (c) (upward
arrows). Photon counting imaging was performed as described under
"Materials and Methods," and the scale bars and
pseudocoloring are as in Fig. 3. Images of single cells (a-c,
insets) correspond to photon production during 30-s integration
beginning at the time points indicated by the left and
right downward arrows.
-cell
heterogeneity (21).
-cells highly reproducibly (Fig. 8b; Table I).
Furthermore, simultaneous addition of high glucose and high
K+ provided a luminescence increase that was larger and
more rapid than the response to glucose alone (Fig. 8; Table
I).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-toxin-permeabilized INS-1
cells to consecutive increases in [ATP]. However, permeabilization approaches are fraught with difficulties arising from the gradual leakage of luciferase, which can occur at very different rates for
different individual cells. These authors (19), who made no adjustment
for intracellular [Mg2+] or [CoA], obtained an apparent
Km for ATP of luciferase in excess of 10 mM. Whether our own values with extracted luciferase or
those of Maechler et al. (19) provide the closest estimate of the true in vivo Km of luciferase for
ATP must be a matter of conjecture. Consequently, caution needs to be
exercised when using in vitro determined
Km values for precise estimates of intracellular
[ATP].
-Cell Subcompartments--
Our data suggest
that the concentration of [ATP] (predominantly as MgATP) is similar
in three discrete domains of living islet
-cells, prior to
elevations in extracellular glucose concentration, at around 1.0 mM. These values for cytosolic [ATP], which are lower
than those of around 8 mM obtained by Maechler et
al. (19), highlight a number of important features of
-cell
metabolism and glucose sensing. First, given the electrogenic nature of
the mitochondrial adenine nucleotide translocase (10), the similarity of [ATP]m and [ATP]c suggests that the free
ADP concentration of mitochondria is likely to be considerably higher
than in the bulk cytosol. Second, the data presented do not support the
prediction that [ATP]pm may be substantially lower than
[ATP]c in the resting
-cell, despite the requirement
for active ion pumping, as demonstrated by the apparent increase in
[ATP]pm in response to ouabain. This finding is congruent
with data recently obtained by electrophysiological approaches in
Xenopus oocytes (52). It therefore follows that inhibition
of the KATP channel by ATP must involve changes in the
concentration of this nucleotide in the mM range. Because the sensitivity of this channel to ATP is in the low µM
range in inside-out patches (3), this might imply either that its ATP
sensitivity is dramatically altered in vivo or that other factors lead to a decrease in its open-state probability at high glucose concentrations. Very recent data (53, 54) suggest that
phosphatidyl inositol-4,5-bisphosphate is a likely candidate molecule.
-cell, some
enhancement of ATP synthesis must occur in order to close
KATP channels and hence increase
[Ca2+]c and
[Ca2+]m. This should then enhance NADH
production by mitochondrial dehydrogenases and hence substrate supply
to the respiratory chain (12, 13, 56) and possibly the glycerol
phosphate shuttle (55, 57-60). Furthermore, increases in
[Ca2+]m may enhance respiratory chain
capacity directly (61, 62). Consistent with this model, increases in
[Ca2+]c have previously been shown to provoke
elevations in the NAD(P)H/NAD+ couple in
-cells
(63)2 and other cell types
(64, 65). Our observations suggest that an increase in
[Ca2+]m in response to elevated glucose
further enhances the increase in mitochondrial proton motive force
provoked by elevated substrate supply alone. Indeed, when
[Ca2+]m was increased with no change in
extracellular glucose (stimulation with K+ the presence of
extracellular Ca2+, Fig. 5) we observed a parallel increase
in both
, and [ATP]m. These data imply a permissive
role for Ca2+ ions in glucose-induced free [ATP]
increases. Lending further support to this view, high
[K+] accelerated but did not substantially affect the
maximum amplitude of glucose-induced increases in [ATP]c
and [ATP]m in MIN6 cells, whereas it both accelerated and
amplified the [ATP]pm change (Table I). Similarly, in
primary
-cells, simultaneous addition of K+
substantially increased the magnitude of the [ATP]c
response to high glucose (Table I). However, in MIN6 cells, longer
incubations (>400 s) with K+ plus glucose led to a small
decrease in [ATP]pm compared with values in the presence
of glucose alone (results not shown), consistent with accelerated
Ca2+ pumping in the sub-plasma membrane region and a
gradual decline in mitochondrial proton motive force.
-cell, given the
significant compartmentalization of ATP (for example, within an inert
secretory granule pool) (69, 70), and the cellular heterogeneity of the
islet (21). Second, when monitored dynamically in the present study,
the effects of elevated K+ on the response to glucose of
[ATP] in each compartment were distinct and, in the case of
[ATP]pm, time-dependent. Finally, exocytosis
from intact islets (or upstream steps such as vesicle mobilization)
(71) may be more vigorous than in either isolated
-cells or the
transformed MIN6 cell line. This would provide a greater ATP drain
relative to ATP synthesis.
, and [ATP]m, as
well as [ATP]pm, presumably due to increased consumption
of ATP by plasma membrane Na+/K+ and other
ATPases. It follows, therefore, that the increases in
[ATP]m prompted by K+ in the presence of
external Ca2+ are smaller than the overall increase in the
rate of ATP synthesis, due to enhanced ATP consumption. However, an
important exception to the close correlation between
[ATP]m and
, occurs during glucose stimulation in
the presence of external Ca2+. Here, increases in
lag behind those of [ATP]m (Fig. 4). One possible
explanation is that in these circumstances, a gradually increasing rate
of increasing ATP synthesis is matched by increasing ATP consumption.
-cell,
[ATP]c changes are likely to occur secondarily to
[ATP]m increases. Intriguingly, the responses to
extracellular glucose of [ATP]c and [ATP]m
were found to differ in two important respects. First, elevated glucose
provoked only a transient increase in [ATP]c compared the
sustained increase seen in [ATP]m. Second, [ATP]c but not [ATP]m increases were
dependent upon increases in [Ca2+]c. These
observations suggest that activation of mitochondrial ATP/ADP exchange
may require elevations in intra- or extramitochondrial [Ca2+]. It might be noted in this context that
Ca2+ may increase dramatically the activity of the ADP/ATP
translocase (72), a phenomenon possibly involved in the creation of
nonspecific pores in the mitochondrial membrane during hypoxia and
other cellular stresses (the "mitochondrial permeability
transition") (73). The current data suggest that changes in
intramitochondrial or cytosolic [Ca2+] might also have a
role in the regulation of ATP/ADP translocase activity under
nonpathological conditions. However, this would only be apparent when
cytosolic [Ca2+] is increased, consistent with the fact
that removal of extracellular Ca2+, which is likely to
cause a small decrease in resting intracellular [Ca2+],
was without effect on [ATP]c or [ATP]m.
-cell to
profoundly increase ATP usage for processes such as secretory granule
mobilization (71), exocytosis (74), extrusion of Ca2+
across the plasma membrane (75) and into intracellular stores (76), and
activation of gene expression (77). Here, we provide evidence that
during these events there may be preferential delivery of mitochondrial
ATP to the sub-plasma membrane domain. Thus, the time course of changes
in [ATP]pm following increases in extracellular glucose
matched closely those of [ATP]m. One explanation of the sustained elevation of [ATP]pm may therefore be that
mitochondria at the cell periphery are exposed to more sustained
[Ca2+]m increases, as a result of
glucose-activated Ca2+ influx, than those deeper within the
cell. This would tend to prolong the activation of ATP synthesis in
peripherally located mitochondria. Consistent with this, the return of
[ATP]c to basal levels after K+ stimulation
was more rapid than that of [ATP]m or
[ATP]pm. Therefore, mitochondria located beneath the
plasma membrane may provide a privileged domain of cytosolic [ATP],
confined to this region of the cell. Such a role for strategically
arranged mitochondria may also provide an explanation for the
activation of exocytosis by the addition of certain mitochondrial
substrates to permeabilized cells, despite the presence of high added
MgATP concentrations (78). Alternatively, it is also conceivable that
there are differences in the time course for the activation of
ATP-consuming events at the plasma membrane (fusion of secretory
vesicles or ion pumping), compared with those within the cytosol.
Intriguingly, control of [ATP]pm in the
-cell would
appear to be distinct from that in heart cells, where ATP derived from
glycolysis appears preferentially to inhibit KATP channels
(79, 80). On the other hand, recent data (81) suggest that mitochondria
may play an important part in generating ATP microdomains close to the
endoplasmic reticulum Ca2+-ATPase of BHK-21 cells.
-cells release insulin in an oscillatory
fashion is not well understood (7). We occasionally observed
oscillations in [ATP]pm, although such oscillations were
rare (data not shown). An important question is therefore whether
oscillations in [ATP]pm and
[Ca2+]pm are causally linked.
-cell, perhaps the simplest model
to explain our own observations is that an initial, small (Ca2+-independent) increase in [ATP]m, and
subsequently in [ATP]pm, may begin to close
KATP channels, causing Ca2+ influx. This may
then begin a "feed-forward" process, involving the further
activation by Ca2+ of mitochondrial ATP synthesis and hence
further closure of KATP channels (Fig.
9).
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Fig. 9.
Scheme illustrating how
peripherally localized mitochondria may participate in a
glucose-activated feed-forward loop, generating locally high
concentrations of ATP and Ca2+ beneath the plasma membrane,
thus prompting exocytosis.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. J. I. Miyazaki for the
provision of MIN6 -cells. We also thank the Medical Research Council
for providing an Infrastructure Award to establish the Bristol School
of Medical Sciences Cell Imaging Facility, Profs. R. M. Denton and
A. P. Halestrap for useful discussion, and Sara Ellis and Eleanor
Basham for technical assistance.
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FOOTNOTES |
---|
* We thank for financial support the Wellcome Trust, the Medical Research Council (United Kingdom), the Royal Society, the British Diabetic Association, the Christine Wheeler Bequest, Italian "Telethon" (project no. 850), the "Biomed" program of the European Union, and the Italian University Ministry (to R. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A Bristol University Research Scholar.
¶ To whom correspondence should be addressed. Tel.: 44-117-928-9724; Fax: 44-117-928-8274; E-mail: g.a.rutter{at}bris.ac.uk.
2 E. K. Ainscow and G. A. Rutter, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
[Ca2+]m, [Ca2+]c,
and [Ca2+]pm, free Ca2+
concentration in the mitochondrial matrix, cytosol, and plasma membrane
regions, respectively;
cLuc, cytosolic firefly luciferase;
mLuc, mitochondrial matrix firefly luciferase;
pmLuc, plasma membrane firefly
luciferase;
, mitochondrial membrane potential, TMREE,
tetramethylrhodamine ethyl ester.
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REFERENCES |
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