Effects of P266T mutation on the characteristics of ATP-sensitive potassium channel

ATPAIM: The activity of ATP-sensitive potassium ion channel (K_ATP) is related to the myocardial function and its electrophysiology. In this study, we investigated the effect of the modulation of ATP-sensitive potassium channel on the electrophysiology of ATP-sensitive potassium channel.METHODS: cDNA of Kir6.2 and its mutation type P266T were transfected into the embryonic kidney cells to express K_ATP. The electrophysiology of K_ATP was investigated by using the whole-cell patch-clamp method. RESULTS: The open-state probability of P266T was twice as high as the wild-type channel, while its channel density was only 20% of the wild-type. P266T mutation significantly reduced the ATP sensitivity and the pH sensitivity.     

Effect of simvastatin on function of mouse pancreatic beta cells and its mechanisms

AIM: To observe the influence of simvastatin on insulin secretion function of mouse pancreatic beta cell line MIN6 and to explore its possible mechanisms.
METHODS: MIN6 cells were randomly divided into normal control group and low-, middle-and high-concentration simvastatin treatment groups, which were cultured for 48 h with high-glucose DMEM containing 15% fetal bovine serum plus 0, 2, 5 and 10 μmol/L simvastatin, respectively. The insulin secretion of MIN6 cells was measured by radioimmunoassay. The content of ATP in MIN6 cells was measured by biochemiluminescence method. The mRNA and protein expression levels of inwardly rectifying potassium channel 6.2 (Kir6.2), voltage-dependent calcium channel 1.2 (CaV1.2) and glucose transporter 2 (GLUT2) were detected by real-time fluorescence quantitative PCR and Western blotting, respectively.
RESULTS: Compared with normal control group, middle-and high-concentration simvastatin treatment markedly decreased the synthesis and secretion of insulin in MIN6 cells (P<005). The content of ATP in MIN6 cells was markedly decreased in simvastatin treatment groups (P<005). The mRNA expression level of Kir6.2 in MIN6 cells was significantly up-regulated in simvastatin treatment groups (P<001), while the mRNA expression levels of CaV1.2 and GLUT2 were significantly down-regulated in middle-and high-concentration simvastatin treatment groups (P<001). The protein expression of Kir6.2 was significantly increased but that of CaV1.2 was significantly decreased in middle-and high-concentration simvastatin treatment groups (P<001), and the protein expression level of GLUT2 was markedly decreased in high-concentration simvastatin treatment group (P<001).
CONCLUSION: Simvastatin inhibits insulin synthesis and secretion in mouse pancreatic beta cell line MIN6 via suppressing ATP production, up-regulating the expression of Kir6.2 and down-regulating the expression of CaV1.2 and GLUT2.     

Effect of zacopride on angiotensin Ⅱ-induced myocardial fibrosis

AIM:To investigate the effect of inwardly rectifying potassium channel (I_K1) agonist zacopride (Zac) on angiotensin Ⅱ (Ang Ⅱ)-induced viability and apoptosis of cardiac fibroblasts (CFb) and to explore the underlying anti-fibrosis mechanism.METHODS:The ventricular fibroblasts from neonatal SD rats were isolated and cultured by tissue digestion and differential adherence methods. The model of rat cardiac fibroblast activation induced by angiotensin Ⅱ was established. The CFb were randomly divided into control group, Ang Ⅱ model group, Ang Ⅱ+Zac group, Ang Ⅱ+Zac+BaCl₂ group, AngⅡ+Zac+chloroquine group and Ang Ⅱ+captopril group. CCK-8 assay was used to detect the effect of Zac on the viability of CFb. The amount of collagen I and collagen Ⅲ secreted by CFb was determined by ELISA. The apoptosis of the CFb was analyzed by flow cytometry. The protein expression of Kir2.1 was determined by Western blot. RESULTS:Compared with the control group, the viability and collagen synthesis of the CFb were significantly increased, along with decreased Kir2.1 expression (P<0.05). Compared with the Ang Ⅱ model group, Zac treatment inhibited the viability and collagen synthesis of the CFb, induced apoptosis and up-regulated Kir2.1 expression (P<0.05). I_K1 blockers BaCl₂ and chloroquine reversed the effect of Zac. CONCLUSION:By enhancing I_K1 (Kir2.1) expression, Zac attenuates Ang Ⅱ-induced ventricular fibrosis, in response to the inhibition of cell viability and induction of apoptosis.     

Nuclear calcium oscillation in cultured neonatal rat myocytes

AIM: To determine whether nuclear Ca²⁺ is independently regulated from the cytosolic Ca²⁺ and nuclear Ca²⁺ oscillation induced by many modulating factors in cultured rat neonatal myocytes and its mechanism. METHODS: Rat neonatal cardiac myocytes were cultured, and fluo-4/AM was loaded as calcium probe. The changes of cytosolic and nuclear Ca²⁺ were observed by confocal laser microscopy. RESULTS: Calcium fluorescent intensity oscillated slightly in myocardiocytes and the average intensity was much higher in the nucleus than that in the cytosole. Ca²⁺ oscillation in nucleus and cytosole induced by norepinephrine, isoproperenol, ATP were completely blocked by Ca²⁺-ATPase antagonist thapsigargin (10^-6 mol/L),L-type Ca²⁺ channel blocker verapermil (500 μmol/L) and KCl (20 mmol/L). Ca²⁺-ATPase antagonist thapsigargin completely blocked the propagation of Ca²⁺ waves and simutaneouly induced a temporary Ca²⁺ increase followed by a magnificient drop and loss of response to norepinephrine. CONCLUSIONS: The results suggested that generation and maintenance of calcium oscillation both in cytosole and nucleus depended on extracellular Ca²⁺ influx, membrane potential, Ca²⁺ release and uptake of cytosolic and nuclear calcium stores. The difference between cytosolic calcium and nuclear calcium indicated that calcium regulating system relatively independent of cytosole may exist in nucleus.     

Relationship between 936C/T polymorphism in 3’-untranslated region of vascular endothelial growth factor gene and diabetic retinopathy in China

AIM: To study the distribution of 936C/T polymorphism in 3’- untranslated region of vascular endothelial growth factor (VEGF) gene in Chinese Han population and to analyze the relationship between the polymorphism and diabetic retinopathy (DR). METHODS: Two hundred and fifty-four patients with type 2 diabetes mellitus recruited in this study were divided into NPDR group (non-proliferative diabetic retinopathy), PDR group (proliferative diabetic retinopathy) and DM (diabetes without retinopathy) group. The normal control group consisted of 120 subjects. Genotypes were identified by PCR-RFLP among all the subjects, while other clinical parameters were measured. Serum levels of VEGF were tested by the method of ELISA. RESULTS: The frequencies of both genotype CC and allele C were significantly higher in NPDR group and PDR group than those in NC group (²=9.934, ²=4.899, P<0.05 and ²=10.895, ²=5.714, P<0.05) and DM group (²=7.490, ²=4.448, P<0.05 and ²=8.333, ²=5.227, P<0.05). However, the frequencies of genotype (TT+CT) and allele T were significantly lower in NPDR group and PDR group than those in NC group (²=9.934, ²=10.895, P<0.01 and ²=4.899, ²=5.714, P<0.05) and DM group (²=7.490, ²=8.333, P<0.01 and ²=4.448, ²=5.227, P<0.05). Multivariate logistic regression analysis showed that the levels of glycohemoglobin(HbA1c), total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C) and plasma VEGF presented a positive correlation with DR, respectively, and the 936C/T mutation of VEGF exhibited a negative correlation with DR (β=-1.027, OR=0.343, P<0.01, CI: 0.157-0.723). CONCLUSION: Allele 936C of VEGF may be a genetic marker susceptible to DR, while allele 936T may be a protective genetic marker of DR. The 936C/T mutation of VEGF may be a protective factor against DR.     

Blocking effects of extracellular Cd2+ on the inward rectifier potassium channel

AIM:To study the blocking effects of extracellular Cd²⁺ on the inward rectifier potassium channel (IRK1) expressed in the Xenopus oocytes. METHODS:Two-microelectrode voltage clamp (TEV) method was used. RESULTS:Cd²⁺ can concentration-, time- and voltage dependently block IRK1 instantaneous currents ( 1.5 ms after voltage applied ) when external Cd²⁺ concentration is 0, 0.1, 0.15, 0.3, 0.9, 2.7 or 5.4 mmol/L with K⁺ concentration fixed to 90 mmol/L. Cd²⁺ almost has no effect on the gating property and outward currents of IRK1. Cd²⁺ can concentration-dependently increase the normalized conductance of IRK1. IRK1 can not permeate Cd²⁺ because reverse potential did not change. Three exponential fitting analyze indicates that time constant is not changed with the change in Cd²⁺ concentration. This shows that the inhibitory effects of Cd²⁺ may be caused by surface potential or the blocking site of Cd²⁺ is at the surface of the channel. Because external Cd²⁺ can not inhibit IRK1 macroscopic currents more powerfully when external Cd²⁺ concentration is lower. These mean that external Cd²⁺ does not work through surface potential mechanism.CONCLUSION:Cd²⁺ is considered as one of the fast open channel blockers of IRK1 and its blocking site is at the surface of the channel.     

Change of L-type voltage-gated Ca2+ channel current during focal brain ischemia in diabetic rats

AIM: To observe the change of L-type voltage-gated Ca²⁺ channel current during focal brain ischemia in the normal rats and the rats with diabetes mellitus (DM). METHODS: Combination of high-fat diet with streptozotocin (STZ) was used to establish DM animal model. The operation of middle cerebral artery occlusion (MCAO) with monofilament on the rats was performed. The animals were divided into sham operation group, MCAO 1 h group, MCAO 3 h group, MCAO 6 h group, MCAO 24 h group, DM sham operation group, DM+MCAO 1 h group, DM+MCAO 3 h group, DM+MCAO 6 h group and DM+MCAO 24 h group. The score of neural function was determined to judge the degree of palsy in the rats in MCAO 24 h group and DM+MCAO 24 h group. The changes of L-type voltage-gated Ca²⁺ channel current of cortex neurons during ischemia were measured using the whole-cell patch clamp technique. RESULTS: The rats in DM+MCAO 24 h group awaked slowly, and the degree of semiplegia was more serious than that in the rats in MCAO 24 h group. The score of neural function in DM+MCAO group was higher than that in MCAO group (P<0.05). The longer the ischemic time was, the higher L-type voltage-gated Ca²⁺ channel current was observed in MCAO group and DM+MCAO group (P<0.05). L-type voltage-gated Ca²⁺ channel current in DM+MCAO group was higher than that in MCAO group at each time point(P<0.05). CONCLUSION: The aggratation of ischemic injury during DM+MCAO is probably associated with Ca²⁺ overload induced by calcium channel opening and current increasing.     

Bio-effects of salusins on isolated rat heart and neonatal cardiomyocytes

AIM: To investigate the bio-effects of salusins on rat heart and cardiomyocytes. METHODS: The cardiac function was determined by multipurpose polygraph in isolated rat heart treated with various concentrations of salusin-α or salusin-β.[⁴⁵Ca²⁺] and[³H]-Leu incorporation were determined in cultured neonatal rat cardiomyocytes with β-liquid scintillation counter. RESULTS: 10^-12-10^-7mol/L salusin-α and salusin-β had no effects on isolated rat cardiac function. However, salusin-α and salusin-β stimulated uptake and[³H]-Leu incorporation. The [⁴⁵Ca²⁺] uptake induced by salusins were inhibited by nicardipine, and were synergistically increased by endothelin-1. The[³H]-Leu incorporation induced by salusin-α and salusin-β was inhibited by nicardipine, FK506 (a special inhibitor of carcineulin), PD₉₈₀₅₉ (inhibitor of MAPK) and chelerthine (inhibitor of PKC). The effects of salusin-β[⁴⁵Ca²⁺] on uptake was stronger than those of salusin-α. But there were no statistical difference in[³H]-Leu incorporation between salusin-α and salusin-β. CONCLUSIONS: Salusin-α and salusin-β did not affect directly cardiac function in rat hearts. But salusins improved calcium uptake and protein synthesize in neonatal rat cardiomyocytes. Those effects of salusins were related with calcium channel, carcinuelin, MAPK and PKC signal pathways. Salusins may be the regulatory factors for myocardium growth and hypertrophy.     

Effect of exogenous H2S and ATP-sensitive potassium channels on colonic hypermotility in a rat model of chronic stress

AIM: To investigate the potential role of exogenous hydrogen sulfide (H₂S) and ATP-sensitive potassium (K_ATP) channels in chronic stress-induced colonic hypermotility.METHODS: Male Wistar rats were divided into water avoidance stress (WAS) group and sham WAS (SWAS) group. Organ bath recordings were used to test the contractile activity of colonic strips. The effects of H₂S donor NaHS and pretreatment with glibenclamide on the contractions of colonic smooth muscle were studied and the IC₅₀ of NaHS was calculated. The localization and expression of the subunits of K_ATP channels were determined by the methods of immunohistochemistry and Western blotting.RESULTS: WAS increased contractile activity of colonic strips. NaHS concentration-dependently inhibited the spontaneous contractions of strips from the SWAS and WAS rats. The IC₅₀ of NaHS for longitudinal muscle (LM) and circular muscle (CM) of the WAS rats was 0.2033 mmol/L and 0.1438 mmol/L, significantly lower than those of the SWAS rats. Glibenclamide significantly increased the IC₅₀ of NaHS for LM and CM from the SWAS and WAS rats. In both SWAS and WAS rat colon, Kir6.1, Kir6.2 and SUR2B were expressed on the plasma membrane of the smooth muscle cells. WAS treatment resulted in up-regulation of the expression of Kir6.1 and SUR2B in the colon devoid of mucosa and submucosa.CONCLUSION: The increased expression of Kir 6.1 and SUR2B in colonic smooth muscle cells may be a defensive response to chronic WAS. H₂S donors may have potential clinical effect on treating chronic stress-induced colonic hypermotility.     

Effect of Ba2+ concentration on L-type Ca2+ channel in hypothalamic neurons

AIM:To investigate the effect of Ba²⁺ concentration on L-type of Ca²⁺ channel in hypothalamic neurons.METHODS:The cell acute isolation technique and cell-attached patch-clamp technique were used.RESULTS:The slope conductance of L-type Ca²⁺ channel were 28.6 pS (110 mmol/L) and 19.1 pS (10 mmol/L), and the open probability (NP₀) obviously different with different Ba²⁺ concentration as carrier. CONCLUSION:Ba²⁺ concentration had the obvious effect on the L-type Ca²⁺ channel.     

Role and regulation of calcineurin in angiotensin Ⅱ-stimulated cardiac myocyte hypertrophy of rats

AIM: To study the role and regulation of calcineurin(CaN) in angiotensin II(AngⅡ)-stimulated cardiacmyocyte hypertrophy of rats. METHODS: Using AngⅡ to induce the cultured cardiac myocyte hypertrophy of rats, and investigating the effect of CaN inhibitor on [³H]-leucine incorporation of AngⅡ-stimulated cardiomyocytes and the regulation of various factors on CaN activity in cardiomyocytes.RESULTS: AngⅡ can stimulate the CaN activity in cultured neonatal rat cardiomyocytes in a dose- and time-dependent manner. In cardiac myocytes incubated with 10, 100, 1000 nmol·L^-1 of AngⅡ for 12h, the CaN activities increased respectively by 13%,57%(P＜0.05) and 228%(P＜0.01) compared with that in non-stimulated cardiomyocytes. The CaN activities in AngⅡ-stimulated cardiomyocytes were significantly inhibited by losartan(50 μmol·L^-1), H₇(50 μmol·L^-1)and Fura-2/AM(4 μmol·L^-1),while no effect was observed with PD98059(50 μmol·L^-1).The [³H]-leucine incorporation in AngⅡ-stimulated cardiomyocytes increased by 46%(P＜0.01) compared with that in control group, which was dramatically inhibited by cyclosporin A(0.5~5μg/mL). CONCLUSIONS: Calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, may play an important role in AngⅡ-induced cardiac myocyte hypertrophy. The activation of CaN may dependent on the sustained increases of [Ca²⁺]i and be regulated by some protein kinases (such as PKC,etc.).     

《中国木本植物种子》

AIM: To test whether ischemic preconditioning (IPC) delays ischemia-induced electrical uncoupling by activation of mitochondrial ATP-sensitive potassium channels (mitoK_ATP). METHODS: Adult rat hearts perfused on a Langendorff apparatus were subjected to 40 min ischemia followed by 30 min reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. RESULTS: IPC delayed the onset of uncoupling campared to ischemic control; Blocking mitoK_ATP channels before the IPC protocol abolished the delay of uncoupling. The specific mitoK_ATP channel opener diazoxide mimicked the protective effect of IPC. The delay induced by diazoxide was reduced by 5-HD, L-type Ca²⁺ channel inhibitor verapamil and a free radical scavenger N-(2-mercaptopropionyl)glycine. CONCLUSIONS: IPC delays the onset of cellular electrical uncoupling induced by acute ischemia, in which activation of the mitoK_ATP channels may be involved.     

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.     

Using antisense nucleic acid technology to study the influence of POLκ on genetic stability

AIM:To establish cell line FL-POLκ^- and to study the role ofPOLκ(polymerase kappa) on genetic stability.METHODS:A mammalian expression vector expressing antisense POLκ gene fragment pMAMneo -amp^--POLκ was constructed by cloning the 1 690-1 918 fragment of POLκ gene into the mammalian expression vector pMAMneo-amp^- in antisense orientation. FL cells were fransfected with this antisense RNA expressing vector and selected by G418. Based on the shuttle-plasmid pZ189, the mutation assay was made.RESULTS:The spontaneous mutation frequency of supF tRNA gene in the plasmid replicated in the FL- POLκ^- was 11.2×10^-4, while it was 4.9×10^-4 and 3.7×10^-4 in the control cells FL and FL-M, respectively.CONCLUSION: POLκ playes an important role in maintenance of genetic stability.     

Clinical significance of Kir2.1/KCNJ2 and MRP1/ABCC1 protein expression in small-cell lung cancer

AIM: To investigate the expression of inward rectifying potassium channel 2.1 (Kir2.1/KCNJ2) and multidrug resistance-associated protein 1 (MRP1/ABCC1) in small-cell lung cancer (SCLC), and the correlation between them. METHODS:The mRNA and protein expression of MRP1/ABCC1 and Kir2.1/KCNJ2 in H69 and H69AR cell lines was detected by real-time PCR and Western blotting. Immunohistochemistry (EnVision) method was used to detect the expression of Kir2.1/KCNJ2 and MRP1/ABCC1 proteins in 44 cases of SCLC tissues. The interaction of Kir2.1/KCNJ2 with MRP1/ABCC1 was tested by co-immunoprecipitation (co-IP). RESULTS:The mRNA and protein expression of MRP1/ABCC1 and Kir2.1/KCNJ2 in H69AR cells was all significantly higher than that in H69 cells (P<005). The positive expression rates of Kir2.1/KCNJ2 and MRP1/ABCC1 in the 44 cases of SCLC tissues were 47.7% (21/44) and 52.3% (23/44), respectively. A positive correlation was found between the expression of Kir2.1/KCNJ2 and MRP1/ABCC1 in SCLC tissues (r=0.853, P<0.01). Co-IP results showed that Kir2.1/KCNJ2 could bind MRP1/ABCC1 in H69AR cells. CONCLUSION: Kir2.1/KCNJ2 is related to the multidrug resistance of SCLC. Kir2.1/KCNJ2 and MRP1/ABCC1 may have common function roles. Therefore, Kir2.1/KCNJ2 may promote the multidrug resistance of SCLC by regulating the expression of MRP1/ABCC1.     

Overexpression of integrin-linked kinase improves survival and proliferation of cardiac c-Kit+ cells

AIM: To investigate the effects of integrin-linked kinase (ILK) overexpression on survival and proliferation of cardiac c-Kit⁺ cells, and the role of ILK-overexpressing c-Kit⁺ cell transplantation in cardiac function in a rat myocardial infarction (MI) model.METHODS: Cardiac c-Kit⁺ cells were isolated from the hearts of neonatal Sprague-Dawley (SD) rats and cultured to prepare the ILK-c-Kit⁺ cells by infected with recombinant adenoviral vector harboring human wild-type ILK cDNA. The survival and proliferation of cardiac c-Kit⁺ cells were detected by cell counting and CCK-8 assay at 48 h after infection, respectively. The protein levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) in the cardiac c-Kit⁺ cells were examined by Western blot. MI was induced by coronary artery ligation in 40 adult rats. After 15 min, ILK-c-Kit⁺ cells were transplanted into the hearts by myocardial injection at 3 different sites in the infracted zone and border zone. All rats were randomly divided into 4 groups:sham group, MI plus saline injection group (MI group), MI plus null vector-infected cardiac c-Kit⁺ cell injection group (Ad-null-c-Kit⁺ cell group), and MI plus ILK-overexpressing cardiac c-Kit⁺ cells injection group (ILK-c-Kit⁺ cell group), with 10 rats in each group. At 2 weeks after MI, the protein levels of c-Kit in MI hearts were investigated by immunohistochemical assay. At 4 weeks, left ventricular function was examined by hemodynamic measurement.RESULTS: The survival and proliferation of cardiac c-Kit⁺ cells and the protein levels of cyclin D1 and PCNA were enhanced by ILK overexpression compared with Ad-null group. In MI rat model, the number of c-Kit⁺ cells was increased by ILK-c-Kit⁺ cell injection compared with Ad-null-c-Kit⁺ cell group at 2 weeks after MI. Cardiac function was significantly improved in ILK-c-Kit⁺ cell-transplanted rats.CONCLUSION: ILK overexpression improves survival and proliferation of cardiac c-Kit⁺ cells by increasing the protein levels of cyclin D1 and PCNA. ILK-c-Kit⁺ cell transplantation enhances the therapeutic efficiency of cardiac c-Kit⁺ cells in the post-MI hearts of rats.     

Effect of glibenclamide on viability and acid-base equilibrium of glioblastoma cells

AIM: To investigate the effect of glibenclamide (Glib) on the viability and acid-base equilibrium of glioblastoma cells. METHODS: U251 cells and U87 cells were treated with Glib at different concentrations. The inhibitory rates were detected by CCK-8 assay. The effective dose was screened and the experiment was divided into control group and drug treatment groups. The migration ability was monitored by wound healing assay, and intracellular pH was detected by pH indicator fluorescent probe. The protein expression levels of inwardly-rectifying potassium channel 4.1 (Kir4.1) and monocarboxylate transport protein 1 (MCT1) were determined by Western blot. RESULTS: The half maximal inhibitory concentrations (IC₅₀) of Glib for 48 h exposure of U251 cells and U87 cells were 400.20 μmol/L and 553.70 μmol/L, respectively. The effective inhibition doses of Glib for U251 cells were from the ranges of 100 μmol/L to 1 600 μmol/L, and those for U87 cells were from 50 μmol/L to 1 600 μmol/L in a concentration-dependent manner (P<0.05). Glib not only inhibited the migration (P<0.05) of U251 cells and U87 cells, which was negatively correlated with drug concentration (P<0.05), but also reduced the intracellular fluorescence intensity in experimental group (P<0.05), suggesting that with the increase in drug concentration, the intracellular pH decreased gradually (P<0.05). The protein expression of Kir4.1 and MCT1 was down-regulated by treatment with Glib, and was negatively correlated with concentration of Glib. CONCLUSION: Glib, a kind of potassium channel blocker, induces intracellular acidification via down-regulating the expression of Kir4.1 and MCT1, thus inhibiting the growth of glioblastoma in a certain dose range.     

Interaction of PKCε with the CaR in hypoxic post-conditioning protects the cardiomyocytes in neonatal rat

AIM: To study the interaction of PKC_ε with the CaR in hypoxic post-conditioning for protecting the cardiomyocyte of neonatal rat. METHODS: The ventricular cardiomyocytes of Wistar neonatal rat (3-7 d after birth) were incubated for about 3-5 d, then randomly divided them into 7 groups: (1) Sham control group (N group); (2) Hypoxic/re-oxygenation group (H/Re group); (3) Hypoxic post-conditioning group (HPC group); (4) HPC+GdCl₃, NiCl₂, CdCl₂ group; (5) HPC+caffeine, GdCl₃, NiCl₂, CdCl₂ group; (6) HPC+PKC_ε inhibitor group (PKCI group); (7) HPC+PKCI+GdCl₃, NiCl₂, CdCl₂ group. The neonatal cells were incubated in the D-Hanks solution (pH=6.8) which was saturated with N₂ gas for 1 h at least and then re-incubated in the DMEM solution containing 20% new-born calf serum to establish a model of H/Re. The viability of cardiomyocytes was assayed by MTT, the activity of LDH and the content of MDA were determined, the expression of CaR and PKC_ε of the membrane in each group was analyzed by Western blotting, PKC_ε interaction with CaR in the membrane was detected by immunoprecipitation, and the concentration of intracellular calcium ([Ca²⁺]_i) was measured by laser confocal scanning microscope (LCSM), apoptotic cells were measured by TUNEL assay. RESULTS: The viability of cardiomyocytes in H/Re, GdCl₃ and PKCI groups was lower than that in N and HPC groups, while the activity of LDH and the content of MDA were significantly higher than those in N and HPC groups. Meanwhile, the quantitative expression of CaR in GdCl₃, caffeine and PKCI+GdCl₃ groups was higher than that in HPC and PKCI groups, and so were the [Ca²⁺]_i and the apoptosis index. The quantitative expression of PKCε in PKCI and PKCI+GdCl₃ groups was lower than that in H/Re, HPC, GdCl₃ and caffeine groups. Immunoprecipitation of cell membrane PKCε revealed the interaction of PKC_ε with CaR. CONCLUSION: In the cardiomyocytes of HPC, PKC_ε translocates to the membrane and interacts with CaR to reduce [Ca²⁺]_i, which protects the cardiomyocytes of neonatal rat during hypoxic/oxygenation.