Hypoxia/Ischemia-Induced Disturbance Of Ionic Homeostasis And Attenuation By Humanin

Humanin (HN) is a 24-aa peptide encoded by a newly identified gene cloned from an apparently normal brain region from patients with Alzheimer's disease (AD) in 2001. From those initial observations it seemed that HN was a selectively neuroprotective factor rescuing neurons from Alzheimer's disease-related insults, hence it is defined as AD-related neuroprotive peptide. However, later efforts suggested a broader spectrum of its survival-promoting activity. Several lines of studies provided evidence for the argument. Prior works in our laboratory proved that HN treatment provided long-term neuroprotection against hypoxia insults in primary cortical neurons. Yet effects of HN when given acutely on early neuronal damage during hypoxia/ischemia remain unclear. And the mechanisms of HN neuroprotection remain to be fully illustrated, especially because of a paucity of electrophysiological studies.It is generally accepted that that neuronal cell death can be described as three stages: (1) early intracellular ionic and chemical changes; early onset of acute hypoxia or ischemia is characterized by changes of ion channel activity and membrane potential, and major disturbances in neuronal ionic homeostasis, including significant rises in intracellular Na⁺, Ca²⁺, and extracellular K⁺ (2) activation of damaging enzymes and (3) changes in cellular functions and structures, eventually leading to cell death. The delayed before cell death occurs varies greatly (from minutes to hours or weeks), depending on the nature of the insults and the cell types. A large number of the damaging changes occurring in the neurons are secondary to loss of ion homeostasis (Lipton 1999), the pathological changes vary greatly, including the mode of final cell death. These early ionic alterations play important roles in hypoxia/ischemia induced neuronal damages: (a) the accumulation of Ca²⁺ and Na⁺ in nerve cells during hypoxia has been shown by many investigators to be deleterious; (b) the main changes in neuronal function early in brain hypoxia or ischemia are almost solely related to changes in ionic homeostasis and (c) it is likely that these early changes dictate not only cell fate in the short term, such as cell injury and necrosis, but also long term changes such as activation of damaging enzymes and programmed cell death. A large number of the damaging changes occurring later in the neurons are secondary to disturbed ion homeostasis. Therefore, it is of great importance to investigate mechanisms of ionic imbalance during early hypoxia/ischemia insults. Ion transmembrane flux is controlled by multiple different mechanisms under physiology conditions. However, changes in ionic channel activities by hypoxia are the major pathway for redistribution of ions across the membrane and the membrane depolarization, contributing to succeeding cell death. To examine the effects of HN on early hypoxia/ischemia induced disturbance of ionic homeostasis, using hypoxia model and glutamate as simulated-ischemia conditions, we investigate the effects of HN on ischemia-related insults induced disturbance of membrane ionic currents and the intracellular calcium ([Ca²⁺]i) deregulation using whole-cell patch clamp technique and calcium imaging.Using freshly isolated hippocampal CA1 neurons and patch clamp techniques, primary cultured neurons and ion imaging techniques, we investigate effects of hypoxia-induced disturbance of sodium and potassium currents and effects of HN. Given the crucial roles of excitotoxicity and dysfunction of calcium homeostasis in various neuropathologic lesions including hypoxia/ischemia, we investigated the effects of HN on Ca²⁺ deregulation induced by toxic concentration of glutamate exposure and its possible mechanisms. The results showed that:1) acute hypoxia induced disturbance of voltage-gated potassium and sodium currents (IK,INa); 2) HN (5μM) co-application with hypoxia solution attenuated hypoxia–induced disturbance of potassium current (IK) and persistent potassium current (INaP); and 3) HN (5μM) pretreatment attenuated Ca²⁺ deregulation during glutamate exposure through attenuated calcium entry via glutamate receptor channels and preserved mitochondrial buffering capacity.Our results showed that disturbance of ionic homeostasis, including enhancement of IK, INaP and [Ca²⁺]i, were the early changes induced by hypoxia and played important roles in hypoxic pathology through actions on membrane potential, excitability. HN was able to maintain ionic homeostasis via attenuating disturbance of ion channels and stabilizing mitochondrial function, and thus protect neurons against early hypoxia/ischemia insults. Furthermore, HN provided neuronal protection by ionic mechanisms acting at the very beginning of insults, i.e., that it is protective against acute hypoxia injury. These results provide further evidence for the broader spectrum of neuroprotective effects of HN against insults other than AD. Partâ… : Effects of HN on Hypoxia-Induced Disturbance of Voltage-Dependent Potassium Currents in Rat Hippocampal CA1 NeuronsDisruption of ionic homeostasis induced by disturbance of ion channel activity has been generally regarded as an initial and key alterations in anoxia/ischemia-induced neuronal injury. Enhanced ef?ux of K⁺ has been shown to be closely associated with anoxia-induced depolarization, which is believed to be a crucial factor leading to neuronal death. The anxia depolarization is mimicked by elevation of the extracellular potassium concentration. The loss of intracellular K⁺ causes cell shrinkage (apoptotic volume decrease) and creates a permissible environment for apoptosis by relieving the inhibition of endogenous caspases and nucleases. Sustained exposure to elevated extracellular K⁺ causes signi?cant neuronal death even under conditions of normoxia and abundant glucose supply, whereas blockade of K⁺ ef?ux has been shown to attenuate hypoxia and ischemia-induced neuronal death. These results suggest that maintaining K⁺ channels functions and K⁺ distribution across plasmamembrane may be of therapeutic bene?t in the treatment of ischemia-related insults. Among the five types of plasmalemma potassium channels, a great deal of researches focus on KATP and KCa channels, while voltage-gated potassium channels get less attention.Mammalian neurons react rapidly to a lack of oxygen with alterations of ion channels activities, being either adaptive or deleterious responses. Of particular interest is the effects that hypoxia has on potassium channels since these channels are one of the fundamental factors that regulate membrane potential and neuronal excitability, in addition there are major alterations in K⁺ ions homeostasis during early hypoxia. Thus K⁺ channels and K⁺ homeostasis may play important roles in the initiation and development of hypoxia/ischemia. Among the five classes of K⁺ channels identified in excitable cells, penetrating researches have been focused on roles of KATP and KCa channels; while changes of voltage-gated potassium channels during hypoxia/ischemia got much less attentions.HN was originally identified as an endogenous peptide that protects neuronal cells from death caused by Alzheimer's disease (AD)-related genes and amyloid-β(Aβ). While recent studies suggested that HN might have a broader spectrum of protections. And the mechanisms underlying the protections of HN remain to be illustrated. Preliminary works in our laboratory showed that HN protected neurons from hypoxia-insults with mechanisms including anti-apoptosis and preserving mitochondrial functions.To illustrate the effects of HN during early hypoxia/ischemia insults and illuminate underlying electrophysiological mechanisms for its potential neuroprotections, we investigated the response of voltage-gated K⁺ (KV) channels (total potassium current, transient potassium current, and delayed rectifier potassium current) to hypoxia and the effects of HN in freshly isolated hippocampal CA1 neurons from rats aged 10～12 days.Major results as following: 1) acute exposure to hypoxia (3 min) significantly enhanced IK currents. In five neurons exposed to hypoxia, the amplitude of IK at +70 mV was 1.96±0.15 times the amplitude of control group (P<0.05, n=6). While the amplitude of IA at +70 mV was 1.05±0.05 times of controls group, i.e. not significantly different from control (P>0.05, n=6); 2) HN (5μM) alone had no effect on IK at constant normoxic PO2, the amplitude of IK were 1240.96±171.71 pA and 1609.63±353.49 pA before and after HN application, respectively (P>0.05, n=6); HN, however, significantly increased IA amplitude under both hypoxia and nomoxia, the amplitude of IA increased from 2250.33±608.77 pA to 3051.47±781.62 pA after HN treatment (P<0.05, n=6); 3) HN co-application abolished the effect of hypoxia on IK. In the presence of HN, hypoxia no longer increased IK current amplitude. The IK current amplitude in the presence of HN was 0.89±0.23 times of control (P<0.05, n=11).These results indicated that: 1) acute hypoxia induced an enhancement of the IK channel in central neurons, which may be the major pathway for K⁺ efflux and results in hypoxia-induced changes and other pathological changes;2) transient IA currents were not influenced by acute hypoxia; 3) HN attenuated hypoxia-induced disturbance of IK, this effect might be neuroprotective during ischemia by attenuated the disturbance of potassium ionic distribution that occured. 4) HN increases IA current amplitude; the physiological and pathological significance of this fact remain unclear. These results suggested that HN may attenuate hypoxia-induced changes of IK currents and thus maintain K⁺ homeostasis, protect neurons against hypoxia-induced damages. Partâ…¡: Effects of HN on Hypoxia-Induced Disturbance of Voltage-Dependent Sodium Currents in Rat Hippocampal CA1 NeuronsDuring hypoxia, cells undergo alterations in membrane potential and detrimental changes in their intracellular environment, including an early increase in intracellular Na⁺ that contributes to the pathophysiology of neuronal death. There are mounting studies illustrating the relation between Na⁺ channels and hypoxia. A large number of the damaging changes occurring in the neurons are secondary to an increase in intracellular Na⁺ levels (Lipton 1999). In dissociated cells and cell cultures, hypoxia raised intracellular Na⁺ concentration ([Na⁺]i). Increased [Na⁺]i may aggravate neuronal lessons by imposing an energy demand on cells due to stimulation of the plasmalemmal Na⁺, K⁺-ATPase. In addition, it contributes to an impairment of Ca²⁺ homeostasis by driving Ca²⁺ into the cells via the Na⁺-Ca²⁺ exchanger, and to the development of other acute dysfunctions. Drugs that selectively block this current might reduce damage to cells during ischemia or hypoxia.HN is a recently described endogenous peptide that antagonizes neurotoxicity caused by Alzheimer's disease (AD) relevant insults, while recent studies suggest that HN may have a broader spectrum of protection. To illustrate the electrophysiological mechanism of HN on neurons and the possible neuroprotection against hypoxia insults, whole-cell patch clamp techniques were applied on isolated hippocampal neurons to study the possible effects of HN (5μM) on persistent and transient sodium current under normoxia and hypoxia.The major results as following: 1) After 2 min hypoxia stimulation, persistent sodium current (INaP) increased significantly to 2.65±0.40 times control group (P < 0·01, n = 5), transient sodium current (INaT) decreased significantly to 65.4±7% of baseline (P<0.05, n= 7); 2) After application of HN (5μmol/l) alone, the current amplitude of INaP remained unchanged; 3)After co-application of HN and hypoxia, current density of INaP increased to 1.44±0.24 times control group, significantly different compared with the value of hypoxia group (P<0.05).These results indicated that: (1) During early hypoxia, enhanced INaP amplitude constitutes the primary pathway of Na⁺ influx, and hence resulted in Na⁺ and Ca²⁺ imbalance, delayed membrane depolarization and cell death; (2)Acute hypoxia inhibited transient INaT; (3) HN could attenuate hypoxia-induced increased of INaP and thus mitigate Na⁺ imbalance and secondary lessons. The physiological and pathological significance of INaT changes remain unclear. Partâ…¢: Effects of HN on Ca²⁺ Dysregulation during Excitotoxic Glutamate Exposure and its MechanismsLines of evidence suggested that HN provided neuroprotection against hypoxia insults and glutamate excitotoxicity. Due to the central involvement of the activation of glutamate receptors and calcium overload in ischemic brain injury, the aim of this work was to investigate whether and how HN can prevent calcium homeostasis deregulation induced by glutamate (500μM) in cultured cortical neurons, and the potential involvements of membrane receptors and mitochondrial mechanisms.In the present study, intracellular Fluo-3 Ca²⁺-imaging was conducted with laser confocal microscopy in cultured cortical neurons. Mitochondrial uncoupler FCCP was used as a tool to collapse the mitochondrial membrane potential and thus block mitochondrial Ca²⁺ uptake, in addition release Ca²⁺ stored within,. Direct estimates of effects of HN on glutamate receptor channels were conducted with whole-cell path clamp in acutely isolated neurons.The major results as following: 1) glutamate (500μM) induced marked increase in intracellular calcium ions in cultured cortical neurons. The mean changes in [Ca²⁺]i at the steady-state level stimulated by 500μM glutamate were 2.29±0.38. 2) HN pretreatment significantly attenuated the increase of intracellular calcium responses upon excitotoxic concentration of glutamate exposure. The mean changes in [Ca²⁺]i stimulated by glutamate were 1.38±0.19 in HN-treated neurons, nearly 40% inhibition compared with control neurons (P<0.01). 3) to investigate the potential involvement of mitochondrial mechanisms in the antagonisms of dysregulation of HN, we used uncouplers FCCP to semi-quantify the amount of Ca²⁺ taken up by mitochondria. Cells were exposed to glutamate until the steady-state phase (plateau) at which time point 1μM FCCP was applied to rapidly dump any Ca²⁺ stored in the mitochondrion back into the cytosol. FCCP application resulted in a significantly greater [Ca²⁺]i increase in HN-treated cells. The changes in Fluo-3 fluorescence were 1.64±0.15 and 2.83±0.29 for control neurons and HN-treated ones, respectively (P< 0.05), indicating an HN-induced increase in the mitochondrial calcium ([Ca²⁺]m) load during glutamate exposure; 4) when FCCP was applied before glutamate exposure, the glutamate-induced [Ca²⁺]i increase was 1.29±0.14 and 1.09±0.35 in control group and HN-pretreated group, respectively (P<0.05). Effects of HN were not abolished in the presence of FCCP, indicating involvement of other mitochondrial-independent mechanisms in antagonism of dysregulation of Ca²⁺; 5) electrophysiological experiments showed that HN significantly inhibited glutamate receptor-mediated currents. The mean peak currents were significantly decreased to 53.1±7.4% (P <0.01, n=13) and 67.1±3.6% (P <0.01, n=12) of initial value for glutamate (200μM)- and NMDA (100μM)-induced currents in neurons treated with HN for 2 min.These results indicated that: 1) HN (5μM) attenuated intracellular calcium dysregulation during excitotoxic glutamate exposure; 2) this attenuation was resulted from preserved mitochondrial functions and calcium buffering capacity and 3) decreased Ca²⁺ entry via glutamate receptor channels.Taken together, we can draw conclusions blow:(1) Acute hypoxia induced disturbance of voltage-gated sodium and potassium channels, suggesting important implications of these channels in early hypoxia.(2) Acute hypoxia induced an enhancement of the delayed rectifier voltage-dependent K⁺ channel in central neurons, which might be a deleterious response that constitutes the pathway for K⁺ efflux and resulted in K⁺ homeostasis deregulation during early hypoxia;transient IA currents were not influenced by acute hypoxia.(3) During early hypoxia, enhanced INaP amplitude constituted the primary pathway of Na⁺ influx during early hypoxia, and hence resulted in Na⁺ and Ca²⁺ imbalance, delayed membrane depolarization and cell death;(4) HN pre-application could attenuated hypoxia-induced increased of IK and INaP and thus mitigate K⁺, Na⁺ imbalance and secondary lessons. The physiological and pathological functional implication of actions of HN on IA remains unclear.(5) Glutamate induced intracellular calcium overload and damage neurons; humanin attenuated intracellular calcium dysregulation during excitotoxic glutamate exposure and thus might mitigate intracellular calcium overload during hypoxia./ischemia.(6) HN attenuated delayed calcium deregulation via preserved mitochondrial buffering capacity and inhibited Ca²⁺ influx through glutamate receptor channels.