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J. Biol. Chem., Vol. 277, Issue 18, 16313-16323, May 3, 2002

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Molecular Basis for Pacemaker Cells in Epithelia^*

M. Fatima Leite, Keiji Hirata^§, Thomas Pusl^§, Angela D. Burgstahler^§, Keisuke Okazaki^¶, J. Miguel Ortega, Alfredo M. Goes, Marco A. M. Prado^**, David C. Spray, and Michael H. Nathanson^§§§

From the  Department of Physiology and Biophysics, Universidade Federal de Minas Gerais (UFMG), 31270-901 Belo Horizonte, Brazil, the ^§ Departments of Medicine and Cell Biology, Yale University, New Haven, Connecticut 06520-8019, the ^¶ Department of Surgery, University of Occupational and Environmental Health, Kitakyushu, 8078555, Japan, the  Department of Biochemistry and Immunology, UFMG, Belo Horizonte, Brazil, the ^** Department of Pharmacology, UFMG, Belo Horizonte, Brazil, and the  Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461

Received for publication, September 24, 2001, and in revised form, February 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intercellular signaling is highly coordinated in excitable tissues such as heart, but the organization of intercellular signaling in epithelia is less clear. We examined Ca²⁺ signaling in hepatoma cells expressing the hepatocyte gap junction protein connexin32 (cx32) or the cardiac gap junction protein cx43, plus a fluorescently tagged V_1a vasopressin receptor (V_1aR). Release of inositol 1,4,5-trisphosphate (InsP₃) in wild type cells increased Ca²⁺ in the injected cell but not in neighboring cells, while the Ca²⁺ signal spread to neighbors when gap junctions were expressed. Photorelease of caged Ca²⁺ rather than InsP₃ resulted in a small increase in Ca²⁺ that did not spread to neighbors with or without gap junctions. However, photorelease of Ca²⁺ in cells stimulated with low concentrations of vasopressin resulted in a much larger increase in Ca²⁺, which spread to neighbors via gap junctions. Cells expressing tagged V_1aR similarly had increased sensitivity to vasopressin, and could signal to neighbors via gap junctions. Higher concentrations of vasopressin elicited Ca²⁺ signals in all cells. In cx32 or cx43 but not in wild type cells, this signaling was synchronized and began in cells expressing the tagged V_1aR. Thus, intercellular Ca²⁺ signals in epithelia are organized by three factors: 1) InsP₃ must be generated in each cell to support a Ca²⁺ signal in that cell; 2) gap junctions are necessary to synchronize Ca²⁺ signals among cells; and 3) cells with relatively increased expression of hormone receptor will initiate Ca²⁺ signals and thus serve as pacemakers for their neighbors. Together, these factors may allow epithelia to act in an integrated, organ-level fashion rather than as a collection of isolated cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells within excitable tissues such as the heart must act in a coordinated fashion to carry out organ-level functions such as muscle contraction to maintain blood flow. Excitation-contraction coupling in the heart is coordinated by anatomically defined pacemaker cells, and is mediated by cytosolic Ca²⁺ signaling in individual myocytes. Intercellular Ca²⁺ waves can be observed in the intact heart (1), and these Ca²⁺ signaling patterns spread from cell to cell via gap junctions (2) and may relate to normal and abnormal cardiac function (1). In individual myocytes, intracellular Ca²⁺ release occurs principally via the ryanodine receptor (RyR)¹ (3). Although RyR are spread throughout myocytes, focal clusters generate Ca²⁺ sparks that can initiate cell-wide Ca²⁺ signals (4-6). Intra- and intercellular Ca²⁺ signaling follows a different paradigm in non-excitable cells (3). Ca²⁺ signaling in epithelial organs and in many other tissues instead occurs principally via inositol 1,4,5-trisphosphate (InsP₃) and the InsP₃ receptor (InsP₃R) (3). As in the heart, cell-to-cell spread of Ca²⁺ waves has been observed in epithelial organs, including the liver (7-9) and salivary gland (10). Intercellular Ca²⁺ signaling follows a complex pattern in the liver, much as it does in the heart. For example, vasopressin induces Ca²⁺ waves that spread from pericentral to periportal hepatocytes, opposite to the direction of blood flow (7, 9), and this may help direct canalicular motility and bile flow (11). Altered intercellular Ca²⁺ signaling may contribute to the pathophysiology of certain disease states, since agents that block gap junction conductance also alter tissue function (12-14), and because certain cholestatic liver diseases are characterized by decreased expression of gap junctions and impaired transmission of intercellular Ca²⁺ signals (15). Here we examined the conditions necessary to organize intercellular Ca²⁺ signals in a cell system in which Ca²⁺ signaling is mediated entirely via InsP₃ and the InsP₃R.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [Arg⁸]vasopressin, tetracycline, penicillin, and streptomycin were obtained from Sigma; fluo-3/AM, fluo-4/AM, fura-2/AM, rhodamine-conjugated phalloidin, and caged compounds (NPE-caged InsP₃ and DM-nitrophen-EDTA) were obtained from Molecular Probes (Eugene, OR). Dulbecco's modified Eagle's medium, Liebovitz 15 (L-15) medium, and other tissue culture reagents were from Invitrogen (Basel, Switzerland). All other chemicals were of the highest quality commercially available.

Antibodies-- InsP₃ receptors were labeled using an antibody from affinity-purified specific rabbit polyclonal antiserum directed against the 18 COOH-terminal residues of the rat type II InsP₃ receptor (16), which was kindly provided by Richard Wojcikiewicz (SUNY, Syracuse, NY). Vasopressin V_1a receptors were labeled using an affinity-purified polyclonal antibody directed against the rat hepatocyte V_1a receptor (7), which was kindly supplied by Carlos Gonzalez (Universidad Austral de Chile) and Juan Saez (Catholic University, Santiago). Commercially available monoclonal antibodies (Chemicon, Temecula, CA) were used to label connexin32 (cx32) and connexin43 (cx43).

Cell Culture-- Wild type SkHep1 cells, SkHep1 cells stably transfected with cx32 (17, 18) or cx43 (19), and CHO cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin/streptomycin (100 µg/ml). Cells were plated onto glass coverslips, and experiments were performed 2-3 days after plating. Coverslips containing the cells were transferred to L-15 medium supplemented with 10% fetal calf serum 1 h before each experiment, then loaded with a fluorescent Ca²⁺ indicator and studied as described below. Expression of cx32 was under the control of a tetracycline-sensitive promoter (17), so tetracycline (1 µg/ml) was either added to or removed from the medium 24 h before experiments using these cells. Primary cultures of rat hepatocytes were isolated from liver by collagenase perfusion, then plated onto glass coverslips and used within 2-4 h as described previously (20).

Immunoblot Analysis-- Wild type and SkHep1 cells transfected with cx32 or cx43 were harvested, dissolved in SDS, and the protein concentration of cell homogenate was determined as described previously (21). Proteins collected from the cells were separated by SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel and subsequently transferred to protein nitrocellulose membranes. The membranes were blocked at 4 °C overnight and probed for 2 h with the primary antibody. The membranes then were washed, incubated for 1 h with peroxidase-conjugated secondary antibody, and revealed by enhanced chemiluminescence (Amersham Bioscience, Arlington Heights, IL).

Reverse Transcription and PCR Amplification-- Total RNA from SkHep1 cells was isolated using Trizol reagent according to the manufacturer (Invitrogen, Grand Island, NY). First strand cDNA was synthesized using the primer oligo-d(T)₁₆ and Moloney murine leukemia virus-reverse transcriptase. Degenerate primers were used to amplify a 530-bp product from a portion of the 3' region common to the three known RyRs (22-24). PCR amplification was performed in a PTC-100 automated thermocycler (MJ Research, Watertown, MA) using 2 µl of the first-strand cDNA reaction, 200 nM of each degenerate primer, 200 µM dNTPs, 2.5 mM MgCl₂, and 2.5 units of AmpliTaq DNA polymerase for a total volume of 100 µl. The PCR samples were subjected to a hot start (2 min at 94 °C), followed by 30 cycles of 45 s at 94 °C, 1 min at 50 °C, and 1 min at 72 °C. The reaction was followed by a final extension at 72 °C for 10 min. The PCR product was analyzed by agarose gel electrophoresis.

V_1aR-RFP Construction and Transfection-- The gene encoding the V_1a vasopressin receptor (V_1aR) was fused in-frame to the amino terminus of red fluorescence protein (RFP) in a pDsRed1-N1 vector (CLONTECH, Palo Alto, CA). The V_1aR open reading frame initially was amplified by PCR from a plasmid containing the full-length cDNA using oligonucleotide primers to introduce HindIII/Kozak consensus and BamHI sites at the 5' and 3' end of the cDNA, respectively. A typical Kozak consensus sequence (underlined) was introduced into the receptor coding sequence to improve translation. The forward primer was 5'-GCGCAAGCTTGCCGCCACCATGAGTTTCCCGCGAGGCTCC-3' and the reverse primer was 5'-CGGTGGATCCCGGAATAAGAAGTCTGTCTTTCGGCTCATGC-3'. To generate the V_1aR-RFP chimera construct, the resulting PCR product was digested with HindIII and BamHI and ligated into the HindIII and BamHI site of pDsRed-N1 followed by transformation in Escherichia coli DH5. Positive clones were identified by miniplasmid preparation and restriction enzyme analysis. Selected clones were used for maxiplasmid preparation and subsequent transfection. Subconfluent monolayers of SkHep1 cells were transfected with 1 µg of pV_1aR-RFP DNA using effectene (Qiagen, Valencia, CA) according to the manufacturer's instructions. Cells were examined by time lapse confocal microscopy 48 h after transfection. To obtain a pV_1aR construct expressing the V_1a receptor without fusion to RFP, the pV_1aR-RFP plasmid was digested with NotI, followed by gel purification of the linearized band for further digestion with BamHI. The final digestion product was purified and the extremities were turned blunt through Klenow DNA polymerase treatment. After ligation and E. coli DH5 transformation, positive clones were identified by loss of the BamHI restriction site. The selected clone was transfected into 5 × 10⁵ CHO cells using effectene, and suspensions of fura-2-loaded cells were used for cytosolic Ca²⁺ measurements 48 h after transfection.

Confocal Immunofluorescence Microscopy-- Immunofluorescence was performed on SkHep1 cells 2-3 days after plating, and on isolated rat hepatocyte couplets 2-4 h after isolation. SkHep1 cells were fixed with acetone, blocked with phosphate-buffered saline containing 1% bovine serum albumin, then labeled with primary antibody for 1 h. SkHep1 cells were labeled with anti-InsP₃R antibodies to determine the subcellular distribution of InsP₃Rs, or with anti-cx32 or anti-cx43 antibodies to confirm that transfected SkHep1 cells expressed the intended gap junction protein. Primary rat hepatocytes were fixed with Bouin's fixative for labeling with anti-V_1a receptor antibodies (7), but were otherwise processed similarly. After primary antibody incubation the cells were rinsed with phosphate-buffered saline and incubated with Alexa 488-conjugated secondary antibody (Molecular Probes). Specimens were co-labeled with rhodamine-phalloidin to facilitate the identification of the plasma membrane. Negative controls were stained with secondary antibodies alone, along with rhodamine-phalloidin. Specimens were examined using a Zeiss LSM 510 Laser Scanning Confocal Microscope equipped with a krypton/argon laser (Thornwood, NY). To ensure specificity of staining, images were obtained using confocal machine settings at which no Alexa 488 fluorescence was detectable in negative control samples labeled with secondary antibodies alone. Specimens were serially excited at 488 nm and observed at 505-550 nm to detect Alexa 488, then excited at 568 nm and observed at >585 nm to detect rhodamine. This approach eliminated bleed-through of Alexa 488 fluorescence into the longer wavelength (rhodamine) detection channel.

Cytosolic Ca²⁺ Measurements-- Cytosolic Ca²⁺ was measured in wild type SkHep1 cells and in SkHep1 stably transfected with cx32 or cx43 using time lapse confocal microscopy (20, 25). Cells were incubated for 1 h at 37 °C with fluo-3/AM or fluo-4/AM (6 µM). Coverslips containing the cells were transferred to a custom-built perfusion chamber on the stage of a Bio-Rad MRC-1024 confocal microscope (Bio-Rad, Hercules, CA) and observed using a ×63, 1.4 N.A. objective. The 488 nm line of a krypton/argon laser was used to excite the dye, and emission signals between 505 and 550 nm were collected. In experiments in which some cells expressed V_1aR-RFP, the 568-nm line of the laser was used to excite RFP, while emission signals above 585 nm were collected. Using this approach, cells expressing the V_1aR-RFP could be identified while Ca²⁺ signaling in those and other cells could be monitored simultaneously. Neither autofluorescence nor background signals were detectable at the machine settings that were used. Cells were stimulated either by perfusion with vasopressin, or else by flash photolysis (uncaging) of second messengers, and were observed at a rate of 2-10 frames/s. During two-photon flash photolysis, software constraints limited data collection to a rate of 1 frame/s.

Cytosolic Ca²⁺ was measured in wild type CHO cells and in CHO cells transiently transfected with pV_1aR or pV_1aR-RFP using spectrofluorometry (26). For these Ca²⁺ measurements, CHO cells were loaded with fura-2/AM (5 µM) for 1 h at 37 °C. Cell suspensions loaded with fura-2 were then transferred to a cuvette to monitor changes in the fura-2 fluorescence ratio in response to addition of vasopressin (100 nM), using an Hitachi F-2000 spectrofluorometer (Danbury, CT).

Flash Photolysis Studies of Caged InsP₃ and Ca²⁺-- The mechanical stimulation associated with microinjection induces transient Ca²⁺ signals in epithelial cells (20), including in SkHep1 cells.² Therefore, cells here were loaded with a caged rather than the active form of InsP₃ or Ca²⁺. Caged InsP₃ was microinjected into cells, then uncaged by UV flash photolysis, while a cell-permeant form of caged Ca²⁺ was loaded into cells, then uncaged by two-photon flash photolysis. For InsP₃ studies, NPE-InsP₃ (1 mM) was dissolved in an intracellular-like buffer (150 mM KCl plus 1 mM Hepes), along with Texas Red (0.4 mg/ml) as a marker of successful microinjection. A series 5171 Eppendorf micromanipulator (Westbury, NY) was used for positioning, and an Eppendorf series 5242 microinjector was used for pressure microinjections. Injected cells were allowed to recover for at least 5 min before flash photolysis. NPE-InsP₃ was photolyzed using a mercury arc lamp (75 W) coupled to a 1-mm quartz fiberoptic cable through a high-speed shutter and filterwheel (26). The flash duration varied from 100 to 200 ms, to permit maximal uncaging. For caged Ca²⁺ experiments, cells were loaded for 1 h with the Ca²⁺ cage DM-nitrophen (2 µM) plus fluo-4/AM (6 µM). Ca²⁺ was uncaged by two-photon flash photolysis using a Bio-Rad MRC 1024 confocal microscope (Hercules, CA) adapted for two-photon excitation with a Spectra-Physics Tsunami titanium:sapphire laser and a Millenia X pump laser (Mountain View, CA). The titanium:sapphire laser was mode-locked with a pulse width of 90 fs and tuned to a wavelength of 730 nm, since that wavelength is optimal for two-photon release of Ca²⁺ from DM-nitrophen (27). A pump laser power of 7.2 W was used, which resulted in a power level of 600 mW exiting the titanium:sapphire laser. The beam was passed through a 32% neutral density filter, and a power level of 2-4 mW was detected at the focal plane. The calculated volume of uncaging at the focal point was 0.02-0.03 fl, so caged Ca²⁺ was photoreleased at multiple adjacent points to elevate cytosolic Ca²⁺ in predetermined subcellular regions (28). Ca²⁺ signals induced by uncaged InsP₃ or Ca²⁺ were monitored by time lapse confocal microscopy as described previously (25, 26).

Statistics-- Values listed are mean ± S.E., except where otherwise noted. Statistical comparisons were made using Student's t test, or paired t test where appropriate.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of Ca²⁺ Release Channels in SkHep1 Cells-- Expression of the InsP3R was examined in SkHep1 cells by immunoblot (Fig. 1a). SkHep1 cells were found to express the type II InsP3R, which is the most prevalent isoform in liver (16, 29). The subcellular distribution of the InsP₃ receptor was investigated by confocal immunofluorescence. SkHep1 cells were labeled with the InsP₃R antibody used for immunoblots, and co-labeled with rhodamine phalloidin to identify the actin cytoskeleton (Fig. 1b). The InsP₃R was diffusely distributed throughout the cytosol. No labeling was seen in negative control tissues stained with secondary but not primary antibodies. In contrast, reverse transcriptase-PCR failed to detect RyR expression in SkHep1 cells (Fig. 1, c and d). These findings demonstrate that the InsP₃R is the only intracellular Ca²⁺ release channel in this cell type. This in turn suggests that SkHep1 cells are a model system in which intracellular Ca²⁺ release is mediated entirely by InsP₃Rs, and that Ca²⁺ signaling in these cells would not be influenced by subcellular gradients in InsP₃R expression, as occur in some epithelia (30-33).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   SkHep1 cells as a model for InsP3-mediated Ca2+ signaling. a, the InsP3R is expressed in SkHep1 cells. Western analysis using type II-specific InsP3R receptor antibody CT2 identifies a single band of the same size in lysates from SkHep1 cells and the positive control, rat liver (60 µg each). b, confocal immunofluorescence image demonstrates that the InsP3R is distributed in a punctate fashion throughout the cytosol of SkHep1 cells. InsP3R were labeled with antibody CT2 and counterstained with Alexa 488-tagged secondary antibody, shown in green. The specimens were co-labeled with rhodamine phalloidin, shown in red, to identify the actin cytoskeleton and plasma membrane of SkHep1 cells. Nonspecific staining by the secondary antibody was not evident (not shown). c, SkHep1 cells do not express RyR. Reverse transcriptase-PCR was used to amplify RyR from SkHep1 RNA using degenerate primers that recognize all three RyR isoforms. A positive band for actin was observed (lane 1), indicating the integrity of the SkHep1 RNA. No band was seen using RyR primers (lane 2) or in RNA or DNA negative controls (lanes 3 and 4, respectively). d, a PCR product of 530 bp in heart RNA indicates the presence of RyR (positive control). No band was seen in RNA or DNA negative controls (lanes 2 and 3).

Intercellular Communication in SkHep1 Cells-- The SkHep1 hepatoma cell line normally is communication deficient, but was stably transfected with cx32 under the control of a tetracycline-sensitive promoter (17) or with cx43 (19), as described previously. We verified expression levels of cx32 and cx43 in these cell lines by immunoblot (Fig. 2, a and b). Single bands were detected at 32 kDa in cx32-transfected cells and at 43 kDa in cx43-transfected cells, and cx32 expression occurred only in the absence of tetracycline. The subcellular location of cx32 and cx43 in these cells was verified by confocal immunofluorescence (Fig. 2c). Both connexin isoforms were detected in a punctate distribution almost entirely along cell-to-cell borders, which is the appropriate location for gap junctions (15). Cell-to-cell transmission of microinjected Lucifer Yellow was examined as a functional measure of gap junction expression in each transfected cell line (Fig. 2, d and e). This small fluorescent dye is biologically inactive and is transmitted across cx32 and cx43 gap junctions with similar efficacy (34). Lucifer Yellow injected into cx32 cells spread to 3.04 ± 0.37 neighboring cells within 3 min (mean ± S.E.; n = 26), whereas Lucifer Yellow injected into cx43 cells spread to 3.80 ± 0.45 neighboring cells during the same time interval (n = 40; p > 0.2). Lucifer Yellow injected into non-transfected (wild type) SkHep1 cells spread to no (0.0 ± 0.0) neighboring cells (n = 30; p < 10⁸ relative to both cx32 and cx43 cells), while Lucifer Yellow injected into cx32 cells in tetracycline spread to only 0.29 ± 0.07 cells (n = 49; p < 10⁷ relative to cx32 cells without tetracycline). These studies confirm functional expression of cx32 and cx43 in SkHep1 cells, and suggest that intercellular communication is established in nearly all of these cells.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Intercellular communication in wild type and transfected SkHep1 cells. a, inducible expression of cx32 in SkHep1 cells. Immunoblotting using cx32-specific antibodies identified a single band of the appropriate size. Expression occurs only in the absence of tetracycline, as described previously (17). b, immunoblotting confirmed stable expression of cx43 in a separate SkHep1 cell line, as described previously (19). c, confocal immunofluorescence confirmed localization of cx43 (shown in the micrograph) or cx32 (not shown) to cell-cell borders. Cx43 was labeled with a monoclonal antibody and counterstained with Alexa 488-tagged secondary antibody, shown in green. Cells were co-labeled with rhodamine phalloidin, shown in red, to identify cell-to-cell boundaries. d, expression of cx32 confers the ability to transfer Lucifer Yellow from cell to cell. Injection of Lucifer Yellow into a single cell (arrow) results in dye transfer to neighboring cells if cx32 is expressed (top row) but not if cx32 expression is suppressed (bottom row). Lucifer Yellow fluorescence is detected by time lapse confocal microscopy. e, intercellular communication of Lucifer Yellow occurs only in SkHep1 cells expressing cx32 or cx43. Lucifer Yellow (LY) fluorescence is detected only in the Lucifer Yellow-injected cell in both wild type (wt) and cx32 cells incubated with tetracycline (cx32 ± tet). Values are mean ± S.E. (*, p < 107 relative to cx32 + tet or wt).

Intercellular Signaling by InsP₃ and Ca²⁺ in Unstimulated Cells-- NPE-caged InsP₃ was injected into wild type and cx43- and cx32-transfected SkHep1 cells, then the InsP₃ was liberated by UV flash photolysis. The resulting increase in cytosolic Ca²⁺ in the injected and neighboring cells was detected by time lapse confocal microscopy (Fig. 3). The Ca²⁺ increase spread to 3.2 ± 1.6 cells in the cx32 group (n = 17), 2.0 ± 1.3 cells in the cx43 group (n = 9; p > 0.5 relative to the cx32 group), and to 0.8 ± 0.9 cells in the wild type SkHep1 cells (n = 11; p < 0.0001 relative to both transfected cell lines). The Ca²⁺ increase did not spread to any other cells in cx32 cells treated with tetracycline (n = 8; p < 0.0001 relative to cx32 cells without tetracycline), similar to what was observed in the wild type. InsP₃-induced Ca²⁺ signals spread to neighboring cells within 0.60 ± 0.13 s (n = 26). These findings demonstrate that InsP₃ can mediate the spread of Ca²⁺ signals among SkHep1 cells via gap junctions, similar to what has been shown previously in other cell lines (35). Furthermore, InsP₃-mediated Ca²⁺ signals can spread across either cx32 or cx43, similar to what has been observed in other cell types as well (36, 37).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   InsP3 initiates a Ca2+ wave that spreads to neighboring SkHep1 cells via gap junctions. a, intercellular Ca2+ signaling in SkHep1 cells expressing cx32. Caged InsP3 (1 mM) was co-injected with Texas Red into one of the cells (top panel), identified by confocal microscopy. Sequential confocal images of the Ca2+ dye fluo-4 (bottom panels) identify increases in cytosolic Ca2+ in the injected cell and its neighbors following UV flash photolysis of the InsP3. Fluo-4 fluorescence in this and subsequent confocal images is pseudocolored according to the color scale shown below. b, tracing of the increases in fluo-4 fluorescence in the cells indicated in the previous panel. Ca2+ signals spread to a similar number of cells in each experiment (see panel f), despite variability in the percent increase in fluo-4 fluorescence among experiments. c, expanded time scale of the previous tracing, which represents events from t = 34 to t = 38 s. Observe that an increase in Ca2+ begins in the injected cell, then spreads to its neighbors. d, Ca2+ signaling in SkHep1 cells in which cx32 expression is suppressed by tetracycline. Caged InsP3 is injected into the cell indicated by the arrow, then released by flash photolysis. Serial confocal images demonstrate an increase in Ca2+ in the microinjected cell, which did not spread to neighboring cells. e, tracing of fluo-4 fluorescence in the cells shown in the previous panel. An increase in cytosolic Ca2+ was detected in the microinjected cell (filled circle) but not in neighboring cells (open symbols). f, summary of experiments using caged InsP3. InsP3-induced Ca2+ signals spread to neighboring SkHep1 cells expressing cx32 or cx43 but not in cx32 cells with tetracycline or wild type cells (*, p < 0.0001).

The next group of studies investigated whether Ca²⁺ can act directly as an intercellular messenger. DM-nitrophen-EDTA (caged Ca²⁺) was loaded into wild type and cx43- and cx32-transfected SkHep1 cells, then the Ca²⁺ was liberated by two-photon flash photolysis as Ca²⁺ was observed by time lapse confocal microscopy (Fig. 4, a and b). Use of two-photon excitation allowed uncaging of Ca²⁺ to be restricted to individual cells, even though all cells were loaded with caged Ca²⁺ (28). In contrast to what was observed after photolysis of NPE-InsP₃, uncaged Ca²⁺ never spread to adjacent cells in either the cx32 group (n = 13) or the cx43 group (n = 16). This is consistent with the idea that the range of action of InsP₃ is greater than that of Ca²⁺ (38). To examine the effective range of action of Ca²⁺ in this system in more detail, Ca²⁺ was uncaged in discrete 40 (6.3 × 6.3) µm² regions in SkHep1 cells, and the spread of Ca²⁺ to elsewhere within the cell was monitored (Fig. 5, a and b). Increases in cytosolic Ca²⁺ dissipated rapidly, and extended no more than 8.7 ± 1.1 (n = 8) µm away from the site of uncaging. Together, these findings suggest that InsP₃ rather than Ca²⁺ acts as an intercellular messenger in unstimulated epithelial cells.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Ca2+ does not act as an intercellular messenger in unstimulated SkHep1 cells. a, serial confocal images of SkHep1 cx32 cells before, during, and after Ca2+ is uncaged in a single cell. All of the cells are loaded with the cell-permeant form of the Ca2+ cage DM-Nitrophen (1 µM), but two-photon excitation is used to restrict the region in which Ca2+ is photoreleased to the (rectangular) area indicated. b, tracing of fluo-4 fluorescence in the cell subjected to two-photon flash photolysis, plus two neighboring cells. The increase in Ca2+ is restricted to the cell in which Ca2+ is uncaged. Result is representative of that seen in 29 separate experiments using cells expressing either cx32 or cx43.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Ca2+ does not act as a global intracellular messenger in unstimulated SkHep1 cells. a, illustration of the limited range over which free Ca2+ travels in a single SkHep1 cell. Serial confocal images show a SkHep1 cell before, during, and 240 ms after photorelease of Ca2+ in region 1. Each square is 6 × 6 µm. b, tracing of fluo-4 fluorescence in the regions of the cell shown in the previous panel. A small transient increase in Ca2+ is detected in the region subjected to flash photolysis, and the increase becomes progressively attenuated in regions farther from the flash site. Result is representative of that seen in 8 cells.

Local Ca²⁺ Release Can Trigger Intercellular Ca²⁺ Signals-- Although the range of action of Ca²⁺ is limited in unstimulated cells, Ca²⁺ acts as a co-agonist for the InsP₃ receptor (39-41). As a result, the effects of Ca²⁺ are potentiated in the presence of InsP₃. Therefore, we examined whether local release of Ca²⁺ can trigger intracellular and intercellular Ca²⁺ waves during hormonal stimulation. Ca²⁺ was photoreleased in individual cells both before and during stimulation with a "subthreshold" concentration of vasopressin (50 pM to 1 nM) that was too low to increase Ca²⁺ by itself (Fig. 6, a and b). Photorelease of caged Ca²⁺ increased fluo-3 fluorescence by 25 ± 5% in the absence of vasopressin, and by 236 ± 26% during subthreshold stimulation with vasopressin (n = 31; p < 0.0001 by paired t test). Similarly, the increase in fluo-3 fluorescence lasted 9.1 ± 0.9 s in the absence of vasopressin, and over 2 min during vasopressin stimulation (p < 0.0001 by paired t test). Thus, although release of caged Ca²⁺ induced a small, transient increase in Ca²⁺ in unstimulated cells, it instead triggered a much larger and prolonged increase in Ca²⁺ in cells stimulated with subthreshold amounts of vasopressin. This demonstrates that local, subcellular release of Ca²⁺ increases the sensitivity of individual SkHep1 cells to vasopressin. Next we investigated whether these vasopressin-plus-Ca²⁺-induced Ca²⁺ signals can spread to other cells as well. For these studies, Ca²⁺ was photoreleased in individual cells during stimulation with subthreshold vasopressin, then Ca²⁺ signaling was monitored in those cells as well as in neighboring cells (Fig. 7, a-c). The Ca²⁺ increase spread to 1.7 ± 0.3 cells in the cx32 group (n = 10), and to 2.1 ± 0.3 cells in the cx43 group (n = 19; p = 0.4 relative to the cx32 group), but did not spread to any other wild type SkHep1 cells (n = 6; p < 0.0005 relative to both transfected cell lines). Similarly, the Ca²⁺ increase spread to no other cells in cx32 cells treated with tetracycline (n = 4; p < 0.0005 relative to cx32 cells without tetracycline). The time delay between photorelease of Ca²⁺ in an individual cell and Ca²⁺ signaling in neighboring cells was 2.8 ± 0.3 s in cells expressing cx32 and 2.3 ± 0.3 s in cells expressing cx43 (p > 0.3), which is similar to the time interval required for InsP₃-induced Cai²⁺ signals to spread in these cells (Fig. 3, a-c). Thus, unlike what was observed in unstimulated cells, Ca²⁺ triggered global intracellular as well as intercellular Ca²⁺ waves in cells stimulated with low concentrations of vasopressin. This ability of local Ca²⁺ signals to enable individual cells to act as pacemakers for their neighbors depended upon expression of gap junctions as well.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Ca2+ can trigger intracellular Ca2+ waves during low-level stimulation with vasopressin. a, serial confocal images of a SkHep1 cell (indicated by the arrow) in which Ca2+ is uncaged before and during stimulation with 50 pM vasopressin. This concentration of vasopressin by itself elicits no increase in cytosolic Ca2+ in SkHep1 cells. Prior to stimulation with vasopressin, photolysis results in a small increase in Ca2+ (t = 33 s) that dissipates quickly (t = 56 s). During stimulation with vasopressin, uncaging of Ca2+ instead causes a very large increase in Ca2+ throughout the cytosol (t = 162 s) that persists for many seconds. b, tracing of fluo-4 fluorescence over time in the cell indicated in a. This illustrates the magnitude of the Ca2+-induced Ca2+ release that occurs in the presence of small concentrations of vasopressin (50 pM in this example). The tick marks along the abscissa correspond to the time points at which the images in the previous panel were obtained. Results are representative of those seen in 31 separate cells.

View larger version (43K): [in this window] [in a new window]   Fig. 7.   Ca2+ can trigger intercellular Ca2+ waves during low-level stimulation with vasopressin. a, serial confocal images of SkHep1 cells expressing cx43 in which Ca2+ is uncaged in one of the cells (indicated by the arrow) before and during stimulation with vasopressin (1 nM). Prior to stimulation, photolysis results in a small increase in Ca2+ that is detected at the time of the flash, then dissipates. During stimulation with vasopressin, uncaging of Ca2+ in the same cell now causes a very large increase in Ca2+ throughout the cytosol that persists for many seconds. A sustained increase in Ca2+ now spreads to neighboring cells 1 and 2 as well. This concentration of vasopressin by itself also elicits no increase in cytosolic Ca2+ in SkHep1 cells. b, tracing of fluo-4 fluorescence over time in the cells indicated in the previous panel. The tick marks along the abscissa correspond to the time points at which the images in the previous panel were obtained. This illustrates that Ca2+-induced Ca2+ release can spread to neighboring cells when the cells are primed with small, subthreshold concentrations of vasopressin (50 pM-1 nM). Results are representative of those seen in 29 separate cells. c, summary of intercellular Ca2+ signaling in cells subjected to photolysis of caged Ca2+ in the presence of subthreshold vasopressin. A prolonged increase in cytosolic Ca2+ always was observed in the cell in which Ca2+ was uncaged, but the increase spread only among cells expressing cx32 or cx43 (*, p < 0.0005).

Vasopressin Receptor Expression and Establishment of Pacemaker Cells-- Since increased hormone receptor expression can lead to increased InsP₃ production (42, 43), we expressed V_1a vasopressin receptors in SkHep1 cells as an alternative way to increase their sensitivity to vasopressin. The rat liver V_1a receptor (44) was tagged with RFP to identify cells expressing the receptor. The V_1aR-RFP construct first was transiently expressed in CHO cells, which do not normally express vasopressin receptors. Transfected cells were readily identified by RFP fluorescence, which was concentrated around and beneath the plasma membrane (Fig. 8a). This fluorescence distribution pattern was similar to the subcellular distribution of the native V_1a receptor, as determined by confocal immunofluorescence of freshly isolated rat hepatocytes (Fig. 8b). To determine whether function was preserved in the tagged receptor, CHO cells were loaded with fluo-3 to monitor cytosolic Ca²⁺, then observed during stimulation with vasopressin. Stimulation with vasopressin (100 nM) led to a rapid, prolonged increase in cytosolic Ca²⁺ in cells expressing the V_1aR-RFP, but not in other CHO cells (n = 11 experiments; Fig. 8c). Finally, the magnitude of Ca²⁺ signals evoked by either the tagged receptor or the V_1a receptor cloned from rat liver was compared in populations of CHO cells loaded with the ratiometric dye fura-2 (Fig. 8d). Vasopressin (100 nM) increased cytosolic Ca²⁺ by 139 ± 37 nM (n = 14) in the V_1aR-RFP group and by 180 ± 84 nM (n = 12) in the wild type V_1aR group (p = 0.48). In contrast, vasopressin did not increase cytosolic Ca²⁺ in non-transfected CHO cells (n = 13; p < 0.0001 relative to either transfected group). These findings demonstrate that the V_1aR-RFP construct is similar to the native V_1aR, both in terms of its subcellular distribution and its ability to increase cytosolic Ca²⁺ when it is stimulated. The V_1aR-RFP then was transiently expressed in SkHep1 cells with or without cx32 or cx43, to establish and identify a subpopulation of SkHep1 cells with increased sensitivity to vasopressin. Stimulation of SkHep1 cells with concentrations of vasopressin <1 nM routinely increased cytosolic Ca²⁺ in cells expressing the V_1aR-RFP in n = 109 experiments (Fig. 9, a-c), even though this concentration of vasopressin was too low to increase Ca²⁺ if no cells were transfected with the V_1a receptor (Fig. 6). Moreover, Ca²⁺ signals were detected in neighboring cells in 56% of experiments with cells expressing cx32 (n = 34), and in 33% of experiments in the cx43 group (n = 42; p < 0.05 relative to the cx32 group). In contrast, Ca²⁺ signals were detected in neighboring cells in only 15% of experiments using the wild type SkHep1 cells (n = 33; p < 0.0005 relative to cells expressing cx32 and p < 0.05 relative to the cx43 group; Fig. 9d). Ca²⁺ signals spread to 1.6 ± 0.2 cells expressing cx32, but to only 1.1 ± 0.1 cells expressing cx43 (p < 0.05). Thus, the behavior of cells with increased expression of the V_1a receptor is similar to cells in which Ca²⁺ is uncaged during stimulation with subthreshold vasopressin. This in turn provides direct evidence that a relative increase in hormone receptor expression enables cells to act as pacemakers for their neighbors. Finally, we examined Ca²⁺ signaling in cells stimulated with suprathreshold concentrations of vasopressin (100 nM to 1 µM). Cytosolic Ca²⁺ always increased sooner in cells expressing the V_1aR-RFP than in neighboring cells (Fig. 10). The Ca²⁺ increase occurred 4.6 ± 0.8 s sooner in cells expressing cx32 (n = 11 experiments), and 10.8 ± 0.8 s sooner in cells expressing cx43 (n = 5 experiments). However, the Ca²⁺ increase occurred 26.8 ± 4.3 s sooner in wild type cells (n = 20 experiments), a lag time that was longer than that observed in cells expressing either cx32 or cx43 (p < 0.05). This demonstrates that epithelial pacemaker cells not only initiate Ca²⁺ signaling for their neighbors, but serve to synchronize the response among neighboring cells as well. Moreover, pacemaker cells appear to exert their control over a number of nearby cells, including some that are in contact only through intermediary cells.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   The V1aR-RFP is a functional V1a vasopressin receptor. a, the V1aR-RFP is expressed and targets to the appropriate subcellular location. Combined light transmission and confocal image of transfected CHO cells demonstrates that the receptor is expressed and localizes to the region of the plasma membrane. b, subcellular distribution of the native V1a vasopressin receptor. Freshly isolated rat hepatocytes were labeled with an affinity purified polyclonal antibody directed against the V1a receptor (7), then counterstained with an Alexa 488-tagged secondary antibody and examined by confocal microscopy. The receptor is concentrated along the plasma membrane, with lesser amounts of subplasmalemmal and perinuclear staining, similar to the distribution of the V1a-RFP receptor seen in a. c, the V1aR-RFP is functional. Time-lapse confocal microscopy demonstrates that vasopressin (100 nM) selectively increases cytosolic Ca2+ in a CHO cell expressing the V1aR-RFP. Eight non-transfected neighboring cells did not respond to vasopressin since CHO cells do not express V1aR endogenously. The result is representative of that seen in 11 experiments. d, the V1aR-RFP mobilizes cytosolic Ca2+ to the same extent as the untagged V1aR. Ratio measurements of fura-2 in control and transfected CHO cells demonstrate that both the native and tagged V1aR significantly increase Ca2+ during stimulation with 100 nM vasopressin (*, p < 0.0001 relative to non-transfected controls; p > 0.40 relative to each other).

View larger version (34K): [in this window] [in a new window]   Fig. 9.   Expression of V1aR-RFP increases the sensitivity of SkHep1 cells to vasopressin. a, SkHep1 cells expressing V1aR-RFP respond to vasopressin concentrations that are below the threshold for evoking Ca2+ signals in wild type SkHep1 cells. Cells were stimulated with 50 pM vasopressin (VP). Wild type cells (left column) respond to VP only if they express the V1aR-RFP (arrow). In contrast, among cells expressing cx32 (right column), both V1aR-RFP cell (arrow) and some neighbors respond to VP. Numbers correspond to the tracings in b and c. b, tracing of fluo-4 fluorescence over time in wild type cells in the previous panel. An increase in Ca2+ is observed only in the cell transfected with V1aR-RFP. Results are representative of those observed in 28 experiments. c, tracing of fluo-4 fluorescence over time in cx32 cells in the first panel of this figure. An increase in Ca2+ is observed in the cell transfected with V1aR-RFP, followed by an increase in Ca2+ in four of the neighboring cells. Results are representative of those observed in 19 experiments. d, summary of intercellular Ca2+ signaling in the presence of subthreshold vasopressin among cells expressing V1aR-RFP. A prolonged increase in cytosolic Ca2+ always was observed in cells expressing the tagged receptor, but the increase subsequently occurred in neighboring cells more frequently among cells co-expressing cx32 (*, p < 0.0005) or cx43 (**, p < 0.05).

View larger version (43K): [in this window] [in a new window]   Fig. 10.   Establishment of pacemaker cells by overexpression of V1a vasopressin receptors. a, SkHep1 cells expressing V1aR-RFP act as pacemakers for their neighbors. Cells here express cx32 and are stimulated with 100 nM vasopressin (VP). Serial confocal images demonstrate that an increase in Ca2+ occurs first in the cell expressing the V1aR-RFP (arrow), but all other cells respond with a prompt, sustained increase in Ca2+ soon afterward. b, tracing of the increases in fluo-4 fluorescence in the cells is indicated in a. c, expanded time scale of the previous tracing reveals that the increase in Ca2+ in the cell expressing V1aR-RFP precedes the Ca2+ signal in its neighbors by several seconds. The result is representative of that seen in 16 experiments using cx32 or cx43 cells. The lag time between Ca2+ signaling in V1aR-RFP cells and their neighbors is significantly longer in wild type SkHep1 cells (n = 20 experiments). d, summary of intercellular Ca2+ signaling in the presence of suprathreshold vasopressin (100 nM) among cells expressing V1aR-RFP. An increase in cytosolic Ca2+ always occurred first in cells expressing the tagged receptor, but the delay between the Ca2+ signal in the V1aR-RFP cell and its neighbors was significantly shorter if cells co-expressed cx32 or cx43 (*, p < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The presence of pacemaker cells regulating cell functions in excitable tissues is well known, but the possibility that analogous pacemaker cells coordinate cell signaling in non-excitable tissues such as epithelia has only recently been investigated (43, 45). Studies using isolated rat hepatocytes suggest that pacemaker cells synchronize Ca²⁺ oscillations in pairs and triplets of cells, since oscillations are coordinated in such multiplets (46), and since one of the cells in a multiplet generally appears to set the rate of oscillations for its neighbors (45, 47). Indirect evidence in both isolated hepatocytes (43, 45) and the isolated perfused liver (11, 14) suggests that this pacemaker activity may be driven by hormone receptor gradients. Here we examined this question directly in the liver-derived SkHep1 cell line. Studies were performed using a cell line rather than primary hepatocytes, since hepatocytes lose expression of hormone receptors (48) and gap junctions (49) within hours of isolation, yet both are necessary components of coordinated intercellular signaling in epithelia. Using the SkHep1 model system, three components were identified that are needed for pacemaker cells to organize signaling events among epithelia. The first component is intercellular communication of second messengers via gap junctions. It was first established that second messengers such as Ca²⁺ and InsP₃ can cross gap junctions in isolated rat hepatocyte couplets (36). Subsequent work in an airway epithelial cell line demonstrated that InsP₃-mediated Ca²⁺ signaling in one cell can induce Ca²⁺ signaling in neighboring cells as well, and that this form of intercellular communication requires gap junctions (50). Moreover, Ca²⁺ waves and oscillations are synchronized when communicating epithelia are stimulated, and this integrated response to hormonal stimulation is dependent upon gap junctions as well (46). InsP₃ can serve as the second messenger that coordinates cell-cell Ca²⁺ signaling (10, 35), although Ca²⁺ also may be able to serve this role under certain circumstances (10, 51). Regardless of which second messenger is responsible, the presence of gap junctions is required for coordination of cell-to-cell signaling among epithelia, since Ca²⁺ oscillations become asynchronous in the absence of gap junctions (45). The current work, furthermore, demonstrates that initiation of Ca²⁺ signals becomes asynchronous in cells that do not express gap junctions. Previous studies have shown that intercellular exchange of both positively and negatively charged molecules depends upon which connexin isoforms are expressed (34), and this includes exchange of second messengers as well (52). The current work extends these observations by providing evidence that pacemaker activity is more enhanced by expression of cx32 than by cx43 (Figs. 9d and 10d). Thus, the extent to which Ca²⁺ signals become synchronized may depend in part upon which connexin isoform is expressed, although each type examined leads to some degree of coordination.

Expression of gap junctions is not the only requirement for synchronization of Ca²⁺ signals. Current and previous studies have shown that stimulation of an individual cell, via application of hormone or else direct injection of InsP₃, may fail to induce Ca²⁺ signaling in neighboring cells (45). However, low-level stimulation of multiple cells does allow Ca²⁺ signaling in one of these cells to trigger Ca²⁺ signals in the neighbors (10, 45, 51). This property has been related to the observation that InsP₃Rs enable cytosol to act as an excitable medium in the presence of elevated concentrations of InsP₃ (53, 54). This concept originally was established in Xenopus oocytes, where regenerative Ca²⁺ waves and oscillations could be elicited by activation of InsP₃Rs (53, 54). Subsequent detailed analyses of this behavior have identified elementary Ca²⁺ release events that occur within the cytosol, and have found that certain spatially defined release sites have a higher sensitivity to InsP3, which may reflect local clustering of InsP₃Rs (55-57). This finding also has been extended to mammalian cells (58), and theoretical work suggests that localized subcellular regions require clustering of at least 20-30 InsP₃Rs to exhibit higher sensitivity to InsP₃ (59). This type of regenerative Ca²⁺ signaling activity now has been shown to occur in networks of epithelial cells as well, including in liver (8), pancreas (51), and salivary glands (10). Alternatively, work in Xenopus oocytes suggests that localized photorelease of caged Ca²⁺ can trigger regenerative activity in the presence of low concentrations of InsP₃ by converting localized Ca²⁺ puffs into Ca²⁺ waves (55, 56). This likely reflects the fact that Ca²⁺ acts as a co-agonist for the InsP₃R (40, 60). The current work uses two-photon flash photolysis to demonstrate that highly localized increases in Ca²⁺ can trigger InsP₃-induced Ca²⁺ signals in mammalian cells as well. We, furthermore, found that localized, subcellular release of Ca²⁺ is sufficient to trigger not only global Ca²⁺ signaling within an individual cell, but also cell-cell signaling, as long as the cell network communicates via gap junctions and is primed by InsP₃. The current work, furthermore, establishes SkHep1 cells as a model system for investigating cell-cell signaling via InsP₃ by demonstrating that InsP₃Rs are distributed uniformly throughout each cell, and that RyR are not expressed.

Signaling patterns that occur in organs and tissues in vivo are more complex than can be accounted for simply by gap junctional communication among excitable cells. For example, intercellular Ca²⁺ waves in liver are oriented in specific directions (7, 9). In addition, the direction of such Ca²⁺ waves depends upon which hormone is used to elicit the waves (9). Similarly, cell-cell spread of Ca²⁺ waves among isolated clusters of hepatocytes occurs in a reproducible pattern, but the pattern varies depending on the hormonal stimulus (61). Cells within the group that initiates Ca²⁺ signals have increased hormonal sensitivity, and several indirect lines of evidence suggest this is due to increased hormone receptor expression (7, 42, 43). The current findings directly demonstrate that increased expression of hormone receptor enables a cell to behave as a pacemaker for its neighbors. Such oriented intercellular signaling is crucial for proper tissue function, since defects in intercellular signaling can alter critical functions such as glucose production or bile secretion in liver (14, 62, 63), or amylase release in pancreas (64). Thus, intercellular integration of Ca²⁺ signaling allows cells to exhibit organ-level behavior, rather than to behave merely as a collection of single cells. Moreover, the distribution of pacemaker cells within an organ permits hormones to evoke distinct responses from that particular organ, even though each hormone may activate the same second messenger pathways at the single cell level.

	ACKNOWLEDGEMENTS

We thank Warren Zipfel for advice regarding two-photon flash photolysis, Richard Wojcikiewicz for kindly supplying InsP₃R antibody CT2, and Carlos Gonzalez and Juan Saez for kindly supplying an antibody directed against the V_1a vasopressin receptor.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK45710, DK34989, DK57751, DK41918, RR04224, and TW01452 and an Established Investigator Grant from the American Heart Association.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.

^§§ To whom correspondence should be addressed: 333 Cedar St., Rm. 1080 LMP, Yale University School of Medicine, New Haven, CT 06520-8019. Tel.: 203-737-6060; Fax: 203-785-4306; E-mail: michael.nathanson@yale.edu.

Published, JBC Papers in Press, February 15, 2002, DOI 10.1074/jbc.M109207200

² A. D. Burgstahler and Michael H. Nathanson, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: RyR, ryanodine receptor; InsP₃, inositol 1,4,5-trisphosphate; InsP₃R, inositol 1,4,5-trisphosphate receptor; cx32, connexin32; cx43, connexin43; CHO, Chinese hamster ovary; V_1aR, V_1a vasopressin receptor; RFP, red fluorescence protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES