Evidence for the optical signalling of changes in bicarbonate concentration within the mitochondrial region of living cells

Article abstract of DOI:10.1039/c1cc11853f

Image and spectral intensity from bicarbonate-selective europium(iii) probes localised in the mitochondria of cells is modulated reversibly by variation of external pCO₂, and is suppressed by addition of the carbonic anhydrase inhibitor, acetazolomide.

Full text of DOI:10.1039/c1cc11853f

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COMMUNICATION
Evidence for the optical signalling of changes in bicarbonate
concentration within the mitochondrial region of living cellsw
a
a
a
a
a
David G. Smith, Ga-lai Law, Benjamin S. Murray, Robert Pal, David Parker* and
ab
Ka-Leung Wong
Received 1st April 2011, Accepted 4th May 2011
DOI: 10.1039/c1cc11853f
9
,12
Image and spectral intensity from bicarbonate-selective
europium(III) probes localised in the mitochondria of cells is modu-
mixtures of Eu and Tb complexes of a common ligand.
Initial work with bicarbonate-responsive europium complexes
could not be adapted for intracellular imaging, as the probes
were only localised to the rather acidic endosomes and
2
lated reversibly by variation of external pCO , and is suppressed by
addition of the carbonic anhydrase inhibitor, acetazolomide.
1
0
lysosomes. More recently, a key structural motif in certain
emissive complexes was identified that led to a predominant
A challenging objective in cellular imaging is to define optical
1,2
3,4
1 3+
probes that can target a cell organelle and signal concentration
staining of the cellular mitochondria. Thus, in [EuꢀL ]
it is the amide-linked azaxanthone sensitising moiety that
promotes uptake of the probe into the cell by macropinocytosis
3
,4
changes of a key biochemical species in real time. Progress
has been made with pH and calcium-sensitive probes, for
example using pH-responsive fluorophores localised in the
11
and determines its subsequent trafficking to the mitochondria.
2
,5
more acidic lysosomal compartments. Here, we report systems
that serve to report changes in the local concentration of
bicarbonate in the mitochondrial region of various cell types.
The production of mitochondrial carbon dioxide is a direct
consequence of carbon flux through glycolysis and the citric
acid (Krebs) cycle. A method is required to monitor this
change in real time, using bicarbonate-selective optical probes.
This could offer unprecedented insight into signalling mechanisms
This complex was non-toxic to several cell types (IC50 4 200 mM),
and 412h post-incubation was observed by microscopy to
migrate to the perinuclear lysosomes. With this background
1
1
2
3+
in mind, we have prepared the analogues [LnꢀL ]
and
3
[LnꢀL ], (Ln = Eu, Tb), evaluated their affinity for bioactive
oxy-anions and protein and studied the changes in emitted
light by microscopy, as a function of pCO
are localised to the mitochondria.
2
, whilst the probes
6
that regulate or are regulated through this variation. For
example, changes in bicarbonate concentration stimulate
the activity of the enzyme adenylyl cyclase that produces
cyclic-AMP. Bicarbonate levels, in turn, are regulated by the
mitochondrial carbonic anhydrase enzymes. The Krebs cycle
2
generates local CO , so that soluble adenylyl cyclase has been
postulated as a regulator of oxidative phosphorylation,
allowing respiration to keep pace with changes in nutritional
6
availability.
In recent work, we have developed families of emissive Eu
and Tb complexes that report on changes in the chemical
composition of the environment in various bio-fluids, through
2
3
1
The ligands L and L are analogues of the triamide, L ,
reported earlier, and were prepared similarly (ESI). In their Eu
and Tb complexes they form a short series of differing hydro-
phobicity and steric demand. Such features were expected to
modulate anion and protein affinity, allowing an in vitro study
of the salient competitive equilibria between anions and a
model protein, serum albumin (HSA). Changes in lanthanide
emission were monitored as a function of added bicarbonate,
HSA, citrate, lactate, phosphate and in certain cases, ATP.
Distinctive limiting europium(III) emission spectra were
observed in each case. For each Eu complex, the bicarbonate
adduct was characterised by a DJ = 2/DJ = 1 intensity ratio
of 44 : 1, compared to 2 : 1 in the aqua adduct. With
3
a,4
modulation of spectral form, polarisation or lifetime.
7
Systems designed to report changes in pH, citrate and
8
lactate, urate as well as bicarbonate
9
10,11
have been devised.
Each system allows a ratiometric analysis, by comparing
band intensities either within a europium spectrum or using
a
Department of Chemistry, Durham University, South Road, Durham,
UK DH1 3LE. E-mail: david.parker@dur.ac.uk
Department of Chemistry, Hong Kong Baptist University, Kowloon
Tong, Hong Kong
b
w Electronic supplementary information (ESI) available: Cell localisation
2
3+
images; synthesis and characterisation of [LnꢀL (H
2
O)
2
]
], spectral titrations of lanthanide(III) complexes with
and
3
[
selected oxy-anions. See DOI: 10.1039/c1cc11853f
LnꢀL (H
2
O)
2
1
3+
[EuꢀL ] , lactate binding was distinguished by the emergence
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Chem. Commun., 2011, 47, 7347–7349 7347

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Table 1 Affinity constants of lanthanide(III) complexes for selected
species (298 K, I = 0.1 M NaCl, pH as stated)
citrate or lactate concentration. The terbium systems offer a
calibration role, as changes caused by variation of anion
concentration in the presence of protein were much smaller.
Incubation of each lanthanide complex (20–100 mM, 1 to
a
3ꢁ
42ꢁ
HCO
pH 7.4
HPO
pH 7.4
Lactate
pH 6
Citrate
pH 7.4
HSA
pH 7.4
Complex
4
h) led to selective staining of the mitochondrial region of
1
3+
[
[
[
[
[
[
EuꢀL ]
3.85(04)
2.90(03)
3.62(07)
3.16(03)
2.97(04)
2.85(07)
2.76(06)
2.95(03)
2.65(01)
2.91(01)
2.81(01)
3.35(07)
3.40(05)
3.61(07)
3.72(01)
2.67(01)
3.53(02)
3.59(04)
4.70(03)
5.21(12)
3.17(04)
3.61(07)
nd
1
3+
3+
3+
CHO, NIH-3T3, A549, MCF-7 or HeLa cells, confirmed by
TbꢀL ]
2
b
EuꢀL ]
co-localisation studies with Mitotracker Greent. The percen-
2
b
TbꢀL ]
nd
3.84(04)
nd
3.49(02)
nd
3.59(01)
2
tage of CO in the incubation chamber was lowered from 5%
3
b
EuꢀL ]
3
b
b
to 4% and 3%, and then stepped back to 5%. After each
change, a period of 30 min elapsed before images were
acquired by microscopy (lexc 360 nm). Incubations of
[EuꢀL ] and [EuꢀL ] (20 or 50 mM) each gave rise to an
observed image intensity (using a 550–700 nm band-pass filter
to select Eu emission) that increased proportionately with
TbꢀL ]
nd
a
The pH regime was selected in preliminary experiments studying the
pH dependence of the emission profile at a fixed anion concentration,
b
seeking a flat region of the spectral response/pH profile. Value not
1
3+
2 3+
determined as intensity decrease occurred without sufficient spectral
modulation to allow data fitting.
the % CO and changed reversibly (Fig. 1). Hyper-spectral
2
imaging of the mitochondrial region of NIH-3T3 or HeLa cells
allowed a quantification of this increase and suggested that
there was no major change in the Eu emission spectral form,
notwithstanding the modest spectral resolution observed.
of a shoulder at 624 nm and the citrate adduct showed a 70%
increase in intensity in the DJ = 2 band intensity vs. the DJ =
2
3+
1
manifold. In the lactate and citrate adducts of [EuꢀL ] and
3
[
EuꢀL ], much smaller changes in spectral form were observed,
Parallel experiments examining pCO sensitivity with the Tb
2
consistent with a different constitution in the ternary adduct.
A tentative assignment places the hydroxyl group, rather than
the more polarisable carboxylate, in the capping axial site of
the mono-capped square anti-prismatic polyhedron. The
changes in the relative intensity of the hypersensitive DJ = 2
transitions (around 616 nm) versus the magnetic-dipole
allowed, DJ = 1 transitions (around 592 nm) in most cases
allowed plots of DJ = 2/DJ = 1 intensity ratios to be used to
estimate apparent binding constants (Table 1).
analogues caused less than a 10% variation in observed image
and Tb spectral emission intensity, summing data from pixels
defining the stained mitochondrial region. Incubation of
2
3+
[EuꢀL ]
(20 or 50 mM) in A549 cells in the presence of
1 or 10 mM acetazolomide (a cell permeable non-specific
inhibitor of carbonic anhydrase activity) stepping from
1
4
3% to 5% CO both lowered the overall emission intensity
2
and suppressed the observed Eu emission intensity change; less
than a 10% change was observed on cycling from 5 to 3% and
then back to 5% CO2.
Addition of serum albumin to each europium complex
(
20 mM) led to a large reduction in emission intensity accompanied
The microscopy experiments suggest that the observed
increase of Eu emission intensity with pCO2 in the mito-
chondrial region of 3T3 or HeLa cells may be attributed to
an increase in the steady-state bicarbonate concentration.
by a change in spectral form. Measurements of complex
hydration state in the free and ‘bound’ forms were consistent
with displacement of coordinated water molecules (ESI). The
large reduction in emission can be ascribed to quenching of the
sensitiser triplet state by a charge transfer process from
9
b,13
electron rich residues (e.g. Tyr) in the protein.
Separate
addition of Tyr or O-phospho-Tyr caused a similar quenching
effect. Terbium emission intensity also was reduced on binding
HSA but to a lesser extent; similar apparent binding constants
to those found with the Eu analogues characterised this
interaction (Table 1).
In a fixed background of 0.4 mM serum albumin and
2
0 mM bicarbonate, the effect of added anions on observed
lanthanide emission was examined. For example, with
2
3+
EuꢀL ] addition of HPO4 , ATP, citrate or lactate (up to a
2ꢁ
[
2
mM limit) led to reductions in emission intensity of o5% for
phosphate and lactate, and 85 and 62% for ATP and citrate
respectively. On the other hand, in the presence of 0.4 mM
protein only, adding 5 to 25 mM bicarbonate to a solution of
1
3+
2 3+
3
[
EuꢀL ] , [EuꢀL ]
or [EuꢀL ] caused a 250% increase in
emission intensity and up to a 25% increase in the DJ = 2/DJ = 1
emission intensity ratio, consistent with partial displacement
of the bound protein by the added bicarbonate. With the
terbium analogues, the corresponding changes in intensity
were generally r20%. Taken together, these competition
experiments suggest that binding of bicarbonate and protein
to the Eu centre is competitive in this series, and is not
perturbed significantly by changes in phosphate, ATP,
Fig. 1 upper: Confocal microscopy images of HeLa cells, showing the
1 3+
mitochondrial region stained by [EuꢀL ]
under 3, 4 and 5% CO
2
(1 h incubation, 20 mM [complex], 30 min equilibration period between
images). lower: Variation of lanthanide emission intensity from
hyper-spectral analysis of microscopy images for HeLa cells stained
1
3+
.
with [EuꢀL ]
7
348 Chem. Commun., 2011, 47, 7347–7349
This journal is c The Royal Society of Chemistry 2011

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corresponds well to the normal bicarbonate range in biological
systems of 10 to 30 mM.
In summary, measurements of Eu/Tb emission intensity ratios
report changes in bicarbonate concentrations both in vitro and
in cellulo. As the probe localises to the mitochondrial region,
changes in the steady-state bicarbonate concentration may now
be able to be monitored by microscopy, as a function of
perturbation to cell homeostasis.
We thank the Royal Society (KLW), the ERC (FCC:
2
66804) and EPSRC for support (DGS).
Notes and references
1
2
V. P. Torchillin, Annu. Rev. Biochem., 2006, 8, 343.
M. H. Kim and B. R. Cho, Acc. Chem. Res., 2009, 42, 863.
Fig. 2 Calibration curve showing the ratio of the overall DJ = 2 Eu
emission band intensity (centred at 616 nm) to the DJ = ꢁ1 Tb
3 (a) C. P. Montgomery, E. J. New, R. Pal and D. Parker,
Acc. Chem. Res., 2009, 42, 925; (b) E. J. New, A. Congreve and
D. Parker, Chem. Sci., 2010, 1, 111.
4 E. J. New, D. Parker, D. G. Smith and J. W. Walton, Curr. Opin.
Chem. Biol., 2010, 14, 238; D. Parker, Aust. J. Chem., 2011, 64,
1
3+
emission band intensity (centred at 545 nm) for [LnꢀL ]
(3 : 2
mixture of complexes; 0.4 mM HSA, 0.1 M NaCl, pH 7.4, 1 mM
2
ꢁ
, 1 mM ATP, 2 mM lactate, 0.13 mM citrate).
HPO
4
2
39.
5
6
P. M. Haggle and A. S. Verkman, J. Biol. Chem., 2009, 284, 7681.
R. Acin-Perez, E. Salazar, M. Kamenetsky, J. Buck, L. R. Levin
and G. Manfredi, Cell Metab., 2009, 9, 265.
Increases of pCO2 did not lead to an increase in observed
emission intensity when carbonic anhydrase activity was sup-
pressed by acetazolomide. These observations and conclusions
accord with the in vitro control experiments. The absence of
change in observed image and spectral emission intensity for
7 R. Pal and D. Parker, Org. Biomol. Chem., 2008, 6, 1020.
8
R. Pal, L. C. Costello and D. Parker, Org. Biomol. Chem., 2009, 7,
525.
1
9
(a) R. A. Poole, F. Kielar, S. L. Richardson, P. A. Stenson and
D. Parker, Chem. Commun., 2006, 4084; (b) G.-L. Law, D. Parker,
S. L. Richardson and K.-L. Wong, Dalton Trans., 2009, 8481.
the Tb analogues suggests that incubations of mixtures of
n 3+
[
LnꢀL ]
(n = 1, 2; Ln = Eu, Tb), under varying pCO
2
1
0 Y. Bretonniere, M. J. Cann, D. Parker and R. J. Slater, Org.
Biomol. Chem., 2004, 2, 1624.
conditions, can be used to calibrate the observed intensity
1
variations observed by microscopy.
5
Such a procedure
1
1 B. S. Murray, E. J. New, R. Pal and D. Parker, Org. Biomol.
Chem., 2008, 6, 2085.
assumes that the Eu and Tb complexes exhibit identical
cellular distributions. This is a reasonable assumption given
their near-identical constitution and very similar anion and
protein affinity profiles (Table 1).
12 M. S. Tremblay, M. Halim and D. Sames, J. Am. Chem. Soc., 2007,
29, 7570.
1
1
1
1
3 F. Kielar, C. P. Montgomery, E. J. New, D. Parker, R. A. Poole,
S. L. Richardson and P. A. Stenson, Org. Biomol. Chem., 2007, 5,
2975.
4 S. Parkkila, H. Rajaniemi, A.-K. Parkkila, J. Kivela, A. Waheed,
S. Pastorekova, J. Pastorek and W. S. Sly, Proc. Natl. Acad. Sci.
U. S. A., 2000, 97, 2200.
5 C. T. G. Flear, S. W. Roberts, S. Hayes, J. C. Stoddart and
A. K. Covington, Clin. Chem., 1987, 33, 13. At equilibrium (298 K,
760 mm Hg, pH 7.4), the Henderson–Hasselbach equation (pH =
1
3+
Moreover, in vitro spectral calibration curves for [LnꢀL ]
2
3+
and [LnꢀL ] that model this change accord with this hypo-
thesis. The variation of the Eu/Tb emission intensity ratio
(
3 : 2 mixture of complexes, examining bands centred at
ꢁ
6
16/545 nm, i.e. intensity area ratios) as a function of [HCO
3
]
1
3+
2 3+
increased by 45% and 42% for [LnꢀL ]
respectively, between 13.6 and 22.7 mM [HCO
These values correspond to calculated concentrations of
and [LnꢀL ]
0
ꢁ
]/spCO
bicarbonate concentration accompanying pCO
pK + log {[HCO
3
2
} allows a calculation of the dissolved
variation. It
ꢁ
3
] (Fig. 2).
2
requires that a value of 6.1 is assumed for the practical coefficient,
0
ꢁ
15
HCO3 for 3% and 5% CO in the atmosphere at pH 7.4,
2
pK , associated with ionisation of carbonic acid. Calculated values
are 13.6, 18.1 and 22.7 mM bicarbonate for 3, 4 and 5% CO
2
,
and were established in a background of 0.4 mM protein
assuming that the solubility coefficient, s, of CO gas in the
2
2
medium is 0.0307 mM per mm Hg pCO , and the atmospheric
pressure is 760 mm Hg.
2ꢁ
(
HSA), 1 mM HPO4 , 1 mM ATP, 2 mM lactate and
.13 mM citrate. The linear range of these calibration curves
0
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 7347–7349 7349