Role of endothelium/nitric oxide in atypical β-adrenoceptor-mediated relaxation in rat isolated aorta
Introduction
Stimulation of β-adrenoceptors in smooth muscle is widely held to involve activation of adenylyl cyclase with a subsequent increase in intracellular cAMP concentration (Kukovetz et al., 1981). Although β-adrenoceptor-mediated vasodilatation has been regarded as endothelium-independent in accordance with this mechanism (Furchgott and Vanhoutte, 1989), there are conflicting reports regarding the role of endothelium. For example, in support of an endothelium-independent effect of isoprenaline, removal of endothelium was found to have to have no inhibitory effect on isoprenaline-induced relaxation in rat aorta Konishi and Su, 1983, Moncada et al., 1991, Satake et al., 1996, canine coronary arteries Cohen et al., 1983, Cohen et al., 1984, White et al., 1986, Macdonald et al., 1987, rat carotid artery (Oriowo, 1994) or human internal mammary artery (Molenaar et al., 1988). In contrast, other studies found that removal of endothelium reduced the relaxations induced by isoprenaline in rat thoracic aorta Grace et al., 1988, Kamata et al., 1989, Dainty et al., 1990, Gray and Marshall, 1992, Delpy et al., 1996, Toyoshima et al., 1998, Trochu et al., 1999, canine coronary arteries (Rubanyi and Vanhoutte, 1985) and human umbilical vein (Ferro et al., 1999). Additional evidence supporting a role for endothelium came from studies showing that relaxations to isoprenaline in rat aorta were inhibited by methylene blue and by haemoglobin (Grace et al., 1988), previously shown to block endothelium-dependent relaxation (Martin et al., 1985). Nitric oxide (NO) synthase inhibitors have also been shown to inhibit isoprenaline-induced relaxations in endothelium-intact rat aorta Gray and Marshall, 1992, Delpy et al., 1996, Toyoshima et al., 1998, Trochu et al., 1999, rat mesenteric arteries (Graves and Poston, 1993), rat pulmonary arteries (Priest et al., 1997), rat carotid artery (MacDonald et al., 1999) and human umbilical vein (Ferro et al., 1999).
It seems therefore that the presence of endothelium can, in some situations at least, affect the response to isoprenaline. β-adrenoceptors have been identified in cultured endothelial cells by radioligand binding (Steinberg et al., 1984) and the presence of endothelial β-adrenoceptors in some blood vessels has also been shown by autoradiography Stephenson and Summers, 1987, Molenaar et al., 1988 raising the possibility that endothelial β-adrenoceptors may release NO. However, Grace et al. (1988) found that, in aortic rings with or without endothelium, isoprenaline elevated cAMP levels but had no effect on cGMP levels. They concluded that isoprenaline did not release NO, but that the presence of endothelium amplified the response to isoprenaline via a synergistic interaction between cAMP and cGMP. This conclusion is supported by Delpy et al. (1996) who suggested that cGMP may potentiate cAMP-dependent relaxation by inhibition of cGMP-inhibited phosphodiesterase (phosphodiesterase 3). In contrast, Gray and Marshall (1992) and others (Iranami et al., 1996) found that isoprenaline increased both cAMP and cGMP levels in endothelium-intact aortic rings and that removal of endothelium abolished these increases. Gray and Marshall (1992) proposed that isoprenaline produces relaxation by stimulating endothelial β-adrenoceptors to raise cAMP and thus release NO, which then evokes relaxation via an increase in smooth muscle cGMP. Direct support for an ability of β2-adrenoceptors to activate NO synthase via cAMP elevation in human umbilical vein endothelial cells has recently been provided (Ferro et al., 1999).
Thus, endothelium may have a role in modulation of β-adrenoceptor-mediated vasorelaxation although the mechanism is unclear. Vascular β-adrenoceptors were originally classified as β2- (Lands et al., 1967), and this appears to be the predominant type in rat aorta although β1-adrenoceptors are also present O'Donnell and Wanstall, 1984, Brawley et al., 2000a. In addition, recent studies have provided evidence for the presence of atypical β-adrenoceptors, not conforming to the β1-/β2-classification, in rat aorta Oriowo, 1995, Sooch and Marshall, 1997, Brawley et al., 2000a. The nature of the atypical β-adrenoceptor is not fully understood, although our previous results suggest that the atypical β-adrenoceptor in rat aorta does not correspond to the β3-adrenoceptor, but is similar to the putative β4-adrenoceptor (Brawley et al., 2000a). The present study was carried out to compare the role of endothelium in modulating classical and atypical β-adrenoceptor-mediated effects. Preliminary accounts of some of these results have been presented in abstract form Brawley et al., 1997, Brawley et al., 1998.
Section snippets
Tissue preparation
Male Wistar rats (150–300 g), were stunned and killed by cervical dislocation followed by exsanguination. The thoracic aorta was isolated, removed carefully to prevent endothelium damage and cleared of fat and connective tissue. The thoracic aorta was cut into 3-mm ring segments, which were mounted on stainless-steel wires in 20-ml organ baths containing Krebs' medium with the following composition (mM): NaCl, 119; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 25; KH2PO4, 1.2; d-glucose, 11.1. The
Isoprenaline and l-NAME
Isoprenaline produced a concentration-dependent relaxation of noradrenaline constricted rings (−log EC50, 6.59±0.09; % maximum relaxation, 93±3, n=17) (Fig. 1a). Incubation with l-NAME (10 and 100 μM) significantly reduced the % maximum relaxation induced by isoprenaline to 57±1 (P<0.001, n=13) and 43±0.5, (P<0.001, n=8), respectively, and shifted the relaxant responses rightward (−log EC50 values: l-NAME (10 μM), 5.97±0.05, P<0.001; l-NAME (100 μM), 5.78±0.02, P<0.001) (Fig. 1a). The effect of
Discussion
In the present study, acetylcholine-induced relaxations of the pre-constricted rat aortic rings were greatly reduced following l-NAME pretreatment or completely abolished by endothelium removal confirming the role of endothelium-derived NO in acetylcholine-induced vasodilatation Furchgott, 1988, Furchgott and Zawadzki, 1980, Ignarro et al., 1988. Endothelium removal was more effective in reducing acetylcholine-induced relaxant responses than inhibition of endothelial nitric oxide synthase
Acknowledgements
L.B. was supported by a Glasgow Caledonian University studentship. We are grateful to Novartis Pharma (CGP 12177A) and Zeneca (ZD 2079, ZM 215001) for gifts of drugs.
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