| Recent studies suggest that glia cells play an important role in nervesystem. Like astrocytes in central nerve system, satellite glia cells (SGCs)participate in the physiological as well as the pathological process ofperipheral nerve system. SGCs affect neuronal activity through glia-neuroninteractions. Recent studies suggest that the volumn activated chloride channel(VACC) may be vital to the communication between neuron and satellite glialcell, possibly mediated by release of glutamate which regulate the interactionbetween neuron and satellite glial cell.As a subtype of chloride channel, the VACC have been a focus of study.These channels are involved in a variety of cell functions, including cardiacrhythm, cell proliferation and differentiation, cell volume regulation and celldeath through apoptosis. Despite its relatively well characterizedelectrophysiological properties, the molecular basis and regulation mechanismof the VACC are not totally elucidated. Research on VACC of nerve systemwill help us understand the basic pathology process of some diseases such aspain.Part one Expression and molecular basis of swelling-activated chloridechannel in satellite glia cellObjective：To study the expression and the molecular basis of VACC inrat DRG satellite glia cells.Meteod：(1) The VACC was recorded by whole-cell patch clamp.(2)Pharmacological tools were used to envalue the roles of calcium andpurinergic receptors in activation of VACC.(3) siRNA was used to envalue theroel of TMEM16A in the moleular basis of VACC.Results：(1) Rat SGCs expresses VACCThe whole-cell patch clamp recordings showed that hypertonic pipette solution induced an inward current when the membrane was held at-60mV.This inward current was blocked by100μM tannic acid, a chloride channelblocker. This inward current disappeared when the Cl-in the hypertonicpipette solution was replaced by SO2-4. Thus SGCs expresses VACC.(2) The activation of VACC invoves release of intracellular Ca2+The VACC in rat SGCs were reduced by96.9%±2.5%(P<0.01, n=9) and80.8%±17.2%(P<0.01, n=8), respectively when the low concentration ofintracellular EGTA in hypertonic pipette solution was replaced with highconcentrations of BAPTA or EGTA.(3)The activation of VACC does not involve activation of P2ATPreceptorsThe VACC in rat SGCs were not significantly affected by100μMsuramin (P>0.05, n=5), a P2receptor blocker.(4)TMEM16A is possibly the molecular basis of VACCThe VACC in SGCs transfected with si-RNA for TMEM16A werereduced by79.6%±10%compared with the control (P<0.01, n=8).Conclution: The results from whole-cell patch clamp recordings suggestthat the rat SGCs expresses VACC. The VACC are activated by the increaseof cell volume involving release of intracellular calcium. And TMEM16Aproteins appear to be a crucial component of the VACC in rat SGCs.Part Two Expression of phospholipase C in rat dorsal root ganglionObjective： To study the expression profile of phospholipase Cβsubfamily (PLC-β) in rat dorsal toot ganglion (DRG). To establish afoundation for future studying the role of PLC-β in the activation of VACC.Method: Real time PCR technique (qPCR);Immunohistochemistry-Frozen section technique; Western blotting.Result:(1) The results of qPCR showed that PLC-β3was detected withparticularly strong expression in rat DRG. PLC-β4and PLC-β1were alsodetected with a relatively high expression level. The expression of PLC-β2was low. (2) The result obtained with the Immunohistochemistry-Frozen sectiontechnique showed that PLC-β3, PLC-β4were expressed in most of the DRGneurons, however, the expression of PLC-β1and β2was rare.(3) The results of Western blotting showed that the proteins of PLC-β3and PLC-β4were expressed in a high level, but that of PLC-β1and β2waslow.Conclution: The above results indicate that the major isoforms of PLC-βfamily expressed rat DRG are PLC-β3and PLC-β4. |
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