Calciumandpolyamineregulatedcalcium-sensing receptors
in cardiac tissues
Rui Wang
1
, Changqing Xu
2
, Weimin Zhao
1
, Jing Zhang
1
, Kun Cao
1
, Baofeng Yang
2
and Lingyun Wu
3
1
Department of Physiology, University of Saskatchewan, Saskatoon, SK, Canada;
2
Department of Pathophysiology, Harbin Medical
University, Harbin, P.R. China;
3
Department of Pharmacology, University of Saskatchewan, Saskatoon, SK, Canada
Activation of a calcium-sensing receptor (Ca-SR) leads to
increased intracellular calcium concentration and altered
cellular activities. The expression of Ca-SR has been iden-
tified in both nonexcitable and excitable cells, including
neurons and smooth muscle cells. Whether Ca-SR was
expressed and functioning incardiac myocytes remained
unclear. In the present study, the transcripts of Ca-SR were
identified in rat heart tissues using RT-PCR that was further
confirmed by sequence analysis. Ca-SR proteins were
detected in rat ventricular and atrial tissues as well as in
isolated cardiac myocytes. Anti-(Ca-SR) Ig did not detect
any specific bands after preadsorption with standard Ca-SR
antigens. An immunohistochemistry study revealed the
presence of Ca-SR in rat cardiac as well as other tissues. An
increase in extracellular calcium or gadolinium induced a
concentration-dependent sustained increase in [Ca
2+
]
i
in
isolated ventricular myocytes from adult rats. Spermine
(1–10 m
M
) also increased [Ca
2+
]
i
. Pre-treatment of cardiac
myocytes with thapsigargin or U73122 abolished the extra-
cellular calcium, gadolinium or spermine-induced increase in
[Ca
2+
]
i
. The blockade of Na
+
/Ca
2+
exchanger or voltage-
dependent calcium channels did not alter the extracellular
calcium-induced increase in [Ca
2+
]
i
. Finally, extracellular
calcium, gadolinium and spermine all increased intracellular
inositol 1,4,5-triphosphate (IP
3
) levels. Our results demon-
strated that Ca-SR was expressed incardiac tissue and car-
diomyocytes and its function was regulated by extracellular
calcium and spermine.
Keywords: calcium-sensing receptor; heart; IP
3
;RT-PCR;
spermine.
Calcium ions were the first identified endogenous substance
to function as both a first and second messenger via the
stimulation of an extracellular calcium sensing receptor (Ca-
SR). The binding of extracellular calcium (first messenger) to
Ca-SR in plasma membrane activates G
q
proteins, stimulates
phospholipase C (PLC)-b activity, and increases intracellular
IP
3
levels, leading to intracellular calcium release (second
messenger) [1,2]. The expression of Ca-SR has been identified
in parathyroid [2], thyroid [3], kidney [4], bone [5] and GI
tract [6], the organs involved in systemic calcium home-
ostasis. Defective Ca-SRs are involved in genetic diseases
linked to calcium homeostasis. Ca-SR and its isoforms or
homologous receptors may represent novel clinical targets
for treatment of these diseases and others like osteoporosis.
Calcium handling is essential for the homeostatic control
of cardiovascular functions, which may not couple directly
to systemic calcium homeostasis. Whether Ca-SR has a
functional role to play in the cardiovascular system is
unclear. Ca-SR proteins were detected, but not the corres-
ponding transcripts, in mesenteric resistant artery tissues [7].
This observation led to the conclusion that Ca-SR was
actually expressed in perivascular nerves with the corres-
ponding mRNA residing in the neuronal soma away from
the isolated blood vessel wall. Interestingly, a recent study
claimed that Ca-SR was present in smooth muscle cells
of spiral modiolar artery of gerbils [8]. The functions of
Ca-SR in these smooth muscle cells were not studied.
Cardiac tissue is very sensitive to calcium homeostasis.
An increased intracellular calcium concentration, either due
to the increased extracellular calcium entry through voltage-
gated calcium channels or the increased intracellular
calcium release, would trigger the contraction of myocytes.
Overloading of cellular calcium, on the other hand, leads to
cell death and heart injury. To date, the expression of Ca-SR
in cardiomyocytes had not been reported, less alone the
function of these receptors. Several lines of evidence are
presented in this communication that demonstrate the
existence of Ca-SR in rat heart by identifying the mRNA
and proteins of Ca-SR incardiactissuesand by delineating
the functional regulation of Ca-SR incardiac myocytes.
Ca-SR may present itself as a novel target by which the
cardiac functions can be modulated.
Materials and methods
RT-PCR analysis of the Ca-SR
Male Sprague–Dawley rats (10–12 weeks old) were used
with an approved protocol (University Committee on
Animal Care and Supply of University of Saskatchewan).
Correspondence to: R. Wang, Department of Physiology,
University of Saskatchewan, Saskatoon, SK, Canada S7N 5E5.
Fax: + 1 306 966 6532, Tel.: + 1 306 966 6592,
E-mail: wangrui@duke.usask.ca
Abbreviations: Ca-SR, calcium-sensing receptor; IP
3
, inositol 1,4,5-
triphosphate; TG, Thapsigargin.
(Received 9 March 2003, revised 10 April 2003,
accepted 30 April 2003)
Eur. J. Biochem. 270, 2680–2688 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03645.x
Total RNA was extracted from isolated tissue with an
RNeasy Total RNA Kit (Qiagen) and treated with RNase-
free DNase I (Ambion). First-strand cDNA was made by
reverse transcribing 2 lg of DNase I-treated total RNA
with MMuLV reverse transcriptase (Perkin-Elmer) using
random hexamers in a total volume of 20 lL. The RT
reaction was carried out at room temperature for 15 min
followed by incubation at 42 °C for 1 h. Five microliters of
RT reaction mixture were used for PCR amplification in a
volume of 50 lL using Advanced PCR II mixer (Clontech)
with gene specific primers designed on reported sequences of
rat Ca-SR (GenBank accession number U20289). Portions
of the Ca-SR cDNA were amplified using the primer pairs
that were Ca-SR (forward), 5¢-ttcggcatcagctttgtg-3¢;and
Ca-SR (reverse), 5¢-tgaagatgatttcgtcttcc-3¢. PCR amplifica-
tion consisted of 35 cycles of denaturation at 94 °Cfor20s,
annealing at 60 °C for 20 s, and polymerization at 68 °Cfor
30 s. Aliquots (5 lL) of PCR reactions were electropho-
resed through ethidium bromide-stained 1.2% agarose gels.
Nucleotide sequence analysis
Gel purified PCR-amplified Ca-SR products were cloned
into the pCR2.1 TA cloning vector (Invitrogen). The
automated sequence analysis was performed on three
independent clones using an ABI-373A (Applied Biosys-
tems Inc.) sequencer.
Western blot analysis
Membrane proteins were prepared as described previously
[9]. Briefly, tissues were homogenized with a Polytron
homogenizer in 1 mL Tris-buffered saline (10 m
M
Tris,
0.3
M
sucrose and 1 m
M
EDTA) containing protease
inhibitor mixture [9]. The homogenate was centrifuged at
6000 g for 15 min at 4 °C to remove nuclei and undisrupted
cells. The supernatant was further centrifuged at 40 000 g
for 1 h at 4 °C. Resulting pellets were then washed and
resuspended with the same Tris-buffered saline without
sucrose. Protein concentration was determined using a
Bio-Rad protein assay solution with BSA as standard.
Membrane proteins (20 lg) were electrophoresed through
standard 10% SDS-PAGE in Tris-glycine electrophoresis
buffer [125 m
M
Tris, 959 m
M
glycine (pH 8.3), and 0.5%
SDS] and blotted onto nitrocellulose membrane in transfer-
ring buffer [39 m
M
glycine, 48 m
M
Tris (pH 8.3) and 20%
methanol] at 80 mA for 1.5 h in a water-cooled transfer
apparatus. The membrane was blocked in a blocking buffer
NaCl/P
i
containing 3% skimmed milk at room temperature
for 2 h. The membrane was then incubated overnight at 4 °C
with 1 : 500 diluted affinity-purified polyclonal antibody
against Ca-SR in blocking buffer. Unless otherwise specified,
anti-(Ca-SR) Igs were from Alpha Diagnostic International
Inc. (San Antonio, TX, USA).
After the membrane was washed five times in NaCl/P
i
,it
was incubated with goat anti-(rabbit IgG) Ig conjugated
with horseradish peroxidase diluted to 1 : 5000 in the
blocking buffer for 2 h at room temperature. Antibody–
antigen complexes were detected by chemiluminescence
using chemiluminescent substrate kit (NEN Life Sciences).
As a control, immunoblotting was carried out as described
above without anti-(Ca-SR) Ig. Anti-actin Ig (Chemicon
International) was used at a dilution of 1 : 400 to detect the
expression level of a-actin in the isolated tissues as the
house-keeping internal control.
Immunohistochemistry study
Sprague–Dawley rats were anasthetized by intraperitoneal
injection of sodium pentobarbital (60 mgÆkg
)1
body
weight). The rats were perfused through the left ventricle
with ice cold NaCl/P
i
(pH 7.4) for 1 min and ice cold 4%
paraformaldehyde in NaCl/P
i
for 2 min. The tissues were
removed and fixed in 4% paraformaldehyde in NaCl/P
i
at
4 °C overnight. Specimens were dehydrated with 20%
sucrose in NaCl/P
i
for 24 h. Cryostat sections (5 lm) were
cut on a Micron cryostat at )20 °C and thaw-mounted onto
ethanol-cleaned slides coated with 1% gelatin. Sections were
postfixed in 4% paraformaldehyde for 20 min, followed by
15 min incubation in 5 lgÆmL
)1
proteinase K (Ambion) for
antigen retrieval at 37 °C. After washing with NaCl/P
i
,the
sections were blocked with 5% normal horse serum in
NaCl/P
i
for 1 h at room temperature and then incubated
with 1 : 500 polyclonal Ig against Ca-SR (Alpha Diagnostic
International) in NaCl/P
i
containing 2.5% normal horse
serum and 0.1% Triton X-100 overnight at 4 °C. After
rinsing with NaCl/P
i
, staining was performed with the
Vectastain Universal Elite ABC Kit (Vector Laboratories,
Burlington) according to manufacturer’s instructions.
Briefly, after washing three times in NaCl/P
i
, sections were
incubated for 30 min with diluted biotinylated universal
secondary IgG. After washing with NaCl/P
i
, the sections
were exposed to Vector ABC reagent (avidin coupled to
biotinylated horseradish peroxidase) for 30 min. Sections
were washed again in NaCl/P
i
and visualized by incubating
with horseradish peroxidase substrate containing 0.02%
diaminobenzidine, 0.3% nickel ammonium sulfate and
0.002% hydrogen peroxide (Vector Laboratories). The
appearance of reaction product was monitored and photo-
graphed under bright-field illumination. As a control, some
sections were not incubated with primary antibody.
Adult rat myocyte isolation
Adult (6–8 weeks old) male Sprague–Dawley rats were
anesthetized with pentobarbital sodium (50 mgÆkg
)1
, i.p.).
The heart was removed and firstly perfused via the aorta at
37 °C with standard Tyrode’s solution for about 5 min until
the effluent was clear. Standard Tyrode’s solution was
composedof(inmM):NaCl,136;KCl,5.4;NaH
2
PO
4
,0.33;
MgCl
2
,1.0;CaCl
2
, 2.0; dextrose, 10 and Hepes, 10 (pH
adjusted to 7.4 with NaOH), and was maintained at room
temperature and equilibrated with 95% O
2
and 5% CO
2
.
Then the heart was perfused with Ca
2+
-free Tyrode’s
solution for 5 min and Ca
2+
-free Tyrode’s solution con-
taining 120 UÆmL
)1
collagenase for 70 min. Ventricular
tissues (2–3 mm in diameter) were excised and placed in a
high [K
+
] solution composed of (in mM): glutamic acid, 70;
taurin, 15; KCl, 30; KH
2
PO
4
, 10; Hepes, 10; MgCl
2
,0.5;
EDTA, 0.5 and glucose, 10 (pH adjusted to 7.3–7.4 with
KOH). Myocytes were isolated by trituration with a Pasture
pipette and collected by centrifuging at 600 r.p.m. for 1 min
at room temperature. Cells were re-suspended in the high
[K
+
] solution and kept at room temperature [10].
Ó FEBS 2003 Calcium-sensingreceptorsin heart (Eur. J. Biochem. 270) 2681
Fura-2 measurements of [Ca
2+
]
i
Single ventricular myocytes attached to the glass bottom of
Petri dishes coated with laminin (10 lgÆmL
)1
,500lLper
dish, with blow-drying). Cells were loaded with 2 l
M
fura
2-AM (Sigma) for 60 min at room temperature in a Hepes
buffer composed of (in mM): NaCl, 125; KCl, 3.0; MgSO
4
,
1.2; Na
2
PO
4
,2.0;CaCl
2
, 1.8; dextrose, 10.5; Hepes, 32 and
0.1% BSA (pH 7.4). Thereafter, myocytes were rinsed with
normal Hepes buffer twice to remove the remaining dye and
then equilibrated for 30 min at room temperature. The
tested compounds were added directly to petri dishes to
reach the desired final concentrations. The fura-2 loaded
myocytes were alternatively excited at 340 and 380 nm from
a monochromator (SpectraMASTER, Olympus America,
Melville, NY, USA). Fluorescent images of ventricular
myocytes were observed through an inverted phase-contrast
microscope (Olympus IX70, Tokyo) and video images of
fluorescence at 510 nm emission were collected at 2 Hz
using an intensified CCD camera system (AstroCam,
Olympus Life Science resources, Cambridge, UK) with
the output digitized at 768 · 512 pixels. The ratio of the
fluorescence intensities at 340 : 380 nm excitations was
monitored and processed with computer software (
ULTRA-
VIEW
, PerkinElmer Life Sciences Inc., Boston, MA, USA).
Measurement of IP
3
formation
Isolated rat ventricular myocytes were incubated for 4 h in
serum-free and inositol-free DMEM, to which 5 lCiÆmL
)1
myo-2-[
3
H]inositol (Du Pont Canada Inc.) were added. The
cells were subjected to different stimuli for 60 mins, and the
reaction was terminated by adding 0.9 mL methanol/
chloroform/HCl (40 : 20 : 1, v/v/v). The initial inositol-
phosphate (IP) pool of the aqueous phase composed of
inositol 4-phosphate, inositol 4,5-biphosphate and IP
3
was
eluted consecutively by ion-exchange chromatography
(AG1-X8 resin, Bio-Rad Laboratories). The lipid phase
was counted to measure the phosphatidylinositol phosphate
(PIP) lipid pool. IP
3
was expressed as a relative value of
(IP
3
/PIP) · 10
3
(arbitrary units) to correct for the variation
in the labeling of the lipid pool.
Chemicals and data analysis
Thapsigargin (TG) was purchased from Calbiochem.
U73122, U73343, spermine, nifedipine, CdCl
2
and other
chemicals were from Sigma. Data were expressed as
means ± SEM. Differences between treatments in the
same cells were evaluated by paired Student’s t-test or in
conjunction with Newman–Keuls test. A significant level
of difference was determined when P <0.05.
Results
Transcriptional expression of
Ca-SR
in rat cardiac tissues
Expression of Ca-SR mRNA was examined using RT-
PCR. A cDNA fragment of 234 bp corresponding to the
selected Ca-SR mRNA sequence was detected in both rat
atrium and ventricle (Fig. 1A). In the absence of reverse
transcriptase, no PCR-amplified fragment could be detec-
ted, indicating the tested RNA samples were free of
genomic DNA contamination. This 234 bp PCR fragment
was gel-purified, subcloned into plasmid vectors, and
sequenced. The derived sequences from three independent
clones were identical to the Ca-SR cDNAs from rat
parathyroid, kidney [4] and brain [11]. The expression
level of Ca-SR mRNA in thyroid appears to be much
greater than that incardiac tissues. However, these results
were derived from RT-PCR, which is a qualitative rather
than quantitative mRNA assay. Therefore, it would be
inappropriate to predict the protein levels based on
RT-PCR results shown in Fig. 1A.
Protein expression of Ca-SR in rat cardiac tissues
The expression of Ca-SR protein was examined using
Western blotting on whole-tissue extract. Ca-SR proteins
with a relative molecular mass between 120 and 140 kDa
were detected in rat atrium and ventricle (Fig. 1B) or in
whole heart tissues (Fig. 1C). The same 120–140 kDa band
was also detected in thyroid, liver, parathyroid and kidney
tissues, which serve as positive control. While the band of
PCR product for atrium was faint (Fig. 1A), the expression
levels of Ca-SR proteins were similar between atrium and
ventricular tissues (Fig. 1B), which may indicate a relative
instability of Ca-SR mRNA in rat atrium. In the absence of
antibody, no positive band was identified (Fig. 1B). Fur-
thermore, preadsorption of anti-Ca-SR antibody with
standard Ca-SR antigen eliminated the 140 kDa band
(Fig. 1C). Together, these results indicate the specificity of
the anti-(Ca-SR) Ig.
Immunohistochemistry study on the expression
of Ca-SR protein in different tissues
Strong immunostaining was observed in liver cells (Fig. 2A
and B) as reported by Canaff et al. [12]. In heart, deep
brown immunostaining was present throughout all cardio-
myocytes (Fig. 2D,E), indicating the expression of Ca-SR at
protein level in rat heart. Lack of specific staining was
demonstrated in control sections in the absence of anti-
Ca-SR antibody (Fig. 2C,F).
Protein expression of Ca-SR in isolated rat cardiac
myocytes
To confirm that Ca-SR was expressed incardiac myocytes,
rather than neuronal or other types of cell in heart tissue,
ventricular and atrial myocytes were isolated separately and
the expression of Ca-SR proteins in these cells was
examined. Similar to the observations on whole heart
tissue, Ca-SR proteins were identified in the isolated
myocytes (Fig. 3). Compared to ventricular and atrial
membrane preparations, membrane preparation from liver
cells had a low protein content as evidenced by low actin
level (Figs 1 and 3). Preadsorption of the anti-Ca-SR Igs
with standard Ca-SR antigen completely eliminated the
140 kDa band (Fig. 3B). In these experiments, the anti-
(Ca-SR) Ig was from Affinity BioRegents, Inc. (Golden,
CO, USA) at dilution of 1 : 400. In all other Western blot
and immnunostaining studies, anti-(Ca-SR) Ig from Alpha
Diagnostic International were used. The same results using
2682 R. Wang et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the anti-(Ca-SR) Igs from different suppliers further validate
the specificity of Ca-SR proteins detected in rat cardio-
myocytes.
Extracellular calcium, gadolinium and spermine induced
changes in intracellular calcium concentration
Elevating [Ca
2+
]
o
from 0 m
M
to > 1.5 m
M
evoked an
increase in intracellular calcium concentration in more than
90% of isolated ventricular myocytes in a given observation
field (Fig. 4A). The maximal increase in intracellular
calcium concentrations was obtained with 5–10 m
M
extra-
cellular calcium (Fig. 4B). After changing extracellular
calcium concentration back to 0 m
M
, the increased intra-
cellular calcium concentration declined gradually (Fig. 4C).
Consecutive exposure of freshly isolated rat ventricular
myocytes to extracellular gadolinium also induced a
concentration-dependent increase in intracellular calcium
(Fig. 4D).
With 1 m
M
Ca
2+
in the bath solution, spermine from
1–10 m
M
induced a time- and concentration-dependent
increase in intracellular calcium (Fig. 5). At 10 m
M
,sper-
mine produced a Ôcalcium burstÕ in a total of 27 cells from
five dishes (P < 0.05). In less than 1 min after spermine
application, all cells in the observation field contracted and
quickly ÔexplodedÕ (Fig. 5A and B). This Ôcalcium burstÕ,
however, was not observed after calcium was removed
completely from the bath solution. As shown in Fig. 5C,D,
spermine still increased intracellular calcium but in a less
dramatic way and all cells survived from this spermine
treatment.
The role of intracellular calcium release and the
phospholipase C (PLC) pathway in the extracellular
calcium-induced increase in [Ca
2+
]
i
Isolatedmyocyteswerepretreatedfor10minwith10l
M
TG that inhibits the refilling of the IP
3
-sensitive calcium
release pools [12,13]. Subsequently, extracellular calcium
was changed from 0–1.5 m
M
, which failed to elicit any
increase in [Ca
2+
]
i
. This effect was observed in a total of 25
cells from six Petri dishes (n ¼ 6, P < 0.05) (Fig. 6A).
Preincubation of myocytes with TG also abolished 0.3 m
M
Gd
3+
-induced (n ¼ 8) or 5 m
M
spermine-induced (n ¼ 6)
increase in the [Ca
2+
]
i
level (not shown). U73122 is a
phosphatidylinositol-specific PLC blocker [3,14]. Pretreat-
ment with U73122 for 10 min eliminated the effect of
extracellular calcium-induced intracellular calcium release
Fig. 1. Expression of Ca-SR in rat cardiac
tissues. (A) Detection of Ca-SR mRNA by
RT-PCR in rat heart in the presence or
absence of reverse transcriptase (RT). M,
DNA marker; bp, base pairs. Similar results
were obtained in four other experiments. (B)
Detection of Ca-SR proteins by Western blot
in various rat tissues using anti-(Ca-SR) Ig
(left) or in the absence of anti-(Ca-SR) Ig
(right). (C) Detection of Ca-SR proteins by
Western blot in various rat tissues using anti-
(Ca-SR) Ig without preadsorption (left) or
after incubation with excess Ca-SR antigens
overnight at 4 °C (right).
Ó FEBS 2003 Calcium-sensingreceptorsin heart (Eur. J. Biochem. 270) 2683
(n ¼ 4) (Fig. 6B). This treatment also abolished 0.3 m
M
Gd
3+
-induced (n ¼ 4) or 5 m
M
spermine-induced (n ¼ 4)
increase in intracellular calcium (not shown). Under the
same condition but without TG or U73122 pretreatment,
extracellular calcium induced significant increase in [Ca
2+
]
i
(Fig. 4C). On the other hand, pretreatment of cells with
U73334 at 10 l
M
, an inactivated analogue of U73122 [8],
for 10 min did not prevent the increase in [Ca
2+
]
i
induced
by extracellular calcium (n ¼ 5, P < 0.05) (Fig. 6C). These
results suggest that activation of Ca-SR resulted in stimu-
lation of PLC pathway, and the subsequent production of
IP
3
stimulated the TG-sensitive IP
3
receptors, leading to
intracellular calcium increase.
Involvement of extracellular calcium entry in the
extracellular calcium-induced increase in [Ca
2+
]
i
To examine whether the increased [Ca
2+
]
i
was related to the
activity of Na
+
/Ca
2+
exchanger, NiCl
2
(10 m
M
)was
applied to the isolated myocytes [15]. Under this condition,
increasing [Ca
2+
]
o
from 0–1.5 m
M
significantly increased
[Ca
2+
]
i
(data not shown). Thus, the activity of Na
+
/Ca
2+
exchanger in plasma membrane could not explain the
increase in [Ca
2+
]
i
upon the stimulation of Ca-SR. In
another series of experiments, myocytes were pretreated
with 200 l
M
CdCl
2
for 10 min. CdCl
2
treatment alone did
not alter [Ca
2+
]
i
.WithCdCl
2
pretreatment, an increase in
[Ca
2+
]
i
induced by extracellular calcium was again observed
(Fig.7A).Furthermore,increasing[Ca
2+
]
o
from 0–1.5 m
M
still significantly increased intracellular calciumin 30 cells
from five Petri dishes in the presence of nifedipine (10 l
M
)
(not shown).
Changes in intracellular IP
3
levels in response
to different Ca-SR stimuli
An increased IP
3
formation in rat ventricular myocytes was
observed after incubation with 3 m
M
calcium, 0.3 m
M
gadolinium, or 1 m
M
spermine (Fig. 7B). The largest IP
3
response was induced by extracellular calcium when com-
pared with the effects of gadolinium and spermine.
Discussion
Expression of Ca-SR in cells with functions unrelated to
systemic calcium homeostasis has been demonstrated in
many cases. For instance, expression of Ca-SR in neurons
suggests the coupling of [Ca
2+
]
o
to neuronal activities [11].
Fig. 2. Immunohistochemical detection of Ca-SR in rat cardiac tissues. Tissue sections of rat liver (A–C) and rat heart (D–F) were processed in the
presence (A,B,D,E) or absence of anti-Ca-SR Ig (C and F). Magnification was · 95 (A,D,F); · 190 (B,C); · 380 (E). Representative results were
shown from three different experiments.
2684 R. Wang et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Identification of Ca-SR in spiral modiolar artery, located
between the eighth cranial nerve and the bond of the
cochlear modiolus, also indicates that the changes in
[Ca
2+
]
o
may somehow affect smooth muscle functions.
The involvement of Ca-SR in diverse cellular functions
implies broad physiological functions beyond the regulation
of systemic calcium homeostasis. Our present study for the
first time demonstrated the existence of Ca-SR in cardiac
myocytes. This conclusion is based on several lines of
evidence: (a) transcripts of Ca-SR were clearly detected in
cardiac tissue and the sequences of these transcripts were
confirmed as identical to the known sequence of Ca-SR; (b)
Ca-SR proteins were identified incardiac tissue as well as in
isolated atrial and ventricular myocytes; (c) Immunohisto-
logical staining clearly located Ca-SR proteins in cardiac
tissues; (d) increase in [Ca
2+
]
o
increased intracellular free
calcium levels, which was not mediated by extracellular
calcium entry through either voltage-gated Ca
2+
channels
or a Na+/Ca
2+
exchanger. Release of intracellular calcium
from thapsigargin-sensitive calcium pools after activation of
PLC pathway was responsible for the extracellular calcium-
induced [Ca
2+
]
i
;(e)[Ca
2+
]
i
increase in isolated ventricular
myocytes was induced by spermine at concentrations
between 1–10 m
M
, which was the concentration range used
in many other studies to elucidate the presence of Ca-SR in
different preparations [6,12,16].
Ca-SR incardiac cells senses the changes
in extracellular calcium concentrations
An increase from 0–1.5 m
M
in [Ca
2+
]
o
triggered an increase
in intracellular calciumand this effect was maximal at
Fig. 4. Extracellular calcium-induced intracellular calcium increase in
freshly isolated rat ventricular myocytes. (A) The same groups of ven-
tricular myocytes were exposed consecutively to different [Ca
2+
]
o
.
Changes in the density of pseudo-greyscale indicate different levels of
intracellular calcium concentrations with the black representing lower
[Ca
2+
]. (B) Concentration dependent effects of extracellular calcium
on [Ca
2+
]
i
in ventricular myocytes. Changes in 4–6 cells in each culture
dish were analysed and a total of four culture dishes were used at each
calcium concentration. *P < 0.05 vs. data obtained at 0 m
M
of
extracellular calcium. (C) Reversibility of the extracellular calcium-
induced [Ca
2+
]
o
change. (D) The same groups of ventricular myocytes
were exposed consecutively to different [Gd
3+
]
o
.
Fig. 3. Detection of Ca-SR receptor in isolated rat atrial and ventricular
myocytes using the anti-(Ca-SR) Igs (Affinity BioRegents, Inc.). (A) Ca-
SR proteins were detected in ventricular and atrial myocytes as well as
in liver. (B) Anti-(Ca-SR) Igs were incubated with excess Ca-SR
antigens overnight at 4 °C before being used in Western blot experi-
ments.
Ó FEBS 2003 Calcium-sensingreceptorsin heart (Eur. J. Biochem. 270) 2685
5m
M
extracellular calcium. The physiological relevance
of this narrow range of [Ca
2+
]
o
in cardiac myocytes should
be commented on. Under what circumstances would the
extracellular calcium be in the range of 0–1.5 m
M
in heart?
Intracellular calcium changes have been observed in para-
thyroid hormone-releasing and calcitonin cells in response
to [Ca
2+
]
o
changes from 0.75– 3 m
M
[17]. Brown et al.
described a steep dose–response relationship of the activa-
tion of Ca-SR by extracellular calciumin parathyroid cells
[2]. The plasma levels of ionized Ca
2+
are between 1.0–
1.3 m
M
[6]. The [Ca
2+
]
o
can be significantly lowered within
the interstitial fluid of the beating heart [18], especially
within the T-tubular system of heart. This system is a
restricted plasma membrane invagination and the calcium
content therein is limited. The sustained membrane depo-
larization of heart membrane has been reported to lead to
calcium depletion in T-tubular system [19]. The lowering of
[Ca
2+
]
o
would reduce the activity of Ca-SR in cardio-
myocytes, lowering [Ca
2+
]
i
and protecting cardiac muscles
from sustained contraction. Upon repolarization, [Ca
2+
]
o
can be restored to a physiological level around 1.5 m
M
.The
consequent re-activation of Ca-SR would then restore
normal contractility of cardiac muscles by normalizing
[Ca
2+
]
i
.Can[Ca
2+
]
o
be further elevated from 1.5–5 m
M
in
cardiac muscle? Similar to our results incardiac myocytes,
Ca-SR in human antral gastrin cells has been reported to
be sensitive to extracellular calcium concentrations ranged
from 1.8–5.4 m
M
[6]. Under certain in vivo conditions, the
luminal surface of the gastrin cells can be exposed to 15 m
M
extracellular calcium [20]. As high as 40 m
M
of extracellular
calcium in the direct vicinity of bone-osteoclasts has been
observed [8]. There are several scenarios for which [Ca
2+
]
o
in the vicinity of cardiac myocytes, especially in T-tubule
system, may temporarily increase, such as the extrusion of
intracellular calcium from the excited myocytes and the
Fig. 5. Extracellular spermine-induced [Ca
2+
]
i
in freshly isolated rat
ventricular myocytes. Changes in the density of pseudo-greyscale
indicate different levels of [Ca
2+
]
i
with black representing lower cal-
cium levels. (A) A sudden exposure of ventricular myocytes to 10 m
M
spermine triggered an intracellular calcium burst and cell death with
1m
M
calcium in the bath solution. (B) Time course of the increase in
[Ca
2+
]
i
induced by a sudden exposure to 10 m
M
spermine with 1 m
M
calcium in the bath solution. (C) Spermine induced a gradual increase
in [Ca
2+
]
i
with 0 m
M
calcium in the bath solution. All cells survived
under this gradual spermine exposure condition. (D) Time course of
theincreasein[Ca
2+
]
i
induced by various concentrations of spermine
with 0 m
M
calcium in the bath solution (total 10 cells from three dif-
ferent Petri dishes).
Fig. 6. Signal transduction pathways involved in the extracellular cal-
cium-induced increase in [Ca
2+
]
i
in isolated rat ventricular myocytes. (A)
Thapsigargin blocked the effect of extracellular calcium-induced
increase in [Ca
2+
]
i
. (B) Pretreatment of cells with 10 l
M
U73122
eliminated the effect of extracellular calcium-induced intracellular
calcium release. (C) Pretreatment of cells with 10 l
M
U73343 did not
prevent the increase in [Ca
2+
]
i
. induced by extracellular calcium.
2686 R. Wang et al. (Eur. J. Biochem. 270) Ó FEBS 2003
release of calcium from necrotic myocytes. The healthy
myocyte in the neighborhood of necrotic myocytes would
face relatively high [Ca
2+
]
o
and increase their Ca-SR
activity. Thus, the contractility of these healthy myocytes
would be increased to maintain the pump function
by increasing their intracellular calcium levels. Contrary
to the conventional thought of a static extracellular calcium
level, [Ca
2+
]
o
in heart tissues may undergo fluctuations
depending on the activity of the heart. The presence of
Ca-SR incardiac myocytes may co-ordinate cellular
activities with the dynamic changes in [Ca
2+
]
o
in the
vicinity of cardiomyocytes [1].
The naturally occurring polyamines, including spermine,
spermidine and putrescine, are involved in the synthesis of
nucleic acids and proteins in eukaryotic and prokaryotic
cells. They play an important role in the regulation of
cellular proliferation and differentiation [21]. For the
regulation of cardiac function, polyamines are also import-
ant. Previous studies have provided evidence that polyam-
ines promote cardiac hypertrophy [22,23]. In spontaneously
hypertensive rats, an increased left ventricular mass [24] or
cardiac hypertrophy [25] was associated with increased
spermine and spermidine contents. The molecular mecha-
nisms underlying the cellular actions of polyamines include
the activation of different plasmalemmal ion channels
[26–28] as well as the stimulation of Ca-SR [29]. In our
study, spermine elicited an extracellular calcium-dependent
intracellular calcium response in isolated cardiomyocytes. In
the absence of extracellular calcium, the spermine-induced
[Ca
2+
]
i
increase was less dramatic than that in the presence
of 1 m
M
extracellular calciumand no Ôcalcium burstÕ and
cellular destruction were observed. Similar extracellular
calcium dependency of the effects of spermine on Ca-SR has
been noticed in other previous studies [6,29].
The physiological concentration of plasma spermine is in
the low micromolar range [29,30]. In the study by Quinn
et al. [29], spermine was used at concentrations from
0.1–1 m
M
to test the Ca-SR-mediated intracellular calcium
response in Ca-SR-expressing HEK cells. Ray et al.[6]
reported the effect of spermine on Ca-SR at concentrations
between 0.1–1 m
M
. In hepatocytes, spermine-induced
[Ca
2+
]
i
increase was manifested at spermine concentrations
from 1.25–10 m
M
[12]. Similarly, in our study, a spermine
response was observed at concentrations between 1–10 m
M
.
No effect was observed when spermine concentration was
lower than 1 m
M
. Nevertheless, the physiological signifi-
cance of this spermine effect at these concentrations can still
be appreciated. Polyamine secretion from some neurons has
been indicated [31], presenting the possibility that local
concentration of spermine can be much higher than the
circulating concentration. Moreover, the tissue spermine
content of ventricular tissues was increased from
68 pmolÆmg
)1
in normotensive Wistar–Kyoto rats to
376 pmolÆmg
)1
in spontaneously hypertensive rats [24].
This observation may also shed light on the pathophysio-
logical significance of the effect of spermine at relatively
high concentrations on Ca-SR in hearts. The spermin-
induced increase in [Ca
2+
]
i
alone may not suffice to
demonstrate conclusively the involvement of Ca-SR but
when taken in conjunction with the effects of extracellular
calcium and gadolinium on [Ca
2+
]
i
, as well as the detection
of Ca-SR at mRNA and protein levels, does provide a line
of evidence for the presence and function of Ca-SR in
cardiac myocytes. The physiological importance of Ca-SR
in cardiomyocytes can be better understood by comparing
the structure and function of hearts with or without Ca-SR
deficiency. Ca-SR knock-out mice provide an avenue for
this kind of study. However, cardiovascular functions of
Ca-SR knock-out mice have not been reported to our
knowledge. Loss of Ca-SR in parathyroid gland in knock-
out mice results in hyperparathyroidism, hypercalcemia,
and growth retardation [32]. These alterations may also
significantly and indirectly affect cardiac function, mingled
with any potential direct cardiac outcome due to the lack of
cardiac Ca-SR. Therefore, organ-selective or heart-selective
inactivation or activation of Ca-SR in living animals should
be engineered, which may help to better determine the direct
cardiac outcome of cardiac Ca-SR deficiency.
In summary, Ca-SR may play an important physiological
role in the modulation of cardiac functions under both
physiological and pathophysiological conditions. Increased
local calcium concentration is sensed by myocytes via
Ca-SR and lead to increased cardiac activity. Increased
extracellular polyamine concentration in heart, on the other
hand, may stimulate Ca-SR on cardiomyocytes to promote
cardiac hypertrophy. Due to the limited access to specific
Fig. 7. Changes in intracellular calciumand IP
3
levels. (A) Effect of
CdCl
2
on the extracellular calcium-induced increase in [Ca
2+
]
i
.in
isolated rat ventricular myocytes (total of 32 cells from five Petri
dishes). [Ca
2+
]
i
. was determined when the changes reached the maxi-
mum levels. (B) IP
3
formation in isolated rat ventricular myocytes.
n ¼ 5 for each group. *P < 0.05 compared with control group.
Ó FEBS 2003 Calcium-sensingreceptorsin heart (Eur. J. Biochem. 270) 2687
blockers of Ca-SR, whether polyamine-induced cardiac
hypertrophy is mediated by Ca-SR cannot be readily tested
at the moment. Nevertheless, delineation of the interaction
among extracellular calcium levels, polyamine concentra-
tions, functional status of Ca-SR, and myocyte apoptosis
and proliferation would help better understand the mech-
anisms of cardiac hypertrophy as well as its management.
Acknowledgements
This study was supported by an operating grant from Canadian
Institutes of Health Research (CIHR). R. Wang is an Investigator of
CIHR. L. Wu is a New Investigator of CIHR.
References
1. Brown, E.M. & MacLeod, R.J. (2001) Extracellular calcium
sensing and extracellular calcium signaling. Physiol. Rev. 81,
239–297.
2. Brown,E.M.,Gamba,G.,Riccardi,D.,Lombardi,M.&Butters,
R. (1993) Cloning and characterization of an extracellular Ca
2+
-
sensing receptor from bovine parathyroid. Nature 366, 575–580.
3. McGehee, D.S., Aldersberg, M., Liu, J.P., Hsuing, S., Heath, M.J.
& Tamir, H. (1997) Mechanisms of extracellular Ca
2+
receptor-
stimulated hormone release from sheep thyroid parafollicular
cells. J. Physiol. (Lond.) 502, 31–44.
4. Riccardi, D., Park, J., Lee, W S., Gamba, G., Brown, E.M. &
Hebert, S.C. (1995) Cloning and functional expression of a rat
kidney extracellular calcium/polyvalent cation-sensing receptor.
Proc. Natl. Acad. Sci. USA. 92, 131–135.
5. Zaidi, M., Alam, A.S., Huang, C.L., Pazianas, M., Bax, C.M.,
Bax, B.E., Moonga, B.S., Bevis, P.J. & Shankar, V.S. (1993)
Extracellular Ca
2+
sensing by the osteoclast. Cell Calcium 14,
271–277.
6. Ray,J.M.,Squires,P.E.,Curtis,S.B.,Meloche,M.R.&Buchan,
A.M.J. (1997) Expression of the calcium-sensing receptor on
human antral gastrin cells. J. Clin. Invest. 99, 2328–2333.
7. Bukoski, R.D., Bian, K., Wang, Y. & Mupanomunda, M. (1997)
Perivascular sensory nerve Ca
2+
receptor and Ca
2+
-induced
relaxation of isolated arteries. Hypertension 30, 1431–1439.
8. Wonneberger, K., Scofield, M.A. & Wangemann, P. (2000) Evi-
dence for a calcium-sensing receptor in the vascular smooth
muscle cells of the spiral modiolar artery. J. Membr. Biol. 175,
203–212.
9. Xu,C.,Lu,Y.,Tang,G.&Wang,R.(1999)Expressionofvol-
tage-dependent K
+
channel genes in mesenteric artery smooth
muscle cells. Am. J. Physiol. 277, G1055–G1063.
10. Lu, Y., Yue, L., Wang, Z. & Nattel, S. (1998) Effects of the
diuretic agent indapamide on Na
+
, transient outward, and
delayed rectifier currents in canine atrial myocytes. Circ. Res. 83,
158–166.
11. Ruat, M., Molliver, M.E., Snowman, A.M. & Snyder, S.H. (1995)
Calcium sensing receptor: molecular cloning in rat and localiza-
tion to nerve terminals. Proc. Natl. Acad. Sci. USA 92, 3161–3165.
12. Canaff,L.,Petit,J.L.,Kisiel,M.,Watson,P.H.,Gascon-Barre
´
,M.
& Hendy, G.N. (2001) Extracellular calcium-sensing receptor is
expressedinrathepatocytes.J. Biol. Chem. 276, 4070–4079.
13. Breitwieser, G.E. & Gama, L. (2001) Calcium-sensing receptor
activation induces intracellular calcium oscillations. Am. J. Phy-
siol. 280, C1412–C1421.
14. Godwin, S.L. & Soltoff, S.P. (2002) Calcium-sensing receptor-
mediated activation of phospholipase C-gamma1 is downstream
of phospholipase C-beta and protein kinase C in MC3T3–E1
osteoblasts. Bone 30, 559–566.
15. Janiak, R., Lewartowski, B. & Langer, G.A. (1996) Functional
coupling between sarcoplasmic reticulum and Na/Ca exchange in
single myocytes of guinea-pig and rat heart. J. Mol. Cell. Cardiol.
28, 253–264.
16. Sanders, J.L., Chattopadhyay, N., Kifor, O., Yamaguchi, T.,
Butters, R.R. & Brown, E.M. (2000) Extracellular calcium-sensing
receptor expression and its potential role in regulating parathyroid
hormone-related peptide secretion in human breast cancer cell
lines. Endocrinology 141, 4357–4364.
17. Chattopadhyay, N., Mithal, A. & Brown, E.M. (1996) The cal-
cium-sensing receptor: a window into the physiology and patho-
physiology of mineral ion metabolism. Endocri. Rev. 17, 289–307.
18. Bers, D.M. (1983) Early transient depletion of extracellular Ca
during individual cardiac muscle contractions. Am. J. Physiol.
244, H462–H468.
19. Almers, W., Fink, R. & Palade, P.T. (1981) Calcium depletion in
frog muscle tubules: the decline of calcium current under main-
tained depolarization. J. Physiol. (Lond.) 312, 177–207.
20. Fordtran, J.S. & Locklear, T.W. (1996) Ionic constituents and
osmolality of gastric and small intestinal fluids after eating. Am. J.
Dig. Dis. 11, 503–521.
21. Bartolome, J., Huguenard, J. & Slotkin, T.A. (1980) Role of
ornithine decarboxylase incardiac growth and hypertrophy.
Science 210, 793–794.
22. Flamigni, F., Rossini, C., Stefanelli, C. & Caldarera, C.M. (1986)
Polyamine metabolism and function in the heart. J. Mol. Cell.
Cardiol. 18, 3–11.
23. Pegg, A.E. & Hibasami, H. (1980) Polyamine metabolism during
cardiac hypertrophy. Am. J. Physiol. 239, E372–E378.
24. Ibrahim, J., Hughes, A.D., Schachter, M. & Sever, P.S. (1996)
Enhanced tissue polyamine content in the spontaneously
hypertensive rat. Clin. Exp. Pharmacol. Physiol. 23, 410–414.
25. Ruskoaha, H. & Raunio, H. (1987) Altered cardiac polyamine
biosynthesis in spontaneously hypertensive rats. Am. J. Physiol.
253, H262–H269.
26. Gamberucci, A., Fulceri, R., Marcolongo, P., Pralong, W.F. &
Benedetti, A. (1998) Histone and basic polypeptides
activate Ca
2+
/cation influx in various cell types. Biochem. J. 331,
623–630.
27. Weiger, T.M., Langer, T. & Hermann, A. (1998) External action
of di- and polyamines on maxi calcium-activated potassium
channels: an electrophysiological and molecular modeling study.
Biophys. J. 74, 722–730.
28. Williams, K. (1997) Interactions of polyamines with ion channels.
Biochem. J. 325, 289–297.
29. Quinn, S.J.YeC.P., Diaz, R., Kifor, O., Bai, M., Vassilev, P. &
Brown, E.M. (1997) The Ca
2+
-sensing receptor: a target for
polyamines. Am. J. Physiol. 273, C1315–C1323.
30. Ventura, C., Ferroni, C., Flamigni, F., Stefanelli, C. & Capogrossi,
M.C. (1994) Polyamine effects on [Ca
2+
]
i
homeostasis and con-
tractility in isolated rat ventricular cardiomyocytes. Am. J. Physiol.
267, H587–H592.
31. Fage, D., Voltz, C., Scatton, B. & Carter, C. (1992) Selective
release of spermine and spermidine from the rat striatum by
N-methyl-
D
-aspartate receptor activation in vivo. J. Neurochem.
58, 2170–2175.
32. Ho, C., Conner, D.A., Pollak, M.R., Ladd, D.J., Kifor, O.,
Warren,H.B.,Brown,E.M.,Seidman,J.G.&Seidman,C.E.
(1995) A mouse model of human familial hypocalciuric
hypercalcemia and neonatal severe hyperparathyroidism. Nat.
Genet. 11, 389–394.
2688 R. Wang et al. (Eur. J. Biochem. 270) Ó FEBS 2003
. Calcium and polyamine regulated calcium- sensing receptors in cardiac tissues Rui Wang 1 , Changqing Xu 2 , Weimin Zhao 1 , Jing Zhang 1 , Kun Cao 1 , Baofeng Yang 2 and Lingyun Wu 3 1 Department. changes in [Ca 2+ ] o in the vicinity of cardiomyocytes [1]. The naturally occurring polyamines, including spermine, spermidine and putrescine, are involved in the synthesis of nucleic acids and. IP 3 receptors, leading to intracellular calcium increase. Involvement of extracellular calcium entry in the extracellular calcium- induced increase in [Ca 2+ ] i To examine whether the increased