| Ghrelin,an endogenous brain-gut peptide,is the only endogenous ligand of the growth hormone secretagogue receptor(GHS-R).Ghrelin is secreted by the stomach,hypothalamus,pituitary and many tissues,and its receptors are also widely distributed in the human body.After combining with GHS-R,ghrelin exerts a series of biological effects through activating multiple signal transduction pathways.Studies in recent years have shown that,ghrelin has the functions of promoting growth hormone secretion,enhancing appetite,regulating energy metabolism,and affecting the function of the cardiovascular system.Moreover,ghrelin is also involved in regulating the functions of the central nervous system and plays an important role in learning and memory,sleep arousal,mood regulation,reward behavior,and neurodegenerative diseases.Parkinson's disease(PD)is a common neurodegenerative disease in daily life.PD patients can exhibit motor symptoms such as resting tremors,muscle rigidity,and bradykinesia.The pathological feature of PD is the degeneration of dopamine(DA)neurons on substantia nigra pars compacta(SNc)and the decrease of DA in striatum.There is still no effective method to treat the progressive loss of DAergic neurons in the pathological process of PD.As an excitable neuron,the excitability of DAergic neurons in the SNc that supply the striatum with DA determines the function of nigrostriatal systemfor motor coordination.Therefore,elucidating the mechanism of regulation and influencing factors of the electrical activity of substantia nigra neurons has important implications for the further study of the pathogenesis of PD and the screening of natural drugs or the development of synthetic drugs.Our previous findings have revealed a novel mechanism by which ghrelin enhances DAergic neuronal firing by inhibiting native Kv7/KCNQ/M channelsthrough GHS-R-PLC-PKC pathway.However,the native Kv7/KCNQ/M current in DAnegic neurons is small,only about 30 p A.Whether the other potassium channels are involved remain unknown.It is known that there are various voltage-dependent potassium channels on the DAergic neurons of the substantia nigra.Among them,the transient K+ channel,namely the A-type potassium channel,is a key factor affecting the spontaneous discharge of DAergic neurons.Previous studies show that ghrelin inhibits A-type potassium channel on cardiomyocytes by activating the PLC-PKC signaling pathway,thereby prolonging the duration of action potentials.However,whether A-type potassium channels are also involved in the ghrelin-induced excitability of DAergic neurons still remain unclear.In this study,we focus on A-type potassium channels(IA),which has a wide expression on dopaminergic neurons and play a key role in pacemaker control.Brain slices of the SNc were prepared from C57BL/6 mice of postnatal 15-20 days.The effects of ghrelin on discharge frequency and IA current of dopaminergic neurons were observed by whole cell patch clamp technique.The results are as follows:1.On substantia nigra DA neurons,application of 100 n M ghrelin increases excitability of dopaminergic neurons.The spike frequency increased from1.84±0.24 Hz to 2.63±0.44 Hz(P(27)0.05).Pretreatment with 4-aminopyridine(4-aminopyride,4-AP,1 m M),a specific inhibitor of A-type potassium channel,the spike frequency increased from1.46±0.21 Hz to 2.71±0.98 Hz(P(27)0.001),further co-application of 4-AP(1m M)and ghrelin(100n M)had no additional effect on neuronal firing(the frequency of neurons was 2.74±0.46 Hz)2.On substantia nigra DA neurons,application of 100 n M ghrelin significantly suppressed the current amplitude of IA from 333.68±143.76 p A to 153.43±167.87 p A(P(27)0.001).Pre-application of 4-AP reduced the amplitude of IA currents on DA neurons from 363.67±154 p A to 77±41 p A(P<0.001),further co-application of 4-AP and ghrelin had no effect on IA(the IA current was 64±23 p A).3.The use of 50 ?M GHS-R1 a blocker D-[Lys3]-GHRP-6 alone had no effect on the amplitude of IA current on DA neurons and could block the inhibitory effect of ghrelin on IA.4.The use of 1 mM PKA inhibitor H89 alone had no effect on IA current amplitude and spontaneous firing frequency of DA neurons,and it could not block the effect of ghrelin on IAcurrent amplitude and spontaneous firing frequency.5.The use of 10 ?M PLC inhibitor U-73122 alone had no effect on the amplitude of IA current on DA neurons,and could not block the inhibitory effect of ghrelin on IA.6.The use of 5 ?M PKC blocker GF109203 X alone had no effect on the amplitude and spontaneous firing frequency of IA on DA neurons,and could block the effect of ghrelin on IA current amplitude and spontaneous firing frequency.7.The use of 10 ?M PKC? isoforms inhibitor Rottlerin alone,had no effect on IA current amplitude and spontaneous firing frequency of DA neurons,and could block the effects of ghrelin on IA current amplitude and spontaneous firing frequency.8.When MES23.5 cells were treated with 10 ?M Rottlerin and 100 n M ghrelin was added for 1 h,the phosphorylation level of PKC?was significantly lower than that of ghrelin alone,which further proved that PKC?participates in the regulation of ghrelin neurons.The above results show that ghrelin enhances neuronal excitability by inhibiting A-type potassium channels on substantia nigra DA neurons.The possible mechanisms are as follows: Ghrelin combines with its receptor GHS-R1 a and activates intracellular PLC-PKC signaling pathway,thereby inhibiting A-type potassium channel current,and further confirmed that ghrelin exerts a regulatory role by activating a novel PKC? subtype in PKC,While the c AMP-PKA signaling pathway is not involved the effect of ghrelin on IA current amplitude and spontaneous firing frequency.In summary,this study aimed at the detailed study of the mechanism of ghrelin's regulation of neuronal electrical activity,and provided a new experimental basis for further elucidating the neuroprotective effect of ghrelin in PD.It will have a great application prospect in the treatment of PD. |
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