ARTICLE |
Correspondence to: Nicholas J. Severs, Cardiac Medicine, National Heart and Lung Inst., Imperial College School of Medicine, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK.
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Summary |
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Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cellcell communication through gap junctions. Gap junction proteins expressed in the endothelium include connexin37, connexin40, and connexin43. To investigate whether individual endothelial cells in vivo express all three connexin types and, if so, whether multiple connexins are assembled into the same gap junction plaque, we used affinity-purified connexin-specific antibodies raised in three different species to permit multiple-label immunoconfocal and immunoelectron microscopy in the rat main pulmonary artery. Immunoconfocal microscopy showed a high incidence of co-localization between connexin43 and connexin40, but lower incidences of co-localization between connexin37 and connexin40 or connexin43. Immunoelectron microscopy revealed that 83% of gap junction profiles contained all three connexins, with the proportion of connexin40 labeling being significantly higher than that of connexin37 or connexin43. The presence of three different connexin types of distinct properties in vitro provides potential for complex regulation and functional differentiation of endothelial intercellular communication properties in vivo. (J Histochem Cytochem 47:683691, 1999)
Key Words: connexin, gap junction, endothelium, immunogold, confocal microscopy
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Introduction |
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Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cellcell communication through gap junctions (
The gap junction channel is constructed from a pair of hemichannels termed connexons, each connexon being assembled from six connexin molecules. The connexins form a multigene family of conserved proteins, at least 13 members of which are expressed in mammalian cells (
In common with many other tissues, endothelia may express more than one connexin type (
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Materials and Methods |
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Tissue Preparation
All studies were conducted on the main pulmonary artery obtained from adult male SpragueDawley rats (320450 g). Samples for immunoconfocal microscopy were obtained by rapidly dissecting arterial samples from animals sacrificed by dislocation of the neck. After rinsing with PBS containing heparin (10 U/ml), the samples were quickly cut into two segments, frozen immediately in isopentane cooled with liquid nitrogen, and stored in liquid nitrogen. Ten-µm-thick cryosections of the arterial segments were cut transversely, mounted on poly-L-lysine-coated slides, and dried overnight in a -20C freezer before immunolabeling. Samples for postembedding immunogold thin-section electron microscopy were prepared by perfusion-fixation. The rats were anesthetized by IP injection of midalozamhypnorm and perfusion-fixed with 2% paraformaldehyde in PBS retrogradely via the abdominal aorta for 15 min, and the main pulmonary arteries dissected out. Fixation of small tissue samples was continued in the same solution for a further 15 min at room temperature (RT). The fixed samples were dehydrated in 30% ethanol at 4C for 30 min, followed by 50% and 70% ethanol at -20C for 30 min and 1 hr, respectively, and then 90% and absolute ethanol three times at -30C with 1 hr for each step. Infiltration with Lowicryl K4M (Agar Scientific; Stansted, UK) was carried out using 1:1 and then 2:1 Lowicryl:ethanol mixtures, followed by pure Lowicryl K4M overnight. After being placed in a new batch of Lowicryl K4M for 1 hr, the samples were embedded in fresh Lowicryl K4M in gelatin capsules and polymerized with UV light at -30C for 16 hr, and then at RT for 72 hr in a Balzers FSU 010 low temperature-embedding unit. Preparation of rat tissues was conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986.
Anti-connexin Antibodies and Secondary Antibody Detection Systems
Three primary antibodies were used for immunohistocytochemical detection of the gap junction proteins Cx37, Cx40, and Cx43. Those against Cx37 and Cx40 were affinity-purified polyclonal antisera raised in rabbits and guinea pigs, respectively. The Cx37 antiserum Y16Y(R4) was raised against a synthetic peptide corresponding to residues 266281 of the cytoplasmic C-terminal tail of the rat Cx37 sequence. The Cx40 antiserum V15K(GP319) was produced against a synthetic peptide corresponding to residues 256270 of the cytoplasmic C-terminal tail of the rat Cx40 sequence. These antibodies were affinity-purified and fully characterized by Western blotting and immunolabeling of transfected cells (
For immunoconfocal microscopy, the working dilution of the anti-Cx37 antibody was 1:60 (0.5 µg/ml) and that of the anti-Cx40 antibody was 1:100 (0.3 µg/ml). The working solution of the commercial anti-Cx43 antibody was 1:1000. The secondary antibody detection systems used were donkey anti-mouse, anti-guinea pig, and anti-rabbit immunoglobulins conjugated to either Cy3 or Cy5 (Chemicon; dilution 1:500). Both these primary antibodies and secondary antibody detection systems were diluted in 0.5% bovine serum albumin (BSA) in PBS.
For immunoelectron microscopy, the optimal dilution of the anti-Cx37 antibody was 1:30 (1 µg/ml) and that of the anti-Cx40 antibody was 1:100 (0.3 µg/ml), used in 0.5% BSA in PBS. The working solution of the commercial anti-Cx43 antibody was 1:500. The secondary antibody detection systems used were 5-nm goldgoat anti-rabbit complexes, 10-nm goldgoat anti-guinea pig complexes, and 15-nm goldgoat anti-mouse complexes (BioCell; Cardiff, UK). All secondary antibodies were diluted 1:50 in PBS.
Immunoconfocal Microscopy
Cryosections were blocked with 0.5% BSA in PBS for 30 min and incubated in the primary antibody of choice. Double labeling for Cx37 with Cx40, or for Cx37 with Cx43, was done by first incubating sections with the anti-Cx37 primary antibody overnight at RT and then with the anti-Cx40 primary antibody for 1 hr or with the anti-Cx43 primary antibody for 2 hr at 37C. For Cx40/Cx43 double labeling, the sections were incubated in a mixture of anti-Cx40 and anti-Cx43 antibodies overnight at RT. The sections were washed in PBS and incubated in the matching secondary antibody or mixture of secondary antibodies for 1 hr at RT before final washing and mounting. Negative controls included (a) omission of the primary antibody, and (b) for double labeling, using each primary antibody with both matching and nonmatching secondary antibodies. All secondary antibodies were confirmed to be species-specific to their individual primary antibody. Peptide inhibition controls were performed for the polyclonal antibodies (
The immunofluorescence-labeled sections were examined by confocal laser scanning microscopy using a Leica TCS 4D equipped with an argonkrypton laser and fitted with the appropriate filter blocks for detection of Cy3 and Cy5 fluorescence. The images were taken using simultaneous dual-channel scanning and were transformed into projection views using sets of five consecutive single optical sections taken at 1-µm intervals. All specimens were examined within 24 hr of immunolabeling.
Five dual-channel projection images from each of five rats were randomly selected to evaluate the distribution and co-localization of connexins on pulmonary arterial endothelial gap junctions. The dual-channel images were further split into two separate single-channel images corresponding to each of the connexin types. Fifty immunolabeled spots were randomly selected from each dual-channel image and the component connexins of each spot were determined by analyzing the corresponding split images. A spot on the dual-channel image that was visualized as a corresponding spot on each of the two split images (i.e., both connexins were labeled) was classified as showing co-localization. If a spot was present on only one of the pair of split images, the spot was classified as containing only the single connexin type identified. In each set of dual-channel and corresponding split images, the percentage of spots showing co-localization was determined. A further analysis involved taking those spots that were positive for a given connexin type and determining the proportion of these spots that were also positive for the second connexin type. Data are presented as mean percent values ± SEM.
Postembedding Immunogold Thin-section Transmission Electron Microscopy
Ultrathin sections of the Lowicryl K4M-embedded specimens were prepared using glass knives. The sections were picked up on nickel grids and incubated at RT successively in 1% BSA in PBS for 5 min, 1% gelatin in PBS for 10 min, and 0.02 M glycine in PBS for 3 min. Sections were immunolabeled as follows. For single labeling, the sections were incubated in the primary antibody, washed with PBS, and then labeled with the appropriate matching secondary antibodygold complex. The optimal incubation periods determined for the anti-Cx37, anti-Cx40, and anti-Cx43 primary antibody treatments were overnight, 2 hr, and 4 hr, respectively. For double labeling of Cx37 with Cx40 and of Cx37 with Cx43, sections were incubated in anti-Cx37 primary antibody overnight and then in anti-Cx40 primary antibody for 2 hr or in anti-Cx43 primary antibody for 4 hr. For double labeling of Cx40 with Cx43, the two primary antibodies were used as a mixture, with a treatment period of 4 hr. For triple labeling, sections were first incubated in anti-Cx37 primary antibody overnight and then in a mixture of anti-Cx40 and anti-Cx43 primary antibodies for 4 hr. For double and triple labeling, the secondary antibodies were applied as mixtures. An incubation period of 11.5 hr was used for the secondary antibody step for single, double, and triple labeling protocols. All immunolabeling steps were carried out at RT. After immunolabeling, the sections were washed with PBS, incubated in 1.25% glutaraldehyde for 3 min, further washed with distilled water, dried, and then stained with uranyl acetate and lead citrate. All sections were examined in either the Philips EM301 or the Hitachi 900 electron microscope. Negative controls consisted of (a) omission of primary antibodies, (b) application of irrelevant primary antibodies or serum [mouse monoclonal anti-dystrophin MAb (
To evaluate the distribution and co-localization of gold label for the different connexins in individual endothelial gap junction plaques, 35 gold-labeled gap junctions were randomly selected from five triple labeled sections. For each gap junction, gold markers of each size were counted and the proportions of each calculated. Results are presented as mean percent values ± SEM, and data were compared using the Wilcoxon rank-sum test with statistical significance defined as p<0.05.
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Results |
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Immunoconfocal Microscopy
By immunoconfocal microscopy, rat main pulmonary artery endothelial gap junctions were visualized as distinct punctate labeling at the luminal side of the internal elastic laminae (Figure 1). Whereas Cx40 and Cx43 endothelial labeling was abundant and evenly distributed, Cx37 labeling was less marked and was heterogeneously distributed. The spatial distribution of each pair of the three connexin types, as assessed by double labeling and dual-channel recording, showed a high incidence of co-localization between Cx40 and Cx43 spots (78.4 ± 0.64%) but only lower incidences of co-localized Cx37 and Cx40 spots (44.8 ± 1.78%) and of Cx37 and Cx43 (38.7 ± 1.88%) spots. Analysis of the proportions of co-localized and single labeled spots for each connexin type demonstrated that most Cx37-positive spots were also positive for Cx40 (84.4 ± 0.56%) and for Cx43 (75.7 ± 1%). The majority of Cx40-positive spots were also positive for Cx43 (88.5 ± 0.32%), although only about half the Cx40-positive spots were also positive for Cx37 (49.9 ± 2%). For Cx43 spots, a pattern was observed similar to that of Cx40 (87.2 ± 2.7% positive for Cx40 and 44.4 ± 11.4% positive for Cx37).
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Postembedding Immunogold Thin-section Transmission Electron Microscopy
Thin sections of Lowicryl K4M-embedded rat main pulmonary arteries revealed endothelial gap junctions as typical pentalaminar structures formed from the adjacent cell membranes of neighboring cells (Figure 2 and Figure 3). Under the optimized conditions used, the majority of gap junctions in the single labeled preparations were heavily decorated with gold particles specifically along the junctional membranes, with minimal background labeling elsewhere in the section (Figure 2). Negative controls consistently showed no labeling. The few gap junction profiles that remained unlabeled in the experimental samples may have represented junctions within the section, unexposed at the surface and thus inaccessible to antibodies. The single labeling experiments showed that the three different sizes of gold markers used to discriminate each connexin type were readily distinguishable from one another and therefore suitable for multiple labeling.
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Double labeling using the same sets of gold markers for each connexin type as those used in single labeling clearly demonstrated co-localization of Cx40 with Cx37, Cx43 with Cx37, and Cx40 with Cx43 within individual gap junctions (Figure 3A3D). The different-sized gold markers were readily distinguishable and showed no segregation transversely across the junction or major lateral aggregation, suggesting that mixtures of connexins were present more or less evenly throughout each of the junctional membranes.
On triple labeling, 83% of the labeled gap junction membranes viewed demonstrated the presence of all three sizes of gold markers (Figure 3E). Some variation was apparent in the relative amounts of gold markers for the different connexin types (Figure 4). The proportion of Cx4010-nm gold labeling (52.7 ± 4.0%) was significantly higher than that of Cx375-nm gold labeling (20.5 ± 2.6%; p = 0.0001) and Cx4315-nm gold labeling (26.8 ± 3.8%; p = 0.003). Of the few gap junction membranes containing only one or two sizes of gold markers, one gap junction contained markers for only one connexin type (Cx43) and five contained markers for two connexin types (one Cx37 with Cx40; four Cx40 with Cx43; n = 35). For all multiple-labeling experiments, background labeling was negligible, and double labeling for each pair of primary antibodies with all three secondary antibodygold complexes confirmed specificity of the localization.
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Discussion |
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This study demonstrates that Cx40, Cx43, and Cx37 are simultaneously expressed in endothelial cells of the main pulmonary artery of the rat and that, in this tissue, the majority of individual gap junction plaques contain all three connexins. Although a number of tissues are known to express more than one connexin type [e.g., Cx40 and Cx45 in the cardiac conduction system (
To address this issue, the present study has built on our earlier studies (
In contrast to our finding that Cx43 is widely expressed in the endothelium of rat pulmonary artery (this study) and aorta (
In culture systems, expression of Cx37 and Cx43 appears to be differentially regulated according to growth status. Cx43 levels become elevated during growth but decline at confluency, whereas Cx37 shows the reverse pattern (
A question raised by our findings concerns the precise organization of the three connexins at the level of the gap junction channel. Our observation that the three sizes of gold markers were visualized with equal frequency on each side of the junction membrane raises the possibility of molecular arrangements involving heteromeric connexons (containing mixtures of connexins within the connexon) (
In conclusion, the present findings indicate that the connexin make-up of main pulmonary artery endothelial gap junctions, involving three connexin types capable of conferring different properties in vitro, provides inherent potential for complex regulation, functional differentiation, and versatility of endothelial intercellular communication properties in vivo. A clearer idea of the molecular structure of individual channels in terms of connexin composition, and their functional correlates, remains a challenge for future work.
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Acknowledgments |
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Supported in part by project grants from the British Heart Foundation (grant nos. PG 97175 and 93136) and the Wellcome Trust (grant no. 046218/Z/95). Dr Yu-Shien Ko is a cardiologist from Chang Gung Memorial Hospital, Taipei, Taiwan, and gratefully acknowledges personal support from its Overseas Biomedical Scholarship Award.
We wish to thank Dr Robert Gourdie (Medical University of South Carolina, Charleston, SC) for providing the crude Cx40 antiserum and Dr J-P. Briand (IBMC, Strasbourg, France) for the gift of the Cx37 peptide.
Received for publication September 18, 1998; accepted December 22, 1998.
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