1 SDSU Heart Institute and Department of Biology, San Diego State University, San Diego, California 92182-4614; 2 Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641; and 3 Consiglio Nazionale delle Ricerca Institute of Neuroscience, Section of Muscle Biology and Physiopathology, Department of Biomedical Sciences, University of Padua, 35121 Padua, Italy
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ABSTRACT |
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Calcium channels are important in a variety of cellular events including muscle contraction, signaling, proliferation, and apoptosis. Sphingolipids have been recognized as mediators of intracellular calcium release through their actions on a calcium channel, sphingolipid calcium release-mediating protein of the endoplasmic reticulum (SCaMPER). The current study investigates the expression and function of SCaMPER in cardiomyocytes. Northern analyses and RT-PCR cloning and sequencing revealed SCaMPER expression in both human and rat cardiac tissue. Immunofluorescence and Western blot analyses demonstrated that SCaMPER is abundant in cardiac tissue and is localized to the sarcotubular junction. This was confirmed by the colocalization of SCaMPER with dihydropyridine and ryanodine receptors by confocal microscopy. Purified T tubules were shown to contain SCaMPER and immunoelectron micrographs suggested that SCaMPER is located to the junctional T tubules, but a junctional SR localization cannot be ruled out. The sphingolipid ligand for SCaMPER, sphingosylphosphorylcholine (SPC), initiated calcium release from the cardiomyocyte SR. Importantly, antisense knockdown of SCaMPER mRNA produced a substantial reduction of sphingolipid-induced calcium release, suggesting that SCaMPER is a potentially important calcium channel of cardiomyocytes.
sphingolipid calcium release-mediating protein of the endoplasmic reticulum; calcium channel; sphingosylphosphorylcholine; sphingolipids and cardiomyocytes
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INTRODUCTION |
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RECENTLY, sphingolipids have emerged as important signaling molecules with diverse effects on the cardiovascular system (8). These effects include promotion of angiogenesis (7, 11), atherogenesis (27, 28), coronary vasoconstriction, and arrhythmias (9, 13, 24). Additionally, sphingolipids have been shown to mediate a variety of cellular events such as proliferation, apoptosis, and calcium signaling (10). The diverse effects of sphingolipid mediators are in part explained by the finding that these signaling molecules have direct intracellular actions as well as acting on surface membrane receptors (4).
Sphingolipid mediators such as sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) act as extracellular signaling molecules (3). For example, many of the actions of extracellular S1P have been attributed to activation of a family of G protein-coupled receptors (GPCRs) belonging to the Edg family of receptors (12). We recently showed that Edg-1 is expressed on the surface of cardiomyocytes and is responsible for S1P's ability to deregulate calcium levels (17) and promote apoptosis in cardiomyocytes (31). Many important physiological actions of S1P and SPC are independent of Edg receptors (26). However, the search for Edg-independent sites of sphingolipid action has remained problematic. The present work was initiated with the intent of identifying other potential protein targets responsible for sphingolipid effects in heart cells.
Recently, S1P and SPC have been recognized as mediators of intracellular calcium release through their actions on a calcium channel, sphingolipid calcium release-mediating protein of the endoplasmic reticulum (SCaMPER) (5). Proof that SCaMPER is a calcium channel came from the finding that a SCaMPER cDNA obtained from a Madin-Darby canine kidney (MDCK) expression library was expressed in oocytes and was subsequently found to mediate sphingolipid-induced intracellular calcium release (14). We previously showed (1) SCaMPER expression in heart, skeletal muscle, and brain tissue and suggested that it may play a calcium regulatory role in the heart; however, we had not determined which cell type in the heart expressed SCaMPER and had not elucidated its functional role in heart cells.
Consequently, this study was initiated to determine the expression, localization, and function of SCaMPER in sphingolipid signaling and calcium regulation of heart cells. We report here that SCaMPER is expressed in cardiomyocytes. Furthermore, immunological techniques localize SCaMPER to the transverse tubules, an important site of calcium regulation. Using antisense expression constructs, we demonstrate SCaMPER's role in calcium signaling in cardiomyocytes.
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MATERIALS AND METHODS |
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RT-PCR, cloning, and DNA sequencing. Kidney, brain, skeletal, and heart tissue isolated from adult rats and adult human heart tissue were used. The tissue was either used fresh or flash frozen, and RNA was isolated with the Qiagen Rneasy Mini Kit (Valencia, CA). Amplification of the sequence was with a Qiagen One-Step RT-PCR kit using the standard protocol. Primer sets included sense primer 5'-CCAGGATTCATCATATGTTAAAAG-3' and antisense primer of 5'-ATCAGTGGGTGCATCAGTAGC-3' for open reading frame (ORF) (designed from GenBank accession no. U33628). Amplification utilized the kit's recommended cycling with an optimization of 40 PCR cycles, which featured 45-s denaturation at 95°C, 45 s of annealing at 50°C, and 1 min of polymerization at 72°C. The SCaMPER PCR products were subcloned into pCR3.1 TOPO (Invitrogen, Carlsbad, CA) with standard techniques. Sequencing with a T7 primer was performed at the San Diego State University DNA Core Facility (San Diego, CA) and was used to diagnose plasmids as sense or antisense orientations. Plasmids were amplified and purified for subsequent transfection studies with QIAfilter plasmid MAXI and MEGA kits (Qiagen).
SCaMPER antibody development. A putative antigenic site for SCaMPER was determined by PC GENE and MCVECTOR software on the basis of its hydrophilicity, surface probability, backbone flexibility, and predicted tertiary structure of the protein (GenBank accession no. U33628). The identified sequence was APDLKIRDPKIEKLYC, which is located near the NH2 terminus of the putative cytosolic domain. A search of the GenBank protein data bank via BLAST revealed that this sequence is unique for the SCaMPER protein. The peptide corresponding to the sequence was synthesized by Sigma-Genosys (Woodlands, TX). A rabbit polyclonal anti-SCaMPER antibody was generated with standard techniques and titered via ELISA. The serum from the final bleed was purified by protein A column chromatography (Bio-Rad, Hercules, CA).
Immunological methods. Microsomal fractions of adult and neonatal rat ventricles were isolated as previously described (2). Sarcoplasmic reticulum (SR) fractions were isolated from total microsomal fractions by iterative loading with calcium oxalate and multistep sucrose density gradient centrifugation as previously described (20). Designated amounts of sample for Western blot analysis were incubated with Laemmli buffer (Bio-Rad), run on SDS-PAGE (18%), and transferred to nitrocellulose. Primary antibody dilutions and incubation times of 1:10,000 SCaMPER IgG for 45 min, 1:1,000 sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2 ATPase and phospholamban IgG2a (ABR, Golden, CO) and 1:1,000 Na+/Ca2+ exchanger (cardiac) IgG1 (a kind gift from Kenneth Philipson, UCLA, Los Angeles, CA) for 1 h were used. Secondary antibody dilutions of 1:5,000 anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated (Invitrogen) antibodies for 1 h were used. The blots were developed with the ECL technique according to the manufacturer's protocol (Amersham Pharmacia Biotech, Piscataway, NJ). For the peptide blocking experiments, blotted protein was preincubated for 1 h with 10 µg/ml of peptide (used for SCaMPER antibody immunizations) before addition of the anti-SCaMPER antibody. For the deglycosylation experiments, an enzymatic deglycosylation kit (Glyko, Novato, CA) was used with the kit protocol.
Immunofluorescence microscopy was performed on 6-µm-thick frozen cryostat sections of adult rat ventricle tissue as previously described (31) and on isolated adult and neonatal cardiomyocytes (17). Antibody dilutions and incubation times were 1:150 anti-SCaMPER IgG for 1 h and 1:500 anti-rabbit-FITC or 1:500 anti-mouse-rhodamine red (Jackson, West Grove, PA) for 1 h. Colocalization experiments used an anti-dihydropyridine receptor (DHPR) antibody at 1:500 dilution (ABR), an anti-ryanodine receptor (RyR) antibody at 1:500 dilution (ABR), and an anti-SERCA2 antibody at 1:500 dilution (ABR) along with anti-mouse-rhodamine red-X antibody (Jackson) at a dilution of 1:500. An antinuclear pore O-linked glycoprotein antibody (ABR) was used at a dilution of 1:100. Confocal images were taken on a Leica TCS SP2 confocal inverted light microscope. In other experiments, immunofluorescence was performed with a Nikon Labophot-2 microscope. Immunoelectron microscopy of cardiac tissue was performed essentially as previously described by Ralston and Ploug (19) with slight modification. Briefly, a fresh rat heart was excised, cannulated, and perfused with 60 ml of ice-cold fixative (4% paraformaldehyde in 0.1 M phosphate buffer). The left ventricle was then cut into thin strips and allowed to incubate further in the fixative for 5 h on ice. Small bundles of cardiac fibers were teased away in 50 mM glycine and incubated in blocking solution (50 mM glycine, 0.25% BSA, 0.1% saponin in 1× PBS) for 30 min. The fibers were then incubated with anti-SCaMPER antibody or preimmune serum at a 1:100 dilution in blocking solution overnight at room temperature. Fibers were washed three times for 30 min in blocking solution and incubated for 4 h with a goat anti-rabbit 10-nm gold-conjugated secondary antibody (BB International). The fibers were then washed twice in blocking solution and once in 1× PBS. Glutaraldehyde fixation, silver enhancement, and further processing for electron microscopy were performed as previously described (19) with a Philips 410A electron microscope. Imaging was performed with the PGT IMIX Image Analysis System.Northern analysis.
Adult rat heart ventricular RNA was isolated with a Qiagen Rneasy MAXI
kit using the kit's standard protocol for isolation from animal
tissue. mRNA was isolated with the PolyATtract mRNA isolation system
(Promega). Ten micrograms of mRNA were fractionated on a 1%
agarose-18% formaldehyde gel and blotted onto Zeta-Probe GT genomic
membranes (Bio-Rad). A 350-bp probe consisting of the SCaMPER ORF was
radioactively labeled with Random Primer Labeling Reaction
(Invitrogen). Hybridization was performed in 20 ng/ml 32P-labeled probe (specific activity of 1-109
cpm/µg) in hybridization solution (5× Denhardt's, 6× SSC, 0.5%
SDS) with 100 µg/ml herring sperm (Sigma, St. Louis, MO) at 55°C
overnight. The blot was washed in 1× SSC-0.5% SDS twice for 20 min
and with 0.1× SSC-0.5% SDS twice for 20 min at 55°C.
Autoradiography was performed for 24 h on Kodak Biomax film at
70°C.
Expression methods. The SCaMPER ORF was cloned into pcDNA3.1-HIS/V5 TOPO TA cloning vector (Invitrogen) and used for expression in an in vitro rabbit reticulocyte lysate system for molecular weight determination. The TNT Quick Coupled Transcription/Translation System (Promega) was used for expression with the Transcend nonradioactive translation detection system (Promega) utilizing biotinylated lysine incorporation. Protein expression was performed and subjected to Western blotting and detection with the kit protocol. Analysis of cotranslational and initial posttranslational processing events including core glycosylation was with addition of canine pancreatic microsomal membrane (Promega) to the transcription/translation system.
Bacterial expression was from a pRHA-driven expression vector induced with 10 mM rhabanose for 2 h in exponentially growing Escherichia coli MG1655 (a kind gift from Mpex BioScience, San Diego, CA). Bacteria were collected at an OD600 of 1.0, lysed in Laemmli buffer, separated by SDS-PAGE (18%), transferred to nitrocellulose, and probed with the SCaMPER antibody as described above. For the antisense stress response assay, neonatal cardiomyocytes were transfected (as described below) with previously published plasmids (25). Briefly, either GRP78-ERSE-Luc encoding the active ERSE from the human GRP78 cDNA driving SV40 luciferase or a pGL2 control vector (Promega) was cotransfected either with the antisense-SCaMPER construct (used in calcium analysis) or with empty vector pCDNA3.1. The cardiomyocytes were also cotransfected with a SV40-Cell culture methods. Cardiomyocytes were isolated from neonatal rat hearts or from adult Sprague-Dawley rat hearts with previously published techniques (6, 23) with modifications including the use of pyrogen-free collagenase (Liberase blendzyme; Roche, Indianapolis, IN) for the digestion of the adult rat hearts. The cardiomyocytes were plated on 5 µg/ml or 25 µg/ml fibronectin-coated cell culture plates or glass coverslips, respectively. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM)-F-12 with 10% fetal bovine serum for 24-48 h for neonatal cells and 1 h for adult cells. All procedures involving experimental animals were performed in accordance with institutional and NIH guidelines for the ethical treatment of animals.
Cell transfections. For neonatal cardiomyocytes, pCR3.1 containing a reverse SCaMPER insert (1,200 bases of the ORF and partial 3' end) was used for antisense expression and a nonsense (not recognized against any specific databased sequence) insert of 1,200 bases was used for control (nonsense) expression. Cardiomyocyte transfections were performed by electroporation, with 3 million neonatal cardiomyocytes/cuvette, 5 µg of pGreen Lantern (Invitrogen), and 20 µg of pcDNA3.1 empty vector, nonsense, or antisense SCaMPER. Electroporations were at 0.55 mV as previously described (31).
Calcium assays. Calcium measurements were measured as described previously (31) with indo 1-AM (Sigma). Results are presented as the ratio of indo 1 fluorescence emission of 405 vs. 485 nm rather than as absolute calcium concentrations. Cardiomyocytes were washed once with Tyrode solution (in mM: 140 NaCl, 5.4 KCl, 0.5 MgCl2, 1 CaCl2, 10 HEPES, 0.25 NaH2PO4; pH 7.3), loaded with 3 mM indo 1-AM for 25 min, and washed again. The coverslips were mounted into a chamber with 1 ml of Tyrode solution and placed in position for calcium analysis with the PTI system. Positively transfected cardiomyocytes were identified with green fluorescent protein (GFP; Photon Technologies) fluorescence at 480 nm and selected for calcium analysis. Selected cardiomyocytes were electrically paced for 20 s at 0.3 Hz with 10-ms stimuli with no delay at a monophasic output. Pacing was turned off, and at the designated time point, 50 µM SPC (final concentration) was carefully added. SPC in CHCl3-MeOH (2:1) was dried down with argon and resuspended in 100 µl of BSA (1 mg/ml) and Tyrode solution to a 100 µM SPC working solution. At the designated time points, 10 mM caffeine (final concentration) was added to elicit the release of calcium from the SR membranes. In selected experiments designed to test for the effects of antisense treatment on phasic calcium release, we compared the calcium transients of electrically paced cells transfected either with empty vector or with the antisense expression construct. The calcium transients were then overlaid to determine whether there was an effect on normal excitation-contraction (EC) coupling.
Statistical analyses were performed with Student's t-test. Means ± SE are shown in all results. ![]() |
RESULTS |
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SCaMPER expression in cardiac tissue.
To investigate SCaMPER expression, total RNA was isolated
from rat heart and subjected to RT-PCR. The RT-PCR procedure was carried out with primers against the MDCK clone of SCaMPER
(see MATERIALS AND METHODS). For both human and rat cardiac
tissue, RT-PCR yielded a product of 1.2 kb (accession nos. AY163814 for
human and AY163813 for rat) . Additional RT-PCR analyses demonstrated
the expression of SCaMPER mRNA in various other tissues including rat kidney, brain, and skeletal muscle (Fig.
1A).
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Subcellular localization of SCaMPER.
The prominent punctate nature of SCaMPER staining seen in Fig.
2C suggested that SCaMPER was not distributed uniformly
along the sarcotubular system and that it could be at the junctional regions where the SR and T tubules are located. Accordingly, confocal immunomicroscopy was used to colocalize SCaMPER with two
well-recognized junctional proteins, the DHPR of the junctional T
tubules and the RyR of the junctional SR. Other antibodies of interest
were used, including an antibody to SERCA, a prominent protein of the longitudinal SR. The data in Fig.
3A show a strong
colocalization of the SCaMPER antibody with the DHPR and a weaker
colocalization with the RyR. Because colocalization of SCaMPER with
SERCA was not observed, SCaMPER is not likely associated with the
longitudinal SR. Together, these data suggest that SCaMPER is localized
to the sarcotubular junction, either the junctional SR or T tubules.
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SCaMPER regulates cardiac cell calcium. The expression and localization of SCaMPER to the sarcotubular junction where EC coupling is initiated suggests that SCaMPER may play an important role in the regulation of calcium in cardiomyocytes. Consequently, we examined the ability of SCaMPER to alter calcium signaling in cultured neonatal cardiomyocytes. Overexpression of SCaMPER in neonatal cardiomyocytes proved to be toxic to these cultured cells (data not shown) and thus precluded us from using SCaMPER expression constructs in our calcium studies. Such cellular toxicity was also reported for MDCK cells overexpressing SCaMPER (21). These workers observed severe alterations in morphology, followed by cell death when SCaMPER was overexpressed. Consequently, we turned to antisense techniques to knock down endogenous SCaMPER expression to deduce the role of SCaMPER in calcium signaling. This was achieved by transfection of isolated neonatal ventricular cardiomyocytes with expression plasmids containing the SCaMPER ORF in antisense orientation.
The ability of SCaMPER to mediate calcium signaling was studied by real-time calcium transient analysis. Neonatal cardiomyocytes were incubated with the ratiometric calcium dye indo 1-AM and then monitored for real-time alterations of calcium signaling as described previously (17). The positively transfected cells were electrically paced for 20 s to ensure that they were able to release calcium from the SR. After electrical pacing was discontinued, the calcium transients ceased and at 30 s SPC was added to elicit a calcium release, as previously demonstrated in adult cardiomyocytes (1). Routinely, 10 mM caffeine (final concentration) was added at 120 s to demonstrate whether or not the SR calcium store had been mobilized by the sphingolipid. Shown in Fig. 4, A and B, are typical responses to SPC in control (empty vector) and antisense-transfected cardiomyocytes. As reported previously in adult cardiomyocytes (1), the neonatal cardiac cells released nearly all of their SR calcium in response to SPC. Resting (diastolic) calcium levels increased dramatically, producing asynchronous and spontaneous calcium transients in the absence of electrical pacing. Eventually, the cell reached calcium overload. By 90 s after SPC addition, the SR was essentially devoid of calcium, as evidenced by the lack of a subsequent caffeine response.
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DISCUSSION |
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This report is the first demonstration of a possible role of the sphingolipid-modulated calcium channel SCaMPER in cardiomyocytes. With RT-PCR, Northern blotting, and a variety of immunological methods, we demonstrate that SCaMPER is expressed in both adult and neonatal cardiomyocytes as well as in adult cardiac tissue.
Several findings presented in this study suggest that SCaMPER is localized subcellularly to the junctional regions of the sarcotubular membrane system where connections between the T tubules and the SR function to regulate cardiac cell calcium levels. Evidence for the sarcotubular localization comes first from the finding that the anti-SCaMPER antibody colocalizes with the DHPR, a known component of the junctional T tubules, and with a junctional SR marker, the RyR. Confocal imaging was confirmed by the demonstration that T tubule-enriched membrane subfractions display the immunoreactive SCaMPER protein in contrast to longitudinal SR membranes, which do not. Finally, immunoelectron microscopy suggests a T tubular localization of SCaMPER. Together these data suggest that SCaMPER may be localized to the T tubules. On the other hand, it is also possible that SCaMPER could be a junctional SR protein, as evidenced by the apparent colocalization with the RyR. The resolution of indirect fluorescence is not sufficiently robust to enable us to distinguish junctional T tubules from junctional SR.
Because SCaMPER has been shown to be a calcium channel expressed by human basophilic leukemia cells and canine MDCK cells (4, 5, 14), localization of this channel to the junctional regions of cardiac cells suggested that it might have an important role in cardiac calcium control. Accordingly, we demonstrated that SPC, the well-recognized sphingolipid ligand for SCaMPER, elicits profound calcium release from the cardiac SR. Moreover, the finding that antisense expression constructs for SCaMPER significantly reduce the calcium response suggests that SCaMPER is the target of SPC action in controlling SR calcium. We had previously demonstrated (1) that the SPC response could be eliminated by pretreating cardiomyocytes with thapsigargin, an agent that depletes SR calcium. However, until this study, it had not been definitively proven that SCaMPER mediates the sphingolipid-triggered calcium response in heart cells. Surprisingly, endogenous knockdown of SCaMPER expression did not alter phasic calcium release, indicating that SCaMPER does not influence normal EC coupling or alter SR calcium content.
Using a SCaMPER expression construct obtained from canine basophilic leukemia cells, Kindman and colleagues (5) demonstrated that patch-clamped oocytes overexpressing SCaMPER displayed a functional SPC-modulated calcium channel. These workers subsequently described SCaMPER as having a 546-bp ORF (14). However, Zacchetti's group (21) recently argued that the MDCK cDNA is substantially smaller. We have confirmed Zacchetti's group's findings in that the rat and human cardiac SCaMPER cDNA is 333 bp in length. We obtained the clone from Kindman and discovered that the sequence of the clone used in his functional expression studies was in fact 333 bp, suggesting that a sequencing error led Kindman et al. to believe that the ORF was larger than it was. It is important to emphasize that the expression construct used in the current work to prove that SCaMPER is a calcium channel came from the clone that was used in the oocyte expression work previously published (14). Thus we confirm in the present study that SCaMPER is likely a calcium channel or a calcium channel modulator. However, it is likely that the shorter ORF reported here is a single-pass membrane protein rather than the double-pass membrane protein suggested previously (14).
Calcium regulation is an essential feature of cardiac function. Because sphingolipids are gaining increasing attention as important signaling molecules in the cardiovascular system (8), an understanding of the role of SCaMPER in cardiac function will be of great interest in explaining the growing list of physiological effects on the heart attributed to sphingolipid mediators such as SPC and S1P. For example, it was recently shown that SCaMPER's putative ligand, SPC, is a normal constituent of serum (9) and that it may be involved in cardiac hypertrophy (22) and in the regulation of cardiac chronotropy and inotropy (9). Our current findings and our previous work (1) demonstrating that skeletal and cardiac calcium is modulated by SPC are consistent with the well-established role of SPC in regulating cellular calcium in other cell types (15, 29).
One might infer from the finding that SCaMPER is expressed on the surface membranes (i.e., T tubules) that some of the extracellular actions attributed to SPC as an extracellular signaling molecule (16) may be explained by the mediator's effects on SCaMPER. However, additional work is needed to elucidate this and to distinguish extracellular from intracellular actions of sphingolipids in the heart. In addition, it has been demonstrated that SPC is a possible ligand for Edg and other GPCRs (18, 30), some of which are expressed by rat cardiac cells (17). Thus it becomes important to distinguish SCaMPER from the GPCRs as putative targets of sphingolipid action in the heart.
The antisense knockdown approach used in this study provides clear demonstration that the SCaMPER protein has a potential physiological role in mediating SPC effects in heart cells. We have shown not only that SCaMPER is expressed in cardiomyocytes, but more importantly, that it is a modulator of calcium regulation in these cells. Experiments in progress will determine the oligomeric nature of SCaMPER and how it functions as a calcium channel. The expression and localization of SCaMPER to the sarcotubular junction set the stage for this protein to play an important role in the regulation of cardiac calcium.
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ACKNOWLEDGEMENTS |
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We thank Doriana Sandoná for help with the design of the peptides for antibody development.
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FOOTNOTES |
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This work was supported by grants from Medlyte, Inc. (R. A. Sabbadini), Consiglio Nazionale delle Ricerca, Italy (R. Betto), and Telethon Italy (no. 1268 to R. Betto).
Address for reprint requests and other correspondence: R. A. Sabbadini, Dept. of Biology, San Diego State Univ., San Diego, CA 92182-4614 (E-mail: rsabba{at}sunstroke.sdsu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 6, 2002;10.1152/ajpcell.00382.2002
Received 22 August 2002; accepted in final form 5 November 2002.
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