Effects Of Glial Cell Line-derived Neurotrophic Growth Factor On Substance P Release From Cultured Dorsal Root Ganglion Neurons And Its Related Mechanisms

Glial cell line-derived neurotrophic factor(GDNF),a member of the transforming growth factor-βfamily,was reported as a multifunctional trophic factor for neurons which has extensive expression in vivo.GDNF has been shown to have neurotrophic actions on different types of central and peripheral nervous system neurons in vitro.Several lines of evidence converged to indicate that GDNF participates in structural and functional plasticity of the dorsal root ganglia(DRG) and spinal cord.Glial cell line-derived neurotrophic growth factor(GDNF)may induct the intracellular events,such as mitochondrial transport or accumulate at regions of focal GDNF stimulation,and intracellular Ca²⁺homeostasis.However, it's not clear that GDNF has a special action on normal DRG neurons.Capsaicin could depolarize DRG neurons and evoke the release of the neuropeptides such as substance P(SP)and calcitonin gene-related peptide(CGRP). The capsaicin-evoked release of SP and CGRP also increased in a dose-dependent manner.Vanilloid receptor(VR1)is extensively expressed in DRG neurons.The expression pattern of VR1 may be associated to the capsaicin responsiveness of sensory neurons and pain sensation.SP,one of peptidic neurotransmitters inprimary sensory neurons,could be found in the central and peripheral nervous system,activates the upper sensory neurons to transmit pain signal from peripheral to central nervous system.It is also involved in inflammatory responses.The function of DRG neurons could be shown by the amount of the released neurotransmitter.The low-dose of capsaicin could cause peptidic neurotransmitter release.However,GDNF's actual physiological role in the release of SP remains obscure.Therefore,in this study we exert different concentrations of GDNF on dissociated cultured DRG neurons to determine the effects of GDNF on SP release from cultured DRG neurons.Primary cultured dissociated DRG cells were from Wistar rat embryos at 15d of gestational age.DRG neurons were cultured without or with GDNF(5 ng/ml,10 ng/ml,50 ng/ml)for 72 h.The living cells were observed with an inverted contrast microscope.At 72 h of culture age,the cultures were processed for microtubule associated protein 2(MAP2)and 4',6-Diamidino-2-phenylindole(DAPI)fluorescent double staining to count the neuron numbers.The amount of SP basal release and evoked by capsaicin was analyzed by radioimmunoassay(RIA).The expression of SP peptide and VR1 protein was examined by Western blot.In the present study,it is discussed that how could GDNF effect on the release of SP in DRG neurons.The results are as follows:(1)The results of the morphology character of DRG neurons observed by inverted phase contrast microscope:The single layered DRG neurons sent neurites to form a network in normal control group;The living status of DRG cells with GDNF incubation was better than that no GDNF incubated cultures(2)The results of MAP2 and DAPI double staining:Fluorescent photomicrographs of MAP2 and DAPI double staining of dissociated DRG neurons at 72 h of culture age show that the more GDNF in cultures,the more number of DRG they have.(3)The results of DRG neurons count:The number of DRG neurons per visal field increased in GDNF treated cultures compared with that in the cultures without GDNF(P＜0.05).(4)The results of the amount of SP release:The amount of SP basal release and evoked by capsaicin increased significantly in GDNF treated cultures compared with that in the cultures without GDNF,and the increased amount of SP release evoked by capsaicin is more than the increased amount of SP basal release.GDNF increased the capsaicin-evoked SP release in a dose-dependent manner.(5)The results of SP expression:The levels of SP peptide expression increased in GDNF treated cultures compared with that in the cultures without GDNF.(6)The results of VR1 expression:The levels of VR1 protein expression increased after GDNF treatment.These results indicated that GDNF may improve the survival of cultured DRG neurons and promote SP basal release and sensitize capsaicin evoked SP release from DRG neurons.