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Am J Physiol Heart Circ Physiol 287: H2677-H2686, 2004. First published August 19, 2004; doi:10.1152/ajpheart.00016.2004
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Disruption of smooth muscle gap junctions attenuates myogenic vasoconstriction of mesenteric resistance arteries

Scott Earley, Thomas C. Resta, and Benjimen R. Walker

Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131

Submitted 9 January 2004 ; accepted in final form 18 August 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Communication between vascular smooth muscle (VSM) cells via low-resistance gap junctions may facilitate vascular function by synchronizing the contractile state of individual cells within the vessel wall. We hypothesized that inhibition of gap junctional communication would impair constrictor responses of mesenteric resistance arteries. Immunohistochemical experiments revealed positive staining for connexin 37 (Cx37) in both endothelium and smooth muscle of rat mesenteric arterioles, whereas connexin 43 (Cx43) immunoreactivity was not detected in the mesenteric vasculature. Administration of the gap junction inhibitory peptide Gap27, which targets Cx37 and Cx43, significantly diminished myogenic vasoconstriction (8.6 ± 3.8% of passive diameter at 100 Torr) and changes in vessel wall intracellular [Ca2+] of mesenteric resistance arteries compared with vessels treated with either vehicle (physiological saline solution) (33.5 ± 6.1%) or a control peptide (32.1 ± 6.5%). Administration of 18{alpha}-glycyrrhetinic acid, structurally distinct from Gap27, also significantly attenuated myogenic constriction compared with its vehicle control (DMSO) (9.6 ± 3.2% vs. 23.8 ± 4.6%). In contrast, phenylephrine-induced vasoconstriction was not altered by gap junction blockers. Attenuated myogenic vasoconstriction resulting from inhibition of gap junctions persisted after disruption of the endothelium. In additional experiments, VSM cell membrane potential was recorded in mesenteric resistance arteries pressurized to 20 or 100 Torr. VSM membrane potential was depolarized at 100 Torr compared with 20 Torr. However, VSM cells in arteries treated with Gap27 were significantly hyperpolarized (–48.6 ± 1.4 mV) at the higher pressure compared with vehicle (–41.4 ± 1.5 mV) and Gap20-treated (–38.4 ± 0.7 mV) vessels. Our findings suggest that inhibition of smooth muscle gap junctions attenuates pressure-induced VSM cell depolarization and myogenic vasoconstriction.

connexin; membrane potential; mechanosensitivity

CELLS WITHIN the vascular wall are coupled via low-resistance gap junctions that allow both electrical transmission (46) and intercellular movement of small signaling molecules (35). Gap junction hemichannels are formed by several types of connexins (Cx) that are expressed by vascular smooth muscle (VSM) and endothelial cells (13). Both homocellular (7) and myoendothelial (36) gap junctions exist in many vascular beds. Recent studies strongly suggest that gap junctions play a central role in normal vascular function. For example, inhibition of intercellular communication attenuates conducted vasodilatory and vasoconstrictor responses (15, 17, 18, 39, 45) as well as endothelium-derived hyperpolarizing factor-type vasodilation (4) in some vascular beds. In addition, disruption of gap junctions impairs both pressure (32) and agonist-induced (6, 8) vasoconstrictor responses. These later findings imply that synchronization of the contractile state of VSM cells within the vessel wall contributes to typical constrictor function by allowing coordinated generation of force (7).

Conclusive demonstration of an important contribution of intercellular communication to vascular function has been hindered by a lack of pharmacological agents that selectively block gap junctions. Many studies use glycyrrhetinic acid (GA) derivatives, such as 18{alpha}-GA, to disrupt intercellular communication within the vasculature (11). Although these drugs are effective in blocking transmission of electrical information (46, 47) among vascular cells, they can potentially directly affect proteins, such as myosin light-chain kinase, involved in vasoconstrictor mechanisms (21). Furthermore, a recent study (41) concluded that GA derivatives can influence several processes in vascular cells, including ion transport and ACh-induced hyperpolarization of endothelial cells, thus limiting the utility of these compounds. The long-chain alcohols heptanol and octanol have been successfully used as gap junction uncouplers (25), but these agents may also have nonspecific effects to mediate VSM relaxation, particularly at high concentrations (3). More recently, inhibitory peptides homologous to extracellular loops of Cx proteins have been used to block intercellular communication (2, 19). Although it has been proposed that these peptides act by blocking hemichannel docking between adjacent cells, a recent study (1) reports that administration of inhibitory peptides does not prevent the formation of gap junction plaques. Thus, although the mechanism of action of these agents remains controversial, inhibitory peptides are potentially more selective than pharmacological agents.

To further examine the importance of cell synchronization in the vasculature, we employed a gap junction inhibitory peptide to test the hypothesis that intercellular communication within the vessel wall is required for normal vasoconstrictor responsiveness. The effects of gap junction inhibitors on myogenic and agonist-induced vasoconstriction, changes in vessel wall intracellular [Ca2+] ([Ca2+]i), and stretch-induced VSM cell depolarization of mesenteric resistance arteries were examined. Assessment of the Cx profile showed that Cx37, but not Cx43, appears to be expressed by mesenteric resistance artery VSM cells. Therefore, functional studies employed the inhibitory peptide Gap27, selective for Cx37 and Cx43 (3). In addition, the nonselective gap junction uncoupler 18{alpha}-GA was used in confirmatory experiments. We found that pressure but not agonist-induced, vasoconstriction was attenuated by gap junction inhibitors and that this effect was independent of the endothelium.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

A total of 56 male Sprague-Dawley rats (250–350 g body wt; Harlan Industries) were used for these studies. Animals were provided with fresh bedding, rat chow, and drinking water and maintained under a 12:12-h light-dark cycle. Before experimentation, animals were deeply anesthetized with pentobarbital sodium (50 mg ip) and euthanized by exsanguination after the vessels were harvested according to a protocol approved by the Institutional Animal Care and Use Committee of the University of New Mexico Health Sciences Center.

Immunohistochemistry for Cx Proteins

Tissue preparation. The mesenteric arcade (second- to fifth-order vessels) and segments of the small intestine (~1 cm in length) were isolated from anesthetized rats, rinsed in ice-cold physiological saline solution (PSS; 129.8 mM NaCl, 5.4 mM KCl, 0.83 mM MgSO4, 19 mM NaHCO3, 1.8 mM CaCl2 and 5.5 mM glucose and 10–4 M papaverine), and cryopreserved in OCT compound (Tissue-Tek) cooled by liquid nitrogen. Longitudinal cryosections (10 µm thick, –24°C) were thaw mounted onto Superfrost Plus slides (Fisher Scientific), air dried, and stored at –20°C. Before being immunolabeled, the sections were permeabilized in PBS (0.05 M Na2HPO4, 0.14 M NaCl, pH 7.40) containing 0.1% Triton X-100 (TX). Sections designated for Cx37 and Cx43 immunostaining were then blocked with PBS-TX containing 0.5% bovine serum albumin for 30 min at room temperature. The sections were then incubated for 2 h at 37°C with the following Cx primary antibodies: rabbit anti-mouse Cx 37 polyclonal (Alpha Diagnostic Cx37A11-A; 1:100), mouse monoclonal antibody generated against rat Cx 43 (Chemicon Int. MAB3068; 1:250), and mouse anti-rat Cx 43 monoclonal (BD Transduction Laboratories 610062; 1:100). Some sections were additionally labeled with mouse anti-smooth muscle {alpha}-actin monoclonal (Sigma A2547; 1:200) and mouse anti-human von Willebrand factor (vWF) monoclonal antibodies (Serotec MCA127T; 1:200) to selectively label the vascular smooth muscle and endothelium, respectively. The immunogenic peptide sequence for the Cx37 antibody shows no significant homology with other Cx proteins according to the manufacturer (Alpha Diagnostic). For double labeling of Cx37 with {alpha}-actin or vWF, sections were incubated simultaneously with both primary antibodies. All primary antibodies were prepared in PBS-TX. Immunohistochemical labeling was demonstrated by incubation with Alexa 488-conjugated goat anti-mouse and/or Alexa 546-conjugated goat anti-rabbit secondary antibodies (Molecular Probes; 1:500) in PBS for 45 min at 37°C. Sections were rinsed in PBS, mounted using a Prolong Antifade Kit (Molecular Probes), and stored at –20°C until imaged.

For antibody preadsorption negative control experiments, primary antibodies were incubated with excess immunogen (50–80 fold excess) for 2 h at 37°C, followed by 24 h at 4°C with agitation. Antigen/antibody solutions were then centrifuged at 15,000 g for 30 min at 4°C, and the supernatant applied to sections instead of primary antibody. Additional negative controls were performed using sections treated only with secondary antibodies, or in samples treated with mouse IgG or rabbit serum instead of primary antibodies. All chemicals were obtained from Sigma unless otherwise indicated.

Laser-scanning confocal microscopy. Mesenteric arteries were imaged with a Zeiss LSM 510 confocal imaging system equipped with argon and helium-neon lasers, appropriate filter blocks for detection of Alexa 488 and 546, and a Zeiss Axioplan 2 microscope at the University of New Mexico Cancer Center Fluorescence Microscopy Facility. Images were collected at 0.5–1 µm steps and processed using system software.

Characterization of Cx37 Antibody by Western Blot Analysis

Because preliminary experiments revealed immunostaining for Cx37 but not Cx43 within the mesenteric arterial wall, Western blot analyses were performed using a Cx37-transfected rat insulinoma (Rin-mCx37) cell line in which Cx37 expression is inducible by doxycycline (gift from Dr. Janis M. Burt, University of Arizona) to confirm the specificity of the Cx37 antibody used for immunohistochemistry protocols. Rin-mCx37 cells were generated from a Rin-104638 cell line that was stably transfected with a Tet-on vector (Clontech) and with pTre2 containing the coding sequence for mouse Cx37. Cells were grown to ~70% confluency in RPMI-1640 medium supplemented with 10% fetal bovine serum (Hyclone), geneticin (300 µg/ml), and hygromycin (100 µg/ml) in a humidified incubator (6% CO2, balance air) at 37°C. Cx37 expression was induced by treatment with doxycycline (4 µg/ml; 48 h), whereas vehicle-treated cells served as a negative control. Cell lysates were prepared in Laemmli buffer (Bio-Rad) and sample protein concentrations determined by the Bradford method (Bio-Rad Protein Assay). A molecular weight standard (Bio-Rad Precision Plus) was added to each gel, and sample proteins (30 µg/lane) were separated by SDS-PAGE (12% Tris·HCl gels, Bio-Rad) and transferred to polyvinylidene difluoride membranes (Bio-Rad). Blots were blocked overnight at 4°C with 5% nonfat milk, 3% bovine serum albumin, and 1% Tween 20 (Bio-Rad) in TBS containing 10 mM Tris·HCl and 50 mM NaCl (pH 7.5). Blots were then incubated for 2 h at room temperature with a rabbit anti-mouse Cx 37 affinity-purified polyclonal antibody (Cx37A11-A; 1:100, Alpha Diagnostic). This antibody does not exhibit cross-reactivity with endothelial Cx40 or Cx43 (38, 40). For immunochemical labeling, blots were incubated for 2 h at room temperature with goat anti-rabbit IgG-horseradish peroxidase (1:7,500) (Stressgen). After chemiluminescence labeling (ECL, Amersham), Cx37 bands were detected by exposing the blots to chemiluminescence-sensitive film (Kodak). All reagents were purchased from Sigma unless otherwise noted.

Gap Junction Inhibitors

Two structurally distinct agents were used to disrupt gap junctions. Our initial experiments employed a peptide, Gap27 (amino acid sequence SRPTEKTIFII) (3) that is homologous to an extracellular loop of Cx37 and Cx43. A BLAST search of the GenBank protein sequence database did not detect homology between Gap27 and Cx40 or Cx45. This peptide was chosen as a putative inhibitor of VSM gap junctions based on previous reports (26, 48) showing expression of Cx37 and/or 43 within these cells in mesenteric resistance arteries. Controls for these experiments consisted of the biologically inactive peptide Gap20 (amino acid sequence EIKKFKYGC), homologous to an intercellular loop of Cx43 (4), and the vehicle for both peptides, PSS. Gap peptides were synthesized by the Tufts Medical School Protein Chemistry Facility (Boston, MA) and were dissolved in PSS at a final concentration of 300 µM. 18{alpha}-GA was used to block gap junctions in separate experiments. 18{alpha}-GA was prepared daily at a concentration of 10 mM in anhydrous DMSO and was diluted with PSS to 20 µM before administration. DMSO (diluted 1/500 in PSS) was used as the solvent control for 18{alpha}-GA experiments.

Isolated Vessel Preparation

Pressurized mesenteric resistance arteries were studied in isolation. The mesenteric arcade was excised from anesthetized animals and transferred to ice-cold dissecting solution [3 mM MOPS (pH 7.4), 145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2.5 mM CaCl2, 1 mM KH2PO4, 0.02 mM EDTA, 2 mM pyruvate, 5 mM glucose, and 1% bovine serum albumin]. Fifth-order vessel segments [passive inner diameter (ID) at an intraluminal pressure of 60 Torr = 175.1 ± 4.0 µm, n = 36] were cleaned of adipose tissue, isolated, and transferred to a vessel chamber (Living Systems). The proximal end of the vessel was cannulated with a glass micropipette and secured, blood was gently rinsed from the lumen, and the distal end of the vessel cannulated and secured. Vessels were slowly pressurized to 60 Torr with PSS using a servo-controlled peristaltic pump (Living Systems) and superfused (5 ml/min) with warmed (37°C) PSS aerated with a normoxic gas mixture (21% O2-6% CO2-balance N2). After a 30-min equilibration period, intraluminal pressure was slowly increased to 120 Torr, vessels were stretched to remove bends, and pressure was reduced to 60 Torr for an additional 30-min equilibration period. Pressurized resistance arteries were loaded with the cell-permeant ratiometric Ca2+-sensitive fluorescent dye fura-2 AM (Molecular Probes). Immediately before being loaded, fura-2 AM (1 mM in anhydrous DMSO) was mixed with 0.5 volumes of a 20% solution of pluronic acid in DMSO, and this mixture was diluted with dissecting solution to yield a final concentration of 2 µM fura-2 AM and 0.05% pluronic acid. Vessels were incubated in this solution for 45 min at room temperature in the dark. Administration of fura-2 to the abluminal surfaces of pressurized small arteries has been shown to preferentially load VSM cells (30). Under the conditions used for the current study, changes in the fura signal are representative of, but not necessarily identical to, changes in VSM intracellular [Ca2+]. However, ACh mediates vasodilation and decreases in vessel wall [Ca2+]i in this preparation as determined in preliminary experiments from our laboratory, thus indicative of preferential VSM loading. Vessels were equilibrated for 20 min with warmed, aerated PSS after the loading period to wash out excess dye and to allow for hydrolysis of AM groups by intracellular esterases. Fura-loaded arteries were incubated in the presence of Gap peptides (300 µM), 18{alpha}-GA, or appropriate vehicles for 1 h before experimentation. The incubation time and peptide concentration employed for the inhibitory peptide studies were selected based on a prior report (3). A single artery was studied from each rat; thus values of n refer to the number of animals used for a particular experimental group.

Myogenic and Agonist-induced Vasoconstrictor and Ca2+ Responsiveness

The effects of gap junction inhibitors on myogenic and agonist-induced vasoconstrictor and Ca2+ responses were examined. Fura-loaded vessels were subjected to a series of pressure steps between 20 and 120 Torr, and spontaneous myogenic tone was allowed to develop at each step for 3 min. After completion of pressure-response curves, vessels were reequilibrated at 60 Torr for 10 min and then superfused with PSS containing increasing concentrations of the {alpha}1-adrenergic agonist phenylephrine (PE) (0.01–100 µM). Completion of myogenic and PE curves required ~45 min. ID was continuously monitored using video microscopy (total magnification x200, numerical aperture 0.75) and edge-detection software (Ionoptix). In addition, fura-loaded vessels were alternatively excited at 340 and 380 nm at a frequency of 10 Hz and the respective 510 nm emissions were quantified using a photomultiplier tube and recorded with the use of Ionwizard software (Ionoptix, version 4.4). Photometric data were collected from the entire arterial segment under study. Vessel wall [Ca2+]i, representative of VSM cell [Ca2+]i, was expressed as the mean F340/F380 ratio from the background-subtracted 510 nm signal collected over ~3 min. After completion of the pressure-response and PE curves, intraluminal pressure was maintained at 60 Torr and vessels were superfused with Ca2+-free PSS (129.8 mM NaCl, 5.4 mM KCl, 0.83 mM MgSO4, 19 mM NaHCO3, 5.5 mM glucose, and 3 mM EGTA) for 1 h. The pressure-response curve was then repeated under Ca2+-free conditions to obtain passive responses. Myogenic tone was calculated as the percent difference in ID observed for Ca2+-containing versus Ca2+-free PSS at each pressure. PE-induced vasoconstriction was calculated as the percent change in ID relative to baseline. Change in vessel wall [Ca2+]i was calculated as the difference in F340/F380 and was compared with that of vessels pressurized at 20 Torr for myogenic curves or baseline F340/F380 of vessels pressurized at 60 Torr for PE curves. Myogenic tone, PE-induced vasoconstriction, and change in vessel wall [Ca2+]i were determined in the presence of vehicle (PSS) (n = 5), Gap27 (n = 5), or Gap20 (n = 6). In separate experiments, myogenic tone and PE-induced vasoconstriction were recorded in the presence of 18{alpha}-GA or its solvent, DMSO (n = 5 for both groups).

Disruption of Endothelium

Experiments were performed to determine whether endothelial cell function contributes to attenuated myogenic vasoconstriction associated with gap junction inhibition. The endothelium of small mesenteric arteries was disrupted by perfusing the lumen with ~2 ml of air at an intraluminal pressure of 60 Torr. To demonstrate effective disruption of the endothelium, arteries were preconstricted with PE (10 µM) and administered the endothelium-dependent vasodilator ACh (1 µM).

VSM Cell Resting Membrane Potential

The effect of gap junction inhibitors on pressure-induced VSM cell depolarization was investigated. Fifth-order mesenteric resistance arteries were isolated and pressurized to 20 or 100 Torr, and VSM cells were impaled through the adventitia with glass intracellular microelectrodes (tip resistance 100–200 M{Omega}). To allow visual identification of the cell type from which recordings were obtained, the tip of the electrode was filled with a 2% solution of Lucifer Yellow (Sigma) dissolved in 1% LiCl and backfilled with 1 M KCl (10). A Neuroprobe Model 1600 amplifier (A-M Systems) was used for recording membrane potential (Em). Analog output from the amplifier was low-pass filtered at 1 kHz and routed to a Tektronix RM502A oscilloscope and a Gould chart recorder. Criteria for acceptance of Em recordings were the following: 1) an abrupt negative deflection of potential as the microelectrode was advanced into a cell; 2) stable membrane potential for at least 1 min; and 3) an abrupt change in potential to ~0 mV after the electrode was retracted from the cell. After completion of Em recordings, vessels were examined under epifluorescent illumination to visualize dye-loaded cells. For some experiments, recordings from several VSM cells were made for each animal. The mean potential of all VSM cells recorded for an individual rat was considered as a single replicate for statistical purposes. VSM cell Em was recorded in the presence of vehicle, Gap27 or Gap20 at intraluminal pressures of 20 and 100 Torr (n = 5 animals for vehicle and Gap20, n = 7 for Gap27).

Calculations and Statistics

All data are means ± SE. Values of n refer to number of animals employed for each group. Comparisons of vasoconstrictor and vessel wall [Ca2+] responses between vehicle-, Gap20-, and Gap27-treated vessels were made by one-way ANOVA. Comparisons of VSM cell Em for arteries at different intraluminal pressure were made by two-way ANOVA. If differences were detected by ANOVA, individual groups were compared with the use of the Student-Newman-Keuls post hoc test for all pairwise comparisons. Differences between 18{alpha}-GA and vehicle-treated arteries and Gap20 and Gap27 endothelium-disrupted vessels were evaluated using Student’s unpaired t-tests. A level of P ≤ 0.05 was accepted as statistically significant for all comparisons.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cx Proteins Expressed by Small Mesenteric Arteries

Immunohistochemistry for Cx37 in second- through fifth-order mesenteric arteries outside the gut wall revealed fine punctate staining in the medial layer (Fig. 1, A and B), which exhibited frequent colocalization with immunoreactive smooth muscle {alpha}-actin (Fig. 1A). Larger Cx37-immunoreactive plaques were identified in the endothelial layer (Fig. 1, A and B) as determined by costaining with vWF (Fig. 1B). Similar patterns of Cx37 staining were observed in mesenteric arteries from two animals. Cx43 was present only in the inner circular layer of gastrointestinal (GI) smooth muscle and was not detected in the outer longitudinal GI smooth muscle layer or vascular tissue (Fig. 1C). An identical pattern of staining for Cx43 was observed for both Cx43 monoclonal antibodies. A similar lack of Cx43 staining was observed in mesenteric arteries outside the gut wall in two animals and in submucosal arteries of the gut wall in four rats. Staining for all three Cx antibodies was eliminated when primary antibodies were preadsorbed with appropriate blocking peptides. Furthermore, no specific staining was observed in sections treated only with secondary antibodies or in samples treated with mouse IgG or rabbit serum instead of primary antibodies. These results validated the use of Gap27 as an inhibitor in subsequent functional studies, as this peptide is homologous to extracellular loops of Cx37 and Cx43 (3).



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  		Fig. 1. A: dual labeling for connexin 37 (Cx37; red) and smooth muscle {alpha}-actin (green) in a transverse section of a mesenteric arteriole (a). Fine punctate staining for Cx37 (arrows) was observed in vascular smooth muscle (vsm) cells, with larger Cx37-immunoreactive plaques present in the endothelial (e) layer. B: dual labeling for Cx37 (red) and endothelial von Willebrand factor (vWF; green) in a transverse section of a mesenteric arteriole. Cx37 staining (arrows) exhibited colocalization with vWF. C: Cx43 staining in a longitudinal section of small intestine. Cx43 immunoreativity was detected in the inner circular layer of gastrointestinal (GI) smooth muscle (arrow) but not in mesenteric arterioles. Scale bars = 20 µm.

 
Characterization of Cx37 antibody. The Cx37 antibody detected a single band at 37 kDa in doxycycline-induced Rin-mCx37 cell lysates but not in lysates from noninduced cells (Fig. 2).



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  		Fig. 2. Representative Western blot for Cx37 in doxycycline-induced rat insulinoma (Rin)-mCx37 cells (Cx37+) and noninduced cells (Cx37–). The positions of molecular mass standards are indicated at the left side of the blot. Cx37 was identified as a single band at 37 kDa in induced cells.

 
Effects of Gap Junction Inhibitors on Myogenic Vasoconstrictor and Ca2+ Responses

Myogenic vasoconstriction of arteries treated with the inhibitory peptide Gap27 was greatly attenuated compared with both vehicle and Gap20-treated vessels (Fig. 3, A–C). Consistent with this finding, increases in vessel wall [Ca2+]i associated with elevation of intraluminal pressure were diminished for small mesenteric arteries treated with Gap27 compared with Gap20- and vehicle-treated vessels (Fig. 3, A, B, and D). Pressure-induced constrictor and vessel wall [Ca2+]i responses did not differ between vessels treated with Gap20 or vehicle (Fig. 3, C and D). Administration of 18{alpha}-GA also decreased myogenic vasoconstriction of mesenteric resistance arteries compared with vehicle (Fig. 4).



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  		Fig. 3. Representative traces of changes in inner diameter (ID) and vessel wall intracellular [Ca2+] ([Ca2+]i; expressed as F340/F380) in response to increases in intraluminal pressure for small mesenteric arteries treated with either vehicle (A) or Gap27 (B). Intraluminal pressure (in Torr) is indicated by arrows. C: mean data ± SE for myogenic tone. D: mean data ± SE for changes in vessel wall [Ca2+]i as a function of intraluminal pressure for mesenteric arteries treated with vehicle (n = 5), Gap27 (n = 5), or Gap20 (n = 6). *P ≤ 0.05 vs. vehicle and Gap20-treated vessels.

 


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  		Fig. 4. Myogenic tone as a function of intraluminal pressure for small mesenteric arteries treated with 18{alpha}-glycyrrhetinic acid (18{alpha}-GA) or DMSO. n = 5 for both groups. *P ≤ 0.05 vs. DMSO.

 
Effects of Gap Junction Inhibitors on PE-Induced Vasoconstrictor and Ca2+ Responses

In contrast to the effects gap junction inhibitors on myogenic constriction, mean PE-induced vasoconstriction (Fig. 5C) and increases in vessel wall [Ca2+]i (Fig. 5D) did not differ between arteries treated with Gap27, Gap20, or vehicle. Interestingly, we found that oscillations in ID and vessel wall [Ca2+]i observed for control (Gap20 treated) vessel after administration of high concentrations of PE (Fig. 5A) were attenuated for vessels treated with Gap27 (Fig. 5B). We also found that PE-induced vasoconstriction did not differ between 18{alpha}-GA and vehicle-treated vessels (Fig. 6).



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  		Fig. 5. Representative traces of phenylephrine (PE)-induced changes in inner diameter (ID) and vessel wall [Ca2+]i (expressed as F340/F380) for vehicle (A) and Gap27 (B)- treated mesenteric resistance arteries. The administered concentration of PE (in µM) is indicated by arrows. C: mean data ± SE for PE-induced vasoconstriction. D: mean data ± SE for PE-induced change in vessel wall [Ca2+]i for small mesenteric arteries treated with vehicle (n = 5), Gap27 (n = 5), or Gap20 (n = 6). There were no significant differences.

 


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  		Fig. 6. PE-induced vasoconstriction for mesenteric resistance arteries treated with 18{alpha}-GA or DMSO (n = 5 for both groups). There were no significant differences.

 
Effects of Disruption of the Endothelium

Before disruption of the endothelium, ACh (1 µM) administration reversed PE-induced tone by 96.7 ± 0.7% for Gap20-treated vessels and 94.7 ± 2.6% for Gap27-treated vessels. ACh-induced vasodilation was nearly abolished (1.48 ± 0.5% for Gap20, 4.8 ± 1.6% for Gap27) after the lumen had been perfused with air, demonstrating that this procedure effectively disrupts endothelial cell function (Table 1). Gap junction inhibitors had similar effects on vasoconstrictor and Ca2+ responses of endothelium-disrupted vessels compared with endothelium-intact vessels. Pressure-induced constriction and increases in vessel wall [Ca2+]i were diminished for arteries treated with Gap27 compared with Gap20-treated vessels (Fig. 7, A and B), whereas PE-induced responses did not differ between these groups (Fig. 7, C and D).


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  		Table 1. Effects of endothelial disruption on agonist-induced vasoconstriction and vasodilation of mesenteric arteries

 


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  		Fig. 7. Myogenic tone (A) and changes in vessel wall [Ca2+]i (expressed as F340/F380) (B) as a function of intraluminal pressure for endothelial-disrupted mesenteric resistance arteries treated with Gap20 or Gap27 (n = 5 for both groups). *P ≤ 0.05 vs. Gap20-treated vessels. PE-induced vasoconstriction (C) and changes in vessel wall [Ca2+]i (D) for endothelial-disrupted mesenteric resistance arteries treated with Gap20 or Gap27 (n = 5 for both groups). There were no significant differences.

 
Comparisons of myogenic and PE-induced vasoconstriction of endothelium-intact and endothelium-disrupted arteries are shown in Table 2. Myogenic vasoconstriction was not different between endothelium-intact or endothelium-disrupted vessels treated with Gap20 or Gap27. In contrast, agonist-induced vasoconstriction of endothelium-disrupted arteries was greater than that of endothelium-intact vessels at [PE] of 1, 10, and 100 µM for both Gap20- and Gap27-treated arteries. In addition, oscillations in ID and vessel wall [Ca2+]i after administration high concentrations of PE in endothelium-intact control (Gap20 and vehicle treated) vessels (Fig. 5A) were not present for endothelium-disrupted arteries.


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  		Table 2. Effects of endothelial disruption on myogenic and PE-induced vasoconstriction of Gap20- and Gap27-treated arteries

 
Effects of Gap Junction Inhibitors on VSM Cell Em

Lucifer yellow loading allowed visual identification of the cell type within the vascular wall from which Em recordings were obtained. Dye-labeled VSM cells were easily identified by their characteristic spindle-shaped morphology and perpendicular orientation relative to the long axis of the artery (Fig. 8A). In agreement with an earlier report (16), lucifer yellow loading was confined to a single VSM cell, suggesting that gap junctions formed by these cells are unable to transport the dye. VSM cell Em was significantly (P < 0.05) depolarized when vessels were pressurized at 100 Torr compared with 20 Torr within all treatment groups (Fig. 8E). However, VSM cells in vessels treated with Gap27 and pressurized to 100 Torr were hyperpolarized compared with arteries treated with Gap20 or vehicle at this pressure (Fig. 8, B–E). Interestingly, some (2 of 7) VSM cells in mesenteric resistance arteries treated with Gap27 and pressurized to 100 Torr exhibited an unusual Em with steady oscillation between –34 and –43 mV at a frequency of ~0.5 Hz (Fig. 8F). Because these recordings apparently constituted a second population of cells, they were excluded from the mean data shown in Fig. 8E. In addition, vessels treated with Gap20 were slightly depolarized compared with Gap27- and vehicle-treated arteries at intraluminal pressures of 20 Torr (Fig. 8E). This may be a nonspecific effect related to the relatively high (300 µM) peptide concentration employed for these studies.



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  		Fig. 8. A: example of a Lucifer yellow-loaded VSM cell in a pressurized mesenteric resistance artery (bar = 100 µm). B–D: representative VSM cell membrane potential (Em) recordings for mesenteric resistance arteries pressurized to 100 Torr and treated with vehicle (B), Gap20 (C), or Gap27 (D). E: mean VSM cell Em for vessels treated with vehicle, Gap20, or Gap27 and pressurized to 20 or 100 Torr (n = 5 animals for all groups). VSM cells were depolarized when recordings were obtained at 100 Torr compared with 20 Torr for all treatment groups. *P ≤ 0.05 vs. vehicle-treated vessels at 20 Torr; #P ≤ 0.05 vs. vehicle and Gap20-treated vessels at 100 Torr. F: representative trace of oscillatory VSM cell Em observed for 2 of 7 recordings obtained for Gap27-treated arteries pressurized to 100 Torr.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The primary goal of this study was to examine the contribution of intercellular communication to myogenic and agonist-induced vasoconstriction of small arteries. Our major findings include the following: 1) both VSM and endothelial cells of small mesenteric arteries appear to express Cx37, although Cx43 immunoreactivity was not detected in these arteries; 2) gap junction inhibitors greatly attenuate myogenic vasoconstriction and pressure-induced increases in vessel wall [Ca2+]i; 3) mean PE-induced constrictor and Ca2+ responses are not altered by gap junction inhibitors; 4) blunted myogenic vasoconstriction associated with gap junction inhibitory peptides persists after disruption of the endothelium; and 5) depolarization of VSM cells associated with increases in intraluminal pressure is attenuated for arteries treated with a gap junction inhibitory peptide. These findings suggest that VSM intercellular communication via Cx37-dependent gap junctions contributes to myogenic, but not agonist-induced, vasoconstriction of small mesenteric arteries. Furthermore, attenuated myogenic vasoconstriction associated with gap junction inhibition is independent of endothelial cell function.

Studies of Cx37 and Cx43 expression within the rat peripheral circulation have revealed substantial heterogeneity in localization depending on the vascular bed and vessel size examined. Consistent with our present findings, Hill and colleagues (24, 34) identified strong expression of Cx37 in the endothelium of rat caudal and basilar arteries but only fine punctate Cx37 immunoreactivity in the medial layer of these vessels using the same Cx37 antibody employed in the current study. However, these investigators detected Cx43 within the endothelium but not media of these same arteries, which is in contrast to the complete lack of Cx43 immunostaining observed in rat mesenteric arteries (Fig. 1C). Furthermore, whereas both Cx43 and Cx37 were expressed within the endothelial layer of rat hepatic (5) and coronary arteries (50), neither Cx37 nor Cx43 was identified in the medial layer of coronary arteries, but staining for both of these Cxs was observed in the smooth muscle layer of hepatic arteries. Discrepancies have also been revealed with respect to Cx37 and Cx43 localization in rat mesenteric arteries. For example, Gustafsson et al. (22) detected both Cx37 and Cx43 plaques in the endothelium of mesenteric resistance arteries but no Cx staining in the media. In contrast, a separate study found relatively low levels of Cx37 and Cx43 in the medial layer of elastic but not muscular rat mesenteric arteries, with strong expression of Cx37 in the endothelium (29). The reason for these apparent discrepancies in the literature is not clear but may reflect differences in methodology and heterogeneity in Cx expression between vessel types and sizes.

Our finding that administration of either gap junction inhibitory peptides or pharmacological agents that block intercellular communication greatly attenuates pressure-induced vasoconstriction of mesenteric resistance arteries is in agreement with those of Lagaud et al. (32), showing loss of myogenic tone after administration of heptanol or 18{alpha}-GA to pressurized cerebral arteries. Myogenic behavior results primarily from stretch-induced VSM cell depolarization associated with elevations in intraluminal pressure (23). In addition, Ca2+ sensitization also contributes to this response in cerebral (31) and mesenteric (42) arteries. Smooth muscle depolarization elicits activation of voltage-dependent Ca2+ channels, Ca2+ influx, and vasoconstriction (30). Our findings clearly demonstrate that administration of either Gap27 or 18{alpha}-GA nearly abolishes myogenic vasoconstriction (Fig. 3C) and that this response is associated with blunted VSM cell depolarization (Fig. 8E) and diminished increases in pressure-induced vessel wall [Ca2+]i (Fig. 3D). Given that earlier studies have shown that pressure-induced vasoconstriction is blocked by inhibitors of voltage-dependent Ca2+ channels (30), these findings suggest that attenuated myogenic vasoconstriction after gap junction inhibition may result from diminished voltage-dependent Ca2+ influx. To examine the possibility that blunted myogenic vasoconstriction during blockade of intercellular gap junctional communication was due to increased production endothelial cell-derived hyperpolarizing or vasodilatory factors, additional experiments were performed using vessels with dysfunctional endothelium. However, attenuated myogenic constriction associated with gap junction inhibitors persisted after disruption of the endothelium (Fig. 7, A and B), demonstrating that interrupted smooth muscle communication is central to this response. Furthermore, because the Gap27 inhibitory peptide is selective for Cxs37 and 43, and that Cx37 but not 43 was detected by immunostaining in VSM cells of mesenteric arteries, we conclude that smooth muscle gap junctions containing Cx37 appear to be required for myogenic vasoconstriction of mesenteric resistance arteries.

In contrast to the dramatic effects of gap junction inhibitors on myogenic responsiveness, mean PE-induced vasoconstrictor and vessel wall [Ca2+]i responses were not altered by inhibitors of intercellular communication (Fig. 5). This finding is in disagreement with earlier reports suggesting that the agonist-induced vasoconstriction of aortic rings is attenuated by inhibition of gap junctions. For example, Christ et al. (8) demonstrated that administration of heptanol relaxed aortic rings that had been precontracted with PE, whereas rings constricted with 60 mM extracellular KCl were not affected by heptanol. A similar study by this group also reported heptanol-induced relaxation of aortic ring precontracted with endothelin-1,5-hydroxytryptamine and prostaglandin F2{alpha} (9). The discrepancy between the current findings and prior reports may be due to heterogeneity between conduit and resistance arteries in the relative significance of gap junctions in these responses. In addition, diffusional distances and barriers are much greater in large arteries containing more concentric layers of VSM compared with resistance vessels. Therefore, a greater proportion of VSM cells in large arteries may not be directly activated by vasoconstrictor agents and therefore must be stimulated indirectly via gap junctions. In contrast, in smaller vessels, it appears that most cells are directly influenced by vasoconstrictor agonists. PE-induced vasoconstriction results from complex intracellular signaling events after agonist binding to G protein coupled {alpha}1-adrenoreceptors. Ca2+-influx (33), Ca2+-release from intracellular stores (27), and increased sensitivity of the contractile apparatus to intracellular [Ca2+] (37) all contribute to PE-induced vasoconstriction. Our findings show that gap junction inhibitors have no effect on PE-induced vasoconstriction of mesenteric resistance arteries, suggesting that these signaling events are not influenced by interrupted intercellular communication. Furthermore, these findings imply that most if not all VSM cells with the vessel wall are equally sensitive to stimulation by this agonist.

Interestingly, we found that although administration of gap junction blockers did not alter mean PE-induced vasoconstriction, administration of an inhibitory peptide reduced oscillations in artery diameter and vessel wall [Ca2+]i associated with high concentrations of PE (Fig. 5, A and B). These findings are consistent with a report by Chaytor and co-workers (3) demonstrating a similar loss of PE-induced oscillations of mesenteric artery rings treated with the Gap27 peptide. In addition, similar to prior studies (14, 49) we found that disruption of the endothelium increased PE-induced vasoconstriction of mesenteric arteries. Furthermore, in agreement with an earlier report (28), we found that disruption of the endothelium also decreased diameter and Ca2+ oscillations associated with PE administration (not shown). These findings are consistent with the hypothesis that in mesenteric resistance arteries, myoendothelial communication involving gap junctions may contribute to oscillations in diameter and vessel wall [Ca2+]i associated with PE administration.

Depolarization of VSM cells associated with elevated intraluminal pressure may result from activation of mechanosensitive ion channels (12, 43, 44). Recent studies have shown that VSM cells express stretch-activated nonselective cation channels (43) and that downregulation of these channels using an antisense oligonucleotide approach attenuates myogenic vasoconstriction (44). These data support the hypothesis that these channels play a central role in pressure-induced vasoconstriction. Given that mechanosensitivity appears to be inherent to VSM cells (12), if all cells within the vascular wall responded equally to mechanical stimuli, it would be predicted that inhibition of gap junction communication would not alter stretch-induced changes in VSM cell Em and myogenic vasoconstriction. However, the current study shows that inhibition of VSM intracellular communication greatly attenuated pressure-induced VSM cell depolarization, Ca2+ influx and vasoconstriction. Thus our findings are consistent with the possibility that heterogeneity in stretch sensitivity exists among VSM cells. Differential VSM cell depolarization may result from heterogeneity in inherent mechanosensitivity or from differences in mechanical stretch sensed by various smooth muscle layers within the vascular wall. Consistent with this hypothesis, heterogeneity in the pressure-induced deformation of smooth muscle cell layers has been reported (20). Layers that are subjected to relatively higher levels of deformation may depolarize to greater extent than cells experiencing lower levels of mechanical stress. This hypothesis predicts that some VSM cells strongly depolarize after vessel wall stretch and transmit this response to adjacent, less mechanosensitive cells through gap junctions, thereby eliciting coordinated vasoconstriction. Recordings obtained from vessels pressurized to 100 Torr and treated with a gap junction inhibitory peptide that exhibit an unusual oscillating membrane potential (Fig. 8F) may represent these strongly depolarizing cells. Although additional work is needed to fully develop and verify this hypothesis, the proposed differences in response to mechanical stress among VSM cells could have significant implications for the understanding of vascular disease.

In conclusion, this study demonstrates that inhibition of gap junctions attenuates myogenic vasoconstriction of rat mesenteric resistance arteries by blunting stretch-induced VSM cell depolarization and increases in vessel wall [Ca2+]i. This effect was independent of endothelial cell function, suggesting that VSM intercellular communication via gap junctions is required for normal myogenic behavior.


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 ABSTRACT
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This study was supported by National Institutes of Health Grants HL-58124 and HL-63207 (both to B. R. Walker), RR-16480 and HL-077876, a Scientist Development Grant from the American Heart Association, and a Parker B. Francis Fellowship in Pulmonary Research (all to T. C. Resta).

Images of connexin immunostaining were generated in the University of New Mexico Cancer Center Fluorescence Microscopy Facility, which received support from National Center for Research Resources Grants1 S10 RR14668, P20 RR11830, S10 RR19287, S10 RR016918, National Science Foundation Grant MCB9982161, National Cancer Institute Grant R24 CA88339, the University of New Mexico Health Sciences Center, and the University of New Mexico Cancer Center.


   ACKNOWLEDGMENTS
 
The authors thank Tasha Nelson (University of Arizona, Department of Physiology) for technical assistance with Western blot analysis experiments for Cx37. The Rin-mCx37 cell line was a generous gift of Dr. Janis M. Burt (University of Arizona, Department of Physiology).

Present address for S. Earley: Dept. of Pharmacology, University of Vermont College of Medicine, 89 Beaumont Ave., Burlington, VT 05405 (E-mail: Scott.Earley{at}uvm.edu).


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. R. Walker, Dept. of Cell Biology and Physiology, MSC08 4750, 1 Univ. of New Mexico, Albuquerque, NM 87131-0001 (E-mail: bwalker{at}salud.unm.edu)

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.


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