Effects of quinine, quinidine and chloroquine on human muscle nicotinic acetylcholine receptors

The genus $\textit {Cinchona}$ is known for a range of alkaloids, such as quinine, quinidine, cinchonine, and cinchonidine. $\textit {Cinchona}$ bark has been used as an antimalarial agent for more than 400 years. Quinine was first isolated in 1820 and is still acknowledged in the therapy of chloroquine-resistant falciparum malaria; in lower dosage quinine has been used as treatment for leg cramps since the 1940s. Here we report the effects of the quinoline derivatives quinine, quinidine, and chloroquine on human adult and fetal muscle nicotinic acetylcholine receptors (nAChRs). It could be demonstrated that the compounds blocked acetylcholine (ACh)-evoked responses in $\textit {Xenopus laevis}$ oocytes expressing the adult nAChR composed of $\alpha$$\beta$$\epsilon$$\delta$ subunits in a concentrationdependent manner, with a ranked potency of quinine (IC$_{50}$ = 1.70 $\mu$M), chloroquine (IC$_50$ = 2.22 $\mu$M) and quinidine (IC$_50$ = 3.96 $\mu$M). At the fetal nAChR composed of abgd subunits, the IC$_50$ for quinine was found to be 2.30 $\mu$M. The efficacy of the block by quinine was independent of the ACh concentration. Therefore, quinine is proposed to inhibit ACh-evoked currents in a non-competitive manner. The present results add to the pharmacological characterization of muscle nAChRs and indicate that quinine is effective at the muscular nAChRs close to therapeutic blood concentrations required for the therapy and prophylaxis of nocturnal leg cramps, suggesting that the clinically proven efficacy of quinine could be based on targeting nAChRs

RNA-Seq analysis of human trigeminal and dorsal root ganglia with a focus on chemoreceptors

The chemosensory capacity of the somatosensory system relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. Knowledge of the complete repertoire of the chemoreceptors expressed in human sensory ganglia is lacking. This study employed the next-generation sequencing technique (RNA-Seq) to conduct the first expression analysis of human trigeminal ganglia (TG) and dorsal root ganglia (DRG). We analyzed the data with a focus on G-protein coupled receptors (GPCRs) and ion channels, which are (potentially) involved in chemosensation by somatosensory neurons in the human TG and DRG. For years, transient receptor potential (TRP) channels have been considered the main group of receptors for chemosensation in the trigeminal system. Interestingly, we could show that sensory ganglia also express a panel of different olfactory receptors (ORs) with putative chemosensory function. To characterize OR expression in more detail, we performed microarray, semi-quantitative RT-PCR experiments, and immunohistochemical staining. Additionally, we analyzed the expression data to identify further known or putative classes of chemoreceptors in the human TG and DRG. Our results give an overview of the major classes of chemoreceptors expressed in the human TG and DRG and provide the basis for a broader understanding of the reception of chemical cues

Deorphanization and characterization of the ectopically expressed olfactory receptor OR51B5 in myelogenous leukemia cells

The ectopic expression of olfactory receptors (ORs) in the human body has been of major interest in the past decade. Several studies have reported the expression of ORs not only in healthy tissues such as heart, sperm or skin cells, but also in cancerous tissues of the liver, prostate or intestine. In the present study, we detected the expression of OR51B5 in the chronic myelogenous leukemia (CML) cell line K562 and in white blood cell samples of clinically diagnosed acute myelogenous leukemia (AML) patients by reverse transcription-PCR and immunocytochemical staining. The known OR51B5 ligand isononyl alcohol increased the levels of intracellular $Ca^{2+}$ in both AML patient blood cells and K562 cells. With calcium imaging experiments, we characterized in greater detail the OR51B5-mediated signaling pathway. Here, we observed an involvement of adenylate cyclase and the downstream L-type and T-type calcium channels. In addition, the activation of OR51B5 leads to an inhibition of cell proliferation in K562 cells. In western blot experiments, we found that incubation with isononyl alcohol led to a reduction in p38-MAPK (mitogen-activated protein kinase) phosphorylation that might be responsible for the decreased cell proliferation. In the present study, we characterized the OR51B5-mediated signaling pathway downstream of the activation with isononyl alcohol, which leads to reduced proliferation and therefore provide a novel pharmacological target for CML and AML, the latter of which remains difficult to treat

Characterization of the olfactory receptor OR10H1 in human urinary bladder cancer

Olfactory receptors (ORs) are a large group of G-protein coupled receptors predominantly found in the olfactory epithelium. Many ORs are, however, ectopically expressed in other tissues and involved in several diseases including cancer. In this study, we describe that one OR, OR10H1, is predominantly expressed in the human urinary bladder with a notably higher expression at mRNA and protein level in bladder cancer tissues. Interestingly, also significantly higher amounts of OR10H1 transcripts were detectable in the urine of bladder cancer patients than in the urine of control persons. We identified the sandalwood-related compound Sandranol as a specific agonist of OR10H1. This deorphanization allowed the functional characterization of OR10H1 in BFTC905 bladder cancer cells. The effect of receptor activation was morphologically apparent in cell rounding, accompanied by changes in the cytoskeleton detected by $\beta$-actin, T-cadherin and $\beta$-Catenin staining. In addition, Sandranol treatment significantly diminished cell viability, cell proliferation and migration and induced a limited degree of apoptosis. Cell cycle analysis revealed an increased G1 fraction. In a concentration-dependent manner, Sandranol application elevated cAMP levels, which was reduced by inhibition of adenylyl cyclase, and elicited intracellular $Ca^{2+}$ concentration increase. Furthermore, activation of OR10H1 enhanced secretion of ATP and serotonin. Our results suggest OR10H1 as a potential biomarker and therapeutic target for bladder cancer

Expression and functionality of TRPV1 in breast cancer cells

Transient receptor potential (TRP) channels contribute to the regulation of intracellular calcium, which can promote cancer hallmarks in cases of dysregulation of gene transcription and calcium-dependent pro-proliferative or anti-apoptotic mechanisms. Several studies have begun to elucidate the roles of TRPV1, TRPV6, TRPM8, and TRPC1 in cancer progression; however, no study has examined the expression profiles of human TRP channels in breast cancer on a large scale. This study focused on the expression and functionality of TRPV1, a nonselective cation channel that was found to be expressed in different carcinoma tissues. Next-generation sequencing analyses revealed the expression of TRPV1 in several native breast cancer tissues, which was subsequently validated via reverse transcriptase-polymerase chain reaction. Activation of TRPV1 by its ligand capsaicin was associated with the growth inhibition of some cancer cell types; however, the signaling components involved are complex. In this study, stimulation by the TRPV1 agonist, capsaicin, of SUM149PT cells, a model system for the most aggressive breast cancer subtype, triple-negative breast cancer, led to intracellular calcium signals that were diminished by the specific TRPV1 antagonist, capsazepin. Activation of TRPV1 by capsaicin caused significant inhibition of cancer cell growth and induced apoptosis and necrosis. In conclusion, the current study revealed the expression profiles of human TRP channels in 60 different breast cancer tissues and cell lines and furthermore validated the antitumor activity of TRPV1 against SUM149PT breast cancer cells, indicating that activation of TRPV1 could be used as a therapeutic target, even in the most aggressive breast cancer types

In vivo\textit {In vivo} monitoring of chemically evoked activity patterns in the rat trigeminal ganglion

Albeit lacking a sense of smell, anosmic patients maintain a reduced ability to distinguish different volatile chemicals by relying exclusively on their trigeminal system (TS). To elucidate differences in the neuronal representation of these volatile substances in the TS, we performed voltage-sensitive dye imaging (VSDI) in the rat trigeminal ganglion (TG) $\textit {in vivo}$. We demonstrated that stimulus-specific patterns of bioelectrical activity occur within the TG upon nasal administration of ten different volatile chemicals. With regard to spatial differences between the evoked trigeminal response patterns, these substances could be sorted into three groups. Signal intensity and onset latencies were also dependent on the administered stimulus and its concentration. We conclude that particular compounds detected by the TS are represented by (1) a specific spatial response pattern, (2) the signal intensity, and (3) onset latencies within the pattern. Jointly, these trigeminal representations may contribute to the surprisingly high discriminative skills of anosmic patients

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