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

OBJECTIVETo investigate the expressions of protein kinase C (PKC) isoforms in X-ray-exposed HepG2 cells and identify the PKC isoforms that induce radioresistance in HepG2 cells.

METHODSCultured HepG2 cells were divided into control group and 6 Gy radiation group for corresponding treatments. The fluorescence intensity (FI) and the percentage of positive cells were determined using flow cytometry.

RESULTSThe FI of PKCalpha and PKCdelta were 2.28 and 5.05 in the radiation group, respectively, significantly higher than those in the control group (P<0.05). The percentages of PKCalpha- and PKCdelta -positive cells were significantly higher in the radiation group than in the control group (P<0.05). The FI and the percentages of PKC zeta, gamma, epsilon, zeta positive cells were rather low and showed no significant differences between the two groups (P>0.05); PKCbeta expression was not detected in the two groups of cells. The apoptosis rates of the control and radiation groups were 1.73% and 20.90%, respectively.

CONCLUSIONPKCalpha and PKCdelta may be involved in protecting HepG2 cells from radiation-induced apoptosis.

Apoptosis ; physiology ; radiation effects ; Hep G2 Cells ; Humans ; Isoenzymes ; classification ; metabolism ; Protein Kinase C-alpha ; metabolism ; Protein Kinase C-delta ; metabolism ; Radiation Tolerance ; Signal Transduction ; drug effects ; physiology

Protein kinase C (PKC) is a critical molecule in cellular signal transduction in mammals. It is involved in many biological processes in embryonic development, including nuclear remodeling, cell cycle adjustment and cellular polarity regulation. The present study aimed to observe the location of PKC&alpha;, an important isozyme of PKC, in fertilized, parthenogenetic and tetraploid preimplantation embryos, and compare the expression of PKC&alpha; during embryonic compaction in Kunming mice. The location of PKC&alpha; was detected by immunochemistry and laser confocal microscopy. Western blot was performed to quantify PKC&alpha; expression during embryonic compaction in the three kinds of embryos. In the experiment, fertilized embryos were flushed from oviduct or uterus at 45, 52, 69, 76 and 93 h after injection of human chorionic gonadotrophin (hCG); parthenogenetic embryos were collected by SrCl2 activation of oocytes for 6 h; and tetraploid embryos were produced by electrofusion of 2-cell embryos. Embryos were fixed at different developmental stages for immunofluorescent staining. 8-cell/4-cell embryos and morula were lysed for Western blot. The results showed that PKC&alpha; had similar location pattern in different embryos. It was distributed mainly in the nuclear aggregating around chromatin at different developmental stages. However, PKC&alpha; expressed strongly in the interphase than in mitotic blastomere. Before embryonic compaction, PKC&alpha; was localized at the blastomere boundary. At late blastocyst stage of fertilized embryos, PKC&alpha; was localized only in the polar trophoblast, but not in other trophoblast. At late stage of pathenogenetic blastocyst, there was no clear PKC&alpha; signal in the polar trophoblast. Tetraploid embryos had larger blastomere than other embryos and compacted after 4-cell stage, but not after 8-cell stage. Meanwhile, there was PKC&alpha; signal at the blastomere boundary at 4-cell stage. Our results showed that the expression of PKC&alpha; lasted through all the preimplantation stage. Although there were different expression levels among different stages, the expression increased around embryonic compaction. Quantification of expression of PKC&alpha; by Western blot demonstrated that the expression increased after compaction, indicating that the compaction was possibly dependent on the relocation of PKC&alpha;. Moreover, it was shown that the second relocation of PKC&alpha; occurred during the blastocyst formation. PKC&alpha; had different expression patterns in the three kinds of preimplantation embryos. However, the effects of PKC&alpha; on embryonic development started in early stage. There must be a necessary connection between PKC&alpha; relocation and cell adhesion starting at embryonic compaction.
Animals ; Embryonic Development ; Female ; Mice ; Parthenogenesis ; Pregnancy ; Protein Kinase C-alpha ; metabolism ; Tetraploidy ; Trophoblasts ; enzymology

AIMTo investigate the effects of sulfated polymannuroguluronate (SPMG), a novel candidate anti-AIDS drug in Phase II clinical trial, on Tat-induced release of proinflammatory cytokines (i.e., TNFalpha, IL-1beta and IL-6) and its related mechanism.

METHODSThe effects of SPMG on Tat induced TNFalpha (4 h), IL-1beta and IL-6 (6 h) secretion in THP-1 cells were measured by ELISA. Western blotting analysis was used to study the effects of SPMG on Tat induced PKCzeta, PKCtheta and PKCsigma phosphorylation.

RESULTSSPMG (50 to 100 microg x mL(-1)) markedly suppressed TNFalpha, IL-1beta and IL-6 secretion in Tat activated THP-1 cells. In THP-1 cells the phosphorylation levels of PKCzeta, PKCtheta and PKCsigma significantly increased following Tat stimulation, and only PKCsigma phosphorylation levels was inhibited by SPMG (50 to 100 microg x mL(-1)).

CONCLUSIONSPMG suppresses the secretion of proinflammatory cytokines in THP-1 cells may be by inhibiting PKCsigma activation.

Cell Line, Tumor ; Gene Products, tat ; pharmacology ; Humans ; Interleukin-1beta ; secretion ; Interleukin-6 ; secretion ; Isoenzymes ; metabolism ; Phosphorylation ; Polysaccharides ; pharmacology ; Protein Kinase C ; metabolism ; Protein Kinase C-delta ; metabolism ; Protein Kinase C-theta ; Tumor Necrosis Factor-alpha ; secretion

Endothelial dysfunction is implicated in a variety of cardiovascular diseases although the detailed mechanisms are not yet completely understood. A relationship has been suggested to exist between inflammation and endothelial dysfunction. TNF-&alpha; serves as one of the most important pro-inflammatory cytokines. The main objectives of the present study were to explore the effect of PKC-ζ on TNF-&alpha;-impaired endothelial function as well as the underlying mechanisms. Acetylcholine-induced endothelium-dependent vasodilation of mouse thoracic aorta stimulated by TNF-&alpha; was initially determined. PKC-ζ deficient mice and the specific inhibitor of NADPH oxidase were respectively applied to elucidate their roles in TNF-&alpha;-induced endothelial dysfunction. In vitro superoxide generation in HAECs was detected by DHE staining after administration of TNF-&alpha;. Meanwhile, the regulatory p47(phox) subunit of NADPH oxidase was evaluated by Western blotting and RT-PCR. The results showed that TNF-&alpha; conspicuously impaired endothelium-dependent vasodilation and the impairment was attenuated by either depleting PKC-ζ or inhibiting NADPH oxidase. In vitro TNF-&alpha; increased superoxide production and p47(phox) expression in HAECs, and such increases could be ameliorated by the specific PKC-ζ inhibitor. Our findings suggest that superoxide over-production triggered by PKC-ζ-dependent NADPH oxidase activation contributes to TNF-&alpha;-induced endothelial dysfunction.
Animals ; Endothelium, Vascular ; metabolism ; Male ; Mice ; NADPH Oxidases ; metabolism ; Protein Kinase C ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study the expression of protein kinase C (PKC) alpha subtype in Chinese hamster ovary (CHO) cells.

METHODSThe PKC alpha primer pairs were designed based on the GenBank sequence of PKC alpha of a species with the highest homology to Chinese hamster identified using EMBL Data Library Clustalw tool. The sequence coding for PKC alpha, amplified from the CHO cells using RT-PCR, was ligated to the pGEM-T plasmid vector, and the recombinant vector was transformed into E.coli DH5alpha with the positive colones selected by blue/white screening. Restriction enzyme digestion, gel electrophoresis analysis, followed by sequencing of the digestion products were performed for identification of the recombinant. Western blotting was used to analyze the PKC alpha expression in the CHO cells.

RESULTSThe presence of PKC alpha mRNA was detected in the CHO cells by RT-PCR. Western blotting also identified PKC alpha expression in the cells.

CONCLUSIONSPKC alpha expression has been identified in the CHO cells, which may facilitate further structural and functional study of PKC alpha and investigation of its role in the intracellular signal transduction pathways.

Animals ; Blotting, Western ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Female ; Protein Kinase C-alpha ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

The previous study indicated that aquaporin 4 (AQP4) deficiency attenuated opioid physical dependence. However, the underlying mechanism remains unknown. In the present study, the effects of AQP4 deficiency on the expression of three factors, protein kinase C (PKC) &alpha;, PKCγ and c-Fos in the spinal cord, which are known to be concerned with spinal neuronal sensitization and opiate dependence, were investigated in AQP4 knockout mice using Western blotting analysis. It was observed that AQP4 deficiency reduced the score of naloxone-precipitated abstinent jumping after repeated morphine administration compared with wild-type (P < 0.001). Meanwhile, the protein levels of PKC&alpha; and c-Fos in the spinal cord of AQP4 knockout mice were significantly higher than those in the wild-type mice; while the expression of PKCγ was decreased remarkably by AQP4 knockout during the withdrawal (P < 0.01). These data suggest that AQP4 deficiency-attenuated morphine withdrawal responses may be partially attributed to the changes in the spinal expression of PKC&alpha;, PKCγ or c-Fos.
Analgesics, Opioid ; pharmacology ; Animals ; Aquaporin 4 ; deficiency ; genetics ; Mice ; Mice, Knockout ; Morphine ; pharmacology ; Naloxone ; pharmacology ; Protein Kinase C ; metabolism ; Protein Kinase C-alpha ; metabolism ; Spinal Cord ; metabolism ; Substance Withdrawal Syndrome ; metabolism

AIMTo investigate whether free fatty acid impair insulin signaling proteins to inhibit insulin action in rat islet cells.

METHODSAfter rat islet cells were incubated with palmitate (0.25 mmol/L) or oleate (0.125 mmol/L) for 12 hours, 24 hours and 36 hours, western bolt was used to assess the protein abundance of cPKCalpha, Grb2 and FERK2.

RESULTSThe protein content of cPKCalpha were significantly upregulated and the protein abundance of Grb2 and ERK2 was decreased comparing with control in rat islet cells after incubated with free fatty acids.

CONCLUSIONSFree fatty acids may inhibit insulin activity in rat islet cells through up-regulating the expression of cPKCalpha or down-regulating the expression of Grb2 and ERK2.

Animals ; Cells, Cultured ; Fatty Acids, Nonesterified ; blood ; GRB2 Adaptor Protein ; metabolism ; Insulin ; metabolism ; Islets of Langerhans ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Protein Kinase C-alpha ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

Our previous studies showed that spinal neurons sensitization was involved in morphine withdrawal response. This study was to investigate the roles of spinal protein kinase C (PKC) alpha, gamma in morphine dependence and naloxone-precipitated withdrawal response. To set up morphine dependence model, rats were subcutaneously injected with morphine (twice a day, for 5 d). The dose of morphine was 10 mg/kg in the first day and was increased by 10 mg/kg each day. On day 6, 4 h after the injection of morphine (50 mg/kg), morphine withdrawal syndrome was precipitated by an injection of naloxone (4 mg/kg, i.p.). Chelerythrine chloride (CHE), a PKC inhibitor, was intrathecally injected 30 min before the administration of naloxone. The scores of morphine withdrawal symptom and morphine withdrawal-induced allodynia were observed. One hour after naloxone-precipitated withdrawal, Fos protein expression was assessed by immunohistochemical analysis and Western blot was used to detect the expression of cytosol and membrane fraction of PKC alpha and gamma in the rat spinal cord. The results showed that intrathecal administration of CHE decreased the scores of morphine withdrawal, attenuated morphine withdrawal-induced allodynia and also inhibited the increase of Fos protein expression in the spinal cord of morphine withdrawal rats. The expression of cytosol and membrane fraction of PKC alpha was significantly increased in the spinal cord of rats with morphine dependence. Naloxone-precipitated withdrawal induced PKC alpha translocation from cytosol to membrane fraction, which was prevented by intrathecal administration of CHE. During morphine dependence, but not naloxone-precipitated withdrawal, PKC gamma in the spinal cord translocated from cytosol to membrane fraction, and intrathecal administration of CHE did not change the expression of PKC gamma in the spinal cord of naloxone-precipitated withdrawal rats. It is suggested that up-regulation and translocation of PKC in the spinal cord contribute to morphine dependence and naloxone-precipitated withdrawal in rats and that PKC alpha and gamma play different roles in the above-mentioned effect.
Animals ; Male ; Morphine Dependence ; physiopathology ; Naloxone ; pharmacology ; Protein Kinase C ; metabolism ; physiology ; Protein Kinase C-alpha ; metabolism ; physiology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; metabolism ; physiopathology ; Substance Withdrawal Syndrome ; enzymology ; physiopathology

OBJECTIVETo observe the effects of low power microwave radiation on lens hydration and lens epithelial cells in vitro, and detect the expression of PKC-alpha, c-fos and c-jun in lens epithelial cells.

METHODSRabbit lens were exposed to microwave radiation with frequency of 2450 MHz and power density of 0.5, 2.0 and 5.0 mW/cm(2) in vitro. The hydration of lens was measured after 8 hours. Morphological changes of lens epithelial cells were observed using a phase-contrast microscope and Hoechst 33258 staining. Expression of PKC-alpha, c-fos and c-jun were analyzed using gel electrophoresis and western blot analysis.

RESULTSAfter 2.0 and 5.0 mW/cm(2) microwave radiation, the hydration of lens was increased compared to control groups (P<0.05), the shape of lens epithelial cells showed shrinking and disorder and cells nuclei appeared chromatin condensation. There was no change of lens and lens epithelial cells after 0.5 mW/cm(2) microwave radiation. The expression of PKC-alpha was significantly increased in cell membrane, however, decreased in cell cytoplasm after 2.0 mW/cm(2) microwave radiation for 2, 4, 6 and 8 hours. There was significantly increased expression of c-fos and c-jun protein compared with control groups (P<0.05, P<0.01).

CONCLUSIONLow power microwave radiation higher than 2.0 mW/cm(2) can activate PKC-alpha by increasing its expression in cell membrane, then induce high expression of c-fos and c-jun, which may relate to cellular signaling pathway of microwave radiation injury to lens and lens epithelial cells.

Animals ; Epithelial Cells ; metabolism ; pathology ; radiation effects ; In Vitro Techniques ; Lens, Crystalline ; metabolism ; pathology ; radiation effects ; Protein Kinase C-alpha ; metabolism ; Rabbits ; Transcription Factors ; metabolism