Effects Of L Type Calcium Channels On Iron Accumulation And Dopamine Neuron Degeneration In The Subtantia Nigra

Iron is an important co-factor for the maintenance of cellular function,and it extensively participates in the metabolic processes in the body.However,when the dynamic balance of iron homeostasis is damaged,it will lead to mitochondrial dysfunction,lipid peroxidation,DNA breakage and protein damage.Growing evidences indicate that iron accumulation in the substantia nigra?SN?is involved in the pathogenesis of Parkinson's disease?PD?.To date,the precise mechanisms underlying iron selective accumulation in the SN and iron-mediated neuronal toxicity remains unclear.Excessive iron can react with hydrogen peroxide to produce hydroxyl radicals by Fenton reaction and thus aggravating oxidative stress.In addition,growing evidences indicate that iron accumulation is associated with neurodegenerative diseases.There is evidence that L-type Ca²⁺channels?LTCCs?might mediate Fe²⁺entry into DA neurons.Blockage of LTCCs by specific antagonists may delay the onset of PD.However,the effects and underlying mechanisms of LTCCs on the pathogenesis of PD are not fully understood.It is known that midbrain dopamine?DA?neurons are autonomic pace-making when there is no excitatory postsynaptic current input.The autonomic pace-making mainly depend on L-type Cav1.3 Ca²⁺channels.It has been reported that LTCCs may mediate iron entry into cardiomyocytes under high-iron condition.To study the underlying mechanisms of LTCCs mediated Fe²⁺entering into DA neurons,and the effects of Ca²⁺on Fe²⁺-induced neurotoxicity,in the present study,we investigated the effects of Fe²⁺and/or Fe²⁺/Ca²⁺co-treatment on neuronal toxicity in primary cultured ventral mesencephalon?VM?neurons and dopaminergic MES23.5 cells.We also tested the neuroprotective effects of isradipine,an specific antagonist of LTCCs on Fe²⁺and/or Fe²⁺/Ca²⁺co-treatment induced neurotoxicity.The cells were pre-incubated with 5?mol/L MPP⁺for 24 h and then treated with Fe²⁺and/or Fe²⁺/Ca²⁺.The changes of nuclear morphology were observed by immunofluorescence labeling.The changes of reactive oxygen species?ROS?and mitochondrial transmembrane potential???m?were detected by flow cytometry method.The expressions of cleaved caspase-3 were detected by western blot.The iron content and calcium concentration in VM neurons were detected by laser scanning confocal microscope.The results are as follows:1.Hoechst33258 fluorescent dye staining was used to observe the nuclear morphology in MES23.5 cells.Severe fragmentation of chromatin and hyper-condensed?brightly stained?nuclei were observed in MPP⁺/Fe²⁺group and MPP⁺/Ca²⁺/Fe²⁺group.The Hoechst-staining positive cells were significantly increased in MPP⁺/Fe²⁺group compared with MPP⁺group?P<0.001?.Co-treatment with isradipine could partly alleviate this effect(P<0.01,compared with MPP⁺/Fe²⁺group).The Hoechst staining positive cells in MPP⁺/Fe²⁺/Ca²⁺group were markedly increased than that in MPP⁺/Fe²⁺group?P<0.01?.Co-treatment with isradipine could alleviate this effect(P<0.01,compared with MPP⁺/Ca²⁺/Fe²⁺group).2.The nuclear morphology of VM neurons were detected by MAP2 and Hoechst double staining.Compared with MPP⁺group,the double staining positive cells in MPP⁺/Fe²⁺group and MPP⁺/Ca²⁺/Fe²⁺group were significantly increased?P<0.001?.Isradipine conferred on a significant protection on the damage of VM neurons induced by Fe²⁺?P<0.05?.The double staining positive cells in MPP⁺/Ca²⁺/Fe²⁺group were markedly increased compared with that in MPP⁺/Fe²⁺group?P<0.05?.Co-treatment with isradipine could alleviate this effect(P<0.05,compared with MPP⁺/Ca²⁺/Fe²⁺group).3.The changes in cellular??m of MES23.5 cells were detected by flow cytometry method.The??m of MES23.5 cells was decreased by 25%in MPP⁺/Fe²⁺group compared with MPP⁺group?P<0.001?.Co-treatment with isradipine could alleviate this effect(P<0.01,compared with MPP⁺/Fe²⁺group).The??m was decreased by 12%in MPP⁺/Ca²⁺/Fe²⁺group compared with MPP⁺/Fe²⁺group?P<0.05?.Co-treatment with isradipine could alleviate this effect(P<0.001,compared with MPP⁺/Ca²⁺/Fe²⁺group).4.We also detected the changes of??m in VM neurons.The??m in VM neurons was decreased by 26%in MPP⁺/Fe²⁺group compared with MPP⁺group?P<0.001?.This effect can be partly reversed by isradipine(P<0.01,compared with MPP⁺/Fe²⁺group).The??m in VM neurons in MPP⁺/Fe²⁺/Ca²⁺group decreased by 11%compared with MPP⁺/Fe²⁺group?P<0.01?.This effect may be blocked by isradipine(P<0.001,compared with MPP⁺/Ca²⁺/Fe²⁺group).5.We used fluorescent dye H₂DCF-DA to detect the intracellular ROS production in the VM neurons by flow cytometry method.The intracellular ROS production in group were up-regulated to 1.8 folds respectively compared with MPP⁺group?P<0.001?.Isradipine markedly inhibited Fe²⁺induced ROS production in VM neurons?P<0.05?.The intracellular ROS production in MPP⁺/Fe²⁺/Ca²⁺group was increased 1.28 folds compared with MPP⁺/Fe²⁺group?P<0.05?.This effect can be reversed by isradipine(P<0.001,compared with MPP⁺/Ca²⁺/Fe²⁺group).6.The expressions of cleaved caspase-3 in MES23.5 cells were detected by western blot.The expressions of cleaved caspase-3 in MPP⁺/Fe²⁺group were significantly increased2.3 folds compared with MPP⁺group?P<0.001?.This increase could be partly reversed by isradipine(P<0.05,compared with MPP⁺/Fe²⁺group).The expressions of cleaved caspase-3 in MPP⁺/Ca²⁺/Fe²⁺group were significantly increased 1.3 folds compared with MPP⁺/Fe²⁺group?P<0.05?.Co-treatment with isradipine could alleviate this effect(P<0.001,compared with MPP⁺/Ca²⁺/Fe²⁺group).7.The expressions of cleaved caspase-3 in VM neurons in MPP⁺/Fe²⁺group were up-regulated by 3.02 folds compared with MPP⁺group?P<0.001?.Co-treatment with isradipine could partly reverse this effect(P<0.05,compared with MPP⁺/Fe²⁺group).The expressions of cleaved caspase-3 in MPP⁺/Ca²⁺/Fe²⁺group were much higher than that in MPP⁺/Fe²⁺group in VM neurons?P<0.01?.Co-treatment with isradipine could alleviate this effect(P<0.001,compared with MPP⁺/Ca²⁺/Fe²⁺group).8.The primary cultured VM neurons were pre-treated with 5?mol/L MPP⁺.Intracellular fluorescence were indicated by calcein.The fluoresence intensity in the MPP⁺-treated neurons was significantly decreased when perfusion with 100?mol/L FeSO₄.The decrease in the fluoresence intensity was partly inhibited by 10?mol/L isradipine.However,there was a significant decrease in the fluorescence intensity with 10?mol/L Bayk8644 perfusion.By laser scanning confocal microscope,we examined the effect of Fe²⁺on intracellular Ca²⁺in VM neurons.We could not observe a significant difference in intracellular Ca²⁺levels in the MPP⁺group and MPP⁺/Ca²⁺?2.5 mmol/L?group compared with the control.However,intracellular Ca²⁺levels were significantly increased when perfusion with FeSO₄ in the K-H buffer.Compared with MPP⁺/Fe²⁺co-application group,a dramatical increase in intracellular Ca²⁺levels was observed when perfusion with 2.5 mM CaCl₂ in the K-H buffer.Bath application of isradipine may partly block iron-induced elevation of intracellular Ca²⁺levels.In summary,activation or inactivation of LTCCs may alter intracellular Fe²⁺,which implicate that LTCCs may mediate iron transport.Elevated cellular iron may induce hydroxyl radicals production by Fenton reaction,which in turn leads to oxidative stress,caspase-3 activation and ultimately cell apoptosis.In addition,the neurons were more stressful to iron and calcium co-application,which indicated that iron and calcium may have a synergistic effect.Iron may delay the inactivation of calcium channels,results in more calcium influx.Elevated intracellular Ca²⁺may also aggravate iron-induced neurotoxicity to the cells.This work may provide a useful framework to advance our knowledge of LTCCs-mediated DA neurons degeneration and might permit the development of a novel alternative approach to treat PD.