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  • The molecular mechanisms underlying VSMC ETB receptor upregu


    The molecular mechanisms underlying VSMC ETB receptor upregulation have been studied after 24–48h organ culture in both coronary and cerebral arteries. The increased expression of contractile ETB receptor at these time points has been shown to depend on transcriptional mechanisms and PKC and the extracellular signal-regulated kinases 1 and 2 (ERK1/2) have been identified as upstream mediators of the increased ETB receptor gene expression (Henriksson et al., 2004, Nilsson et al., 2008, Sandhu et al., 2010). However, the detailed time course of the ETB receptor upregulation in rat coronary SC 560 synthesis at time points earlier than 24h after start of organ culture has hitherto not been studied. Moreover, it is not clear from the earlier reports whether the critical factor triggering the ETB receptor upregulation during organ culture of coronary arteries is serum-deprivation or low oxygen tension, both of which are prominent characteristics of the DMEM in vitro organ culture model hitherto used. Consequently, the aim of the present study was to investigate whether an ETB receptor-mediated vasoconstrictor response developed during short-term incubation of isolated LAD as well as septal coronary artery (SCA) segments in oxygenated buffer in a myograph. Furthermore, we aimed at investigating the detailed time course and molecular mechanisms behind this putative early ETB receptor plasticity.
    Materials and methods
    Results The average diameters of LAD segments (340±16μm) were larger than those of SCA segments (260±13μm). However, maximal contractile capacity (KPSS with 10μM PGF2α and 10μM 5-HT) was not significantly different between LAD (6.4±0.8mN/mm) and SCA (7.0±0.5mN/mm) segments. In most experiments, LAD had a slightly higher basal tone compared to SCA, and since the basal tone in LAD was highly dependent on the pH in the buffer, the pH was kept tightly in a range between 7.35 and 7.45.
    Discussion The present study demonstrates for the first time that only 7h incubation of isolated coronary artery segments in oxygenated physiological buffer in a myograph induces a significant upregulation of contractile ETB receptors. This rapid upregulation of ETB receptors is important to keep in mind, if tissue bath experiments on coronary arteries persist for more than 4h, due to a potentially interference of these emerging ETB receptors. This rapid functional upregulation is associated with increased ETB receptor immunoreactivity in VSMC and is abolished by the transcription inhibitor actinomycin D and the MEK1/2 inhibitor U0126. Mounting evidence suggests that under different pathological circumstances including atherosclerosis (Dagassan et al., 1996a, Pernow et al., 2000), ischemic heart disease (Dimitrijevic et al., 2009, Wackenfors et al., 2004), and focal cerebral ischemia (Stenman et al. 2002), the balance of vasoconstriction and relaxation by endothelin receptor activation is skewed towards a contractile phenotype with increased levels of vasoconstrictive ETB receptors in VSMCs. In vitro organ culture of isolated artery segments (Adner et al. 1998a) induces upregulation of contractile endothelin receptors resembling the pattern seen in cardiovascular diseases (Wackenfors et al. 2004), indicating that organ culture can be used as a convenient in vitro model to study this pathophysiological phenomenon. Previous studies have shown that fresh isolated rat coronary arteries display negligible ETB receptor-mediated contractions (Goodwin et al., 1999, Harrison et al., 1992), whereas a strong ETB receptor-mediated contractile response appears after 24h of organ culture in serum-free DMEM (Eskesen and Edvinsson, 2006, Ghorbani et al., 2010, Johnsson et al., 2008). In the present study, we studied for the first time the early time course of contractile ETB receptor upregulation during incubation in aerated buffer in a myograph. We demonstrate a rapid and strong increase in S6c mediated contractile responses in coronary arteries already after 7h of incubation, which is a considerably more rapid response than what has earlier been demonstrated in both mesenteric (Moller et al. 2002) and cerebral arteries (Henriksson et al. 2003). The specific involvement of ETB receptors in the rapidly upregulated contractile response in coronary arteries was confirmed by the demonstration of a strong parallel rightwards shift of the S6c concentration–response curves using the competitive selective ETB receptor antagonist BQ788 (Ishikawa et al. 1994).