Alpha-2 adrenoceptor agonists, in particular clonidine and dexmedetomidine, are reported to improve posthypoxic function of several organs, including heart, central nervous system and kidneys, in animal models and humans.1–5 The mechanisms underlying this effect are not fully understood. The central effects of alpha-2 adrenoceptor agonists as sympatholysis or the modulation of the stress reaction are often proposed to explain the mechanism. But several studies performed on isolated organs confirm the protective effect of alpha-2 adrenoceptor agonists, demonstrating a local action with a direct beneficial effect on the posthypoxic function.
Blood vessels, and especially endothelial cells located at the blood–organ interface, are particularly exposed to hypoxia–reoxygenation injuries. They constitute an interesting target for studying the alpha-2 adrenoceptor agonists' effects in hypoxia–reoxygenation. Posthypoxic endothelial dysfunctions enhance hypoxia–reoxygenation injuries in many organs.6–8 A competent blood vessel motricity during the early posthypoxic period is essential for better organ perfusion and functional recovery.6–8 Consequently, preservation of the endothelial functions and vasomotricity during reperfusion is a key target in the protection of an organ against hypoxia–reoxygenation injuries.
Stimulation of alpha-2 adrenoceptors activates numerous pro-survival intracellular pathways such as phosphatidyl-inositide-3-kinase/Akt pathways, nitrogen oxide synthase or prostaglandin synthesis.9–11 These pathways could improve posthypoxic vasomotricity and contribute to the improvement of the posthypoxic organ's function.
The present study first intends to investigate a direct effect of clonidine on posthypoxic vasomotricity in a rodent model of hypoxia–reoxygenation and to identify subtypes of alpha-2 adrenoceptors implicated in these effects. Second, this study will try to investigate the implication of the nitrogen oxide synthase and arachidonic acid pathway in the clonidine-induced effect.
Animals and aortic ring preparation
All experiments were conducted according to the National and European Community Guidelines for the use of experimental animals. Normal chow and tap water were given ad libitum. After local animal ethics committee approval (UCL MD 2007–018), young male Wistar rats (n = 96, 250–300 g body weight, 10–12 weeks old) were incorporated in the study.
The methodology used for aortic ring preparation and hypoxic challenge was described by other authors.12,13 After intraperitoneal (i.p.) injection of thiopental sodium (50 mg kg−1, Pentothal; Hospira Enterprises, Hoofddorp, The Netherlands) and subcutaneous morphine (10 mg kg−1, Sterop, Brussels, Belgium), the descending thoracic aorta was quickly removed and placed in warmed Krebs–Henseleit buffer (37°C, composition in mmol l−1: NaCl 118, NaHCO3 25, KCl 4.8, KH2PO4 1.2, MgCl2 1.2, CaCl2 2.5 and Glucose 11). Adherent tissues were carefully removed and the vessels were cut into four rings of 3 mm. These vessel rings were mounted onto two stainless steel supports, suspended in a bath filled with Krebs–Henseleit buffer bubbled with 95% O2/5% CO2 to maintain a piO2 between 650 and 700 mmHg and pH 7.4. The filled baths were maintained at 37°C. Rings were attached to a micrometer connected to an isometric force transducer (Power Laboratory 400; AD Instruments, Casterhill, New South Wales, Australia). It was linked to an amplifier and to a computerized acquisition system (Acknowledge Software, MP100WSW; Biopac System Inc., Santa Barbara, California, USA) to allow recordings of the changes in isometric force. Each aortic ring was equilibrated for 60 min under an initial resting tension of 2 g prior to completion of experimental protocol. During this equilibration period, Krebs–Henseleit buffer was changed every 15 min. Preliminary studies in our laboratory showed that a preload of 2 g corresponds to the optimal length for tension development in the aortas from our rat population. After equilibration, test doses of phenylephrine (10−4 mol l−1) and acetylcholine (10−6 mol l−1) were added to the rings to ensure reproducibility of contraction and endothelial integrity. After precontraction reached a plateau (phenylephrine 10−4 mol l−1), endothelium-dependent relaxation was produced with acetylcholine (10−6 mol l−1). In some experiments, endothelium was removed by gently rubbing the luminal surface of the vessels with a piece of stainless steel wire. The endothelium removal was confirmed by the lack of relaxation in response to acetylcholine in precontracted aorta.
The vasomotricity of the aorta participates little in the organ perfusion and in the vascular peripheral resistance. However, alpha-2 adrenoceptors, their expression of different subtypes in the vascular wall and the related intracellular pathways were extensively studied in the rat aorta.9–11,14–16 To investigate the effect of prehypoxic administration of clonidine on the posthypoxic vasomotricity, we relied on these previous works to understand and study its effects. Therefore, we chose segments of rat aorta for this study.
The methodology used was the same in all groups. Two baths were randomly chosen to receive the studied drugs and two were used as control. After 15 min of incubation, all baths were washed three times using warmed Krebs–Henseleit buffer with an interval of 5 min between rinsings. After that, the hypoxia challenge began at once.
In all groups, hypoxia was induced for 20 min by changing the gas mixture to 95% N2/5% CO2 (piO2 in the bath: 36–38 mmHg after 3 min and remained stable during all the periods of hypoxia, pH 7.4). Reoxygenation was performed for 45 min. During this period, each ring was placed under a resting tension of 2 g. The Krebs–Henseleit buffer was changed every 15 min. At the end of reoxygenation, posthypoxic vasomotricity was tested.
Different experiments were conducted in order to identify a clonidine effect, the subtypes of alpha-2 adrenoceptors implicated in this effect and the implication of nitrogen oxide synthase and cyclooxygenase:
- Experiment I: clonidine (10−5 mol l−1) was added to two randomized baths.
- Experiment II: clonidine (10−5 mol l−1) was added to two randomized baths, but endothelium was removed before the experiment was carried out in all groups.
- Experiment III: UK14.304 (10−5 mol l−1), a specific alpha-2 adrenoceptor agonist, was studied in two randomized baths.
- Experiment IV: rauwolscine (10−6 mol l−1), a nonselective alpha-2 adrenoceptor antagonist, was added to all baths 15 min before clonidine (10−5 mol l−1) in the randomized baths to block all alpha-2 adrenoceptor subtypes.
- Experiment V: BRL44408 (10−6 mol l−1), an alpha-2A subtype adrenoceptor antagonist, was added to all baths 15 min before randomization. Clonidine (10−5 mol l−1) was added to two randomized baths.
- Experiment VI: ARC239 (10−6 mol l−1), an alpha-2B/C subtype adrenoceptor antagonist, was added to all baths 15 min before randomization. Clonidine (10−5 mol l−1) was added to two randomized baths.
- Experiment VII: L-NAME (10−5 mol l−1), Nω-nitro-L-arginine methyl ester, a nitrogen oxide synthase inhibitor, was added to all baths 15 min before clonidine was added in the randomized baths.
- Experiment VIII: indometacin (10−5 mol l−1), a cyclooxygenase inhibitor, was added to all baths 15 min before clonidine was added in the randomized baths.
Analysis of posthypoxic vasomotricity
Posthypoxic endothelium-dependent dilatation was investigated on precontracted aortic rings (phenylephrine 10−4 mol l−1) in groups I, III–VIII and X. Posthypoxic endothelium-independent dilatation was tested on precontracted aorta only in group I. After a stable plateau was reached, aortic rings were exposed respectively to cumulative concentrations of acetylcholine (10−10–10−4 mol l−1) or sodium nitroprusside (10−10–10−4 mol l−1). Relaxations were expressed as the percentage of the maximal contraction induced by phenylephrine.17
Posthypoxic blood vessel contractility was assessed in groups I–VI and IX–X. Aortic rings were exposed to cumulative concentrations of phenylephrine (10−10–10−4 mol l−1). Phenylephrine-induced contraction was expressed as the percentage of prehypoxic maximal contraction in response to phenylephrine (10−4 mol l−1).
All concentrations of the drugs used in isolated aortic ring experiments are expressed as the final molar concentration in Krebs–Henseleit solution in organ baths. L-NAME and nitroprusside were purchased from Sigma-Aldrich (Bornem, Belgium). Clonidine, rauwolscine, BRL44408, ARC239, UK14.304, phenylephrine, indometacin and acetylcholine were obtained from Tocris Bioscience (Avonmouth, Bristol, UK).
The percentage of maximal contraction or dilatation is expressed as means ± SDs. The effect of clonidine in each of the 10 experiments was analysed by regression with generalized estimating equations (GEEs).18 This method allows the simultaneous influence of two independent variables to be studied, that is, the presence or absence of clonidine or one of its substitutes (main variable) and the varying concentration of acetylcholine or phenylephrine according to time (covariate), on the measure of contraction or dilatation of aortic rings (dependent variable). Contrary to classical linear regression, GEE regression is valid when the observations are not independent, by taking into account the multiplicity of intercorrelated measurements in each aortic ring. From a technical viewpoint, the chosen GEE models used a normal probability distribution, an identity link function and an exchangeable working correlation matrix structure. They were performed by the Repeated Measures using Generalized Estimating Equations (RMGEE) program.
Effect of prehypoxic clonidine administration on posthypoxic aortic vasomotricity (experiments I and II)
Clonidine enhances posthypoxic endothelium-dependent dilatation (clonidine group: −30.9 ± 5.0% versus control group: −25.4 ± 4.0%, P = 0.002, Fig. 1a). Clonidine has no effect on endothelium-independent dilatation (clonidine group: −59.0 ± 7.2% versus control group: −58.0 ± 12.3%, P = 0.703). Posthypoxic contractility is higher in the clonidine group than in the control group (87.8 ± 29.2 versus 76.5 ± 24.0%, respectively, P = 0.001, Fig. 1b). Without endothelium, the difference observed on posthypoxic contractility disappears (clonidine group: 111.6 ± 27.0% versus control group: 112.4 ± 24.2%, P = 0.687).
Alpha-2 adrenoceptors in the effect of prehypoxic clonidine administration on the posthypoxic aortic vasomotricity (experiments III and IV)
In contrast with clonidine, UK14.304, a specific alpha-2 adrenoceptor agonist, has no effect on the posthypoxic endothelium-dependent dilatation (clonidine group: 33.40 ± 9.1% versus control group: 33.4 ± 8.4%, P = 0.954, Fig. 2a). Nevertheless, as for clonidine, UK14.304 improves posthypoxic contractility in comparison with controls (UK14.304 group: 73.7 ± 18.2% versus control group: 67.0 ± 18.0%, P = 0.005, Fig. 2b). When rauwolscine, an alpha-2 adrenoceptor antagonist, was used, no difference was found concerning posthypoxic endothelium-dependent relaxation (clonidine + rauwolscine: −41.9 ± 8.5% versus control + rauwolscine: −41.7 ± 9.7%, P = 0.803) as well as contractility (clonidine + rauwolscine: 73.8 ± 17.5% versus control + rauwolscine: 70.3 ± 21.5%; P = 0.917).
Alpha-2 adrenoceptor subtypes in the effect of prehypoxic clonidine administration on the posthypoxic aortic vasomotricity (experiments V and VI)
When BRL44408, an alpha-2A adrenoceptor antagonist, is added, clonidine decreases the posthypoxic endothelium-dependent dilatation in comparison with the control group (clonidine + BRL44408 group: −25.6 ± 9.0% versus control group – BRL alone: −32.9 ± 7.1%, P = 0.001, Fig. 3a). BRL44408 prevents the effect of clonidine on the posthypoxic contractility. No difference was found between treated and control groups (54.0 ± 22.4 versus 59.1 ± 29.7%, respectively, P = 0.204, Fig. 3b). ARC239, an alpha-2B/C antagonist, prevents the clonidine effect on the endothelium-dependent dilatation (clonidine + ARC239 group: −28.3 ± 4.2% versus control group – ARC239 alone: −25.7 ± 4.6%, P = 0.236, Fig. 4a) as well as on the posthypoxic contractility (clonidine + ARC239 group: 53.7 ± 18.7% versus control group – ARC239 alone: 61.2 ± 24.5%, P = 0.118, Fig. 4b).
Nitrogen oxide synthase in the effect of prehypoxic clonidine administration on the posthypoxic aortic vasomotricity (experiment VII)
L-NAME decreases acetylcholine-induced dilatation by nitric oxide synthase inhibition. Concerning posthypoxic endothelium-dependent dilatation, no difference was found between clonidine and the control groups (−17.99 ± 7.33% and −17.61 ± 7.70%, respectively, P = 0.993). In contrast, posthypoxic contractility in the clonidine and control groups was significantly different. Posthypoxic contractility was improved by prehypoxic clonidine administration (clonidine group: 90.73 ± 24.31% versus control group: 60.81 ± 30.02%, P = 0.026, Fig. 5).
Cyclooxygenase and its products in the effect of prehypoxic clonidine administration on the posthypoxic aortic vasomotricity (experiment VIII)
In the presence of the cyclooxygenase inhibitor indometacin, clonidine does not induce any difference in the posthypoxic endothelium dilatation between the clonidine baths and control baths (indometacin + clonidine group: −26.94 ± 9.29% versus control group: −28.14 ± 12.00%, P = 0.678). Concerning posthypoxic contractility, no difference was found between the indometacin + clonidine and indometacin alone control groups (75.94 ± 22.39 and 73.00 ± 31.80%, respectively, P = 0.851).
The present study was designed to investigate the specific effect of clonidine on posthypoxic vasomotricity in isolated aorta rings. The methodology and drug concentrations have been validated and used by other authors.12,13,15 Our results show that clonidine improves both posthypoxic blood vessel contractility and posthypoxic endothelium-dependent dilatation. Endothelium-independent dilatation is weakly affected by hypoxia–reoxygenation and is not modified by clonidine.12
During the early posthypoxic period, preservation of a competent blood vessel motricity is essential to maintain perfusion and organ function.6–8 Endothelium plays an essential role in the control of vascular tone by the production of endothelium relaxing and contracting factors. A reduced bioavailability of nitrogen oxide, an alteration in the production of prostanoids (prostacyclin, thromboxane A2), an impairment in endothelium-dependent hyperpolarization and an increased release of endothelin-1 can contribute to endothelial dysfunction and can compromise the organ's vascularization.19
Effect of prehypoxic clonidine administration in posthypoxic blood vessel contractility
We have observed that rauwolscine, endothelium removal and specific alpha-2A or alpha-2B/C-adrenoceptor antagonists suppress the clonidine-induced improvement of posthypoxic contractility. Alpha-2B and alpha-2C adrenoceptors cannot be studied separately regarding the lack of specificity of the antagonists available. UK14.304 reproduces the clonidine effect. It suggests that clonidine maintains posthypoxic blood vessel contractility by at least two endothelial subtypes of alpha-2 adrenoceptors: alpha-2A and alpha-2B and/or alpha-2C subtypes. Even if the presence of alpha-2 adrenoceptor is confirmed on the vascular smooth muscular cells of rat, endothelium removal abolishes the clonidine effect. It suggests the presence of diffusing substances produced by endothelium and acting on vascular smooth muscles. It probably acts before hypoxia, which influences the posthypoxic contractility. Several molecules have been suggested, among them nitrogen oxide or prostanoids. The nitrogen oxide synthase activity is enhanced by alpha-2A adrenoceptor stimulation in rat mesenteric arteries and aorta.9,14,16 We show that blocking this subtype of alpha-2 adrenoceptor abolishes the clonidine effect on posthypoxic contractility. However, the inhibition of nitrogen oxide synthase by L-NAME does not abolish the clonidine effect, suggesting that nitrogen oxide is not involved in the clonidine-induced improvement of the posthypoxic blood vessel contractility. Alpha-2 adrenoceptor activation modulates the vascular tone by the production of prostanoids.10,20,21 We administered indometacin, a cyclooxygenase inhibitor, and showed that the clonidine effect on posthypoxic blood vessel contractility is prevented. It suggests that metabolites of the arachidonic acid support contractile mechanism activity, even if these have opposite effects during hypoxia/reoxygenation. The balance tends to increase protection. Thromboxane A2 increases hypoxia/reoxygenation injuries, whereas other prostaglandins, especially E2 and I2 (prostacyclin), decrease them. Prostaglandin E2 and prostaglandin I2 decrease the production of free radicals during reperfusion, preventing leucocyte migration, regulating the production of inflammatory cytokines and cell adhesion molecules.22,23 Nebigil and Malik21 showed that the alpha-2C adrenoceptor subtype stimulation of vascular smooth muscles of rabbit aorta led to prostaglandin I2 production. This inhibition by indometacin could abolish the pro-survival effect of prostaglandin I2 and affect the posthypoxic blood vessel contractility.
Effect of prehypoxic clonidine administration in posthypoxic endothelium-dependent dilatation
Vascular tone is a continual balance between contracting and relaxing factors. Endothelium-dependent relaxing factors play an essential role. In our study, clonidine improves posthypoxic endothelium-dependent dilatation. Alpha-2 adrenoceptor stimulation seems to be inescapable because the clonidine effect disappears either if alpha-2 adrenoceptors are blocked by rauwolscine or if the alpha-2B or alpha-2C adrenoceptor is blocked by ARC239. Moreover, the effect of clonidine stimulation is reduced when the alpha-2A adrenoceptor is blocked by BRL 44408 compared with the control group. The fact that the alpha-2B adrenoceptor subtype is positively coupled to the L-type Ca2+ channels can explain the dramatic effect of clonidine when the alpha-2A adrenoceptor is blocked.24 It allows intracellular calcium overload during hypoxia/reoxygenation and increases related cellular injuries. Contrary to previous results, when alpha-2A adrenoceptors are free, clonidine can activate the pro-survival metabolic pathways in the same way as nitrogen oxide synthase or prostanoids. Nitric oxide and prostaglandins seem essential in the induction of the clonidine effect. This is shown by the selective inhibition of nitrogen oxide synthase by L-NAME or prostaglandin production by indometacin. Each one prevents it. As previously described, the alpha-2C adrenoceptor is coupled with a production of prostaglandin I2, which has a pro-survival effect during hypoxia/reoxygenation.21 Taken together, these results suggest a balance between pro-survival effects of the alpha-2A and alpha-2C adrenoceptor stimulations and negative effects of the alpha-2B adrenoceptor. Surprisingly, the stimulation by a specific alpha-2 adrenoceptor agonist such as UK14.304 does not mimic the clonidine effect. Previous studies show that clonidine and UK14.304 have a similar order of potency on the alpha-2 adrenoceptor.15 Clonidine possesses alpha-1 adrenoceptor and imidazoline agonist affinities.25 The affinity of the clonidine for the other receptors could be necessary to improve posthypoxic endothelium-dependent dilatation.
In this study, we have not specifically investigated the contribution of endothelium-derived hyperpolarizing factor (EDHF) in the prehypoxic administration of clonidine on posthypoxic vasomotricity. EDHF is present in rat aorta, but, although alpha-2 adrenoceptors lead to the production of EDHF in rat mesenteric artery, no study has demonstrated it in rat aorta.26,27 The basic mechanism of the EDHF-mediated response is that increased intracellular calcium in endothelium activates the SKCa and IKCa channels causing K+-efflux and membrane hyperpolarization. The opening of K+Ca channels, leading to a reduction in intracellular Ca2+, is probably involved.28 Hyperpolarization of endothelium is transferred to vascular smooth muscle through synthesis or generation of signals capable of diffusing through membranes or myoendothelial gap junctions. In vascular smooth muscle, EDHF activates K+-channels, causing hyperpolarization.28 Hyperpolarization decreases hypoxia/reoxygenation-induced intracellular calcium overload.29,30 Calcium overload during hypoxia and reoxygenation exerts deleterious effects in endothelial and smooth muscle cells. A greater tolerance of the vascular wall towards hypoxia could explain not only the best acetylcholine-induced vasodilatation observed in clonidine groups but also the improved phenylephrine-induced contraction. Further studies are needed to investigate the potential role of EDHF in the clonidine-induced effect on posthypoxic vasomotricity.
This study shows several limitations. First, the aorta is a large vessel that participates little in vascular peripheral resistance and in the distribution of blood flow. However, it is sensitive to hypoxia/reoxygenation injuries. These injuries are not limited only to an effect on vasomotricity, but induce an important inflammatory reaction, which can affect the organs perfused.31 The modulation of aortic inflammation induced by hypoxia/reoxygenation can have a positive effect on them. No study has so far evaluated the potential of clonidine administration to preserve endothelial function and vasomotricity in the aorta. Second, the alpha2 adrenoceptors are widely distributed in the vascular tissues. However, their distribution varies between vascular beds, between species and between large and small vessels. The alpha-2A adrenoceptor appears to be expressed at a higher concentration in large arteries, whereas the alpha-2B adrenoceptors contribute more to vascular tone in small arteries and veins. The heterogeneity of the expression of the alpha-2 adrenoceptors on the surface of vascular smooth muscle and endothelial cells between various levels of vascularization indicates that the clonidine effect on posthypoxic vasomotricity could be different on smaller vessels from that on resistance level vessels. Further studies on these vessels are needed. Third, isolated blood vessel studies provide useful mechanistic information. In the intact animal, clonidine interacts with the sympathetic nervous system. It decreases sympathetic output and the release of catecholamine through central mechanisms. A lower rate of circulating catecholamine limits the in-vivo effect of the improvement of posthypoxic phenylephrine-induced contraction and promotes vasodilatation. Therefore, the overall effect of clonidine on the response to ischemia–reperfusion probably is much more complex than examined in this study.
In summary, our study shows that prehypoxic clonidine administration supports both posthypoxic endothelium-dependent dilatation and contractility in young rat aorta. These effects involved alpha-2A and alpha-2B and/or alpha-2C subtype adrenoceptors. Our results suggest that multiple interactive pathways such as nitrogen oxide synthase and prostaglandin synthesis are necessary. Concerning posthypoxic endothelium-dependent dilatation, it seems that other receptors such as the alpha-1 adrenoceptor or imidazoline receptors are also necessary.
The authors declare that they have no conflicts of interest.
This work was supported by institutional and departmental funds only.
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