• Biomolecules & Therapeutics
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• v.32 no.3
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• pp.361-367
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• 2024

In this study, we investigated the efficacy of kaempferol (a flavonoid found in plants and plant-derived foods such as kale, beans, tea, spinach and broccoli) on vascular contractibility and aimed to clarify the detailed mechanism underlying the relaxation. Isometric contractions of divested muscles were stored and linked with western blot analysis which was carried out to estimate the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to estimate the effect of kaempferol on the RhoA/ROCK/CPI-17 pathway. Kaempferol conspicuously impeded phorbol ester-, fluoride- and a thromboxane mimetic-derived contractions regardless of endothelial nitric oxide synthesis, indicating its direct effect on smooth muscles. It also conspicuously impeded the fluoride-derived elevation in phospho-MYPT1 rather than phospho-CPI-17 levels and phorbol 12,13-dibutyrate-derived increase in phospho-CPI-17 and phospho-ERK1/2 levels, suggesting the depression of PKC and MEK activities and subsequent phosphorylation of CPI-17 and ERK1/2. Taken together, these outcomes suggest that kaempferol-derived relaxation incorporates myosin phosphatase retrieval and calcium desensitization, which appear to be modulated by CPI-17 dephosphorylation mainly through PKC inactivation.

• BMB Reports
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• v.34 no.2
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• pp.182-187
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• 2001

Phospholiapse D (PLD), and phosphatidic acid generated by it, have been implicated in receptor-mediated intracellular signaling. Carbachol (CCh) is known to activate PLD1, and protein kinase C (PKC) is known to mediate in this signaling pathway In recent reports (Kim et al., 1999b; Kim et al., 2000), we published our observations of the direct phosphorylation of PLD1 by PKC and we described the phosphorylation-dependent regulation of PLD1 activity. In this study, we investigated the phasphorylation and compartmentalization of PLD1 in terms of CCh signaling in M3 muscarinic receptor (M3R)-expressing COS-7 cells. CCh treatment of COS-7 cells transiently coexpressing PLD1 and M3R stimulated PLD1 activity and induced direct phosphorylation of PLD1 by PKC. The CCh-induced activation and phosphorylation of PLD1 was completely blocked upon pretreatment of the cells with PKC-specific inhibitors. We looked at the localization of the PLD1 phosphorylation by PKC and found that PLD1 was mainly located in the caveolin-enriched membrane (CEM) fraction. Based on these results, we conclude that CCh induces the activation and phosphorylation of PLD1 via PKC and that the phosphorylation of PLD1 occurs in caveolae.

• Korean Journal of Veterinary Research
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• v.44 no.4
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• pp.497-505
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• 2004

The proliferation of mesangial cells has been associated with the development of diabetic nephropathy. The cell proliferation has been regulated by diverse growth factors. Among them, insulin like growth factors(IGFs) are also involved in the pathogenesis of diabetic nephropathy. However, it is not yet known about the effect of high glucose on IGF-I and IGF-II secretion and the relationship between high glucose-induced secretion of IGFs and PKC or oxidative stress in the mesangial cells. Thus, we examined the mechanisms by which high glucose regulates secretion of IGFs in mesangial cells. High glucose(25 mM) increased IGF-I and IGF-II secretion. High glucose-induced increase of IGF-I and IGF-II secretion were blocked by taurine($2{\times}10^{-3}$ M), N-acetyl cystein(NAC, $10^{-5}M$), or GSH($10^{-5}M$) (antioxidants), suggesting the role of oxidative stress. High glucose-induced secretion of IGF-I and IGF-II were blocked by H-7, staurosporine, and bisindolylmaleimide I(protein kinase C inhibitors). On the other hand, high glucose also increased lipid peroxide (LPO) formation in a dose dependent manner. In addition, high glucoseinduced stimulation of LPO formation was blocked by PKC inhibitors. These results suggest that PKC is responsible for the increase of oxidative stress in the action of high glucose-induced secretion of IGF-I and IGF-II in mesangial cells. In conclusion, high glucose stimulates IGF-I and IGF-II secretion via PKCoxidative stress signal pathways in mesangial cells.

• Microbiology and Biotechnology Letters
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• v.33 no.3
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• pp.184-188
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• 2005

IgE-dependent histamine-releasing factor (HRF) is found extracellularly to regulate the degranulation process of histamine in mast cells and basophils and known to play a predominant role in the pathogenesis of chronic allergic disease. HRF has been also identified in the intracellular region of the cell. Previously, we reported that HRF interacts with the 3rd cytoplasmic domain of the alpha subunit of Na,K ATPase and inhibits Na,K-ATPase activity. The predicated phosphorylation site in HRF by PKC was mapped to one serine residues (S98) by the computer analysis. In this study, we identified that S98 residue of HRF was phosphorylated using anti-HRFpS98 antibody which specifically recognizes the phosphorylated serine residue of HRF and HRFS98A mutant construct. We also performed $^{86}Rb^{+}-uptake$ assay to understand the role of HRF wild-type and HRFS98A mutants on the regulation of Na,K-ATPase activity. Dephosphorylation of HRF at serine 98 residue recovers slightly the inhibitory function of HRF, suggesting that phosphorylated HRF at serine 98 may not suppress the Na,K-hfpase activity.

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

• Biomolecules & Therapeutics
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• v.30 no.2
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• pp.145-150
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• 2022

In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of denuded aortic muscles were recorded and combined with western blot analysis which was performed to measure the phosphorylation of phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) and to evaluate the effect of galangin on the RhoA/ROCK/CPI-17 pathway. Galangin significantly inhibited phorbol ester-, fluoride- and thromboxane mimetic-induced vasoconstrictions regardless of endothelial nitric oxide synthesis, suggesting its direct effect on vascular smooth muscle. Galangin significantly inhibited the fluoride-dependent increase in pMYPT1 and pCPI-17 levels and phorbol 12,13-dibutyrate-dependent increase in pERK1/2 level, suggesting repression of ROCK and MEK activity and subsequent phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that galangin-induced relaxation involves myosin phosphatase reactivation and calcium desensitization, which appears to be mediated by CPI-17 dephosphorylation via not PKC but ROCK inactivation.

• The Korean Journal of Pain
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• v.29 no.4
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• pp.229-238
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• 2016

Background: The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta. Methods: The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ($[Ca^{2+}]_i$) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting. Results: Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in $[Ca^{2+}]_i$. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation. Conclusions: These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics.

• The korean journal of orthodontics
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• v.26 no.6
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• pp.667-676
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• 1996

We have examined the in vitro stage-related chondrogenic potential of human mandibular and limb bud mesenchyme cells using micromass culture. Our results indicate that limb bud mesenchyme cells as early as stage 16 by Carnegie system (37 days), well before the initiation of in vivo chondrogenesis, have chondrogenic potential which is expressed in micromass culture. These results are correlated with stage-related chondrogenic potential of human limb bud in vivo as a result of Alcian blue staining. The proliferation of chondrogenic cells increased in the first 3 days after culture and then decreased. These results were correlated with the cell cycle analysis of which the number of $G_0^1/G_1$ phase increased markedly after 3 days of culture, while the percentage of cells in S phase was decreased. On the other hand, it was rarely differentiated in the mandible. We examined the effects of two PKC modulators such as phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, and staurosporine (STSN), an inhibitor of PKC. PMA inhibited the chondrogenesis, whereas STSN promoted the chondrogenesis in a dose dependent manner. In addition, PMA exerted no inhibitory effect when the cells were pretreated for 24 h with STSN, implying that the chondrogenic events might be settled at an early step in vitro and FKC may act as a negative modulator. Collectively, these results demonstrate, for the first time, the stage-related chondrogenic potential of human mandibular and limb bud mesenchyme cells and the role of PKC during chondrogenesis in vitro & in vivo.

• Korean Journal of Veterinary Research
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• v.39 no.6
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• pp.1073-1080
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• 1999

Previous studies suggested that the inhibition of thyroxine ($T_4$) release by ${\alpha}_1$-adrenoceptor and muscarinic receptor stimulation results in activated protein kinase C (PKC) from mouse and guinea pig thyroids. In the present study, the effect of carbachol, methoxamine, phorbol myristate acetate (PMA), and R59022 on the release of $T_4$ from the mouse, rat, and guinea pig thyroids was compared to clarify the role of PKC in the regulation of the release of $T_4$. The thyroids were incubated in the medium containing the test agents, samples of the medium were assayed for $T_4$ by EIA kits. Forskolin, an adenylate cyclase activator, chlorophenylthio-cAMP sodium, a membrane permeable analog of cAMP, and isobutyl-methylxanthine, a phosphodiesterase inhibitor, like TSH (thyroid stimulating hormone), enhaced the release of $T_4$ from the mouse, rat, and guinea pig thyroids. Methoxamine, an ${\alpha}_1$-adrenoceptor agonist, inhibited the TSH-stimulated release of $T_4$ in mouse, but not rat and guinea pig thyroids. In contrast, carbachol, a muscarinic receptor agonist, inhibited the release of $T_4$ in guinea pig, but not mouse and rat thyroids. These inhibition were reversed by prazosin, an ${\alpha}_1$-adrenoceptor antagonist or atropine, a muscarinic antagonist or $M_1$- and $M_3$-muscarinic antagonists, in mouse or guinea pig thyroids. In addition, staurosporine, a PKC inhibitor, reversed methoxamine or carbachol inhibition of TSH stimulation. Furthermore, PMA, a PKC activator, and R59022, a diacylglycerol (DAG) kinase inhibitor, inhibited the TSH-stimulated release of $T_4$ in mouse, rat, and guinea pig thyroids. These inhibition were blocked by staurosporine. These findings suggest that the activation of receptor or DAG inhibits TSH-stimulated $T_4$ release through a PKC-dependent mechanism in thyroid gland.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.5
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• pp.261-267
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• 2002

The aim of the present study was to investigate the interaction of $estradiol-17{\beta}-bovine$ serum albumin $(E_2-BSA)$ and calcitropic hormones, such as parathyroid hormone, calcitonin, and vitamin D, in regulation of $Ca^{2+}$ uptake in primary cultured renal proximal tubule cells. Statistically significant increase in $Ca^{2+}$ uptake was found from 2 hours after $(E_2-BSA)\;(10^{-9}\;M)$ treatment, while $estradiol-17{\beta}\;(10^{-9}\;M)$ did not affect. Treatment of the cells with $(E_2-BSA)\;(10^{-9}\;M)$ together with parathyroid hormone (PTH) $(10^{-8}\;M),$ vitamin D $(10^{-8}\;M),$ or calcitonin $(10^{-8}\;M)$ significantly stimulated $Ca^{2+}$ uptake by 32.50%, 29.30%, or 27.75%, respectively, compared with the control. However, calcitropic hormones did not exhibit any synergistic effect on the E2-BSA-induced stimulation. $E_2-BSA$ significantly increased cAMP generation and PKC activity. The stimulatory effect of cotreatment of $E_2-BSA$ and PTH or vitamin D was blocked by SQ22536 (an adenylate cyclase inhibitor) and staurosporine (a PKC inhibitor), but the effect of cotreatment of $E_2-BSA$ and calcitonin was not blocked. Furthermore, 8-Br-cAMP and TPA (an artificial PKC promoter) increased $Ca^{2+}$ uptake by 25.51% and 16.47%, respectively, compared with the control. In conclusion, $E_2-BSA$ combined with calcitropic hormones regulated $Ca^{2+}$ uptake partially via cAMP and PKC-dependent mechanisms in renal proximal tubule cells.