Effects Of N-n-buytl Haloperidol Liodide On Tranlocation Of PKCε, α And Changes Of Intracellular Calcium Of Myocardial Cells In Rats After Hypoxia/Reoxygenation

Protein kinase C (PKC), a serine/threonine kinase family, is widely distributed in eukaryotic cells, especially mammalian cells. Its activity depends on phospholipids activation. As one of the important signal transduction molecules, it is involved in cell proliferation, differentiation, apoptosis, gene expression, ion channel regulation, cell secretion, membrane transport and signal transduction which related to a wide range of physiological process and activated after extracellular signaling is passed to the intracellular second messenger. Meanwhile, abnormal PKC activity is indicated to be closely related to some pathological process for example the cancer, inflammation, viral infections, immune and central nervous system disorders, cardiovascular abnormalities, diabetes complications such as cardiovascular disease and insulin resistance. Under resting state, PKC mainly locates in the cytoplasm in inactive form. When cells are stimulated, PKC translocates from cytoplasm to membrane or other membrane components. This process is called translocation generally as a sign for activation of PKC. Our previous study showed that N-butyl haloperidol iodide (F₂) protects the myocardial cells through inhibiting the expression of early growth response gene-1 (Egr-1) in myocardial injury after ischemia and reperfusion (I/R). Is PKC, as one of upstream regulatory factors to Egr-1, involved in the protective effects of F₂ on myocardial cells. Using hypoxia-reoxygenation (H/R) injury model of primary cultured myocardial cells, we observed the effects of F₂ on changes of PKCεand PKCαinduced by H/R in myocardial cells. Indirect immunofluorescence staining and laser scanning confocal microscope were used in this experiment to clarify whether the protective effects of F₂ on myocardial cells is due to its regulation at PKCεand PKCα.Hempel found that nifedipine (NIF),another calcium antagonist, prevent ischemia-induced endothelial cell permeability independent on calcium channels. Our previous experiments also showed that F₂ can reduce H/R injury in cardiac microvascular endothelial cells(hereinafter referred to as endothelial cells). Because some type of endothelial cells lack L-type calcium channels, in order to explore whether F₂ can reduce H/R injury through the non-calcium-dependent mechanism, we established the myocardial cells H/R injury model in the absence of extracellular calcium and observed the effects of F 2 on the changes of intracellular calcium induced by H/R. Meanwhile, cardiac microvascular endothelial cells were stimulated by high concentration of potassium ions to verify whether there were L-type calcium channel in it.MethodsPart I1. Primary myocardial cells from the Sprague Dawley rats were cultured. Cells were seeded in glass coverslips preplaced in the 24-well plate to be myocardial cells climbing films. Myocardial cells morphology and beating status were observed by inverted phase contrast microscop.2. Preparation of H/R injury modelAfter replacing the initial medium with hypoxic buffer,the myocardial cells were incubated in an air-tight chamber gassed with pure nitrogen for 2 h of hypoxia at 37℃. The buffer was then replaced with oxygenated culture medium and the plates were transferred into a normoxic incubator for 0.5h of reoxygenation, caused H/R injury.3. Experimental groupsCells cultured for 3-4 days were randomly divided into one of four groups: Control, H/R, F₂+H/R and PMA. Mitochondria was stained firstly, and then the cells were stimulated with 1mmol/L PMA in PMA Group; Myocardial cells were pre-administrated with F₂ (1×10^-5mol/L) for 30min and incubated with F₂ (1×10^-5mol/L) duaring H/R treatment in F₂+H/R group; Mitochondria were also stained when H/R treatment were conducted in control group, H/R group and F₂+H/R group.4. PKCεand PKCαwere labled by using indirect immunofluorescence staining in different groups. Laser confocal microscope was used to observe the localization of PKCεand PKCαin myocardial cells.Part II 1. Primary myocardial cells and endothelial cells from the Sprague Dawley rats were cultured and seeded in the petri dishes. Cells morphology were observed by inverted phase contrast microscop.2. Preparation of H/R injury model in the absence of extracellular calcium Cells were incubated in calcium-free hypoxic buffer saturated by nitrogen for 2h followed by calcium-free Esumi buffer saturated by oxygen for 48min which caused H/R injury.3. Experimental groupsMyocardial cells cultured for 3-4 days were randomly divided into one of two groups: H/R, F₂+H/R. Cells were incubated in calcium-free Esumi buffer saturated by oxygen for 40min and then stimulated by H/R in H/R group; Cells were incubated in calcium-free Esumi buffer saturated by oxygen for 10min followed by calcium-free Esumi buffer which contained F₂ (1×10^-5mol/L) and saturated by oxygen, and then stimulated by H/R which buffers both contained F₂ (1×10^-5mol/L).4. Potassium stimulation to cellsMyocardial cells or endothelial cells cultured for 3-4d were stimulated by KCl solution of final concentration of 60mmol/L. Myocardial cells were incubated in the buffers contained verapamil (1×10^-5mol/L) after potassium stimulation.5. High-speed calcium imaging system were used to record the intracellular calcium changes in different groups.ResultsPart I1. Primary myocardial cells cultureMyocardial cells cultured for 3-4d gradually adhered to the glass coverslip to be spreaded and showed irregular polygon shape with 3-6 pseudopodia. Some cells clustered together beating in the same rhythm because of the contacts with each other pseudopodia networks.2. PKCεlocalizationPKCεscattered in the cytoplasm and part of them colocalized with mitochondria in Control group. There were almost no distribution of PKCεon the cell membrane. H/R group showed a punctate distribution of PKCεand more co-localization of PKCεwith mitochondria compared with the Control group. There were no PKCεon membrane in Control group; F₂+H/R group showed a punctate distribution of PKCεand more co-localization of PKCεwith mitochondria compared with the Control group. There were no PKCεon membrane in F₂+H/R group. There were no significant differences about PKCεbetween H/R group and F₂+H/R group; In PMA group, PKCεdecreased in cytoplasm and partly colocalized with mitochondria but there were significant increase of PKCεon membrane.3. PKCαlocalizationPKCαscattered in the cytoplasm and part of them colocalized with mitochondria in Control group. There were almost no distribution of PKCαon the cell membrane. H/R group showed a punctate distribution of PKCαand more co-localization of PKCαwith mitochondria compared with the Control group. There were no PKCαon membrane in Control group; F₂+H/R group showed a punctate distribution of PKCαand more co-localization of PKCαwith mitochondria compared with the Control group. There were no PKCαon membrane in F₂+H/R group. There were no significant differences about PKCαbetween H/R group and F₂+H/R group; In PMA group, PKCαdecreased in cytoplasm and partly colocalized with mitochondria but there were significant increase of PKCαin cytoplasmPart II1. Endothelial cells cultureEndothelial cells were exhibited with"spindle"after 1-3h culture,"spindle"or"polygonal"after 6h,"cobblestones"after 3-5d on morphology and arranged without overlapping.2. Effects of F₂ on changes of intracellular calcium in myocardial cells of rats after H/R in the absence of extracellular calciumIntracellular calcium increased significantly and stabilized at a certain level duaring hypoxia, and then returned to normal levels after reoxygenation in H/R group. There were no significant difference between H/R group and F₂+H/R group about the changes of intracellular calcium duaring H/R.3. Effects of potassium stimulation on intracellular calciumAfter potassium stimulation, intracellular calcium transiently decreased and then gradually recovering to the level before stimulation in endothelial cells; After potassium stimulation, intracellular calcium rapidly increased and stabilized at a certain level and then gradually fell back to resting level after intervention by VER in myocardial cells.Conclusions1. H/R caused PKCεand PKCαtranslocate from cytoplasm to the mitochondria.2. F₂ had no effects on translocation of PKCεand PKCαcaused by H/R.3. F₂ had no effects on changes of intracellular calcium of myocardial cells induced by H/R in rats in the absence of extracellular calcium.4. L-type calcium channel do not exist in endothelial cells.