RESUMO
Tissue injury generates endogenous factors that heighten our sense of pain by increasing the response of sensory nerve endings to noxious stimuli. Bradykinin and nerve growth factor (NGF) are two such pro-algesic agents that activate G-protein-coupled (BK2) and tyrosine kinase (TrkA) receptors, respectively, to stimulate phospholipase C (PLC) signalling pathways in primary afferent neurons. How these actions produce sensitization to physical or chemical stimuli has not been elucidated at the molecular level. Here, we show that bradykinin- or NGF-mediated potentiation of thermal sensitivity in vivo requires expression of VR1, a heat-activated ion channel on sensory neurons. Diminution of plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) levels through antibody sequestration or PLC-mediated hydrolysis mimics the potentiating effects of bradykinin or NGF at the cellular level. Moreover, recruitment of PLC-gamma to TrkA is essential for NGF-mediated potentiation of channel activity, and biochemical studies suggest that VR1 associates with this complex. These studies delineate a biochemical mechanism through which bradykinin and NGF produce hypersensitivity and might explain how the activation of PLC signalling systems regulates other members of the TRP channel family.
Assuntos
Bradicinina/fisiologia , Fator de Crescimento Neural/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Receptores de Droga/fisiologia , Animais , Linhagem Celular , Eletrofisiologia , Ativação Enzimática , Feminino , Temperatura Alta , Masculino , Camundongos , Nociceptores/metabolismo , Oócitos/fisiologia , Dor , Proteína Quinase C/metabolismo , Receptor trkA/fisiologia , Receptores de Droga/genética , Transdução de Sinais , Fosfolipases Tipo C/fisiologia , Xenopus laevisRESUMO
Mice deficient in src and fyn or src and yes move and breathe poorly and die perinatally, consistent with defects in neuromuscular function. Src and Fyn are associated with acetylcholine receptors (AChRs) in muscle cells, and Src and Yes can act downstream of ErbB2, suggesting roles for Src family kinases in signaling pathways regulating neuromuscular synapse formation. We studied neuromuscular synapses in src(-/-); fyn(-/-) and src(-/-); yes(-/-) mutant mice and found that muscle development, motor axon pathfinding, clustering of postsynaptic proteins, and synapse-specific transcription are normal in these double mutants, showing that these pairs of kinases are not required for early steps in synapse formation. We generated muscle cell lines lacking src and fyn and found that neural agrin and laminin-1 induced normal clustering of AChRs and that agrin induced normal tyrosine phosphorylation of the AChR beta subunit in the absence of Src and Fyn. Another Src family member, most likely Yes, was associated with AChRs and phosphorylated by agrin in myotubes lacking Src and Fyn, indicating that Yes may compensate for the loss of Src and Fyn. Nevertheless, PP1 and PP2, inhibitors of Src-class kinases, did not inhibit agrin signaling, suggesting that Src class kinase activity is dispensable for agrin-induced clustering and tyrosine phosphorylation of AChRs. AChR clusters, however, were less stable in myotubes lacking Src and Fyn but not in PP1- or PP2-treated wild-type cells. These data show that the stabilization of agrin-induced AChR clusters requires Src and Fyn and suggest that the adaptor activities, rather than the kinase activities, of these kinases are essential for this stabilization.
Assuntos
Agrina/metabolismo , Junção Neuromuscular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Agregação de Receptores/fisiologia , Receptores Colinérgicos/metabolismo , Agrina/farmacologia , Animais , Axônios/fisiologia , Células Cultivadas , Diafragma/citologia , Diafragma/embriologia , Diafragma/inervação , Diafragma/metabolismo , Laminina/metabolismo , Laminina/farmacologia , Camundongos , Camundongos Mutantes , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Músculo Esquelético/inervação , Músculo Esquelético/metabolismo , Junção Neuromuscular/embriologia , Fosforilação/efeitos dos fármacos , Subunidades Proteicas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Proteínas Proto-Oncogênicas c-yes , Receptores Colinérgicos/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transcrição Gênica , Quinases da Família src/metabolismoRESUMO
DNA topoisomerase IIbeta is shown to have an unsuspected and critical role in neural development. Neurogenesis was normal in IIbeta mutant mice, but motor axons failed to contact skeletal muscles, and sensory axons failed to enter the spinal cord. Despite an absence of innervation, clusters of acetylcholine receptors were concentrated in the central region of skeletal muscles, thereby revealing patterning mechanisms that are autonomous to skeletal muscle. The defects in motor axon growth in IIbeta mutant mice resulted in a breathing impairment and death of the pups shortly after birth.