Pathways that control, or can be exploited to alter, the increase in airway smooth muscle (ASM) mass and cellular remodeling that occur in asthma are not well defined. Here we report the expression of odorant receptors (ORs) belonging to the superfamily of G-protein coupled receptors (GPCRs), as well as the canonical olfaction machinery (Golf and AC3) in the smooth muscle of human bronchi. In primary cultures of isolated human ASM, we identified mRNA expression for multiple ORs. Strikingly, OR51E2 was the most highly enriched OR transcript mapped to the human olfactome in lung-resident cells. In a heterologous expression system, OR51E2 trafficked readily to the cell surface and showed ligand selectivity and sensitivity to the short chain fatty acids (SCFAs) acetate and propionate. These endogenous metabolic byproducts of the gut microbiota slowed the rate of cytoskeletal remodeling, as well as the proliferation of human ASM cells. These cellular responses in vitro were found in ASM from non-asthmatics and asthmatics, and were absent in OR51E2-deleted primary human ASM. These results demonstrate a novel chemo-mechanical signaling network in the ASM and serve as a proof-of-concept that a specific receptor of the gut-lung axis can be targeted to treat airflow obstruction in asthma.
Recent studies have highlighted the important roles that “sensory” receptors (olfactory receptors, taste receptors, and orphan “GPR” receptors) play in a variety of tissues, including the kidney. Although several studies have identified important roles that individual sensory receptors play in the kidney, there has not been a systematic analysis of the renal repertoire of sensory receptors. In this study, we identify novel renal sensory receptors belonging to the GPR (n = 76), olfactory receptor (n = 6), and taste receptor (n = 11) gene families. A variety of reverse transcriptase (RT)- PCR screening strategies were used to identify novel renal sensory receptors, which were subsequently confirmed using gene-specific primers. The tissue-specific distribution of these receptors was determined, and the novel renal ORs were cloned from whole mouse kidney. Renal ORs that trafficked properly in vitro were screened for potential ligands using a dual-luciferase ligand screen, and novel ligands were identified for Olfr691. These studies demonstrate that multiple sensory receptors are expressed in the kidney beyond those previously identified. These results greatly expand the known repertoire of renal sensory receptors. Importantly, the mRNA of many of the receptors identified in this study are expressed highly in the kidney (comparable to well-known and extensively studied renal GPCRs), and in future studies it will be important to elucidate the roles that these novel renal receptors play in renal physiology.
A primary function of the kidney is to maintain homeostasis. Recent studies have shown that specialized G‐protein coupled receptors, such as olfactory receptors (ORs), play essential roles in modulating kidney function. However, ORs are often orphan receptors with unknown ligands. In a recent RNA seq screen we identified 11 ORs as expressed in the renal cortex, including 5 ORs we had previously published as being expressed in the kidney, and 6 novel ORs. We have previously published the results of ligand screening for 4 of these ORs (Olfr31, Olfr78, Olfr1392, Olfr1393), but have not yet explored the ligands of the remaining 7 (Olfr56, Olfr90, Olfr461, Olfr558, Olfr1033, Olfr1034, Olfr1396). In order to better understand the physiological relevance of these ORs in the kidney, we cloned these 7 receptors from the kidney and attempted to determine ligands that activate these receptors to provide insight into physiological function. Ligand‐screening studies were performed using a cAMP dependent luciferase reporter assay, for which it is crucial that the ORs traffic to the cell surface. Because surface expression is often problematic for heterologously expressed ORs, we first performed immunofluorescence imaging utilizing an N‐terminal flag tag to determine the surface expression of each OR. Surface labeling for flag antibody in live, unpermeabilized cells revealed that 5 ORs (Olfr90, Olfr461, Olfr558, Olfr1034, Olfr1396) demonstrated robust surface trafficking, while 2 ORs (Olfr56 and Olfr1033) were very poorly trafficked to the cell surface. Thus, we screened the 5 ORs which trafficked to the cell surface using a ligand library of 85 compounds with diverse functional groups, divided into 3 categories: 1) compounds known to activate a large percentage of isolated olfactory sensory neurons, 2) sibling ligands, or compounds known to activate ORs within the same sub‐family as these uncharacterized ORs, particularly those known to be present in biofluids including blood and urine, and 3) other odorant and small molecules known to exist in the body produced either endogenously or by microorganisms. Olfr558 has previously reported ligands, and we were able to confirm activation by both butyric acid and nonanoic acid using our luciferase assay (p<0.05 vs control media); to date, none of the other chemicals screened have activated this receptor. Using this library of odorant compounds, we identified 10 novel ligands that activated Olfr90 (p<0.05 vs control media) in a dose dependent manner, none of which activated unrelated ORs or cells transfected with vector only (1‐octen‐3‐ol, 2‐methyl‐4‐propyl‐1,3‐oxathiane, 2‐pentylfuran, allyl benzene, amyl acetate, cinnamaldehyde, 2‐octanone, 3‐octanol, benzyl cyanide, linalool). Interestingly, 6 out of 10 of these ligands are known to be of fungal origin, strongly suggesting that this receptor senses fungal metabolites. To date, none of the compounds screened against Olfr461, Olfr1034, or Olfr1396 have resulted in OR activation. In sum, we have examined 7 ORs expressed in the renal c...
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