Odorant Receptor 51E2 Agonist beta-ionone Regulates RPE Cell Migration and Proliferation

The odorant receptor 51E2 (OR51E2), which is well-characterized in prostate cancer cells and epidermal pigment cells, was identified for the first time as the most highly expressed OR in human fetal and adult retinal pigment epithelial (RPE) cells. Immunofluorescence staining and Western blot analysis revealed OR51E2 localization throughout the cytosol and in the plasma membrane. Additionally, immunohistochemical staining of diverse layers of the eye showed that the expression of OR51E2 is restricted to the pigment cells of the RPE and choroid. The results of Ca2+-imaging experiments demonstrate that activation of OR51E2 triggers a Ca2+ dependent signal pathway in RPE cells. Downstream signaling of OR51E2 involves the activation of adenylyl cyclase, ERK1/2 and AKT. The activity of these protein kinases likely accounts for the demonstrated increase in the migration and proliferation of RPE cells upon stimulation with the OR51E2 ligand beta-ionone. These findings suggest that OR51E2 is involved in the regulation of RPE cell growth. Thus, OR51E2 represents a potential target for the treatment of proliferative disorders

The Effect of Pungent and Tingling Compounds from Piper nigrum L. on Background K+ Currents

Black peppercorns (Piper nigrum L.) elicit a pungent and tingling oral impression. Their pungency is partially explained by the agonist activity of some of their active principles, especially piperine, on TRP channels. However, we recently showed that piperine, as well as other pungent compounds, also possess a marked effect on two-pore domain (KCNK, K2P) K+ channels. Members of this family play a key role in maintaining the resting membrane potential of excitable cells. Interestingly, tingling compounds have been shown to induce neuronal excitation by inhibiting KCNK channels. We addressed the question of whether it was plausible that KCNK channels could constitute a physiologically relevant target for the sensory active compounds present in black peppercorns. Because previous studies have demonstrated that mouse trigeminal neurons respond to several pungent compounds, to which humans are also sensitive, we used a primary culture of mouse trigeminal neurons to investigate whether the effect of piperine on these cell types could also be mediated by KCNK channels. We observed that even in the presence of classical TRP-antagonists, piperine was still able to activate a fraction of trigeminal neurons. Furthermore, our results showed that piperine is capable of inducing neuronal depolarization by a mechanism that does not require extracellular Na+ or Ca2+. This depolarization was mediated by the inhibition of a background K+ conductance, most likely corresponding to the KCNK channels of the TASK subfamily. We then performed a screening with 12 other pungent and/or tingling chemosensates isolated from black peppercorns. These compounds were evaluated on Xenopus laevis oocytes expressing the human orthologues of KCNK3, KNCK9 and KCNK18, which we previously showed to be inhibited by piperine. Remarkably, almost all of the isolated chemosensates inhibited the basal activity of hKCNK3, with 1-(octadeca-2E,4E,13/12Z-trienoyl)pyrrolidine acting as one of the most potent natural blockers for hKCNK3 found to date. Our results suggest that KCNK channels, especially KCNK3, are likely to play a complementary role to TRP channels in the complex orosensory impression elicited by black peppercorns, while they also help to expand the pharmacological knowledge of KCNK channels

Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis

The Stimulatory Gαs Protein Is Involved in Olfactory Signal Transduction in Drosophila

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology, constituting a key difference between the olfactory systems of insects and other animals. While heteromeric insect ORs form ligand-activated non-selective cation channels in recombinant expression systems, the evidence for an involvement of cyclic nucleotides and G-proteins in odor reception is inconsistent. We addressed this question in vivo by analyzing the role of G-proteins in olfactory signaling using electrophysiological recordings. We found that Gαs plays a crucial role for odorant induced signal transduction in OR83b expressing olfactory sensory neurons, but not in neurons expressing CO2 responsive proteins GR21a/GR63a. Moreover, signaling of Drosophila ORs involved Gαs also in a heterologous expression system. In agreement with these observations was the finding that elevated levels of cAMP result in increased firing rates, demonstrating the existence of a cAMP dependent excitatory signaling pathway in the sensory neurons. Together, we provide evidence that Gαs plays a role in the OR mediated signaling cascade in Drosophila

Comparison of ouabain-sensitive and -insensitive Na/K pumps in HEK293 cells

AbstractThe Na/K pump current Ip of single HEK293 cells either untransfected (endogenous Ip) or transfected with the α1 subunit of the rat Na/K pump (exogenous Ip) was investigated in Na-containing solution by means of whole-cell recording at 30°C. The endogenous Ip was irreversibly blocked by 10−4 M ouabain or 2·10−4 M dihydro-ouabain (DHO). Its density amounted to 0.33 pA pF−1 at 0 mV and 5.4 mM Ko. It was half maximally activated at 1.5 mM Ko and increased linearly with depolarization over the entire voltage range studied (−80 to +60 mV). In contrast, HEK293 cells stably transfected with cDNA for the cardiac glycoside-resistant α1 subunit of the rat Na/K pump showed an Ip in the presence of 10−4 M ouabain and 2·10−4 M DHO, respectively. This exogenous Ip was reversibly blocked by 10−2 M ouabain. Half maximal activation of the exogenous Ip occurred at 1.7 mM Ko. Its amplitude increased linearly with depolarization at negative voltages but remained almost constant at positive membrane potentials. Comparison with the Ip of isolated rat cardiac ventricular myocytes strongly suggests that the exogenous Ip in HEK293 cells is generated by the α1 subunit of the rat Na/K pump since it displays identical properties. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycoside-resistant Na/K pumps by means of whole-cell recording

Timberol® Inhibits TAAR5-Mediated Responses to Trimethylamine and Influences the Olfactory Threshold in Humans.

In mice, trace amine-associated receptors (TAARs) are interspersed in the olfactory epithelium and constitute a chemosensory subsystem that is highly specific for detecting volatile amines. Humans possess six putative functional TAAR genes. Human TAAR5 (hTAAR5) is highly expressed in the olfactory mucosa and was shown to be specifically activated by trimethylamine. In this study, we were challenged to uncover an effective blocker substance for trimethylamine-induced hTAAR5 activation. To monitor blocking effects, we recombinantly expressed hTAAR5 and employed a commonly used Cre-luciferase reporter gene assay. Among all tested potential blocker substances, Timberol®, an amber-woody fragrance, is able to inhibit the trimethylamine-induced hTAAR5 activation up to 96%. Moreover, human psychophysical data showed that the presence of Timberol® increases the olfactory detection threshold for the characteristic fishy odor of trimethylamine by almost one order of magnitude. In conclusion, our results show that among tested receptors Timberol® is a specific and potent antagonist for the hTAAR5-mediated response to trimethylamine in a heterologous system. Furthermore, our data concerning the observed shift of the olfactory detection threshold in vivo implicate that hTAAR5 or other receptors that may be inhibited by Timberol® could be involved in the high affinity olfactory perception of trimethylamine in humans

Modulatory Effects of Sex Steroids Progesterone and Estradiol on Odorant Evoked Responses in Olfactory Receptor Neurons

The influence of the sex steroid hormones progesterone and estradiol on physiology and behavior during menstrual cycles and pregnancy is well known. Several studies indicate that olfactory performance changes with cyclically fluctuating steroid hormone levels in females. Knowledge of the exact mechanisms behind how female sex steroids modulate olfactory signaling is limited. A number of different known genomic and non-genomic actions that are mediated by progesterone and estradiol via interactions with different receptors may be responsible for this modulation. Next generation sequencing-based RNA-Seq transcriptome data from the murine olfactory epithelium (OE) and olfactory receptor neurons (ORNs) revealed the expression of several membrane progestin receptors and the estradiol receptor Gpr30. These receptors are known to mediate rapid non-genomic effects through interactions with G proteins. RT-PCR and immunohistochemical staining results provide evidence for progestin and estradiol receptors in the ORNs. These data support the hypothesis that steroid hormones are capable of modulating the odorant-evoked activity of ORNs. Here, we validated this hypothesis through the investigation of steroid hormone effects by submerged electro-olfactogram and whole cell patch-clamp recordings of ORNs. For the first time, we demonstrate that the sex steroid hormones progesterone and estradiol decrease odorant-evoked signals in the OE and ORNs of mice at low nanomolar concentrations. Thus, both of these sex steroids can rapidly modulate the odor responsiveness of ORNs through membrane progestin receptors and the estradiol receptor Gpr30

Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice

International audienceSpinal cord injury (SCI) often causes severe and permanent disabilities due to the regenerative failure of severed axons. Here we report significant locomotor recovery of both hindlimbs after a complete spinal cord crush. This is achieved by the unilateral transduction of cortical motoneurons with an AAV expressing hyper-IL-6 (hIL-6), a potent designer cytokine stimulating JAK/STAT3 signaling and axon regeneration. We find collaterals of these AAV-transduced motoneurons projecting to serotonergic neurons in both sides of the raphe nuclei. Hence, the transduction of cortical neurons facilitates the axonal transport and release of hIL-6 at innervated neurons in the brain stem. Therefore, this transneuronal delivery of hIL-6 promotes the regeneration of corticospinal and raphespinal fibers after injury, with the latter being essential for hIL-6-induced functional recovery. Thus, transneuronal delivery enables regenerative stimulation of neurons in the deep brain stem that are otherwise challenging to access, yet highly relevant for functional recovery after SCI