Imaging histamine in live basophils and macrophages with a fluorescent mesoionic acid fluoride

Article abstract of DOI:10.1039/c2cc32292g

Histamine is a biogenic amine with fundamental roles in circulatory and immune systems. We report a fluorescent small molecule (Histamine Blue) for imaging intracellular histamine in live basophils and macrophages. Histamine Blue is a fluorescent mesoionic acid fluoride that turns on upon reaction with histamine. The selective response of Histamine Blue enabled the visualization of intracellular histamine under different physiological conditions.

Full text of DOI:10.1039/c2cc32292g

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Imaging histamine in live basophils and macrophages with a fluorescent
mesoionic acid fluoridew
a
b
Nicola Kielland,z Marc Vendrell,z Rodolfo Lavilla*cd and Young-Tae Chang*be
Received 30th March 2012, Accepted 29th May 2012
DOI: 10.1039/c2cc32292g
Histamine is a biogenic amine with fundamental roles in circulatory
and immune systems. We report a fluorescent small molecule
(Histamine Blue) for imaging intracellular histamine in live basophils
and macrophages. Histamine Blue is a fluorescent mesoionic acid
fluoride that turns on upon reaction with histamine. The selective
response of Histamine Blue enabled the visualization of intracellular
histamine under different physiological conditions.
mesoionic acid fluoride. Our discovery has been achieved by
combining the synthetic methodology of multicomponent
reactions (MCR),16 which can provide a straightforward
access to complex heterocycles in a single step,17 and the
systematic screening of fluorescent molecules.18 We recently
described a new MCR involving azines 1, isocyanides 2 and
fluorinated anhydrides 3 to yield unprecedented mesoionic
adducts 4 in good yields19 (Scheme 1) and prepared a set of
12 compounds (4a–4h and 5a–5d, Scheme S1 in Electronic
Supporting Information (ESIw)) based on the isoquinoline
scaffold. These derivatives exhibited absorption bands compatible
with commercially available excitation sources (i.e., xenon and
mercury lamps as well as violet lasers), blue fluorescence emission
wavelengths (from 402 to 450 nm) and moderate-to-low quantum
yields (0.24 as average) with potential to behave as fluorescent
turn-on probes (Table S1 in ESIw). We examined their spectral
properties in the presence of a broad panel of biomolecules
(e.g. proteins, saccharides, nucleic acids, peptides and small
molecule metabolites) and observed that acid fluorides 4a–4d
(Scheme S1 in ESIw) displayed a distinct fluorescence increase
after incubation with histamine (Fig. S1 in ESIw). In particular,
the reaction of histamine with 4a resulted in the bathochromic
shift of its emission spectrum (i.e., from 406 to 432 nm) and a
significant fluorescence increase that was distinguishable to the
naked eye under a 365 nm lamp (Fig. 1).
Histamine is a ubiquitous biogenic amine, synthesized by
histidine decarboxylation and with multiple functions in the
physiology of the body.1 Histamine is especially abundant in mast
cells and basophils, where it is stored and released under chemical
stimulation,2 and plays essential roles in gastric acid secretion,3
allergic and inflammatory responses,4 neurotransmission,5
thermoregulation6 and cognition and behavioural events.7
A major challenge in elucidating the function and dynamics
of histamine in all these processes is represented by the lack of
tools for tracking histamine in live cells. Conventional methods
to determine histamine levels8–12 (e.g. chromatography, capillary
electrophoresis, fluorimetry, immunoassays) rely on multistep
protocols that employ cell extracts, which are limited in spatial
and temporal resolution. While the localization of histamine in
the sympathetic nervous system has been demonstrated by
immunohistochemistry in permeabilized samples13 and Imato’s
group described fluorescent nickel-based complexes to monitor
histamine levels in macrophages,14,15 there is a demand for new
fluorescent probes to image intracellular histamine in real-time
and in a variety of live cells under different conditions.
As mesoionic acid fluorides were found to be remarkably
stable to hydrolysis in neutral and basic solutions, we monitored
the reaction between 4a and histamine in phosphate buffer saline
(PBS) by HPLC-MS (Fig. S2 in ESIw). After 20 min, 4a was fully
converted to the highly fluorescent conjugate 5i (Fig. 1). We
hypothesize that the formation of the amide 5i may block the
quenching electron transfer from the mesoionic core to the
electron-deficient fluoride and lead to the observed fluorescence
turn-on effect. In order to confirm the exact structure of
the fluorescent species formed, we synthesized different amide
We have developed a simple and rapid method to image
histamine in live basophils and macrophages using a fluorescent
a Institute for Biomedical Research, Barcelona Science Park, Spain.
b Laboratory of Bioimaging Probe Development, Singapore
Bioimaging Consortium, A*STAR, Singapore.
c Barcelona Science Park, University of Barcelona, Spain.
d Laboratory of Organic Chemistry, Faculty of Pharmacy,
University of Barcelona, Spain. E-mail: rlavilla@pcb.ub.es;
Fax: +34 4021896; Tel: +34 4037126
e Department of Chemistry & MedChem Program of Life Sciences
Institute, National University of Singapore, Singapore.
E-mail: chmcyt@nus.edu.sg; Fax: +65 6779 1691;
Tel: +65 6516 6774
w Electronic supplementary information (ESI) available: Synthetic
procedures and characterization, protocols for cell imaging, extended
screening analysis, cell viability studies and additional imaging data.
See DOI: 10.1039/c2cc32292g
z These authors contributed equally to this work.
Scheme 1 General MCR scheme for mesoionic isoquinoline adducts 4.
Chem. Commun., 2012, 48, 7401–7403 7401
c
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very weak fluorescent response against other intracellular
metabolites (e.g. glutathione, glucose, ATP, ADP or cAMP),
which asserted its suitability for imaging intracellular histamine
(Fig. S4 in ESIw). In view of this selectivity and the fluorescent
colour of the resulting histamine adduct, we named 4a as
Histamine Blue.
The fast kinetics of Histamine Blue prompted us to investigate
the mechanisms of its preferential reactivity. Hence we examined
the role of the sp2 nitrogen of histamine by comparing the
condensation rates of Histamine Blue with two similar primary
amines including or not an sp2 heterocyclic nitrogen (i.e., 2-(pyridin-
2-yl)ethylamine and 2-phenylethylamine respectively). The reactions
of Histamine Blue with both amines proceeded at a similar rate,
around 20-fold slower than histamine (Fig. S5 in ESIw). These
results suggested that the role of the sp2 nitrogen of the heterocycle
was not the main factor in controlling the rate determining
step of the reaction. Moreover, the unique catalytic effect
observed with histamine might be more related to the imidazole
group acting as a proton transfer moiety than as a nucleophilic
catalyst (Scheme S3 in ESIw).20,21
In view of the characteristics of Histamine Blue, we evaluated its
application for imaging histamine in live RBL-2H3 basophils and
RAW 264.7 macrophages. Histamine Blue stained the cytoplasm of
RBL-2H3 basophils, where histamine-storing granules are located,
after incubating the cells with the dye (20 mM) for 15 min at 37 1C
(Fig. 3). The fluorescence of Histamine Blue did not co-localize with
DRAQ5 nuclear staining and matched the high levels of
intracellular histamine in basophils, in the range of nmol
histamine per 106 cells (Fig. 4d).22,23 Moreover, the treatment
of basophils with Histamine Blue did not affect their morphology
or induced any cytotoxicity (Fig. S6, S7 in ESIw).
Fig. 1 Reaction of 4a with histamine in PBS. Fluorescence spectra of
4a before (black) and after (blue) reaction with histamine; exc: 340 nm.
Fluorescent images of PBS solutions of 4a and 5i under a 365 nm lamp.
conjugates by the reaction of mesoionic acid fluorides with
histamine, imidazole and N-Boc-histamine (Scheme S2 in ESIw).
Mesoionic amides from histamine were highly fluorescent, while
amides from imidazole and N-Boc-histamine were non-fluorescent
due to the quenching PeT (photoinduced electron transfer) of the
imidazole group (Fig. S3 in ESIw). This observation indicates that
the linkage between 4a and histamine is through the primary
amine and not through the nitrogen of the imidazole.
Next we evaluated the selectivity of 4a against histamine
and different signaling molecules, mainly neurotransmitters
and hormones. 4a displayed a very good selectivity towards
histamine with a maximum 14-fold fluorescence increase
(Fig. 2). The combination of both imidazole and amine groups
of histamine appeared to be critical for this response, as only
minor enhancements were observed with other primary amines.
The kinetic analysis of these reactions indicated that 4a exhibited
an optimum selectivity toward histamine at incubation times
between 15 and 30 min (Fig. 2 inset). Furthermore, 4a showed a
We also examined the ability of Histamine Blue to image the
uptake and de novo synthesis of histamine in RAW 264.7
macrophages, which do not contain high levels of intracellular
Fig. 2 Fluorescent response of 4a (Histamine Blue) (10 mM) after
incubation with different signaling molecules (5 mM) for 30 min in
PBS (pH: 7.3). Inset: Kinetic analysis of the reactions of Histamine
Blue with histamine (blue), GABA (red), adenosine (green), glutamate
(orange), inositol (purple) and others (grey); exc: 370 nm. Values are
represented as means (n = 4) and errors bars as standard deviations.
Fig. 3 RBL-2H3 basophils after incubation with Histamine Blue.
(a) Bright field image, (b) DRAQ5 nuclear counterstaining (TXR),
(c) Histamine Blue staining (DAPI), (d) merged a, b and c. Scale bar:
20 mm. Pearson’s co-localization coefficient for TXR and DAPI
channels: 0.16.
c
7402 Chem. Commun., 2012, 48, 7401–7403
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upon condensation with histamine. Histamine Blue displays
very good selectivity over a broad range of signalling molecules
and metabolites due to the catalytic effect of the imidazole group,
and is the first fluorescent small molecule for imaging histamine
in live RBL-2H3 basophils. Histamine Blue can also monitor the
uptake and de novo synthesis of histamine in live RAW 264.7
macrophages and its fluorescence intensity correlates well with
the histamine levels in cell extracts. This discovery may assist the
investigation of the roles that histamine plays in mammalian
physiology and a number of pathologies.
The authors acknowledge the financial support from DGICYT
(CTQ 2009-07758), the Generalitat de Catalunya (2009/
SGR/208), Barcelona Science Park, Grupo Ferrer (Barcelona,
Spain) and the intramural funding and grant 10/1/21/19/656
from A*STAR (Singapore). N. K. thanks DGICYT for a PhD
fellowship.
Notes and references
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Fig. 4 RAW 264.7 macrophages after treatment with Histamine Blue.
Bright field (upper) and fluorescent (lower) images after incubation with
Histamine Blue of: (a) RAW 264.7, (b) RAW 264.7 after histamine
uptake and (c) RAW 264.7 after treatment with thapsigargin. Scale bar:
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Histamine Blue (black) and quantification of histamine levels per 106
cells (grey) in RAW 264.7 before and after chemical treatment and in
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histamine under normal conditions. Histamine Blue did not
stain non-treated macrophages (Fig. 4a), while brightly
stained them after the uptake of histamine (Fig. 4b). Titration
experiments showed that Histamine Blue stained RAW
264.7 cells in a histamine uptake-dependent manner (Fig. S8
in ESIw). These results are consistent with previous reports
indicating that RAW 264.7 macrophages can reach histamine
levels similar to basophils after incubation with 10 mM
histamine for 2 h.24 Furthermore, we determined the amount
of histamine in extracts of RBL-2H3 basophils and RAW
264.7 macrophages by the conventional method using
o-phthaldialdehyde.25 In vitro quantification of the histamine
levels correlated well with the fluorescence intensity of
Histamine Blue in live cell imaging (Fig. 4d). Finally we
induced de novo synthesis of histamine in RAW 264.7 by
treatment with thapsigargin, an endomembrane Ca2+-ATPase
inhibitor.26 Histamine Blue stained thapsigargin-treated
macrophages (Fig. 4c) more brightly than non-treated macrophages
(Fig. 4a). Altogether we proved that Histamine Blue can be
used for imaging histamine in live basophils and macrophages
under different physiological conditions.
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In summary, we developed a new fluorescent probe (Histamine
Blue) for imaging histamine in live cells. Histamine Blue is a
mesoionic acid fluoride showing a 14-fold fluorescence increase
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 7401–7403 7403