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GAT-1and GAD65mRNA Expressions in Different Brain Regions at Brain Propofol Uptake Equilibrium in Dogs

Author: YangJingJing
Tutor: LinChunShui
School: Southern Medical University,
Course: Anesthesiology
Keywords: Propofol GABA Plasma Tranaport Proteins glutamate decarboxylase2 Brain Dogs
CLC: R614
Type: Master's thesis
Year: 2013
Downloads: 3
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Abstract

Propofol has been widely used in clinical anesthesia and clinic short surgery for the advantages of rapid onset, short action time and good controllability. But the mechanism of propofol-induced anesthesia remains unclear.The release and transfer of neurotransmitter is the basis of the normal function of central nervous system (CNS). Strengthen inhibitory neurotransmition was one of the main mechanisms of propofol-induced anesthesia. Gamma aminobutyric acid (GAB A) is the most important inhibitory neurotransmitter in CNS. The GAB A receptors are widely distributed in CNS, but each has its own pharmacology and neural electrophysiology. Studies using whole cell patch clamp technique indicaded that propopol activated Cl-channel by three-dimensional allosteric action of GABA and GABAA receptors complex so as to produce the central nervous inhibition. GABAA receptors can be divided into the synaptic and extrasynaptic, excited the synaptic GABAA receptor will produce a type of phasic inhibitory current, and excited extrasynaptic GABAA receptor will produce a type of tonic inhibitory current, both currents are affected by the adjustment of the concentration of GABA. The concentration of GABA depends largely on the synthesis by glutamic acid decarboxylase (GAD) and the uptake and release by gamma aminobutyric acid transporter (GAT). GAD is the speed limit synthetic enzyme of GABA, and there are two subtypes as GAD65and GAD67in the brain. The in vitro an in vivo research about GAD65were mainly by using genetically modified technology, and achieved the following findings:GAD65plays an important role in GABA synthesis under physiological conditions while GAD67in certain pathological conditions; the tonic inhibitory current decreases obviously and the reactivity to propofol but not ketamine declined in GAD65gene knockout mice. The studies above showed that GAD65plays a dominant role in physiological GABA synthesis and GAD65mediated GABA synthesis plays an important role during propofol-induced anesthesia. GAT which including vesicle transporter and membrane transporter is an important glycoprotein that regulate the activities of GABA neurons. There are four subtypes of membrane GAT namely GAT-1, GAT-2, GAT-3and GAT-4(or BGT-1). GAT-1is the most important GABA transporter subtype that is widely distributed throughout the brain. GAT-1predominantly participates in GABA uptake and release through a non-vesicle antiporter, thus playing the most important role in regulating presynaptic GABA homeostasis. Wu and his colleagues found that phasic inhibitory current and tonic inhibitory current could be regulated independently and played a complementary role in nerve excitability controlling, and the balance of GAT was the key factor in the regulating ambient GABA concentration and tonic inhibitory current Studies had shown that propofol could inhibited GABA uptake by presynaptic mechanism:the uptake of [3H]GABA by purified synaptic corpuscle striatum could be inhibited reversibly and dose-dependently by propofol, the release of [3H]GABA from striatum nerve endings stimulated by K+didn’t affected by propofol, either the spontaneity or the K+stimulated GABA release in mouse cortex synapses could be reinforcement by propofol. Other studies showed that0.3-100μM propofol did not affect GABA uptake and Ca2+-independent release of GABA in rat cortical. The studies above mainly focused on the function of GAT-1in regulating the activity of GABA nerve and the effects of propofol on GABA uptake and release,, the effect of propofol on GAT-1hadn’t been reported by now.Our previous study had shown that the concentrations of GABA in deep anesthesia group were significantly higher than that in light anesthesia group in each brain region at brain propofol uptake equilibrium in dogs, and the change rates of GABA in thalamus and hypothalamus were higher than that in other brain regions. In this study, we detected the mRNA expression of GAT-1and GAD65in different brain regions (hypothalamus, sub thalamus, dorsal thalamus, hippocampus, pons, parietal lobe and frontal lobe) in dogs using quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the effects of propofol on the amino acid transporters and the synthesis enzymes.Materials and methods1Animal preparation and study designEighteen healthy Chinese rural dogs (aged12-18months) of either sex weighing10-12kg were randomly divided into either the control group (group C, n=6), the low dose group (group L, n=6) or the high dose group (group H, n=6). Anesthesia was carried out by intravenous injection of propofol at5.5mg/kg (group L) or7.0mg/kg (group H) for15s each, followed by an intravenous infusion of propofol at constant rates of55mg/kg/h (group L) or70mg/kg/h (group H) for50min using a syringe pump. Tracheal intubation was implemented when an appropriate depth of anesthesia (as indicated by loss of eyelash reflex and tail-pinch withdrawal reflex) was achieved. End-tidal carbon dioxide pressure (PEtCO2) was maintained at30-38mmHg by mechanical ventilation, and the mean artery pressure (MAP) and the pulse rate (PR) were monitored. Blood samples were taken from the internal carotid artery and jugular vein after propofol was infused for50min. Two milliliters of each blood sample was placed in an Eppendorf (EP) tube containing heparin (50I.U.), which was then frozen and stored below4℃for an HPLC-UV drug assay to be performed later. Simultaneously, dogs were sacrificed by rapid intravenous injection of10%KCl2mg/kg. Dogs in group C were sacrificed by rapid intravenous injection of10%KCl2mg/kg without any previous intervention. Brain tissue samples of the hypothalamus, sub thalamus, dorsal thalamus, hippocampus, pons, parietal lobe and frontal lobe were collected from each dog, placed in EP tubes and stored below-80℃for subsequent measurement of the mRNA levels of GAT-1and GAD65using quantitative real-time polymerase chain reaction (qRT-PCR).2Measurement of propofol plasma concentrations by HPLC-UVEach blood sample was centrifuged at3500rpm (10min,4℃). The supernatant (200μl) was added to acetonitrile (400μl), vortexed at1000rpm (2min), and centrifuged again at10000rpm (10min). The supernatant was analyzed using HPLC-UV. The following columns were used for detection:the chromatogram column was Shim-pack VP-ODS (250mmx4.6mm) and the guard column was Shim-pack GVP-ODS (10mm x4.6mm). A15:85ratio of mobile phase A (water) and B (methanol) was used. The automatic injection volume was20μl, and both columns were kept at4℃with a mobile phase flow rate of1ml/min. A270nm wavelength was used for detection.3Brain region selection schemeThe samples of the same brain areas of the6dogs in each group were pooled quality equality to be one sample. The quality of each sample was0.1g. After treated with above method we would have21samples. Detected the GAT-1mRNA and GAD65mRNA expression of the21samples by application of qRT-PCR and screened out significant brain areas for further testing.4GAT-1mRNA and GAD65mRNA expressions detection by qRT-PCR Total RNA was extracted from brain tissue samples using the RNA extraction Kit. A total of1μg of total RNA was used for reverse transcriptase (RT) reactions using a reverse transcriptase kit. A total of1μg of cDNA was used to set up qRT-PCR reactions. The qRT-PCR reactions used the SYBR green qPCR kit in a fluorescent temperature cycler. The following protocol was used for PCR reactions:incubate at95℃for10min, amplification and quantitation repeated for40cycles (95℃for10s,60℃for30s, with a single fluorescence measurement), a melting curve program (95℃for10s,55-95℃with a heating rate of0.1℃/s and continuous fluorescence measurement). Relative gene expression was quantified according to the comparative Ct method using β-actin as an internal standard and samples from the control group as calibrators. The gene expression levels were analyzed with the analysis software MXPro4.01(Stratagene, USA) and quantified by the2-ΔΔCt method.5Statistical analysisData were analyzed by using SPSS13.0software and expressed as the means±SD. Statistical analysis was performed using paired-Samples t-test, an independent-samples t-test and a one-way ANOVA. Values of P<0.05were considered to be statistically significant.Results1General conditionThe sex, age, and weight of the dogs were similar among the three groups. Both of the dosages of propofol chosen produced moderate or deep anesthesia by the end of the infusion in all dogs. When propofol continued infusion at a constant rate for50minutes, the mean arterial pressure (MAP) was (77.00±6.90) mmHg and (54.17±5.98) mmHg respectively in group L and group H and the pulse rate (PR) was (115.50±5.17) bpm and (89.00±4.10) bpm. There was significant difference between the two groups both of MAP and PR (P<0.05)2Plasma concentrations of propofol The mean propofol plasma concentrations in the internal carotid artery and jugular vein were3.18±0.06μg/ml and3.12±0.09μg/ml in group L and6.21±0.07μg/ml and6.13±0.11μg/ml in group H. There was little difference between artery and vein propofol concentrations in either of the anesthesia groups (P>0.05). However, the mean propofol plasma concentrations in group H were higher than those in group L for both the internal carotid artery and the jugular vein (P<0.01).3Brain regions screening resultsThe mRNA expression levels of GAT-1and GAD65in hypothalamus, dorsal thalamus and hippocampal differd obviously in group L and group H compared to group C. There was no significant difference in other brain regions.4The effect of propofol on the mRNA expression levels of GAT-1and GAD65In the hypothalamus and the hippocampus, propofol increased the GAT-1mRNA levels in group L (1.76±0.08,1.85±0.41) and group H (1.90±0.21,2.03±0.33) compared to the control group (1.09±0.46,1.03±0.27; P<0.05&P<0.01). In the dorsal thalamus, propofol increased the GAD65mRNA levels in group L (1.69±0.28) and group H (1.75±0.30) compared to the control group (1.09±0.43; P<0.01). There was no significant difference of the GAT-1mRNA levels in the dorsal thalamus and the GAD65mRNA levels in the hypothalamus and the hippocampus among the three groups. The change rates of GAT-1mRNA levels in hypothalamus and hippocampus were61.26%±7.17%and79.34%±39.95%in group L, and were74.64%±19.63%and97.12%±32.31%in group H. The changes between the two brain regions had no significant difference in both group(P>0.05). The change rates of GAD65mRNA levels in the dorsal thalamus were59.28%±27.02%in group L and60.98%±27.85%in group H. The levels of each gene showed little difference between group L and group H with in the same brain regions.Conclusion 1Brain uptake of propofol is in equilibrium when propofol continued infusion at a constant rate for50minutes. Then, propofol can increase GAT-1mRNA expression in hypothalamus and hippocampus, and increase GAD65mRNA expression in dorsal thalamus.2The effect of propofol on GAT-1mRNA and GAD65mRNA in brain in dogs is a non-dose-dependent manner in this experiment.

GAT-1and GAD65mRNA Expressions in Different Brain Regions at Brain Propofol Uptake Equilibrium in Dogs

Abstract

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