The Effect Of Activation Of TRPV4 Receptor On Hippocampal Neuron Voltage-gated Sodium Channel Current And ?-aminobutyric Acid Activation Current And Its Mechanism

BackgroundTransient receptor potential vanilloid 4(TRPV4)is a member of transient receptor potential superfamily.TRPV4 is widely expressed in the nervous system.For example,TRPV4 is abundant in the nociceptors in the peripheral nervous system and is expressed in hippocampus,cortex,thalamus and cerebellum in the central nervous system.TRPV4 is a multipolar-activated receptor that can be activated by cell edema,mild heat,arachidonic acid and its metabolism,etc.Activation of TRPV4 induces the inward current that is mainly carried by calcium and results in an increase of intracellular free calcium concentration and the depolarization of cell membrane.TRPV4 has been proven to participate in the modulation of resting membrane potential of hippocampal neurons.Application of TRPV4 agonist increases the spontaneous firing frequency in retinal ganglion cells and the evoked action potential number in trigeminal ganglion neurons,indicating that activation of TRPV4 may increases the cellular excitability.However,the mechanisms underlying TRPV4-increased cellular excitability remain unclear.Voltage-gated sodium channel(VGSC)is of great importance in initiating and propagating action potential(AP)in excitable cells.According to the sensitivity to tetrodotoxin(TTX),VGSC is divided into TTX-sensitive(TTX-S)channels and TTX-resistant(TTX-R)channels.In neurons,TTX-S plays a primary role in the depolarization of AP.It has been proven that activation of TRPV4 can modulate VGSC in the nociceptors.By now,there is lack of the evidence about the effect of TRPV4 activation on VGSC in the central nervous system.Besides the voltage-gated ion channels,the balance between excitatory and inhibitory neurotransmitter systems is also important for the modulation of neuronal excitability in the central nervous system.Glutamate is the major excitatory neurotransmitter in the central nervous system and in our previous study activation of TRPV4 may enhance the function of glutamate receptors and facilitate the excitatory synaptic transmission.?-aminobutyric acid(GABA)is the major inhibitory neurotransmitter in the adult CNS.GABAA receptors are ligand-gated chloride ion channels that mediate most of the inhibitory activity in the brain.Blockage of GABAA receptor results in an increase of neuronal excitability.It has been reported that protease-activated receptor-2(PAR-2)agonist and hyperthermia,two factors that are related to the activation of TRPV4,reduce GABA-mediated current and GABAergic synaptic transmission.However,there is no direct evidence for TRPV4-induced modulation of GABA receptors.VGSC and GABAA receptors can be modulated by phosphoryaltion.For example,VGSC can be inhibited by activation of protein kinase A(PKA)or protein kinase C(PKC)signaling pathway.Additional modulation of VGSC by activation of p38 mitogen-activated protein kinase(p38 MAPK)is also reported in neurons or transfected mammalian cells.GABAA receptor subunits contain phosphorylation sites for PKC,PKA,Ca2+/calmodulin-dependent protein kinase ?(CaMK?)and phosphatidyl inositol 3 kinase(PI3K).We previously reported that PKA,PKC and CaMK? signaling pathways were involved in TRPV4-induced modulation of some voltage-gated ion channels and glutamate receptors.We also found that activation of TRPV4 increased the protein level of p38 MAPK and decrease that of phosphorylated protein kinase B(p-Akt).Based on the above reports and our previous study,the present study proposes that activation of TRPV4 may modulate VGSC and/or GABAA receptor through intracellular signaling pathway(PKA,PKC,CaMK?,PI3K,etc.).The results are expected to reveal the mechanism underlying TRPV4-regulated neuronal excitability.Objective1.To determine the effect of TRPV4 activation on voltage-gated sodium current(INa)in hippocampal CA1 pyramidal neurons and its underlying mechanisms.2.To determine the effect of TRPV4 activation on GABA-evoked current(IGABA)in hippocampal CA1 pyramidal neurons and its underlying mechanisms.Part ?.Effect of TRPV4 activation on VGSC in hippocampal CA1 pyramidal neurons and its underlying mechanismsMaterials and Methods1.Whole cell patch clamp recording:hippocampal slices were prepared and INa was recorded to study the effect of TRPV4 activation on INa.2.Drug treatment:TRPV4 agonist GSK1016790A was intracerebroventricularly injected to prepare for the in vivo model of TRPV4 activation.3.Western blot:Western blot analysis was performed to examine the changes of VGSC subunits(Nav 1.1,Nav 1.2,Nav 1.3,Nav 1.6 and Nav ?1)expression in GSK1016790A-injected mice.Results1.INa was inhibited by acute application of GSK1016790A and 4?-PDD in hippocampal CA1 pyramidal neurons.In the presence of TRPV4 agonists,voltage-dependent inactivation curve shifted to the hyperpolarizing direction,whereas voltage-dependent activation curve remained unchanged.2.TRPV4 agonist-induced inhibition in INa was blocked by TRPV4 antagonist and tetrodotoxin.3.Blockage of PKA markedly attenuated GSK1016790A-induced inhibition in INa,whereas antagonist of PKC or p38 MAPK had no effect on GSK1016790A-action.4.The protein levels of Nav 1.1,Nav 1.2 and Nav 1.6 in hippocampus increased in GSK1016790A-injected mice,while that of Nav 1.3 or Nav ?1 almost unchanged.ConclusionINa is inhibited by acute activation of TRPV4 through PKA signaling pathway in hippocampal pyramidal neurons,which is not involved in TRPV4-increased neuronal excitability;the protein expression for ?-subunit of VGSC is increased by sustained activation of TRPV4,which provides a possibility for hyper-excitability upon sustained TRPV4-activation.Part ?.Effect of TRPV4 activation on GABAA receptor in hippocampal CA1 pyramidal neurons and its underlying mechanismsMaterials and methods1.Whole cell patch clamp recording:hippocampal slices were prepared and IGABA was recorded to study the effect of TRPV4 agonist on IGABA.2.Western blot:hippocampal slices were incubated with GSK1016790A for 15min,30min,1h and 2h,respectively.Western blot analysis was performed at different time point after GSK1016790A treatment to examine the effect of TRPV4 activation on changes in Adenosine5'-monophosphate-activated protein kinase(AMPK),phosphorylated AMPK(p-AMPK),protein kinase B(Akt)and phosphorylated protein kinase B(p-Akt)protein levels in hippocampi.Results1.IGABA was inhibited by the application of synthetic(GSK1016790A and 4?-PDD)and endogenous agonists(5,6-EET)of TRPV4.2.TRPV4 agonist-induced inhibition in IGABA was blocked by specific antagonists of TRPV4 and of GABAA receptors.3.GSK1016790A increased the phosphorylated AMP-activated protein kinase(p-AMPK)and decreased the phosphorylated protein kinase B(p-Akt)protein levels,which was attenuated by the removal of extracellular calcium or by treatment with a calcium/calmodulin-dependent protein kinase kinase-?antagonist.Additionally,the GSK1016790A-induced decrease in p-Akt protein level was sensitive to an AMPK antagonist.4.GSK1016790A-inhibited IGABAwas blocked by either an AMPK antagonist or a PI3K agonist.GSK1016790A-induced inhibition of IGABA was also significantly attenuated by a PKC antagonist but was unaffected by PKA or CaMK?antagonists.ConclusionTRPV4 activation inhibits GABAa receptor function,which may be mediated by activation of AMPK and subsequent down-regulation of PI3K/Akt signaling and activation of PKC signaling.Inhibition of GABAa receptors may account for the increase in neuronal excitability caused by TRPV4 activation.