Am J Physiol Regul Integr Comp Physiol 300: R251–R263, 2011.
First published November 17, 2010; doi:10.1152/ajpregu.00736.2009.
Review
Nuclear membrane receptors for ET-1 in cardiovascular function
Ghassan Bkaily,1 Levon Avedanian,1 Johny Al-Khoury,1 Chantale Provost,1 Moni Nader,1
Pedro D’Orléans-Juste,2 and Danielle Jacques1
Faculty of Medicine, Departments of 1Anatomy and Cell Biology and 2Pharmacology, Université de Sherbrooke, Sherbrooke,
Quebec, Canada
Submitted 6 November 2009; accepted in final form 15 November 2010
endothelin-1; endothelin-1 receptors; calcium; nuclear receptors; reactive oxygen
species; GPCR
IN THE LAST TWENTY YEARS, most studies addressing the role of
endothelin-1 (ET-1) on modulation of heart and vascular
smooth muscle (VSM) functions have focused on sarcolemma
endothelin type A (ETARs) and endothelin type B (ETBRs)
receptors. Among those studies, several were directed toward
ET-1 modulation of cytosolic Ca2⫹ during excitation-contraction coupling in conditions of health and disease. However,
growing evidence in the literature raises new paradigms related
to a cross talk between cytosolic and nuclear Ca2⫹ (1, 2, 4,
10 –26, 34, 42, 43, 45, 50, 57, 63, 68, 71, 74, 75; for review, see
Refs. 11, 22, 25, 29) and the presence of G protein(s)-coupled
receptors at the nuclear envelope membranes (8, 10 –23, 25, 26,
30, 42, 55, 57, 68, 71, 74; for review, see Refs. 11 and 13),
such as ET-1 receptors (10 –15, 19 –22, 24, 25, 28, 49, 51, 56,
57, 68, 71). Stimulation of nuclear membrane receptors contributes to nuclear signaling processes, including nuclear free
Ca2⫹ level (1–3, 8, 10 –26, 29, 31, 42, 49, 56, 57, 68, 71, 74),
cell cycle, gene expression (36, 47, 55, 74), activation of
Address for reprint requests and other correspondence: G. Bkaily, Dept. of
Anatomy and Cell Biology, Faculty of Medicine, Université de Sherbrooke,
3001- 12th Ave. North, Sherbrooke, Quebec, Canada J1H 5N4 (e-mail: ghassan.
[email protected]).
http://www.ajpregu.org
nuclear kinases (73) and phosphateases, as well as DNA repair,
break down of the nuclear envelope, and apoptosis (70; for
review, see Refs. 11, 22, 29, 36, 47). Studies using isolated
nuclear membrane vesicles did not take into consideration the
cross talk that may exist between the nuclear membrane nuclear receptors and ion transporters on one hand, and the
surrounding cytosolic environment of the living cell on the
other hand (30). It was not until the development of ionic
fluorescent probes, mainly Ca2⫹ probes, and real three-dimensional (3D) confocal microscopy techniques, that several laboratories, including ours, rediscovered the nucleus as a cell
within a cell (3, 10 –26, 35, 36, 44, 47, 51, 56, 68, 80).
Furthermore, the nucleus possesses nuclear structures (11,
12, 25, 41, 47, 64) that may largely participate in ET-1
regulation of life and death of the cell (70). Thus the use of
real 3D confocal microscopy imaging in living cells allowed
several laboratories, including ours, to visualize and quantify cytosolic and nuclear ET-1 and ET-1 receptor trafficking along with nuclear Ca2⫹ movements and signaling (3,
10 –15, 19 –22, 24, 25, 28, 56, 57, 68, 71).
In the present review, we emphasize the contribution of
ET-1 and ET-1 nuclear membrane receptors to overall cytosolic and nuclear signaling. We show that plasma membrane
0363-6119/11 Copyright © 2011 the American Physiological Society
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Bkaily G, Avedanian L, Al-Khoury J, Provost C, Nader M, D’Orléans-Juste
P, Jacques D. Nuclear membrane receptors for ET-1 in cardiovascular function.
Am J Physiol Regul Integr Comp Physiol 300: R251–R263, 2011. First published
November 17, 2010; doi:10.1152/ajpregu.00736.2009.—Plasma membrane endothelin type A (ETA) receptors are internalized and recycled to the plasma membrane, whereas endothelin type B (ETB) receptors undergo degradation and subsequent nuclear translocation. Recent studies show that G protein-coupled receptors
(GPCRs) and ion transporters are also present and functional at the nuclear
membranes of many cell types. Similarly to other GPCRs, ETA and ETB are present
at both the plasma and nuclear membranes of several cardiovascular cell types,
including human cardiac, vascular smooth muscle, endocardial endothelial, and
vascular endothelial cells. The distribution and density of ETARs in the cytosol
(including the cell membrane) and the nucleus (including the nuclear membranes)
differ between these cell types. However, the localization and density of ET-1 and
ETB receptors are similar in these cell types. The extracellular ET-1-induced
increase in cytosolic ([Ca]c) and nuclear ([Ca]n) free Ca2⫹ is associated with
an increase of cytosolic and nuclear reactive oxygen species. The extracellular
ET-1-induced increase of [Ca]c and [Ca]n as well as intracellular ET-1-induced
increase of [Ca]n are cell-type dependent. The type of ET-1 receptor mediating the
extracellular ET-1-induced increase of [Ca]c and [Ca]n depends on the cell type.
However, the cytosolic ET-1-induced increase of [Ca]n does not depend on cell
type. In conclusion, nuclear membranes’ ET-1 receptors may play an important role
in overall ET-1 action. These nuclear membrane ET-1 receptors could be targets for
a new generation of antagonists.
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ET-1 receptor-mediated cytosolic signaling is transferred to the
nucleus through the nuclear pore and/or via the signaling of
nuclear membrane ET-1 receptors.
Critical Technical Issues of Fluorescence Real 3D Imaging
of Cytosolic and Nuclear ET-1 Receptors
Fig. 1. Presence and distribution of endothelin type A (ETAR) and endothelin type B (ETBR) receptors in cardiovascular cells. A: 3-dimensional (3D)
reconstruction top-view image of a cardiomyocyte labeled with a fluorescent probe coupled to endothelin-1 (ET-1). The fluorescence is spread from the plasma
membrane to the cytosol and the nucleus (white arrow indicates the nucleus). B: a cardiomyocyte labeled with fluorescent probe tagged to ET-1, presented in
a “look-through” mode with a cross-sectional cut at the top. The figure demonstrates that the fluorescence is not limited to the plasma membrane. C–E: double
labeling of a human vascular smooth muscle cell with anti-ETA and anti-ETB antibodies. The receptors’ fluorescence is presented in the surface-plot mode, with
a ⫺4° tilt. ETA is present at the plasma membrane, the cytosol, and the nuclear membranes (C). However, it is absent in the nucleoplasm. ETBR is found
throughout the cell (sarcolemma membrane and cytosol), including the nucleus (nuclear envelope membranes and nucleoplasm). The merge of ETAR and ETBR
fluorescence in E demonstrates colocalization of the fluorescence of the two receptors at the sarcolemma membrane and absence of colocalization in the nucleus.
The pseudocolor scale represents fluorescence intensity according to an arbitrary scale from 0 nm (black, absence of fluorescence) to 255 nm (white, maximum
fluorescence). The scale of the white bar is in ␮m (modified from Refs. 14 and 22).
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The development and use of real 3D confocal microscopy
coupled to high-performance hardware and software systems
has provided scientists with the capacity to overcome some of
the limitations of standard microscopy imaging measurements
and image deconvolution (19, 20). However, to optimize this
technique, one should be familiar with the basic approaches
and limitations of confocal microscopy. For further information on the latter topic, the reader is invited to refer to key
studies (11, 19, 20, 22, 25).
To overcome the limitation of using fluorescence imaging
for the detection of ET-1 receptors and measurement of Ca2⫹
signals in the cytosol and nucleus, the following should be
considered: 1) cell phenotypes should be stable, and those cells
should be plated at low density and well attached to a glass
cover slip; 2) laser line intensity, photometric gain, photomultiplier tube settings, and filter attenuation should be set below
the autofluorescence level of the cell and these settings kept
rigorously constant throughout all of the experiments; 3) for
ionic probes, optimal loading should be ensured by determining the relative concentration of the bound and nonbound
probe; 4) for ET-1 and ET-1 receptor fluorescence density
measurement, specificity and selectivity of the antibody should
be ensured using different controls, such as blocking peptides,
deletion of the receptors’ genes (small-interfering or shorthairpin RNA), as well as ensuring that Western blot reveals
only the band corresponding to the target protein; 5) in simultaneous double, triple, or quadruple labeling, absence of cross
reaction between the different antibodies used should be ensured; 6) for real 3D imaging, the cell should be quiescent;
7) in studying nuclear transport of macromolecules, as well as
nuclear internalization and de novo synthesis of receptors, the
extranuclear milieu should not contain any cell debris; 8) access to
the nuclear membrane receptors and channels should be gained
by either extracting the nucleus or by perforating the sarco-
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NUCLEAR MEMBRANE ET-1 RECEPTORS IN PHYSIOPATHOLOGY
Nuclear Localization of ET-1 System Components in Human
Cardiomyocytes, hEECs, hVSMCs, and hVECs
Short-term exposure of human or chick single heart cells to
extracellular ET-1 fluorescent probe (25) revealed the presence
of ET-1 receptors in the plasma membrane in a cluster-like
fashion (Fig. 1A). However, longer exposure of the same single
cell to the extracellular ET-1 fluorescent probe also showed
labeling in the cytosol as well as at the nuclear envelope
membranes. Figure 1B shows an example of a “look-through”
real 3D fluorescence image (25). Presence of the fluorescent
probe at the cytosolic level indicates that the receptor at the
plasma membrane was internalized along with its fluorescent
ligand (25). Furthermore, presence of fluorescent labeling at
the nuclear membrane levels teaches us that the fluorescent
ET-1, internalized along with its receptors, binds to ET-1
receptors of the nuclear membranes (11, 25). Washout of the
extracellular ET-1 fluorescent probe did not remove the labeling of ET-1 receptors. However, pretreatment with nonlabeled
ET-1 prevented labeling of ET-1 receptors by the ET-1 fluorescent probe (14, 25). Cytosolic application of the ET-1
fluorescent probe showed labeling of ET-1 receptors at the
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Fig. 2. Presence and distribution of ETAR and ETBR in human (h) vascular
smooth muscle cells (VSMCs), human vascular endothelial cells (VECs), and
human endocardial endothelial cells (EECs). A and B: histograms showing the
relative density of ETAR and ETBR (in volume, per ␮m3) in cytosol (including
the plasma membrane, PM) and nucleus (including the nuclear envelope
membranes, NEMs) of hVSMCs, hVECs, and hEECs detected by indirect
immunofluorescence. The values are presented as means ⫾ SE; n represents
the no. of different experiments (5–12 for A and 5–10 for B). *P ⬍ 0.05
nucleus vs. cytosol; ***P ⬍ 0.001 nucleus vs. cytosol; P ⬍ 0.05 vs. hVSMC
cytosol; P ⬍ 0.01 vs. hVSMC cytosol; P ⬍ 0.01 vs. hVSMC nucleus;
P ⬍ 0.001 vs. hVSMC nucleus; ⫹P ⬍ 0.05 hVEC vs. hEEC; ⫹⫹P ⬍ 0.01
hVEC vs. hEEC; ⫹⫹⫹P ⬍ 0.001 hVEC vs. hEEC (modified from Refs. 6, 14,
and 57).
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nuclear envelope membranes (21). This clearly suggests that
extracellular ET-1 is internalized along with its receptors, and
it is made available to bind to its receptors in the nuclear
envelope membranes.
Antibodies directed toward ETAR and ETBR revealed the
presence of the latter proteins at the plasma membrane, cytosol,
nuclear envelope membranes, as well as at the nucleoplasm of
hVSMCs (Fig. 1, C–E), human cardiomyocytes, hEECs, and
hVECs (11, 14, 22, 25, 57). Figure 1, C–E, shows examples of
a surface plot of fluorescent double labeling of ETAR and
ETBR in hVSMCs, whereas Fig. 2 quantifies the results (11,
14, 22, 57). The ETAR density is higher in the cytosol and in
the cell membrane than in the nucleus (including the nuclear
membranes) in hVSMCs and hEECs (11, 14, 22, 57). However, in hVECs, the density of ETARs is higher in the nucleus
than in the cytosol (Fig. 2A) (6). These results show that ETAR
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lemma membrane (1, 12, 15, 20, 21, 26); 9) a minimum of 36
serial sections, and optimal 75–150 serial sections of the cell
(depending on the morphology), should be taken for real 3D
imaging; and 10) transiently occurring phenomena should be
only monitored and subsequently disclosed using “rapid scan”
of a cell section. This will permit measuring temporal changes
of density and redistribution. Measurement of amplitude of the
temporal changes of contracting cells is not possible because of
continuous change of the position of the cell section; 11) small,
fast, and instantaneously occurring changes in kinetics of
cytosolic and nuclear events should be recorded using the “line
scan” technique, which exclusively measures temporal changes; 12) temporal changes, including Ca2⫹ changes during cell
contraction, can be measured using rapid scan; 13) for measurement of cytosolic and nuclear fluorescence intensities, at
the end of the experiment, labeling of the nucleoplasm should
be done using nuclear probes, such as Syto-11; 14) because of
variations in volumes between cells or between intracellular
organelles, the fluorescence intensity should be normalized and
expressed per cubic micrometer; 15) measurement in a single
section of a cell represents fluorescence intensity of a surface
area that should be expressed per square micrometer and not
represent the absolute level of fluorescence intensity of the
whole cell; and 16) it is recommended to determine the
distribution of the fluorescent probe when a new type of cell is
used. Fluo 3 as well as other Ca2⫹ probes such as indo 1, fluo
4, Ca2⫹ green, yellow cameleon, and fura 2 are equally
distributed in the cytosol and the nucleus of cardiomyocytes,
vascular smooth muscle cells (VSMCs), vascular endothelial
cells (VECs), and endocardial endothelial cells (EECs) of
human (h), rat, rabbit, hamster, and mouse origins (14, 25).
These fluorescent probes can be used safely in these previously
mentioned cell types.
All of these above-described aspects, in addition to others
(for details, see Refs. 19 and 20), should be established and
well controlled throughout the experiment to accurately determine the fluorescence distribution and density of a fluorescent
probe.
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NUCLEAR MEMBRANE ET-1 RECEPTORS IN PHYSIOPATHOLOGY
Similar to intact cell, isolated nuclei of hVSMCs (11, 22)
also showed the presence of nuclear membrane ETBRs at the
outer and inner nuclear membranes (Fig. 3, A and B) in a
cluster-like fashion. Furthermore, these receptors are also
present within the nucleus (Fig. 3C), possibly at both the
nucleoplasm (11) and nuclear T tubule (NTTs) membrane
levels (5, 11, 41). Because NTTs are distributed throughout the
nucleoplasm (5), the ETBRs present at the NTT membranes
may contribute to acceleration of propagation of signaling of
these receptors deeper within the nucleoplasm (5, 41). Although the presence of ET-1 at the level of the cytosol is
expected, the presence of the ligand in the nucleoplasm is also
sought (Fig. 4) (11, 22), since it can be made available for its
receptors present at the membranes of both the nuclear envelope (11, 22, 25) and NTTs (5, 11, 41). Surprisingly, the
density of ET-1 is higher in the nucleus than in the cytosol of
both ET-1-secreting cells, hVECs and hEECs (Fig. 4) (11). A
Fig. 3. Presence of ETBR in isolated nuclei of hVSMCs. Distribution of ETBRs in an isolated nucleus of hVSMCs. A: frontal view of external membrane ETBR
(white arrows) distribution and density. B: front and look-through views of external (white arrows) and internal (black arrow) nuclear envelope membrane ETBR
distribution and density. C: internal view of ETBR presence (black arrows) within the nucleus, suggesting that these receptors are localized on structures inside
the nucleus that could be extensions of the nuclear envelope membranes, probably nuclear T tubules (NTTs). D: nucleic acid stain of the same view as in C.
All representations from A to D are from the same isolated nucleus. E: negative control studies with blocking peptides for the anti-ET-1, anti-ETA, and anti-ETB
antibodies from Figs. 1 to 4 and the corresponding grayscale images of the nuclei. The pseudocolor scale represents the fluorescence intensity according to an
arbitrary scale from 0 nm (black color, absence of fluorescence) to 255 nm (white color, maximum fluorescence). The scale bar is in ␮m (modified from Refs.
11 and 22).
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is also present at the nuclear level; however, its density depends on the cell type. In contrast to ETAR, ETBR density is
higher in the nucleus than in the cytosol of all human cell types
tested (Figs. 1D and 2B) (6, 11, 14, 22, 52).
The order of density of nuclear ETARs in the different
cardiovascular cells is: hVEC ⬎ hVSMCs ⬎ hEECs, whereas,
for nuclear ETB, it is: hVEC ⬎ hEEC ⬎ hVSMCs (6, 11, 14,
22, 57). However, the orders of density of ETAR and ETBR in
the cytosol (including the cell membrane) are, respectively:
hVEC ⫽ hVSMC ⬎ hEEC and hVSMC ⫽ hEECs ⬎ hVEC (6,
11, 14, 22, 57). Thus the density of ETAR or ETBR at the cell
membrane plus cytosol is similar in different cell types of the
same species. However, we cannot make such a generalized
conclusion for nuclear ET-1 receptors. Hence, it is postulated
that the cell-type-dependent effect of ET-1 is due, at least
partly, to the differences in nuclear ET-1 receptor densities of
different cell types.
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NUCLEAR MEMBRANE ET-1 RECEPTORS IN PHYSIOPATHOLOGY
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similar high density of nuclear ET-1 (73–93% of total cell, Fig. 4)
would imply that this nuclear ligand plays an important role not
only in its own secretion process but also in regulation of nuclear
function and, consequently, in the modulation of cell functions.
Transcellular Trafficking of ET-1 and Its Receptors
In addition to its conventional cytoplasmic membrane localization, the endothelin-converting enzyme-1a is also targeted
to the nuclear membrane (58). Furthermore, receptors such as
ETB present in the nucleoplasm (Figs. 2 and 3) (6, 11, 22)
could be targeted to nuclear membranes. Thus nuclear membrane receptors may originate from the nucleoplasm (Fig. 5)
(55). The activation of these nuclear membrane ET-1 receptors
by their respective cytosolic or perinucleoplasmatic ligands
would cause nuclear second messenger formation, such as
inositol trisphosphate (IP3) (59, 63) and PKC activation (73).
These ET-1 receptor activation products may directly regulate
nuclear membrane ionic transporters (11, 42, 53, 80) as well as
nuclear pore complex trafficking of macromolecules (34). The
schematic model in Fig. 5 suggests that internalization of ET-1
with its receptors ETA and ETB (Fig. 5, A–F) will lead the
ET-1/ETAR and ET-1/ETBR complexes to recycling and degradation, respectively (9, 11, 51). Both phenomena would
release ET-1 in the cytosol (9, 11, 16, 22, 51) and make it
available for activation of nuclear membrane ET-1 receptors.
Hence cytosolic free ET-1 can activate outer nuclear membrane ETBRs, as was reported in cardiomyocytes using ET-1
fluorescent probe (21, 22, 25). We also hypothesize that, during
increases of cytosolic Ca2⫹, cytosolic ET-1, normally present
in secretory vesicles (Fig. 5, in yellow), is simultaneously
released extracellularily and in the nuclear envelope space via
the same exocytosis process (31). The ET-1 released in the
perinucleoplasm will be available for activation of the inner
nuclear membrane ETBRs (Fig. 5) (11, 14, 25). If we consider
that the nucleus is a cell within a cell (11, 22) and because
ET-1 is also present in the nucleoplasm, this process may also
take place from the nucleoplasm toward the nuclear envelope
space (Fig. 5). According to this hypothesis, the increase of
AJP-Regul Integr Comp Physiol • VOL
nucleoplasmic free Ca2⫹ would promote exocytosis (exonucleosis) of nucleoplasmic ET-1 to the perinucleoplasm,
which in turn activates ET-1 receptors located at the inner
nuclear membrane (Fig. 5). We do not exclude that the increase
of perinucleoplasmic free Ca2⫹ will promote exocytosis (exoperinucleosis) of perinucleoplasmic ET-1 and liberation of this
peptide in the cytosol, thus activating ET-1 receptors located at
the outer nuclear membrane (Fig. 5). Activation of these
nuclear membrane receptors would further promote transnuclear membrane Ca2⫹ influx and release of nucleoplasmic
Ca2⫹ via nuclear ryanodine receptor and IP3 receptor pools
(12, 59, 64, 80). Thus it is possible that activation of nuclear
membrane ET-1 receptors may follow the activation of plasma
membrane ET-1 receptors. In addition, nuclear membrane
ET-1 receptors could be activated by its ligand(s) locally
released from the nucleus, independent of plasma membrane
ET-1 receptor activation (Fig. 5). The increase of nucleoplasmic ETBR density following activation of plasma membrane
ETBRs (Fig. 5) could be in part due to both nuclear translocation (11) and/or nuclear mobilization and/or de novo nuclear
synthesis of the receptor (Fig. 5) (11, 55). Thus the density of
a particular type of ET-1 receptor is not identical in plasma and
nuclear membranes, and the nuclear membranes may possess a
higher density for one particular subtype of a given receptor
relative to plasma membrane (Fig. 2) (6, 11, 22). Because ET-1
receptors exist at both the nuclear membranes and the cell
membranes, we postulate that a cross talk exists between cell
membrane receptors and nuclear membrane receptors of the
same cell (Fig. 5). Such a cross talk may imply that nuclear
ET-1 receptors also play a role of relay for their receptors
present at the cell membrane, thus perpetuating their signaling
to the nucleoplasm.
In conclusion, our recent results and ongoing work strongly
support the hypothesis that many biochemical events occurring
at the plasma membrane and cytosolic levels also occur in a
parallel manner at the nuclear membranes and within the
nucleus. Such nuclear events may occur dependently and
independently of those occurring at plasma membrane and
cytoplasmic levels (Fig. 5).
Extracellular ET-1 Modulation of Cytosolic and Nuclear
Free Ca2⫹ Signaling
In hVSMCs, hVECs, and cardiomyocytes, increasing the
concentration of extracellular ET-1 from 10⫺17 up to 10⫺5 M
induced a dose-dependent increase of both sustained basal
cytosolic free Ca2⫹ ([Ca]c) and nuclear free Ca2⫹ ([Ca]n).
Figure 6 shows a compilation of different published results (6,
11, 13, 21, 24, 57). The EC50 of the extracellular ET-1-induced
increase of sustained basal [Ca]c and [Ca]n is different in the
three different cell types. The order of potency of ET-1 on
[Ca]c and [Ca]n is as follows: cardiomyocytes (⫾10⫺13 M) ⬎
hVECs (⫾10⫺12 M) ⬎ hVSMCs (⫾10⫺10 M) (Fig. 6). Within
each cell type, there is nearly no difference between EC50 of
the [Ca]c and [Ca]n increase induced by extracellular ET-1
(Fig. 6). Moreover, the increase of nuclear Ca2⫹ by extracellular ET-1 seems to be mainly, but not exclusively, the result
of the stimulation of Ca2⫹ influx through the sarcolemma
membrane (21, 25, 57).
The type of receptor mediating the ET-1-induced increase of
[Ca]c and [Ca]n depends on the cell type (6, 11, 12, 21, 56, 57).
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Fig. 4. Presence and distribution of ET-1 in human vascular and endothelial
cells. Histograms showing the relative density of ET-1 fluorescence (in
volume, per ␮m3) in cytosol (including the PM) and nucleus (including the
NEMs) of hVECs and hEECs detected by indirect immunofluorescence. The
values are presented as means ⫾ SE; n (7–15) represents the no. of different
experiments. *P ⬍ 0.05 nucleus vs. cytosol; ***P ⬍ 0.001 nucleus vs. cytosol;
P ⬍ 0.001 vs. hVEC cytosol;
P ⬍ 0.001 vs. hVEC nucleus (modified
from Refs. 6 and 57).
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The effect of ET-1 on [Ca]c and [Ca]n of the hVSMCs was
reversed by the ETAR antagonist BMS-182874 and the ETBR
antagonist A-192621 (Fig. 7, A–H). Similar results were reported in cardiomyocytes and left ventricular hEECs (12, 21,
57). However, in hVECs and right ventricular hEECs, only the
ETAR is responsible for the ET-1-induced increase of [Ca]c
and [Ca]n (6, 56). Thus it is not possible to make a generalization in relation to the type of ET-1 receptor responsible for
the modulation of cytosolic and nuclear Ca2⫹.
In isolated nuclei of hVSMCs, ET-1 (10⫺11 M) induced an
increase of [Ca]n (Fig. 8A). Furthermore, extranuclear ETBR
agonist IRL-1620 induced an increase of [Ca]n (Fig. 8B). In
cardiomyocytes and hVSMCs, cytosolic ET-1 as low as 10⫺15
M induced a sustained increase in [Ca]n (Fig. 9). A steady-state
effect takes place in heart cells at 10⫺10 M, whereas, in
hVSMCs, it is only reached at a concentration of 10⫺6 M (Fig.
9). Accordingly, the EC50 of cytosolic ET-1 on [Ca]n is lower
in cardiomyocytes (⫾10⫺14 M) than that in hVSMC nuclei
(⫾10⫺11 M). Furthermore, the EC50 of cytosolic ET-1 on [Ca]n
of both cardiomyocytes and hVSMCs was lower than that of
extracellular ET-1. These results demonstrate that nuclear
membrane ET-1R has intrinsically higher affinity to cytosolic
AJP-Regul Integr Comp Physiol • VOL
ET-1 than to those located at the cell membrane. The difference between the EC50 of extracellular and cytosolic ET-1
could be partly due to differences of composition between the
cell and nuclear membranes, which may permit a better interaction of ET-1 with its receptor. Alternatively, there could be
a different endogenous agonist that stimulates these nuclear
membrane receptors, and/or the nuclear receptors and their
pharmacology are different than those of the cell membrane.
Because the nuclear envelope receptors are more sensitive to
ET-1 than those present at the sarcolemma membrane, we
postulate that ET-1 internalized with its plasma membrane
receptor could be snatched by the nuclear membrane ET-1
receptors (14), thus activating the latter. All of these aspects
should be further investigated in the future.
In both intact cells and isolated nuclei (11, 12, 14), the effect
of ET-1 on [Ca]c and [Ca]n was completely blocked by the
Ca2⫹ chelator EGTA (13). In intact cells, the ET-1 increase of
[Ca]c is due to an increase of Ca2⫹ influx through the sarcolemma membrane, whereas the increase of [Ca]n is at least in
part due to cytosolic Ca2⫹ buffering by the nucleus (12, 21, 22,
25, 57). However, in isolated nuclei, the increase in [Ca]n by
cytosolic ET-1 is not exclusively the result of the stimulation of
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Fig. 5. Schematic model representing presence, distribution, and transcellular trafficking of ETBR
(A–E) and ETAR (F and G) upon their stimulation
with extracellular ET-1 in hVSMCs. In the absence
of ET-1, both ETA and ETB are present in the
sarcolemma and the cytosol (A and F), but only ETB
is present at the nuclear membranes and in the
nucleoplasm (A and E). Stimulation of sarcolemma
ETAR and ETBR induces their internalization (B–D).
This is followed by recycling of ETA to the sarcolemma (G) as well as degradation (C) and nuclear
accumulation (E) of ETB. Stimulation of sarcolemma ETAR and ETBR induces an increase of
cytosolic and nuclear Ca2⫹, which promotes exocytosis of perinucleoplasmic and nucleoplasmic ET-1.
The released peptide activates nuclear membrane
ETBRs and further promotes an increase of intracellular free Ca2⫹. ET-1 in black contoured ovals
signifies vesicularized peptide while the noncontoured ET-1-ovals signify free peptides. The
pseudocolor bar represents intensity of fluorescence
from 0 (black) to 255 (white). A–G are 3D reconstruction top-view images of ETA and ETB densities
and distribution. NPC, nuclear pore complex. Each
panel is a different cell (modified from Ref. 22).
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ET-1 and Cytosolic and Nuclear Reactive Oxygen Species
Generation
Fig. 6. Dose response of the effect of extracellular ET-1 on cytosolic free Ca2⫹
([Ca]c) and nuclear free Ca2⫹ ([Ca]n) of different cardiovascular cells. Stimulation of intact hVSMCs, hVECs, and cardiomyocytes with different extracellular concentrations of ET-1 induced a dose-dependent increase in sustained
basal cytosolic (A) and nuclear (B) Ca2⫹. The EC50 values differ between cell
types. The no. of experiments for hVSMC, hVEC, and cardiomyocytes is 5,
3–7, and 3–10, respectively (modified from Refs. 13, 21, 24, and 57).
Ca2⫹ influx through nuclear envelope membranes (11, 13, 57,
68). In fact, in isolated nuclei, in absence of cytosolic Ca2⫹,
cytosolic ET-1 induced an increase in [Ca]n (25). Therefore,
stimulation of ET-1 receptors of the nuclear envelope membranes induced both nuclear Ca2⫹ influx (11, 13, 25) and
mobilization of nucleoplasmic Ca2⫹ (25).
Cytoplasmic ET-1 Modulation of Ca2⫹ Buffering Capacity of
the Nucleus in Cardiomyocytes and VSMCs
Extracellular and cytosolic ET-1 induced a steady-state increase in [Ca]c and [Ca]n in both cardiomyocytes and VSMCs
via stimulation of Ca2⫹ influx through the steady-state resting
R-type Ca2⫹ channel and Ca2⫹ release from the sarcoplasmic
reticulum (25). Cytosolic Ca2⫹ buffering by the nucleus was
determined by a gradual change of cytosolic Ca2⫹ concentration from 100 to 2,000 nM (14, 21). A visible increase of [Ca]n
was detected starting from 200 nM extranuclear free Ca2⫹
AJP-Regul Integr Comp Physiol • VOL
It is well established that the major products of superoxide
metabolism, such as hydrogen peroxide (H2O2), have multiple
actions on cell function (38, 44, 61, 77, 78). Compelling
evidence shows that extracellular ET-1 induces NADPH oxidase to stimulate production of superoxide from several cell
types (38, 77). Over the past few years, growing evidence has
implicated both ET-1 and cytosolic reactive oxygen species
(ROS) as pathological mediators of cardiovascular diseases
(38, 61, 77, 78). However, little attention has been given to the
nucleus.
Similar to H2O2, extracellular ET-1 (10⫺9 M) induced a
sustained increase of both cytosolic and nuclear ROS (71) in
hEECs (Fig. 10, A–E) (71) and hVSMCs (71). In the presence
of ET-1, superfusion with glutathione (10 mM) reversed the
ET-1-induced sustained increase of cytosolic and nuclear ROS
in both cell types (Fig. 10, A–C and E) (71). In addition, the
resting basal level of ROS in the nucleus of both hEECs and
hVSMCs is higher than in the cytosol (71). This again demonstrates that, like Ca2⫹ and Na⫹ (11, 14), in resting as well
as stimulated states, ROS have a higher density in the
nucleus than in the cytosol (71). Hence, ET-1 was found to
induce an increase of both ROS and Ca2⫹ in both the cytosol
and the nucleus of all cells tested, including hEECs and
hVSMCs (25, 71).
Extranuclear ET-1 (10⫺11 M) induced a sustained increase
in nuclear ROS (Fig. 10F) (71). In the presence of cytosolic
(extranuclear) ET-1, addition of H2O2 (50 ␮M) to the extranuclear milieu did not further increase the level of nuclear ROS
(71). However, the increase of nuclear ROS by cytosolic ET-1
(in the presence of H2O2) was reversed by glutathione (10 mM)
(Fig. 10F). Thus ROS levels in the nucleus can be increased by
cytosolic ET-1, and further increase of cytosolic H2O2 does not
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concentrations (24, 25). The lowest concentration of [Ca]c
needed to initiate nuclear Ca2⫹ uptake was called the minimal
Ca2⫹-buffering capacity of the nucleus (25). Elevation of
extranuclear free Ca2⫹ up to 1,200 nM increased [Ca]n in a
concentration-dependent manner (14, 25). However, further
elevation of extranuclear free Ca2⫹ up to 1,600 and 2,000 nM
did not cause an additional increase of [Ca]n (14, 25). Thus,
1,200 nM cytosolic Ca2⫹ was called the maximal Ca2⫹buffering capacity of the nucleus (25). In the presence of 1,600
or 2,000 nM of extranuclear free Ca2⫹, superfusion with
extranuclear medium containing ET-1 (100 nM) induced a
further increase in [Ca]n. In the presence of nearly normal
concentrations of [Ca]c (100 nM), superfusion with extranuclear ET-1 (100 nM) solution induced a significant increase in
[Ca]n of both cardiomyocytes (16) and VSMCs. In the presence of cytosolic ET-1, superfusion with sequentially increasing concentrations of extranuclear free Ca2⫹ from 200 to 1,200
(or 2,000) nM did not further increase ET-1-induced sustained
elevation of [Ca]n. Thus activation of nuclear membrane ET-1
receptors seems to not only increase nuclear Ca2⫹ influx and
nuclear Ca2⫹ release but also to decrease the maximal cytosolic Ca2⫹ buffering of the nucleus. Such a condition will shield
the nucleus from further uptake of cystosolic Ca2⫹ and/or
cytosolic Ca2⫹ overload (1, 2, 4, 11, 14, 15, 20, 25, 26) and
protect the nucleus from cytosolic Ca2⫹ accumulation.
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NUCLEAR MEMBRANE ET-1 RECEPTORS IN PHYSIOPATHOLOGY
amplify the cytosolic ET-1 response. This suggests that the
cytosolic ET-1-induced increase of nuclear ROS can be mediated via the H2O2 pathway (71). Furthermore, cytosolic H2O2
(50 ␮M) induces a sustained increase of nuclear ROS, whereas
cytosolic ET-1 (10⫺11 M) does not further raise the H2O2induced increase of ROS (71). The increase of ROS by H2O2
or by ET-1 is reversed with glutathione (71). Thus the nucleus
can generate its own ROS, and nuclear ET-1 receptor activation contributes directly to nuclear ROS generation.
Role of Nuclear ET-1 Receptors in Physiology and
Pathophysiology
ET-1 is also known as a cardiac hypertrophic factor (39).
Because, as in cell membranes, functional ET-1 receptors are
also present at the nuclear membranes of all types of cells
tested, and because the density of ETBRs is higher in the
nucleus than in the cell membrane and the cytosol (31), we thus
postulate that the ET-1-induced hypertrophy is not only cytoplasmic but nuclear as well. Such a phenomenon seems to exist
in human cardiomyocytes as shown, for example, in Fig. 11.
As seen in Fig. 11, treatment with 10⫺7 M ET-1 for 48 h
AJP-Regul Integr Comp Physiol • VOL
induced an increase of both cytosolic and nuclear volumes of
these cell types (Fig. 11). These results clearly demonstrate that
the ET-1-induced hypertrophy takes place also at the nuclear
level. Furthermore, these results showed that ET-1 is not only
a cardiomyocyte hypertrophic factor, but it also induces nuclear hypertrophy.
Nuclear ET-1 receptor activation and generation of nuclear
ROS may also contribute to the ET-1-induced generation of
ROS in cardiovascular pathology.
The activation of nuclear membrane ET-1 receptors and the
resulting increase of nuclear Ca2⫹ shield the nucleus from
cytosolic Ca2⫹ overload that characterizes several cardiovascular pathologies, such as reperfusion-induced increase of
intracellular Ca2⫹, hypertension, and hypoxia.
Discussion
In conclusion, our results as well as those of others reported
in the literature clearly demonstrate that ET-1 receptors are
present at both the plasma membrane and nuclear envelope
membranes. These receptors may contribute to maintaining not
only Ca2⫹ influx through the plasma membrane but also
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Fig. 7. Reversal effects of nonpeptidic ETAR
and ETBR antagonists on sustained Ca2⫹ increase induced by ET-1. Effect of ETAR and
ETBR nonpeptidic antagonists BMS-182874
(BMS) (A) and A-192621 (B) on ET-1-induced
cytosolic and nuclear Ca2⫹ increase in
hVSMCs. ET-1 at a concentration of 10⫺9 M
produced a sustained increase in [Ca]c and
[Ca]n (Ab and Bb). Addition of the nonpeptidic
ETAR antagonist BMS-182874 (10⫺6 M; Ac)
or the nonpeptidic ETBR antagonist A-192621
(10⫺7 M; Bc) reversed the effect of ET-1 on
[Ca]c and [Ca]n. Nucleic acid probe Syto-11
labeling in Ad and Bd represents demarcation
of nucleus at the end of the experiments. The
images are 3D top-view reconstructions. The
pseudocolor scale represents the fluorescence
intensity of the fluo 3-Ca2⫹ complex at an
arbitrary scale from 0 nm (black color) to 255
nm (white color), or from zero free Ca2⫹
(black) to 45 ␮M of Ca2⫹ (white). The scale of
the white bar is in ␮m (modified from Ref. 12).
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NUCLEAR MEMBRANE ET-1 RECEPTORS IN PHYSIOPATHOLOGY
participate in controlling the basal nucleoplasmic free Ca2⫹ in
resting cells (11, 14, 22, 25). As shown in the literature as well
as in our published work (10 –26, 57, 68, 71), several highly
active circulating compounds, such as ET-1, may directly act at
the nuclear envelope membrane level. Consequently, this
Fig. 9. Dose response of the effect of cytosolic ET-1 on [Ca]n in cell membrane
perforated hVSMCs and cardiomyocytes. Following perforation of sarcolemma membranes with ionomycin, stimulation with various concentrations of
cytosolic ET-1 induced a dose-dependent increase of sustained basal [Ca]n in
hVSMCs and cardiomyocytes. Please note that the EC50 values differ between
the two cell types. The no. of experiments for hVSMCs and cardiomyocytes
are 3 and 3–11, respectively (modified from Refs. 13, 20, and 24).
AJP-Regul Integr Comp Physiol • VOL
Fig. 10. Effect of ET-1 on cytosolic reactive oxygen species (ROS) concentration ([ROS]c) and nuclear ROS concentration ([ROS]n) in hEECs. A: real 3D
image of an hEEC loaded with ROS probe carboxy-H2DCFDA (DCF).
Treatment with ET-1 (10⫺9 M) (B) induced a sustained increase of ROS that
was reversed by glutathione (10 mM) (C). D: labeling of the nucleus with the
live nucleic acid stain Syto-11. E: ROS fluorescence intensity measurements in
cytosol and nucleus of hEECs in a set of experiments as in A–C. Values are
expressed as means ⫾ SE. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs.
corresponding control condition. ⫹⫹P ⬍ 0.01 cytosol vs. nucleus. N, no. of
different experiments. Measurements were taken when the effect reached a
steady-state level. F: during a rapid scan performed on the confocal microscope, stimulation of the nucleus with ET-1 (10⫺9 M) caused an increase of the
[ROS]n level. In the presence of ET-1, hydrogen peroxide (50 ␮M) did not
further increase [ROS]n. Glutathione (10 mM) decreased the [ROS]n back to
the control level. The insets show images of the nucleus at each experimental
condition. The pseudocolored bars represent fluorescence intensity according
to an arbitrary scale from 0 nm (black, absence of fluorescence) to 255 nm
(white, maximum fluorescence), and they do not apply to the Syto-11 image.
The scale bar is in ␮m (modified from Ref. 71).
would promote both Ca2⫹ entry in the nucleus as well as
nucleoplasmic Ca2⫹ release, thus increasing [Ca]n and shielding the nucleoplasm from sustained nucleoplasmic Ca2⫹ overload (11, 25). Thus, ET-1 receptor-mediated Ca2⫹ signals are
not only limited to the cytoplasm but also take place in the
nucleus via nuclear uptake of cytosolic Ca2⫹ (16, 21, 25)
and/or via ET-1-stimulated IP3-induced nucleoplasmic Ca2⫹
release (5, 49, 79). Alternatively, this phenomenon may also
take place independent of cytosolic Ca2⫹ and IP3 changes, by
direct activation of nuclear ET-1 receptors by ET-1 present in
the cytosol (11, 13, 14, 16). Thus changes in extracellular
and/or intracellular and/or nucleoplasmic levels of ET-1 as
well as changes in cell membrane and/or nuclear membrane
ET-1 receptor density would either indirectly or directly affect
nuclear Ca2⫹ signaling. In addition to the cytosolic Ca2⫹buffering property of the nucleus (16, 25) and its regulation by
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Fig. 8. Direct activation of nuclear envelope membrane ET-1 receptors induces
increase of [Ca]n. A: 3D imaging of Ca2⫹ (control) in a nucleus isolated from
hVSMC and loaded with the free Ca2⫹ probe fluo 3. Superfusion with 10⫺11
M ET-1 induced an increase of [Ca]n. Live nucleic acid stain Syto-11 labels the
nucleoplasm. B: during a rapid scan of an isolated intact nucleus loaded with
fluo 3 Ca2⫹ probe, direct stimulation of nuclear membrane ETBR by ETBR
agonist IRL-1620 (10⫺10 M) induced a transient increase of [Ca]n. Insets of
colored images show distribution and the level of free Ca2⫹ of the isolated
intact nucleus. The pseudocolor scale represents the fluorescence intensity of
the fluo 3-Ca2⫹ complex at an arbitrary scale from 0 nm (black color) to 255
nm (white color), or from zero free Ca2⫹ (black) to 45 ␮M of Ca2⫹ (white).
White bar is in ␮m (modified from Refs. 14 and 21).
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NUCLEAR MEMBRANE ET-1 RECEPTORS IN PHYSIOPATHOLOGY
Fig. 12. Schematic representation of various components present in both a cell and its nucleus that directly or indirectly modulate both cytosolic and nuclear Ca2⫹.
TT, T tubule; NTT, nuclear T tubule; NR, nucleoplasmic reticulum; RyR, ryanodine receptor; IP3R, inositol trisphosphate (IP3) receptor; ER, endoplasmic
reticulum; SR, sarcoplasmic reticulum; R, ET-1 receptor; NO, nitric oxide; NOX, NADPH oxidase; PKG, protein kinase G; sGC, soluble guanylate cyclase;
puffs, IP3-generated Ca2⫹ wave; spark, RyR-generated Ca2⫹ wave (modified from Ref. 11).
AJP-Regul Integr Comp Physiol • VOL
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Fig. 11. ET-1 induces an increase in the cell volume of cardiac cells.
A and B: confocal microscopy top-view 3D images of human cardiac cells
before (A, control) and after (B) 48 h of treatment with 10⫺7 M ET-1. Note the
marked hypertrophy induced by ET-1 in the treated cardiac cell (B) compared
with the two cardiac cells in the control condition (A). C: measurement of cell
volume (in ␮m3) using the 3D analysis software Image space shows a
significant increase in cell volume following ET-1 treatment in both the cytosol
and the nucleus of cardiac cells. The white scale bar is in 5 ␮m. ***P ⬍ 0.001
ET-1-treated vs. control cells. n, No. of different cells (G. Bkaily and D.
Jacques, unpublished observations).
ET-1 receptor stimulation (25), the slow time course of Ca2⫹
release by the nucleus (21) helps to promote and/or maintain
for a longer period of time the high level of cytoplasmic Ca2⫹,
thus allowing longer muscle contraction and secretion. Such a
nuclear increase of free Ca2⫹ would as well activate other
phenomena that are Ca2⫹-dependent, such as Ca2⫹-dependent
protein kinases (65, 69, 72, 76) and nuclear Ca2⫹-binding
proteins (such as calmodulin and calpains) (7, 60) that are
known to regulate both cell function and gene expression (46).
Current knowledge dealing with plasma membrane ET-1
receptors teaches us that the presence of either ETAR or ETBR
is cell type dependent (26, 32, 37). However, recent work
investigating nuclear membrane ET-1 receptors shows that the
presence of a certain type of ET-1 receptor in a cell is not
determined only by its presence in the cell membrane but also
by its presence in the nuclear membranes. The presence of
ETARs in two types of endothelial cells, the hVECs and
hEECs, demonstrates that at least human endothelial cells
possess ETARs and that ETBRs are not the only type of ET-1
receptor present in these types of cells (6, 57). The role of
ETBRs as ET-1 clearance receptor is well documented in the
literature (9, 32), whereas the role of ETARs seems to be
related to modulation of intracellular Ca2⫹ (6). Whether nuclear membrane ETAR and ETBR play other roles in regulating
cell function remains to be elucidated. It is nonetheless possible to postulate that the ETAR, which seems to be highly
present at the nuclear level of endothelial cells, may contribute
to the secretion process via regulating [Ca]c and [Ca]n homeostasis (6, 57). The presence of this type of receptor on the
nuclei of both hEECs (6, 57) and hVSMCs (11) as well as
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NUCLEAR MEMBRANE ET-1 RECEPTORS IN PHYSIOPATHOLOGY
AJP-Regul Integr Comp Physiol • VOL
in the plasma membrane (11, 14, 22, 25). Such a difference
between cytosolic and nuclear Ca2⫹ levels becomes more
apparent in highly active and stimulated cells (25) as reported
in some diseases, such as hypertrophy. In some specific conditions that mimic cardiovascular dysfunctions, such as overexpression of the cell membrane and nuclear membranes, ANG
II type 1 receptors (26), and in cardiomyopathy (G. Bkaily and
D. Jacques, unpublished observation), there is no difference
between [Ca]c and [Ca]n. In conclusion, endothelin regulation
of cell function cannot anymore be attributed only to activation
of plasma membrane ET-1 receptors and their downstream
signaling but also to activation of nuclear membrane ET-1
receptors and their nuclear signaling.
Perspectives and Significance
The presence of functional ET-1 receptors at the nuclear
membranes opens a new perspective in the field of endothelin.
This new area of ET-1 research will help us to better understand the complex mechanisms underlying the role of ET-1 in
cell function. There is no doubt that several aspects remain to
be elucidated on the roles of each type of nuclear membrane
ET-1 receptor and their contribution to overall ET-1 action in
health and diseases. The growing evidence regarding the role
of nuclear membrane ET-1 receptors will allow the optimal
identification of new targets toward the development of specific nuclear membrane ET-1 receptor antagonists that act
specifically on overstimulation of the nuclear membrane ET-1
receptors. Further studies should, however, be performed in
this new field of nuclear membranes’ G protein-coupled receptors, and specifically those involved in ET-1-induced cellular
events.
ACKNOWLEDGMENTS
We thank Helene Morin for secretarial assistance and G. B. Bkaily and C.
Perreault for technical assistance.
GRANTS
This study was supported by grants received from the Canadian Institutes of
Health Research, the Natural Sciences and Engineering Research Council of
Canada, and the Heart and Stroke Foundation of Quebec (grants awarded to G.
Bkaily and D. Jacques).
DISCLOSURES
No conflicts of interest are declared by the authors.
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