BACKGROUND AND OBJECTIVES: Protein kinase C (PKC) is known to be related with development of various cells. In the heart, each isoform reacts differentially against agonists and the reaction changes during development. In this study, the roles of PKC isoforms (alpha, beta, gamma, delta, epsilon, zeta) were investigated through the localization of mRNA expression in the developing rat heart with in situ hybridization histochemistry. MATERIALS AND METHOD: The mRNA expression pattern of PKC isoforms (alpha, beta, gamma, delta, epsilon, zeta) was investigated with in situ hybridization histochemistry in developing and adult rat hearts. Whole body parasagittal sections were used for embryonal day 14 (E14), E16, E18 and heart sections were used for just born (P0), postnatal day 7 (P7), P14, P21 and adult rat. RESULTS: The expression of PKC alpha was found from E14, peaked at P7, and gradually decreased to adult level. The expression of PKC beta was observed from P14, peaked at P21, and decreased to adult level. The expression of PKC delta in the heart was observed from E14, peaked at P0, and abruptly disappeared at P14. The expression of PKC epsilon was observed from E14, peaked at P0, after that gradually decreased and disappeared at adult rat heart. The expression of PKC gamma and zeta was not found from any stage of developing rat heart. CONCLUSION: From these results, it is suspected that each PKC isoform may be differentially related with development of heart. The strong expression of PKC alpha, delta, epsilon around perinatal period, rapidly developing stage, suggests that PKC alpha, delta, epsilon may be related with rapid development of rat heart. And the late postnatal expression of PKC beta suggests that PKC beta may be related with maturation of rat heart.
Adult ; Animals ; Heart* ; Humans ; In Situ Hybridization ; Protein Isoforms ; Protein Kinase C* ; Protein Kinase C-epsilon ; Protein Kinases* ; Rats* ; RNA, Messenger*

OBJECTIVETo observe the role of PKC-potentiated inhibitory protein for protein phosphatase 1 of 17 x 10(3) (CPI-17) in vascular calcium sensitivity regulated by protein kinase Calpha (PKCalpha) and Cepsilon (PKCepsilon) in rats with hemorrhagic shock (HS).

METHODSEight Wistar rats were used to reproduce 2 h HS model. Superior mesenteric artery (SMA) rings from HS rats were randomly divided into 2 h shock group (without treatment), PKCalpha agonist group (with addition of thymelea toxin into the nutrient solution), CPI-17 antibody + PKCalpha agonist group [incubation with thymelea toxin and CPI-17 antibody (1:800)], PKCepsilon agonist group (with addition of carbachol into the nutrient solution), and CPI-17 antibody + PKCepsilon agonist group [incubation with carbachol and CPI-17 antibody (1:800)]. SMA rings from another eight normal rats were used as normal control group. Calcium sensitivity indices (Emax, pD2) of SMA rings were measured by isolated organ perfusion system. Hypoxic VSMCs in primary culture were randomly divided into 2 h hypoxia group, PKCalpha agonist group (with above-mentioned treatment), PKCepsilon agonist group (with above-mentioned treatment), normal VSMCs were used as normal control group. Protein expression and phosphorylation of CPI-17 were measured via Western blot.

RESULTSEmax and pD2 in all the experimental groups were lower than those in normal control group (P < 0.01). Emax in PKCalpha agonist group and PKCepsilon agonist group was increased (5.8 +/- 0.8, 5.8 +/- 0.9 mN, respectively) as compared with that of 2 h shock group (4.1 +/- 0.6 mN, P < 0.01). Protein expression and phosphorylation of CPI-17 in VSMC were significantly decreased in 2 h hypoxia group, compared with those in normal control group (P < 0.05), and those in PKCalpha agonist and PKC agonist groups (P < 0.05 or P < 0.01).

CONCLUSIONSPKCalpha and PKCepsilon may regulate vascular calcium sensitivity through change in protein expression and activity of CPI-17 in HS rats.

Animals ; Calcium ; blood ; pharmacology ; Female ; Male ; Muscle Proteins ; metabolism ; Phosphoproteins ; metabolism ; Phosphorylation ; Protein Kinase C-alpha ; metabolism ; Protein Kinase C-epsilon ; metabolism ; Rats ; Rats, Wistar ; Shock, Hemorrhagic ; metabolism

BACKGROUND AND OBJECTIVES: Ischemic injury is the most common and important cause of myocardial damage. Over past decades, a number of studies have identified a protective mechanism known as ischemic preconditioning, which can block or delay cell death from ischemic injury. Protein kinase C (PKC), especially theepsilonisoform has been proposed as a key factor in the signaling pathway of ischemic preconditioning. However, whether PKCepsilon expression in cardiomyocytes can offer such protection from acute ischemia has not been explored. MATERIALS AND METHODS: To demonstrate a direct effect of PKCepsilon expression, a lentiviral vector system was established. Using the lentiviral vector, PKCepsilon was introduced to neonatal rat ventricular myocytes (NRVM) cultured under ischemic conditions, and also to adult rat myocardium subject to left coronary artery ligation. RESULTS: Compared to control, PKCepsilon expression in cultured NRVM under ischemia resulted in preserved cell density and morphology, and a reduction in cell death (77.6+/-12.8% vs 58.1+/-7.2%, p<0.05). In adult rats, the infarcted area after coronary artery ligation was markedly reduced in myocardium injected with PKCepsilon vector compared to control (11.4+/-5.3% vs 20.5+/-11.3%, p<0.01). CONCLUSION: These results provide direct evidence that PKCepsilon is a central player in protection against cell death from acute ischemia.
Adult ; Animals ; Cell Count ; Cell Death ; Coronary Vessels ; Humans ; Ischemia ; Ischemic Preconditioning ; Lentivirus ; Ligation ; Muscle Cells ; Myocardium ; Myocytes, Cardiac* ; Protein Kinase C ; Protein Kinase C-epsilon ; Protein Kinases* ; Rats

The mRNA expression of protein kinase C (PKC) isozymes (alpha, beta, gamma, delta, epsilon and zeta) in the rat nervous system was investigated with in situ hybridization histochemistry. In the central nervous system of rat, each PKC isozyme mRNAs was expressed in isozyme-specific pattern. PKC alpha mRNA was highly expressed in the olfactory bulb, piriform cortex, hippocampus, substantia nigra compacta, and inferior olive. The expression of PKC beta was highest in the olfactory tubercle, piriform cortex, caudate putamen, accumbens nucleus, neocortex, hippocampus, basolateral amygdaloid nucleus, pontine nucleus, and cerebellum. PKC gamma mRNA was distributed in the caudate putamen, hippocampus and cerebellum and PKC delta was expressed in the thalamus. PKC epsilon had widespread distribution, with relatively high levels in the anterior olfactory nucleus, olfactory tubercle, tinea tecta, piriform cortex, dorsal lateral septal nucleus, neocortex, hippocampus and cerebellum. PKC zeta had widespread and low expression. The spacially differential expression of PKC isozymes (alpha, beta, gamma, delta, epsilon and zeta) suggests that each PKC isozyme may be related with specific cellular function in the nervous system.
Animals ; Brain* ; Central Nervous System ; Cerebellum ; Gene Expression ; Hippocampus ; In Situ Hybridization ; Isoenzymes ; Neocortex ; Nervous System ; Olea ; Olfactory Bulb ; Olfactory Pathways ; Protein Kinase C* ; Protein Kinase C-epsilon ; Protein Kinases* ; Putamen ; Rats* ; RNA, Messenger ; Septal Nuclei ; Substantia Nigra ; Thalamus ; Tinea

OBJECTIVETo investigate the effect of phosphorylated protein kinase C epsilon (pPKC epsilon) on apoptosis of 32D cells induced by sera from patients with aplastic anemia (AA).

METHODSThe expression of pPKC epsilon and apoptosis in 32D cells were measured by Western blotting and flow cytometry after incubation with sera from healthy individuals (controls, n = 8), patients with severe AA ( SAA, n = 8)and non severe AA (NSAA, n = 6).

RESULTSAfter incubation for 0, 12, 24, 36 and 48 hours in the presence of serum and for another 4 hours in medium deprived of serum, the levels of pPKC epsilon in cells in SAA and NSAA group increased gradually, peaked at 24 hours, and then declined (P < 0.05). Compared with that in control group (0.54 +/- 0.08), pPKC epsilon was overexpressed in both SAA group (0.90 +/- 0.10) and NSAA group (0.64 +/- 0.08) (P < 0.05) after 24 hours incubation with serum and subsequent 4 hours without serum. pPKC epsilon level was higher in SAA group than in NSAA group (P < 0.05). A greater proportion of 32D cells showed apoptosis after 24 hours incubation with sera from SAA patients [(4.05 +/- 1.05)%] and subsequent 4 hours incubation without serum than that in controls [(2.45 +/- 0.51)%, P < 0.05], which was correlated with the same serum-induced expression of pPKC epsilon (r = 0.869, P < 0.05). Although the mean level of pPKC epsilon expression was higher in NSAA group than in control group, no significant difference of apoptosis was found between the two groups [(2.45 +/- 0.51)% vs (3.24 +/- 0.56)%, P > 0.05].

CONCLUSIONSera from both SAA and NSAA patients could upregulate the expression of pPKC epsilon in 32D cells. The SAA sera induce apoptosis in 32D cells significantly, but the latter do not.

Adolescent ; Adult ; Anemia, Aplastic ; enzymology ; pathology ; Apoptosis ; Case-Control Studies ; Cells, Cultured ; Child ; Female ; Humans ; Male ; Middle Aged ; Phosphorylation ; Protein Kinase C-epsilon ; blood ; Young Adult

The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKC&epsilon; inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKC&epsilon; cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.
Animals ; Female ; Ganglia, Spinal ; physiology ; Male ; Patch-Clamp Techniques ; Protein Kinase C-epsilon ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; physiology ; Signal Transduction ; Substance P ; physiology

BACKGROUND/AIMS: Hepatic fibrosis in rat induced by thioacet amide shares similar morphological and biochemical characteristics with human liver cirrhosis. Thioacetamide (T AA) initially induces accumulation of collagen in Disse space and eventually leads to macro- and micronodular cirrhos is. Ito cell was believed to play a main role in hepatic fibrosis. And it s activity was known to be regulated by the expression of various genes. But little has been discovered about the upstream signal trans duction pathway of these genes in hepatic fibrosis. The expression of genesrelated to Ito cell activity was regulated by many transcription factors , the activity of which was regulated by protein kinase C( PKC) is oforms. So it is s upposed that PKC could be as s ociated with fibrosis in liver. METHODS: We investigated the correlation of PKC is oforms and It ocell activity in the course of hepatic fibrosis using TAA induced rat liver cirrhosis model. We used six week- old male rats , and administered 0.03% TAA in drinking water. The animals were sacrificed at 9, 20, and 30 weeks after TAA administration. The degree of hepatic fibrosis was evaluated by measuring the total amount of collagen.-SMA immunohist ochemical st aining of liver tissue was done to determine the Ito cell activity. The expression pattern of PKC isoforms was investigated by West ern blotting. RESULTS: In TAA- treated group, collagen cont ent and Ito cell activity did not increase until 30 weeks and 20 weeks of treatment , respectively, while in control group collagen cont ent and Ito cell activity were not detected. Collagen content showed linear correlation with Ito cell activity. This implied that the proliferation of activated Ito cells was prior to the increase of collagen content. In view of expression pattern of PKC is oforms, PKC alpha showed no difference in TAA- treated group and control group. In TAA-treated group, PKCbeta1 exhibited increased level of expression in both particulate and cytosolic forms at 9 weeks, while PKCdelta and PKC epsilon showed striking shift to particulated form. After 20 weeks, all of the PKC beta1, delta, and epsilon degenerated and showed remarkably decreased level of expression. This suggested PKC alpha had no relation to hepatic fibrosis,while PKC beta1, delta, and epsilon, showing activity at 9 weeks, were related to fibrosis og liver. In response to fibrogenic factors, molecules engaged in intracellular signal transduction pathway like PKC beta1, delta, and epsilon, began to change prior to the increase of Ito cell activity, morphologic changes and alterations of collagen content. CONCLUSION: Our results strongly suggest that the activity of PKC isoforms play an important role in early step of hepatic fibrosis, while accompanying Ito cell activity do in later step.
Animals ; Collagen ; Cytosol ; Drinking Water ; Fibrosis ; Hepatic Stellate Cells ; Humans ; Liver Cirrhosis* ; Liver* ; Male ; Protein Isoforms ; Protein Kinase C-epsilon ; Protein Kinases ; Rats ; Signal Transduction ; Strikes, Employee ; Thioacetamide ; Transcription Factors

OBJECTIVETo study the relationship between Nanog-promoted metastasis of breast cancer and ezrin(T567) phosphorylation, and explore the possible mechanism by which Nanog regulates ezrin(T567) phosphorylation.

METHODSA siRNA construct targeting Nanog was transfected in breast cancer cells to knock down Nanog expression, and the changes in the cell invasion was detected using Transwell assay. The expression levels of Nanog and PKC and the phosphorylation level of ezrin(T567) were detected using Western blotting and immunofluorescent staining; the protein interaction between PKC&epsilon; and ezrin was assayed by co-immunoprecipitation and Western blotting.

RESULTSNanog knockdown significantly decreased the expression of PKC&epsilon; protein, phosphorylation level of ezrin(T567) and the invasion ability of breast cancer cells. PKC&epsilon; knockdown obviously decreased the phosphorylation level of ezrin(T567) in the cells, and PKC&epsilon; and ezrin were co-immunoprecipitated.

CONCLUDIONSNanogcan can upregulate the expression of PKC&epsilon; to promote the phosphorylation of ezrin(T567), which can be a new mechanism by which Nanog promotes tumor metastasis.

Blotting, Western ; Breast Neoplasms ; metabolism ; Cytoskeletal Proteins ; metabolism ; Gene Knockdown Techniques ; Homeodomain Proteins ; metabolism ; Humans ; Nanog Homeobox Protein ; Neoplasm Invasiveness ; Phosphorylation ; Protein Kinase C-epsilon ; metabolism ; RNA, Small Interfering ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

BACKGROUND: It is known that cigarette smoke (CS) causes cell death. Apoptotic cell death is involved in the pathogenesis of CS-related lung diseases. Some members of the protein kinase C (PKC) family have roles in cigarette smoke extract (CSE)-induced apoptosis. This study was conducted to investigate the role of PKC epsilon in CSE-induced apoptosis in human lung fibroblast cell line, MRC-5. METHODS: Lactate dehydrogenase release was measured using a cytotoxicity detection kit. The MTT assay was used to measure cell viability. Western immunoblot, Hoechst 33342 staining and flow cytometry were used to demonstrate the effect of PKCepsilon. Caspase-3 and caspase-8 activities were determined using a colorimetric assay. To examine PKCepsilon activation, Western blotting was performed using both fractions of membrane and cytosol. RESULTS: We showed that CSE activated PKCepsilon by demonstrating increased expression of PKCepsilon in the plasma membrane fraction. Pre-treatment of PKCepsilon peptide inhibitor attenuated CSE-induced apoptotic cell death, as demonstrated by the MTT assay (13.03% of control, 85.66% of CSE-treatment, and 53.73% of PKCepsilon peptide inhibitor-pre-treatment, respectively), Hoechst 33342 staining, and flow cytometry (85.64% of CSE-treatment, 53.73% of PKCepsilon peptide inhibitor-pre-treatment). Pre-treatment of PKCepsilon peptide inhibitor reduced caspase-3 expression and attenuated caspase-3, caspase-8 activity compared with CSE treatment alone. CONCLUSION: PKCepsilon seem to have pro-apoptotic function and exerts its function through the extrinsic apoptotic pathway in CSE-exposed MRC-5 cells. This study suggests that PKCepsilon inhibition may be a therapeutic strategy in CS-related lung disease such as chronic obstructive pulmonary disease.
Apoptosis ; Benzimidazoles ; Blotting, Western ; Caspase 3 ; Caspase 8 ; Cell Death ; Cell Line ; Cell Membrane ; Cell Survival ; Fibroblasts ; Flow Cytometry ; Humans ; L-Lactate Dehydrogenase ; Lung ; Lung Diseases ; Membranes ; Protein Kinase C ; Protein Kinase C-epsilon ; Pulmonary Disease, Chronic Obstructive ; Smoke ; Smoking ; Tobacco Products

This study is to observe the effect of gross saponins of Tribulus terrestris (GSTT) on protein kinase Cepsilon (PKCepsilon) and apoptosis-associated protein in the apoptosis of cultured cardiocyte apoptosis induced by hydrogen peroxide (H2O2), and to explore the mechanisms of GSTT against myocardial apoptosis. Primary cardiocytes were isolated and cultured. Myocardial apoptosis was induced by H2O2 and analyzed with flow cytometry. Protein content of phospho-PKCepsilon, Bcl-2, and Bax were detected with Western blotting analysis. Cleaved caspase-3 protein content was determined with immunocytochemical technique. After the pretreatment of 100 mg x L(-1) GSTT, compared with H2O2 group, GSTT could not only decrease the apoptotic percentage in cardiocytes damaged by H2O2 (P < 0.01), but also reduce protein contents of Bax and cleaved caspase-3 (P < 0.01), and increase protein content of phospho-PKCepsilon and Bcl-2 significantly (P < 0.01). PKC inhibitor chelerythrine (Che) could prevent partly the effect of GSTT against myocardial apoptosis (P < 0.05 and P < 0.01). Mechanisms of GSTT against myocardial apoptosis might be associated with inhibition of mitochondrial apoptosis pathway after PKCepsilon activation.
Animals ; Apoptosis ; drug effects ; Benzophenanthridines ; pharmacology ; Caspase 3 ; metabolism ; Cells, Cultured ; Dose-Response Relationship, Drug ; Enzyme Activation ; Female ; Hydrogen Peroxide ; toxicity ; Male ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Oxidative Stress ; Phosphorylation ; Plants, Medicinal ; chemistry ; Protein Kinase C ; antagonists & inhibitors ; Protein Kinase C-epsilon ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Wistar ; Saponins ; administration & dosage ; isolation & purification ; pharmacology ; Tribulus ; chemistry ; bcl-2-Associated X Protein ; metabolism