Effect of compound salvae-dropping-pill on intracellular free calcium concentration of cultured rat myocardial cells in case of hypoxia and reoxygenation

AIM:To examine the effect of compound salvae-dropping-pill (CSDP) on intracellular free calcium in cultured rat myocardial cells subjected to hypoxia and reoxygenation.METHODS:The Fluo- 3/AM was applied to probe intracellular calcium concentration and the fluorescent intensity was detected using laser confocal microscopy technique.RESULTS:Fluorescent intensity in hypoxia plus CSDP group was significantly lower (1 217.78±312.07) than that of hypoxia group (1 509±508.48), and the Fluorescent intensity of hypoxia/reoxygenation plus CSDP group was also markedly lower (1 567.91±577.61) than that of hypoxia/reoxygenation group (1 617.60±477.53).CONCLUSION:The cultured rat myocardial cells could be effectively protected by administration of CSDP in case of hypoxia and reoxygenation through decreasing the intracellular calcium concentration.     

Salvia miltiorrhiza antagonized the alterations of contraction and intracellular calcium of rat ventricular cardiomyocytes induced by anoxia and reoxygenation

AIM:To study the effect of salvia miltiorrhiza (SM) on cell contraction and intracellular calcium of enzymatically isolated rat ventricular myocytes during normoxia and anoxia/reoxygenation.METHODS:Contraction and intracel ular calcium were determined with video tracking system and spectrofluorometric method,and the chemical anoxic method was employed. RESULTS:The ±dL/dt_max, dL of cell contraction and the amplitude of [Ca²⁺]_i in the cardiomyocytes following SM treatment were decreased in a dose-dependent manner. During anoxia, the ±dL/dt_max, dL and amplitude of [Ca²⁺]_i were decreased, while the diastolic Ca²⁺ level was elevated compared with control group. All the contractile parameters and the diastolic Ca²⁺ level were back toward pretreatment values during reoxygenation, but could not return to control level. After the treatment with SM (3 g/L), ±dL/dt_max and dL of cell contraction and the amplitude of [Ca²⁺]_i were higher and the diastolic Ca²⁺ level was lower than those in anoxia/reoxygenation group. CONCLUSION:SM antagonized effects of anoxia and reoxygenation on cell contraction and intracellular calcium in isolated ventricular myocytes.     

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.     

Effect of experimental acute necrotizing pancreatitis on sodium and L-type calcium current in rat cardiomyocytes

AIM: To study the effect of experimental acute necrotizing pancreatitis (ANP) on sodium and L-type calcium current in rat cardiomyocytes. METHODS: I_Na and I_Ca-L were recorded using whole cell patch-clamp techniques from left ventricular myocytes in ANP model established by retrograde injection of 3.5% sodium taurocholate 2.5 mL/kg into pancreatic duct. RESULTS: Peak I_Na current density (at -30 mV) was significantly reduced in ANP [(12.45±2.26)pA/pF,n=16] compared with sham [(25.32±3.31)pA/pF,n=14], P<0.01; I_Ca-L current density (at +10 mV) was also significantly reduced in ANP [(3.63±0.65)pA/pF,n=16] compared with sham [(5.46±1.03)pA/pF,n=12], P<0.05. CONCLUSIONS: There were changes in both I_Na and I_Ca-L in cardiomyocytes of ANP. These changes may underlie the altered excitability and abnormally short transmembrane action potentials and repolarization of cardiomyocytes, thus contributing to arrhymias in ANP.     

Role of cyclooxygenase-2 in injury induced by hypoxia/reoxygenation in cultured rat cortical neurons

AIM: To observe the role of cyclooxygenase-2 (COX-2) in injury induced by hypoxia and reoxygenation in cultured rat cortical neurons and protective effects of COX-2 specific inhibitor NS398.METHODS: Primary rat cortical neuronal cells were cultured. Experiments were divided into control group, hypoxia/reoxygenation group and hypoxia/reoxygenation with COX-2 inhibitor group. Cell viability was measured by MTT assay. COX-2 protein expression was examined by Western blotting. Apoptosis was measured by DNA agarose electrophoresis.RESULTS: The expression levels of COX-2 increased significantly after neurons were treated with hypoxia and reoxygenation, compared with control group and hypoxia/reoxygenation with COX-2 inhibitor group (P＜0.05). COX-2 specific inhibitor NS398 protected neurons from death (P＜0.05 and P＜0.01), DNA fragmentation analysis showed DNA fragmentation was inhibited significantly by NS398.CONCLUSION: COX-2 is involved in the pathogenesis of neuron apoptosis induced by hypoxia/reoxygenation. COX-2 specific inhibitor significantly protects cortical neurons against hypoxia/reoxygenation injury and inhibits apoptosis induced by hypoxia.     

Protective effect of quercetin on rat cardiomyocytes against anoxia/reoxygenation injury is mediated by PKCε signaling pathway

AIM：To investigate whether quercetin (Que) protects cardiomyocytes from anoxia/reoxygenation (A/R) injury through protein kinase C epsilon (PKCε) pathway. METHODS：Primary cardiomyocytes were isolated from neonatal SD rats and exposed to A/R (3 h of anoxia followed by 2 h of reoxygenation) as well as Que and/or εV1-2 (a selective PKCε inhibitor) preconditioning. The expression of PKCε in the cells was detected by Western blotting. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) in cell culture supernatants, the reactive oxygen species (ROS) and mitochondrial membrane potential in the cells, the opening of mitochondrial permeability transition pore (mPTP) and the cell viability and apoptosis were also measured. RESULTS：The expression of PKCε protein was significantly increased in the cardiomyocytes pretreated with 40 μmol/L Que 72 h before A/R (P<0.01 vs A/R group). Meanwhile, Que preconditioning could increase cell survival rate, decrease ROS production and cell apoptosis, alleviate the loss of mitochondrial membrane potential and inhibit the opening of mPTP induced by A/R injury (P<0.01 vs A/R group). However, pretreatment with Que and εV1-2 attenuated these protective effects of Que (P<0.01 vs Que+A/R group). CONCLUSION：One of the mechanisms underlying the cardioprotective effect of Que might be the increase in PKCε protein expression and the activation of its downstream pathway.     

Preparation of glycine liposomes and it's effect on cardiomyocyte injury induced by hypoxia/reoxygenation

AIM: To prepare glycine liposome microparticle and observe the effect of glycine liposomes on cardiomyocyte injury induced by hypoxia/reoxygenation. METHODS: （1） Reverse-phase evaporation method was used to produce glycine liposomes, the effects of different organic solvents: aether, chloroform and two mixtures of aether/chloroform on entrapment efficiency were evaluated, transmission electron microscope was used to detect the particle diameter of glycine liposomes. （2） A cardiomyocyte injury model was established by using hypoxia/reoxygenation, the activities of lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase isoenzyme (CK-MB) of each group were detected. RESULTS: The entrapment efficiency of glycine liposomes prepared with the mixtures of aether/chloroform is highest compared with other organic solvents （64.8％, P<0.01）; all of glycine, glycine liposomes and blank liposomes inhibited the release of LDH, CK and CK-MB induced by hypoxia/reoxygenation in cultured cardiomyocytes, and the inhibition by glycine liposomes was the most obvious （P<0.05，P<0.01）. CONCLUSIONS: The mixtures of aether/chloroform can be used to prepare glycine liposomes, which can get higher entrapment efficiency; glycine liposomes protects cultured cardiomyocytes against hypoxia/reoxygenation-induced injury, and the protective effect of glycine liposomes is better than that of glycine.     

Effects of glycine on intracellular of free calcium and tumor necrosis factor-α of cardiomyocytes during hypoxia/reoxygenation

AIM: To observe the influence of glycine on intracellular free calcium, the concentration of tumor necrosis factor-α and the survival rate of myocardial cells during hypoxia/reoxygenation (H/R). METHODS: The simulated model of myocardial ischemia-reperfusion with the primary cultured cardiomyocytes of neonatal rats was established, and the cultured cardiomyocytes were divided into seven groups, control group, hypoxia/reoxygenation group, glycine (0.5 mmol/L) plus hypoxia/reoxygenation group, glycine (1.0 mmol/L) plus hypoxia/reoxygenation group, glycine (2.0 mmol/L) plus hypoxia/reoxygenation group, glycine (4.0 mmol/L) plus hypoxia/reoxygenation group, 4.0 mmol/L glycine group. RESULTS: Within certain concentration (0.5-2.0 mmol/L), the glycine could inhibit the calcium overload resulting from hypoxia/reoxygenation injury in cells in a dose-dependent manner with the optimal inhibitory effect at 2.0 mmol/L. Glycine inhibited the secretion of tumor necrosis factor-α from myocardial cells and increased the survival rate of myocardial cells. CONCLUSION: Glycine has a protective effect on hypoxia/reoxygenation myocardial cells, which may be related to inhibiting calcium overload and decreasing the production of tumor necrosis factor-α.     

Roles of ERKs and intracellular free calcium in cardiomyocyte hypertrophic response induced by endothelin-1

AIM: To study the roles and mechanisms of ERKs and intracellular free calcium in cardiomyocyte hypertrophic response induced by endothelin-1(ET-1). METHODS: (1) Neonatal rat cardiomyocyte hypertrophic response was assayed by measuring cell surface area and protein content; (2) ERKs activity was determined by Whatman Paper Filter method; (3) Intracellular free calcium concentration ([Ca²⁺]i) was measured using Fura-2/AM as a fluorescent indicator. RESULTS:(1)ET-1 could increase total protein production,surface area,ERKs activity and[Ca²⁺]i in cultured cardiomyocyte in dose-dependent manner at concentrations ranging from 10^-9to 10^-7mol/L.And this effect could be abolished by BQ123,an antagonist of ETA receptor,partly inhibited by PTX,but not by BQ788,an antagonist of ETB receptor.(2)The activation of ERKs and the increase of[Ca²⁺]i induced by ET-1 were obviously in hibited by PD98059,a selective ERKs kinase inhibitor,and nifedipine,a calcium channel blocker,respectively.Both antagonists partialy inhibited ET-1-stimulated cardiomyocyte hypertrophic response.(3)Staurosporine,a selective PKC inhibitor,could inhibit ET-1-stimulated cardiomyocyte hypertrophic response and increase of[Ca²⁺]i,but not af ect the activation of ERKs.CONCLUSION: Cardiomyocyte hypertrophic response induced by ET-1 is mediated by ET_A receptor coupled to PTX-sensitive G-protein, which involves at least two signalling pathways: PKC-mediated increase of [Ca²⁺]i , and PKC-independent activation of ERKs.     

Effects of nitric oxide on mitochondrial damage caused by exogenous calcium

AIM: To study the effects of nitric oxide (NO) on mitochondrial damage caused by exogenous calcium. METHODS: Normal myocardial mitochondria were divided into three groups; L-arginine control group (CG), Ca^2＋-damaged group (DG) and L-NAME-preserved group (PG). Mitochondria of all groups were incubated at 30 ℃ with reaction medium containing 20 μmol/L EDTA, 100 μmol/L CaCl₂ and 1 μmol/L L-NAME with 100 μmol/L CaCl₂ respectively. Then the NO₂^-/NO₃^- contents, mitochondrial viability and membrane potential were investigated. RESULTS: The NO₂^-/NO₃^- contents of DG was obviously higher than that of CG and PG, meanwhile, there was no obvious difference between CG and PG. Mitochondrial viability and membrane potential of DG were significantly lower than that of CG and PG, and negatively related to NO^-₂/NO^-₃ contents (r=-0.5297, P＜0.01; r=-0.6041, P＜0.01). But, the mitochondrial viability and membrane potential of PG were still lower than that of CG. CONCLUSION: Exogenous calcium could activate mitochondrial nitric oxide synthase resulting in NO production and the latter play an important role in decreasing mitochondrial viability and membrane potential.     

Effects of diltiazem on platelet activation and cytosolic calcium during percutaneous transluminal coronary angioplasty

AIM: To evaluate effects of diltiazem on platelet hyperreactivity in situations associated with endothelial injury and their possible relationship to cytosolic calcium concentration. METHODS: Blood samples were collected at 7 time points from 35 patients undergoing percutaneous transluminal coronary angioplasty (PTCA) who received combined diltiazem and aspirin/ticlopidine therapy or aspirin/ticlopidine therapy alone. Platelet expression of glycoprotein Ⅱb/Ⅲa and cytosolic calcium concentration were measured, respectively, by whole blood flow cytometry and fluorospectrophotometry. The effects of diltiazem of different concentrations on expression of glycoprotein Ⅱb/Ⅲa were also studied in vitro in blood samples from patients with chronic stable angina. RESULTS: Of the two treatments, aspirin/ticlopidine therapy did not prevent an acute increase of expression of glycoprotein Ⅱb/Ⅲa 5 minutes and 10 minutes after first inflation and 10 minutes after PTCA, whereas combined diltiazem and aspirin/ticlopidine therapy had a significant inhibitory effect. In the group receiving aspirin/ticlopidine therapy, there was a short-term elevation of platelet i immediately following PTCA which was significantly reduced by diltiazem treatment. Expression of glycoprotein Ⅱb/Ⅲa was significantly inhibited in vitro by diltiazem in the concentration of 200 μg/L or higher, but not 50 μg/L. CONCLUSIONS: Combined diltiazem and aspirin/ticlopidine therapy significantly inhibited platelet activation that continued in the presence of conventional aspirin/ticlopidine treatment. Antiplatelet effects of diltiazem were probably a consequence of reduction of platelet i and may only be achieved in higher than therapeutic concentrations.     

Effects of acetal hairy holly extractive compound R4 on rat cardiomyocyte injury induced by hypoxia/reoxygenation

AIM: To observe the effects of acetal hairy holly extractive compound R4(AHHECR4) on myocardial cell injury induced by hypoxia/reoxygenation. METHODS: The model of rat myocardial cell injury was induced by hypoxia/reoxygenation. The activity of superoxide dismutase (SOD) in the myocardial cells was measured by the method of xanthine oxidase. The content of malondialdehyde (MDA) was determined by the method of thiobarbituric acid. The activity of dehydrogenase (A) in mitochondria was detected by MTT assay. The activity of lactate dehydrogenase(LDH) and the content of NO in the culture medium were also evaluated. RESULTS: AHHECR4 at concentrations of 5, 10 and 20 μmol/L remarkably increased SOD activity and the value of A, and significantly inhibited MDA production and LDH leakage. Greatly increased content of NO in the culture medium was also observed. CONCLUSION: The results indicate that AHHECR4 has a protective effect on myocardial cells under the condition of hypoxia/reoxygenation injury.     

Effects of Tribulus terrestris L. saponin on apoptosis and changes in cytosolic calcium induced by hypoxia/reoxygenation in rat cortical neurons

AIM: To study the effect of Tribulus terrestris L. saponin (TTLS) on apoptosis and changes in cytosolic calcium concentration induced by hypoxia/re-oxygenation in rat cortical neurons. METHODS: Rat cortical neurons in primary culture were used, and a apoptosis model was induced by hypoxia/reoxygenation. LDH releasing rate was detected by spectrophotometry. The apoptosis rate of cortical neurons was analyzed quantitatively by flow cytometry with Annexin V-FITC and PI staining. Intracellular free Ca2+(［Ca2+］i) was observed with a confocal laser-scanning microscope and determined by mean fluorescent value with Fluo-3 fluometry. RESULTS: Compared to control group, three hours of hypoxia and twelve hours of reoxygenation group induced cortical neuronal apoptosis and significantly increased the intracellular free Ca2+ concentration（P<0.01). Compared with model group, TTLS decreased the percentage of neuronal apoptosis and reduced neuronal ［Ca2+］i（P<0.01).CONCLUSION: TTLS could obviously reduce hypoxia/reoxygenation-induced apoptosis and alleviate the damage degree of rat cortical neurons.The mechanism might be related to inhibiting the calcium overload induced by hypoxia/reoxygenation in rat cortical neurons.     

Effect of AMPKα2 gene silencing by shRNA recombinant plasmid on chloride-mediated anoxia/reoxygenation injury in rat cardiomyocytes

AIM: To explore the role of AMP-activated protein kinase α2 subunit (AMPKα2) gene in chloride-mediated anoxia/reoxygenation (A/R) injury by transfection of short-hairpin RNA (shRNA) expression vector targeting to AMPKα2 gene into H9c2 cardiomyocytes. METHODS: Recombinant shRNA expression vector pSuper-AMPKα2 targeting to AMPKα2 gene was constructed and transfected into H9c2 cardiomyocytes. The protein expression of AMPKα2 was determined by Western blotting. The cells were divided into 5 groups: control group, A/R group, Cl^--free A/R group, pSuper+Cl^--free A/R group and pSuper-AMPKα2 shRNA+Cl^--free A/R group. After treatment, the cell viability was detected by MTT assay. LDH activity was analyzed with an automatic biochemical analyzer. The apoptotic rate and the level of intracellular ROS was measured by flow cytometry. The activity of SOD and GSH-Px was analyzed by a colorimetric method. RESULTS: The result of sequencing proved that the recombinant plasmid pSuper-AMPKα2 shRNA was correctly constructed. The protein level of AMPKα2 significantly decreased after the plasmid was transfected into the cardiomyocytes. Compared with A/R group, the cell viability and the activity of SOD and GSH-Px were significantly increased, while the activity of LDH, apoptotic rate and ROS production were significantly decreased in Cl^--free A/R group. The protective effect of Cl^--free solution on the A/R-induced injury of cardiomyocytes was abolished, and the ROS production was increased and the activity of SOD and GSH-Px was decreased after the cells were transfected with pSuper-AMPKα2 shRNA. CONCLUSION: Recombinant plasmid pSuper-AMPKα2 shRNA is successfully constructed, and silencing of AMPKα2 gene abolishes the protective effect of Cl^--free solution on A/R injury.     

The characteristics of hypertrophic cardiomyocyte injury and the effect of intervention on energy metabolism after different time of hypoxia/reoxygenation

AIM:To investigate the characteristics of pathological injury and its relationship with the transformation of energy metabolism of hypertrophic cardiomyocytes after hypoxia-reoxygenation. METHODS:Cultured rat cardiomyocytes were induced to be hypertrophy by angiotensin Ⅱ (Ang Ⅱ) and norepinephrine (NE). Glucose oxidation rate (GOR), glucolysis rate (GLR) and fatty acid oxidation rate (FOR) were determined by liquid scintillation counting, and cell apoptosis was detected by TUNEL. RESULTS:(1) Compared with the normal cardiomyocytes (NC), the GOR and GLR were slightly higher and the FOR was slightly lower in the group of hypertrophic cardiac cells (HC) than that in the group of normal cardiomyocytes cultured under the normal oxygen partial pressure. The apoptosis rate had no difference between the two groups. (2) The apoptosis rate of hypertrophic cardiomyocytes after hypoxia was significantly higher than that of hypertrophic cardiomyocytes in normal culture. It was higher and moreover, some necrosis cardiomyocytes appeared after reoxygenation. (3) GOR and FOR in both group (NC and HC) were slightly lower in a time-dependent manner after hypoxia than that in each group in normal culture condition. GLR had no difference in both group. The GOR was more lower in both NC and HC group when reoxygenation than that at the point of hypoxia for 2 hours, but the GLR and FOR were significantly higher in HC than that in NC when reoxygenation. (4) The GOR was significantly higher and the GLR and FOR were significantly lower in the hypertrophic cardiomyocytes group (HC) with dichloroacetate (DCA, 1 000 μmol/L) or trimetazidine (TMZ, 1 μmol/L) treated respectively than that in the responded hypertrophic cardiomyocytes after stimulation by hypoxia-reoxygenation. In the meanwhile, the apoptosis rate also was markedly lower in the treated hypertrophic cardiomyocytes group. CONCLUSION:The transformation of energetic metabolism pathway plays an important role in the pathogenesis (mainly the apoptosis) of the hypertrophic cardiomyocytes after hypoxia-reoxygenation.     

Regulation of LPS-induced elevation of Ca2+ intracellular level of alveolarmacrophages in chronic bronchitis by Angelica Sinensis and nifedipine

AIM: To explore regulation of lipopolysaccharide (LPS)-induced elevation of Ca²⁺ intracellular level in alveolar macrophages(AMs) from patients with chronic bronchitis by Angelica Sinensis and nifedipine.METHODS:AMs was obtained from 7 patients with chronic bronchitis and 6 normal controls by bronchoalveolar lavage and intracellular Calevel was detected after adding Angelica Sinensis, nifedipine or LPS to the supernatant of AMs loaded by Fura-2. RESULTS: In contrast with normal control group (99.65±32.21 nmol/L), intracellular Ca²⁺ level in AMs from chronic bronchitis group (189.47±23.69 nmol/L) was increased significantly in the absence of extracellular Ca²⁺ but not 1 mmol/L. Intracellular Ca²⁺ level in AMs from chronic bronchitis group were significantly increased by adding 10 μg/mL LPS to the supernatant of AMs. LPS-induced elevation of intracellular Ca²⁺ level in AMs from chronic bronchitis group was completely inhibited by Angelica Sinensis or nifedipine.CONCLUSION: Both Anelica Sinensis and nifedipine may inhibit activation of AMs from patients with chronic bronchitis by reducing LPS-induced elevation of intracellular Ca²⁺ level in AMs, suggested that these two medicines may inhibit non-specific inflammation of airways in chronic bronchitis.     

Effects of calmodulin antagonist and slow calcium channel antagonist on reperfusion injury of isolated rat hearts in calcium paradox

AIM: To observe the protective effect of calmodulin antagonist trifluoperazine(TFP) and slow calcium channel antagonist verapamil (VP) upon calcium overload injury. METHODS: The calcium content of the myocardium, lactate dehydrogenase (LDH) release, protein loss and coronary flow were measured after the isolated rat hearts were perfused by TFP and VP. RESULTS: TFP could significantly reduce the tissue calcium , LDH release and protein loss of the myocardium ( P <0.01) while VP had no such effects.The above indexes were further reduced when TFP and VP were used simutaneously( P <0.01).CONCLUSION:TFP can effectively alleviate the severity of the myocardium injury during calcium paradox while VP has no such effects.The simultanous use of VP and TFP is more effective in preventing tissue damage of the heart than the use of TFP alone.The massive influx of Ca²⁺ in calcium paradox is mainly a calmodulin related process.     

Protective role of losartan in reversion of hypertensive calcium overload and glomerular injury

AIM:To investigate the relation between cytosolic calcium and glomerular injury in hypertension.METHODS:The normotensive control (WKY) and spontaneously hypertensive rat group(SHR) with or without treatment (losartan 20 mg·kg^-1·d^-1) were compared. Intralymphocytic free calcium level and ultrastructural changes in glomerulus were observed at three and eight months, respectively.RESULTS: The results demonstrated early impairment in glomerulus and elevation of cytosolic calcium at three months in SHR group, at eight months, aggravation of impairment in glomerulus correlating with calcium elevation was shown. Losartan significantly attenuated the above pathologic changes. CONCLUSION:Calcium-overload state was not only related to blood pressure and vessel impairment, but also associated with glomerular injury, which could be reversed by losartan.     

Effects of PKC and ROCK on vasodilation induced by nifedipine

AIM: To investigate the effects of Rho-associated kinase (ROCK) and protein kinase C (PKC) on the relaxation of isolated rat aortic rings induced by nifedipine and the mechanisms. METHODS: The changes of tension in vascular rings induced by nifedipine under the basic condition and pre-contracted by norepinephrine (NE, 10^-6 mol/L) or KCl (60 mmol/L) were observed. The effects of ROCK and PKC on the vasodilation induced by nifedipine were studied using the vascular ring perfusion device. RESULTS: Nifedipine (10^-10 mol/L, 10^-9 mol/L, 10^-8 mol/L, 10^-7 mol/L, 10^-6 mol/L and 10^-5 mol/L) had no significant relaxation effect on isolated aortic rings under basic condition. Nifedipine induced dose-dependent relaxation in both endothelium-intact and endothelium-denuded aortic rings pre-contracted by 10^-6 mol/L NE and 60 mmol/L KCl (P<0.05). No obvious difference between endothelium-intact group and endothelium-denuded group was observed. After incubation of the PKC inhibitor staurosporine (STA, 10^-8 mol/L) and PKC agonist phorbol 12-myristate 13-acetate (PMA, 10^-7 mol/L), STA increased the relaxation induced by nifedipine, while PMA reduced the effect of nifedipine on blood vessels (P<0.05). After the incubation of the ROCK inhibitor fasudil (10^-6 mol/L) and ROCK agonist angiotensin Ⅱ (Ang-Ⅱ, 10^-9 mol/L), fasudil increased the relaxation induced by nifedipine, while Ang-Ⅱ reduced the effect of nifedipine on blood vessels (P<0.05). The relaxation induced by nifedipine was not statistically inhibited by BaCl₂ (10^-4 mol/L), tetraethylammonium (10^-3 mol/L), glibenclamide (10^-5 mol/L) and 4-aminopyridine (10^-3 mol/L). In calcium-free and high-potassium solution, pre-treatment with nifedipine (10^-9 mol/L, 5×10^-8 mol/L and 10^-6 mol/L) inhibited calcium-induced contraction of the aortic rings (P<0.05). However, nifedipine pre-treatment did not affect the contraction induced by NE in Ca²⁺-free medium. CONCLUSION: Nifedipine exhibits vasodilatation effect in a dose-dependent manner and the vasodilatation activity is endothelium-independent. The vasodilatation effect of nifedipine may be related to the inhibition of extracellular calcium influx, and inhibition of PKC and ROCK enhances the vasodilatation effect of nifedipine.     

Effects of fructose-1,6-diphosphate on adriamycin-induced calcium and sarcoplosnic reticulum Ca2+-ATPase activity in cardiomyocytes of rats

AIM: To study the effects of fructose-1,6-diphosphate (FDP) on adriamycin (ADR)-induced calcium and sarcoplasmic reticulum Ca²⁺-ATPase activity in cardiomyocytes of rats. METHODS: Rats were treated with ADR by intraperitoneal injection (2.5 mg·kg^-1 body weight) once every two days for 11 days, and then ADR-treated rats were intervened by FDP at different dosages (ip) once every other day for 41 days. Enzyme linked immune absorption assay (ELISA) was employed to detect froponin I (CTnI). CK-MB was examined by monoclonal antibody. Intracellular free calcium concentration was measured on fluorescent spectrophotometry and SRCa²⁺-ATPase activity was examined by inorganic phosphate. RESULTS: FDP (300, 600, 1 200 mg·kg^-1) significantly reduced the levels of CTnI and CK-MB in serum. Decreased calcium and increased SRCa²⁺-ATPase activity in cardiomyocytes were also observed when ADR-treated rats were intervened by FDP (P＜0.01). CONCLUSION: FDP reduced the injury of cardiotoxicity induced by ADR via decreasing intracellular free calcium and increasing SRCa²⁺-ATPase activity in cardiomyocytes.