Changes of intracellular potassium activity and membrane potential in guinea pig ventricular myocardium during superfusion with low sodium solutions and effects of low pH

AIM:To investigate the changes in intracellular potassium activity(aⁱK) and membrane potential(V_m) induced by low external sodium infusion (Low [Na]_o) and to detect the mechanisms involved and the relationship between aⁱK and V_m. METHODS:aⁱK and V_m were measured in infusion with different sodium concentrations using methods of convenient and ion selective microelectrodes in guinea pig ventricular myocardium. RESULTS:Low [Na]_o resulted in a decrease in aⁱK and an increase in V_m in a Na⁺ concentration-dependent manner.At the same time,contraction and resting potential increased, and action potential duration decreased markedly,but action potential amplitude was not affected. A change of the pH from 7.4 to 7.0 in low [Na]_o solution reduced the decrease in aⁱK, but did not affect the increase in V_m.CONCLUSION:A better linear relationship appeared between the changes in aⁱk and [Na]_o or in V_m and [Na]_o,while during each low [Na]_o the change in both aⁱK or V_m may reach a new balance level.     

Role of potassium channels in the regulation of intracellular free Ca2+ concentration of pulmonary artery smooth muscle cells in rats

AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration ( [Ca²⁺]_i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition, [Ca²⁺]_i was (156.91±8.60) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (294.01±16.81) nmol/L. 2. In normoxic condition, the voltage-dependent K⁺-channel antagonist 4-aminopyridine (4AP), but not the Ca²⁺-activated K⁺-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K⁺-channel antagonist glibenclamide (Glib) increased [Ca²⁺]_i. 3. In hypoxic condition, 4AP and TEA caused the rise in [Ca²⁺]_i , but Glib had no effect on [Ca²⁺]_i. 4. MTT assay showed that 4AP increased the value of absorbing light degree (A value) in normoxic and hypoxic condition (0.582±0.062,0.873±0.043,respectively, P＜0.01), TEA increased A value only in hypoxic condition, and Glib had no effect on the proliferation of PASMCs. CONCLUSIONS: K_V plays an important role in the regulation of [Ca²⁺]_i and proliferation of PASMCs. K_Ca serves as distinct responsive roles in the regulation of proliferation of PASMCs in hypoxic condition. K_ATP has no effect on [Ca²⁺]_i and proliferation of PASMCs in normoxic and hypoxic conditions.     

Protective effect of rapid ischemic preconditioning against spinal cord ischemic injury in rabbits

AIM: To evaluate the protective effect of rapid phase of ischemic preconditioning against spinal cord ischemic injury in rabbits. METHODS: Thirty six male New Zealands white rabbits were randomly assigned to 3 groups (12 in each group): ischemia and reperfusion injury group (IR group), ischemic preconditioning + IR group (IPC+IR group) and sham operation group (sham). In IR group, spinal cord ischemia was induced by an infrarenal aorta clamping for 20 min; The rabbits in IPC+IR group underwent a 6 min ischemic preconditioning followed by 30 min of reperfusion before the 20 min clamping; The rabbits in sham group underwent the same procedures as the IR group except for infrarental aortic unclamping. Neurologic status was scored at 8, 12, 24 and 48 h after reperfusion. All animals were sacrificed at 48 h after reperfusion and the spinal cords (L_5-7) were removed for histopathologic study and determination of the activity of Na⁺, K⁺-ATPase. RESULTS: The neurologic function scores in sham group and IPC+IR group at each observation interval were higher than those in IR group (P＜0.01). Compared to IR group, there were more normal neurons in anterior horn of spinal cord in sham group and IPC+IR group (P＜0.01); the activity of Na⁺, K⁺-ATPase in sham group and IPC+IR group were higher than those in IR group (P＜0.01). CONCLUSION: The rapid phase of ischemic preconditioning has a protective effect against spinal cord ischemic injury in rabbits, and this neuroprotection may be related to the maintenance of Na⁺, K⁺-ATPase activity.     

Effect of 2, 3-butanedione monoxime (BDM) on myocardial protection in cold cardioplegia

AIM: The effects of BDM on isolated rat heart in cold cardioplegia were studied. METHODS: Rat heart were subjected to cold cardioplegia at 4℃ for 8, 18 and 24 h.Then each heart was perfused (90 cm H₂O) in Langendorff model at 37℃ for 40 min. In the high K⁺ group(n=24) the hearts were preserved in St.Thomas cardioplegic solution, in BDM group(n=24) hearts were preserved in K-H solution with BDM 30 mmoL/L. RESULTS: After 18 h, heart rate and the coronary flow in BDM group were significantly higher than in high K⁺ group(P＜0.05). Activity of Na⁺-K⁺-ATPase in cell membrane and in mitochondrial membrane in the BDM group was significantly higher than high K⁺ group(P＜0.01). After 24 h, all hearts in high K⁺ group were dead, but were alive in BDM group. CONCLUSION: Under given experiment conditions, BDM did enhance the tolerance to cold ischemia significantly. The results showed that BDM may become a useful agent for prolong the storage period of heart in cold cardioglegia.     

Relaxation effect of isoliensinine on isolated mouse airway smooth muscle

AIM: To study the relaxation effect of isoliensinine on high K⁺-induced isolated mouse airway smooth muscle (ASM) and the underlying mechanism. METHODS: The muscle tension transducer was used to detect the effects of isoliensinine on high K⁺-induced precontraction and Ca²⁺ influx in ASM. The technique of patch-clamp and calcium imaging system were respectively used to examine the effects of isoliensinine on LVDCC currents and[Ca²⁺]_i of the ASM cells (ASMCs). RESULTS: Isoliensinine significantly relaxed precontracted ASM induced by high K⁺ in a concentration-dependent manner. The maximum relaxation ratio was(95.3±3.9)% by isoliensinine at 100 μmol/L. In addition, LVDCC currents were measured using the whole-cell patch-clamp technique, which were abolished by isoliensinine. High K⁺-induced 340/380 nm fluorescence ratio of Fura-2 was 0.63±0.10 in ASMCs, while it decreased to 0.36±0.05 after the addition of isoliensinine (P<0.01). When isoliensinine was added at the peak point of[Ca²⁺]_i, the ratio rapidly decreased from 0.74±0.02 to 0.42±0.05 (P<0.01). Moreover, isoliensinine inhibited high K⁺-induced Ca²⁺ influx-mediated contraction of ASM. CONCLUSION: Isoliensinine inhibits LVDCC currents, terminates Ca²⁺ influx and reduces[Ca²⁺]_i, eventually resulting in relaxation of the ASM, indicating isoliensinine might be a potential bronchodilator.     

Interleukin-2 altered the frequency -dependent relationship of intracellular calcium in rat ventricular myocytes

AIM:We examined the effect of interleukin-2 (IL-2) on calcium handling of rat cardiomyocytes. METHODS:The effects of steady state and transient changes in stimulus frequency on the intracellular calcium transient were investigated in the isolated ventricular myocytes with spectrofluorometry technique. RESULTS: Under the steady state (0.2 Hz), IL-2 at 2×10⁵U/L decreased the peak [Ca²⁺] i and amplitude of the [Ca²⁺]i transient, increased the diastolic calcium level, and prolonged the decay of the calcium transient. At 1.25 mmol/L of extracellular [Ca²⁺], when increasing the stimulus frequency from 0.2 to 1.0 Hz, diastolic calcium level and peak [Ca²⁺] i as well as the amplitude of the transient were increased. The positive frequency relationship was blunted in the IL-2-treated myocytes and this was not normalized by increasing extracellular [Ca²⁺] to 2.5 mmol/L. The caffeine induced Ca²⁺ release was increased with increase in stimulus frequency. IL-2 inhibited the frequency relationship of caffeine induced Ca²⁺ release. The restitution was not different between control and IL-2 groups at the 1.25 mmol/L of extracellular [Ca²⁺], which was slowed in IL-2-treated myocytes when the extracellular [Ca²⁺] was increased to 2.5 mmol/L. CONCLUSIONS:It is concluded that the blunted frequency response of IL-2-treated myocytes was resulted from the decrease in SR Ca²⁺ release, which was related to depression of SR function. Despite the evidence of depressed SR Ca²⁺ uptake, the restitution of calcium transient at 1.25 mmol/L of extracellular remains unchanged, which maybe due to the increase in the Na⁺/Ca²⁺ exchanger activity.     

Protective effect of hydrogen sulfide on PC12 cells injured by ATP

AIM: To prove the purinergic signaling mechanism of the neuroprotective action of hydrogen sulfide by observing the effects of sodium hydrosulfide (NaHS), a donor of hydrogen sulfide, on the cell viability, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the change of membrane permeability in the PC12 cells injured by adenosine triphosphate (ATP). METHODS: PC12 cells in logarithmic growth phase were randomly divided into 4 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treated with ATP after cultured for 24 h. In NaHS+ATP group, the cells were incubated with NaHS for 30 min before treated with ATP, and NaHS always existed in the reaction system. In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, and the other treatments were as the same as those in NaHS+ATP group. The cell viability was assessed by MTT assay. The [Ca²⁺]_i was detected by Fura-2/AM staining. The membrane permeability was observed by staining with fluorescent dye YO-PRO-1.RESULTS: ATP at concentration of 0.3 mmol/L showed no injury effect on the cells. However, the cell viability was dropped gradually in a dose-dependent manner as the ATP at doses of 1, 3, 5 and 10 mmol/L. The decline of cell viability by ATP was obviously reversed by 200 μmol/L of NaHS in the PC12 cells (P<0.05), but exasperated by 800 μmol/L of NaHS (P<0.05). At the same time, ATP evoked the increase in [Ca²⁺]_i in a dose-dependent manner, which was inhibited by NaHS (P<0.05). Furthermore, the YO-PRO-1 uptake induced by ATP in a dose-dependent and time-dependent manner was also reduced by NaHS (P<0.05). CONCLUSION: Hydrogen sulfide has protective effect on the PC12 cells injured by ATP. The mechanism may be related to the reverse of the increased [Ca²⁺]_i and YO-PRO-1 uptake.     

Calcium-sensing receptor modulates pulmonary artery tension through G-protein-PLC-IP3 pathways

AIM: To observe the role of calcium-sensing receptor (CaSR) in the regulation of pulmonary artery tension. METHODS: The intracellular calcium concentration ([Ca²⁺]_i) was detected by laser-scanning confocal microscopy, and the pulmonary artery tension was determined by the pulmonary arterial ring technique. RESULTS: Increased levels of [Ca²⁺]_o or Gd³⁺ (an agonist of CaSR) induced the increase in [Ca²⁺]_i and pulmonary artery constriction in a concentration-dependent manner. Additionally, the effects of Ca²⁺ and Gd³⁺ were inhibited by U73122 and D609 (specific inhibitor of PLC), and 2-APB and heparin (specific antagonist of IP₃ receptor). However, U73343 (U73122 inactive analogue) did not take effect. CONCLUSION: CaSR may be involved in the regulation of pulmonary artery tension by increasing [Ca²⁺]_i through G-protein-PLC-IP₃ pathway.     

Effects of glucose and Mg2+ in the neurons damaged by glutamate

AIM and METHODS: To observe the effects of glucose-free and Mg²⁺-free in the extracellular fluid on the changes of [Ca ²⁺]_i in the cerebro-cortical neurons damaged by 1mmol/L glutamate using laser confocal scanning microscope. RESULTS: Both frequency and amplitude of neuronal calcium oscillation induced by glutamate were lowered in glucose-free and Mg²⁺-free buffers. The basic [Ca²⁺]_i concentration was lowered in the former case , but it was elevated in the latter case. CONCLUSION: Mg²⁺-free aggravates [Ca²⁺]_i overload induced by 1mmol/L glutamate ,under certain conditions the glucose-free might resist damage role of glutamate and Mg²⁺-free.     

Effects of hydrogen peroxide on sodium current in acutely isolated rat hippocampal CA1 neurons

AIM and METHODS: The effects of hydrogen peroxide on Na⁺ currents were studied in freshly dissociated rat hippocampal CA1 neurons using the whole-cell patch-clamp techinique. RESULTS: ①H₂O₂ caused a dose-dependent and voltage-dependent increase in the voltage-activated Na⁺ currents. The amplitudes of Na⁺ currents were increased (48.0±4.2)% and (88. 2±5. 1)% (n=10) by H₂O₂ at 10 μmol/L and 100 μmol/L, respectively. ②H₂O₂ (10 μmol/L) did not affect the activation process, but changed the inactivation process significantly. Before and after application of 10 μmol/L of H₂O₂, the half-inactivation voltage was (-64.58±1.22)mV and (-53.55±0.94)mV (n=10, P<0.01), but the slope factor was not changed. CONCLUSION: As a product of oxidation metabolism, H₂O₂ is related to some diseases in the central nervous system.     

Effect of interleukin-2 on intracellular calcium levels in rat ventricular myocytes during anoxia and reoxygenation

AIM: To investigate the effect of interleukin-2(IL-2) on the intracellular calcium in electrically stimulated adult rat ventricular myocytes during anoxia and reoxygenation. METHODS: The isolated cardiac ventricular myocytes were exposed to 5 min anoxia followed by 10 min reoxygenation. Chemical anoxia was introduced by Krebs-Henseleit(K-H) solution containing 10^-3 mol/L sodium dithionite. The spectrofluorometric method was used to verify intracellular calcium transient with fura-2/AM as calcium fluorescence probe. RESULTS: It was shown that during anoxia, the amplitude of Ca²⁺ transient was decreased, diastolic [Ca²⁺]_i, time to peak and time to relaxation of Ca²⁺ transient were increased. All the parameters were got back but did not returned to the pre-anoxia level during reoxygenation. IL-2 at 2×10⁵ U/L administrated during anoxia aggravated the effect of rexoxygenation on [Ca²⁺]_i transient. Pretreatment with a specific κ opioid antagonist, nor-BNI(10^-8 mol/L), abolished the effect induced by IL-2 during anoxia on the [Ca²⁺]_i transients, whereas specific δ opioid antagonist, naltrindole(10^-6 mol/L), did not cancel the effect. CONCLUSION: It is concluded that administration of IL-2 during anoxia aggravated the effect of reoxygenation on the [Ca²⁺]_i transients of isolated ventricular myocytes, which was mediated by cardiac κ opioid receptor pathway.     

Rapid preparation of hypoxic Krebs-Henseleit solution with sodium sulfite for in vitro model of hypoxic pulmonary vasoconstriction

AIM: To investigate the feasibility of using sodium sulfite (Na₂SO₃) to prepare hypoxic Krebs-Henseleit (KH) solution for hypoxic pulmonary vasoconstriction (HPV) model in vitro. METHODS: Different doses of Na₂SO₃ were added into 0.5 L KH solution at 37°C. An i-STAT portable clinical analyzer was used to measure the oxygen partial pressure (PO₂), carbon dioxide partial pressure (PCO₂), pH value and the concentration of sodium (Na⁺) in these KH-Na₂SO₃ solutions 1 min after administration. Then the dose of Na₂SO₃ suitable for HPV model was dissolved in 0.5 L KH solution and the above indexes in the solution were monitored at various time points at 37°C under atmospheric pressure. RESULTS: More than 0.2 g (including 0.2 g) Na₂SO₃ reduced the PO₂ of 0.5 L KH solution in a dose-dependent manner (P<0.01). In addition, 1.5 g Na₂SO₃ reduced the PO₂ of 0.5 L KH solution to 20~40 mmHg and maintained the hypoxic state for at least 90 min (suitable for HPV model in vitro), but had nearly no effect on the PCO₂, pH and Na⁺ levels. CONCLUSION: The hypoxia solution for HPV model could be reached by Na₂SO₃ in open air and the method is simple, easily feasible and stable.     

平邑甜茶根系 NO 和活性氧对钠与氯离子胁迫的反应

 以平邑甜茶（Malus hupehensis）实生苗为试材,在水培条件下研究了氯化钠（NaCl）、钠离 子（Na+）和氯离子（Cl-）对根系一氧化氮（NO）和活性氧（ROS）生成速率以及膜脂过氧化的影响。 结果表明：在相同浓度等渗透势NaCl、Na⁺和Cl^-盐溶液处理下,平邑甜茶根系NO 和超氧阴离子 产生速率以及丙二醛（MDA）和过氧化氢（H₂O₂）含量均升高,升高数值由大到小均为NaCl>Cl^- >Na⁺ 处理,并且NaCl 处理下的所测各指标的数值均小于Na+和Cl-单独处理下两者的加和,显示在NaCl 胁迫 诱导平邑甜茶根系积累ROS 和NO 进而引起氧化伤害的过程中,Cl^-的作用大于Na⁺,NaCl 引起的胁迫效 应并不是Na⁺和Cl^-胁迫效应的简单叠加。     

Role of K+ channels in hypoxic pulmonary vasoconstriction in normal and chronic hypoxic rats

AIM:To investigate the role of K⁺ channels in the decreased hypoxic pulmonary vasoconstriction(HPV) in chronic hypoxic rats. METHODS:Blockers of three kinds of K⁺ channels, 4-AP(voltage dependent K⁺ channel blocker), TEA(Ca²⁺ activated K⁺ channel blocker), GLIB(ATP sensitive K⁺ channel blocker) were used in isolated perfused rat lungs to detect the role of K⁺ channels in HPV. RESULTS:In normal rats, 4-AP and TEA, but not GLIB, both elicited a significant increase in pulmonary artery baseline pressure, and also potentiated the acute hypoxic pulmonary vasoconstriction. In chronic hypoxic rats, the HPV is significantly decreased, while 4-AP, TEA, GLIB all elicited a significant but smaller increase in pulmonary artery baseline pressure. Additionally, all these three blockers potentiated the HPV stronger in chronic hypoxic rats than in control rats. CONCLUSION:The opening of Kv, K_Ca, K_ATP might modulate the hypoxic pulmonary vasoconstriction in isolated rat lungs, and the increase in this modulation by potassium channel in chronic hypoxic rats might play a role in its decrease in HPV.     

百合在NaHCO3胁迫下的生理响应机制

比较了耐盐碱的岷江百合（Lilium regale Wilson）和对盐碱敏感的东方百合‘索邦’（‘Sorbonne’）在不同浓度的碱性盐NaHCO₃胁迫下叶片的渗透调节物质、活性氧清除物质、离子调节物质（Na⁺、K⁺）及有机酸含量的变化,探讨百合耐盐碱的生理机制。研究发现,两种百合叶片中各种物质的变化趋势基本相似,但整体上岷江百合的各种物质含量或者活性均高于‘索邦’。随着NaHCO₃胁迫浓度的增加,两种百合叶片中的可溶性蛋白、脯氨酸含量增加,可溶性糖含量先升后降;SOD、GR的活性和AsA的含量先上升后下降,APX的活性和GSH的含量在低浓度胁迫时无明显变化,高浓度胁迫时显著下降;‘索邦’Na⁺主要积累在成熟叶和新生叶中,岷江百合则主要积累在茎和成熟叶中,且能维持根、成熟叶和新生叶中较高水平的K⁺/Na⁺值;岷江百合中的草酸、乳酸和乙酸含量都一直增加或高于对照,‘索邦’中的苹果酸和乙酸含量一直增加。与‘索邦’相比,岷江百合在低浓度NaHCO₃胁迫下提高SOD活性和AsA含量,高浓度NaHCO₃胁迫下增加可溶性蛋白的含量并保持可溶性糖的稳定性,是其耐盐碱性较强的原因之一。     

Role of calcium ion in the cyotoxicity and DNA fragment induction in HL-60 cells by 6F isolated from Pteris semipinnata L.

AIM: To investigate the changes of cytosolic free calcium concentration([Ca²⁺]_i) and expression of Bcl-2 in HL-60 cells treated by 6F isolated from Pteris semipinnata L.(PSL), and to discuss the relations between calcium ion and cytotoxicity and DNA fragment induction effects of 6F. METHODS: HL-60 cells were used as in vitro model. [Ca²⁺]_i was measured on fluorescent spectrophotometry using Fura-2/AM as Ca²⁺ indicator. Bcl-2 expressing level was measured by flow cytometry. Tetrazolium salt(MTT) and diphenylamine staining methods were applied for cytotoxicity assay and DNA fragmentation detection, respectively. RESULTS: [Ca²⁺]_i increased obviously in a dose and time dependent manner after treated HL-60 cells with 6F. 6F decreased the expressing level of Bcl-2. Adding 2 mmol/L Ca²⁺ to the medium, or 1 mmol/L EDTA to chelate Ca²⁺, or 4 μmol/L calcium ionophore A 23187 to increase the concentration of cytosolic Ca²⁺, the DNA fragment induction by 6F was not affected, whereas the cytotoxicity of 6F was enhanced. 250 μmol/L Zn²⁺ attenuated the DNA fragment induction, and the cytotoxicity of 6F against HL-60 cells was enhanced significantly. CONCLUSION: It was speculated that the decreased expressing of Bcl-2 by compound 6F was related to increased [Ca²⁺]_i in HL-60 cells, and DNA fragment induction was possibly catalyzed by Ca²⁺ - independent DNase.     

ATPase activities and Ca2+/calmodulin alterations in hippocampi of rats with PTSD-like behaviors

AIM: To explore the pathophysiological bases in the pathogenesis of the lasting emotional behavioral disorders following posttraumatic stress disorder(PTSD). METHODS: 240 male Wistar rats were divided randomly into 3 groups. Group SE(n =96) for rats with PTSD-like behavior by constant pulsating current of 100 μA with intratrain frequencies of 16 Hz, pulsating duration of 1 ms, train duration of 10 s and interstimulus interval of 7 min for 5 days with 8 times per day. Group CE(n =96) for control with electrode implanted in hippocampus without stimulation, and Group NC(n =48) for normal control. The activities of Na⁺-K⁺-ATPase and Ca²⁺ -ATPase, levels of intracellular calcium and free calmodulin(CaM), and the total CaM expression were detected in hippocampi of experimental rats. RESULTS: The activities of Na⁺-K⁺-ATPase and Ca²⁺ -ATPase in mitochondria of hippocampal cells in Group SE rats were significantly decreased at 48 h and 72 h after the last stimulation, respectively. The intracellular free calcium levels were increased, and the mean channel fluorescence of intracellular free CaM decreased remarkably at 72 h poststimulation, while the expression of total CaM was significantly elevated at 48 h after the last stimulation in hippocampi of Group SE rats. CONCLUSION: The lasting increased levels of intracellular free calcium and expression of Ca²⁺ -CaM in hippocampus, as well as the dysfunction of Na⁺-K⁺ pump and Ca²⁺ -ATPase in mitochondria may play important roles in the long-term neuropsychological sequelae in PTSD.     

Inhibitory effect of ATP on proliferative signaling in immortalized human fibroblasts

AIM: To investigate the inhibitory effects of ATP on proliferation signaling in immortalized human fibroblasts. METHODS: Immortalized human fibroblasts were treated with ATP, ATP conbined with calcium or potassium channel antagonists, respectively. The intracelluar-free calcium ([Ca²⁺]i), inositol 1,4,5-trisphosphate(IP₃) levels and cell viability were detected at different time points. RESULTS: ATP significantly increased the [Ca²⁺]i and decreased the IP₃ level in immortalized human fibroblasts, especially at initial stage (P＜0.01) . Compared to ATP alone, the proliferation rates remarkably increased when calcium or potassium channel antagonists were used (P＜0.01, respectively) with ATP. CONCLUSION: The calcium and potassium channels and IP₃ involved in the inhibitory effects of ATP on the proliferative signaling in immortalized human fibroblasts.     

Metallothionein inhibits homocysteine-induced proliferation of rat vascular smooth muscle cells

AIM:To investigate the effect of metallothionein(MT) on proliferation of rat vascular smooth muscle cells (VSMCs) stimulated by homocysteine and its mechanism. METHODS:VSMCs proliferation was measured by [³-H]-TdR incorporation, mitogen-activated protein kinase(MAPK)activity were determined by immunoprecipitation method, the intracellular contents of MT and malondialdehyde (MDA)were assayed by -hemoglobin saturation method and TBA reaction, respectively, and lactate dehydrogenase (LDH) leakage was measured by NADH oxidation. RESULTS:Hcy(10^-6-10^-4 mmol/L) stimulated [³-H]-TdR incorporation by the VSMCs in a concentration-dependent manner. Compared with control, [³-H]-TdR incorporation in VSMCs treated with 0.1 mmol/L Hcy was increased by 4.2 fold (P＜0.01). Meanwhile, Hcy enhanced MAPK activity, MDA formation and LDH release (P＜0.01)in a concentration-dependent manner. Treatment of VSMCs with MT alone did not change above parameters, compared with control. However, MT (10^-6-10^-4 mol/L)attenuated significantly Hcy-stimulated proliferation of VSMCs (P＜0.01)in a concentration-dependent manner. And MT inhibited obviously Hcy-induced activation of MAPK activity, MDA formation and LDH release. Preincubation of VSMCs with 0.5 mmol/L ZnCl₂ for 6 h induced an increase cellular MT content by 5.7-fold (P＜0.01). The MT-overexpressed VSMCs resisted Hcy-stimulating action on MAPK activity, MDA formation and LDH leakage (P＜0.01). CONCLUSION:These results show that MT has an inhibitory effect on Hcy-induced VSMCs proliferation, and that MT could inhibit Hcy-stimulated MAPK activity and lipid peroxidation.     

Importance of mitochondrial calcium uniporter in high glucose-induced cardiac myocyte apoptosis

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in high glucose(HG)-induced apoptosis of cardiac myocytes. METHODS: Cardiac myocytes were exposed to normal glucose (5.5 mmol/L glucose+ 19.5 mmol/L mannitol), HG (25 mmol/L glucose), or HG combined with 5 μmol/L spermine for 72 h. Mitochondrial free Ca²⁺ concentration ([Ca²⁺]_m), MCU at mRNA and protein levels, pyruvate dehydrogenase (PDH) activity, mitochondrial membrane potential (Δψ_m), the levels of ATP and reactive oxygen species (ROS), and apoptosis were determined. RESULTS: The [Ca²⁺]_m, the mRNA and protein levels of MCU, PDH activity, ATP levels, and Δψ_m were reduced (P<0.05), while ROS content and the protein levels of caspase-9 and caspase-3 were increased in HG group (P<0.05). Adding 5 μmol/L spermine returned these parameters toward control levels (P<0.05). Moreover, apoptosis was reduced by adding spermine and HG treatment (P<0.05). CONCLUSION: HG-induced cardiac myocyte apoptosis may be associated with the decreased MCU expression and activity, abnormal mitochondrial Ca²⁺ handling, deviant mitochon-drial respiratory chain, and mitochondrial dysfunction.