• The Korean Journal of Physiology and Pharmacology
• /
• v.17 no.1
• /
• pp.51-56
• /
• 2013

Many intracellular proteins and signaling cascades contribute to the sensitivity of N-methyl-D-aspartate receptors (NMDARs). One such putative contributor is the serine/threonine kinase, protein kinase C (PKC). Activation of PKC by phorbol 12-myristate 13-acetate (PMA) causes activation of extracellular signal-regulated kinase (ERK) and promotes the formation of new spines in cultured hippocampal neurons. The purpose of this study was to examine which PKC isoforms are responsible for the PMA-induced augmentation of long-term potentiation (LTP) in the CA1 stratum radiatum of the hippocampus in vitro and verify that this facilitation requires NMDAR activation. We found that PMA enhanced the induction of LTP by a single episode of theta-burst stimulation in a concentration-dependent manner without affecting to magnitude of baseline field excitatory postsynaptic potentials. Facilitation of LTP by PMA (200 nM) was blocked by the nonspecific PKC inhibitor, Ro 31-8220 ($10{\mu}M$); the selective $PKC{\delta}$ inhibitor, rottlerin ($1{\mu}M$); and the $PKC{\varepsilon}$ inhibitor, TAT-${\varepsilon}V1$-2 peptide (500 nM). Moreover, the NMDAR blocker DL-APV ($50{\mu}M$) prevented enhancement of LTP by PMA. Our results suggest that PMA contributes to synaptic plasticity in the nervous system via activation of $PKC{\delta}$ and/or $PKC{\varepsilon}$, and confirm that NMDAR activity is required for this effect.

• The Korean Journal of Physiology and Pharmacology
• /
• v.26 no.2
• /
• pp.87-94
• /
• 2022

Myocardial infarction promotes cardiac remodeling and myocardial fibrosis, thus leading to cardiac dysfunction or heart failure. Peiminine has been regarded as a traditional anti-fibrotic Chinese medicine in pulmonary fibrosis. However, the role of peiminine in myocardial infarction-induced myocardial injury and fibrosis remained elusive. Firstly, rat model of myocardial infarction was established using ligation of the left coronary artery, which were then intraperitoneally injected with 2 or 5 mg/kg peiminine once a day for 4 weeks. Echocardiography and haemodynamic evaluation results showed that peiminine treatment reduced left ventricular end-diastolic pressure, and enhanced maximum rate of increase/decrease of left ventricle pressure (± dP/dt max) and left ventricular systolic pressure, which ameliorate the cardiac function. Secondly, myocardial infarction-induced myocardial injury and infarct size were also attenuated by peiminine. Moreover, peiminine inhibited myocardial infarction-induced increase of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α production, as well as the myocardial cell apoptosis, in the rats. Thirdly, peiminine also decreased the myocardial fibrosis related protein expression including collagen I and collagen III. Lastly, peiminine reduced the expression of p38 and phosphorylation of extracellular signal-regulated kinase 1/2 in rat model of myocardial infarction. In conclusion, peiminine has a cardioprotective effect against myocardial infarction-induced myocardial injury and fibrosis, which can be attributed to the inactivation of mitogen-activated protein kinase pathway.

• Journal of Applied Biological Chemistry
• /
• v.50 no.2
• /
• pp.58-62
• /
• 2007

Identification of Dual-specificity protein phosphatase (DSP) substrates is essential in revealing physiological roles of DSPs. We isolated VHZ-interacting proteins from extracts of 293T cells overexpressing a VHZ (C95S, D65A) mutant known to be substrate- trapping mutant. Analysis of specific proteins bound to VHZ by 2D gel electrophoresis and mass spectroscopy revealed that these proteins contained Chaperonin containing TCP1, Type II phosphatidylinositol phosphate kinase ${\gamma}$, Intraflagellar transport 80 homolog, and Kinesin superfamily protein 1B. VHZ-interacting proteins showed that VHZ is involved in many important cellular signal pathways such as protein folding, molecular transportation, and tumor suppression.

• Nutrition Research and Practice
• /
• v.18 no.1
• /
• pp.1-18
• /
• 2024

BACKGROUND/OBJECTIVES: Endoplasmic reticulum (ER) stress in adipose tissue causes an inflammatory response and leads to metabolic diseases. However, the association between vitamin D and adipose ER stress remains poorly understood. In this study, we investigated whether 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) alleviates ER stress in adipocytes. MATERIALS/METHODS: 3T3-L1 cells were treated with different concentrations (i.e., 10-100 nM) of 1,25(OH)₂D₃ after or during differentiation (i.e., on day 0-7, 3-7, or 7). They were then incubated with thapsigargin (TG, 500 nM) for an additional 24 h to induce ER stress. Next, we measured the mRNA and protein levels of genes involved in unfold protein response (UPR) and adipogenesis using real-time polymerase chain reaction and western blotting and quantified the secreted protein levels of pro-inflammatory cytokines. Finally, the mRNA levels of UPR pathway genes were measured in adipocytes transfected with siRNA-targeting Vdr. RESULTS: Treatment with 1,25(OH)₂D₃ during various stages of adipocyte differentiation significantly inhibited ER stress induced by TG. In fully differentiated 3T3-L1 adipocytes, 1,25(OH)₂D₃ treatment suppressed mRNA levels of Ddit3, sXbp1, and Atf4 and decreased the secretion of monocyte chemoattractant protein-1, interleukin-6, and tumor necrosis factor-α. However, downregulation of the mRNA levels of Ddit3, sXbp1, and Atf4 following 1,25(OH)₂D₃ administration was not observed in Vdr-knockdown adipocytes. In addition, exposure of 3T3-L1 preadipocytes to 1,25(OH)₂D₃ inhibited transcription of Ddit3, sXbp1, Atf4, Bip, and Atf6 and reduced the p-alpha subunit of translation initiation factor 2 (eIF2α)/eIF2α and p-protein kinase RNA-like ER kinase (PERK)/PERK protein ratios. Furthermore, 1,25(OH)₂D₃ treatment before adipocyte differentiation reduced adipogenesis and the mRNA levels of adipogenic genes. CONCLUSIONS: Our data suggest that 1,25(OH)₂D₃ prevents TG-induced ER stress and inflammatory responses in mature adipocytes by downregulating UPR signaling via binding with Vdr. In addition, the inhibition of adipogenesis by vitamin D may contribute to the reduction of ER stress in adipocytes.

• The Korean Journal of Pharmacology
• /
• v.32 no.2
• /
• pp.243-257
• /
• 1996

CCK and cholinergic agonist stimulate enzyme release from the pancreatic acini via G-protein-mediated activation of phospholipase C, In contrast secretin and related peptides increase the level of cAMP and activate cAMP-dependent protein kinase. Camostat, a synthetic protease inhibitor, causes pancreatic hypertrophy and hyperplasia by increasing the CCK release. In this study, the secretagogue-induced changes of intracellular proteins were examined in the dispersed pancreatic acini of rats with or without camostat treatment. Camostat(FOY-305, 200 mg/kg, p.o.) was given for 4 days twice daily and the dispersed acini were prepared at 12 bouts after last treatment. The profiles of Intracellular phosphoproteins were analyzed by two-dimensional gel electrophoresis after incubating the acini with $^{32}P$. The amylase release from the dispersed acini was measured. The pancreatic weight was increased to 126% of control, while amylase activity per mg acinar protein decreased to 41% of control, The maximum response of amylase release from dispersed acini to CCK-8 or carbachol was markedly decreased(65% or 46% of control, respectively). The group of intracellular proteins(24 kD, pI $4.5{\sim}8.5$) was increased in quantity by camostat. CCK-8 or secretin increased phosphorylation of a protein(34 kD, pI 4.7) in camostat-treated as well as control rats. CCK-8 increased tyrosine phosphoryiation in the acini of control rats. However, in camostat-treated rats, the basal level of tyrosine phosphorylation was increased and it was rather decreased by CCK-8. Secretin had no effect on the level of tyrosine phosphorylation in acini. These results indicate that both phospholipase C and adenylate cyclase induce phosphorylation of an intracellular acinar protein(34 kD, pI 4.7) and camostat treatment increases the basal level of tyrosine phosphorylation in acinar cells. And these results suggest that not only serine/threonine protein kinase but also protein tyrosine kinase/phosphatase are involved in the process of CCK receptor mediated stimulation-secrelion coupling.

• Asian-Australasian Journal of Animal Sciences
• /
• v.15 no.7
• /
• pp.1045-1050
• /
• 2002

The aims of this study were to detect spontaneously occurring apoptosis in cultured porcine ovarian cells, to examine the role of growth hormone (GH), tyrosine kinase (TK), protein kinase G (PKG) and cyclin-dependent kinase (CDK) in the control of this process, and to determine whether the effect of GH on apoptosis is mediated by TK-, PKG- and cdc2-dependent intracellular mechanisms. We studied the action of pGH (10 ng/ml), blockers of TK (genistein, lavendustin, both 100 ng/ml), PKG (Rp-Br-PET-cGMPS, 50 nM; KT5823, 100 ng/ml) and CDK (olomoucine, $1{\mu}g/ml$), as well as combinations of GH with these blockers, on the onset of apoptosis in cultured granulosa cells isolated from antral (3-6 mm) porcine follicles. The functional characteristics of an early apoptotic event, DNA fragmentation, were determined using terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL), whilst morphological signs of advanced apoptosis such as pyknosis, chromatin marginalization, shrinkage and fragmentation of nucleus, were detected using routine light microscopy. After culture, some ovarian granulosa cells exhibited DNA fragmentation, which in some cases was associated with morphological apoptosis-related changes (pyknosis, shrinkage and fragmentation of the nucleus). GH significantly reduced the proportion of TUNEL-positive cells. Neither TK nor CDK blockers when given alone, significantly affected the percentage of TUNEL-positive cells although both PKG blockers significantly increased this index. Furthermore, TK and PKG blockers given together with GH, prevented or reversed the inhibitory effect of GH on apoptosis, whilst the CDK blocker olomoucine promoted it. These observations demonstrate apoptosis in porcine ovaries and suggest the involvement of GH, TK, PKG and CDK in the control of this process. They also suggest that the effect of GH on ovarian apoptosis is mediated or regulated by multiple signalling pathways including TK-, PKG- and CDK-dependent intracellular mechanisms.

• Molecules and Cells
• /
• v.42 no.6
• /
• pp.470-479
• /
• 2019

Interstitial cells of Cajal (ICCs) are pacemaker cells that exhibit periodic spontaneous depolarization in the gastrointestinal (GI) tract and generate pacemaker potentials. In this study, we investigated the effects of ghrelin and motilin on the pacemaker potentials of ICCs isolated from the mouse small intestine. Using the whole-cell patch-clamp configuration, we demonstrated that ghrelin depolarized pacemaker potentials of cultured ICCs in a dose-dependent manner. The ghrelin receptor antagonist [D-Lys] GHRP-6 completely inhibited this ghrelin-induced depolarization. Intracellular guanosine 5'-diphosphate-${\beta}$-S and pre-treatment with $Ca^{2+}$-free solution or thapsigargin also blocked the ghrelin-induced depolarization. To investigate the involvement of inositol triphosphate ($IP_3$), Rho kinase, and protein kinase C (PKC) in ghrelin-mediated pacemaker potential depolarization of ICCs, we used the $IP_3$ receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C, the Rho kinase inhibitor Y-27632, and the PKC inhibitors staurosporine, Go6976, and rottlerin. All inhibitors except rottlerin blocked the ghrelin-induced pacemaker potential depolarization of ICCs. In addition, motilin depolarized the pacemaker potentials of ICCs in a similar dose-dependent manner as ghrelin, and this was also completely inhibited by [D-Lys] GHRP-6. These results suggest that ghrelin induced the pacemaker potential depolarization through the ghrelin receptor in a G protein-, $IP_3$-, Rho kinase-, and PKC-dependent manner via intracellular and extracellular $Ca^{2+}$ regulation. In addition, motilin was able to depolarize the pacemaker potentials of ICCs through the ghrelin receptor. Therefore, ghrelin and its receptor may modulate GI motility by acting on ICCs in the murine small intestine.

• Journal of Integrative Natural Science
• /
• v.10 no.2
• /
• pp.95-104
• /
• 2017

Proviral Integration site of Moloney (Pim) murine Leukemia virus kinases is a serine/threonine specific protein kinase. It is largely involved in cell survival and proliferation. Pim-1 phosphorylates multiple cellular substrates to inhibit apoptosis and promote cell cycle progression. Over expression of Pim-1 kinase is observed in a range of malignancies and various solid cancers. High level of Pim-1 expression is seen in myeloma, acute myeloid leukemia, prostate cancer and liver carcinomas. Hence, Pim-1 is considered as an interesting cancer target. In the present study, we have performed region-focused CoMFA study on a series of imidazopyridazine derivatives as Pim-1 kinase inhibitors. A statistically acceptable region-focused CoMFA model ($q^2=0.571$; ONC=3; $r^2=0.909$) was developed. The model was then validated using Bootsrapping and progressive sampling. The contour map highlighted the regions favorable to increase the activity. Bulky substitutions in $R^2$ position of the phenyl ring could increase the activity. Similarly, small negative substitution in the $R^1$ position of the Pyridine ring could increase the activity considerably. Our results will be useful to design novel Pim-1 kinase inhibitors of this series.

• Molecules and Cells
• /
• v.37 no.3
• /
• pp.248-256
• /
• 2014

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is a N-acetylhexosamine kinase that belong to the sugar kinase/heat shock protein 70/actin superfamily. In this study, we investigated both the expression and function of NAGK in neurons. Immunohistochemistry of rat brain sections showed that NAGK was expressed at high levels in neurons but at low levels in astrocytes. Immunocytochemistry of rat hippocampal dissociate cultures confirmed these findings and showed that NAGK was also expressed at low levels in oligodendrocytes. Furthermore, several NAGK clusters were observed in the nucleoplasm of both neuron and glia. The overexpression of EGFP- or RFP (DsRed2)-tagged NAGK in rat hippocampal neurons (DIV 5-9) increased the complexity of dendritic architecture by increasing the numbers of primary dendrites and dendritic branches. In contrast, knockdown of NAGK by shRNA resulted in dendrite degeneration, and this was prevented by the co-expression of RFP-tagged NAGK. These results suggest that the upregulation of dendritic complexity is a non-canonical function of NAGK.

• IMMUNE NETWORK
• /
• v.6 no.4
• /
• pp.204-210
• /
• 2006

Background: Nitric oxide (NO) in articular chondrocytes regulates dedifferentiation and inflammatory responses by modulating MAP kinases. In this study, we investigated whether the Src kinase in chondrocytes regulates NO-induced dedifferentiation and cyclooxygenase-2 (COX-2) expression. Methods: Primary chondrocytes were treated with various concentrations of SNP for 24 h. The COX-2 and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin $E_2\;(PGE_2)$ was determined by using a $PGE_2$ assay kit. Expression and distribution of p-Caveolin and COX-2 in rabbit articular chondrocytes and cartilage explants were determined by immunohistochemical staining and immunocytochemical staining, respectively. Results: SNP treatment stimulated Src kinase activation in a dose-dependent manner in articular chondrocytes. The Src kinase inhibitors PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine], a significantly blocked SNP-induced p38 kinase and caveolin-1 activation in a dose-dependent manner. Therefore, to determine whether Src kinase activation is associated with dedifferentiation and/or COX-2 expression and $PGE_2$ production. As expected, PP2 potentiated SNP-stimulated dedifferentiation, but completely blocked both COX-2 expression and $PGE_2$ production. And also, levels of p-Caveolin and COX-2 protein expression were increased in SNP-treated primary chondrocytes and osteoarthritic and rheumatoid arthritic cartilage, suggesting that p-Caveolin may playa role in the inflammatory responses of arthritic cartilage. Conclusion: Our previously studies indicated that NO caused dedifferentiation and COX-2 expression is regulated by p38 kinase through caveolin-1 (1). Therefore, our results collectively suggest that Src kinase regulates NO-induced dedifferentiation and COX-2 expression in chondrocytes via p38 kinase in association with caveolin-1.