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

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 pD₂ 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 bacterial contamination of the saline solution or mechanical stimulation, played a role in the observed induction of NOS2 as a potential moderate induction of NOS2 was already visible in arteries incubated with Krebs solution alone or in the presence of DMSO or U46619 as shown in Figure 7.

Instead of using selective antagonists for the different oestrogen receptors (ERα, ERβ and G protein‐coupled oestrogen receptor, GPER), which are available, the authors used ICI 182,780, which according to the authors is a ‘pure oestrogen receptor antagonist’. This description of ICI 182,780, taken from an earlier publication of the authors, is not entirely correct. ICI 182,780 (fulvestrant, Faslodex®) is a selective oestrogen receptor down‐regulator (SERD as opposed to the SERM raloxifene), which also degrades oestrogen receptor protein. ICI 182780, however, also acts as an agonist for GPER and, like the selective GPER agonist G‐1, has been shown to cause rapid arterial relaxation. It is also surprising that the authors used equimolar concentrations of the SERD ICI 182,780 to ‘block’ the response to the SERM raloxifene, which is pharmacologically illogical, given their similar affinities. On a final note, the non‐specificity of SERMSs with regard to their targets should be noted: ICI 182,780 has been shown to exert agonist activity on GPER (Meyer et al., 2010), and raloxifene has been newly identified as an inverse agonist for cannabinoid CB₁ receptors (Kumar and Song, 2013). The latter is likely a SERM‐class effect since it has also been described for tamoxifen and its active metabolite, 4OH‐tamoxifen (Prather et al., 2013). Finally, raloxifene has also been shown to inhibit xanthine oxidase and aldehyde oxidase (Weidert et al., 2014). The data presented by Wong et al. must thus be interpreted with caution. Furthermore, the authors fail to provide any explanation for the lack of relaxant responses to 17β‐estradiol and tamoxifen, which have been described in many studies.

At this point it remains unclear whether the observed effects only involve oestrogen receptors (and not other or additional pathways). It also appears, contrary to the authors' statements, that the relaxation responses to raloxifene are far more rapid than the proposed mechanism, in keeping with the many reports on rapid vascular effects of raloxifene and possibly other SERMs.

Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015a,b,c).

Conflict of interest

M.B. and E.R.P. are inventors on a U.S. patent application for the therapeutic use of compounds targeting GPER. E.R.P. is an inventor on U.S. patent numbers 7875721 and 8487100 for GPER‐selective ligands and imaging agents.

Barton, M. , and Prossnitz, E. R. (2017) Rapid vasodilation to raloxifene: role of oestrogen receptors and off‐target effects. British Journal of Pharmacology, 174: 4201–4202. doi: 10.1111/bph.14020.