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Department of Diagnostic Radiology, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff CF14 4XN, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26, 43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 µM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 µM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor 40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.
gap junctions; connexin; endothelium-derived hyperpolarizing factor
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IN ARTERIES AND
VEINS from the rabbit, guinea pig, pig, and mouse, direct
intercellular communication via gap junction channels may play a
central role in the mediation of endothelium-dependent relaxations that
do not involve nitric oxide (NO) or prostanoids and are widely
attributed to an endothelium-derived hyperpolarizing factor (EDHF)
(6, 9, 11, 14, 15, 19, 23, 31, 34). Gap junction channels
are formed by the docking of two connexon hemichannels, each
constructed from six connexin protein subunits that cross the cell
membrane four times with two loops exposed extracellularly
(28), and cell-cell coupling is thought to occur
predominantly at sites where several hundred such channels cluster in
plaquelike structures in the cell membrane (7, 27). Different connexin subtypes are conventionally classified according to
their molecular weight (in kDa), with endothelial and smooth muscle
cells expressing Cx37, Cx40, and Cx43, according to vessel type and
species (32). In the rabbit mesenteric artery, Cx43 is the
only connexin detectable in smooth muscle cells on Western blot
analysis, and EDHF-type relaxations and hyperpolarizations evoked by
ACh are attenuated by a synthetic undecapeptide homologous to the Gap
27 domain of the second extracellular loop of this connexin
(9). This peptide may be denoted 37,43Gap 27, inasmuch as the sequence is also conserved in Cx37 (Table 1). Substitution of three amino acids in
the 37,43Gap 27 sequence, so as to confer homology with the
corresponding domain of Cx40 (denoted 40Gap 27; Table 1),
abolishes its ability to inhibit EDHF-type relaxations in rabbit
arteries, thereby confirming a high degree of connexin specificity
(10).
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37,43Gap 27 also inhibits EDHF-type hyperpolarizations in guinea pig and porcine arteries but is inactive in the rat hepatic artery, leading to the suggestion that gap junctions play a negligible role in EDHF-type relaxations in this vessel type but, rather, that K+ ions released from the endothelium promote smooth muscle hyperpolarization and relaxation by activating inwardly rectifying K+ channels and an Na+-K+-ATPase (13-15). It is possible, however, that restricted expression of Cx43 in the rat hepatic artery (22) accounts for this apparent insensitivity to 37,43Gap 27. Indeed, expression of Cx43 in rat arteries is vessel specific, being present in the endothelium and media of elastic arteries, but confined to the endothelium or completely absent from the vessel wall in muscular arteries (22, 32, 35). Cx37 and Cx40 similarly exhibit wide regional variations in expression in the rat vasculature (24, 26, 30). To evaluate the possible contributions of these different connexins to EDHF-type responses in the rat hepatic artery, we have therefore exploited similarities and differences in their Gap 26 and Gap 27 domains to generate a series of connexin-specific peptides that were administered alone and in combination in isolated arterial rings. Mechanical studies were conducted in tandem with dye transfer experiments in A7r5 cells, a rat aortic smooth muscle line that expresses Cx40 and Cx43 (21). Specific antibodies were used to define the distribution of different connexin subtypes in the arterial wall and A7r5 monolayers. Collectively, the findings provide evidence that Cx37, Cx40, and Cx43 each contribute to EDHF-type relaxations in the rat hepatic artery.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolated arterial rings. Rings of hepatic artery 1-1.5 mm wide, obtained from male Wistar rats (200-250 g) killed by cervical dislocation, were mounted in a Mulvany myograph (Danish Myo Technology) containing gassed (95% O2-5% CO2, pH 7.4, 37°C) Holman's solution of the following composition (in mM): 120 NaCl, 5 KCl, 2.5 CaCl2, 1.3 NaH2PO4, 25 NaHCO3, 11 glucose, and 10 sucrose. Tension was initially set at 0.25 g and readjusted as necessary during an equilibrium period of 1 h.
The rings were precontracted with 300 nM phenylephrine (PE) in the presence of NG-nitro-L-arginine methyl ester (300 µM) and indomethacin (10 µM), and cumulative concentration-relaxation curves to ACh were constructed. After washout for 1 h, rings were preincubated for 40 min with one of the gap junction peptides, 37,40Gap 26, 43Gap 26, 37,43Gap 27, or 40Gap 27, at a concentration of 600 µM or employed as a time-matched control, and after reconstriction, further concentration-relaxation curves were obtained. A similar protocol was used to investigate the effects of the six possible paired peptide combinations, with each component being administered at 300 µM. In the case of the 40Gap 27 + 43Gap 26 combination, experiments were also conducted with each component at 500 µM. In a further series of experiments, rings were incubated with the triple combination 37,43Gap 27 + 40Gap 27 + 43Gap 26 at 300 µM each. In separate experiments, the effects of apamin + charybdotoxin (500 and 100 nM, respectively), a combination of K+ channel blockers that has previously been shown to inhibit EDHF-type relaxation in the rat hepatic artery (36), were also evaluated.Immunohistochemistry.
Freshly isolated rat hepatic arteries were cryopreserved in OCT
compound (BDH) cooled by liquid nitrogen. Cryosections of transverse
rings (8-10 µm thick) were prepared and mounted onto poly-L-lysine-coated slides (Surgipath), air-dried, and
stored at 
20°C. Immediately before the sections were immunostained, they were fixed in 20°C methanol for 10 min and then rehydrated in
PBS (120 mM NaCl and 2.7 mM
Na2PO4 · 2H2O, pH 7.4) for
10 min. Permeabilization was performed in PBS containing 0.1%
(vol/vol) Triton X-100 for 30 min, and the sections were blocked with
PBS containing 0.5% (wt/vol) BSA for 30 min at room temperature.
Sections were stained with the following primary antibodies: for Cx43, a monoclonal antibody generated against amino acids 252-279 on the
COOH-terminal tail (Chemicon; 4 µg/ml) was used, and for Cx45, Cx40,
and Cx37, rabbit polyclonal antipeptide antibodies, prepared against 14 (Cx45) and 19 (Cx37 and Cx40) amino acid sequences on the COOH-terminal
tail of each connexin, were used (Alpha Diagnostics; 5 µg/ml). The
endothelial cell layer was also stained with factor VIII-conjugated
FITC (Serotec; 1:100 dilution). Primary antibodies were incubated for
2 h at 37°C and then washed for 30 min at room temperature in
PBS. Secondary antibodies of goat anti-mouse conjugated Alexa 488 or
goat anti-rabbit conjugated Alexa 546 (Molecular Probes; 1:500
dilution) were incubated for 45 min at 37°C and then extensively
washed to remove unbound antibody. The sections were then mounted under
Fluorsave (Calbiochem).
Confocal laser scanning microscopy. Rat hepatic artery sections and A7r5 cells were imaged with a Bio-Rad MRC 1024ES laser scanning system. For costained samples, images were taken using simultaneous dual-channel screening with emission filters set at 488 and 546 nm for Alexa 488 and Alexa 546, respectively. For the artery sections, images were collected at 0.5-µm steps and then processed using the Bio-Rad Lasersharp software to obtain a three-dimensional single-maximum optical projection view for each section.
Dye transfer.
A7r5 cells were seeded at a density of 1 × 106 cells
per 60-mm2 tissue culture dish and cultured as described
above. Confluent cell monolayers were washed twice with PBS and then
preincubated in Leibowitz L-15 medium for 90 min at 37°C. In some
experiments, 43Gap 26 or 40Gap 27 peptide (600 µM) or a combination of these peptides at 300 or 500 µM each were
present during this incubation period. At 15 min after microinjection
of Lucifer yellow CH (charge 2, 457 Da), cells were fixed in 4%
paraformaldehyde, and the percentage of injections resulting in dye
transfer to 0, 1-4, 5-10, and >10 neighboring cells was
evaluated as previously described (10, 11).
Drugs. All reagents were obtained from Sigma Chemical unless otherwise stated. The purity of the gap peptides was >95%.
Statistics. Values are means ± SE. Concentration-relaxation curves to ACh were evaluated by one-way ANOVA, with Bonferroni multiple comparisons test as a further method of analysis. Concentrations of ACh causing half-maximal relaxation (EC50) and maximal relaxations (expressed as percent reversal of PE-induced constriction) were compared by a Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Inhibition of EDHF-type relaxations by gap junction peptides and
apamin + charybdotoxin.
EDHF-type relaxations were maximal at 10 µM ACh, with a maximal
response of ~80% of PE-induced tone (Fig.
1A, Table
2). Preincubation with
37,40Gap 26, 43Gap 26, 37,43Gap 27, and 40Gap 27 (all at 600 µM) did not significantly alter
maximal relaxation to ACh or EC50 compared with control
(n = 5 for all; Figs. 1B and
2, Table 2). Preincubation with
37,40Gap 26 + 40Gap 27 or
37,43Gap 27 + 43Gap 26 (300 µM for each
component) was similarly without effect (n = 5 and 4, respectively; Figs. 1C and 3,
A and B, Table 2). However, after preincubation
with 37,40Gap 26 + 43Gap 26 or
37,43Gap 27 + 40Gap 27 (300 µM for each
peptide), maximal EDHF-type relaxations to ACh were significantly
reduced (P < 0.05, n = 5 for both;
Fig. 3, C and D, Table 2). Although
EC50 values were not altered after preincubation with
37,40Gap 26 + 43Gap 26, there was a
significant rightward shift after incubation with 37,43Gap
27 + 40Gap 27 (P < 0.05; Table 2).
Preincubation with 37,40Gap 26 + 37,43Gap
27 (300 µM each) inhibited maximal relaxations (P < 0.01, n = 5; Fig. 3E, Table 2) with a
significant rightward shift in EC50 values
(P < 0.05; Table 2). Washout for 1 h restored
ACh-evoked relaxation (Fig. 3E, Table 2). 40Gap
27 + 43Gap 26 (300 µM each) attenuated the maximal
response to ACh (P < 0.001, n = 5;
Figs. 1D and 3F, Table 2) with a significant shift in EC50 values (Table 2; P < 0.01).
After a 1-h washout, maximal relaxations and EC50 values
were partially restored (n = 4; Fig. 3F,
Table 2). Increasing the concentrations of 40Gap 27 and
43Gap 26 to 500 µM each caused a further shift in the
concentration-relaxation curve to ACh (P < 0.001, n = 5; Fig. 3F, Table 2). Preincubation with
37,43Gap 27 + 40Gap 27 + 43Gap 26 (300 µM each) before or after constriction to PE
(300 nM) effectively abolished EDHF-type relaxations (n = 4; Figs. 1E and 3F, Table 2). Apamin + charybdotoxin attenuated maximal relaxations to ACh from 88.5 ± 6.8 to 12.3 ± 3.6% of PE-induced constriction (P < 0.001, n = 4; Fig. 1F) with a significant
rightward shift in EC50 values from 0.18 ± 0.08 to
35.7 ± 2.1 µM (P < 0.001).
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Effects of gap junction peptides and apamin + charybdotoxin on PE-induced tone. PE (300 nM) evoked constrictions of 1.6 ± 0.1 g (n = 42) that were not significantly affected by preincubation with gap peptides, alone or in paired combinations (Table 2). After constriction by 300 nM PE, 37,43Gap 27 + 40Gap 27 + 43Gap 26 (300 µM each) did not significantly alter tone, whereas apamin + charybdotoxin caused a sustained increase from 1.6 ± 0.2 to 2.1 ± 0.1 g (P < 0.01, n = 4 in both cases; Fig. 1, E and F).
Connexin distribution in rat hepatic artery.
Immunostaining of connexin proteins in the wall of the hepatic artery
revealed the typical punctate distribution of gap junction plaques and
demonstrated considerable heterogeneity in the expression of Cx37,
Cx40, and Cx43 (Fig. 4,
A-F). Cx37 was widely expressed in the endothelium and
media, but whereas Cx43-containing plaques were similarly found in the
endothelium, in the media they were restricted to the adventitial
border. Studies at high magnification showed Cx37-containing gap
junction plaques at the smooth muscle-endothelium interface (Fig.
4G). Expression of Cx40 appeared to be restricted to the
endothelium as confirmed by costaining with factor VIII (Fig. 4,
H and I).
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Characterization of connexin expression in A7r5 cells.
Immunocytochemical analysis demonstrated that A7r5 cells express Cx43
and Cx40 in gap junction plaques at sites of cell-cell contact.
Costaining with Cx43 and Cx40 antibodies showed almost complete
coexpression of the two connexin subtypes (Fig.
6).
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Dye transfer in A7r5 cell monolayers.
Under control conditions, 87 ± 5% of cells transferred dye to
10 neighboring cells and 6 ± 4% of cells transferred dye to 5-10 neighboring cells. After preincubation of the cells with 600 µM 43Gap 26 or 40Gap 27, no significant
difference in the ability of cells to transfer dye to >10 cells was
evident (79 ± 13 and 72 ± 16%, respectively). When cells
were incubated with both peptides at 300 µM each, only 14 ± 5%
of cells transferred dye to >10 cells in association with an increase
in the number of cells transferring dye to <10 cells. At up to 500 µM each, only 8 ± 6% of cells transferred dye to >10 cells
(Figs. 7 and
8).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, short connexin-mimetic peptides homologous to the extracellular loops of Cx37, Cx40, and Cx43 have been employed to investigate the contribution of gap junctional communication to NO- and prostanoid-independent relaxations of the rat hepatic artery. The findings provide evidence that direct cell-cell coupling underpins EDHF-type responses in this vessel type.
In the presence of NG-nitro-L-arginine methyl ester and indomethacin, maximum EDHF-type relaxations evoked by ACh were on the order of 80% of PE-induced tone. At 600 µM, none of the peptides, 37,40Gap 26, 43Gap 26, 37,43Gap 27, or 40Gap 27, significantly affected the initial constrictor response to PE or maximal relaxation and EC50 values for the concentration-relaxation curves constructed for ACh. Furthermore, in experiments with two peptides at individual concentrations of 300 µM each, combinations targeted principally to Cx40 (37,40Gap 26 + 40Gap 27) or to Cx43 (43Gap 26 + 37,43Gap 27) were also inactive. By contrast, paired peptide combinations targeted specifically to either of the Gap 26 and Gap 27 domains of Cx37, Cx40, and Cx43 (37,40Gap 26 + 43Gap 26 and 37,43Gap 27 + 40Gap 27) or a combination targeted to both of these domains (37,40Gap 26 + 37,43Gap 27) attenuated maximum relaxations to ACh by 30-40% and caused two- to fivefold rightward shifts in the EC50 for relaxation. A peptide combination that should in theory exert no effect on gap junctions constructed from Cx37 (43Gap 26 + 40Gap 27) similarly attenuated maximum relaxations to ACh by ~50% when both components were administered at 300 or 500 µM each, although the higher-concentration combination caused larger (~25- vs. ~2-fold) increases in the EC50 for relaxation. EDHF-type responses to ACh were effectively abolished by 37,43Gap 27 + 43Gap 26 + 40Gap 27 when each component was administered at 300 µM. The observation that this triple combination suppressed the response to ACh at a total peptide concentration of 900 µM, whereas 43Gap 26 + 40Gap 27 resulted in only partial inhibition at a total concentration of 1,000 µM, suggests that a spectrum of gap junction channels constructed from Cx37, Cx40, and Cx43 contributes to EDHF-type relaxations in the rat hepatic artery.
To confirm that specific combinations of inhibitory peptide may be
necessary to attenuate intercellular communication between cells
coupled by more than one connexin subtype, dye transfer studies were
performed in an A7r5 cell system that was shown to coexpress Cx40 and
Cx43 in gap junction plaques. At individual concentrations of 600 µM,
neither 43Gap 26 nor 40Gap 27 affected the
spread of Lucifer yellow in confluent A7r5 monolayers, whereas combined
peptide administration at 300 or 500 µM each decreased the number of
cells transferring dye to 10 neighboring cells from ~80% to 10%.
These findings contrast with the effects of connexin-mimetic peptides
in COS-7 cells, a fibroblast line that expresses Cx43 as its only
functional connexin protein. Predictably, in this system
37,43Gap 27 attenuates intercellular diffusion of Lucifer
yellow, whereas 40Gap 27 is without effect, thus
emphasizing the connexin selectivity of these peptides
(10). Although the molecular mechanism through which such
agents interrupt intercellular communication remains to be established
(2), the inability of four different peptides to affect
relaxation in the rat hepatic artery individually and their complete
lack of effect on PE-induced tone in combinations that inhibited
relaxations to ACh exclude nonspecific actions against other mechanisms
involved in the EDHF phenomenon. The activation of
Ca2+-activated K+ channels, for example, is an
essential component of EDHF-type responses that are abolished by the
peptidergic K+ channel blockers charybdotoxin and apamin
when administered in combination (5, 12, 13, 36). Unlike
connexin-mimetic peptides, however, coadministration of these agents
induces pronounced vascular smooth muscle constriction, as confirmed in
the present study in the rat hepatic artery.
Immunohistological staining revealed the typical punctate appearance of
gap junction plaques in the rat hepatic artery and demonstrated that
different connexin subtypes were distributed heterogeneously in the
vascular wall. Cx37, Cx40, and Cx43 were each present in large
interendothelial plaques, whereas in the smooth muscle cells of the
media there was wide expression of Cx37 but limited expression of Cx43
in pockets of gap junction plaques near the adventitia and no
detectable expression of Cx40. Inasmuch as Cx40 and Cx43 were absent
from subintimal smooth muscle but predominant in the endothelium, the
ability of 43Gap 26 and 40Gap 27 to attenuate
EDHF-type relaxations when administered in combination implies an
important role for both connexins in endothelial signaling via gap
junctions in the rat hepatic artery. Indeed, previous studies with
layered "sandwich" preparations of endothelium-intact and
endothelium-denuded arterial strips have provided evidence that direct
endothelium-smooth muscle communication is central to EDHF-type
responses in rabbit arteries (9). Although analogous experiments remain to be performed with rat vessels, it seems unlikely
that direct intercellular communication within the endothelial monolayer plays a major role in the initiation of EDHF-type relaxations via chemical signalling mechanisms, inasmuch as each cell will be
exposed to an identical concentration of ACh in organ chamber experiments. While 37,43Gap 27 inhibits the propagation of
Ca2+ waves in coupled respiratory epithelial cells
(4), we previously showed that this peptide does not
inhibit ACh-evoked NO production by the endothelium, which, like the
synthesis of the putative EDHF, is Ca2+ dependent (9,
18). Furthermore, uncoupling of endothelial cells with
18
-glycyrrhetinic acid, a lipophilic aglycone that disrupts gap
junction plaques (20), does not affect direct endothelial cell hyperpolarization induced by ACh, which is a major stimulus to
Ca2+ influx in the electrically nonexcitable endothelial
cell (33). It is nevertheless conceivable that the large
~150-fold increase in the electrical resistance of the endothelial
monolayer that follows uncoupling of its component cells
(33) impairs its ability to function as a low-resistance
current source able to transmit hyperpolarizing current into the media
via passive electrotonic mechanisms. It remains to be determined
whether inhibition of homocellular smooth muscle communication via gap
junctions, and thus in theory the relay of chemical and/or electrical
signals from the endothelium through successive layers of smooth muscle cells in the media, contributes to the ability of connexin-mimetic peptides to attenuate EDHF-type relaxations, a possibility suggested by
observations that 43Gap 26 and 37,43Gap 27 abolish rhythmic contractile activity in endothelium-denuded rings of
rabbit superior mesenteric artery (8).
Specialized triple immunostaining techniques have previously documented colocalization of Cx37, Cx40, and Cx43 in the endothelium of the rat aorta and coronary artery (35), and in the present study, quantitative analysis with double-antibody staining demonstrated that the majority of Cx43 in the endothelium of the rat hepatic artery was associated with Cx40 or Cx37. Although the antibody combinations available did not permit direct analysis of the overlap between Cx37 and Cx40, cross-correlation of the observed staining patterns indicates that Cx40 and Cx37 may also colocalize in endothelial gap junction plaques in the rat hepatic artery. By contrast, Cx37 and Cx43, the only connexin subtypes present in the media, showed little overlap in smooth muscle gap junction plaques. Recent serial electron-microscopic studies have definitively demonstrated the existence of myoendothelial gap junction plaques in rat mesenteric arteries and have shown that these are more than five times smaller than plaques coupling endothelial cells (29). In the present study, small plaques could be identified at the endothelium-smooth muscle interface in the rat hepatic artery, although the fluorescent antibody technique possesses insufficient spatial resolution to classify these as myoendothelial with certainty. In theory, such plaques could contain homotypic (Cx37/Cx37), heterotypic (Cx40/Cx37, Cx43/Cx37), and heteromeric (each hemichannel containing a mixture of different connexin proteins) gap junctions (3, 16, 21) constructed from Cx37, Cx40, and/or Cx43 in the endothelium and Cx37 in smooth muscle. Further research is necessary to define the precise connexin composition of such channels, inasmuch as differences in their susceptibility to connexin-mimetic peptides could potentially explain why EDHF-type relaxations and hyperpolarizations in the rabbit mesenteric artery are sensitive to inhibition by 37,43Gap 27 alone (9, 11), whereas peptide combinations are necessary in the rat hepatic artery. Further research is also necessary to determine the extent to which NO- and prostanoid-independent responses result from direct intercellular diffusion of chemical signals into the vessel wall and/or the spread of hyperpolarizing current from the endothelium. Although passive conduction is not thought to contribute to EDHF-type relaxations in thick-walled conduit arteries because of electrical mismatching between the large mass of the media and the endothelial monolayer (1), electrotonic mechanisms may nevertheless play a role in the response to ACh in microvessels where the mass of the two cell layers is comparable (17).
In summary, we have provided evidence that intercellular communication via myoendothelial gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type relaxations of the rat hepatic artery. Apparent species and/or vessel heterogeneity in the contribution of gap junctions to agonist-induced responses may reflect variations in connexin expression in the endothelial and medial layers of the vascular wall, rather than fundamental differences in the mechanisms that ultimately mediate vasorelaxation.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Rachel Errington for advice on confocal analysis and Sharon Davies for technical assistance.
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FOOTNOTES |
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This work was supported by the Medical Research Council.
Address for reprint requests and other correspondence: T. M. Griffith, Dept. of Diagnostic Radiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, Wales, UK (E-mail: Griffith{at}Cardiff.ac.uk).
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.
Received 13 December 2000; accepted in final form 9 January 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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