Uncovering the Physiological Role of Olfactory Receptor 558 (Olfr558) in the Vasculature

Olfactory receptors are not exclusively expressed in the nose; they are also observed and play pivotal roles outside of the olfactory sensory neurons as ecnomotopic (out of the usual/conventional place) receptors in other tissues. We have previously reported that renal olfactory receptor 78 (Olfr78) detects two major short‐chain fatty acids (SCFAs), acetate and propionate, to modulate blood pressure regulation (Pluznick et al. 2013). We recently found that the most closely related receptor to Olfr78, Olfr558, is primarily activated by butyrate, the other major SCFAs produced by the gut microbiota (Halperin Kuhns et al. 2019). To expand on these findings, here we performed both real‐time cAMP and calcium imaging assays to measure the activation of Olfr558 and its human ortholog, OR51E1. Butyrate dose‐dependently (0.1, 0.2, 0.5, 1, and 2 mM) and time‐dependently (1–10 min) increases cAMP production, with significant enhancement at the 10 min time point by all detected doses (p<0.05) for the murine and the human receptor. To further verify that butyrate activates OR51E1, olfactory G protein (Golf) and OR51E1 were transiently co‐transfected into HEK293T cells. Butyrate (2 mM) evokes Ca 2+ responses in 12.13% of cells (160 of 1319 cells), but failed to activate cells transfected with Golf alone (0.19%, 2 of 1046 cells) or non‐transfected (0%, 0 of 505 cells), with weak activation seen in OR51E1 alone transfected control cells (6.29%, 91 of 1446 cells). We also find that although isobutyrate does not increase cAMP production (2 mM), it does evoke Ca 2+ responses of 6.93% (82 of 1183 cells) in OR51E1 and Golf double transfected cells. Little or no activation was seen in cells transfected with OR51E1 alone, Golf alone, or non‐transfected cells. These results suggest that butyrate and isobutyrate activate OR51E1 by different signaling pathways. Next, we looked into the expression and distribution pattern of Olfr558 in 8‐week‐old C57BL/6 mice. RNAscope reveals that Olfr558 mRNA is expressed in renal vasculature and co‐localizes with alpha‐smooth muscle actin (α‐SMA, blood vessel marker). Further exploration of Olfr558 expression shows that Olfr558 is also expressed in the heart and aorta vasculature, but little to no expression is seen in the large intestine, brain, lung, cecum, or liver by both qPCR and RNAscope assays (n=6, 3 male and 3 female). Exclusive expression of Olfr558 in the vasculature indicates a possible role of Olfr558 in blood pressure regulation. To test this possibility, we compared Olfr558 expression in mice treated with minipumps infusing either saline or Angiotensin II (AngII) for 4 weeks (400 ng.kg −1 .min −1 ). qPCR data indicates that Olfr558 mRNA expression significantly increases in AngII‐treated mice in the heart (1.5 fold increase, p<0.05, n=12 for each treatment, 6 male and 6 female), but not the kidney (n=6 for each treatment, 3 male and 3 female). These findings were confirmed by RNAscope (n=3 for each treatment, male). In summary, these results demonstrate that Olfr558 is a potential modulator of blood pressure regulation; the ligand profile of Olfr558 suggests that it may also tie blood pressure regulation to gut microbial metabolism. Support or Funding Information Funding: R01HL128512, R01DK107726