Rapid vasodilation to raloxifene: role of oestrogen receptors and off‐target effects

Abstract

For decades, raloxifene has been recognized as mediating the rapid, non-genomic relaxation of endothelium-intact and endothelium-denuded arteries of many species. In their recent article, Wong et al. (2017) report a number of observations and conclusions that are difficult to reconcile, both methodologically and mechanistically. The authors state that in endothelium-denuded aortic rings of male Sprague-Dawley rats, raloxifene at concentrations of 1 μM caused 'a time-dependent relaxant response for 3 h' that was mediated by inducible NOS (iNOS or NOS2).According to information provided in the 'Materials' section, the authors did not individually determine optimal length-tension curves (by repeated exposure to high K + ) but simply added a passive tension of 1 g. This is substantially below the normally employed tension for rat aorta (typically between 2 and 4 g), and no repeated passive-tension curves to KCl were performed for each individual ring to determine its optimal passive tension. Moreover, due to the lack of an internal contractility standard for each ring, the degree of precontraction induced by each drug relative to KCl cannot be ascertained, and it is therefore possible that given the concentrations of U46619 used, the contractions exceeded those evoked by 60 mM KCl, which would have the effect of directly affecting (i.e. reducing) relaxant responses as the artery was initially 'overcontracted'. This may, in part, even explain the delayed relaxant response to raloxifene, which in previous studies has been shown to occur within minutes and, as with oestradiol or ICI 182,780 (Meyer et al., 2010), is usually reached within 40 min. Furthermore, the authors' claim that the relaxation response requires a full 3 h is not supported by the data: As seen in Figure 4B, C, the onset of relaxation is observed by 20 min and the pD 2 is reached after about 40 min (and 50-60 min in Figures 2, 3 and 5). The time courses of the 'original tracings' also reveal that the authors did not wait until a stable contraction plateau was reached but added drugs or solvent upon reaching the maximum contraction, without allowing the vessel to equilibrate. Thus, the reported responses could have represented either a simple decrease of tone during the plateauing phase or a spontaneous loss of tone prior to reaching the plateau, which is frequently observed in this preparation. Also, the original tracings of the raloxifene response are in fact identical in three figures (2A, 3A and 4A), and the onset of relaxation in these 'representative' tracings is not mirrored by the analysed cumulative data presented. Furthermore, as there was little additional relaxation to raloxifene observed beyond 80-90 min, waiting a further 90 min to measure NOS2 expression makes little sense. Increased NOS2 expression at 3 h post-stimulation does not explain relaxant responses observed as early as 20 min following raloxifene treatment, a time when changes in NOS2 protein expression would not be expected. It is also possible that additional factors, such as...