Nerve Growth Factor Sensitizes The Release Of Substance P Evoked By Capsaicin From Dorsal Root Ganglion Neurons

Among many neurotrophic factors that act on sensory neurons, nerve growth factor (NGF) has been studied extensively. NGF initially interested neurobiologists because of its effects in the developing nervous system of the survival, differentiation and maturation. It is now clear that NGF functions throughout the life of the animal with a wide repertoire of actions. In the course of the last years, several lines of evidence converged to indicate that NGF participates in structural and functional plasticity of the dorsal root ganglion (DRG). NGF may induct the intracellular events, such as mitochondrial transport or accumulate at regions of focal NGF stimulation, and intracellular Ca²⁺ homeostasis. Recently, NGF was suggested to exert an acute effect on nociceptive sensory neurons in addition to its trophic effect. NGF produces sensitization of nociceptive responses and increases capsaicin sensitivity of primary sensory neurons. Levels of NGF are increased in inflamed tissues, and this effect may contribute to the role of NGF in inflammatory hyperalgesia. Neuropeptide release induced by capsaicin is also related to pain sensition. The modulation function of capsaicin is through the activation of Vanilloid receptor 1 (VR1) which expressed in the plasma membrance of primary sensory neurons to cause peptidic neurotransmitter release. Substance P (SP), one of peptidic neurotransmitters in primary sensory neurons, acitivates the upper sensory neurons to transmit pain sensition signal from peripheral to central nervous system. Apart from involved in nociceptive transmission, SP is also involved in inflammatory responses. In the course of inflammatory responses, the relationship between inflammatory hyperalgesia contributed by NGF and neuropeptide release induced by capsaicin is unclear at this moment. Dissociated DRG cell culture model was established in the present study. DRG cultures were exposed to different concentrations of NGF. Seventy-two hours after incubation, cultured DRG neurons were processed for RT-PCR to analyze SP mRNA and VR1 mRNA levels, radioimmunoassay (RIA) to detect the amount of the SP release before and after capsaicin stimulation, Confocal laser scanning microscopy (CLSM) to monitor intracelluar calcium levels before and after capsaicin stimulation. The actions and mechanisms of NGF on SP release from DRG neurons evoked by capsaicin were analyzed according to the data obtained in this experiment.The results are as follows: (1) The networks formed by nerve fibers in NGF incubated DRG cultures were more densed than that in control group without NGF. (2) The number of double labeled DRG neurons by microtubule-associated protein 2 (MAP2) and 4',6-Diamidino-2-phenylindole (DAPI) increased in NGF incubated DRG cultures as compared with that in control group without NGF. (3) The amount of SP mRNA expression in NGF-treated DRG neurons increased as compared with that in control group without NGF. In NGF-treated DRG neurons, the amount of SP mRNA expression increased in a dose-dependent manner. (4) The amount of VR1 mRNA expression in NGF-treated DRG neurons increased as compared with that in control group without NGF. In NGF-treated DRG neurons, the amount of VR1 mRNA expression increased in a dose-dependent manner. (5) In NGF-treated DRG neurons, capsaicin-evoked SP release increased as compared with that in control group. The amount of SP release evoked by capsaicin increased in a dose-dependent manner with NGF treatment. (6) The intracelluar calcium fluorescent intensity in DRG neurons increased significantly after capsaicin treatment.The results indicated that (1) NGF could promote neuronal growth and survival and increase the number of neurons in dissociated DRG cell cultures. (2) NGF could promote SP mRNA expression and may increase SP synthesis and SP content in DRG neurons. (3) NGF could promote VR1 mRNA expression and may increase capsaicin sensitivity of DRG neurons. (4) Capsaicin had a highly significant effect on SP release and the addition of NGF significantly affected the response to capsaicin. The mechanisms may be that NGF improved the functional status, elevated SP levels, and increased capsaicin sensitivity of DRG neurons. (5) Capsaicin could stimulated intracelluar calcium concentration increase which may promote peptidic neurotransmitter release from DRG neurons.DRG neurons had responsiveness to NGF. The mechanisms of NGF increased neurotransmitter release evoked by capsaicin may be that: (1) NGF increased the number of DRG neurons, (2) NGF improved the neuronal status and promoted neurotransmitter synthesis, and (3) NGF promoted VR1 expression or might increase VR1 sensitivity.