RESUMO
Proalgesic sensitization of peripheral nociceptors in painful syndromes is a complex molecular process poorly understood that involves mobilization of thermosensory receptors to the neuronal surface. However, whether recruitment of vesicular thermoTRP channels is a general mechanism underlying sensitization of all nociceptor types or is subtype-specific remains controversial. We report that sensitization-induced Ca(2+)-dependent exocytotic insertion of transient receptor potential vanilloid 1 (TRPV1) receptors to the neuronal plasma membrane is a mechanism specifically used by peptidergic nociceptors to potentiate their excitability. Notably, we found that TRPV1 is present in large dense-core vesicles (LDCVs) that were mobilized to the neuronal surface in response to a sensitizing insult. Deletion or silencing of calcitonin-gene-related peptide alpha (αCGRP) gene expression drastically reduced proalgesic TRPV1 potentiation in peptidergic nociceptors by abrogating its Ca(2+)-dependent exocytotic recruitment. These findings uncover a context-dependent molecular mechanism of TRPV1 algesic sensitization and a previously unrecognized role of αCGRP in LDCV mobilization in peptidergic nociceptors. Furthermore, these results imply that concurrent secretion of neuropeptides and channels in peptidergic C-type nociceptors facilitates a rapid modulation of pain signaling.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Exocitose/fisiologia , Nociceptores/metabolismo , Canais de Cátion TRPV/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Inativação Gênica , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Ratos , Ratos Wistar , Substância P/genéticaRESUMO
Ca(2+) channel ß subunits determine the maturation, biophysical properties and cell surface expression of high voltage-activated channels. Thus, we have analysed the expression, regional distribution and subcellular localization of the Ca(v) ß subunit family in mice from birth to adulthood. In the hippocampus and cerebellum, Ca(v) ß(1), Ca(v) ß(3) and Ca(v) ß(4) protein levels increased with age, although there were marked region- and developmental stage-specific differences in their expression. Ca(v) ß(1) was predominantly expressed in the strata oriens and radiatum of the hippocampus, and only weakly in the cerebellum. The Ca(v) ß(3) subunit was mainly expressed in the strata radiatum and lucidum of the hippocampus and in the molecular layer of the cerebellum. During development, Ca(v) ß(3) protein expression in the cerebellum peaked at postnatal days (P) 15 and 21, and had diminished drastically by P60, and in the hippocampus increased with age throughout all subfields. Ca(v) ß(4) protein was detected throughout the cerebellum, particularly in the molecular layer, and in contrast to the other subunits, Ca(v) ß(4) was mainly detected in the molecular layer and the hilus of the hippocampus. At the subcellular level, Ca(v) ß(1) and Ca(v) ß(3) were predominantly located post-synaptically in hippocampal pyramidal cells and cerebellar Purkinje cells. Ca(v) ß(4) subunits were detected in the pre-synaptic and post-synaptic compartments of both regions, albeit more strongly at post-synaptic sites. These results shed new light on the developmental regulation and subcellular localization of Ca(v) ß subunits, and their possible role in pre- and post-synaptic transmission.
Assuntos
Química Encefálica , Canais de Cálcio Tipo L/biossíntese , Canais de Cálcio/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Animais , Animais Recém-Nascidos , Química Encefálica/genética , Cerebelo/química , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Hipocampo/química , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Camundongos , Frações Subcelulares/química , Frações Subcelulares/metabolismoRESUMO
We investigated the temporal and spatial expression of SK2 in the developing mouse hippocampus using molecular and biochemical techniques, quantitative immunogold electron microscopy, and electrophysiology. The mRNA encoding SK2 was expressed in the developing and adult hippocampus. Western blotting and immunohistochemistry showed that SK2 protein increased with age. This was accompanied by a shift in subcellular localization. Early in development (P5), SK2 was predominantly localized to the endoplasmic reticulum in the pyramidal cell layer. But by P30 SK2 was almost exclusively expressed in the dendrites and spines. The level of SK2 at the postsynaptic density (PSD) also increased during development. In the adult, SK2 expression on the spine plasma membrane showed a proximal-to-distal gradient. Consistent with this redistribution and gradient of SK2, the selective SK channel blocker apamin increased evoked excitatory postsynaptic potentials (EPSPs) only in CA1 pyramidal neurons from mice older than P15. However, the effect of apamin on EPSPs was not different between synapses in proximal or distal stratum radiatum or stratum lacunosum-moleculare in adult. These results show a developmental increase and gradient in SK2-containing channel surface expression that underlie their influence on neurotransmission, and that may contribute to increased memory acquisition during early development.
Assuntos
Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/crescimento & desenvolvimento , Região CA1 Hipocampal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Canais de Potássio Ativados por Cálcio de Condutância Baixa/biossíntese , Animais , Animais Recém-Nascidos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sinapses/fisiologiaRESUMO
P-glycoprotein (P-gp) is a plasma membrane glycoprotein that has been signaled as a primary cause of multidrug resistance (MDR) in tumors. We performed a yeast 2-hybrid screen using the C-terminal domain of P-gp and identified 2 small GTPases involved in vesicular trafficking, Rab4 and Rab14, which complex with P-gp. The overexpression of GFP-Rab4, either transiently or stably, but not of Rab14, in K562ADR cells decreased the presence of P-gp in the cell surface. As a result, expression of this GTPase reduced the MDR phenotype of K562ADR cells, by augmenting the intracellular accumulation of daunomycin (DNM). This effect was mimicked by the constitutively active Rab4Q72L mutant, but not by the dominant negative Rab4S27N mutant. Rab4 regulated excocytotic P-gp trafficking to the plasma membrane from intracellular compartments, and this modulation required the interaction of both proteins and the GTPase activity. Noteworthy, K562ADR cells exhibited a significant reduction of Rab4 levels, but not of other Rab GTPases, as compared with the sensitive parental cell line, suggesting that the development of the MDR phenotype in these cells involves upregulation of P-gp and a concomitant downregulation of proteins that regulate its surface expression. Attenuation of endogenous Rab4 levels in K562ADR by RNA interference enhanced the expression of P-gp in the cell surface, and reduced the uptake of DNM. Accordingly, these findings substantiate the notion that modulation of the temporal and spatial distribution of P-gp in cancer cells may be a valid therapeutic strategy to alleviate the MDR phenotype, and signal to Rab4 as a potential target.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacologia , Western Blotting , Membrana Celular/efeitos dos fármacos , Daunorrubicina/metabolismo , Daunorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Endossomos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Células K562 , Microscopia Confocal , Dados de Sequência Molecular , Ligação Proteica , Interferência de RNA , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/genéticaRESUMO
ThermoTRP channels (thermoTRPs) define a subfamily of the transient receptor potential (TRP) channels that are activated by changes in the environmental temperature, from noxious cold to injurious heat. Acting as integrators of several stimuli and signalling pathways, dysfunction of these channels contributes to several pathological states. The surface expression of thermoTRPs is controlled by both, the constitutive and regulated vesicular trafficking. Modulation of receptor surface density during pathological processes is nowadays considered as an interesting therapeutic approach for management of diseases, such as chronic pain, in which an increased trafficking is associated with the pathological state. This review will focus on the recent advances trafficking of the thermoTRP channels, TRPV1, TRPV2, TRPV4, TRPM3, TRPM8 and TRPA1, into/from the plasma membrane. Particularly, regulated membrane insertion of thermoTRPs channels contributes to a fine tuning of final channel activity, and indeed, it has resulted in the development of novel therapeutic approaches with successful clinical results such as disruption of SNARE-dependent exocytosis by botulinum toxin or botulinomimetic peptides.
RESUMO
The design and synthesis of a library of novel families of 3-oxopiperazinium and perhydro-3-oxo-1,4-diazepinium derivatives is reported. The library was composed of 44 3-oxopiperazinium derivatives (11 of these compounds had a spiranic skeleton) and 22 perhydro-3-oxo-1,4-diazepinium compounds. The synthetic procedure involved a 6-step sequence carried out in solution, along with the use of solid-phase linked scavengers and microwave activation for the rapid removal of the excess of amine reagents. A final cyclization step performed under mild conditions led to the charged heterocyclic moiety. Screening of this library in two biological assays identified active compounds that inhibit the activity of the vanilloid receptor TRPV1 and modulators of the multidrug resistance phenomenon. Thus, this synthetic sequence represents a facile and convenient entry to unprecedented libraries of this sort of tetraalkylammonium derivatives that may be of use for identification of novel scaffolds of diverse biological activity.