Bivalent angiotensin II suppresses oxidative stress-induced hyper-responsiveness of angiotensin II receptor type i

Article abstract of DOI:10.1016/j.ejmech.2013.02.041

Angiotensin II receptor type I (AT₁R) is a G-protein coupled receptor involved in regulation of body water-electrolyte balance and blood pressure. Oxidative stress promotes AT₁R oligomerization and hyper-responsiveness to its cognate ligand Ang II. In this study, bivalent Ang II, synthesized by linking with aminocaproic acid (Acp) at the N-terminus, was used to induce AT₁R dimerization and hyper-responsiveness in AT ₁R-expressed human embryonic kidney (AT₁R-HEK) cells, determined using image correlation spectroscopy (ICS) and by measuring AT ₁R-mediated change in intracellular Ca²⁺ concentration, respectively. In addition, ICS was employed to determine distribution pattern of cell-surface AT₁R and its degree of aggregation when stimulated by monomeric (monovalent) and bivalent Ang II under oxidative stress (100 μM H₂O₂) condition in comparison with normal (unoxidized) AT₁R-HEK cells. Bivalent Ang II induced cell-surface AT₁R aggregation/clustering but maintained AT₁R normal signaling response under oxidative stress condition, whereas stimulation by monomeric Ang II or a mixture of monomeric and Acp-modified Ang II (used in the synthesis of bivalent form) resulted in AT₁R hyper-responsiveness. These results suggest that bivalent ligand (viz. Ang II) provides another strategy in the development of novel drugs specifically designed for attenuating aberrant responsiveness of cognate receptor (AT₁R) under pathological (oxidative stress) conditions.

Full text of DOI:10.1016/j.ejmech.2013.02.041

European Journal of Medicinal Chemistry 63 (2013) 629e634
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Bivalent angiotensin II suppresses oxidative stress-induced
hyper-responsiveness of angiotensin II receptor type I
Titiwat Sungkaworn a, Chutima Jiarpinitnun b, Pongkorn Chaiyakunvat b,
,
Varanuj Chatsudthipong a
*
a Department of Physiology and Research Center of Transport Protein for Medical Innovation, Faculty of Science, Mahidol University, Rama VI Road,
Ratchathewi, Bangkok 10400, Thailand
b Department of Chemistry and Center for Innovation in Chemistry (PERCH-CIC), Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi,
Bangkok 10400, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Angiotensin II receptor type I (AT1R) is a G-protein coupled receptor involved in regulation of body
water-electrolyte balance and blood pressure. Oxidative stress promotes AT1R oligomerization and
hyper-responsiveness to its cognate ligand Ang II. In this study, bivalent Ang II, synthesized by linking
with aminocaproic acid (Acp) at the N-terminus, was used to induce AT1R dimerization and hyper-
responsiveness in AT1R-expressed human embryonic kidney (AT1R-HEK) cells, determined using image
correlation spectroscopy (ICS) and by measuring AT1R-mediated change in intracellular Ca2þ concen-
tration, respectively. In addition, ICS was employed to determine distribution pattern of cell-surface AT1R
and its degree of aggregation when stimulated by monomeric (monovalent) and bivalent Ang II under
Received 20 September 2012
Received in revised form
7 January 2013
Accepted 25 February 2013
Available online 14 March 2013
Keywords:
Oxidative stress
Angiotensin receptor
Dimeric ligand
Image correlation spectroscopy
Aggregation
oxidative stress (100
mM H2O2) condition in comparison with normal (unoxidized) AT1R-HEK cells.
Bivalent Ang II induced cell-surface AT1R aggregation/clustering but maintained AT1R normal signaling
response under oxidative stress condition, whereas stimulation by monomeric Ang II or a mixture of
monomeric and Acp-modified Ang II (used in the synthesis of bivalent form) resulted in AT1R hyper-
responsiveness. These results suggest that bivalent ligand (viz. Ang II) provides another strategy in the
development of novel drugs specifically designed for attenuating aberrant responsiveness of cognate
receptor (AT1R) under pathological (oxidative stress) conditions.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
promotes vasoconstriction, maintains vascular tone and electrolyte
homeostasis. However, AT1R hyper-function can lead to patholog-
Angiotensin II receptor type I (AT1R), the predominant subtype
of angiotensin II receptors, plays a significant role in regulating
body watereelectrolyte balance and blood pressure [1]. AT1R is a
member of G-protein coupled receptor (GPCR) family. Upon bind-
ing by its cognate ligand angiotensin II (Ang II), activated AT1R
transduces signals through Gaq-IP3-calcium signaling pathway,
leading to activation of renineangiotensin system (RAS), which
ical conditions such as hypertension and atherosclerosis [2].
Western blot analysis of AT1R on monocyte from normotensive
and hypertensive subjects showed that expression levels of AT1R in
both groups are comparable [3]. Interestingly, while normotensive
subject expresses monomer receptor, AT1R from hypertensive
group is present in a dimer form [3]. H2O2-induced oxidative stress
also causes AT1R dimerization [4]. Using image correlation spec-
troscopy (ICS), we have previously shown that in AT1R-HEK cells
exposed to 100
mM H2O2 for 3 h, cell-surface AT1R exists in an
aggregated state, in which AT1R becomes hyper-responsive to Ang
II stimulation [5]. Thus, the ability to manipulate cell-surface AT1R
oligomerization provides a potential approach for treatment of
atherosclerosis and hypertension.
Utilization of synthetic bivalent ligands to target and manipu-
late GPCR signaling, in particular those involving dimerization, has
been reported [6]. A bivalent ligand consists of two monomeric
ligands joined together by a spacer molecule in order to provide
Abbreviations: Acp, aminocaproic acid; AT1R, angiotensin II receptor type I;
[Ca2þ]i, intracellular calcium ion concentration; CD, cluster density; DA, degree of
aggregation; DIEA, N,N-diisopropyl ethylamine; DMSO, dimethyl sulfoxide; EGFP,
enhanced green fluorescent protein; EGTA, ethylene glycol tetraacetic acid; GPCR,
G-protein coupled receptor; HBSS, Hanks’ balanced salt solution; HEK, human
embryonic kidney; ICS, image correlation spectroscopy; RAS, renineangiotensin
system.
* Corresponding author. Tel.: þ66 2201 5614; fax: þ66 2354 7154.
E-mail address: varanuj.cha@mahidol.ac.th (V. Chatsudthipong).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.041

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T. Sungkaworn et al. / European Journal of Medicinal Chemistry 63 (2013) 629e634
flexibility for ligand binding to its cognate receptor. Studies,
particularly in GPCR families, have demonstrated that synthetic
bivalent ligand can significantly alter ligand potency, binding af-
finity and selectivity, through induction of receptor dimerization
and thereby impacting on receptor signaling responses and sub-
sequent cellular activities [7,8]. Interestingly, binding of a dimeric
antagonist of gonadotropin-releasing hormone receptor results in
receptor dimerization and agonistic signal transduction [9]. The
ability of bivalent ligand to induce changes in receptor oligomeric
state by bringing monomeric receptors into close proximity has
been proposed to account for the unexpected receptor activation
and agonistic response, suggesting that physical interactions be-
tween receptors are sufficient for induction of receptor activation
[10]. Bivalent agomelatine, a potent agonist of melatonin receptor
(MR), enhances binding affinity to both melatonin receptor type I
(MT1R) and type II (MT2R), with a 75-fold increase in selectivity to
MT2R but alters MR pharmacological characteristics from an
agonistic to antagonistic response of both MT1R and MT2R [11].
There have been a number of studies reporting the use of
bivalent ligands to manipulate GPCR pharmacological properties
[6,12,13]. Dimerization by transglutaminase-mediated crosslinking
of intracellular C-terminus of two adjacent AT1R molecules en-
hances receptor-mediated intracellular Ca2þ level ([Ca2þ]i) [3],
however, the synthesis and pharmacological properties of a biva-
lent Ang II targeting AT1R have not as yet been investigated. We
hypothesized that changes in AT1R distribution pattern could
modulate AT1R-mediated signaling response and thereby AT1R-
mediated cellular functions. In order to test this notion, we have
synthesized bivalent Ang II and examined its ability to perturb
oligomeric state of cell-surface AT1R and its trans-membrane
signaling properties. In addition, experiments were conducted to
examine these effects in oxidative stress condition, which has been
reported to cause clustering of cell surface AT1R [4,5].
not essential for receptor binding or activation, their side chains are
difficult to modify chemically. As the N-terminal Asp residue is not
crucial for Ang II function [17], modification at this site should not
compromise its physiological properties. Replacement of Ang II N-
terminal Asp with sarcosine even resulted in an increase in
agonistic activity [18]. Thus we chose Ang II N-terminus as the site
to attach a linker to generate the bivalent ligand.
Various types of compounds have been used as linkers in the
synthesis of bivalent ligands [6,19]. We opted for aminocaproic acid
(Acp), a methylene-based linker which is widely-used in the syn-
thesis of bivalent ligands [20e23], as the chemical structure of Acp
allows the ability to extend linker lengths by a simple amide bond
formation. Accordingly, Acp was covalently attached to the N-ter-
minus of Ang II to generate Acp-Ang II (Acp-Asp-Arg-Val-Tyr-Ile-
His-Pro-Phe). Acp-Ang II still possesses a functional amine group at
the N-terminus, allowing linking to the N-terminus of another Ang
II via reaction with the dimethyl squarate (3,4-dimethoxy-3-
cyclobutene-1,2-dione) attached to the N-terminus of the second
Ang II to yield bivalent Ang II (2) (Scheme 1). Dimethyl squarate was
chosen as a linkage for two N-terminal amines because the reaction
occurs under mild condition, i.e., in organic solvent or even in
aqueous buffer [24e26]. In addition, the conjugation of one amino
group could be selectively achieved by adjusting pH [24,27].
2.2. AT1R-mediated signaling response from stimulation by
monovalent and bivalent Ang II in AT1R-HEK cells under oxidative
stress
Transmembrane signaling from binding of Ang II to AT1R results
in Gaq dissociation and phospholipase C activation which sequen-
tially produces secondary messengers diacylglycerol (DAG) and
inositol triphosphate (IP3), generating a variety of cellular re-
sponses, such as vasoconstriction, aldosterone release and increase
in [Ca2þ]i [28]. As the latter event is an early step in AT1R-mediated
signaling pathway, we monitored [Ca2þ]i in AT1R-HEK cells pre-
loaded with Ca2þ-responsive Indo-1 dye and stimulated by
monovalent or bivalent Ang II, then compared the results with
2. Results
2.1. Design and synthesis of bivalent Ang II
oxidatively stressed cells (exposure to 100 mM H2O2 for 3 h).
In order to synthesize bivalent Ang II, the peptide was subjected
to chemical modification. Structureeactivity relationship of Ang II
for binding to AT1R indicated that the C-terminus carboxyl group of
Ang II is crucial for electrostatic interaction required for high-
affinity binding between Ang II and AT1R [14]. Any modification
to the C-terminus would result in severe perturbations to ligande
receptor binding and ability to activate its cognate receptor [15]. In
addition to the C-terminus, side-chains of Arg, His and Tyr are also
key components for high-binding affinity [16]. Although several
amino acid residues in Ang II sequence (viz., Val, Ile, and Pro) are
Stimulation of AT1R-HEK cells by monomeric Ang II, Acp-Ang II,
or bivalent Ang II (2) increased [Ca2þ]i 10-fold compared to non-
stimulated control cells. (Fig. 1). As expected, Acp-Ang II behaved
similarly to unmodified Ang II, but stimulation by Ang II-squarate
(1) significantly decreased [Ca2þ]i. However, the presence of squa-
rate in the linker of bivalent Ang II (2) did not compromise its ability
to increase [Ca2þ]i to the level obtained with Ang II and Acp-Ang II
(Fig. 1).
Oxidative-stressed AT1R-HEK cells, which were induced by H2O2
treatment, exhibited AT1R hyper-function in response to Ang II or to
Scheme 1. Pathway of chemical synthesis of Ang II-squarate (1) and bivalent Ang II (2).

T. Sungkaworn et al. / European Journal of Medicinal Chemistry 63 (2013) 629e634
631
Fig. 1. [Ca2þ]i response of AT1R-HEK cells induced by Ang II, Acp-Ang II, Ang II-squarate
(1) and bivalent Ang II (2). AT1R-HEK cells (2 ꢀ 106 cell/mL HBSS supplemented with
1% (w/v) BSA and 1 mM CaCl2), pre-loaded with Indo1-AM (6 mg/mL), were stimulated
with 10 nM Ang II and analogs at 37 ꢁC. Results are presented as [Ca2þ]i from at least
three independent experiments performed in triplicate. *p < 0.05 compared with basal
condition, #p < 0.05 compared with Ang II stimulation.
a 1:1 mixture of Ang II and Ang II-squarate (1) (Fig. 2), as previously
reported [5]. Interestingly, stimulation of oxidative-stressed cells
with bivalent Ang II (2) abrogated AT1R hyper-function, whereas a
mixture of Ang II and Ang II-squarate (1), at the same equivalent
Ang II concentration as in the bivalent Ang II (2), was still able to
stimulate AT1R hyper-responsiveness (Fig. 2).
2.3. AT1R distribution pattern induced by monovalent and bivalent
Ang II
In order to investigate the underlying mechanism of the
inability of bivalent Ang II (2) to induce AT1R hyper-function in
oxidative-stressed AT1R-HEK cells, we employed ICS [28,29] to
characterize the distribution pattern (mean fluorescence intensity,
cluster density (CD) and degree of aggregation (DA)) of EGFP-
tagged AT1R on cell surface of oxidized and non-oxidized cells
following monovalent Ang II, mixture of Ang II and Ang II-
squarate (1), or bivalent Ang II stimulations. Stimulation of
oxidative stressed and non-stressed cells by Ang II or a mixture of
Ang II and Ang II-squarate (1) significantly increases mean fluo-
rescence intensity of EGFP-tagged AT1R when compared to
unstimulated control cells (p < 0.05) (Fig. 3A), indicating an
Fig. 3. Cell-surface distribution pattern of EGFP-tagged AT1R induced by monovalent
and bivalent Ang II under H2O2-mediated oxidative condition. HEK cells expressing
EGFP-tagged AT1R were treated as described in legend of Fig. 2. EGFP-tagged AT1R on
HEK cell surface, before and after exposure to 5 nM monovalent Ang II, 5 nM (total)
monovalent Ang II þ Ang II-squarate (1) and 2.5 nM bivalent Ang II (2) were processed
for ICS examination as described in Experimental section. A) Mean fluorescence in-
tensity, B) cluster density (CD), C) degree of aggregation (DA). *p < 0.05 compared with
non-Ang II stimulation (baseline) group, #p < 0.05 compared with non-Ang II stim-
ulation (baseline) in H2O2 pre-treated group.
increase in number of AT1R at cell surface. These results are in
agreement with the previous report of increased mean fluores-
cence intensity of EGFP-tagged AT1R on the cell surface upon Ang
II stimulation [5].
In non-oxidized cells, both Ang II and mixture of Ang II and Ang
II-squarate (1) produced a significant increase in CD values
compared to non-stimulated cells (p < 0.05), whereas bivalent Ang
II (2) had no effect on CD value (Fig. 3B). However, CD values of
oxidative stressed cells stimulated by Ang II or Ang II-squarate
mixture were comparable to those obtained from non-stimulated
cells. This result is in agreement with the previous report [5]. On
the other hand, CD value of bivalent Ang II (2)-stimulated oxidized
cells is significantly lower than that of non-stimulated cells
(p < 0.05) (Fig. 3B).
DA values mirrored those of CD, namely. All peptides stimula-
tion caused significant increase in DA of oxidized cells (Fig. 3C).
Surprisingly, in non-oxidized cells, a significant increase in DA was
observed with bivalent Ang II (2) stimulation (p < 0.05) while
monomeric Ang II (singly or 1:1 mixture) could not alter DA under
control condition (Fig. 3C).
Fig. 2. [Ca2þ]i response of AT1R-HEK cells induced by monovalent and bivalent Ang II
under H2O2-mediated oxidative condition. AT1R-HEK cells (2 ꢀ 106 cell/ml) were
treated with 100 m
M H2O2 for 3 h prior to measurement of [Ca2þ]i as descried in legend
of Fig. 1. Cells were stimulated by 5 nM indicated peptide (based on Ang II equivalent).
*p < 0.05 compared with control group of each tested peptide, #p < 0.05 compared
with control group of Ang II stimulation.

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T. Sungkaworn et al. / European Journal of Medicinal Chemistry 63 (2013) 629e634
3. Discussion
under pathological (oxidative stress) conditions indicates another
approach in the design and development of therapeutic com-
pounds targeting AT1R implicated in such diseases as atheroscle-
rosis and hypertension.
We have previously demonstrated using ICS that H2O2-induced
oxidative stress changes the distribution pattern of cell-surface
AT1R resulting in hyper-responsiveness of AT1R to Ang II stimula-
tion [5]. In this study, we employed bivalent Ang II (2), synthesized
by linking N-terminus of two Ang II peptides with aminocaproic
acid (Acp), which upon binding to AT1R-HEK cells should generate
AT1R dimers on cell surface. Unexpectedly, bivalent Ang II (2)
stimulation did not induce AT1R hyper-responsiveness, as deduced
from the similarity of [Ca2þ]i in bivalent Ang II (2)-stimulated cells
with that of cells stimulated by monomer Ang II, alone or in a 1:1
mixture with Acp-Ang II (Fig. 1).
Nevertheless, ICS showed in non-oxidized cells that bivalent
Ang II (2), at the same equivalent of monomeric Ang II concen-
tration, was able to induce aggregation and a decrease in cluster
density of cell-surface AT1R compared to stimulating by mono-
meric Ang II or non-stimulated control cells (Fig. 3B and C). This
evidence solidly supports the abilities of bivalent ligand to induce
receptor dimerization [6,12]. On the other hand, 1:1 mixture of
Ang II and monovalent Ang II-squarate (1), the chemical compo-
nents of bivalent Ang II (2), yielded similar cluster density and
degree aggregation to those when stimulated by Ang II (Fig. 3B
and C). The observations coincided with previous reports on other
GPCRs studies, demonstrating that monomeric ligands are unable
to cause alteration in distribution or oligomerization of their
5. Experimental
5.1. Materials
Human Ang II (DRVYIHPF; C50H71N13O12, MW ¼ 1046.20) and its
aminocaproic acid analog Acp-Ang II (Acp-DRVYIHPF; C56H82N14O13
;
MW ¼ 1159.36) were custom synthesized by GL Biochem, Shanghai,
China; 3,4-dimethoxy-3-cyclobutene-1,2-dione from Sigma Aldrich,
Missouri, USA; Dulbecco’s modified Eagle medium (DMEM), Hank’s
balanced salt solution (HBSS), and Geneticin from Invitrogen, Cali-
fornia, USA; fetal bovine serum (FBS), penicillin and streptomycin
from HyClone, Thermo Scientific, Utah, USA; Indo-1 acetoxymethyl
(Indo-1/AM) from Molecular probes, Invitrogen, California, USA; and
EGTA and ionomycin from Calbiochem, New Jersey, USA. Human
embryonic kidney cell line containing stably expressed enhanced
green fluorescent protein (EGFP)-tagged rat Ang II receptor type I
(AT1R-HEK) was kindly provided by Dr. Tamás Balla, National In-
stitutes of Health, USA [36]. All other commercial chemicals used
were of analytical grade.
5.2. Cell culture
cognate receptors [30], such as, M3 muscarinic receptor [31],
k
opioid receptor [32],
receptor [34].
d
opioid receptor [33], and adenosine A1
AT1R-HEK cells were cultured in DMEM, supplemented with 10%
FBS,100 U/ml penicillin and 100 m
g/ml streptomycin, at 37 ꢁC under
Under H2O2-induced oxidative stress condition, all monovalent
forms of Ang II, including Ang II-squarate (1) and 1:1 mixture of
Ang II and Ang II-squarate (1), were able to enhance AT1R
responsiveness, manifested by elevation of [Ca2þ]i, compared to
that of the non-oxidized cells (Fig. 2), in agreement with the pre-
vious report [5]. Interestingly, bivalent Ang II (2), did not alter AT1R
responsiveness under H2O2-induced oxidative stress condition
(Fig. 2). These results can be attributed to the ability of bivalent
Ang II (2) to induce clustered AT1R on cell-surface stemming from
the prior exposure to H2O2 (Fig. 3C). Taken together, the
AT1R aggregation found in oxidative-stressed cells with bivalent
Ang II stimulation resulted from decreasing in CD together with
increasing in DA (Fig. 3B, C). This pattern of aggregation was also
reported for epidermal growth factor receptor at low temperature
[35].
Thereby, under H2O2-induced oxidative stress condition, the
AT1R-mediated signaling response, [Ca2þ]i level, was indifferent
from the result obtained under control condition. This unique
property of the bivalent ligand to retain AT1R-mediated signaling
response under pathological oxidative stress condition might be
beneficial for the development of novel angiotensin-based thera-
peutic for AT1R implicated diseases.
humidified atmosphere of 5% CO2. In order to maintain continuous
EGFP-tagged AT1R expression, every two weeks AT1R-HEK cells
were subjected to selection with 500 mg/ml Geneticin.
5.3. Chemical synthesis
Synthesis of Ang II-squarate (1): Custom synthesized Acp-
DRVYIHPF, dissolved in a small volume of dimethyl sulfoxide
(DMSO), was added to a solution containing two equivalents of 3,4-
dimethoxy-3-cyclobutene-1,2-dione (dimethyl squarate) in dichlo
romethane (CH2Cl2), and pH of the resulting mixture was adjusted
a value of 7 by addition of N,N-diisopropyl ethylamine (DIEA). The
reaction mixture was stirred at ambient temperature overnight
under an inert N2 atmosphere. Then, the solution was concentrated
under reduced pressure and lyophilized. The product was charac-
terized using high resolution mass spectrometry obtaining a
[M
61H85N15O15).
Synthesis of bivalent Ang II (2): Synthesized Ang II-squarate (1)
þ
H]þ of 1269.61 (calculated 1267.63 for
a mass of
C
was mixed with custom synthesized Ang II in CH2Cl2 and pH of the
resulting reaction mixture was adjusted to 9e10 by the addition
of DIEA. The reaction mixture was stirred overnight at ambient
temperature under N2 atmosphere and then concentrated
under reduced pressure and lyophilized. The product was charac-
terized using high resolution mass spectrometry obtaining a
[M þ 4H þ 4Na]þ of 663.44 (calculated 663.75 for a mass of
4. Conclusion
We have demonstrated using ICS that bivalent Ang II is capable
of re-organizing AT1R distribution pattern on HEK cell surface.
Remarkably, bivalent Ang II retains normal transmembrane
signaling property of AT1R in pathological (oxidatively stressed)
cells, whereas monovalent Ang II enhances AT1R signaling
response (i.e. hyper-responsiveness). Although further in-
vestigations into the biochemical and biophysical properties of
clustered/aggregated AT1R are required in order to gain an un-
derstanding at the molecular level of their functions under normal
physiological and pathological conditions. The unique property of
bivalent Ang II to retain normal AT1R transmembrane signaling
C110H152N28O26).
5.4. Intracellular calcium measurement
A fluorometric-based assay was employed to determine intra-
cellular calcium content. Confluent AT1R-HEK cells were trypsi-
nized and washed twice with HBSS devoid of calcium. Then, cells
were resuspended in calcium-free HBSS to at
a
m
density of
g/mL Indo1-
2 ꢀ 106 cell/mL and subsequently incubated with 6
AM at 37 ꢁC for 60 min. Cells were then washed twice and

T. Sungkaworn et al. / European Journal of Medicinal Chemistry 63 (2013) 629e634
633
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resuspended with measuring buffer (HBSS supplemented with 1%
(w/v) BSA and 1 mM CaCl2) at 1 ꢀ106 cell/mL. Intracellular baseline
calcium level and that upon ligand stimulation were measured as
ratio of fluorescence emission at 405 nm and 490 nm, excitation at
338 nm. Fluorescence emission ratio was followed for 3 min after
ligand stimulation, unless indicated otherwise. Intracellular cal-
cium content was calculated using the following formula [37]:
ꢀ
ꢁꢀ
ꢁ
h
i
ðR ꢂ Rmin
Þ
Sf2
Ca2þ ¼ Kd
(1)
i
ðRmax ꢂ RÞ Sb2
where,
R
¼
ratio of fluorescent emission at 405 nm and
490 nm, Kd ¼ dissociation constant of Indo1-AM, and (Sf2/
Sb2) ¼ fluorescence ratio of 490 nm emission in the absence of Ca2þ
(presence of 3 mM EGTA) to that at Ca2þ saturation (presence of
5 mM ionomycin) [38]. Calcium level induced by ligand stimulation
was determined from magnitude of resulting fluorescence signal.
5.5. Image correlation spectroscopy (ICS) and image processing
AT1R-HEK cells were cultured in cover glass bottom black dish
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1.5 ꢀ 105 cell/plate and observed using a 488 laser line under 60ꢀ
oil immersion objective, with image acquisition using a 3ꢀ digital
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zoom and Z-stack capture mode at 0.5
mm height for each step
(Olympus, FLUOVIEWÒ FV1000). Final images of 30e40 individual
planar cross-section images were reconstructed to obtain a cell-
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5.6. Statistical analysis
Student’s t-test was used and significant difference is considered
at p < 0.05. All experimental results are expressed as mean ꢃ S.E.M.
from at least three independent experiments performed in
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We appreciatively acknowledge Prof. Prapon Wilairat for valu-
able suggestions and comments on the manuscript. This work was
supported in part by the Royal Golden Jubilee Ph.D. Program (Grant
PHD/0245/2548 to T.S. and V.C.), the National Center for Genetic
Engineering and Biotechnology (BIOTEC), National Science and
Technology Development Agency (NSTDA), Thailand (Grant 3-2548
to V.C.), the Thailand Research Fund and the Office of the Higher
Education Commission (MRG5280040 to C.J.), Center of Excellence
for Innovation in Chemistry (PERCH-CIC), and Mahidol University
under National Research Universities Initiative program.
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