| Endothelium-derived hyperpolarizing factor (EDHF) is a non-nitric oxide (NO), non-prostacyclin (PGI2) autacoids which is product by vascular endothelial cells after stimulated by agonist. The relaxation of vascular is by activated Ca2+-sensitive K+ channel (KCa) induced the hyperpolarization of vascular smooth muscle cells. Endothelium-dependent vasorelaxation are depending on the vessel size, with relatively greater role of NO in conduit arteries and predominant role of EDHF in small resistance arteries, which are more important for the regulation of total peripheral resistance and blood pressure. Epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2) was proven to have no relationship with EDHF in cerebral vascular. The previous research of our laboratory has indicates that H2S may be EDHF in cerebral vascular. Base on this, we used CSE gene knockout (CSE-/-) to investigate the effect of EDHF/H2S in cerebral vascular through cell membrane hyperpolarization experiment on nerve cell.Cerebral ischemic is one of the major cause the mortality,especially the aged group.Deprivation of blood and oxygen supply to an area or organ causes the intracellular calcium overload, oxidative stress and synaptic vesicle release of glutamic acid, calcium protease activation etc. Cerebral ischemia can induce various degrees of nerve damage, affect the function of the brain,especially of learning and memory.This subject we use the CSE-/- mice ischemia reperfusion (I/R) model to investigate the role of endogenous/exogenous of H2S on learning and memory in mice after cerebral ischemia injury.Purpose:1. To research whether KCa channel subtypes mRNA expression in cerebral arteries on endothelial cells;2. To investigate the effect of EDHF/H2S in mice on nerve cells;3. To detect role of H2S on learning and memory function in mice after I/R injury.Methods:1. Endothelial cells were collected from cerebral arteries by using magnetic activated cell sorting (MACS) which analyzed by flow cytometry. To observe KCa channel subtypes mRNA gene expression, we conducted Quantitative real-time (qRT)-Polymerase Chain Reaction(PCR) analysis.2. Intracellular membrane potential was recorded to evaluate the effect of hyperpolarization. In presence of N-nitro-L-arginine-methyl-ester (L-NAME) plus indomethacin (Indo) which inhibit NO and PGI2, the hyperpolarization effect of acetylcholine (ACh) on nerve cell in CSE+/+ mice were observed. The effect of charybdotoxin (ChTX)+ apamin (Apa) or DL-propargylglycine (PPG) on ACh induced nerve cell non NO/PGI2 responses in CSE+/+ mice were tested. The hyperpolarization effect of ACh on nerve cell in CSE-/- mice was detected. The hyperpolarization effect of Sodium hydrosulfide (NaHS) and the effect of Iberiotoxin (IBTX) or ChTX or Apa on NaHS induced hyperpolarization were also recorded.3. Established a global model of cerebral I/R injury by 2-vessle occlusion (2-VO) in CSE+/+ and CSE-/- mice.4. The content of malondialdehyde (MDA) and the activity of lactate dehydrogenase (LDH) in brain tissue were mearsued by automatic enzyme standard instrument on I/R mice in each group.5. Examined the performance of learning and memory by Platform diving experiment in CSE+/+ and CSE-/- mice. The effect of endogenous H2S and NaHS on learning and memory after cerebral I/R injury were studied.6. Examined the performance of learning and memory by the Morris water maze (MWM) in CSE+/+ and CSE-/- mice. The effect of endogenous H2S and NaHS on learning and memory after cerebral I/R injury were recorded.Results:1. MACS analysis results revealed that the purity of CD31+ cells >90%. qRT-PCR analysis indicated that Kca2.3 and Kg,3.1 are the predominant subtypes on cerebral artery endothelial cells while the expression of Kca2.2 lower and almost no expression of KCa2.1.2. The membrane potential experimental results showed that in presence of L-NAME plus Indo which inhibit NO and PGI2, Ach (10-8～10-5.5 M) can induce a hyperpolarization of nerve cell in CSE+/+ mice, the maximum hyperpolarization amplitude up to -8.02±1.18 mv. Remove of MCA/Endo abolished nerve cell non NO/PGI2 hyperpolarization induced by ACh in CSE+/+ mice. Co-application of 100 nM ChTX and 5μM Apa reduced ACh induced endothelium-dependent non NO/PGI2 hyperpolarization, the hyperpolarization amplitude up to -0.24±0.19 mv.100μM PPG (an inhibitor of CSE) markedly inhibit ACh induced concentration dependent non NO/PGI2 hyperpolarization, the hyperpolarization amplitude up to -2.65±0.83 mv. CSE-/- mice abolished nerve cell non NO/PGI2 hyperpolarization induced by ACh, the hyperpolarization amplitude up to -1.7±0.49 mv. NaHS (10-5～10-2.5 M) caused concentration dependent hyperpolarization of nerve cell in CSE+/+ mice, the maximum hyperpolarization amplitude up to -12.82±0.88 mv.100 nM ChTX and 5 μM Apa reduced NaHS induced endothelium-dependent hyperpolarization, the maximum hyperpolarization amplitude up to -7.41±0.98 mv and -6.91±1.62 mv.100 nM IBTX abolished nerve cell hyperpolarization induced by NaHS in CSE+/+ mice.3. Compared with sham group, the activity of LDH in model group mice are reduced.While compared with CSE+/+ model group, the activity of LDH in CSE-/-model group mice are significantly reduced, NaHS (2.4mg/kg,4.8 mg/kg) group significantly inhibit reduce of the activity of LDH compared with CSE-/- model group.4. Compared with sham group, the content of MDA in model mice are increased. While compared with CSE+/+ model group, the content of MDA in CSE-/- model group mice are significantly increased, NaHS (2.4 mg/kg,4.8 mg/kg) group significantly inhibit increase of the content of MDA.5. Platform diving experiment suggested that compared with sham group, the frequency of mistakes was significantly increased and the latency of learning significantly increased while the latency of memory significantly decreased in model group; Compared with CSE+/+ model group, the frequency of mistakes was significantly increased and the latency of learning significantly increased while the latency of memory significantly decreased in CSE-/- model group. Moreover, NaHS (4.8 mg/kg) group significantly decreased the frequency of mistakes and the latency of learning, significantly increased the latency of memory comparing with that of CSE-/- model group.6. During the training of place navigation of MWM testing demonstrated that the escape latency of each group of mice gradually decreased. Compared with sham group, the latency of model group was significantly higher, while that of the NaHS (2.4 mg/kg,4.8 mg/kg) group decreased significantly comparing with CSE-/- model group, improve the ability of Learning; The spatial probe of MWM testing demonstrated that compared with sham group the number of entries as well of the percentage of time spent and swim distance in the target quadrant in model group was significantly decreased,while that of the NaHS (2.4 mg/kg,4.8 mg/kg) group increased significantly compared with CSE-/- model group, improve the ability of memory.Conclusions:1. Kca2.3 and Kca3.1 are the predominant subtypes on cerebral artery endothelial cells.2. EDHF/H2S induced nerve cell hyperpolarization in mice.3. H2S have protective effects on learning and memory function after I/R injury in mice. |
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