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CARDIOVASCULAR
2A-Adrenoceptors Located on the Endothelium
Autonomic Physiology Unit, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland (M.M., C.D., A.W., J.M., C.J.D., J.C.M.); and Sari Medical Faculty, Mazandaran, Iran (M.M.S.)
Received March 7, 2005; accepted May 3, 2005.
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Abstract |
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2-adrenoceptors. In the presence of induced tone, UK-14,304 produced concentration-related vasodilatation that was abolished by rauwolscine, N
-nitro-L-arginine methyl ester (L-NAME), or endothelium removal, indicating that endothelial 2-adrenoceptors can release nitric oxide. In the 2A-adrenoceptor knockout mouse and the D79N mouse, a functional knockout of the 2A-adrenoceptor, these relaxant effects of UK-14,304 were lost, indicating the involvement of the 2A-adrenoceptor. UK-14,304 could also contract aorta: a small contraction occurred at high concentrations, was enhanced by L-NAME, and was absent in the 1D-adrenoceptor knockout mouse, indicating activation of the 1D-adrenoceptor. There was no evidence for a contractile 2-adrenoceptor-mediated response. A fluorescent ligand, quinazoline piperazine bodipy, antagonized the relaxant action of UK-14,304. This compound could be visualized on aortic endothelial cells, and its binding could be prevented by rauwolscine, providing direct evidence for the presence of 2-adrenoceptors on the endothelium. Norepinephrine reduced tone in the 1D-adrenoceptor knockout and controls, an effect blocked by rauwolscine and L-NAME but not by prazosin. This suggests that norepinephrine activates endothelial 2-adrenoceptors. In conclusion, the endothelium of mouse aorta has an 2A-adrenoceptor that responds to norepinephrine; promotes the release of nitric oxide, causing smooth muscle relaxation; and that can be directly visualized. Knockout or genetic malfunction of this receptor should increase arterial stiffness, exacerbated by raised catecholamines, and contribute to heart failure.
2-adrenoceptors have distinct, yet poorly defined, roles in the control of the vascular system. The limited selectivity of agonists and antagonists has therefore prompted the use of transgenic mouse models. The subtypes are 2A, 2B, and 2C: the mouse ortholog of the human 2A-adrenoceptor is sometimes called the 2D- or 2A/D-adrenoceptor; we use the generic term 2A-adrenoceptor (Alexander et al., 2004
). They have two direct pharmacological effects on blood vessels that can modify vascular tone: a direct vasopressor action (for review, see Wilson et al., 1991; Guimaraes and Moura, 2001) and vasodilatation via endothelium-derived relaxant factors (Cocks and Angus, 1983; Vanhoutte, 2001). They also reduce sympathetic traffic centrally and inhibit transmitter release from sympathetic postganglionic nerves (Starke, 2001), although this is not well established as a physiological phenomenon in blood vessels. Available pharmacological data and knockout studies, although not definitive, present evidence for, at least, 2A-, 2B-, and 2C-adrenoceptors for vasoconstriction, 2A- and 2C-adrenoceptors for sympatho-inhibition, and 2A-adrenoceptors for endothelial vasodilatation, although none of the relevant studies has considered all three actions, and each chooses a different example preparation (Bockman et al., 1996; Link et al., 1996; Hein et al., 1999; McCafferty et al., 1999; Chotani et al., 2000; Guimaraes and Moura, 2001; Vanhoutte, 2001).
Thus, there is no consensus for the 2-adrenoceptor subtypes responsible for direct vascular actions, constrictor or dilator. Yet, both phenomena are potentially significant for the therapeutic use of 2-adrenoceptor agonists and antagonists and for the physiological and pathophysiological roles of 2-adrenoceptors in the cardiovascular system.
A pathophysiological role for 2-adrenoceptors has recently emerged from the demonstration that polymorphisms of 2A- and 2C-adrenoceptors are linked with cardiovascular disease (Brede et al., 2002; Small et al., 2002). Hypotheses for the etiology have focused almost exclusively on the concept that malfunction of 2-adrenoceptors regulating the release of neurotransmitters from sympathetic nerves could be deleterious to the cardiovascular system. However, this could just as rationally be attributable to endothelial 2-adrenoceptors. The first specific clinical implication is that synergistic polymorphisms of 
1- and 2C-adrenoceptors, a combination over-represented in the American population of African descent, can increase the risk of congestive heart failure (Small et al., 2002, 2004). Since interpretation of this etiology involves the use of genetically modified mice (Brede et al., 2002; Liggett, 2004), it is timely to elucidate the mechanisms underlying vascular 2-adrenoceptors using this species to establish the physiological and hence potential pathophysiological roles of the different 2-adrenoceptor subtypes and to determine whether endothelial 2-adrenoceptors need to be considered.
There is also controversy surrounding whether the initial step in the release of endothelial relaxant factors is direct activation of receptors on the endothelial cells or indirectly through activation of receptors on smooth muscle cells that then signal to the endothelium via myo-endothelial connections (Dora, 2001). This is compelling since it is consistent with previous vascular localization of receptors by autoradiography, which indicated 2-adrenoceptors in the medial layer but not on endothelium (Stephenson and Summers, 1987).
Analysis of 2-adrenoceptors in the aorta is complicated by the presence of a powerful 1-adrenoceptor-mediated contraction, even when using relatively selective agonists. Vandeputte et al. (2003) showed that, in mouse aorta, the complex response to norepinephrine contains constrictor 1D- and dilator 2A-adrenoceptor components acting in opposition. This factor was accounted for by isolating the 2-adrenoceptor-mediated response in a strain of mice in which the dominant contractile adrenoceptor in this vessel, 1D-adrenoceptor (Daly et al., 2002), was knocked out (Tanoue et al., 2002). "2-Adrenoceptor-selective" agonists are often partial agonists at 1-adrenoceptors (Docherty and McGrath, 1980; Wilson et al., 1991).
The objectives of the present study were to establish the vasodilator phenotype for 2-adrenoceptors in mouse large arteries; identify by two independent transgenic models whether the 2A-adrenoceptor subtype is involved; establish the functional involvement of the endothelium and nitric oxide; determine whether the receptors are located on the endothelium; and demonstrate that the phenomenon is activated by a physiological agonist, norepinephrine.
We demonstrate that 2A-adrenoceptors can account for the entire 2-adrenoceptor-mediated vasodilator response in mouse aorta. A combination of pharmacology, transgenic models, and fluorescent ligand binding shows that the site is on the endothelial cells and that the mechanism involves the 2A-adrenoceptor subtype, which activates the release of endothelial nitric oxide.
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Materials and Methods |
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2-adrenoceptor-mediated vasodilatation (Russell and Watts, 2000) but used strips, which are susceptible to endothelial damage (Furchgott and Zawadski, 1980).
Receptor "knockout" strains were used to simplify pharmacological interpretation where nonselectivity of drugs limits analysis. These were the 2A-adrenoceptor knockout mouse (Altman et al., 1999) and mice harboring the D79N point mutation of the 2A-adrenoceptor, which serves as a "functional knockout" in some systems due to low expression of the mutant receptor and dysfunction due to disengagement of G protein-coupling to potassium but not calcium currents (Surprenant et al., 1992; Ceresa and Limbird, 1994; MacMillan et al., 1996, 1998).
Aortic contraction, by agonists, is a confounding factor. We eliminated this using the 1D-adrenoceptor knockout (Tanoue et al., 2002).
Finally, we set out to make a direct visual demonstration of endothelial 2-adrenoceptors using a fluorescent ligand. Proof of specificity of binding was complicated by the unexpected demonstration of endothelial 1-adrenoceptors: this was overcome by using the knockout of the 1B-adrenoceptor (Cavalli et al., 1997) and selective antagonists of the 1A-adrenoceptor and 1D-adrenoceptor subtypes. Concentrations of these drugs were selected, from pharmacological analysis of 1A- and 1D-adrenoceptor-mediated responses in these same vessels, to be clearly (approximately 10-fold) above the affinity constant for the desired receptor but below that for the others (Daly et al., 2002). We showed that 0.1 µM QAPB antagonized the 2-adrenoceptor-mediated relaxation to UK-14,304 before using this concentration to visualize 2-adrenoceptors on the vascular endothelium.
Myography
Male mice (4 months old) were killed by CO2 inhalation. Descending thoracic aortae were isolated and set up in Krebs' on wire myographs with 5-ml baths to which drugs were added directly, as described previously (Daly et al., 2002). Strains were 2A-adrenoceptor mutant D79N mouse (MacMillan et al., 1996, 1998), backcrossed onto C57/BL6 (gift from Professor Lee Limbird, Vanderbilt University, Nashville, TN); 2A-adrenoceptor knockout C57/BL6 (2A/D-knockout; The Jackson Laboratory, Bar Harbor, ME) (gift from Professor J. R. Docherty, Royal College of Surgeons in Ireland, Dublin, Ireland); 1D-adrenoceptor knockout (Tanoue et al., 2002), background of 129sv/C57/BL6 (gift from Professor Gozoh Tsujimoto, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan); and 1B-adrenoceptor knockout (Cavalli et al., 1997) background of 129sv/C57/BL6 (gift from Professor Susanna Cotecchia, Université de Lausanne, Lausanne, Switzerland). We compared several aspects of adrenergic pharmacology between 129sv/C57/BL6 controls and the C57/BL6 without finding significant differences. Thus, in this study we used the 129sv/C57/BL6 as control. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and with the provisions of the UK Animals (Scientific Procedures) Act 1986.
Endothelium was removed, where appropriate, by rubbing the intimal surface with a roughened metal probe. Reproducible responses were obtained to 0.1 µM norepinephrine, 0.1 µM phenylephrine, or 10 nM U-46619, according to protocol, before commencing experiments.
At the plateau of contraction to 0.1 µM phenylephrine or 0.1 µM norepinephrine, 0.1 µM acetylcholine was added to assess endothelial integrity. Criteria for functional and dysfunctional endothelium were, respectively, >50 and <5% relaxation at start and finish of experiment. N-Nitro-L-arginine methyl ester (L-NAME) (0.1 mM) abolished relaxation to acetylcholine.
Tissues were tested with increasing cumulative concentrations of UK-14,304 in 0.5 log unit increments from 1 nM to 30 µM. After a 60-min rest period, test drugs were added for at least 30 min before construction of a second cumulative concentration-response curve.
Visualization of Endothelial 2-Adrenoceptors
We used the fluorescent ligand QAPB, an analog of prazosin with high affinity for 1-adrenoceptors (pKi = 8.1–8.9) (McGrath et al., 1996; Daly et al., 1998; Mackenzie et al., 2000) but that also has moderate affinity for 2-adrenoceptors (pKi = 7.3–7.8; Dr. C. M. Milligan, personal communication; method of Brown et al., 1993). Binding to the three 1-adrenoceptors was eliminated by using vessels from the 1B-adrenoceptor-knockout mouse and blocking the other two subtypes with selective antagonists: for 1A-adrenoceptors, 5-methylurapidil, (5MeU); and for 1D-adrenoceptors, BMY7378. This allowed us to visualize putative 2-adrenoceptors. We then confirmed that they were 2-adrenoceptors by preventing this binding with rauwolscine.
Laser Scanning Confocal Microscopy
Tissue Preparation. Segments (2–3 mm) of aorta from 1B-knockout mice were incubated for 30 min in 0.1 µM BMY7378 and 0.1 µM 5MeU, with or without 0.1 µM rauwolscine, and then 0.1 µM QAPB was added for 60 min. After incubation, without washing, aortic segments were cut open and placed endothelial side up in the sample well of a slide sealed with a glass coverslip (thickness no. 1.5).
Image Capture. Serial optical sections were collected on a Bio-Rad 1024 and Radiance 2100 confocal laser scanning microscope. Excitation/emission 488/515 nm for QAPB. Laser power, gain, and offset (contrast and brightness) were kept constant. Tissues were visualized using a 40x oil immersion objective numerical aperture 1.00 and therefore optimal pinhole setting 1.5. Image size 512 x 512 pixels equates to a field size of 289 x 289 µm. Each procedure was carried out in triplicate on at least three different mice.
Drugs
All drugs were of analytical grade and were dissolved either in distilled water, ethanol, or DMSO. Phenylephrine (H2O), norepinephrine hydrochloride (23 µM EDTA), acetylcholine chloride (H2O), BMY7378 (H2O), propranolol (H2O), 5-methyl-urapidil (H2O), U-46619 (ethanol), L-NAME (H2O), rauwolscine (H2O) were obtained from Sigma-Aldrich (Poole, Dorset, UK), quinazoline piperazine bodipy (DMSO) was obtained from Molecular Probes (Eugene, OR), and UK-14,304 (DMSO) was obtained from Pfizer Central Research (Sandwich, UK).
Statistics
Values are means ± standard error of the mean from n experiments. Differences in maximal contraction response to agonist in the presence and absence of drugs were compared using one-way analysis of variance or by Student's t test. Statistical and graphical analysis was carried out using Excel 97 (Microsoft, Redmond, WA) and GraphPad Prism 3.00 (GraphPad Software Inc., San Diego, CA).
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2-Adrenoceptor-Mediated Relaxation. Rings were preconstricted with a concentration of U-46619 required to produce 
75% of the maximum contraction. At the preconstriction plateau, 1 µM UK-14,304 was added, causing a marked rapid fall in tone (vasodilatation; Fig. 1a). UK-14,304-induced relaxations were blocked in the presence of rauwolscine (Fig. 1b) but not prazosin (Fig. 1c).
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2-adrenoceptor agonist UK-14,304 versus U-46619 preconstriction had two phases (Fig. 2a). UK-14,304 produced vasodilator responses at low concentrations (1 nM–0.3 µM) and vasoconstriction at higher (>0.3 µM) concentrations. In aorta taken from either 2A-knockout or D79N mice, the vasodilator response was not present (Fig. 2a).
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2-adrenoceptors on the endothelium. In aorta, from 1D-knockout mice, the vasoconstrictor effect of high concentrations of UK-14,304 was absent (Fig. 2b). In a separate series of experiments, a single concentration of 1 µM UK-14,304 was tested against 1 to 10 nM U-46619 in the presence and absence of the nitric oxide synthase inhibitor L-NAME (0.1 mM). In control tissues, UK-14,304 produced a reduction in tone (7.4 ± 4.7%; n = 7). In the presence of L-NAME, UK-14,304 produced an increase in tone (44.2 ± 0.7%; n = 7).
Localization of 2-Adrenoceptors in Aortic Endothelial Cells. QAPB (0.1 µM), a fluorescent -adrenoceptor ligand, inhibited UK-14,304-induced relaxation of mouse aorta (Fig. 3a; p < 0.001). To visualize QAPB binding to 2-adrenoceptor sites, the three 1-adrenoceptor subtypes were first eliminated by using the 1B-adrenoceptor-knockout mouse and incubating vessels in the 1D-adrenoceptor antagonist BMY7378 (0.1 µM) and the 1A-adrenoceptor antagonist 5MeU (0.1 µM). Confocal fluorescence microscopy revealed 0.1 µM QAPB binding to endothelial cells, which line the grooves of the (unpressurized) internal elastic lamina (Fig. 3, b and c). In the presence of 0.1 µM rauwolscine, no QAPB binding could be detected (Fig. 3d).
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-blockade (0.1 µM propranolol) alone or -blockade and 1-blockade (0.1 µM prazosin) to determine the physiological significance of the 2-adrenoceptor-mediated vasodilatation. In the presence of preconstrictor U-46619, -blockade and in the absence of the 1D-adrenoceptors, 1 µM norepinephrine produced a marked vasodilatation (Fig. 4a). Norepinephrine's vasodilator effect was blocked in the presence of 0.1 mM L-NAME (Fig. 4b). In control mice, after -blockade and 1-blockade, 1 µM norepinephrine caused a relaxation of U-46619-induced tone (Fig. 4c). In aorta taken from D79N mice, the vasodilator action of norepinephrine was also absent (Fig. 4d). In 1D-knockout aorta, after -blockade, 0.1 µM rauwolscine significantly inhibited the norepinephrine-induced relaxation (Fig. 4e; p < 0.0001, unpaired t test).
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2-adrenoceptors in the mouse that involves nitric oxide release of endothelial origin. The 2A-adrenoceptor subtype alone is responsible with no evidence for a residual response by another subtype after its elimination. Having established the properties of the phenomenon using the selective synthetic agonist UK-14,304, we demonstrated that the physiological ligand for the receptors, norepinephrine, was highly effective once its other actions had been eliminated. Since conducting arteries are not (or are sparsely) innervated, but the endothelial cells are in intimate contact with the plasma, we propose that circulating catecholamines inhibit tone (stiffness) of these vessels via endothelial 2A-adrenoceptors and that dysfunction of this action would exacerbate vasoconstrictor factors, causing an increased load on the heart.
The aortic phenotype of the vasodilator 2-adrenoceptor contrasts with the prejunctional 2-adrenoceptor in that only one subtype, the 2A-adrenoceptor, was responsible, whereas both 2A- and 2C-adrenoceptors were implicated in the prejunctional modulation of norepinephrine release, and both had to be knocked out to eliminate that response (Hein et al., 1999). There was no evidence of compensatory up-regulation of another 2-adrenoceptor subtype.
The ability to focus on the 2A-adrenoceptor-activated, endothelium/NO-mediated vasodilator response in the mouse and other model species should accelerate appreciation of its role in humans. Endothelial 2-adrenoceptors have not been reported in humans, but we can find no evidence of this having been pursued.
The endothelial 2-adrenoceptors should be considered as possible physiological players since they are activated by norepinephrine. The correct function of 2A-adrenoceptors may be essential to regulation of blood flow in critical vascular beds, in the face of a generalized sympathetic activation in "fight or flight", e.g., nitric oxide released by 2-adrenoceptors protects the kidney from excessive adrenergic vasoconstriction (Zou and Cowley, 2000). Norepinephrine and epinephrine both circulate freely in the plasma and have intimate contact with endothelial cells. In our demonstration that natural ligands can activate endothelial 2-adrenoceptors, we used norepinephrine rather than epinephrine to avoid activating vasodilatory -adrenergic receptors. All evidence points to similar potency of these two catecholamines at 2-adrenoceptors.
Pharmacological analysis of vasodilator responses via 2-adrenoceptors was simplified by the use of selective agonists and receptor knockouts. Aorta had relaxation as its most sensitive response to UK-14,304, and this was susceptible to endothelial removal or inhibition of NOS. This represents the conducting artery vasodilator phenotype of the 2A-adrenoceptor since it was absent in the knockout and the D79N mutation of this receptor. The loss of the response in both of these strains strengthens the case. It produces definitive evidence that an endothelial 2-adrenoceptor response is mediated via the 2A-adrenoceptor and validates the pharmacological analysis in large arteries of the pig (Bockman et al., 1996) of an endothelial 2A-adrenoceptor subtype.
The effect of UK-14,304 in the 1D-adrenoceptor-knockout mouse was interesting because, by eliminating the contractile response, the entire concentration response curve for vasodilatation was isolated.
We visualized the fluorescent ligand QAPB binding to aortic endothelial cells and eliminated this binding with the archetypal 2-adrenoceptor antagonist rauwolscine. We validated this by showing that this fluorescent ligand is a functional antagonist of aortic relaxation to UK-14,304 at the concentration used for visualization. This provides compelling direct evidence for the endothelial location of the 2-adrenoceptors that mediate vasodilatation. This direct proof of 2-adrenoceptor binding sites on endothelial cells suggests that earlier autoradiography, which indicated no endothelial binding of tritiated rauwolscine (Stephenson and Summers, 1987), provided a false negative result due to the small volume of endothelial tissue to which radioligand can bind, coupled with the low receptor expression level on endothelium relative to a high level in the arterial media. We now show that 2-adrenoceptors are located on the endothelium.
Demonstrating that a natural ligand, norepinephrine, could activate the endothelial 2-adrenoceptors was straightforward once the confounding factors had been clarified using the selective agonist, antagonists, and knockout strains. Norepinephrine produces, in vitro, a powerful contractile response that overwhelms its vasodilator actions. Its relaxant endothelial 2-adrenoceptor-mediated effect could be seen clearly in the 1D-adrenoceptor-knockout and was shown to be rauwolscine sensitive. Demonstrating that the receptor involved is the 2A-adrenoceptor is more difficult since the 1D-adrenoceptor is present in the 2A-adrenoceptor knockout strains. However, comparing the normal mouse with the D79N in the presence of prazosin and propranolol, it was possible to show the complete absence of a dilator response in D79N in contrast to a relaxant or multiphasic response in normal mice. Together with the other evidence this strongly supports that norepinephrine activates vasodilator 2A-adrenoceptors.
A peripheral endothelium/nitric oxide-mediated direct vasodilatation to 2-adrenoceptor agonists must now be considered as a potential depressor mechanism to intravenous 2-adrenoceptor agonists in addition to any centrally mediated sympatho-inhibitory effects or prejunctional inhibition of postganglionic sympathetic transmission. We suggest that emphasis on the latter action should be reconsidered. Deletion or mutation of the 2A-adrenoceptors eliminates the reduction in heart rate and blood pressure caused by intravenous 2-adrenoceptor agonists such as UK-14,304 and clonidine in the conscious mouse (MacMillan et al., 1996; Altman et al., 1999). This has been assumed to arise entirely from withdrawal of sympathetic tone, although no evidence for a change in sympathetic tone is available. There is also no evidence for prejunctional 2-adrenoceptors on mouse vascular sympathetic nerves. In the pithed rat, the vascular response to sympathetic nerve stimulation was the least sensitive of several organ systems to inhibition by clonidine (Docherty and McGrath, 1980). Thus, if the mouse is like the rat, inhibition of norepinephrine release from nerve endings in blood vessels may not be a major action of 2-adrenoceptor agonists. In conducting arteries, vasodilatation via endothelial 2-adrenoceptors would reduce blood pressure via a reduced after-load. Thus, there may be an endothelial component to the vasodepressor action of 2-adrenoceptor activation, and this may combine with any sympatho-inhibition that exists.
Large artery endothelial 2-adrenoceptors should be relevant to the hypothesis that 2A-adrenoceptors confer protection from heart failure (Brede et al., 2002). Excess mortality in the 2A-adrenoceptor-knockout strain was attributed to heart failure due to a combination of enhanced left ventricular hypertrophy, fibrosis, and elevated circulating catecholamines (Brede et al., 2002). The present work predicts that in the 2A-adrenoceptor-knockout the loss of large artery vasodilatation via 2A-adrenoceptors combined with unopposed vasoconstriction to elevated catecholamines would create a highly deleterious situation of reduced arterial compliance and consequent increased cardiac work. Lack of innervation makes it unlikely that sympatho-inhibition has a role and likely that the endothelial 2A-adrenoceptor effect will be responsible.
The 2C-adrenoceptor-knockout mouse is susceptible to a similar pathology (Brede et al., 2002). Combined with the link between the polymorphisms of this receptor and susceptibility to heart failure (Small et al., 2002), this has focused attention on this receptor. The present work indicates that 2C-adrenoceptors are not involved in mouse large artery endothelial vasodilatation and the lack of innervation excludes sympatho-inhibition, so there is no evidence for a vasodilator action by this subtype in large arteries. However, 2C-adrenoceptors could be involved in innervated small arteries that regulate blood pressure through the peripheral resistance, either through endothelial or nerve mechanisms. Thus, heart failure may be exacerbated by different mechanisms according to which 2-adrenoceptor is "abnormal".
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: QAPB, quinazoline piperazine bodipy; UK-14,304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; U-46619, 9,11-dideoxy-9,11-methanoepoxy-prostaglandin F2; L-NAME, N-nitro-L-arginine methyl ester; 5MeU, 5-methylurapidil; BMY7378, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione; DMSO, dimethyl sulfoxide.
1 These authors contributed equally to this work. 
Address correspondence to: Professor J. C. McGrath, Autonomic Physiology Unit, Institute of Biomedical and Life Sciences, West Medical Bldg., University of Glasgow, Glasgow G12 8QQ, UK. E-mail: i.mcgrath{at}bio.gla.ac.uk
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