Detection and imaging of zinc secretion from pancreatic, β-cells using a new fluorescent zinc indicator

Article abstract of DOI:10.1021/ja011774y

A novel Zn²⁺-selective visible wavelength fluoroionophore (FluoZin-3, 9) was synthesized. The chelating portion of the molecule resembles known EGTA-based Ca²⁺-selective fluoroionophores, except that one of the N-acetic acid moieties

Full text of DOI:10.1021/ja011774y

Published on Web 01/12/2002
Detection and Imaging of Zinc Secretion from Pancreatic â-Cells Using a New
Fluorescent Zinc Indicator
Kyle R. Gee,*,† Zhang-Lin Zhou,† Wei-Jun Qian,‡ and Robert Kennedy*,‡
Molecular Probes, Inc., 4849 Pitchford AVenue, Eugene, Oregon 97402, and UniVersity of Florida,
Department of Chemistry, GainesVille, Florida 32611-7200
Received July 23, 2001
Scheme 1 a
Zinc is an element critical to life. Some of its roles have been
long known, such as in gene transcription and metalloenzyme
function.1,2 Other functions are just being discovered and investi-
gated, such as zinc’s role in synaptic neurotransmission3 and in
mediating neuronal excitotoxicity.4,5 Understanding the role of free
zinc in living cells has been hindered by a lack of suitable detection
and imaging reagents. Fluorescent sulfonamides of 8-aminoquino-
lines have been used with a moderate degree of success.6-9
However, these probes suffer from relatively low signal levels and
require potentially damaging UV excitation. More recently, visible
wavelength fluorescent probes of [Zn2+] have been reported.10-12
These newer probes have higher affinity for zinc and are brighter
than the first generation UV-based probes, but they suffer from a
different drawback: their chelator moieties all include aliphatic
tertiary amines, which are significantly protonated at physiological
pH. This protonation limits the pH range in which the probes are
useful measures of [Zn2+], and also results in intracellular localiza-
tion to acidic compartments.11 This property is in contrast to the
anionic character of fluorescent calcium indicators, which have been
of enormous benefit for measuring cytosolic [Ca2+] in all areas of
biology.13 Herein we report a novel visible wavelength, zinc-specific
fluorescent probe (9, FluoZin-3) that is tetraanionic and its
a Reagents: a) 1,2-dibromoethane, K2CO3 (77% yield); b) ethylene
glycol, p-TsOH (97%); c) 5-methoxy-2-nitrophenol, K2CO3 (88%); d) H2,
Pt/C (49%); e) DMF, BrCH2CO2CH3, DIEA (40%); f) 4-fluororesorcinol,
MeSO3H (98%); g) p-chloranil (60%); h) KOH (50%).
application in the imaging of zinc secretion from pancreatic â-cells.
The structure of 9 is related to the fluorescent [Ca2+] probes
fluo-314 and fluo-4.15 Fluo-3/4 bind Zn2+ with low nM affinity,
and have Ca2+ Kd ≈ 300 nM.13 We sought to reduce the ion affinity
of the chelator portion of the fluo-3/4 molecule, so as to retain
physiologically relevant Zn2+ affinity while lowering the Ca2+
affinity to a physiologically irrelevant concentration value. This
was accomplished by simply removing one of the N-acetic acid
moieties. Synthesis of the bis-anilino dioxolane 5 proceeds from
appropriately substituted 2-nitrophenols. Subjection of 5 to alky-
lation conditions (hot DMF, excess methyl bromoacetate, diiso-
propylethylamine) gave the benzaldehyde 6. The acetal group in 5
cleaved back to the carboxaldehyde moiety during the reaction,
which inhibits bis-alkylation at the p-anilino nitrogen atom, giving
the desired trialkylated product (6). Cleavage of dioxolanes to their
corresponding carbonyl compounds in the presence of electrophiles
is well established,16-18 and this cleavage is promoted by the
electron-donating p-anilino substituent that stabilizes the incipient
benzylic cation.19 Prolonged reaction times did in fact produce low
yields of the N,N,N′,N′-tetraalkylated product from 5. Acid-mediated
condensation of 6 with 4-fluororesorcinol,20 followed by dehydro-
genative oxidation, gave the tetraester 8, which was converted into
the fluoroionophore 9 by saponification (Scheme 1). The methoxy
group serves to increase the affinity of the chelator toward zinc by
donation of electron density to the p-nitrogen atom. In the absence
of zinc, that is, the presence of the zinc chelator N,N,N′,N′-tetra-
(2-picolyl)ethylenediamine (TPEN), the fluorescence quantum yield
is negligible (<0.005). Titration with increasing concentrations of
buffered Zn2+ solutions gives an apparent Kd of 15 ( 2 nM, with
a Hill coefficient of 1, consistent with a 1:1 binding of Zn2+:9
(Figure 1). The fluorescence reached a maximum near 100 nM Zn2+
(Figure 1). The fluorescence increases several hundred-fold, and a
fluorescence quantum yield of 0.43 ( 0.04 was measured in the
presence of 5 µM Zn2+ 21 In the presence of saturating Zn2+ (5
.
µM), FluoZin-3 has stable fluorescence from pH 6 to 9. The
fluorescence of the complex decreases as the pH is lowered below
6.0 with an apparent pKa of 4.8, and no fluorescence is observed
at pH < 4. This pH dependence correlates exactly with the
fluorescence pH dependence of other 2,7-difluoroxanthen-3-ol-6-
ones,20 which lose their fluorescence as the phenolic hydroxyl group
(pKa ) 4.8) becomes protonated. This behavior indicates that the
pH sensitivity of 9 is due exclusively to protonation of the
fluorescent part of the molecule at low pH, and not of the chelator
portion. The Kd and titration profile are unchanged in the presence
of 1 µM free Ca2+, indicating good selectivity of 9 for zinc over
calcium.
A potential biological application of 9 is for monitoring secretion
from single cells. Secretion assays, especially at the single cell level,
are important in assessing signal transduction of secretion, effects
of new drugs on secretion, and intercellular communication. Zn2+
† Molecular Probes, Inc.
‡ University of Florida.
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776 VOL. 124, NO. 5, 2002 J. AM. CHEM. SOC.
10.1021/ja011774y CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
fluorescent complex. In the presence of 50 µM Zn2+ chelator TPEN,
no fluorescence enhancements were observed from cells following
stimulation confirming that the observed fluorescence enhancements
are dependent on the presence of free Zn2+ and the formation of
the Zn2+:FluoZin-3 complex. The pH independence of the signal
suggests that the fluorescence changes are not associated with local
pH changes during exocytosis. As shown in Figure 2, the total Zn2+
concentration near the cells reached 600 nM at peak response while
the total Zn2+ concentration decreased moving away from the cell
consistent with diffusional dilution of released Zn2+ 30
. The spatial
Figure 1. (a). Fluorescence emission spectra (excitation 488 nm) of
FluoZin-3 (9, 0.5 µM) in buffered Zn2+ solutions with free (unbound) Zn2+
resolution of the imaging technique allows observation of hetero-
geneity in secretion among cells as some cells in the cluster do not
release Zn2+ while others are active. In addition, within individual
cells, some regions of the cell membrane give rise to secretion while
others do not suggesting active zones of secretion on the cell surface.
In previous work29 similar measurements were made using
Zinquin,31 one of the UV-excited Zn2+-specific 8-aminoquinoline
sulfonamide fluorophores. The new dye 9 has several advantages
for this application with the most significant being the improvement
in signal-to-noise (S/N) ratio. The fluorescence enhancement seen
during secretion is >100-fold over baseline with FluoZin-3 while
typical measurements with Zinquin yielded only a 3-5-fold
enhancement over baseline. This improvement can be attributed to
greater sensitivity of the dye and lower background autofluorescence
from both the cell and surrounding solution due to the longer
wavelength for excitation. Furthermore, as a tetraanionic dye
FluoZin-3 is less permeable to the cell than the monoanionic
Zinquin7 and creates lower background fluorescence and less
fluctuations inside the cell. Besides the obvious advantage of
detecting lower levels of Zn2+, the greater S/N allows better
characterization of the cloud of Zn2+ formed by secretion. In
previous work29 Zn2+ was only detected in the immediate vicinity
of the cell (∼1-2 µm away); however, in the image shown in
Figure 2 Zn2+ can be detected at least 15 µm away from the cell,
giving an enhanced view of the transport of Zn2+ away from the
cell. The higher sensitivity may also be translated into better
temporal resolution. Ongoing investigations with this dye have
shown that bursts of Zn2+ secretion due to exocytosis can be
detected with video-rate imaging. Regarding probe ion selectivity,
it should be noted that the cells being imaged with 9 were bathed
-
concentrations of 0, 1, 3, 5, 9, 12, 20, 30, 45, and 80 nM and 10 µM,
respectively. The up-arrow indicates the increase of free [Zn2+] from 0 to
10 µM. The first spectrum almost overlaps with the x-axis indicating the
very low fluorescence in the absence of Zn2+. The spectra were measured
at 22 °C, pH 7.4 in buffered Zn2+ solutions comprising 20 mM HEPES,
135 mM NaCl, 1.1 mM total EGTA, and 0-1.1 mM ZnCl2. Free Zn2+
concentrations were calculated from the equation Kd ) [Zn2+][EGTA]/
[Zn2+-EGTA] using Kd ) 1.1 nM of EGTA for zinc.32 Fluorescence intensity
is in arbitrary units. (b). Normalized fluorescence response as a function of
free [Zn2+]. The fluorescence emission intensities at 515 nm (maximum
emission) were normalized to the full scale response obtained at 10 µM
free Zn2+. An apparent Kd of 15 nM was observed by analyzing the data
with nonlinear least-squares fitting.
Figure 2. Imaging of Zn2+ secretion from pancreatic â-cells. Images are
shown in ratios of fluorescence intensities against a reference image collected
in the beginning of the sequence. The time at which each image was acquired
is indicated as 20, 40, 50, 140 s, respectively. The temporal responses of
Zn2+ secretion were analyzed using the four regions of interest (ROI) (4
µm2) as indicated as 1, 2, 3, 4 in the first image. The traces from top to
bottom correspond to the ROI 1, 2, 3, 4, respectively. Cells were incubated
in Krebs-Ringer buffer containing 2 µM FluoZin-3 (9) and stimulated to
secrete by the application of 20 mM glucose. The bar on top of the traces
indicates the application of stimulation. The details of the imaging
experiments and data analysis are as previously described.29
in Krebs-Ringer buffer containing 2.4 mM Ca2+, 1.2 mM Mg2+
,
118 mM Na+, and 5.4 mM K+, yet no fluorescence was observed
until extracellular Zn2+ was added or Zn2+ secretion was induced,
even though in vitro weak fluorescence could be induced from 9
with 40 µM Ca2+ in the absence of Zn2+. In vitro screening of 9
against various other biologically relevant heavy metals showed
some sensitivity toward Fe2+ and Hg2+, although substantially
higher concentrations than for Zn2+ were needed to induce
fluorescence; 9 was even less sensitive to Cu2+, Cd2+, Ni2+, Co2+
Ba2+, Pb2+, Mn2+, Tb3+, and La3+ than to Ca2+
,
is released from neurons, where it appears to act as a neuro-
transmitter,8,22-24 and from â-cells of the pancreas where it may
serve an autoregulatory role on the â-cell.25 In addition, Zn2+
secretion can serve as an indicator of insulin secretion since insulin
and Zn2+ are co-stored in secretory vesicles and co-released by
exocytosis.26-29
To assess 9 for monitoring secretion of Zn2+, â-cells were bathed
in a solution of 2 µM of the dye while monitoring fluorescence by
laser-scanning confocal microscopy. Cells were stimulated by
applying 20 mM glucose using a micropipet.28 As shown in Figure
2, once glucose is applied large fluorescence signals are observed
around the outside of the cell presumably corresponding to detection
of Zn2+ released from the cell. Zn2+ is secreted by exocytosis to
the extracellular milieu where it reacts with the dye to form the
.
In conclusion, we have reported on the synthesis and fluorescence
properties of an outstanding new visible wavelength Zn2+-selective
fluorescent probe (9), and its application in imaging and quantifica-
tion of Zn2+ secretion from pancreatic â-cells. Further work is
underway to utilize the probe in other cell-secretion assays.
Acknowledgment. K.R.G. and Z.-L.Z. gratefully acknowledge
Nabi Malekzedah and Dr. Iain Johnson for helpful discussions, and
Anca Rothe and Diane Ryan for spectroscopic measurements. This
work was supported in part by NIH Grant 46960 (R.T.K.) and a
fellowship from Eastman Chemical Company (W.-J.Q.). We also
thank Melody Ouellet for editorial assistance in the preparation of
this manuscript.
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C O M M U N I C A T I O N S
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(21) Solutions of 9 and fluorescein were matched by optical density (0.30) at
475 nm and pH 8. After a 10-fold dilution, the integrated fluorescence
emission from 9 in the presence of 5 µM Zn2+ was 47 ( 4% of that from
fluorescein, which has an absolute fluorescence quantum yield of 0.92
(Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer
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Supporting Information Available: Detailed experimental pro-
cedures and analytical data for the synthesis of compounds 2-9; data
from the fluorescence pH sensitivity of the 9:Zn2+ complex; tabulated
fluorescence sensitivity of 9 to other metals, relative to Zn2+(PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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