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Cardiovascular Research 2003 60(3):643-653; doi:10.1016/j.cardiores.2003.09.017
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Copyright © 2003, European Society of Cardiology

Expression of homocellular and heterocellular gap junctions in hamster arterioles and feed arteries

Shaun L. Sandow*,b,1, Robin Looft-Wilsona,1, Beth Dorana, T.Hilton Graysonb, Steven S. Segala and Caryl E. Hillb

aThe John B. Pierce Laboratory and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06519, USA
bDivision of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia

*Corresponding author. Tel.: +61-2-6125-2149; fax: +61-2-6125-8077. Email address: shaun.sandow{at}anu.edu.au

Received 3 August 2003; revised 1 September 2003; accepted 11 September 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objectives: Conduction of vasoconstrictor and vasodilator responses in the microcirculation involves electrical coupling through gap junction channels among cells of the vascular wall. The present study determined whether reported differences in the properties of conduction along the arterioles of the epithelial hamster cheek pouch (CPA) and feed arteries of its retractor skeletal muscle (RFA) result from differences in the expression profile of specific connexin (Cx) isoforms and the gap junctions they comprise. Methods: Real-time PCR, immunohistochemistry and serial section electron microscopy were used to compare wall morphology and the distribution of gap junctions between respective vessels. Results: Expression of mRNA for Cx37, 40, 43 and 45 was similar between CPA and RFA. In the endothelium, Cx37, 40 and 43 proteins were expressed abundantly between adjacent cells while Cx37 was present in the smooth muscle. In both vessels, endothelial and smooth muscle cell (SMC) layers were well connected by myoendothelial gap junctions (MEGJs), which were found near endothelial cell (EC) gap junctions. Conclusions: The absence of differential gap junctional expression between CPA and RFA, in spite of documented differences in cellular conduction pathways, supports the hypothesis that conductance of vascular gap junction channels can be differentially modulated in resistance microvessels.

KEYWORDS Cell communication; Electron microscopy; Endothelial function; Smooth muscle; Vasoconstriction/dilation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Peripheral vascular resistance is determined by the caliber and tone of resistance arteries and the arteriolar networks they supply. In these microvessels, vasomotor responses can be conducted for several millimeters, permitting the control of tissue blood flow to be coordinated among distal and proximal vessel branches [1]. Conduction of vasomotor responses in the microcirculation has been shown to be due to electrical coupling through gap junction channels between adjacent cells in the vascular wall [2,3]. Since local vasoconstrictor and vasodilator responses may arise through the impact of physical or chemical factors acting on smooth muscle cells (SMCs) or on the endothelium, conduction may occur in either cell layer, according to the nature of expression and regulation of gap junction channels. Indeed, selective stimulation or disruption of one cell layer or the other indicates that heterogeneity can exist in the cellular pathway used for conduction in different microvessels [1,4–8].

Gap junctions are comprised of connexon hemichannels, which are each composed of six connexin (Cx) proteins. The respective hexamers dock in the extracellular space to form an intercellular trans-membrane channel [9]. Of the more than 20 mammalian Cxs characterized to date, Cxs37, 40, 43 and 45 have been described in vascular tissue [10]. However, significant heterogeneity occurs in the expression of these four Cx subtypes amongst different arteries (see Ref. [10]). In the endothelium, Cxs 37, 40 and 43 have been described in a number of different vessels [11] (see Table 2 in Ref. [10]). Expression in the media, however, is more controversial. Cx43 is clearly expressed in the media of large elastic arteries, while in large and small muscular arteries, this role is not played by Cx43, but variously by Cx37, 40 and 45 [12,13] (see Table 2 in Ref. [10]). While some of this apparent heterogeneity may arise through comparison of vessels from different animal species, the use of non-selective antisera may provide further complications [12,14].

Selective conductance, gating and permeability properties have been attributed to gap junctions formed from particular Cx isoforms [15]. Heterogeneity in cellular pathways for conduction between different microvessels could thereby arise from corresponding differences in the expression of Cx isoforms in and between the SMC and endothelial cell (EC) layers. For example, in hamster cheek pouch arterioles (CPA), vasodilatory responses initiated by acetylcholine are conducted in either cell layer, while vasoconstrictor responses are conducted along the smooth muscle layer [6–8]. Whereas these results suggest some differences in the Cx makeup of gap junctions in the respective layers, Cxs40 and 43 have been reported in both the endothelium and smooth muscle [16]. Alternatively, in feed arteries supplying the retractor muscle (RFA) of the cheek pouch, vasodilator responses are readily conducted along the endothelium while the smooth muscle layer appears relatively ineffective as a conduction pathway [4,17].

Variation also exists between CPA and RFA in the role played by myoendothelial gap junctions (MEGJs). Electrophysiological measurements indicate that MEGJs are present and functional in RFA in vitro [18], while data obtained from CPA studied in vivo indicate that MEGJs are either absent or closed [6,7]. Nevertheless, in vitro studies of CPA indicate transfer of dyes from the endothelium to smooth muscle [19] and of calcium from smooth muscle to endothelium [5,20]. Taken together, these results suggest that MEGJs can be modulated by experimental or environmental conditions. However, there is a paucity of information concerning the prevalence of MEGJs in microvessels in which conducted vasomotor responses have been described [3,6,7,18,21,22].

The present study was undertaken to determine whether differences in the conduction properties of CPA and RFA of the hamster result from differences in the incidence of MEGJs and in the expression profile of specific Cx isoforms within SMC and EC layers. We investigated mRNA expression for Cxs37, 40, 43 and 45 in these microvessels using real-time PCR with isoform-specific probes and protein expression for the same Cxs using immunohistochemistry and isoform-specific antibodies. Further, serial section electron microscopy was used to investigate the incidence of MEGJs between the cell layers of these two resistance microvessels.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. General procedures
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Experiments were approved by the Institutional Animal Care and Use Committee of The John B. Pierce Laboratory. Male Golden Syrian hamsters (80–125 g, Charles River Laboratories, Wilmington, MA) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Euthanasia was by exsanguination or an overdose of pentobarbital (i.p. injection).

2.2. RNA extraction and real-time PCR
RFA were removed with the attached vein, immersed in RNA preservative (RNAlater, Ambion, Austin, TX), dissected away from the vein and placed in a tube filled with RNAlater. Vessels were collected bilaterally and pooled from 1 to 2 hamsters (4–10 RFA) for each sample. For CPA, the pouch was everted, rinsed with saline, cut close to the mouth, and placed in a dissecting dish filled with RNAlater. Four to eight CPA (1A and 2A branches) were removed bilaterally from the epithelial region of cheek pouches of one hamster and placed in RNAlater. At least nine of these pooled samples (n) were analyzed for each vessel type. Total RNA was isolated from each sample using RNeasy Protect Mini kit (Qiagen, Valencia, CA), including 15 min incubation with DNase and reverse transcribed using Superscript II (Invitrogen, Carlsbad, CA).

Specific primers and probes (Taqman fluorescent probe, Applied Biosystems, Foster City, CA) for real-time PCR were designed for each gene based on the sequence determined by PCR cloning of hamster genes [Cxs37, 40, 43 and 45, smooth muscle {alpha}-actin (SMAA) and PECAM-1] [23]. The 20-µl real-time PCR reaction comprised 900 nM primers, 250 nM probe and 1 µl sample in TaqMan Universal PCR Mastermix (Applied Biosystems; ABI Prism model 7900HT). The protocol consisted of: 50°C, 2 min; 95°C, 10 min; then 40 cycles of 95°C, 15 s; 60°C, 1 min. Following amplification, absolute mRNA concentrations for each gene were determined by comparing the fluorescent signal at threshold (threshold value equal to 10 x S.D. of baseline fluorescence) to that generated by a standard curve using serial dilutions (≥16-fold range) of purified plasmid containing each gene [23].

The concentration of each Cx mRNA was normalized to the total cDNA concentration after the reverse-transcription reaction using a fluorescent nucleic acid stain (PicoGreen, Molecular Probes, Eugene, OR) as an indicator of the total available mRNA for PCR. Cx mRNA was also normalized to mRNA for SMAA (a smooth muscle-specific gene) and PECAM-1 (an EC-specific gene). Measurements of mRNA concentration by real-time PCR were performed in duplicate for each gene in each sample and all of the genes were measured in every sample.

2.3. Immunohistochemistry
RFA were relaxed in situ with sodium nitroprusside (SNP, 10 µM) before excision and immersion in 4% paraformaldehyde in PBS (10 min). Some vessels were fixed in situ. The cheek pouch was exteriorized, immersed in SNP, removed, pinned in a dish and fixed. Single arterioles from the epithelial region were excised. Vessels were washed with PBS, preincubated in 2% BSA/0.2% Triton-X100/PBS (2 h), followed by 0.05% Chicago Sky Blue/PBS (5 min). After washing in PBS, whole mounts were incubated in primary antibody (4 h, room temperature and overnight, 4°C), washed with 1% Triton-X100/PBS, incubated in secondary antibody (1 h, room temperature), washed and mounted (Vectashield, Vector Laboratories, Burlingame, CA). Specimens were imaged with a fluorescence microscope (E800, Nikon, Melville, NY).

Morphological characteristics of ECs from both RFA and CPA were determined from these whole mount preparations using the imaging program MCID (Imaging Research, Ontario, Canada). Length and width of ECs represented the longest and widest points, while area was the two-dimensional surface area facing the lumen or the smooth muscle layers. Eighteen to 24 cells were sampled from 4 different animals.

2.4. Antibodies
Antibodies were raised in rabbits against mouse Cx37 (Cx37 ADI; aa 318–333, Alpha Diagnostic International, TX, USA, 1:400), rat Cx37 (Cx37 AS; aa 229–333, kindly provided by Alex Simon, University of Arizona, 1:150 [24]), rat Cx40 (Cx40 AS; aa 231–331, Alex Simon, 1:200 [24]), mouse Cx40 (Cx40 Chemicon; 19aa, Chemicon International, Temecula, CA, 1:400), human/rat Cx43 (Cx43 Sigma; aa 363–382, Sigma, St. Louis, MO, 1:200) and human Cx45 either raised in rabbits (Cx45 Chemicon; aa 354–367, 1:300) or sheep (Cx45 CH, aa 354–367 [12], 1:250). Secondary antibodies were goat anti-rabbit immunoglobulins conjugated with Alexa 488 (Molecular Probes, Eugene, OR, 1:800) or donkey anti-goat immunoglobulins conjugated with Alexa 546 (Molecular Probes, 1:800).

Antibodies against rat Cxs37 and 40 have been previously characterized [24,25]. The remaining antibodies were tested for cross-reactivity with other Cx isoforms using either Rin cells stably transfected with Cx43 or Cx40 (kindly provided by Janis Burt, University of Arizona) or COS-7 cells transiently transfected with DNA encoding mouse Cxs37, 40, 43 and 45 (kind gift of Klaus Willecke, University of Bonn [26]). In the latter case, the cells were electroporated with plasmid DNA (pcDEF3) constructs encoding each individual Cx gene controlled by the human EF-1{alpha} promoter [27]. Cells were fixed in either cold methanol (5 min) or 4% paraformaldehyde/PBS (pH 7.4, 10 min), before permeabilization (0.2% Triton-X100/PBS) and staining with Cx antibodies (3 h, room temperature). Cells were washed and incubated in secondary antibody (2 h, room temperature).

2.5. Electron microscopy
Anesthetized hamsters were perfusion fixed using standard procedures [28,29]. Short segments of RFA (2.5–3 mm) and CPA from three animals were excised, with care being taken to obtain a consistent region of vessel from each animal, and further fixed (2 h, 4°C). Tissues were embedded in Araldite 502 according to standard procedures. Sets of 50 serial transverse sections (~100 nm each) of each vessel were cut and mounted on 0.5% Formvar and carbon (~10 nm) coated 1 x 2 mm slot grids. Photographs were taken plate film with an Hitachi 7100 transmission electron microscope.

Vessel characteristics were determined from montages of a complete section of each vessel (x 1000–2500). In order to obtain precise cross sections, care was taken to cut each vessel at 90° perpendicular to the knife edge. Vessel circumference and the number of SMCs comprising the thickness of the media were determined as the length of the internal elastic lamina (IEL) and from counting the number of SMC profiles >5 µm in length from four transects 90° apart, respectively [28]. Serial section analysis was performed along 5 µm of the vessel as previously described [28]. Briefly, the IEL of each section in each series was examined (x 10,000) and all projections from ECs and SMCs photographed (x 20,000–40,000). Selected gap junctions between adjacent ECs or between adjacent SMCs, and MEGJs between the respective cell layers, were photographed (x 20,000–40,000).

In order to compare the density of MEGJs between vessels, the number of MEGJs per EC was determined using the appropriate area of the IEL examined and the EC areas measured in RFA and CPA.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Real-time PCR
Patterns of Cx mRNA were similar between CPA and RFA, irrespective of normalization (Fig. 1). Thus, the relative expression of Cx mRNA in both CPA and RFA was Cx43{cong}Cx37>Cx45>>Cx40 (Fig. 1).


Figure 1
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  		Fig. 1 Expression of mRNA for Cxs37, 40, 43 and 45 in hamster retractor feed arteries (RFA; n = 9) and cheek pouch arterioles (CPA; n = 10). Cx mRNA was normalized to smooth muscle {alpha}-actin mRNA (SMAA), PECAM-1 mRNA and cDNA. Data are mean±S.E.M. Values for n indicate the number of samples each containing 4–16 microvessels. aP<0.05 versus Cx37 and Cx43 in same tissue. bP<0.05 versus Cx43 in same tissue.

 
3.2. Immunohistochemistry
In both RFA and CPA, ECs stained positively with antibodies against Cxs37, 40 and 43 (Figs. 2 and 3)Go. In each case, the cell borders between adjacent ECs were highlighted by punctate staining (Figs. 2A,B and 3A–D)Go. Under the same conditions as those used for the other Cx antibodies, no staining was seen in ECs using the two antibodies to Cx45.


Figure 2
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  		Fig. 2 Immunohistochemical staining of Cx37 in hamster microvessels. Extensive punctate staining outlines the ECs in both CPA (A) and RFA (B). The dim appearance in these panels is due to the Cx37 endothelial cell staining being photographed through the multiple layers of smooth muscle cells, which also show Cx37 staining in both CPA (C) and RFA (D). Control images of CPA (E) and RFA (F) incubated in the absence of primary antibody show an absence of Cx staining.

 

Figure 3
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  		Fig. 3 Immunohistochemical staining of Cx40 and Cx43 in hamster microvessels. ECs of both the CPA (A, C) and RFA (B, D) stained brightly with antibodies against Cx40 (A, B) and Cx43 (C, D). No staining of SMCs was apparent.

 
ECs were significantly longer and narrower in the CPA than in the RFA (Table 1). There was no difference in the length of ECs of RFA fixed in situ or following isolation (data not shown).


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  		Table 1 Characteristics of hamster CPA and RFA

 
There was no staining observed in the smooth muscle of either vessel when incubated with antibodies against Cx40 (Fig. 3A,B). On the other hand, weak punctate Cx37 staining was seen in the smooth muscle layer of the RFA and more diffusely in the CPA (Fig. 2C,D). Punctate staining was never seen in smooth muscle using antibodies against Cx43, although diffuse cytoplasmic staining was occasionally observed in both vessels. We found no evidence for staining with the two antibodies against Cx45 in the smooth muscle of either artery under the same staining conditions used for the other Cx antibodies. No staining was seen in either the endothelium or the muscle when the primary antibody was omitted from the incubation solution.

Cells stably transfected with Cx40 did not stain with Cx37 ADI or with Cx43 Sigma, but did show positive staining with Cx40 Chemicon and Cx40 AS (Fig. 4A–D). Cells stably transfected with Cx43 stained with Cx43 Sigma, but not with Cx37 ADI or Cx40 Chemicon or Cx40 AS (Fig. 4E–H). Cx37 ADI stained cells transiently transfected with Cx37, but not cells transfected with Cxs40, 43 or 45 (Fig. 5A–D). Both Cx45 Chemicon (Fig. 5E–H) and Cx45 CH (Fig. 5I–L) stained only cells transfected with Cx45. These experiments confirm the specificity of each antibody.


Figure 4
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  		Fig. 4 Specificity of antibodies. Rin cells stably transfected with either Cx40 or Cx43 failed to demonstrate any staining when incubated with anti-Cx37 ADI (A,E). Cx40 transfected cells, but not Cx43 cells, stained with the anti-Cx40 AS (B, F) and anti-Cx40 Chemicon (C, G), while Cx43 transfected cells, but not Cx40 cells, stained with the anti-Cx43 Sigma (D, H).

 

Figure 5
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  		Fig. 5 Specificity of antibodies. When COS-7 cells transiently transfected with either Cxs37,40,43 or 45 were incubated with antibodies against Cx37 and Cx45, only Cx37 cells stained with anti-Cx37 ADI (Panel A) and only Cx45 cells stained with anti-Cx45 Chemicon (Panel H) or anti-Cx45 CH (Panel L). All other combinations were negative (B–G, I–K).

 
3.3. Electron microscopy
The CPA and RFA were not significantly different in diameter or circumference, although the number of SMC layers was significantly greater in the RFA (P<0.05, Table 1; Fig. 6A,I).


Figure 6
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  		Fig. 6 Characteristics of vessel wall and gap junctions in CPA and RFA. Media of CPA (A) contained less SMC layers than RFA (I). MEGJs were present in the CPA (B–D; arrow and inset) and RFA (J; arrow and inset). Numerous projections, which failed to make gap junctional contact with cells in the adjacent layer, were present in the CPA (data not shown) and RFA (K, L, asterisk; M arrow and inset, N). Large gap junctions between ECs (E–G, for CPA; O, for RFA) and small gap junctions between SMCs (H, for CPA; P, for RFA) were present in both vessels. IEL, internal elastic lamina. Bar; 2 µm for A, I; 250 nm for B–D, J; 500 nm for K–M; 100 nm for N; 50 nm for E–H, O, P.

 
MEGJs were found in both the CPA and RFA, although they were three-fold more common per EC in the CPA. This difference resulted in ~two-fold greater area of IEL and of vessel length per MEGJ in RFA compared to CPA (Table 1). In the CPA, 56% of MEGJs were found on narrow extensions of ECs projecting through the IEL, 24% on SMC projections and 20% were located between projections arising from both layers, while in the RFA 97% of MEGJs were found on narrow ECs projections (Fig. 6B–D,J). Where distances from MEGJs to EC gap junctions could be measured, there was no significant difference between the two vessels (1.1±0.2 µm, n = 14, CPA; 1.2±0.2 µm, n = 4, RFA). In contrast to the CPA where MEGJs were found in almost every section, MEGJs were rare in the first two series of RFA examined. Since the electrophysiological data demonstrated extensive electrical coupling in the RFA [30], we investigated whether there might be regional distribution of MEGJs in the RFA, as previously described in mesenteric arteries [31]. Series of sections were therefore cut from the other end of the RFA blocks ~2.5–3 mm away. In each RFA sample, there was a five-fold difference in the incidence of MEGJs from one end to the other.

For the RFA, in addition to the projections supporting MEGJs, numerous other projections penetrated the IEL and came within ~200 nm of the opposite layer, but failed to make gap junctional contacts (Fig. 6K–M). In a subset of sections, there were approximately 30-fold more of these projections than ones supporting MEGJs. Myoendothelial projections were less common in the CPA, with approximately five-fold more myoendothelial projections compared to projections supporting MEGJs. However, 40% of these projections came within 20 nm of the opposing cell membrane. Such close appositions were rare in the RFA. The vast majority of these projections in both arteries were derived from ECs. In both vessels EC projections were occasionally found opposite SMCs with small regions of isolated pentalaminar membrane (Fig. 6M,N). Large gap junctions between ECs were readily found in both vessels (Fig. 6E–G,O). Where observed, gap junctions between SMCs were very small (Fig. 6H,P); however, the distribution of these structures was not examined systematically.

In both vessels small holes (≤~2 µm wide) in the IEL, in addition to those containing cellular projections, were present. Otherwise, the IEL was continuous in RFA, but discontinuous over long regions (≤~20 µm) in CPA.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The pattern of Cx isoform expression has been shown here to be remarkably similar in CPA and RFA. In both vessels, mRNA was expressed for all four vascular Cxs and the relative abundance amongst the Cx subtypes was similar. At the protein level, punctate labeling between adjacent ECs was found using antibodies against Cx37, 40 and 43, while in the media only Cx37 was present. The similarity in the expression pattern of the vascular Cxs in the face of appreciable differences in properties of conduction within and between the endothelium and smooth muscle of the two vessels suggests that the conductance of vascular gap junction channels within these vessels is under physiological modulation.

While immunohistochemical findings indicate that Cx37, 40 and 43 were all highly expressed in the endothelium of the two microvessels, variation in relative expression has been described in other vessels. In the rat mesentery, Cx37 was consistently expressed in the endothelium of resistance arteries, but not in arterioles [22]. In contrast, in larger vessels, such as the caudal artery and aorta, Cx43 exhibited regional variation in expression while Cx37 and 40 were highly expressed [12,32]. In spite of the similarity in Cx expression in CPA and RFA, differences in the sizes of the ECs were found. Arteriolar ECs in the CPA were significantly longer and narrower than in those of the RFA. Since vasodilation is conducted along the endothelium in both vessels [4,7] and expression of all three Cxs was similar between vessels, such differences in the size of ECs may contribute to regional differences in the properties of conduction, for example in the rate at which the response decays with distance or with the branching of resistance networks [33,34].

Information is limited concerning the expression of Cxs in the media of resistance arteries and arterioles as revealed by immunohistochemistry (see Ref. [10]). In the present study, we only found convincing evidence for expression of Cx37 in the smooth muscle of both vessels. In contrast to a previous report [16], we did not find any expression of Cx40 and no punctate staining was found for Cx43 or for Cx45 in the media. Physiological studies using intracellular recording and light-dye treatment to selectively disrupt the continuity of one or the other of the cell layers indicate that vasoconstriction can be conducted readily along the smooth muscle in CPA [6–8] yet relatively ineffectively along RFA [4,17], suggesting that conduction within the media may be differentially regulated within respective vascular beds. The expression of Cx37 in the smooth muscle may contribute to this ability and offers an explanation for the observation that conducted vasoconstriction to phenylephrine was impaired in arterioles of the cremaster muscle of mice deficient for Cx37, while the conduction of vasodilation to acetylcholine was unaffected [35]. In contrast, the conduction of vasodilation was significantly impaired in arterioles of the cremaster muscle of mice deficient in Cx40 while conducted vasoconstriction remained intact [36], suggesting an impairment in coupling along the endothelium with Cx40 deficiency.

When studied as pressurized segments in vitro, electrical coupling between the two cell layers was robust in RFA, such that hyperpolarization or depolarization which was conducted along the endothelium spread radially into the smooth muscle [4,18]. CPA isolated and studied in vitro also displayed myoendothelial coupling [3,5], although this was not evident in the same vessels in vivo [6–8]. Since MEGJs could be demonstrated in both the CPA and RFA using serial section electron microscopy (Fig. 6), we suggest that the conductance of these heterocellular gap junction channels can be modulated by environmental and/or experimental conditions. Further, the prevalence of projections which did not form MEGJs, and of isolated gap junctional membranes, suggests that these structures may be dynamic. While real-time observations are needed to further investigate this hypothesis, such behavior would be consistent with the dynamic nature of gap junctions, which have half-lives on the order of hours [37].

In contrast to the present results, neither MEGJs nor homocellular gap junctions in the media were found in mesenteric arterioles, in spite of physiological evidence supporting their existence [22]. The lack of these structures could be attributed to their small size (often <100 nm) and low probability of detection in random sections [31,38]. Regional variation in the distribution of MEGJs, as found here in the RFA and in mesenteric arteries [31] could further add to the problem of detecting MEGJs using random sections. Indeed, the identification of MEGJs in studies employing random sections is limited to the carotid artery of the rabbit [39] and cerebral arterioles of humans [40]. However, through serial section electron microscopy these small junctions have been identified in numerous arteries of the rat and mouse, correlating well with their involvement in vascular function [28,29,31,41]. In contrast to gap junctions in EC layers, which are large and commonly observed [29,42], gap junctions within the smooth muscle, like those between the two layers, are small and not commonly found [10,29]. The functional significance of the variation in MEGJ distribution and morphology between the two vessels is unclear at present.

While the relative expression of the four vascular Cxs was similar between the two microvessels studied here, there was poor correlation between mRNA and protein expression. This was particularly so for Cx43 and Cx40, which represented the most and least abundant mRNA species, respectively, but whose protein expression appeared similar in the endothelium of both vessels. The lack of correlation between the mRNA and protein was unlikely to arise from non-specificity of the antibodies against the Cx isoforms, since all the antibodies used in the present study were tested against cells transfected with cDNA expressing the four vascular Cxs and found to stain only the appropriate cell type (Figs. 4 and 5)Go. A mismatch between Cx mRNA and protein has been reported in cultured SMCs [43], although in larger arteries of the rat, such as the thoracic aorta and caudal artery, mRNA and protein expression were well correlated [12]. The surprisingly low Cx40 mRNA expression was confirmed using two different real-time PCR primer/probe sets and by performing semi-quantitative PCR with primer sets designed to other regions of the sequence [23]. Although Cx37, Cx43 and Cx45 proteins have similar short half-lives [44–46], the half-life of Cx40 protein has not been reported and may be such that lower levels of mRNA are sufficient to maintain protein expression. The mismatch between Cx mRNA and protein may therefore suggest differential regulation of respective mRNAs or proteins in resistance microvessels.

In summary, the distribution and presence of gap junctions and their component Cxs shows no appreciable difference within and between the SMC and EC layers of two resistance microvessels from which differential functional coupling data have been obtained. These results provide new evidence for the modulation of cellular coupling in blood vessels by local physiological and experimental conditions. The factors responsible for this modulation await further experimental investigation.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the United States Public Health Service, National Institutes of Health grant RO1-HL41026, the National Health and Medical Research Council and the National Heart Foundation of Australia. SLS was supported by a NH and MRC Peter Doherty Fellowship, RL-W by National Research Service Award F32-HL67626 from the NIH and CEH received a Career Development Award from the American Physiological Society.


   Notes
 
1 These authors contributed equally to this study. Back

Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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