RESUMO
The FERONIA (FER) signaling pathway is known to have diverse roles in Arabidopsis thaliana, such as growth, reproduction, and defense, but how this receptor kinase is involved in various biological processes is not well established. In this work, we applied multiple mass spectrometry techniques to identify metabolites involved in the FER signaling pathway and to understand their biological roles. A direct infusion Fourier transform ion cyclotron resonance (FT-ICR)-MS approach was used for initial screening of wild-type and feronia (fer) mutant plant extracts, and Arabidopsides were found to be significantly enriched in the mutant. As Arabidopsides are known to be induced by wounding, further experiments on wounded and non-wounded leaf samples were carried out to investigate these oxylipins as well as related phytohormones using a quadrupole-time-of-flight (Q-TOF) MS by direct injection and LC-MS/MS. In a root growth bioassay with Arabidopside A isolated from fer mutants, the wild-type showed significant root growth inhibition compared with the fer mutant. Our results therefore implicated Arabidopsides, and Arabidopside A specifically, in FER functions and/or signaling. Finally, matrix-assisted laser desorption/ionization MS imaging (MALDI-MSI) was used to visualize the localization of Arabidopsides, and we confirmed that Arabidopsides are highly abundant at wounding sites in both wild-type and fer mutant leaves. More significantly, five micron high-spatial resolution MALDI-MSI revealed that Arabidopsides are localized to the chloroplasts where many stress signaling molecules are made.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Galactolipídeos/metabolismo , Oxilipinas/metabolismo , Feofitinas/metabolismo , Fosfotransferases/genética , Transdução de Sinais/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Biomarcadores/metabolismo , Cloroplastos/metabolismo , Cromatografia Líquida , Mutação , Fosfotransferases/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Hyperpolarization- and Cyclic Nucleotide-gated (HCN) channels are a family of six transmembrane domain, single pore-loop, hyperpolarization activated, non-selective cation channels. The HCN family consists of four members (HCN1-4). HCN channels represent the molecular correlates of I(h) (also known as 'funny' I(f) and 'queer' I(q)), a hyperpolarization-activated current best known for its role in controlling heart rate and in the regulation of neuronal resting membrane potential and excitability. A significant body of molecular and pharmacological evidence is now emerging to support a role for these channels in the function of sensory neurons and pain sensation, particularly pain associated with nerve or tissue injury. As such, HCN channels may represent valid targets for novel analgesic agents. This evidence will be reviewed in this article. We will then summarize our efforts to develop and validate methods for screening for novel HCN channel blockers.
Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/efeitos dos fármacos , Descoberta de Drogas/métodos , Canais de Potássio/efeitos dos fármacos , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por HiperpolarizaçãoRESUMO
The cloning of novel G protein-coupled receptors and the search for their natural ligands, a process called reverse pharmacology, is an excellent opportunity to discover novel hormones and neurotransmitters. Based on a degenerate primer approach we have cloned a G protein-coupled receptor whose mRNA expression profile indicates highest expression in the dorsal root ganglia, specifically in the subset of small neurons, suggesting a role in nociception. In addition, moderate expression was found in lung, hypothalamus, peripheral blood leukocytes, and ovaries. Guided by a receptor-activation bioassay, we identified adenine as the endogenous ligand, which activated the receptor potently and with high structural stringency. Therefore, we propose to name this receptor as the adenine receptor. Hormonal functions have already been demonstrated for adenine derivatives like 6-benzylaminopurine in plants and 1-methyladenine in lower animals. Here, we demonstrate that adenine functions as a signaling molecule in mammals. This finding adds a third family besides P1 and P2 receptors to the class of purinergic receptors.