ENaC in Cholinergic Brush Cells.

Cholinergic polymodal chemosensory cells in the mammalian urethra (urethral brush cells = UBC) functionally express the canonical bitter and umami taste transduction signaling cascade. Here, we aimed to determine whether UBC are functionally equipped for the perception of salt through ENaC (epithelial sodium channel). Cholinergic UBC were isolated from ChAT-eGFP reporter mice (ChAT = choline acetyltransferase). RT-PCR showed mRNA expression of ENaC subunits Scnn1a, Scnn1b, and Scnn1g in urethral epithelium and isolated UBC. Scnn1a could also be detected by next generation sequencing in 4/6 (66%) single UBC, two of them also expressed the bitter receptor Tas2R108. Strong expression of Scnn1a was seen in some urothelial umbrella cells and in 65% of UBC (30/46 cells) in a Scnn1a reporter mouse strain. Intracellular [Ca2+] was recorded in isolated UBC stimulated with the bitter substance denatonium benzoate (25 mM), ATP (0.5 mM) and NaCl (50 mM, on top of 145 mM Na+ and 153 mM Cl- baseline in buffer); mannitol (150 mM) served as osmolarity control. NaCl, but not mannitol, evoked an increase in intracellular [Ca2+] in 70% of the tested UBC. The NaCl-induced effect was blocked by the ENaC inhibitor amiloride (IC50 = 0.47 µM). When responses to both NaCl and denatonium were tested, all three possible positive response patterns occurred in a balanced distribution: 42% NaCl only, 33% denatonium only, 25% to both stimuli. A similar reaction pattern was observed with ATP and NaCl as test stimuli. About 22% of the UBC reacted to all three stimuli. Thus, NaCl evokes calcium responses in several UBC, likely involving an amiloride-sensitive channel containing α-ENaC. This feature does not define a new subpopulation of UBC, but rather emphasizes their polymodal character. The actual function of α-ENaC in cholinergic UBC-salt perception, homeostatic ion transport, mechanoreception-remains to be determined.

OR2H2 regulates the differentiation of human myoblast cells by its ligand aldehyde 13-13.

Olfactory receptors (ORs) regulate various cellular processes in the human body. The receptors' participation in physiological and pathophysiological processes could be demonstrated in several studies. In addition to the regulation of sperm motility, respiratory physiology, and heart contraction, ORs play a crucial role in cancer cells. In murine myoblasts, mOR23 regulates the myogenesis and branching of skeletal muscle cells. To date, the expression and physiological role of ORs in human skeletal muscle cells have not been thoroughly elucidated. We demonstrate that four different ORs are expressed at the transcript level in differentiated myoblasts, and one other OR is expressed in undifferentiated myoblasts. Moreover, we characterized the expression of OR2H2 in differentiated human myoblasts and identified a specific ligand, aldehyde 13-13. We could observe a concentration-dependent Ca2+ increase in differentiated human myoblasts upon aldehyde 13-13 stimulation, which is mediated by PI3K signaling. Aldehyde 13-13 has a reducing effect on myoblast fusion. We conclude that OR2H2 could have a regulatory role in myoblast differentiation. To the best of our knowledge, this report presents the first verification of the expression of ORs in human myoblasts. OR2H2 might be an interesting candidate for playing a role in the complex mechanism of myogenesis.

Helional-induced activation of human olfactory receptor 2J3 promotes apoptosis and inhibits proliferation in a non-small-cell lung cancer cell line.

Studies within the last decade have localized the functional expression of olfactory receptors (ORs) to cells outside of the olfactory epithelium. In human hepatocarcinoma and prostate cancer cells, the activation of ORs by odors modulates elementary physiological processes and leads to an inhibitory effect on proliferation. Cells of the respiratory tract are in direct contact with the surrounding air, in which a myriad of volatile molecules, especially odors, are present. Non-small-cell lung cancer (NSCLC) has a high prevalence, a high mortality rate and is difficult to treat. NSCLC cells are nearly resistant to common chemotherapeutic approaches, and surgical resection provides the only possible chance of a cure for most patients. New approaches for the treatment of NSCLC are the focus of many current studies. Thus, it is of interest to characterize the functional expression of ORs in cancer cells of the lung and to investigate the impact of ORs on pathophysiological processes. In the present study, we demonstrate that the expression of OR2J3 and cytosolic Ca2+ increase via the activation of the agonist helional in the NSCLC cell line A549. We further investigated the underlying pathway. Helional triggers phoshoinositol-3 kinase (PI3K), signaling the release of intracellular Ca2+ and phosphorylation of ERK. We observed that OR2J3 activation induces apoptosis and inhibits cell proliferation and migration in long-term stimulus experiments with helional. Our study provides the first evidence of the functional expression of an OR in NSCLC cells and its putative therapeutic impact.

Olfactory signaling components and olfactory receptors are expressed in tubule cells of the human kidney.

Cells of the renal tubule system are in direct contact with compounds dissolved in the urine, such as short chain fatty acids (SCFA). Murine OR78, a member of the olfactory receptor (OR) family, is involved in SCFA-related regulation of renal blood pressure in mice. It is still unclear whether OR signaling has an impact on human renal physiology. In our study, we showed that OR51E1 and OR11H7, both of which can be activated by the SCFA isovaleric acid, are expressed in the HK-2 human proximal tubule cell line. We observed a transient increase in intracellular Ca2+ when isovaleric acid and 4-methylvaleric acid were added to HK-2 cells. The isovaleric acid-induced response was dependent on extracellular Ca2+ and adenylyl cyclase (AC) activation. Furthermore, we demonstrated that the canonical olfactory signaling components Gαolf and ACIII are co-localized with OR51E1. The number of cells responding to isovaleric acid correlated with the presence of primary cilia on HK-2 cells. OR51E1 protein expression was confirmed in the tubule system of human kidney tissue. Our study is the first to show the expression of ORs and olfactory signaling components in human kidney cells. Additionally, we discuss ORs as potential modulators of the renal physiology.

Olfactory Receptors Modulate Physiological Processes in Human Airway Smooth Muscle Cells.

Pathophysiological mechanisms in human airway smooth muscle cells (HASMCs) significantly contribute to the progression of chronic inflammatory airway diseases with limited therapeutic options, such as severe asthma and COPD. These abnormalities include the contractility and hyperproduction of inflammatory proteins. To develop therapeutic strategies, key pathological mechanisms, and putative clinical targets need to be identified. In the present study, we demonstrated that the human olfactory receptors (ORs) OR1D2 and OR2AG1 are expressed at the RNA and protein levels in HASMCs. Using fluorometric calcium imaging, specific agonists for OR2AG1 and OR1D2 were identified to trigger transient Ca(2+) increases in HASMCs via a cAMP-dependent signal transduction cascade. Furthermore, the activation of OR2AG1 via amyl butyrate inhibited the histamine-induced contraction of HASMCs, whereas the stimulation of OR1D2 with bourgeonal led to an increase in cell contractility. In addition, OR1D2 activation induced the secretion of IL-8 and GM-CSF. Both effects were inhibited by the specific OR1D2 antagonist undecanal. We herein provide the first evidence to show that ORs are functionally expressed in HASMCs and regulate pathophysiological processes. Therefore, ORs might be new therapeutic targets for these diseases, and blocking ORs could be an auspicious strategy for the treatment of early-stage chronic inflammatory lung diseases.

Helional induces Ca2+ decrease and serotonin secretion of QGP-1 cells via a PKG-mediated pathway.

The secretion, motility and transport by intestinal tissues are regulated among others by specialized neuroendocrine cells, the so-called enterochromaffin (EC) cells. These cells detect different luminal stimuli, such as mechanical stimuli, fatty acids, glucose and distinct chemosensory substances. The EC cells react to the changes in their environment through the release of transmitter molecules, most importantly serotonin, to mediate the corresponding physiological response. However, little is known about the molecular targets of the chemical stimuli delivered from consumed food, spices and cosmetics within EC cells. In this study, we evaluated the expression of the olfactory receptor (OR) 2J3 in the human pancreatic EC cell line QGP-1 at the mRNA and protein levels. Using ratiofluorometric Ca(2+) imaging experiments, we demonstrated that the OR2J3-specific agonist helional induces a transient dose-dependent decrease in the intracellular Ca(2+) levels. This Ca(2+) decrease is mediated by protein kinase G (PKG) on the basis that the specific pharmacological inhibition of PKG with Rp-8-pCPT-cGMPS abolished the helional-induced Ca(2+) response. Furthermore, stimulation of QGP-1 cells with helional caused a dose-dependent release of serotonin that was comparable with the release induced by the application of a direct PKG activator (8-bromo-cGMP). Taken together, our results demonstrate that luminal odorants can be detected by specific ORs in QGP-1 cells and thus cause the directed release of serotonin and a PKG-dependent decrease in intracellular Ca(2.)

Identification of a Novel Gnao-Mediated Alternate Olfactory Signaling Pathway in Murine OSNs.

It is generally agreed that in olfactory sensory neurons (OSNs), the binding of odorant molecules to their specific olfactory receptor (OR) triggers a cAMP-dependent signaling cascade, activating cyclic-nucleotide gated (CNG) channels. However, considerable controversy dating back more than 20 years has surrounded the question of whether alternate signaling plays a role in mammalian olfactory transduction. In this study, we demonstrate a specific alternate signaling pathway in Olfr73-expressing OSNs. Methylisoeugenol (MIEG) and at least one other known weak Olfr73 agonist (Raspberry Ketone) trigger a signaling cascade independent from the canonical pathway, leading to the depolarization of the cell. Interestingly, this pathway is mediated by Gnao activation, leading to Cl(-) efflux; however, the activation of adenylyl cyclase III (ACIII), the recruitment of Ca(2+) from extra-or intracellular stores, and phosphatidylinositol 3-kinase-dependent signaling (PI signaling) are not involved. Furthermore, we demonstrated that our newly identified pathway coexists with the canonical olfactory cAMP pathway in the same OSN and can be triggered by the same OR in a ligand-selective manner. We suggest that this pathway might reflect a mechanism for odor recognition predominantly used in early developmental stages before olfactory cAMP signaling is fully developed. Taken together, our findings support the existence of at least one odor-induced alternate signal transduction pathway in native OSNs mediated by Olfr73 in a ligand-selective manner.

Transcriptome Analysis of Murine Olfactory Sensory Neurons during Development Using Single Cell RNA-Seq.

Mammalian odor reception is achieved by highly specialized olfactory sensory neurons (OSNs) located in the nasal cavity. Despite their importance for the daily survival of most mammals, the gene expression and regulatory profiles of these single neurons are poorly understood. Here, we report the isolation of individual GFP-labeled OSNs from Olfr73-GFP mice at different developmental stages followed by Next Generation Sequencing, thereby analyzing the detailed transcriptome for the first time. We characterized the repertoire of olfactory receptors (ORs) and found that in addition to the highly and predominant detectable Olfr73, 20 additional ORs were stably detectable at lower transcript levels in adult mice. Additionally, OSNs collected from mice of earlier developmental stages did not show any stable OR patterns. However, more than one predominant OR per OSN was detectable.

Identification and functional characterization of TRPA1 in human myoblasts.

The proper function of the skeletal muscle is essential for the survival of most animals. Thus, efficient and rapid repair of muscular damage following injury is crucial. In recent years, satellite cells have emerged as key players of muscle repair, capable of undergoing extensive proliferation after injury, fusing into myotubes and restoring muscle function. Furthermore, it has been shown that Ca(2+)/calmodulin-dependent generation of nitric oxide (NO) is an important regulator of muscle repair. Here, we demonstrate the functional expression of transient receptor potential, subfamily A1 (TRPA1) channel in human primary myoblasts. Stimulation of these cells with well-known TRPA1 ligands led to robust intracellular Ca(2+) rises which could be inhibited by specific TRPA1 antagonists. Moreover, we show that TRPA1 activation enhances important aspects of skeletal muscle repair such as cell migration and myoblast fusion in vitro. Interestingly, TRPA1 levels and inducible Ca(2+) transients decline with ongoing myoblast differentiation. We suggest that TRPA1 might serve as a physiological mediator for inflammatory signals and appears to have a functional role in promoting myoblast migration, fusion, and potentially also in activating satellite cells in humans.

Impact of the Usher syndrome on olfaction.

Usher syndrome is a genetically and clinically heterogeneous disease in humans, characterized by sensorineural hearing loss, retinitis pigmentosa and vestibular dysfunction. This disease is caused by mutations in genes encoding proteins that form complex networks in different cellular compartments. Currently, it remains unclear whether the Usher proteins also form networks within the olfactory epithelium (OE). Here, we describe Usher gene expression at the mRNA and protein level in the OE of mice and showed interactions between these proteins and olfactory signaling proteins. Additionally, we analyzed the odor sensitivity of different Usher syndrome mouse models using electro-olfactogram recordings and monitored significant changes in the odor detection capabilities in mice expressing mutant Usher proteins. Furthermore, we observed changes in the expression of signaling proteins that might compensate for the Usher protein deficiency. In summary, this study provides novel insights into the presence and purpose of the Usher proteins in olfactory signal transduction.

Biochemical Large-Scale Interaction Analysis of Murine Olfactory Receptors and Associated Signaling Proteins with Post-Synaptic Density 95, Drosophila Discs Large, Zona-Occludens 1 (PDZ) Domains.

G protein-coupled receptors (GPCRs) constitute the largest family among mammalian membrane proteins and are capable of initiating numerous essential signaling cascades. Various GPCR-mediated pathways are organized into protein microdomains that can be orchestrated and regulated through scaffolding proteins, such as PSD-95/discs-large/ZO1 (PDZ) domain proteins. However, detailed binding characteristics of PDZ-GPCR interactions remain elusive because these interactions seem to be more complex than previously thought. To address this issue, we analyzed binding modalities using our established model system. This system includes the 13 individual PDZ domains of the multiple PDZ domain protein 1 (MUPP1; the largest PDZ protein), a broad range of murine olfactory receptors (a multifaceted gene cluster within the family of GPCRs), and associated olfactory signaling proteins. These proteins were analyzed in a large-scale peptide microarray approach and continuative interaction studies. As a result, we demonstrate that canonical binding motifs were not overrepresented among the interaction partners of MUPP1. Furthermore, C-terminal phosphorylation and distinct amino acid replacements abolished PDZ binding promiscuity. In addition to the described in vitro experiments, we identified new interaction partners within the murine olfactory epithelium using pull-down-based interactomics and could verify the partners through co-immunoprecipitation. In summary, the present study provides important insight into the complexity of the binding characteristics of PDZ-GPCR interactions based on olfactory signaling proteins, which could identify novel clinical targets for GPCR-associated diseases in the future.