activation of sarcolemma and nuclear membranes et-1 receptors regulates transcellular calcium levels...

SURVEY REVIEW / SYNTHÈSE D’ENSEMBLE

Activation of sarcolemma and nuclear membranesET-1 receptors regulates transcellular calciumlevels in heart and vascular smooth muscle cells1

Ghassan Bkaily, Sanaa Choufani, Sawsan Sader, Danielle Jacques,Pedro d’Orléans-Juste, Moni Nader, Ghada Kurban, and Maud Kamal

Abstract: The use of an ET-1 fluorescent probe in human heart and vascular smooth muscle cells showed that ET-1 re-ceptors are present at both the sarcolemma and nuclear envelope membranes. The use of immunofluorescence studiesshowed that the ETA receptor was mainly present at the sarcolemma and cytosolic levels. However, the ETB receptorwas present at the sarcolemma and the cytosol, as well as the nuclear envelope membranes and the nucleoplasm. In ad-dition, ET-1 immunoreactivity was seen in the cytosol and the nucleus. Using Ca2+fluorescent probes such as Fluo-3,Indo 1, and yellow cameleon, as well as confocal microscopy three-dimensional image measurement technique, stimula-tion of ET-1 receptors at the sarcolemma membranes induced an increase of cytosolic and nuclear free Ca2+ levels.This effect of extracellular ET-1 was blocked by removal of extracellular calcium. Direct stimulation of ET-1 receptorsat the nuclear envelope membranes also induced an increase of intranuclear free Ca2+ level. Our results suggest that thestimulation of sarcolemmal Ca2+ influx by ET-1 seems to be due to the activation of ETA and ETB receptors. However,the increase of nucleoplasmic Ca2+ levels by cytosolic ET-1 seems to be mediated via the activation of ETB receptors.Activation of nuclear membranes ETB receptors seems to prevent nuclear Ca2+ overload and may protect the cell fromapoptosis.

Key words: endothelin-1, endothelin-1 receptors, calcium, nuclear receptors, confocal microscopy.

Résumé : L’utilisation des sondes fluorescentes couplées à l’ET-1 dans les cellules cardiaques et le muscle lisse vascu-laire humain a permis de démontrer que les récepteurs à l’ET-1 sont présents, non seulement au niveau de la sarco-lemme, mais aussi au niveau des membranes de l’enveloppe nucléaire. En utilizant des sondes calciques comme leFluo-3, l’Indo-1 et le cameleon jaune ainsi que la technique de mesure des images en trois dimensions à l’aide de lamicroscopie confocale, nous avons démontré que la stimulation des récepteurs à l’ET-1 de la sarcolemme induit uneaugmentation du calcium libre cytosolique et nucléaire. Cet effet est bloqué par le chelateur calcique, l’EGTA. De plus,la stimulation directe des récepteurs à l’ET-1, au niveau des membranes de l’enveloppe nucléaire, augmente le calciumlibre dans le nucléoplasme. Nos résultats suggèrent que la stimulation de l’influx calcique de la sarcolemme par l’ET-1semble être due à la stimulation des récepteurs ETA et ETB. Par contre, l’augmentation du Ca2+ libre nucléaire induitepar l’ET-1 semble être médiée via l’activation des récepteurs ETB des membranes de l’enveloppe nucléaire. L’activationdu récepteur ETB des membranes nucléaires semble prévenir la surcharge calcique et ainsi la mort cellulaire.

Mots clés : endothelin-1, récepteurs de l’endothelin-1, calcium, récepteurs nucleaire, microscopie confocale.

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Introduction

Until recently, the focus on endothelin-1 modulation ofheart and vascular smooth muscle function has mostly been

concentrated on its sarcolemma ETA and ETB receptors. Alarge part of those studies was directed towards ET-1 modu-lation of cytosolic Ca2+ during excitation–contraction cou-pling. However, little attention was given to cytosolic Ca2+

Can. J. Physiol. Pharmacol. 81: 654–662 (2003) doi: 10.1139/Y03-020 © 2003 NRC Canada

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Received 12 March 2002. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 19 May 2003.

G. Bkaily,2 S. Choufani, S. Sader, D. Jacques, M. Nader, G. Kurban, and M. Kamal. Department of Anatomy and CellBiology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QC J1H 5N4, Canada.P. d’Orléans-Juste. Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QC J1H 5N4, Canada.

1This paper has undergone the Journal’s usual peer review process.2Corresponding author (e-mail: [email protected]).

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regulation by the nucleus (Bkaily 1994; Bkaily et al. 1994,1997a, 1997b, 2000, 2002; Jacques et al. 2000; Naik et al.1998) and the possible presence of G-protein(s)-coupled re-ceptors at the nuclear envelope membranes, such as ET-1 re-ceptors (Bkaily et al. 1997b, 2000, 2002). The stimulation ofthese nuclear envelope membranes receptors may contributeto the overall nuclear signalling process, including the nu-clear Ca2+ level (Bkaily et al. 1997b, 2000, 2002), cell cycle(Means 1994), gene expression (Peunova and Enikilopov1993; Hardingham et al. 1997), and activation of nuclearkinases and phosphatases (Czubryt et al. 1996), as well asDNA repair, break-down of the nuclear envelope (Steinhardtand Alderton 1988), and apoptosis (Nicotera and Rossi1994; Filippatos et al. 2001). Although several studies havemade use of isolated nuclei membrane vesicles, these studiesdid not take into consideration the cross-talk that may existbetween nuclear membranes receptors and channels with thesurrounding cytosolic environment of the living cell. It wasnot until the recent development of ionic fluorescent probes,mainly Ca2+ probes, and 3-D confocal microscopy tech-niques that it was possible to rediscover the nucleus in livingworking cells. Moreover, its possible important contributionbegan to be realized, not only as a locus for DNA synthesisand cell differentiation, but also as an important organellethat may largely participate in ET-1 regulation of Ca2+ ho-meostasis (Bkaily et al. 1997b, 2000) and protein synthesis(Hentze 2001; Iborra et al. 2001). Thus, the recent use ofconfocal microscopy in living cells allowed us to visualizeand quantify ET-1 and ET-1 receptors trafficking along withcalcium movement within the cells.

Currently, we attempt to emphasize the contribution ofET-1 and ET-1 nuclear membranes receptors to the overallintracellular Ca2+ trafficking. We further demonstrate thatsarcolemma ET-1 receptors-mediated calcium signals couldbe transferred to the nucleus, and that these signals could beamplified via stimulation of ET-1 receptors located at thenuclear envelope membranes, and most probably ETB recep-tors.

Materials and methods

The work was performed in accordance with the require-ments of the institutional review committee for the use ofhuman material. Human cells are isolated from normal or-gans of declared donors. The aortas are supplied by QuebecTransplant. The human aortic cultured cells used in thisstudy originated from one donor, however the human cul-tured cardiomyocytes originate from several donors. Allmethods used for cell isolation and culture, confocal micros-copy setting, Fluo-3 loading, nuclear Syto-11 staining, ET-1fluorescent probes, solutions, and sources of all compounds,as well as statistical analysis, were the same as those re-ported recently (Bkaily et al. 1997b, 1999, 2000). Nucleiwere isolated, and their morphological integrity was as-sessed as previously described (Gobeil et al. 2002). In addi-tion, staining with live cell nucleic acid Syto-11 is used atthe end of each experiment to assess the integrity of eachsingle nucleus used in our experiments. As previously de-scribed, the specificity of the fluorescent labelled ET-1 wastested by displacement of the labelled ET-1 by pretreatmentof the cells with unlabelled ET-1 as well by comparing the

dose–response curve of labelled and unlabelled ET-1 on thetension of aortic vascular smooth muscle (Bkaily et al.1997b, 1999, 2002).

Experiments were done using the ratiometric dyes yellowcameleon YC 3.1 and YC 3.1nu, which were a gift fromR.Y. Tsien. The expression of these two ratiometric Ca2+

probes in human aortic vascular smooth muscle cells wasdone using the same technique described by Miyawaki et al.(1999).

For immunofluorescence studies, the cells were fixed for10 min in ice-cold 4% paraformaldehyde (Brismar et al.1998). After washing with phosphate buffer solution (PBS),cells were incubated for 10 min with PBS containing sodiumborohydride (2 mg/mL), then permeabilized and blockedwith 0.1% Triton X-100, 7% normal goat serum (NGS), and5% nonfat dry milk (NFDM) for 30 min (Brismar et al.1998). Finally, the cells were washed twice in PBS and incu-bated overnight at 4°C with rabbit monoclonal anti-ETA andanti-ETB (Alamone Labs, Jerusalem, Israel) in PBS contain-ing 1.4% Triton X-100. After two PBS washes, the cellswere incubated for 1 h at room temperature with rabbit ormice anti-IgG-FITC secondary antibodies (MolecularProbes, Eugene, Ore.). The cells were examined using 3-Dimaging confocal microscopy, as described by Bkaily et al.(1999). Controls for the specificity of ETA and ETBmonoclonal antibodies were assessed using double labellingof ETA and ETB receptors, as well as using blocking stainingwith the peptide used to raise the antibody. All of these con-trols demonstrate a non-cross-staining between the two anti-bodies and complete absence of staining in presence of theirrespective blocking peptides (G. Bkaily, personal communi-cation).

Results

Presence of sarcolemma and nuclear membranes ET-1receptors in heart and aortic vascular smooth musclecells

Using an ET-1 fluorescent probe, developed in our labora-tory, that showed same efficiency as unlabelled ET-1 (Bkailyet al. 1997b, 2002), superfusion of embryonic chick heartcells (Bkaily et al. 1995a, 1995b; Taoudi-Benchekroun1995) with this fluorescent probe immediately showed a la-belling of ET-1 receptors at the sarcolemma membrane in acluster-like fashion (Figs. 1A and 1B). After 30 min, in thepresence of the ET-1 fluorescent probe, very low fluorescentlabelling was also seen in the cytosol, as well as at the nu-clear envelope membranes (Bkaily et al. 1997b, 2002). AsET-1 binding to its receptors is irreversible, the labelled ET-1also bound irreversibly to ET-1Rs, as washout of the ET-1fluorescent probe did not remove the labelling of ET-1 re-ceptors (Bkaily et al. 2002). However, pretreatment with un-labelled ET-1 did prevent the labelling of ET-1 receptors bythe labelled ET-1 (Bkaily et al. 2002). Using the sarcolemmaperforation technique (Bkaily et al. 1997b, 1999, 2000,2002), the cytosolic application of the ET-1 fluorescentprobe showed, within a minute, a high labelling of ET-1 re-ceptors at the nuclear envelope membranes (Fig. 1C). Thevery low labelling of the ET-1 fluorescent probe in the nu-cleus of intact cells (even after 30 min of exposure to la-belled ET-1) and the fast high labelling of the nucleus of

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sarcolemma perforated cells highly suggest that the probedoes not easily cross the plasma membrane and that the la-belling of the nucleus is due to the ET-1 binding to its recep-tors. In addition, the very low labelling observed after30 min of exposure of intact cells to label ET-1 could be dueto internalization of the ET-1 probe along with itssarcolemma receptors.

The use of monoclonal antibody against the ETA receptorshowed the presence of this type of receptor at thesarcolemma and the cystosol of embryonic chick heart cellsand human aortic vascular smooth muscle cells (Fig. 2B).Moreover, the use of the monoclonal antibody against ETBreceptors showed the presence of this type of receptor at thesarcolemma, cytosol, and nuclear envelope membranes, aswell as at the nucleoplasm of human aortic vascular smoothmuscle cells (hVSMCs) (Fig. 2C), ventricular heart cells,and human aortic endothelial cells (hECs) (not shown).Immunoreactivity of ET-1 was also found within the cytosoland the nucleus, and Fig. 2A shows an example. The pres-ence of ET-1 in cultured hVSMCs and hECs could be due tothe peptide synthesized within the cell and (or) to the recep-tor-internalized peptide. These two phenomena seem to takeplace and account for the presence of ET-1 in hVSMCs,hEC, and heart cells (G. Bkaily, personal communication).

Our results highly suggest that the ETA receptor in bothventricular heart cells and human aortic vascular smoothmuscle cells is present at the sarcolemma and the cytosoliclevels. On the other hand, the ETB receptor seems to be pres-ent in both human aortic vascular smooth muscle and heart

cells, with a high density at the nuclear envelope membranesand the nucleoplasm of the two cell types used. Endothelin-1also was found to be present not only at the cytosolic levelbut also within the nucleus. In addition, the difference in thedensity, distribution, and presence of ETA and ETB in differ-ent cell types and one cell type of different species highlydemonstrate that the antibodies used are specific (G. Bkaily,personal communication).

Modulation of cytosolic and nuclear free Ca2+ level byextracellular ET-1

In these series of experiments, using the Ca2+ fluorescentprobe Fluo-3, we tested the effect of different concentrationsof ET-1 (10–15–10–8 M) on the sustained basal cytosolic([Ca]c) and nucleoplasmic ([Ca]n) levels of free calcium inheart and vascular smooth muscle cells.

As can be seen in Fig. 3, superfusion of heart cells with10–15 M of extracellular ET-1 induced an increase of sus-tained basal [Ca]c and [Ca]n. Increasing the concentration ofET-1 up to 10–14 M caused a rise in both sustained basal[Ca]c and [Ca]n. Subsequent addition of ET-1 from 10–13 to10–9 M induced a dose-dependent increase in both sustained

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Fig. 1. Using the fluorescent ET-1 probe, three-dimensional re-construction of ET-1 receptor distribution in intact (A, front lat-eral view and B, rear lateral view) chick heart cells reveal thepresence of ET-1 receptors in the sarcolemma (A and B). Noteabsence of labelling at the nuclear levels. (C, look-through mode,ET-1 fluorescent probe) In sarcolemma perforated heart cells,cytosolic application of the ET-1 fluorescent probe showed label-ling all throughout the cell, including the nuclear envelope mem-branes. The colour scale represents pseudocolor fluorescenceintensity level from 0–255. The scale bar indicates 2 µm (modi-fied from Bkaily et al. 2002).

Fig. 2. Immunofluorescence labelling of ET-1 (A) and its recep-tors subtypes ETA (B) and ETB (C) in human aortic vascularsmooth muscle cells. Note that the labelling is distributedthroughout the cell (A and C), at the plasma membrane, in thecytosol and in the nucleus. However, the ETA receptor immuno-reactivity is mainly observed at the level of the plasma mem-brane (B). These results suggest a different distribution pattern ofendothelin-1 and its receptors subtypes in human aortic vascularsmooth cells. The pseudocolour bar represents fluorescence in-tensity from 0 to 255. The scale bar is in micrometres.



basal [Ca]c and [Ca]n. There was no further increase at aconcentration of 10–8 M. As can be seen in Fig. 3, the EC50of extracellular ET-1-induced increase of sustained basal[Ca]c in intact nonworking heart cells was 5.9 × 10–13 M,and that of sustained basal [Ca]n was 8.5 × 10–13 M.

In another series of experiments, we tested the effect ofextracellular ET-1 on intact embryonic chick heart cells andhuman aortic vascular smooth muscle cells using the ratio-metric Ca2+ dyes Indo-1 and yellow cameleon (YC 3.1 andYC 3.1nu). Figures 4 and 5 show typical experiments. Ascan be seen in these figures, superfusion of heart cellsloaded with Indo-1 and human aortic vascular smooth mus-cle cells expressing cytosolic and nuclear yellow cameleon(YC 3.1 and YC 3.1nu, respectively) with 10–7 M ET-1 in-duced a sustained increase in cytosolic and nuclear Ca2+ in-dependent of the Ca2+ probe used (Figs. 3, 4, and 5). Thecalcium chelator EGTA blocked the ET-1 induced increasein [Ca]c and [Ca]n (Fig. 4). Our results, using heart andVSMCs as well as Fluo-3, Indo 1, and yellow cameleonCa2+ probes, demonstrate that contrary to the results re-ported in Xenopus oocytes (Perez-Terzic et al. 1997), Fluo-3is an excellent probe for Ca2+ measurement, and its fluores-cence characteristics are not influenced by the compositionaldifferences between the nucleoplasm and the cytoplasm.

Using the sarcolemma-perforated technique, and in pres-ence of 100 nM [Ca]c, cytosolic application of ET-1 in heartcells showed a dose-dependent (10–15–10–8 M) increase in[Ca]n. As can be seen in Fig. 3, a sustained rise in basalnucleoplasmic free Ca2+ of heart cells was detected at acytosolic concentration as low as 10–15 M of ET-1. A subse-

quent elevation of cytosolic ET-1 from 10–15 to 10–10 M in-duced a dose-dependent increase in sustained basal [Ca]n, andno further increase was detected in the presence of 10–9 M ofthe peptide. The EC50 of the effect of cytosolic ET-1 on sus-tained basal [Ca]n was 1.1 × 10–14 M.

Using the same protocols and techniques, superfusion ofintact human aortic vascular smooth muscle cells with in-creasing concentration of extracellular ET-1 induced a dose-dependent increase in [Ca]c and [Ca]n. The EC50 of ET-1 in-duced increase of [Ca]c was 3.9 × 10–10 M, and that of [Ca]nwas 3 × 10–10 M (Bkaily et al. 2000). However, usingsarcolemma-perforated single cell technique, and in presenceof 100 nM [Ca]c, elevation of cytosolic ET-1 induced a dose-dependent increase of [Ca]n with an EC50 of 1.7 × 10–11 M(Fig. 6A). In both intact cells and sarcolemma-perforatedcells from human aortic vascular smooth muscle and embry-onic chick heart, the effect of ET-1 on [Ca]c and [Ca]n wascompletely blocked by the Ca2+ chelator EGTA. These re-sults suggest that in intact sarcolemma, the ET-1 increase in[Ca]c and [Ca]n in intact cells is due to the stimulation ofCa2+ influx through the sarcolemma membrane; however, theincrease of [Ca]n could be due in part to uptake of thecytosolic calcium increase by the nucleus (Bkaily et al.1997b, 2000, 2002). In addition, the increase in [Ca]n bycytosolic ET-1 is due to the stimulation of Ca2+ influx throughthe nuclear envelope membranes (Bkaily et al. 1997b, 2000,2002).

Using the sarcolemma perforated technique and in the ab-sence of cytosolic free Ca2+, cytosolic ET-1 was also foundto induce an increase in [Ca]n (Bkaily et al. 1997b). Theseresults suggest that stimulation of ET-1 receptors of the nu-clear envelope membranes induced both Ca2+ influx and mo-bilization of nucleoplasmic Ca2+. The mechanism by whichcytosolic ET-1 induced nucleoplasmic Ca2+ mobilizationshould be identified in the future.

These results suggest that in both heart cells and vascularsmooth muscle cells, extracellular ET-1 induced a dose-dependent increase of both cytosolic and nuclear Ca2+ levels.The increase in nuclear Ca2+ by extracellular ET-1 seems tobe due to the stimulation of Ca2+ influx through thesarcolemma membrane (Bkaily et al. 1997b, 2000, 2002).However, the EC50 of the effect of ET-1 on ET-1 receptors inthe sarcolemma of heart cells seems to be lower than thatfound in human aortic vascular smooth muscle cells (EC50respectively near 10–13 and 10–11 M). This difference couldbe due to higher sensitivity of ET-1Rs to ET-1 and (or)higher density and (or) type of receptor(s) presents in thesetwo types of cells. As for the sarcolemma membrane, theEC50 of ET-1 on nuclear envelope membranes of ET-1 recep-tors of heart cells seems to be lower (near 10–14 M) than thatfound for the nuclei of human aortic vascular smooth musclecells (near 10–11 M). Since only immunoreactivity for ETBreceptor was found in both heart and vascular smooth mus-cle cells, thus it is possible that the ET-1 effect on [Ca]n inboth nuclear preparations could be due to the activation ofETB receptors. This difference between the EC50 of ET-1 onnuclear Ca2+ of nuclei of heart and vascular smooth musclecells could be due in part to a high density and (or) sensitiv-ity of the nuclear envelope membranes ET-1 receptors. Inaddition, the ET-1 receptors at the nuclear envelope mem-

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Fig. 3. Stimulation of intact nonworking cardiac cells with dif-ferent extracellular concentrations induced a dose-dependent in-crease in sustained basal [Ca]c and [Ca]n. Following cytoplasmicmembrane perforation with ionomycin, stimulation with differentconcentrations of cytosolic ET-1 (10–15–10–18 M) induced a dose-dependent increase in sustained basal [Ca]n. n, number of experi-ments. Modified from Bkaily et al. 2002.



branes seem to be functional and may contribute to regula-tion of nuclear Ca2+ homeostasis.

Like the nucleus of sarcolemma perforated cells, ET-1 infM concentrations induced an increase in nuclear free Ca2+

of isolated intact nuclei (Fig. 6B). Thus, cytosolic ET-1 di-rectly modulates Ca2+ entry into the nucleus via activation ofET-1Rs located at the nuclear membranes levels. In addition,these results suggest that nuclear membranes ET-1Rs couldfunction independently of sarcolemma ET-1 receptors. How-ever, it is also possible that the nuclear membranes receptorsstimulation could also be indirectly dependent onsarcolemma ET-1Rs and on the internalization of ET-1 alongwith its receptors. These suggestions should be verified inthe future.

Direct modulation of cytoplasmic Ca2+ bufferingcapacity of the nucleus by endothelin-1 in heart andvascular smooth muscle cells

As was shown previously, extracellular and cytosolicendothelin-1 induced a steady-state increase in [Ca]c and[Ca]n in both heart and vascular smooth muscle cells viastimulation of Ca2+ influx through the steady-state resting R-type Ca2+ channel (Bkaily et al. 1991, 1995a, 1997b; Bkaily1994; Claing et al. 2002). We also showed that ET-1 recep-

tors are distributed in a cluster-like fashion in bothsarcolemma and nuclear membranes of heart and vascularsmooth muscle cells. In addition, in the absence of cytosolicCa2+, cytosolic application of ET-1 induced nucleoplasmicCa2+ release in human aortic vascular smooth muscle cells.We hypothesized that nuclear membranes ET-1 receptors ac-

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Fig. 5. In hVSMC expressing both the cytosolic (YC 3.1) and nu-clear (YC 3.1nu) yellow cameleon (YC) ratiometric Ca2+ probe(obtained from R. Tsien’s group), and using 3-D confocal micros-copy technique, free Ca2+ in the nucleus was found to be higherthan in the cytosol as was shown with Fluo-3 and Indo-1.Superfusion with 10–7 M ET-1 induced an increase in intracellularCa2+ that was higher in the nucleus. The changes in emission ratio535/480 (bound/unbound) were normalized to the effects of fullCa2+ saturation of the probe (Rmax, using ionomycin perforation)which were near 90% increases in ratio over the value at zeroCa2+ (by chelating cytosolic Ca2+ with EGTA). The method oftransfection and ratio calculation is similar to that used by theTsien group (Miyawaki et al. 1999). The last panel shows label-ling of the nucleus with Syto-11. White bar is in micrometres.

Fig. 4. The use of Indo-1 ratiometric Ca2+ dye and 3-D confocalimaging technique confirm the results reported by our laboratoryusing Fluo-3 Ca2+ dye which show that, in resting chick heartcell, the nuclear Ca2+ level is higher than that in the cytosol andextracellular application of ET-1 induced a large increase inintracellular Ca2+ that was higher in the nucleus, and this effectwas blocked by chelating extracellular Ca2+ with EGTA. Thechanges in emission ratio (405/485 nm) were normalized to theeffect of full Ca2+ saturation (Rmax using ionomycin), which werenear 100% increases in the ratio over the value at zero Ca2+

(Rmin using ionomycin plus EGTA). The color scale representsCa2+ ratio images (405/485 nm). The last panel represents label-ling of the nucleus with Syto-11.



tivation may stimulate nuclear membranes Ca2+ influx andmodulate cytosolic Ca2+ buffering capacity of the nucleus(Bkaily et al. 1997b). Using the sarcolemma membrane per-foration technique in heart and human aortic vascularsmooth muscle cells as well as the Fluo-3 Ca2+ confocal mi-croscopy (Bkaily et al. 1997b, 2002), our recent resultsshowed that when cytosolic Ca2+ concentration was gradu-ally changed from 100 to 2000 nM, a visible increase in nu-clear free Ca2+ could be seen starting from a 200 nMextranuclear free Ca2+ concentration (Fig. 7A). Further ele-vation of extranuclear free Ca2+ up to 1200 nM elevated nu-clear free Ca2+ in a concentration dependent manner. Furtherelevation of extranuclear free Ca2+ up to 1600 and 2000 nMdid not cause a further [Ca]n rise. In the presence of 1600 or2000 nM of extranuclear free Ca2+, superfusion of the extra-nuclear medium with ET-1 (100 nM) induced a further in-

crease in [Ca]n (Fig. 7A). However, in the presence of nearlynormal concentrations of cytosolic free Ca2+ (100 nM),superfusion with ET-1 (100 nM) induced a significant in-crease in the nuclear free Ca2+ of both heart and vascularsmooth muscle cells (Figs. 7B and 7C). In the presence ofcytosolic ET-1, superfusion with sequentially increasingconcentrations of extranuclear free Ca2+ from 200 to 1200(or 2000) nM did not further increase ET-1 induced sus-tained elevation of [Ca]n (Figs. 7B and 7C).

Discussion and future perspectives

Using confocal microscopy and Fluo-3 Ca2+ measure-ments, as well as indirect immunofluorescence studies, ourresults here and those recently reported by our laboratorydemonstrate that the nucleus plays an important role in

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Fig. 6. (A) Dose–response of the effect of cytosolic ET-1 on [Ca]n in sarcolemma perforated human aortic vascular smooth musclecells. The number of different experiments was 3. The cytosolic Ca2+ concentration was fixed at 100 nM (Bkaily et al. 2000) duringall the experiments (nucleus EC50 = 1.69 × 10–11 M). (B) In isolated intact nucleus, loaded with Fluo-3 Ca2+ dye, cytosolic applicationof 10–11 M ET-1 induced an increase in nucleoplasmic free Ca2+. The control shows the 3-D Ca2+ free imaging of the nucleus. Thecytosolic Ca2+ concentration is fixed at 400 nM. Last panel represents nuclear staining of the nucleus with Syto-11. The colour scalerepresents the pseudocolour intensity of the Fluo-3 dye-Ca2+ complex from 0 to 255 and calcium concentration from 0 to 40 µM.White bar is in micrometres. In panel B, all records are from the same nucleus. Panel A is modified from Bkaily et al. 2000.



excitation–contraction coupling by controlling cytosolicCa2+ level and waves. All of these results highly suggest thatresting cytosolic and nuclear free Ca2+, which determinesthe resting tension, is largely due to Ca2+ influx through thesarcolemma and nuclear membranes R-type Ca2+ channels.However, the transient increase in [Ca]c and [Ca]n duringexcitation–contraction coupling is mainly due to a calciuminflux through the sarcolemmal T- and (or) L-type Ca2+

channels, as well as to Ca2+ release from the sarcoplasmicreticulum. Since T- and L-type Ca2+ channels are rapidly in-activated during sustained voltage or pharmacological stimu-lation, these types of channels can only contribute to theonset stimulation of Ca2+ influx. However, the R-type Ca2+

channel will contribute to both the onset and sustained ele-vation in cytosolic and nuclear free Ca2+ seen in normal andpathological conditions, depending on the function of thestudied cell type.

Our results using the ratiometric calcium probe Fluo-3, aswell as the ratiometric Ca2+ dyes Indo-1 and yellowcameleon, showed that the level of free calcium in the nu-cleus is higher than that in the cytosol. These results confirmthat the observed effect of ET-1 on [Ca]c and [Ca]n is inde-pendent of the Ca2+ dye used. Moreover, these results dem-onstrate clearly that the calcium signal of Fluo-3 is notaffected by the experimental conditions used in our prepara-tions.

Our present results, as well as those reported in the litera-ture, clearly demonstrate that ET-1 receptors are present atboth the sarcolemma and nuclear envelope membranes lev-els. These receptors may contribute to maintaining not onlyCa2+ influx through the sarcolemmal membrane but also par-ticipate in controlling the basal nucleoplasmic free Ca2+ inresting cells. As shown in our present and recently publishedwork (Bkaily 1994; Bkaily et al. 1997b, 1998, 2000, 2002),several highly active circulating compounds such as ET-1may directly act at the nuclear envelope membranes and pro-mote both Ca2+ entry into the nucleus, as well as nucleo-plasmic Ca2+ release, thus increasing [Ca]n and shielding thenucleoplasm from sustained nucleoplasmic Ca2+ overload.Thus, our results support the fact that ET-1 receptors-mediated Ca2+ signals could be transferred not only to thecytoplasm but also to the nucleus. Alternatively, this phe-nomenon may as well take place independently of cytosolicCa2+ changes once ET-1 is present in the cytosol. In additionto the cytosolic Ca2+ buffering property of the nucleus andits regulation by ET-1 receptors stimulation, the low kineticrelease of Ca2+ (G. Bkaily, personal communication) by thenucleus maintains a high level of cytoplasmic Ca2+, thus al-lowing longer vascular smooth muscle contraction. As well,

it allows activation of other phenomena that are Ca2+ de-pendent, such as Ca2+-dependent protein kinase and nuclearCa2+ binding proteins (such as calmodulin), which areknown to regulate both cell function and gene expression(Gilchrist et al. 1994). The ET-1 receptors located at the nu-clear membranes levels seem to be more sensitive to ET-1than receptors located at the sarcolemma level, as demon-

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Fig. 7. Effect of cytosolic ET-1 on 3-D measurements of intra-nuclear free Ca2+ of plasma membrane perforated human aorticvascular smooth muscle cells (A (n = 4) and B (n = 14)) andchick heart cells (C (n = 3)) before and after the sequential addi-tion of increasing concentrations of Ca2+ to the extranuclear me-dium. The results are expressed in terms of absolute Fluo-3calcium fluorescence intensity. Values are expressed as means ±SE. Measurements were taken when the effect reached a steady-state level. Panels A and B modified from Bkaily et al. (1997b)and panel C modified from Bkaily et al. (2002).



strated in Fig. 3. Such an extremely high sensitivity of nu-clear membrane ET-Rs when compared with those of thesarcolemma was also reported for angiotensin II type 1 re-ceptors (Booz et al. 1992). In addition, our recent resultsshowed that the ET-1-induced increase in sustained [Ca]cand [Ca]n was insensitive to the peptidic ETA and ETB re-ceptors antagonists BQ123 and BQ788 (Bkaily et al. 2002).However, our very recent results showed that contrary to thepeptidic ET-1Rs antagonists, the effect of ET-1 on [Ca]c and[Ca]n on intact cells was blocked by the nonpeptidic antago-nists of ETA (BMS-182874) and ETB (Ro46-8443) receptors.In addition, direct nuclear effect of ET-1 was blocked onlyby the nonpeptidic antagonist of this receptor (G. Bkaily,personal communication). These differences between thepeptidic and nonpeptidic ET-1Rs antagonists could be due tothe short half-life of the peptidic antagonists (Stein et al.1994) and (or) to a population of receptors that are insensi-tive to BQ123 and BQ788 (Hay et al. 1998).

As reported in the literature dealing with ET-1 and ET-1receptors, both ETA and ETB receptors are localized at thesarcolemma of heart and human aortic vascular smooth mus-cle cells. However, our recent results showed that only theETB receptor is present at the nuclear envelope membranesas well as in the nucleoplasm. Since ET-1 undergoes inter-nalization along with its ligand-receptor complexes (Hocheret al. 1992; Bhownick et al. 1998), and since the nuclear en-velope receptors are more sensitive to ET-1 than those pres-ent at the sarcolemma membrane, thus it is possible topostulate that ET-1 of the internalized ligand–receptor com-plex may activate the nuclear membranes ET-1 receptors. Inaddition, ET-1 could be made available for the nuclear mem-branes ET-1 receptors from leaky ET-1 secretory vesiclesand (or) from ET-1 present at the nuclear level (as wasshown in Fig. 3A). This latter phenomenon may take placeupon increase of nucleoplasmic and perinucleoplasmic freeCa2+ which trigger exocytosis and release of nucleoplasmicET-1 into the perinucleoplasm as well as the release of theperinucleoplasmic ET-1 into the cytosol. A rapid increase infree Ca2+ would induce a rapid burst of exocytosis (Rettigand Neher 2002) from the perinucleoplasm into the cytosoland from the nucleoplasm into the perinucleoplasm. The ac-tivation of ET-1 nuclear membranes receptors may explainin part the well-known long-term effect of ET-1. Since ourresults showed that only ETB receptors are present at thenuclear envelope membranes, thus, this type of nuclear mem-branes receptor may promote the effect of sarcolemmal ETAreceptor activation by ET-1. The contribution of nuclear mem-branes ETB receptors to the overall known effect of ET-1 oncell cycle and apoptosis still needs to be verified.

Acknowledgements

This study was supported by the Canadian Institutes ofHealth Research (CIHR) Group grant and the Quebec HeartFoundation. P. D’Orléans-Juste and D. Jacques are scholarsof the Fonds de la recherché en santé du Québec (FRSQ).The authors thank R.Y. Tsien for the gift of yellow cameleonYC 3.1 and YC 3.1nu, Ghassan Bassam Bkaily for cell cul-ture preparations, and Christiane Ducharme for her secre-tarial assistance.

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activation of sarcolemma and nuclear membranes et-1 receptors regulates transcellular calcium levels...

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