Effects of adrenomedullin 2 on proliferation of microvascular endothelial cells from the rat cerebral cortex

AIM: To investigate the effects of adrenomedullin (ADM) 2 (AM2) on proliferation of microvascular endothelial cells from the rat cerebral cortex. METHODS: Microvascular endothelial cells (MEC) were isolated from the cerebral cortex of SD rats and cultured. The cultured cells were identified using immunocytochemistry assay with antibody for factor VIII-related antigen and randomly distributed into eight experimental groups as follows: control, AM2 10-7 mol/L, 10-8 mol/L, 10-9 mol/L, ADM, ADM+AM2, 10% fetal bovine serum (FBS) stimulated, and 10% FBS＋AM2 10^-7 mol/L groups. The proliferation of MEC was detected using ［3H］-TdR incorporation assay. RESULTS: Compared with control, AM2 (10^-7-10^-9 mol/L), ADM (10^-7 mol/L), and AM2 (10^-7mol/L) co-incubated with ADM (10^-7 mol/L) had no effects on ［3H］-TdR incorporation into the MEC (P>0.05). 10% FBS induced ［3H］-TdR incorporation increased by 87.5% (vs control, P<0.05), which was abolished by co-incubated the MEC with 10^-7 mol/L AM2 (P<0.05). CONCLUSION: AM2 inhibits FBS-stimulated proliferation of MEC from the rat cerebral cortex.     

Regulation of estrogen on the expression of KLK6 mRNA and protein in human breast cancer cell line MCF-7

AIM:To investigate the effects of estrogen and tamoxifen on the expression of KLK6 mRNA and protein (hK6) in human breast cancer cell line MCF-7. METHODS:MCF-7 cells were incubated with 17-βE2 and tamoxifen at different concentrations for 72 hours, respectively. The expression levels of kallikrein 6 (KLK6) mRNA and protein were evaluated by fluorescence quantitative RT-PCR and flow cytometry, respectively. RESULTS:Compared with ethanol control, KLK6 mRNA expression levels were significantly decreased when 17-βE2 was added at concentrations of 10^-10 and 10^-8 mol/L (P<0.01). No statistical change was observed when 17-βE2 was at 10^-12 mol/L (P>0.05). Flow cytometry showed the same results. The average fluorescence intensity (AFI) that represents the level of hK6 was decreased after incubated with 17-βE2 (P<0.01). After incubation with tamoxifen, the levels of KLK6 mRNA and hK6 were increased (P<0.01). CONCLUSION:Estrogen down-regulates the expression levels of KLK6 mRNA and protein (hK6), while tamoxifen has an opposite effect.     

Upregulatory effect of isopsoralen on expression of estrogen receptor in human lens epithelial cells

AIM: To investigate the effect of isopsoralen(ISR) on the expression of estrogen receptor (ER) in human lens epithelial cells (HLECs). METHODS: HLECs were cultured and sub-cultured in vitro. The cultured HLECs pretreated with E₂ or ISR were exposed to H₂O₂ at the concentration of 300 μmol/L. The expression of ERα and ERβ in HLECs was analyzed by flow cytometry. RESULTS: The expression of ERα and ERβ in H₂O₂ group was obviously decreased as compared to control group (P<0.01). The expression of ERα and ERβ in the cells treated with E₂ and with ISR at the concentration of 10^-5 mol/L, 10^-6 mol/L or 10^-7 mol/L plus H₂O₂ was obviously increased as compared to the cells treated with H₂O₂ only (P<0.01). A concentration-dependent effect of ISR was observed. CONCLUSION: H₂O₂ decreases the expression of ERα and ERβ in HLECs.E₂ and ISR increase the expression of ERα and ERβ in HLECs treated with H₂O₂ in a concentration-dependent manner, which may account for their antioxidative effect.     

Modulatory roles of muscarinic cholinergic receptor 3 in proliferation and migration of small cell lung cancer

AIM: To study the expression and roles of muscarinic cholinergic receptor 3 (M3R) in human small cell lung cancer (SCLC) cells. METHODS: Human SCLC cell lines SBC3 and H82 were cultured in vitro. RT-PCR and Western blotting were used to investigate the expression of M3R. MTT assay and Boyden chamber assay were carried out to determine the roles of cholinergic receptor agonist acetylcholine iodide (ACh) and M3R antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) in the proliferation and migration of SBC3 cells. RESULTS: M3R was expressed in SBC3 and H82 cells. The relative protein expression of M3R normalized with β-actin in SBC3 was 2.65-fold higher than that in H82. ACh stimulated SBC3 cell proliferation in a dose-dependent manner. Treatment with ACh at concentrations of 10^-4 and 10^-3 mol/L significantly stimulated SBC3 cell growth at 48 h and 72 h (P＜0.01). SBC3 cell proliferation induced by ACh was inhibited by 4-DAMP in a dose-dependent manner. Pretreatment of the cells with 10^-5 mol/L 4-DAMP suppressed the effect of ACh at 48 h (P＜0.05). Pretreatment with 4-DAMP at concentrations of 10^-6, 10^-7 (P＜0.05) and 10^-5 mol/L (P＜0.01) inhibited the effect of ACh at 72 h. Treatment with 10^-5 or 10^-6 mol/L 4-DAMP alone inhibited the cell proliferation at 48 h (P＜0.01) and the inhibitory effect of 4-DAMP at concentration of 10^-5 mol/L was stronger than that of 4-DAMP at concentration of 10^-6 mol/L at 72 h. ACh increased the cell migration towards fibronectin (Fn) in a dose-dependent manner and ACh at concentration of 10^-4 mol/L enhanced the cell migration by about 3 folds. The cell migration stimulated by 10^-4 mol/L ACh was almost completely blocked by pretreatment with 4-DAMP at concentration of 10^-6 or 10^-5 mol/L (P＜0.01). CONCLUSION: M3R is expressed in human SCLC cells. The M3R antagonist inhibits SBC3 cell proliferation and migration.     

Changes of RyR-mediated Ca2+ release in VSMCs is involved in the development of vascular hyporeactivity in hemorrhagic shock rats

AIM: In order to investigate the mechanisms involved in the vascular hyporeactivity after hemorrhagic shock, the changes of Ca²⁺ release from calcium store in vascular smooth muscle cells (VSMCs) with hypoxia were observed and the role of Ca²⁺ release from calcium store in the occurrence of vascular hyporeactivity to norepinephrine (NE) after hemorrhagic shock in rats was further explored.METHODS: A hemorrhagic shock model (40 mmHg for 2 h) in rats and a VSMCs hypoxic model were established. The changes of intracellular Ca²⁺ concentration in VSMCs were evaluated by fura3-AM and the role of IP₃R and RyR mediated Ca²⁺ release from calcium store was further observed. The role of IP₃R and RyR mediated Ca²⁺ release from Ca²⁺ store in the development of vascular hyporeactivity was measured with an isolated organ perfusion system. RESULTS: In the absence of extracellular Ca²⁺, NE upregulated by mobilizing Ca²⁺ release through calcium store. Compared to the normal control, the VSMCs had a slight increase when treated with hypoxia and NE-induced intracellular down-regulated, both without significant difference. Compared to the normal control cells, there was a significant change of Ca²⁺ release from calcium store in hypoxia-treated VSMCs, characterized by the significant increase in triggered by RyR-sensitive Ca²⁺ releasing activator caffeine. However, the increase in triggered by IP₃R-mediated Ca²⁺ release agonist adenophostin A (10^-5 mol/L) and ATP-Na₂ (10^-4 mol/L) had no significant difference in hypoxic VSMCs. Furthermore, the vascular reactivity to NE decreased in abdominal aorta in hemorrhagic shock (40 mmHg, 2 h) rats. The activation of IP₃R mediated Ca²⁺ release with ATP-Na₂ (10^-4 mol/L) did not improve the vascular reactivity to NE, while inhibition of IP₃R mediated Ca²⁺ release with heparin (10⁴ U/L) significantly antagonized the vascular reactivity to NE in hemorrhagic shock rats. In addition, in normal K-H solution (with about 2.2 mmol/L) and Ca²⁺-free K-H solution, RyR antagonist ryanodine (10^-5 mol/L) partly restored the vascular reactivity to NE in hemorrhagic shock rats, while RyR agonist caffeine(10^-3 mol/L) further decreased the vascular reactivity. CONCLUSION: The over-activation of RyR-mediated Ca²⁺ release from calcium store is partly involved in the development of vascular hyporeactivity after hemorrhagic shock in rats.     

Sinomenine inhibits TNF-α-induced VCAM-1 expression on human umbilical vein endothelial cells

AIM: To investigate whether sinomenine(SN) can decrease TNF-α-induced VCAM-1 expression in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were isolated from freshly collected umbilical cords.Positive control samples were stimulated with TNF-α, omitting SN. Negative control samples were treated in the same way, omitting TNF-α and SN. Experiment samples were co-cultured with TNF-α and SN at different concentration (0.25, 0.5, and 1.0 mol/L),or TNF-α and dexamethasone(Dex) at concentration of 1.0×10^-6mol/L.Cells were harvested after cultivation with the drugs for 12 hours. VCAM-1 mRNA expression was detected by real-time quantitative PCR, and VCAM-1 expression was detected by flow cytometry (FCM).RESULTS: VCAM-1 mRNA and VCAM-1 were induced by TNF-α. Compared with the positives, the relative VCAM-1 mRNA expression decreased to varying degrees in the experiment groups (P<0.05), and SN at concentration of 0.5 mol/L and 1.0 mol/L inhibited expression of VCAM-1 (P<0.05). SN at concentration of 1.0 mol/L decreased VCAM-1 expression by 28.8%(P<0.05), and SN at concentration of 0.5 mol/L reduced VCAM-1 expression by 21.68%(P<0.05). But SN at concentration of 0.25 mol/L and Dex at concentration of 1.0×10^-6mol/L didnt depress expression of VCAM-1. CONCLUSION: SN may inhibit TNF-α-induced VCAM-1 expression in HUVECs in vitro.     

Effect of salvianolic acid B on expression of TGF-β1 receptors and Smad2 in activated mesangial cells

AIM: To study the mechanisms of salvianolic acid B (Sal B)antagonizing mesangial cell activation and kidney fibrosis through investigating the effect of Sal B on expression of transforming growth factor-β₁ (TGF-β₁) receptors and Smad2 in TGF-β₁-stimulated renal mesangial cell activation. METHODS: Mesangial cells was isolated and purified from rat kidney. TGF-β₁ was used to establish rat primary mesangial cell activation model and Smad2,Smad7 protein expression was detected. Sal B (10^-6 mol/L and 10^-5 mol/L) was employed to treat the cells; α-smooth muscle actin(α-SMA) expression was analyzed by immunofluorescence staining and Western blotting. Mesangial cells were treated with Sal B alone or additional with TGF-β₁,and TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),Smad2 phosphorylation and Smad2 protein expression was determined by Western blotting. RESULTS: Cell ular model was established by incubating with 5 μg/L TGF-β₁ for 24 h,and in early stage Smad2 was significantly phosphorylated. Sal B (10^-6 mol/L and 10^-5 mol/L) could inhibit α-SMA expression,which was the biomarker of activated mesangial cells. In addition,in Sal B group,the protein expression of TβRⅠand TβRⅡ was significantly down-regulated while Smad2 phosphorylation in mesangial cells was inhibited. CONCLUSION: Sal B inhibits the TGF-β₁-Smad pathway,the protein expression of TβRⅠ,TβRⅡ and Smad2 phosphorylation in mesangial cells,which is probably one of the mechanisms of Sal B alleviating kidney fibrosis.     

Senescence of endothelial cells and gene expression associated with apoptosis induced by angiotensinⅡ

AIM: To study the senescence of human umbilical vein endothelial cells (HUVECs) and Bcl-2, Bax gene expression associated with apoptosis induced by angiotensinⅡ (AngⅡ).METHODS: HUVECs were cultured in vitro and the cell viability was observed by methyl thiazolyl tetrazolium (MTT). HUVECs were intervened by AngⅡ and valsartan (AngⅡ type 1 receptor blocking) and divided into 3 groups: the control group, AngⅡ group (stimulated with AngⅡ10^-6mol/L for 48 h), valsartan group (valsartan was added to cells 1 h before 10^-6mol/L AngⅡ treatment). β-gal staining and cell cycle analysis were used to identify the cell aging status. Morphologic changes and percentage of apoptosis were assayed with Hoechst33258 under fluorescent microscope. The expressions of Bcl-2 and Bax, and the apoptosis-associated genes were detected by immunocytochemical staining, RT-PCR and Western blotting. RESULTS: The cell viability by AngⅡ-induced cells was (81.9%±4.1)%, the positive cell number of β-gal staining was significantly higher in AngⅡ-induced cells (80.10%±6.81)% than that in the control cells. The cell cycle was at G₀-G₁(91.36%±6.45)%, the apoptotic cells significantly increased (31.84±2.86)% under fluorescent microscope. In valsartan group, Bcl-2 mRNA and protein expression increased markedly (P<0.05), but Bax mRNA and protein expression decreased evidently (P<0.05) compared to those in the AngⅡ group.CONCLUSION: Cell apoptosis is possibly an important factor for endothelial cell senescence and vascular aging induced by AngⅡ. One of its molecular mechanisms might be associated with decreasing the expression level of Bcl-2 and increasing that of Bax, which regulate the imbalance between mRNA and protein expression of Bcl-2 and Bax. Valsartan improves endothelial cell aging.     

Calcineurin signal transduction pathway involves in cardiomyocyte hypertrophy induced by prostaglandin F2α

AIM: To study the role of prostaglandin F₂α (PGF₂α) in cardiac hypertrophy and its relation with calcineurin (CaN) signal transduction pathway in vitro. METHODS: The cultured neonatal rat cardiomyocyte was used to observe the hypertrophic effect of PGF2α, and the hypertrophic response was assayed by measuring the cell diameter, protein content and atrial natriuretic factor (ANF) mRNA expression. For mechanism studies, the intracellular free calcium concentration (［Ca²⁺］_i) in cultured cardiomyocytes was measured by using Fura-2/AM as a fluorescent indicator. ANF and CaN mRNA expressions, and the expressions of CaN and its downstream effectors, NFAT₃ and GATA₄ proteins were assayed by RT-PCR and Western blotting, respectively.RESULTS: In cultured cardiomyocytes, PGF₂α induced profound hypertrophic morphology change, the significant increase in cell diameter and protein content in a concentration-dependent manner compared with those in vehicle control (P<0.01). The same result was found in measuring the ［Ca²⁺］_i in cardiomyocytes (P<0.01). PGF₂α at concentration of 10-7 mol/L significantly promoted ANF and CaN mRNA expressions and the protein expressions of CaN/NFAT₃/GATA₄ compared with those in the vehicle control (P<0.05). Cyclosporin A, a CaN inhibitor, markedly inhibited the myocyte hypertrophy (P<0.01), reduced the increased ［Ca²⁺］_i(P<0.01) and decreased the expressions of CaN mRNA and CaN/NFAT₃/GATA₄ proteins (P<0.05) compared with those of only PGF₂α　10^-7 mol/L treatment. CONCLUSION: Cardiomyocyte hypertrophy induced by PGF2α may be, at least in part, mediated by CaN signal transduction pathway activated by increasing ［Ca²⁺］_i.     

Role of PKC-α and δ isoforms in AVP improved contractile response of VSMC after hypoxia and its relations to MLC20 phosphorylation

AIM:The present study was to investigate the roles of protein kinase C α and δ isoforms (PKC-α, δ) in arginine vasopressin (AVP) improved contractile response of vascular smooth muscle cells (VSMC) to norepinephrine (NE) after hypoxia and its relations to myosin light chain (MLC₂₀) phosphorylation, myosin light chain phosphatase (MLCP) and myosin light chain kinase (MLCK) activity.METHODS: Primary cultures of VSMC were obtained from the superior mesenteric artery (SMA) of rats by explanting technique and the cells in third to fifth passage were used in the study. The effects of PKC-α and δ antagonists on AVP induced contractile response of VSMC to NE after 1.5 h hypoxia were observed by measuring the ratio of accumulative infiltration of fluorescent isothiocyanate-conjugated bovine serum albumin with transwell, and their effect on the activity of MLCP/MLCK in VSMC was assayed by enzymatic catalysis. At the same time, with the SMA from hemorrhagic shock rats (30 mmHg for 2 h), the effects of PKC α and δ isoforms in the regulation of AVP on MLC₂₀ phosphorylation of SMA after shock were observed by Western blotting.RESULTS: G 6976 (5×10^-6 mol/L, PKC-α isoform inhibitor) significantly antagonized AVP (5×10^-10 mol/L)-induced increase in the contractile response of VSMC to NE after hypoxia, and rottlerin (10^-5 mol/L, PKC-δ isoform inhibitor) also partly inhibited this effect. Hypoxia resulted in a significant increase in MLCP activity, with a decrease in MLCK activity of VSMC, and at the same time, the MLC₂₀ phosphorylation of SMA following hemorrhagic shock was significantly decreased. AVP inhibited the activity of MLCP and increased the phosphorylation of MLC₂₀, which was inhibited by G 6976, while rottlerin treatment only showed a slightly inhibitory effect. AVP and PKC-α, δ inhibitor had no significant influence on MLCK activity.CONCLUSION:AVP up-regulates vascular reactivity and calcium sensitivity of VSMC possibly through inhibiting the activity of MLCP and increasing the phosphorylation of MLC₂₀ by PKC-α isoform.     

he effects of constant magnetic field on apoptosis,adhesion molecule expression in endothelial cells and their adhesion rates with monocytes

AIM: To investigate the effects of constant magnetic field on apoptosis, secretion and expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC), and their adhesion rates with THP-1 monocytes induced by angiotensin Ⅱ (AngⅡ).METHODS: The third passage of cultured HUVEC was used.There were six groups: control group, Ang Ⅱ (10^-6 mol/L) group, Ang Ⅱ with 1, 5, 10 or 20 gausses of constant magnetic field group.Samples were collected 24 h after incubation with or without magnetic field.Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and propidinm iodide staining with flow cytometry.Secretion and expression of ICAM-1 and VCAM-1 were detected by ELISA and immunocytochemistry, respectively.Adhesion rate between HUVEC and THP-1 was measured by counting method.RESULTS: Ang Ⅱ at concentration of 10^-6mol/L induced apoptosis in HUVECs (P<0.05 vs control), whereas in 1, 5, 10 and 20 gausses group, apoptosis of HUVECs was significantly lower than that in Ang Ⅱ group (P<0.05).Ang Ⅱ at concentration of 10-6 mol/L significantly increased secretion and expression of ICAM-1 and VCAM-1 (P<0.05 vs control), whereas secretion and expression of ICAM-1 and VCAM-1 in 1, 5, 10 and 20 gausses group significantly decreased, compared with Ang Ⅱ group (P<0.05).The adhesion rates between HUVEC and THP-1 significantly increased 24 h after incubation of HUVEC with Ang Ⅱ (P<0.05 vs control), in contrast, the adhesion rates between HUVEC and THP-1 in 1, 5, 10 and 20 gausses group significantly decreased, compaed with Ang Ⅱ group (P<0.05).CONCLUSIONS: One gauss to 20 gausses of constant magnetic field antagonizes the effects of Ang Ⅱ on HUVEC, decreases apoptosis and expression of ICAM-1 and VCAM-1 in HUVEC, and also decreases the adhesion rates between HUVEC and monocytes induced by Ang Ⅱ.     

Effect of inhaled nitric oxide on aquaporin expression in newborn rat lung in acute hyperoxic lung injury

AIM:To investigate the effect of inhaled nitric oxide on aquaporin expression and alveolar epithelial fluid transport in newborn rats with acute hyperoxic lung injury. METHODS:32 newborn SD rats were randomized to breathe for 48 h room air (C), >95%O₂ (O), >95%O₂+10^-5 NO (NO only in the first 24 h, ONO), room air + NO (CN). Then, the rats were killed, the lung wet-to-dry weight ratio (Q_W/Q_D), the histology, and AQP1, AQP5, α₁-NKA, α-ENaC mRNA expressions in the lungs were measured. RESULTS:Compared with C group, the Q_W/Q_D in O group significantly increased (P<0.01), and AQP1 mRNA expression decreased significantly (P<0.01). Compared with O group, ONO group had a lower level of Q_W/Q_D (P<0.05), and AQP1 mRNA expression increased (P<0.05). AQP5 mRNA expression in all groups remained unchanged. CONCLUSION:In newborn rats with acute hyperoxic lung injury, inhaled 10-5 nitric oxide for 24 h may attenuate lung edema and increase AQP1 mRNA expression, suggesting that inhaled 10^-5 nitric oxide for 24 h may promote the AQP1 expression in lung in this model of acute lung injury.     

Deficiency in Na-K-2Cl co-transporter impaired hearing and balance in mice

AIM: We generated transgenic mice of NKCC1^-/- (homozygous mutant),NKCC1^+/- (heterozygous) and NKCC1^+/+ (wild-type) that have a targeted disruption in the NKCC1 gene to investigate the role of Na-K-2Cl (NKCC1) channel in auditory function of the inner ear.METHODS: Hearing threshold and endocochlear potential (EP) were measured in the NKCC1^-/-,NKCC1^+/- and NKCC1^+/+ mice by auditory brainstem response (ABR) and EP recordings,respectively.The inner ears of the mice were removed and examined morphologically with the light microscope.RESULTS: The auditory function of NKCC1^+/+ mice was normal,the mean value for ABR thresholds in response to click sound was ［（23.13±3.78）dB,SPL］,EP was （98±16）mV.The mean value for ABR thresholds to click sound was elevated in NKCC1^+/- mice ［（38.49±12.29） dB,SPL］,relative to that significantly increased in NKCC1^+/+ mice (P<0.01).EP in NKCC1^+/- mice was about （78±7） mV,significantly decreased than that in NKCC1^+/+ mice (P<0.05).NKCC1^-/- mice were completely deaf,the ABR waveform was not observed for even 100 dB SPL sound stimuli used and EP was nearly disappeared (EP,4 mV±6 mV).NKCC1^-/- mice were deaf and demonstrated difficulties in maintaining their balance.NKCC1^-/- mice exhibited a marked atrophy of the stria vascularis,contraction of the endolymphatic compartments and collapse.CONCLUSION: NKCC1 channel plays a critical role in potassium homeostasis of endolymph in the inner ear.Mice lacking of NKCC1 can lead to changing K⁺ concentration in endolymph and influence auditory function subsequently.NKCC1 knockout mice exhibit marked structural abnormalities of the cochlea as well.     

Effects of atorvastatin on cardiac myocyte hypertrophy of neonatal rat induced by angiotensinⅡ in vitro and TLR4 expression

AIM: To assess effects of atorvastatin (Ator) on cardiac myocytes (CM) hypertrophy of neonatal rat induced by angiotensinⅡ (AngⅡ) in vitro and Toll like receptor 4 (TLR4) expression for theoretical bases of preventing and treating myocardial hypertrophy.METHODS: CM of neonatal Sprague-Dawley (SD) rats were isolated with trypsin digestion method and those growth-arrested cells were stimulated with 10^-7 mol/L AngⅡ in the presence of various concentrations of Ator.The method of coomassie brilliant blue was adopted to evaluate the protein contents of CM.The changes in β-MHC,AT₁ receptor and TLR4 mRNA expression were observed by RT-PCR.RESULTS: ① AngⅡ increased the protein content of CM and β-MHC mRNA expression significantly,upregulated AT₁ receptor and TLR4 gene expression.② In a dose-dependent manner,Ator inhibited the increases in the protein contents of CM and β-MHC mRNA expression induced by AngⅡ.③ In a dose-dependent manner,Ator downregulated the AT₁ receptor and TLR4 mRNA expression of CM hypertrophy of neonatal rat induced by AngⅡ.CONCLUSION: Ator inhibits CM hypertrophy,downregulates the AT₁ receptor and TLR4 gene expression.     

Harmful factors affect glycine receptor α1 subunit mRNA expression in the neonatal rat cardiomyocytes

AIM: To investigate the effects of lipopolysaccharide, hypoxia/reoxygenation,isoproterenol and high concentration of glucose on glycine receptor α₁ subunit mRNA expression in the neonatal rat cardiomyocytes. METHODS: Isolation of cardiomyocytes from Sprague-Dawley rats aging 1～3 d were performed. Cardiomyocytes （1×10⁵～5×10⁵ cells·L^-1）were cultured in DMEM medium containing 15% fetal bovine serum at 37 ℃ in 5%CO₂ atmosphere for 72 h. Then, cultured rat cardiomyocytes were treated with lipopolysaccharide, isoproterenol or high concentration of glucose for 24 h, respectively, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell survival rate was measured using CCK-8 reactant and RT-PCR was applied to monitor the expression of glycine receptor α₁ subunit mRNA. RESULTS: Compared with the control group, no significant difference in the cell survival rate was observed （P＞0.05）. The expression of glycine receptor α₁ subunit mRNA was increased （P＜0.01） in lipopolysaccharide（5,10,20,40,80 mg/L）,isoproterenol（20,100,500 μmol/L） or hypoxia/reoxygenation, hypoxia groups, but decreased（P＜0.01）in the group treated with high concentration of glucose（25, 50 mmol/L）. CONCLUSION: Lipopolysaccharide, isoproterenol, hypoxia/reoxygenation or hypoxia upregulates the expression of glycine receptor α₁ subunit mRNA,but high concentration of glucose down-regulates the expression of glycine receptor α₁ subunit mRNA in cultured neonatal rat cardiomyocytes.