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Cardiovascular Research 2004 62(2):388-396; doi:10.1016/j.cardiores.2003.12.024
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Copyright © 2004, European Society of Cardiology

Increased vascular sensitivity and connexin43 expression after sympathetic denervation

David P. Slovuta, Shyamal H. Mehta*,b, Anne M. Dorranceb, Frank C. Brosiusc, Stephanie W. Wattsd and R.Clinton Webbb

aDepartment of Physiology, University of Michigan, Ann Arbor, MI, USA
bDepartment of Physiology, Medical College of Georgia, 1120, 15th Street, Augusta, GA 30912, USA
cDepartment of Nephrology, University of Michigan, Ann Arbor, MI, USA
dDepartment of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA

* Corresponding author. Tel.: +1-706-721-9102; fax: +1-706-721-7299. Email address: shmehta{at}mail.mcg.edu

Received 26 August 2003; revised 4 December 2003; accepted 20 December 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Following denervation, arteries demonstrate a heightened sensitivity to {alpha}-adrenergic agonists and increased oscillatory contractions that may partly result from increased gap junction expression. Hence, we wanted to study the effect of sympathetic denervation on connexin43 (Cx43) expression and agonist-induced contractility in the vascular smooth muscle (VSM). Methods: Effects of denervation with reserpine (3 mg/kg/day, i.p.) or topical 5% phenol–glycerol on VSM contractions and expression of the gap junction Cx43 mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting for Cx43 protein were examined. Wistar–Kyoto (WKY) rat tail arteries were exposed to norepinephrine (NE) (10–9–10–5 M). Reactivity was also examined in the carotid arteries and thoracic aortas from Cx43 heterozygote deficient (KO) mice. Results: The concentration for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (p<0.05). NE-induced oscillatory activity (OA) was seen in 5/5 reserpine- and 5/8 phenol-treated vessels vs. 0/12 controls (p<0.05). Spontaneous OA was observed more frequently in carotid and aortic rings from WT than Cx43 KO rings. Cumulative OA in response to {alpha}-adrenergic stimulation was significantly greater in WT carotid (429±101 vs. 128±7 mN s, p<0.05) and aortic rings (337±85 vs. 134±11 mN s, p<0.05) than in Cx43 KO rings. Following denervation, RT-PCR showed significantly increased levels of Cx43 mRNA (p<0.05). Western blot analysis revealed near doubling of Cx43 protein (p<0.05). Conclusion: We conclude that sympathetic denervation results in increased expression of Cx43, which in turn, contributes to increased spontaneous and agonist-induced OA in VSM.

KEYWORDS Gap junctions; Connexin43; Vascular smooth muscle; Sympathetic denervation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Gap junctions play an important role in intercellular communication. They are formed by hexameric assembly of membrane spanning proteins termed connexins (Cxs) [1] which, in mammals, belong to a family of 14 members [2]. Despite the large number of Cxs that have been identified in the mammalian organ systems, four of these proteins, referred to as Cx43, Cx45, Cx40 and Cx37, are important in the cardiovascular system [4].

The Cxs play an important role in modulating the coordination of dilatation and contraction in the vascular tree [5–8]. In previously published results, our laboratory has shown that vertebral arteries from human hypertensive subjects exhibit a greater incidence of oscillatory contractions in response to serotonin and endothelin than those isolated from normotensive subjects [10]. The increased vascular reactivity observed in hypertension may result partly from enhanced gap junctional activity [9].

The mechanisms regulating the expression of gap junctions remain to be elucidated. Gap junction expression, especially Cx43 is markedly increased in some forms of hypertension. However, significant variation in the expression of Cx43 in the different vascular beds and cell types has been reported in the cardiovascular system [3,11–13]. In addition, sympathetic neurons may play a role in modulating gap junction expression. Westfall et al. [15] have shown that denervation increases gap junction nexuses in the rat vas deferens. Following sympathectomy, isolated resistance arteries from rats demonstrate increased vascular reactivity in response to {alpha}-adrenergic agonists that resembles the heightened vascular reactivity seen in hypertensive subjects [16,17]. Thus, the denervated artery may serve as a useful model for investigating the role of sympathetic nerves in regulating gap junction expression and modulating vascular reactivity.

In the present study, we examined the effects of short-term denervation with reserpine or topical phenol on vascular smooth muscle contractions and expression of Cx43 in rat tail artery. In addition, experiments were also performed on arteries isolated from Cx43 heterozygote deficient mice. We tested the hypothesis that sympathetic denervation increases gap junctional communication, which in turn, leads to increased vascular reactivity and oscillatory contractile activity. The premise for this hypothesis and the experimental approach were based on the following evidence: (1) arteries such as the rat aorta that express Cx43 to a high degree [18] are more sensitive to {alpha}-adrenergic agonists than those (rat tail artery) that express Cx43 at a low level; (2) the rat thoracic aorta has a low density of adrenergic innervation compared to the tail artery of the same species [18]; and (3) oscillatory contractile activity occurs with greater incidence in the rat aorta than in the rat tail artery. We surmised that contractile response of the denervated tail artery would be similar to the response of the aorta, which is functionally denervated. Additional experiments were performed on arteries isolated from Cx43 heterozygote deficient mice to further elucidate the role of Cx43 expression on vascular smooth muscle reactivity.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Vascular reactivity studies using rat tail arteries
All procedures used were in accordance with NIH guidelines for animal care and use at the University of Michigan and the Medical College of Georgia. Adult male Wistar–Kyoto (WKY) rats (275–350 g) were used. In some experiments, rats were sympathetically denervated with intraperitoneal reserpine [3 mg/kg/day for 5 days (n=5)]. In other experiments tail arteries were denervated by topical 5% phenol–glycerol (n=8) [19]. For phenol denervation, each rat was anesthetized with sodium pentobarbital and under sterile conditions a 10-mm portion of the artery was exposed at the origin of the tail. The surface of the vessel was dissected free from surrounding tissue and the artery was painted with 5% phenol and the sheath and tail were closed in layers. Six to ten days following denervation, the rats were anesthetized with sodium pentobarbital 50 mg/kg i.p., the tail artery was removed, cut into strips (reserpine-treated) or rings (phenol-treated), and suspended in tissue baths containing physiologic salt solution (PSS; see below) for measurement of isometric force development. The optimal passive force–tension relationship was determined in response to potassium (122 mmol/l). Baths were oxygenated (95% O2 and 5% CO2) and maintained at 37 °C. The composition of PSS (mmol/l) was as follows: NaCl (130.0); KCl (4.7); KH2PO4 (1.18); MgSO4 7H20 (1.17); NaHCO3 (14.9); CaCl2 (1.6); dextrose (5.5); and CaNa2 EDTA (0.03), pH 7.20. Preparations were allowed to equilibrate for a minimum of 60 min before measurements were performed. Endothelium was removed by mechanical abrasion. Removal of the endothelium was validated by lack of relaxation to acetylcholine (10–6 mol/l) following contraction to phenylephrine (10–7 mol/l).

Norepinephrine (10–9–10–5 mol/l) was added cumulatively to the muscle bath. After a washout period, the arterial strips and rings were contracted again with norepinephrine (10–9–10–5 mol/l) and observed for development of oscillatory activity. In some experiments, after the onset of agonist-induced oscillatory contractions, we added Gap27 (10 mmol/l), a synthetic peptide corresponding to amino acid residues 204–214 on the second extracellular loop of Cx43 (sequence SRPTEKIFII; Genemed Synthesis, San Francisco, CA) to inhibit the oscillations [21]. As a negative control, we added peptide Gap20 to some rings. Gap20, which corresponds to an intracellular loop on the connexin molecule, has been shown to have no effect on vascular smooth muscle tone [21]. Data were digitized at 10 Hz using a 12-bit A/D converter (PowerLab v3.6, ADInstruments, Mountain View, CA).

2.2. Vascular reactivity studies using Cx43 heterozygote deficient (KO) mice
Vascular reactivity studies to further elucidate the role of Cx43 in the development of oscillatory activity were done using WT and Cx43 heterozygote deficient mice (KO) (Jackson Labs, Bar Harbor, ME). The Cx43 KO mice belonged to C57BL strain that were heterozygous for the Gja1tm1Kdr targeted mutation [22,23]. Wild-type C57BL mice were used as controls. Animals were anesthetized using sodium pentobarbital, 50 mg/kg i.p. The carotid artery and thoracic aorta were dissected free, cut into rings with intact endothelium and suspended in PSS (described above) in tissue baths for isometric force generation studies [20] using the Multi Myograph System Model 610M ver.1.5 (Danish Myo Technology, Denmark). Following equilibration in PSS, vascular rings were exposed to cumulative addition of phenylephrine (10–9–10–5 mol/l) and isometric force was measured. At each concentration, oscillatory contractile activity was determined by computing the integral from the lowest tension value during the final 4 min of each recording epoch according to the formula:



Formula

where y is the maximal force generated in mN during the selected time interval, ylow is the lowest value of force generated in the selected time interval and {Delta}t is the selected time interval. Cumulative oscillatory activity was determined by summing the amplitude of contractile activity at each concentration. Results are expressed in mN sec. This calculation method examines the amplitude of oscillatory contractile activity superimposed over any increases in tension in response to phenylephrine (PE).

2.3. Reverse transcriptase polymerase chain reaction (RT-PCR)
RNA was extracted from tail artery of control and phenol-treated rats using a preparative kit (Qiagen, CA), 1 µg of RNA was used to produce cDNA. Each RNA generated and a negative control sample was subjected to first strand cDNA synthesis using oligo dT as a primer. Some RNA samples were subjected to the PCR procedure without prior reverse transcription to control for the presence of contaminating genomic DNA in the sample. PCR amplifications were carried out on a portion of the cDNA produced as previously published [37]. The results were normalized to the expression of the constitutively expressed gene GAPDH. The specific oligoneuclotide primers used are shown in Table 1.


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  		Table 1 Primers used for PCR

 
2.4. Western blot analysis
Endothelium-denuded tail arteries were snap-frozen in liquid nitrogen, pulverized, suspended in cold isotonic buffer {sucrose 250 mM/l, HEPES [pH 7.4] 20 mM, KCl 10 mM, MgCl21.5 mM, EDTA 1 mM, EGTA 1 mM, dithiothreitol (Cleland's reagent) 1 mM, phenylmethylsulfonyl fluoride 1 mmol/l, leupeptin 10 µg/ml, pepstatin A 10 µg/ml, and aprotonin 10 µg/ml}, and centrifuged at 20,000 x g for 45 min at 4 °C. After removing the supernatant, membrane fractions were prepared by resuspending the pellet in cold buffer (Tris–HCl [pH 6.8] 62.5 mM, SDS 2%, glycerol 10%, phenylmethylsulfonyl fluoride 1 mM, leupeptin 10 µg/ml, pepstatin A 10 µg/ml, and aprotonin 10 µg/ml), sonicating 20 s x 2, and centrifuging at 20,000 x g for 5 min to remove cellular debris. Total protein concentration of the supernatant was determined using the bicinchoninic acid assay (Pierce). Supernatant protein (50 µg per lane) was resolved by polyacrylamide gel electrophoresis and immunoblotting as previously described [40]. Polyclonal rabbit anti-Cx43 antibody (1:500, Zymed Laboratories, CA), anti-rabbit peroxidase secondary antibody (1:5000, Sigma-Aldrich, MO) and an enhanced chemiluminescence kit (ECL, Amersham Biosciences, NJ) were used to visualize protein bands. The primary antibody reacts to the third cytoplasmic domain of rat Cx43. Left ventricle and liver served as positive and negative controls, respectively. All results were normalized to β-actin.

2.5. Data analysis and statistical evaluation
Data are expressed as means±S.E.M. Group means were compared using the unpaired two-tailed Student's t-test. The presence or absence of oscillatory contractile activity in control and denervated rat tail arteries and the incidence of oscillatory activity in the Cx43 KO mice studies were analyzed using the chi-square test. Differences were deemed statistically significant when p<0.05. Agonist threshold concentration was defined as the agonist concentration necessary to produce the first measurable arterial contraction. EC50 values (agonist concentration required to produce half-maximal response) were determined by fitting data to the sigmoidal relation:



Formula

Tmin represents minimal tension and Tmax indicates maximal tension. Cx43 expression was quantified using densitometric analysis of autoradiographs (NIH Image) as reported previously [24].


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Denervated tail arteries show increased oscillatory contractions to norepinephrine which is inhibited by gap junction inhibitor, Gap27
Experiments were done with reserpine denervated tail arteries and tail arteries with intact innervation which served as controls. Previous studies have shown that incubation with zero-potassium solution results in near-total inhibition of Na+–K+ATPase, depolarization of vascular adrenergic nerve endings, and release of norepinephrine, which causes tail arteries to contract [25,26]. Absence of a significant contractile response to zero-potassium solution reflects the decreased norepinephrine (NE) levels found at the nerve terminals following denervation. Compared with denervated vessels, control strips subjected to potassium-free salt solution demonstrated an 8-fold increase in isometric tension (17.2±4.5 vs. 138.4±23.2% contraction to phenylephrine 10–6 mol/l, p<0.001).

The reactivity of tail artery strips was assessed with the addition of norepinephrine (10–9–10–5 mol/l) to the muscle. The tail artery strips constricted in response to increasing doses of norepinephrine. The threshold for contraction was approximately 10–8 mol/l NE in the vehicle treated controls and 3 x 10–9 mol/l in the reserpine treated arteries.

Reserpine-denervated tail artery strips showed a greater sensitivity to NE compared to controls as demonstrated by a leftward shift in the concentration–response curve (Fig. 1A) and a lower EC50 value. The EC50 for agonist-induced contraction was significantly lower for reserpine-treated vessels than controls (–log EC50 [mol/l] control 6.89 vs. denervated 7.16, p<0.05, Table 2A).


Figure 1
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  		Fig. 1 Relationship between step-wise addition of norepinephrine (10–9–10–5 mol/l) and active force in rat tail artery strips. (A) Reserpine-denervated tail artery strips ({circ}, n=6) were more sensitive to the contractile effect of NE than vehicle treated control strips (bullet, n=4). (B) Similar observation was made with phenol denervated and control tail artery strips. Phenol-denervated tail artery strips ({circ}, n=5) demonstrated increased sensitivity to NE induced contractions in comparison to controls (bullet, n=4). Force values are expressed as mean±S.E.M. in relation to maximal force induced by NE (*p<0.05 vs. control).

 

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  		Table 2A NE concentration–response characteristics of control and reserpine-treated arterial strips

 
Control and phenol-denervated tail artery rings were also exposed to cumulative addition of norepinephrine (10–9 mol/l–10–5 mol/l). Previously published studies from our laboratory have shown that low potassium-induced contractions are completely abolished by topical phenol denervation [38]. Phenol denervated rings had a markedly lower threshold for contraction than controls (3 x 10–9 vs. 3 x 10–8 mol/l). Phenol-denervated vessels showed an increased sensitivity to NE-induced contractions as evidenced by a leftward shift of the curve (Fig. 1B). As with reserpine-treated vessels, the EC50 for norepinephrine-induced contraction was lower for phenol-treated vessels than for controls (–log EC50 [mol/l] control 6.86 vs. denervated 7.32, p<0.0001, Table 2B). At peak norepinephrine dose, denervated rings contracted 25% more than controls. Thus, both reserpine- and phenol-denervated the tail arteries demonstrated increased sensitivity to the {alpha}-adrenergic agonist, norepinephrine.


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  		Table 2B NE concentration–response characteristics of control and phenol-treated arterial strips

 
Tail artery strips from reserpine-treated rats exhibited oscillatory contractions superimposed on the tonic component of norepinephrine-induced contraction (Fig. 2A). Overall, oscillatory activity was observed in 5/5 reserpine-treated animals and 0/6 controls ({chi}2 test, p<0.01) (Fig. 2B). Peak oscillatory activity occurred between 10–9 M and 10–7 M norepinephrine. Tail artery rings denervated with phenol also oscillated in response to norepinephrine. Oscillatory activity was observed in 5/8 phenol-treated animals and 0/6 controls ({chi}2 test, p<0.05). Addition of 10 mM Gap27, a synthetic peptide corresponding to amino acid residues on the second extracellular loop of Cx43 which acts as a highly specific competitive inhibitor of gap junctions [28] abolished agonist-induced oscillations (Fig. 3) (n=4). Gap20, a peptide which corresponds to an intracellular domain of Cx43 was used as a negative control and had no effect on oscillatory contractions.


Figure 2
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  		Fig. 2 (A) Representative tracing of contractile force generation and oscillatory activity in tail artery strips from vehicle (top) and reserpine-treated rats (bottom). Norepinephrine-induced oscillatory activity in tail artery from reserpine-denervated, but not vehicle treated rats. (B) Incidence of oscillatory activity in reserpine-treated and phenol-treated tail artery strips in comparison to controls was examined. Oscillatory activity was observed in 5/5 reserpine-treated animals vs. 0/6 controls and in 5/8 phenol-treated animals vs. 0/6 controls (*{chi}2 test, p<0.05).

 

Figure 3
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  		Fig. 3 Representative tracing of isometric force in response to NE in phenol-treated tail artery rings. Depolarizing concentration of K+ (122 mmol/l) caused maintained force development without oscillatory contractions. The highly selective gap junction inhibitor, Gap27 (10 mM), markedly attenuated the NE-induced vascular smooth muscle oscillations.

 
3.2. Cx43 mRNA levels and Cx43 protein expression increases following denervation in rat tail arteries
In order to assess the expression of the Cx43 gene in the denervated and intact tail arteries, RNA was extracted from denervated and vehicle-treated tissue. Analysis of the mRNA for Cx43 by RT-PCR showed an increase in message in denervated vessels in comparison to controls (0.484±0.059 vs. 0.20±0.047 a.u., p<0.05) and densitometric analysis of the representative blot confirmed that Cx43 mRNA levels were increased >2-fold in the denervated rats as compared to the vehicle treated controls (Fig. 4). Results are normalized to constitutively expressed GAPDH gene.


Figure 4
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  		Fig. 4 RT-PCR analysis of rat tail artery. Top: RT-PCR analysis revealed that Cx43 mRNA was increased in the denervated rats as compared to controls. Bottom: quantitative assessment confirmed that Cx43 mRNA levels were increased >2-fold in the denervated rats as compared to the vehicle treated controls (*p<0.05 vs. controls). Values represent densitometric measurements of Cx43 and GAPDH mRNAs and are expressed as mean±S.E.M.

 
Western blot analysis was used to measure changes in Cx43 expression in the tail artery after denervation with topical phenol. The polyclonal rabbit anti-connexin 43 recognized three bands, two at approximately 43 kDa and one at 33.9 kDa (Fig. 5). Cx43 levels nearly doubled following denervation (0.80±0.34 vs. 0.41±0.13 a.u., p<0.05). To verify that the anti-Cx43 antibody was specific for Cx43, we preincubated rabbit anti-Cx43 antibody with 2 µg/ml of Cx43-immunizing peptide for 15 min at room temperature before immunoblotting. Preincubation of primary antibody with free peptide resulted in disappearance of all three bands. As expected, Cx43 protein was found in abundance in the left ventricle extracts and not in the hepatocyte extracts. Left ventricle served as a positive control for detection of Cx43 protein due to its abundance in the left ventricle. Results are expressed after normalization to β-actin.


Figure 5
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  		Fig. 5 Expression of Cx43 in the rat tail artery. Left: Western blot analysis of Cx43 from rat tail artery detected as a 43-kDa band was increased in denervated tail arteries (lane 2) as compared to vehicle treated control (lane 1). Left ventricle (lane 3) served as a positive control for Cx43 expression. Lanes 1 and 2 contain 50 µg protein, lane 3 contains 10 µg protein immobilized on a PVDF membrane and blocked with nonfat dry milk. Cx43 was detected using a primary polyclonal antibody, a horseradish-peroxidase-conjugated secondary anti-rabbit IgG antibody, and an enhanced chemiluminescence kit. Right: quantitative assessment confirmed that levels of Cx43 in the denervated rat tail arteries increased >2-fold in comparison to the controls (*p<0.05 vs. controls). Values represent densitometric measurements of Cx43 protein in arbitrary units (normalized to β-actin) and are expressed as mean±S.E.M.

 
3.3. Decreased oscillatory vasoreactivity in Cx43 heterozygote deficient (KO) mice
In this series of experiments, we assessed the oscillatory contractile activity in wild-type (WT) and Cx43 KO mice (n=8) in response to the cumulative addition of phenylephrine (10–9–10–5M). These experiments were performed to examine the effect of gap junction expression on oscillatory contractile activity without the potentially confounding effects of denervation supersensitivity. Spontaneous oscillatory contractile activity was observed in 5/8 WT carotid artery rings vs. 1/7 Cx43 KO carotid artery rings ({chi}2 test, p=0.057) and 5/8 WT aortic rings vs. 1/8 Cx43 KO aortic rings ({chi}2 test, p=0.039). Compared to the Cx43 KO mice carotid artery rings, cumulative oscillatory activity was significantly greater in WT carotid rings (429±101 vs. 128±7 mN sec, p<0.05) in response to phenylephrine. Similar differences were observed with the aortic rings from the Cx43 KO mice vs. WT mice (134±11 vs. 337±85 mN sec, p<0.05). In addition, oscillatory activity was abolished after the addition of 10 mM Gap 27 to the aortic rings from WT mice as compared to the Cx43 heterozygote deficient mice (n=4). Thus, both spontaneous oscillatory activity and cumulative oscillatory activity in response to {alpha}-adrenergic agonist stimulation were seen more commonly in the WT as compared to the Cx43 KO. Hence, these data suggest that Cx43 plays an important role in mediating the oscillatory contractile activity.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Vascular smooth muscle tone is modulated by several mechanisms. Gap junctions provide a pivotal means of coordinating these inputs for regulating vascular smooth muscle tone. Studies have suggested that gap junction activity is important for vascular contraction in response to agonists and for the appearance of contractile oscillations. Elucidating these mechanisms would have important implications in our understanding of hypertension. In some forms of animal and human hypertension, there is an increased response to contractile agonists as well as an increased incidence of oscillatory activity in the vasculature [9]. In addition, an increase in Cx43 in the rat aorta is associated with the two kidney, one clip renal model and DOCA salt model of rat hypertension [27].

We hypothesized that when neural input is abolished, increased gap junction expression enables the vessel to maintain a coordinated response to a contractile stimulus. In the present study, we found that sympathetic denervation of rat tail artery with reserpine or topical phenol produced heightened sensitivity and increased oscillatory contractions in response to {alpha}-adrenergic agonists. The oscillations were abolished by the highly selective gap junction inhibitor, Gap27. Compared with controls, denervated vessels expressed increased levels of mRNA and Cx43 protein. The results suggest that increased gap junction expression and communication play a critical role in permitting vascular smooth muscle cells to act in a coordinated way to facilitate contraction.

Christ et al. [29] suggest that gap junctions may enhance {alpha}1 contractility through various mechanisms by increasing intercellular communication. The crucial role of gap junctions in maintaining agonist-induced contraction is shown when rat aorta is preincubated with heptanol, an inhibitor of gap junction activity, which decreased the magnitude of contraction induced by the partial agonist oxymetolazone and high-efficacy agonist phenylephrine [30]. Several groups have shown that denervated tissues show a greater contractile response to agonists [17,31]. In part, the increase in responsiveness stems from increased receptor affinity [32] and in part, from increased gap junction communication [15]. The latter idea is supported by our results which demonstrate a significant increase in Cx43 mRNA levels and elevated Cx43 protein expression after denervation in the rat tail artery. In principle, increased gap junction communication would enable a smaller fraction of vascular smooth muscle cells to be activated to achieve syncitial tissue activation. Alternatively, it would permit a greater number of cells to be recruited into the functional unit [30]. Either of these explanations may account for the decreased EC50 values to NE observed in reserpine- and phenol-treated vessels observed in the present experiment.

In addition to modulating agonist sensitivity, enhanced gap junction communication may explain the increased agonist-induced oscillatory contractile activity observed following denervation. Calcium mobilization from the sarcoplasmic reticulum has been shown to play a major role in vascular smooth muscle oscillations [33]. At the cellular level, smooth muscle from rat tail artery responds to norepinephrine by producing fluctuations in [Ca2+]i and the phase of [Ca2+]i oscillations varies from cell to cell [34]. Similar findings have been observed in MDCK cells exposed to thrombin, bradykinin and ATP. Stimulation by these agonists produced oscillations in [Ca2+]i which spread from cell to cell and were abolished by octanol, a non-specific inhibitor of gap junctions [35]. Gap junction communication determines how vascular smooth muscle responds as a tissue to oscillations in intracellular calcium. When [Ca2+]i fluctuations are unsynchronized, [Ca2+]i reaches a plateau and induces tonic contraction. With increased gap junction communication, [Ca2+]i fluctuations between cells become synchronized and the vascular smooth muscle develops oscillatory contractile activity. Oscillations in vascular smooth muscle tone have been described in young rat carotid arteries exposed to {alpha}-adrenergic agonists [36], endothelium-denuded rabbit superior mesenteric artery exposed to phenylephrine [21], and vessels from hypertensive subjects [9]. In the latter two studies, the oscillations were attenuated or abolished by various gap junction inhibitors, including Gap27, sucrose, and heptanol. In the present study, denervated vessels demonstrated a greater propensity to developing oscillatory contractions than controls; the oscillations were inhibited by Gap27. In addition, the experiments on Cx43 heterozygote deficient mice have directly demonstrated that low Cx43 levels are associated with decreased incidence of oscillatory contractile activity. Taken together, our results suggest that Cx43 plays a crucial role in propagating synchronous activity between smooth muscle cells and maintaining contractile responses. Our results also suggest that the increased oscillatory activity seen after denervation may not be due to the denervation supersensitivity but an effect of Cx43 upregulation, which is corroborated by our findings with the Cx43 KO mice studies.

The differences in the incidence of oscillatory activity between the intact rat tail arteries and WT mice aortic and carotid rings may be attributable to the differing extents of expression of Cx43 in these tissues, as significant heterogeneity exists in the distribution of gap junctions and connexins in different tissues [8]. In addition, studies by Webb et al. [38] have shown that the aorta and carotid vessels are functionally denervated in comparison to the rat tail artery, as evidenced by activation of adrenergic nerve endings by electrical stimulation.

Possible confounding variables which may have influenced our results include structural changes in the vessel wall and injury from phenol. Dimitriadou et al. [39] found hypertrophy of vascular smooth muscle cells in rabbit cerebral artery 6 weeks following denervation. In rat medial plantar artery, there was no change in both optimal length or active wall tension 10 days following surgical denervation [17]. Although we did not specifically examine vessel morphology, we procured vessels within 10 days of denervation, well before the time when hypertrophy develops. We found similar length–tension relationships for both control and denervated vessels, a finding which suggests that structural changes in the vessel wall are unlikely to account for the enhanced contractility and increased oscillatory activity following denervation. It is possible that injury to the adventitial surface by phenol produced increased Cx43 expression. Yeh et al. [14] have demonstrated increased Cx43 expression in the inner and medial zone of the rat carotid artery following balloon injury of the intima. By 9 days, however, gap junction content in the inner, medial and outer zones had returned to baseline values. Based on these data, we think it unlikely that injury to the adventitia explains the increased Cx43 expression we observed. Moreover, we demonstrated similar levels of increased vascular reactivity and oscillatory contractions in both phenol- and reserpine-treated rats. Reserpine was administered using intraperitoneal injection and thus could not have induced vascular injury.

In summary, denervation results in increased agonist-induced oscillatory activity that is abolished by a highly selective gap junction inhibitor and a greater sensitivity to agonist-induced contraction. The increased sensitivity and oscillatory activity may partly result from increased Cx43 expression and gap junction communication in vascular smooth muscle.


   Acknowledgements
 
This work is supported by NIH grant HL-18575.


   Notes
 
Time for primary review 16 days


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

  1. Haefliger J.A., Meda P. Chronic hypertension alters the expression of Cx43 in cardiovascular muscle cells. Braz. J. Med. Res. (2000) 33:431–438.
  2. Loewenstein W.R. Junctional intercellular communication. The cell-to-cell membrane channel. Phys. Rev. (1981) 6:829–931.
  3. Cottrell G.T., Wu Y., Burt J.M. Cx40 and Cx43 expression ratio influences heteromeric/heterotypic gap junction channel properties. Am. J. Physiol. Cell Physiol. (2002) 282:C1469–C1482.[Abstract/Free Full Text]
  4. Haefliger J.A., Bruzzone R., Jenkins N.A., Gilbert D.J., Copeland N.G., Paul D.L. Four novel members of the connexin family of gap junction proteins: molecular cloning, expression and chromosome mapping. J. Biol. Chem. (1992) 267:2057–2064.[Abstract/Free Full Text]
  5. Segal S.S. Cell-to-cell communication coordinates blood flow control. Hypertension (1994) 23:1113–1120.[Abstract/Free Full Text]
  6. Segal S.S., Duling B.R. Flow control among microvessels coordinated by intercellular conduction. Science (1986) 234:8688–8700.
  7. Larson D.M., Haudenschild C.C., Beyer E.C. Gap junction messenger RNA expression by vascular wall cells. Circ. Res. (1990) 66:1074–1080.[Abstract/Free Full Text]
  8. Hill C.E., Rummery N., Hickey H., Sandow S.L. Heterogeneity in the distribution of vascular gap junctions and connexins: implications for function. Clin. Exp. Pharmacol. Physiol. (2002) 29:620–625.[CrossRef][Web of Science][Medline]
  9. Watts S.W., Webb R.C. Vascular gap junctional communication is increased in mineralocorticoid-salt hypertension. Hypertension (1996) 28:888–893.[Abstract/Free Full Text]
  10. Webb R.C., Schreur K.D., Papadopoulos S.M. Oscillatory contractions in vertebral arteries from hypertensive subjects. Clin. Physiol. (1992) 12:69–77.[Web of Science][Medline]
  11. Haefliger J.A., Castillo E., Waeber G., et al. Hypertension differentially affects the expression of the gap junction protein connexin43 in cardiac myocytes and aortic smooth muscle cells. Adv. Exp. Med. Biol. (1997) 432:71–82.[Web of Science][Medline]
  12. Hong T., Hill C.E. Restricted expression of the gap junctional protein connexin 43 in the arterial system of the rat. J. Anat. (1998) 193:583–593.
  13. Yeh H.I., Rothery S., Dupont E., Coppen S.R., Severs N.J. Individual gap junction plaques contain multiple connexins in arterial endothelium. Circ. Res. (1998) 83:1248–1263.[Abstract/Free Full Text]
  14. Yeh H., Lupu F., DuPont E., Severs N.E. Upregulation of connexin43 gap junctions between smooth muscle cells after balloon catheter injury in the rat carotid artery. Arterioscler. Thromb. Vasc. Biol. (1997) 17:3174–3184.[Abstract/Free Full Text]
  15. Westfall D.P., Millecchia L.L., Lee T.J.F., Corey S.P., Smith D.J., Fleming W.W. Effects of denervation and reserpine on nexuses in the rat vas deferens. Eur. J. Pharmacol. (1977) 41:239–242.[CrossRef][Web of Science][Medline]
  16. Rizzoni D., Perlini S., Mircoli L., et al. Enhanced vascular reactivity in the sympathectomized rat: studies in vivo and in small isolated resistance arteries. J. Hypertens. (2000) 18:1041–1049.[CrossRef][Web of Science][Medline]
  17. Bentzer P., Nielsen N., Arner M., et al. Supersensitivity in rat microarteries after short term denervation. Acta Physiol. Scand. (1997) 161:125–133.[CrossRef][Web of Science][Medline]
  18. Rummery N.M., Hickey H., McGurk G., Hill C.E. Connexin37 is the major connexin expressed in the media of caudal artery. Arterioscler. Thromb. Vasc. Biol. (2002) 22:1427–1432.[Abstract/Free Full Text]
  19. Wood K.M. The use of phenol as a neurolytic agent: a review. Pain (1978) 5:205–229.[CrossRef][Web of Science][Medline]
  20. Mulvany M.J., Halpern W. Contractile properties of small arterial resistance vessels in spontaneously hypertensive and normotensive rats. Circ. Res. (1977) 41:19–26.[Free Full Text]
  21. Chaytor A.T., Evans W.H., Griffith T.M. Peptides homologous to extracellular loop motifs of connexin43 reversibly abolish rhythmic contractile activity in rabbit arteries. J. Physiol. (1997) 50:99–110.
  22. Reaume A.G., de Sousa P.A., Kulkarni S., et al. Cardiac malformations in neonatal mice lacking connexin43. Science (1995) 267:1831–1834.[Abstract/Free Full Text]
  23. Suadicani S.O., Vink M.J., Spray D.C. Slow intracellular Ca+2 signaling in wild type and Cx43-null neonatal mouse cardiac myocytes. Am. J. Physiol. Heart Circ. Physiol. (2000) 279:H3076–H3088.[Abstract/Free Full Text]
  24. Brosius F.C. 3rd, Liu Y., Nguyen N., Sun D., Bartlett J., Schwaiger M. Persistent myocardial ischemia increases GLUT1 glucose transporter expression in both ischemic and non-ischemic heart regions. J. Mol. Cell. Cardiol. (1997) 29:1675–1685.[CrossRef][Web of Science][Medline]
  25. Arvola P., Porsti I., Vuorinen P., Pekki A., Vapaatalo H. Contractions induced by potassium-free solution and potassium relaxation in vascular smooth muscle of hypertensive and normotensive rats. Br. J. Pharmacol. (1992) 106:157–165.[Web of Science][Medline]
  26. Bohr D.F., Bruner C.A., Lamb F.C., Webb R.C. Physiology of vascular smooth muscle in relation to hypertension. Acta Physiol. Scand. Suppl. (1988) 571:15–24.[Medline]
  27. Haefliger J.A., Castillo E., Waeber G., et al. Hypertension increases connexin43 in a tissue-specific manner. Circulation (1997) 95:1007–1014.[Abstract/Free Full Text]
  28. Dora K.A., Martin P.E.M., Chaytor A.T., Evans W.H., Garland C.J., Griffith T.M. Role of heterocellular gap junctional commnication in endothelium dependent smooth muscle hyperpolarization: inhibition by a connexin mimetic peptide. Biochem. Biophys. Res. Commun. (1999) 254:27–31.[CrossRef][Web of Science][Medline]
  29. Christ G.J., Spray D.C., El-Sabban M., Moore L.K., Brink P.R. Gap junctions in vascular tissues: evaluating the role of intracellular communication in the modulation of vascular tone. Circ. Res. (1996) 79:631–646.[Abstract/Free Full Text]
  30. Christ G.J., Brink P.R., Zhao W., et al. Gap junctions modulate tissue contractility and alpha1 adrenergic agonist efficiency in isolated rat aorta. J. Pharmacol. Exp. Ther. (1993) 266:1054–1065.[Abstract/Free Full Text]
  31. Hogestatt E.D., Hammarstrom L.E., Andersson K.E., Holmin T. Contractile effects of various vasoactive agents in small rat portal veins and hepatic arteries and the influence of sympathetic denervation on the noradrenaline response. Acta Physiol. Scand. (1986) 128:309–315.[Web of Science][Medline]
  32. Colucci W.S., Gimbrone M.A. Jr., Alexander R.W. Regulation of postsynaptic alpha-adrenergic receptor in rat mesentric artery. Effeccts of chemical sympathectomy and epinephrine treatment. Circ. Res. (1981) 48:104–111.[Free Full Text]
  33. Berridge M.J., Galione A. Cytosolic calcium oscillators. FASEB J. (1988) 2:3074–3082.[Abstract]
  34. Iino M., Kasai H., Yamazawa T. Visualization of neural control of intracellular Ca2+ concentration in single vascular smooth muscle cells in situ. EMBO J. (1994) 13:5026–5031.[Web of Science][Medline]
  35. Rottingen J.A., Camerer E., Mathiesen I., Prydz H., Iversen J.G. Synchronized Ca2+ oscillations induced in Madin Darby canine kidney cells by bradykinin and thrombin but not by ATP. Cell Calcium (1997) 21:195–211.[CrossRef][Web of Science][Medline]
  36. Eddinger T.J., Ratz P.H. Alpha-adrenoceptor activation induces rhythmic contractile activity in carotid arteries from young, not adult rats. Acta Physiol. Scand. Suppl. (1997) 159:123–129.
  37. Dorrance A.M., Osborn H.L., Grekin R., Webb R.C. Spironolactone reduces cerebral infarct size and epidermal growth factor receptor mRNA in stroke prone rats. Am. J. Physiol. (2001) 281:R944–R950.[Web of Science]
  38. Webb R.C., Johnson J.C., Bohr D.F. Adrenergic neurotransmission in tail arteries from two-kidney, one clip, renal hypertensive rats. Hypertension (1983) 5:298–306.[Abstract/Free Full Text]
  39. Dimitriadou V., Aubineau P., Taxi J., Seylaz J. Ultrastructural changes in the cerebral artery wall induced by long-term sympathetic denervation. Blood Vessels (1988) 25:122–143.[Web of Science][Medline]
  40. Northcott C.A., Poy M.N., Najjar S.M., Watts S.W. Phosphoinositide 3-kinase mediates enhanced spontaneous and agonist-induced contraction in aorta of deoxycorticosterone acetate-salt hypertensive rats. Circ. Res. (2002) 91:360–369.[Abstract/Free Full Text]

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