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1.
Front Cell Neurosci ; 11: 03, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28174521

RESUMO

Several studies have demonstrated that the expression of odorant receptors (ORs) occurs in various tissues. These findings have served as a basis for functional studies that demonstrate the potential of ORs as drug targets for a clinical application. To the best of our knowledge, this report describes the first evaluation of the mRNA expression of ORs and the localization of OR proteins in the human retina that set a stage for subsequent functional analyses. RNA-Sequencing datasets of three individual neural retinae were generated using Next-generation sequencing and were compared to previously published but reanalyzed datasets of the peripheral and the macular human retina and to reference tissues. The protein localization of several ORs was investigated by immunohistochemistry. The transcriptome analyses detected an average of 14 OR transcripts in the neural retina, of which OR6B3 is one of the most highly expressed ORs. Immunohistochemical stainings of retina sections localized OR2W3 to the photosensitive outer segment membranes of cones, whereas OR6B3 was found in various cell types. OR5P3 and OR10AD1 were detected at the base of the photoreceptor connecting cilium, and OR10AD1 was also localized to the nuclear envelope of all of the nuclei of the retina. The cell type-specific expression of the ORs in the retina suggests that there are unique biological functions for those receptors.

2.
Sci Rep ; 6: 32255, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27572937

RESUMO

G protein-coupled receptors (GPCRs) transduce external chemical cues into intracellular signals and are involved in a plethora of physiological processes, but knowledge regarding the function of these receptors in spermatozoa is limited. In the present study, we performed RNA-Seq and analyzed the expression of the all GPCRs except olfactory receptors in human spermatozoa. We revealed the expression of up to 223 different GPCR transcripts in human spermatozoa (FPKM > 0.1) and identified GPR18, a newly described cannabinoid receptor, together with GPR137 and GPR135, as one of the three most highly expressed GPCRs. To date, the expression of GPR18 was completely unknown in human spermatozoa. We confirmed GPR18 expression using RT-PCR and immuncytochemistry experiments and localized the GPR18 protein in the midpiece of human spermatozoa. Stimulation of human spermatozoa with the GPR18 ligand N-arachidonoylglycine induced the phosphorylation of 12 protein kinases, some of them are for example known to be involved in the acrosome reaction. In line with this, N-arachidonoylglycine affected the cytoskeleton by changing levels of F-actin and inducing the acrosome reaction in human spermatozoa in a concentration-dependent manner. Our results indicate that GPR18 might be involved in physiological processes of human spermatozoa, suggesting GPR18 to be a potential player in sperm physiology.


Assuntos
Perfilação da Expressão Gênica/métodos , Receptores Acoplados a Proteínas G/genética , Espermatozoides/metabolismo , Reação Acrossômica/efeitos dos fármacos , Reação Acrossômica/genética , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Ácidos Araquidônicos/farmacologia , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Peça Intermédia do Espermatozoide/metabolismo
3.
Biochem Biophys Res Commun ; 467(4): 1090-6, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26456648

RESUMO

Serotonin receptor type 3 (5-HT3 receptor) is a ligand-gated ion channel that is expressed in the central nervous system (CNS) as well as in the peripheral nervous system (PNS). The receptor plays an important role in regulating peristalsis of the gastrointestinal tract and in functions such as emesis, cognition and anxiety. Therefore, a variety of pharmacologically active substances target the 5-HT3 receptor to treat chemotherapy-induced nausea and vomiting. The 5-HT3 receptors are activated, antagonized, or modulated by a wide range of chemically different substances, such as 2-methyl-serotonin, phenylbiguanide, setrones, or cannabinoids. Whereas the action of all of these substances is well described, less is known about the effect of terpenoids or fragrances on 5-HT3A receptors. In this study, we screened a large number of natural odorous and pungent substances for their pharmacological action on recombinantly expressed human 5-HT3A receptors. The receptors were functionally expressed in Xenopus oocytes and characterized by electrophysiological recordings using the two-electrode voltage-clamp technique. A screening of two odorous mixes containing a total of 200 substances revealed that the monoterpenes, thymol and carvacrol, act as both weak partial agonists and positive modulators on the 5-HT3A receptor. In contrast, the most effective blockers were the terpenes, citronellol and geraniol, as well as the pungent substances gingerol, capsaicin and polygodial. In our study, we identified new modulators of 5-HT3A receptors out of the classes of monoterpenes and vanilloid substances that frequently occur in various plants.


Assuntos
Receptores 5-HT3 de Serotonina/metabolismo , Terpenos/farmacologia , Animais , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Xenopus laevis
4.
PLoS One ; 10(6): e0128951, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26070209

RESUMO

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.


Assuntos
Gânglios Espinais/metabolismo , RNA/biossíntese , Receptores Odorantes/biossíntese , Gânglio Trigeminal/metabolismo , Gânglios Espinais/citologia , Regulação da Expressão Gênica , Humanos , RNA/genética , Receptores Odorantes/genética , Análise de Sequência de RNA , Gânglio Trigeminal/citologia
5.
Front Mol Biosci ; 2: 73, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26779489

RESUMO

The detection of external cues is fundamental for human spermatozoa to locate the oocyte in the female reproductive tract. This task requires a specific chemoreceptor repertoire that is expressed on the surface of human spermatozoa, which is not fully identified to date. Olfactory receptors (ORs) are candidate molecules and have been attributed to be involved in sperm chemotaxis and chemokinesis, indicating an important role in mammalian spermatozoa. An increasing importance has been suggested for spermatozoal RNA, which led us to investigate the expression of all 387 OR genes. This study provides the first comprehensive analysis of OR transcripts in human spermatozoa of several individuals by RNA-Seq. We detected 91 different transcripts in the spermatozoa samples that could be aligned to annotated OR genes. Using stranded mRNA-Seq, we detected a class of these putative OR transcripts in an antisense orientation, indicating a different function, rather than coding for a functional OR protein. Nevertheless, we were able to detect OR proteins in various compartments of human spermatozoa, indicating distinct functions in human sperm. A panel of various OR ligands induced Ca(2+) signals in human spermatozoa, which could be inhibited by mibefradil. This study indicates that a variety of ORs are expressed at the mRNA and protein level in human spermatozoa.

6.
PLoS One ; 8(11): e79523, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24260241

RESUMO

The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, in situ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain.


Assuntos
Gânglios Sensitivos/metabolismo , Gânglios Espinais/metabolismo , Gânglio Trigeminal/metabolismo , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Hibridização In Situ , Masculino , Camundongos , Canais de Potássio/genética , Receptores Acoplados a Proteínas G
7.
PLoS One ; 8(2): e55368, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23405139

RESUMO

Olfactory receptors (ORs) provide the molecular basis for the detection of volatile odorant molecules by olfactory sensory neurons. The OR supergene family encodes G-protein coupled proteins that belong to the seven-transmembrane-domain receptor family. It was initially postulated that ORs are exclusively expressed in the olfactory epithelium. However, recent studies have demonstrated ectopic expression of some ORs in a variety of other tissues. In the present study, we conducted a comprehensive expression analysis of ORs using an extended panel of human tissues. This analysis made use of recent dramatic technical developments of the so-called Next Generation Sequencing (NGS) technique, which encouraged us to use open access data for the first comprehensive RNA-Seq expression analysis of ectopically expressed ORs in multiple human tissues. We analyzed mRNA-Seq data obtained by Illumina sequencing of 16 human tissues available from Illumina Body Map project 2.0 and from an additional study of OR expression in testis. At least some ORs were expressed in all the tissues analyzed. In several tissues, we could detect broadly expressed ORs such as OR2W3 and OR51E1. We also identified ORs that showed exclusive expression in one investigated tissue, such as OR4N4 in testis. For some ORs, the coding exon was found to be part of a transcript of upstream genes. In total, 111 of 400 OR genes were expressed with an FPKM (fragments per kilobase of exon per million fragments mapped) higher than 0.1 in at least one tissue. For several ORs, mRNA expression was verified by RT-PCR. Our results support the idea that ORs are broadly expressed in a variety of tissues and provide the basis for further functional studies.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/genética , Adulto , Éxons , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Pessoa de Meia-Idade , Pseudogenes , RNA Mensageiro/genética , Transdução de Sinais , Olfato/genética , Transcriptoma
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