Effects of Hemin on Ca2+ Permeability in Neurons of C57BL/6 Mouse Brain
Department of Biochemistry, Shiraz University of Medical Sciences, Iran
Submission: May 22, 2019;Published: November 04, 2019
*Corresponding author: Hamid Islampoor, Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
How to cite this article: Hamid Islampoor. Effects of Hemin on Ca2+ Permeability in Neurons of C57BL/6 Mouse Brain. Open Access J Neurol Neurosurg.
2019; 12(1): 555826.
SExcitotoxicity results in a significant increase in Ca2+ influx; essentially from open N-methyl-D-aspartate receptors (NMDARs) channels that cause a secondary rise in the intracellular Ca2+ concentration. It is correlated with neuronal death induced by Ca2+ overload. Dysfunction of NMDARs is associated with excitotoxic neuronal death in neurodegenerative disorders. In this study, the effects of hemin on Ca2+ permeability in neurons of C57BL/6 mouse brain examined. Isolated from 1-dayold C57BL/6 mice, were cultured in serum-free media. Cells were maintained in growth medium at 37°C in 95% air/5% CO2 for 2 weeks in vitro before treatment. Primary neurons were cultured in serum-free media were treated with hemin (0, 12.5, 25, 50, 75,100 μM) for 18 (h). Intensity of calcium fluorescence was reduced in treated cultures with hemin (100, 86, 78.5, 60, 56, 46%, respective to the concentrations stated previously; P<0.05 for all). Hemin increased Ca2+ influx in cultured neurons. NMDAR stimulation by hemin increased the activating of NMDARs and Ca2+ influx in the cultured neurons. Therefore, hemin is cytotoxic due to increase of intracellular Ca2+ influx.
At rest, the cytoplasmic concentration ([Ca2+]i) in mammalian cells is very low (50-100 nM) . Excitotoxicity results in a significant increase in Ca2+ influx essentially from open N-methyl-D- aspartate receptors (NMDARs) channels that cause a secondary rise in the intracellular Ca2+ concentration , Glutamate is capable of to killing neurons with excitotoxicity . This secondary overload is correlated with neuronal death induced by Ca2+ overload . Dysfunction of NMDARs is associated with excitotoxic neuronal death in neurodegenerative disorders . Glutamate excitotoxicity is a critical factor in brain damage leading to brain and neurodegenerative disorders, such as; Alzheimer’s disease. The mechanism of excitotoxicity is causally linked to an intense elevation in cytoplasmic calcium concentration ([Ca2+]c) that leads to cell injury and neuronal death [6-8]. Correspondingly, stabilizing low cytosolic Ca2+ by chelation with BAPTA [(1,2-bis(o-aminophenoxy) ethane-N, N, N, N-tetra acetic acid;)] increases the survival rate of neurons exposed to excitotoxic glutamate [9,10].
In the central nervous system of mammals, 𝛼-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPARs) and NMDARs primarily mediate fast excitatory neurotransmission. Ca2+ permeability; allows NMDARs to play a vital role in braindamage, neuropathology, and neuroplasticity . The glutamate receptors of AMPA and NMDA are permeable to Ca2+ and are expressed in the brain regions responsible for cognitive functions, such as neocortex and hippocampus . The receptors are heterothermies comprising a combination of GluN1, GluN2A-D and GluN3A-B subunits . NMDARs receptors activation leads to opening of an ion channels that is permeable for cations, resulting in the influx of Na+ and Ca2+ ions and efflux of K+ ions . During excitatory neurotransmission, presynaptic release of glutamate activates glutamate receptors in the postsynaptic membrane, resulting in the generation of an excitatory postsynaptic potential . Phosphorylation of glutamate receptor regulates the Ca2+ channels and Ca2+ permeability .
NMDARs have a high affinity to stimulate L-glutamate . L-glutamate is the strongest NMDA agonist (EC50 2.3 μM) . Hemin is an iron–protoporphyrin molecule consisting of four pyrrole rings . Hemin is cytotoxic due to its ability to contribute to the generation of reactive oxygen species . As respects, hemin play an crucial role in correlated with neuronal death induced by Ca2+, In this study, the effects of hemin on Ca2+ permeability in neurons of C57BL/6 mouse brain examined.
Twenty-eight 1day-old C57BL/6 male mice were prepared
from Animal Center of School of Medicine, Shiraz University of
Medical Sciences, Shiraz, Iran. They were kept cages (4 mice per
cage) at room temperature and provided with a balanced diet,
Light cycle with 12h light, 12h dark. The mice randomly divided
into 6 groups; and anesthetized with Ketamine (87 mg/kg) and
Xylazine (13 mg/kg). All mice were killed, and their brain and neocortex
were removed. Neurons isolated by neutrosphere method
and were cultured in Dulbecco’s Modified Eagle Medium (Thermo
Fisher Scientific). Cells were maintained in growth medium at 37
°C in 95% air/5% CO2 for 2 weeks in vitro before treatment. Primary
postnatal neurons cultured in serum-free media were treated
with hemin (Frontier Scientific; 0, 12.5, 25, 50, 75,100 μM) for
18 h .
The neurons were incubated in medium with (Fluo-8 AM)
(ATT Bio quest, Inc) for 1 h at 37°C. The neurons were then
washed twice with Hank’s Balanced Salt Solution (Frontier Scientificcontaining CaCl2 (2 mM) and MgCl2 (1 mM). The intensity
of Flu-8 AM fluorescence was evaluated after adding NMDA (100
μM) and Nifedepine (10 μM) to neurons, with hemin. The intensity
of fluorescence was measured of 482 nm and 505-530 nm
(emission reverse microscopy) (Leica HC PL FLUOTAR 20×/0.50
objective, Lambda DG-5 Plus) .
Statistical analysis was carried out using ANOVA; with SPSS
19. One-Way ANOVA was used to test for differences in mean values
from multiple samples (calcium imaging data) with comparison
tests. Statistical difference between two means was analyzed
by two-tailed unpaired or paired Student’s t-test. Differences
were considered significant if p<0.05. All data are expressed as
Primary postnatal neurons cultured in serum-free media were
treated with hemin (0, 12.5, 25, 50, 75,100 μM) for 18 h. fluorescence
was reduced in treated cultures with hemin (100, 86, 78.5,
60, 56, 46%, respectively) (P < 0/05 for all) (Figure 1 & Table 1).
Ca2+ influx is regulateds by NMDARs subunits . NMDAR
stimulation increases Ca2+ influx . Stimulation of NMDAR activates
Ca2+ signaling pathway in neurons . Conversely, inhibition
of NMDARs inhibits it . Hemin suppresses NMDAR activity
, by regulating the GluN1, GluN2A and GluN2B subunits of
NMDA . NMDAR stimulation by hemin was increased activating
of NMDARs and Ca2+ influx in the cultured neurons.