RESUMEN
Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons. We studied HCN1-4 channel mRNA and protein expression and correlated these findings with I(h) functional properties in rat DRG neurons of different size. Quantitative RT-PCR (TaqMan) analysis demonstrated that HCN2 and HCN1 mRNAs were more abundantly expressed in large diameter (55-80 microm) neurons, while HCN3 mRNA was preferentially expressed in small diameter (20-30 microm) neurons. HCN4 mRNA expression was very low in neurons of all sizes. At the protein level, subunit-selective polyclonal antibodies and immunofluorescence indicated that HCN1 and HCN3 are present in large diameter neurons and small diameter neurons. Staining in small diameter neurons was in IB4-positive (non-peptidergic) and IB4-negative (peptidergic) cells. HCN2 immunofluorescent staining was heterogeneous and predominantly in large diameter neurons and in small diameter IB4-negative neurons. HCN4 was poorly expressed in all neurons. Functionally, I(h) amplitude and density were significantly larger, and activation kinetics faster, in large diameter neurons when compared with small neurons. I(h) activation rates in small and large diameter DRG neurons were consistent with the relative abundance of HCN subunits in the respective cell type, considering the reported HCN channel activation rates in heterologous systems (HCN1>HCN2 approximately HCN3>HCN4), suggesting exclusivity of roles of different HCN subunits contributing to the excitability of DRG neurons of different size. Additionally, a functional role of I(h) in small DRG neuron excitability was evaluated using a computational model.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Ganglios Espinales/citología , Neuronas/clasificación , Neuronas/fisiología , ARN Mensajero/metabolismo , Animales , Células Cultivadas , Simulación por Computador , Canales Catiónicos Regulados por Nucleótidos Cíclicos/clasificación , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Lectinas/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Modelos Neurológicos , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K(d) = 1.13 nm) and NPFF2 (K(d) = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.
Asunto(s)
Arginina/análogos & derivados , Oligopéptidos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Neuropéptido/química , Receptores de Neuropéptido/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Sitios de Unión , Encéfalo/metabolismo , Células COS , Calcio/metabolismo , Mapeo Cromosómico , Clonación Molecular , AMP Cíclico/metabolismo , ADN Complementario/metabolismo , Electrofisiología , Biblioteca de Genes , Humanos , Cinética , Ligandos , Datos de Secuencia Molecular , Oocitos , Fosfatidilinositoles/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Ratas , Receptores de Superficie Celular/química , Receptores de Neuropéptido/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Distribución Tisular , XenopusRESUMEN
Two structurally related, G-protein-coupled receptors were identified as receptors for the neuropeptide, neuromedin U. This peptide is found in highest levels in the gut and genitourinary system where it potently contracts smooth muscle but is also expressed in the spinal cord and discrete regions of the brain. Binding sites for neuromedin U have been characterized in rat uterus, however, little is known about the activity of this peptide in the regions of the central nervous system where it is expressed. The receptors characterized in this report are activated by neuromedin U at nanomolar potency in heterologous expression systems and bind radiolabeled neuromedin U with high affinity. Localization of the receptor RNA by quantitative reverse transcription-polymerase chain reaction in a variety of human tissues shows distinct expression patterns for the two receptors. NMU1 is expressed predominantly in peripheral tissues, whereas NMU2 is more highly expressed in the central nervous system. Identification of neuromedin U receptor subtypes will greatly aid in the determination of the physiological roles of this peptide.