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Am J Physiol Heart Circ Physiol 291: H357-H367, 2006. First published January 20, 2006; doi:10.1152/ajpheart.00681.2005
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Analysis of effects of connexin-mimetic peptides in rat mesenteric small arteries

Vladimir V. Matchkov,1 Awahan Rahman,1 Linda M. Bakker,2 Tudor M. Griffith,2 Holger Nilsson,1 and Christian Aalkjær1

1The Water and Salt Research Center, Institute of Physiology and Biophysics, University of Aarhus, Aarhus, Denmark; 2Department of Diagnostic Radiology, Wales Heart Research Institute, Cardiff University, Cardiff, United Kingdom

Submitted 22 June 2005 ; accepted in final form 13 January 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Synthetic peptides homologous to the extracellular loops of the major vascular connexins represent a novel class of gap junction blockers that have been used to assess the role of direct cellular communication in arteries and veins. However, the specificity of action of such peptides on the coupling between smooth muscle cells (SMCs) has not yet been fully characterized. Isolated third-order rat mesenteric arteries were therefore studied with respect to isometric tension (myography), intracellular Ca2+ concentraton ([Ca2+]i) (Ca2+-sensitive dyes), membrane potential, and input resistance (sharp intracellular glass electrodes). Confocal imaging was used for visualization of [Ca2+]i events in individual SMCs in the arterial wall and membrane currents (patch clamp) measured in individual SMCs isolated from the same arteries. A triple peptide combination (37,43Gap 27 + 40Gap 27 + 43Gap 26) increased intercellular resistance (measured as input resistance) in intact arterial segments without affecting the membrane conductance of individual cells and also interrupted electrical coupling between pairs of rat aortic A7r5 myocytes. In intact arterial segments, the peptides desynchronized [Ca2+]i transients in individual SMCs and abolished vasomotion without suppressing Ca2+ transients in individual cells. They also depolarized SMCs, increased [Ca2+]i, and attenuated acetylcholine-induced, endothelium-dependent smooth muscle hyperpolarization. Experiments with endothelium-denuded arteries suggested that the depolarization produced by the peptides under basal conditions was in part secondary to electrical uncoupling of the endothelium from SMCs with loss of a tonic hyperpolarizing effect of the endothelium. Taken together, the results indicate that connexin-mimetic peptides block electrical signaling in rat mesenteric small arteries without exerting major nonjunctional effects.

gap junctions; acetylcholine; endothelium-derived hyperpolarizing factor

THERE IS GROWING EVIDENCE that direct homocellular and heterocellular signaling between endothelial and smooth muscle cells via gap junctions is essential for normal vascular function (10, 21, 27). These communication pathways are constructed from two connexon hemichannels that dock to form a contiguous channel that links the cytoplasm of coupled cells, thereby conferring electrical continuity and allowing the transfer of small signaling molecules (2). Each connexon is composed of six connexin subunits and may contain single or multiple subtypes. Three principal connexins, Cx37, Cx40, and Cx43, designated according to molecular mass (in kDa), are found in endothelial and vascular smooth muscle cells, and some vessels may also express Cx45, although the expression of these proteins varies widely between species, between individual vascular beds, and even within the same vascular bed (27).

One of the major biological functions of gap junctions is to synchronize cellular activity (2, 27), and in the vascular wall, direct coupling between smooth muscle cells (SMCs) appears essential for the maintenance of oscillations in membrane potential, intracellular calcium concentration ([Ca2+]i), and contractile activity–the phenomenon of vasomotion (5, 24, 40). Coupling between SMCs has been confirmed by dye transfer studies (31) and by the demonstration of electrical signal spread between SMCs in endothelium-denuded arteries (55). In some vessels the presence of an intact endothelium may contribute to the maintenance of rhythmic activity (24, 36, 40), and the existence of myoendothelial gap junction plaques has been confirmed by electron microscopy in rat and rabbit arteries (45, 49). Their presence has also been inferred functionally from attenuation of endothelium-dependent relaxation by gap junction blockers (1, 6–9, 16, 18, 29, 51), signaling from SMCs to the endothelium (3, 14, 15, 47, 56), dye transfer studies (22, 31), and electrical coupling (11, 19, 53, 55). Electrotonic signaling via myoendothelial and homocellular smooth muscle gap junctions, rather than the extracellular transfer of a putative endothelium-derived hyperpolarizing factor, may therefore underpin the phenomenon of nitric oxide-independent, endothelium-dependent smooth muscle relaxation (21, 44). The importance of connexins for vascular development and function has recently been emphasized in connexin-knockout animals (13, 20, 48).

Compounds that have been employed as "established" inhibitors of direct intercellular communication, such as the long-chain alcohol heptanol and 18beta-glycyrrhetinic acid, may nevertheless also exhibit nonjunctional properties (5, 6, 50, 54). Indeed, in a study designed to evaluate the specificity of these agents, we recently demonstrated that they exert major nonjunctional effects at concentrations that result in negligible inhibition of gap junctional communication (34). Both classes of compound altered nonjunctional membrane conductance, hyperpolarized SMCs, inhibited Ca2+ currents, and reduced [Ca2+]i and vascular tone in the rat small mesenteric artery (34). It follows that caution is needed when interpreting the effects of these compounds as a specific response to inhibition of intercellular communication. Because the specificity of gap junction blockers is critical for evaluation of the role of direct intercellular communication in vascular function, in the present study we have evaluated the effects of more recently introduced inhibitory connexin-mimetic peptides on membrane currents, membrane potential, [Ca2+]i, and vascular reactivity at concentrations that inhibit electrical communication. Such peptides are homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of Cxs 37, 40, and 43 and have previously been employed as probes to evaluate the role of gap junctions in vasomotion, endothelium-dependent smooth muscle hyperpolarization, and the myogenic response (5, 8, 21). A triple combination of connexin-mimetic peptides (43Gap 26, 40Gap 27, and 37,43Gap 27) was used to interrupt gap junctional communication, because multiple connexin subtypes can contribute to cell-cell coupling in rat arteries (8). Whereas the molecular mechanism of action of such peptides remains to be clarified, there is now evidence that they fail to prevent the de novo docking of connexons and subsequent formation of gap junction plaques, i.e., aggregates of individual channels at points of cell-cell contact, suggesting that their mode of action could involve effects on channel gating (1, 32). Despite their apparent utility, the hypothesis that connexin-mimetic peptides block electrical coupling in the vascular wall without exerting nonjunctional effects has received limited attention and has therefore been addressed in the present study.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Preparations. All procedures complied with Danish or United Kingdom animal welfare regulations and American Physiological Society principles. Animal facilities were approved by the Danish Inspectorate for Experimental Animals and the Animal Welfare Officer of the Medical Faculty of the University of Aarhus or by the Cardiff University Ethical Review Process. Male Wistar rats, 12–18 wk old, were killed with CO2 for functional studies and stunning followed by cervical dislocation for immunohistochemical studies. The mesentery was removed and placed in ice-cold physiological salt solution, and the superior mesenteric artery and its third-order branches were dissected out as required and cleaned of fat and connective tissue. In some experiments, rat aortic A7r5 cells were used to obtain measurements of membrane capacitance (see Patch clamp to assess membrane capacitance).

Immunohistochemical study. Superior and third-order mesenteric arteries were cryopreserved in OCT compound (Agar Scientific) cooled by liquid N2 and prepared for immunostaining as previously described (51). Sections were labeled with the following primary antibodies: for Cxs 37 and 40 rabbit polyclonal antibodies, prepared against the respective specific sequences of 16 and 19 amino acids (Alpha Diagnostics, 5 µg/ml); for Cx43, a mouse monoclonal antibody generated against amino acids 252–270 (Chemicon, 5 µg/ml). The secondary antibodies employed were goat anti-rabbit conjugated Alexa 546 for Cxs 37 and 40 and goat anti-mouse conjugated Alexa 488 for Cx43 (Molecular Probes, 1:500 dilution). Sections were mounted in Fluorsave (Calbiochem) and imaged with a Leica TCS 4D confocal laser scanning microscope equipped with an argon-krypton laser, with maximum projection images being derived from 8 to 10 optical sections obtained at 0.5-µm intervals. Omission of the primary antibodies for Cxs 37, 40, and 43 did not result in nonspecific staining. The specificity of the antibodies for Cxs 37 and 43 for their target connexin proteins has been previously been confirmed in HeLa cells transfected to express Cx37 (23) or Cx43 (1), and the specificity of the Cx40 antibody was demonstrated in studies with cultured rat aortic endothelial and A7r5 aortic cells (8, 32). Preliminary experiments with a mouse monoclonal antibody generated against amino acid residues 354–367 of Cx45 (Chemicon, 10 µg/ml) and goat anti-mouse conjugated Alexa 488 as the secondary antibody (51) failed to detect this connexin subtype in the endothelium and media of either the third-order arteries or the superior mesenteric artery.

Simultaneous measurements of isometric force and [Ca2+]i. Segments of a mesenteric small artery ~2 mm long were mounted as ring preparations in a standard myograph (Danish Myo Technology, Aarhus, Denmark) and maintained at 37°C for isometric contraction studies. The internal circumference of the mounted artery was normalized on the basis of the passive tension-length curve to a value that gives maximal force development (37).

To visualize [Ca2+]i changes in single SMCs in the arterial wall, laser-scanning confocal microscopy was used as described previously (41). Arteries, mounted in the isometric myograph, were loaded with 3 µM Calcium Green-1 acetoxymethyl ester. Calcium Green-1 AM was dissolved in DMSO with 0.1% (wt/vol) cremophor and 0.02% (wt/vol) pluronic F127, and the arteries were loaded (final DMSO concentration 0.1%) for 1.5 h at 37°C. The myograph was placed on the stage of an inverted confocal laser scanning microscope (ODYSSEY XL, Noran). Confocal optical sections were acquired with a water immersion objective (x60, numerical aperature 1.2, Nikon). A 77 x 58-µm image (640 x 480 8-bit pixels) was obtained every 266 ms by using a 100-ns time scan mode and eight-frame averaging (in some experiments 16-frame averaging with 533-ms time interval was used). The emission signals at 530 nm (after excitation at 488 nm) were stored on a computer together with simultaneous force measurements. For image analysis, the programs Intervision (Noran) and Image Space (Molecular Dynamics) were used. The [Ca2+]i changes within cells were estimated as changes in the mean intensity of Calcium Green-1 fluorescence within regions of interests in which all pixel values were averaged (40).

To obtain ratiometric measurements of wall [Ca2+]i, the artery segments were loaded with 2.5 µM fura 2-acetoxymethyl ester (fura 2-AM) for 1 h (2 x 30 min) at 37°C. Fura 2-AM dissolved in DMSO with 0.1% (wt/vol) cremophor and 0.02% (wt/vol) pluronic F127 was added to the bath solution, maintaining the final DMSO concentration below 0.2%. Previous studies have demonstrated that this procedure only loads SMCs near the adventitia (41). Vessels were excited by a 75-W xenon light source alternately at 340 and 380 nm, and emitted light at 515 nm was measured at 10 Hz, as previously described (35, 42). Fluorescent and simultaneous force signals were collected and stored digitally using Felix software (version 1.11, Photon Technology). [Ca2+]i was expressed as the ratio of fluorescence at 340 and 380 nm (34). To minimize bleaching and washout of dye, the time for experiments with fura 2 was limited to 2 h.

Simultaneous measurements of isometric force and membrane potential. The arterial segments were mounted as described above in a myograph for isometric force measurements and normalized (37). Intracellular recordings of membrane potential were obtained by using glass KCl-filled microelectrodes with resistance in the range 40–100 M{Omega} as previously described (38, 42). An Ag-AgCl electrode in the organ bath was used as a reference electrode. Input resistance was evaluated semiquantitatively by injecting 1-nA current pulses (25 ms) and measuring the subsequent potential change; electrode resistance was routinely compensated by balancing the Wheatstone bridge of the amplifier (Intro-710, WPI) before impalements.

Unless otherwise stated, endothelium-intact arteries were used throughout the study. In experiments with endothelium-denuded arteries, the endothelium was removed with the use of a rat's whisker, as described previously (35). Successful denudation was confirmed by assessing the effect of 10 µM acetylcholine (ACh) on arteries preconstricted with norepinephrine (NE).

Patch-clamp recordings to assess membrane conductance. To assess membrane conductance, SMCs from the mesenteric small arteries were isolated as described previously (33). Briefly, a few branches of the third-order arteries were placed in a microtube containing an enzyme solution and stored overnight at 4°C. The enzyme solution contained (in mM) 110 NaCl, 5 KCl, 2 MgCl2, 0.5 KH2PO4, 0.5 NaH2PO4, 10 NaHCO3, 0.16 CaCl2, 0.49 EDTA, 10 Na-HEPES, 10 glucose, 10 taurine at pH 7.0, as well as 1.5 mg/ml papain, 1.6 mg/ml albumin, and 0.4 mg/ml DL-dithiothreitol. On the following day, the microtube with the vessels was incubated for 5–10 min at 37°C, and single cells were released by trituration with a polyethylene pipette into the bath solution. All patch-clamp recordings were made at room temperature (22–24°C). Whole cell recordings were made with patch pipettes having resistances in the range of 3–7 M{Omega}. Only cells with low access resistance (5–10 M{Omega}) were used. Recordings were made with an Axopatch 200B amplifier (Axon Instruments) in conventional whole cell configuration. Series resistance and capacitative current were routinely compensated. Data acquisition and analysis were performed with the software package Clampex 9 for Windows (Axon Instruments) and Microcal Origin version 5.0 for Windows (Microcal Software). Currents were recorded at holding potential of –60 mV, and current-voltage characteristics were obtained by voltage-step protocols, using steps of 20 mV from –60 to +60 mV with duration of 400 ms. Sustained currents over the last 20 ms of the voltage steps were used for calculations.

Patch clamp to assess membrane capacitance. The uncoupling effect of connexin-mimetic peptides was studied directly on electrically coupled cultured rat aortic SMCs (A7r5), by measurements of membrane capacitance. A7r5 cells, in contrast to freshly isolated smooth muscle cells from rat mesenteric arteries, form electrically coupled cell pairs. The cells were seeded into a 40-ml tissue culture flask and cultured for 5–7 days in DMEM (In Vitro, Denmark) media supplemented with 10% fetal calf serum, 1% L-glutamine, 2 g 0.1% kanamycin, 1 million IU penicillin, and 1 g streptomycin in 20 ml PBS-A (KPS, Dulbecco's solution A, In Vitro). Confluent cells were mechanically scraped from the bottom and pipetted into 35 x 10 mm tissue culture dishes (Falcon, Becton Dickson). After 4–6 h the medium was replaced with bath solution, and paired or single cells were used for patch clamp studies as described above within the next 2–4 h. Membrane capacitance measurements on A7r5 cells were performed by using the Membrane Test tool of Clampex 9.

Solutions. The physiological salt solution (PSS) used in myograph experiments contained (in mM) 119 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4, 25 NaHCO3, 1.6 CaCl2, 0.026 EDTA, and 5.5 glucose, gassed with 5% CO2-95% O2 and pH 7.4.

In patch-clamp experiments the standard bath solution contained (in mM) 135 NaCl, 6 KCl, 10 Na-HEPES, 1 MgCl2, and 0.1 CaCl2 at pH 7.4. The standard pipette solution contained (in mM) 10 NaCl, 122 KCl, 10 K-HEPES, 1 MgCl2, 0.01 CaCl2, 0.1 BAPTA, and 0.1 MgATP at pH 7.4. Free intracellular calcium was estimated below 100 nM by using WEBMAXC v.2.22 (Chris Patton, Stanford University, CA).

Drugs and chemicals. Synthetic connexin-mimetic peptides 37,43Gap 27 (SRPTEKTIFII), 40Gap 27 (SRPTEKNVFIV), and 43Gap 26 (VCYDKSFPISHVR) were obtained from Sigma-Genosys. Fura 2-AM, Calcium Green 1-AM, cremophor, and pluronic F127 were obtained from Molecular Probes (Molecular Probes Europe). All other chemicals were obtained from Sigma (Sigma-Aldrich).

Statistics. All data are given as means ± SE. In the patch-clamp experiments, only one experimental recording was taken from each cell, thus n is the number of cells; in other experiments n denotes the number of arteries studied, each being obtained from a different animal. Differences between means were tested with unpaired or (where indicated) with paired two-tailed Student's t-tests, with P < 0.05 being considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Immunostaining for connexin protein. Superimposed images of autofluorescence and nuclear staining demonstrated that the rat mesenteric small artery possesses three layers of smooth muscle cells and defined the anatomic relationship among its endothelium, media, and internal elastic lamina, which exhibited a folded appearance (Fig. 1). Gap junction plaques containing Cx43 were not detectable by immunostaining in either the endothelium or the media of the third-order vessels, whereas a small number of plaques containing this connexin were evident in both layers of the parent superior mesenteric artery (Fig. 2). Although punctuate staining for Cx37 was evident in the endothelium and the media of both vessels, plaques containing Cx40 were detectable only in their endothelium. It should be noted that myoendothelial plaques cannot be identified by immunostaining because of their small size and anatomic proximity to much larger interendothelial gap junction plaques (23, 25, 45, 46).


Figure 1
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  		Fig. 1. Overlaid images of autofluorescence at 488 nm (green) and propidium iodide labeling of cell nuclei (red) in a rat third-order branch mesenteric artery illustrating relative locations of the endothelium (E), internal elastic lamina (IEL), smooth muscle (SM), and adventitia (A). Bar = 25 µm.

 

Figure 2
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  		Fig. 2. Immunostaining of transverse sections of the rat third-order branch (top) and superior mesenteric artery (bottom) illustrating the expression of connexins (Cxs) 43, 40, and 37 in gap junction plaques in the endothelial and medial layers of these vessels. Autofluorescence of the internal elastic lamina (IEL) was evident at the excitation wavelength of the Alexa 488 secondary antibody used to detect Cx43. L, lumen; SM, smooth muscle. Bars = 25 µm.

 
Effects of connexin-mimetic peptides on oscillations in the mesenteric small artery. Submaximal constrictor concentrations of NE reliably stimulated vasomotion, with synchronized oscillations in [Ca2+]i being evident in individual SMCs in the wall of rhythmically active arteries (n = 7; Fig. 3A). The triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27 inhibited vasomotion and desynchronized but did not abolish the associated [Ca2+]i transients (Fig. 3A). At 100 µM each the peptide combination significantly reduced both the frequency and amplitude of vasomotion (P < 0.001 in each case), whereas concentrations of 300 µM each were required to suppress rhythmic activity completely (Fig. 3, B and C). The finding that nonsynchronized [Ca2+]i transients were still evident in the presence of the peptides suggests that interruption of intercellular communication is directly responsible for the loss of observable rhythmic contractile activity.


Figure 3
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  		Fig. 3. Experiments showing that a triple peptide combination (37,43Gap 27, 40Gap 27, 43Gap 26) inhibited vasomotion and desynchronized intracellular Ca2+ concentration ([Ca2+]i) transients in individual smooth muscle cells (SMCs) in mesenteric small arteries stimulated with a submaximal constrictor concentration of norepinephrine (NE). A: simultaneous recordings of isometric force (top traces) and confocal recordings of [Ca2+]i (bottom traces) in regions of interest corresponding to individual SMCs. Peptide combination was present as indicated at 300 µM each. Traces are typical of 7 such experiments. B and C: concentration-dependent effect of connexin-mimetic peptides on vasomotion frequency and amplitude. Values over each bar denote number of experiments.

 
Connexin-mimetic peptides elevate [Ca2+]i in the vascular wall. To further evaluate the effects of the peptide combination on [Ca2+]i, ratiometric dye measurements were obtained to quantify changes in wall [Ca2+]i. These demonstrated that the 300 µM peptide combination significantly increased wall [Ca2+]i in unstimulated arteries, and functional experiments showed that this elevation in basal [Ca2+]i was accompanied by force development (Fig. 4, A–C). The peptide combination also tended to increase wall [Ca2+]i in the presence of NE, although this trend did not achieve overall statistical significance (Fig. 4, B and C).


Figure 4
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  		Fig. 4. Experiments showing simultaneous recordings of isometric force and ratiometric measurements of [Ca2+]i in wall of mesenteric small arteries. A and B: original recordings of force and wall [Ca2+]i obtained under control conditions and in arteries stimulated with a submaximal constrictor concentration of NE, with measurements then repeated in the presence of a 300 µM triple peptide combination. C: averaged wall [Ca2+]i for a series of 5 such experiments with NE and peptides present as indicated. *Significantly different from peptide-free conditions (P < 0.05).

 
Connexin-mimetic peptides depolarize SMCs and potentiate vasoconstriction. The increase in tone induced by the 300 µM peptide combination in unstimulated arteries developed over 15 to 40 min (32 ± 14 min, n = 7) (Fig. 5A). This was associated with depolarization (Fig. 5B), and the effects of the peptides on force development and depolarization were shown to be concentration dependent (Fig. 5, C and D). The depolarization evoked by NE was also significantly potentiated by the 300 µM peptide combination, such that 10 µM NE depolarized arteries from –57.6 ± 1.3 (resting membrane potential) to –36.6 ± 1.6 mV (n = 22) under control conditions and from –42.6 ± 3.9 to –23.6 ± 1.2 mV (n = 5) in the presence of the peptides (P < 0.05). Correspondingly, the sensitivity of the contractile response to NE increased significantly in the presence of the 300 µM peptide combination (Table 1, experiment a) though maximal force development to NE was not changed significantly (data not shown).


Figure 5
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  		Fig. 5. Effects of a 300 µM peptide combination on force development, membrane potential (Em), and input resistance in unstimulated mesenteric small arteries. A and B: simultaneous recordings of isometric force and Em showing constriction and depolarization in association with an increase in input resistance, assessed continuously as the rapid voltage deflections (in mV) induced by repeated 1-nA current injections into medial SMCs. Note changes in time scale. C: isometric force under control conditions (n = 8), after treatment with 200 µM of each peptide (n = 4), after treatment with 300 µM of each peptide (n = 5), and after 40–60 min washout (n = 5). D: concentration-dependent effects of 300 µM peptide combination on Em (open bars) and input resistance (solid bars) under control conditions in physiological saline solution (n = 8); after treatment with 200 µM of each peptide (n = 4); after treatment with 300 µM of each peptide (n = 5) and after 40–60 min washout (n = 5). *, **, ***Significantly different from control (P < 0.05, P < 0.01, and P < 0.001, respectively); §significantly different from input resistance after treatment with 300 µM of each peptide (P < 0.05).

 

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  		Table 1. Changes of sensitivity (shown as pD2 values) to norepinephrine of mesenteric small arteries after removal of endothelium and/or after treatment with triple combination of connexin-mimetic peptides

 
Connexin-mimetic peptides increase intercellular resistance in the vascular wall and uncouple cultured SMCs. Electrical coupling in the vessel wall was assessed through measurement of input resistance (measured as a voltage deflection to the current injection) (34), which was increased by the peptides in a concentration-dependent manner that correlated with changes in membrane potential and force (Fig. 5D). The effect of the peptides was not readily reversible. Cell input resistance remained significantly elevated after a 1-h washout, although the membrane potential returned toward control levels.

Because a reduction in cell membrane conductance could be responsible for the increase in input resistance, we performed conventional whole cell voltage-clamp experiments on SMCs isolated from the mesenteric arteries to assess possible direct effects of the peptides on membrane currents. However, the current-voltage characteristics of whole cell membrane currents under control conditions and after incubation with the 300 µM peptide combination were identical (Fig. 6), suggesting that the peptides have no significant effects on the membrane conductances of single SMCs.


Figure 6
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  		Fig. 6. Current-voltage relations obtained in isolated smooth muscle cells from rat mesenteric small arteries under control conditions and in presence of 300 µM peptide combination (n = 8). Membrane conductance was unaffected by the peptides.

 
In parallel experiments, measurements of membrane capacitance were obtained as an index of electrical communication between A7r5 cell pairs (12). After 4–6 h following resuspension some cells formed "pairs" (Fig. 7A) while others remained solitary. The membrane capacitance of cell pairs was twice that of the capacitance of solitary cells: 104.1 ± 8.0 pF (n = 4) compared with 53.9 ± 8.9 pF (n = 4), indicating tight electrical coupling between paired cells (Fig. 7C). The 300 µM peptide combination rapidly reduced the capacitance of cell pairs by ~50% within 5 min (Fig. 7B) but did not affect the membrane capacitance of solitary cells, so that the capacitance of single and paired cells became almost identical after peptide treatment (Fig. 7C). Because membrane capacitance is determined by the combined surface area of electrically coupled cells, this observation directly demonstrates electrical uncoupling by connexin-mimetic peptides.


Figure 7
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  		Fig. 7. Effects of triple 300 µM peptide combination on electrical coupling between pairs of cultured A7r5 aortic smooth muscle cells as evaluated by measurements of membrane capacitance. A: image of paired A7r5 cells. B: original recording showing that superfusion of a cell pair with the peptide combination rapidly reduced membrane capacitance. C: capacitance of paired and single cells under control conditions and after 5 min incubation with peptide combination (solid and open bars, respectively; n = 4 in each case). ***Significantly different from control (P < 0.001).

 
Connexin-mimetic peptides uncouple endothelial and smooth muscle cells. The 300 µM peptide combination also attenuated endothelium-dependent ACh-induced hyperpolarization and relaxation in the small mesenteric artery (Fig. 8A). Ten micromolar ACh reduced force development by preconstricted arteries from 6.9 ± 0.7 to 0.4 ± 0.1 mN (n = 3) under control conditions but only from 6.4 ± 0.5 to 4.2 ± 0.4 mN (n = 4) in the presence of the peptides. ACh hyperpolarized SMCs in these arteries by 23.3 ± 3.3 mV (n = 3) under control conditions and by 2.3 ± 3.3 mV (n = 4) in the presence of the peptide combination (Fig. 8B). These inhibitory effects of the peptides on force and membrane potential were both statistically significant (P < 0.01).


Figure 8
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  		Fig. 8. Effects of 300 µM triple peptide combination on endothelium-dependent relaxation and hyperpolarization evoked by ACh in mesenteric small arteries preconstricted with NE. A: Simultaneous measurements of force (top) and Em (bottom). Left and right, control responses and responses in vessels incubated with triple 300 µM peptide combination, respectively. B and C: averaged ACh-induced relaxations and changes in Em under control conditions and in presence of the peptides (n = 3 and 4, respectively). **Significantly different from control (P < 0.01).

 
The ability of the peptide combination to attenuate ACh-induced responses suggests that the depolarization of SMCs evoked by the peptides under unstimulated conditions might reflect uncoupling from the endothelium. To test this hypothesis, we studied the effect of the peptides in endothelium-denuded arteries. Removal of the endothelium significantly depolarized unstimulated SMCs (from –50.8 ± 1.6 to –47.5 ± 1.5 mV; P < 0.05, n = 8). The 300 µM peptide combination produced an additional significant depolarization to –42.5 ± 2.0 mV in endothelium-denuded arteries (P < 0.05, n = 8), and this value was not significantly different from the membrane potential in the presence of the peptides in endothelium-intact arteries (see above). Endothelial denudation also significantly increased sensitivity to NE (Table 1, experiment b). In a separate series of experiments, addition of the triple peptide combination did not significantly change the sensitivity of denuded arteries to NE (Table 1, experiment c). Thus the depolarization induced by the peptides in unstimulated arteries and an associated enhancement of the constrictor response to NE can be partially, but not completely, attributed to loss of a tonic hyperpolarizing influence of the endothelium.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study has highlighted the central contribution of gap junctions to dynamic cell synchronization and force development in rat third-order mesenteric small arteries by using synthetic peptides to interrupt intercellular communication. New physiological findings are 1) that loss of rhythmic vasomotor activity reflects a desynchronizing effect of the peptides on coordinated Ca2+ oscillations in individual smooth muscle cells, rather than suppression of underlying Ca2+ transients, and 2) that the peptides can elevate [Ca2+]i and promote smooth muscle depolarization through a mechanism that is in part attributable to electrical uncoupling of the endothelium from smooth muscle cells.

Immunostaining demonstrated the typical punctuate appearance of gap junction plaques containing Cx37 and Cx40 in the endothelium of the mesenteric small artery, whereas definitive identification of connexin expression in homocellular smooth muscle plaques was limited to Cx37. Indeed, there was an apparent paucity of gap junction plaques containing Cx43 in the endothelium and the media of these third-order vessels, even though plaques containing this connexin subtype were evident in both layers in the parent superior mesenteric artery. This pattern of connexin distribution is broadly consistent with previous reports, including the general observation that the expression of Cx43 is decreased in muscular compared with elastic arteries in the rat (28). Thus Earley and coworkers (17) reported plaques containing Cx37 in the endothelium and media of fifth-order rat mesenteric arteries and an absence of Cx43 from this vessel (Cx40 was not examined), and Kansui and coworkers (30) found abundant expression of Cx37 and Cx40 in the endothelium of second- and third-order order rat mesenteric arteries, whereas endothelial plaques containing Cx43 were very scarce (medial expression of these proteins was not examined). By contrast, Gustafsson and colleagues (23) detected Cxs 37, 40, and 43 in endothelial gap junction plaques of rat mesenteric resistance arteries and arterioles but failed to detect expression of these proteins in smooth muscle gap junction plaques. Because preliminary functional experiments indicated that the combined administration of three peptides 37,43Gap 27, 40Gap 27 and 43Gap 26 was necessary to abolish rhythmic vasomotor activity completely, whereas vasomotion persisted in the presence of 37,43Gap 27 alone or 37,43Gap 27 plus 43Gap 26, the triple combination was employed in all subsequent protocols. The requirement for more than one peptide may in part reflect evidence that the endothelium contributes to the synchronized activity that underpins vasomotion in the rat mesenteric small artery (36, 40), suggesting that full inhibition may require blockade of myoendothelial, intermuscular, and possibly even interendothelial gap junctions. It may also be argued that the apparent absence of detectable punctuate medial staining for Cx40 and Cx43 does not preclude the participation of these connexin subtypes in direct communication between SMCs in the rat small mesenteric artery because there is evidence that electrical coupling can occur between cells that lack morphologically identifiable plaques (52). Further research is therefore necessary to evaluate the correlation between plaque size and direct intercellular communication in the vascular wall. Indeed, in the rabbit iliac artery, which is a thick-walled conduit artery, the inhibitory effects of individual connexin-mimetic peptides on electrotonic signaling via myoendothelial and homocellular smooth muscle gap junctions can be correlated with the anatomic distribution of Cxs 37, 40, and 43 in endothelial and medial gap junction plaques (4).

Previous studies have indicated that the rhythmic contractile activity observed when segments of rat mesenteric small artery are activated by NE at concentrations that induce submaximal force development coincides with the onset of synchronized Ca2+ oscillations in individual SMCs (26, 36, 40). The present results demonstrate that the inhibitory effects of connexin-mimetic peptides on vasomotion is a direct consequence of the desynchronization of such SMC Ca2+ transients, a finding consistent with their ability to suppress synchronized Ca2+ oscillations in confluent rat aortic A7r5 myocytes (32). Importantly, we have now shown that the peptides do not exert nonspecific effects on the mechanisms that ultimately generate [Ca2+]i oscillations in the vessel wall, because desynchronized [Ca2+]i transients persisted in individual SMCs in the presence of the peptide combination. Junctional effects were confirmed by observations that the peptides increased smooth muscle input resistance in intact arterial segments, and measurements of membrane capacitance confirmed the ability of the peptides to abolish electrical continuity between coupled A7r5 cell pairs. Whereas an increase in input resistance could theoretically reflect decreased smooth muscle membrane conductance, this possibility was excluded by the observation that the peptide combination did not affect the membrane conductance of individual myocytes isolated from the third-order vessels. These properties of the peptides contrast markedly with those of less specific gap junction blockers such as 18beta-glycrrhetinic acid and heptanol, which exert substantial nonjunctional effects at concentrations that fail to block gap junctional communication (34). Although the triple peptide combination was shown to uncouple paired A7r5 cells within minutes, in arterial segments its full action was evident only after 15–40 min. This observation could simply reflect the time necessary for diffusion of the peptides into the arterial media. However, because A7r5 cells express Cx40 and Cx43, but not Cx37 (8, 32), an alternative possibility is that blockade of the dominant medial connexin Cx37 by 37,43Gap 27 is relatively slow in onset. Further studies are required to determine whether the desynchronizing effect of connexin-mimetic peptides on [Ca2+]i transients simply reflects reduced electrical uncoupling, or whether intercellular movement of Ca2+ and/or signaling molecules such as inositol 1,4,5 trisphosphate via gap junctions is also necessary to coordinate rhythmic smooth muscle activity.

We also found that the triple peptide combination depolarized SMCs, increased [Ca2+]i, and induced constriction of the rat mesenteric small artery under basal conditions. These effects may in part be explained by uncoupling of the endothelium from the media with the consequent loss of a tonic hyperpolarizing influence on SMCs. This hypothesis is supported by the demonstrated ability of the peptide combination to inhibit endothelium-dependent smooth muscle hyperpolarizations and relaxations induced by ACh. Furthermore, as previously reported (39), we confirmed that endothelial denudation resulted in SMC depolarization, increased basal [Ca2+]i, and enhanced contractile sensitivity to NE similar to the effects of the peptide combination. The peptides nevertheless also evoked a small depolarizing response in endothelium-denuded arteries, and further research is necessary to determine whether this direct smooth muscle effect is nonspecific or junctional in origin, given that the peptides did not affect membrane conductance. One possibility is that it reflects alterations in global ionic homeostasis following administration of the peptides, because gap junctional communication is necessary to equalize the concentration of ions such as Na+ across coupled cell populations, thereby affecting the regulation of Na+-K+-ATPase activity (43). Another possibility would be that even though the connexin-mimetic peptides do not disturb gap junctional plaques (32), they may make individual junctions leaky. Whatever the reason for the depolarization, it did not seem to significantly affect the sensitivity to NE of the endothelium-denuded arteries.

In summary, we have demonstrated that a combination of peptides targeting the three major vascular connexins, Cxs 37, 40 and 43, depresses electrotonic signaling via gap junctions without exerting major nonjunctional effects. Global inhibition of gap junctional communication thus desynchronizes oscillations in [Ca2+]i and leads to an increase in medial input resistance, SMC depolarization, increases in resting [Ca2+]i and sensitivity to NE, and loss of ACh-induced relaxation.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work was supported by the Danish Heart Foundation and the British Medical Research Council. The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond).


   ACKNOWLEDGMENTS
 
Jørgen Andresen and Kirsten Skaarup are thanked for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. V. Matchkov, Institute of Physiology and Biophysics, Universitetsparken, Bldg. 160, Univ. of Aarhus, 8000 Aarhus C, Denmark (e-mail: vvm{at}fi.au.dk)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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