1.Cardiac Hypertrophy in Transgenic Mice with Overexpressed Small-Subunit of Human Myosin Light Chain Phosphatase.
Sung Won BAE ; Won Ha LEE ; Chul Hong KIM ; Haing Kee PARK ; Sang Chol LEE ; Young Hyeh KO ; Seung Woo PARK ; Takuro ARIMURA ; Akinori KIMURA ; Jeong Euy PARK
Korean Circulation Journal 2003;33(12):1165-1173
BACKGROUND AND OBJECTIVES: Recent reports have demonstrated that perturbation of the balance between myo-sin light chain (MLC) phosphorylation and the dephosphorylation status is associated with the development of cardiac hypertrophy. Myosin light chain phosphatase (MLCP) is a key enzyme that regulates the phosphorylation status of the MLC, but its functional roles in cardiac muscle have not been well investigated. Especially, the functions of the small-subunit of MLCP in cardiac muscles are not well elucidated. Here, whether the human heart-specific small-subunit (M21) of MLCP is associated with hypertrophic responses in a transgenic mice model were assessed. MATERIALS AND METHODS: The transgenic mice, overexpressing the human M21, were generated from a cardiac-specific transgenic construct. Cardiac tissues from the transgenic mice were subjected to histology for their morphological examination. The echocardiographic parameters of the murine heart were examined with transgenic mice aged 1, 2 and 3 months, and compared with their non-transgenic littermates. To determine whether the transgenic heart was sensitive to stress, the echocardiographic examination was also performed at the baseline, both before and after the administration of isoproterenol, at a dosage of 30 microgram/g/day, for 2 weeks. RESULTS: The histological analysis of the transgenic heart revealed myocyte disarray and nuclear hypertrophy. No significant differences were observed between the transgenic and non-transgenic mice in relation to the echocardiographic determinants, such as the left ventricular dimensions and the wall thickness. Chronic cardiac stress, induced by isoproterenol infusion, also failed to show any significant differences in relation to the same determinants. CONCLUSION: Overexpression of the human M21 in the murine heart induced myocyte hypertrophy. However, the overall cardiac functions were not affected under normal and stressed conditions.
Animals
;
Cardiomegaly*
;
Cardiomyopathy, Hypertrophic
;
Echocardiography
;
Heart
;
Humans*
;
Hypertrophy
;
Isoproterenol
;
Mice
;
Mice, Transgenic*
;
Muscle Cells
;
Myocardium
;
Myosin Light Chains*
;
Myosin-Light-Chain Phosphatase*
;
Myosins*
;
Phosphorylation
;
Protein Subunits
2.Calcium Sensitization Mechanisms in Gastrointestinal Smooth Muscles.
Journal of Neurogastroenterology and Motility 2016;22(2):213-225
An increase in intracellular Ca2+ is the primary trigger of contraction of gastrointestinal (GI) smooth muscles. However, increasing the Ca2+ sensitivity of the myofilaments by elevating myosin light chain phosphorylation also plays an essential role. Inhibiting myosin light chain phosphatase activity with protein kinase C-potentiated phosphatase inhibitor protein-17 kDa (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation is considered to be the primary mechanism underlying myofilament Ca2+ sensitization. The relative importance of Ca2+ sensitization mechanisms to the diverse patterns of GI motility is likely related to the varied functional roles of GI smooth muscles. Increases in CPI-17 and MYPT1 phosphorylation in response to agonist stimulation regulate myosin light chain phosphatase activity in phasic, tonic, and sphincteric GI smooth muscles. Recent evidence suggests that MYPT1 phosphorylation may also contribute to force generation by reorganization of the actin cytoskeleton. The mechanisms responsible for maintaining constitutive CPI-17 and MYPT1 phosphorylation in GI smooth muscles are still largely unknown. The characteristics of the cell-types comprising the neuroeffector junction lead to fundamental differences between the effects of exogenous agonists and endogenous neurotransmitters on Ca2+ sensitization mechanisms. The contribution of various cell-types within the tunica muscularis to the motor responses of GI organs to neurotransmission must be considered when determining the mechanisms by which Ca2+ sensitization pathways are activated. The signaling pathways regulating Ca2+ sensitization may provide novel therapeutic strategies for controlling GI motility. This article will provide an overview of the current understanding of the biochemical basis for the regulation of Ca2+ sensitization, while also discussing the functional importance to different smooth muscles of the GI tract.
Actin Cytoskeleton
;
Calcium*
;
Gastrointestinal Motility
;
Gastrointestinal Tract
;
Muscle, Smooth*
;
Myofibrils
;
Myosin Light Chains
;
Myosin-Light-Chain Phosphatase
;
Neuroeffector Junction
;
Neurotransmitter Agents
;
Phosphorylation
;
Protein Kinases
;
Signal Transduction
;
Synaptic Transmission
3.Flavone Attenuates Vascular Contractions by Inhibiting RhoA/Rho Kinase Pathway.
Inji BAEK ; Su Bun JEON ; Min Ji SONG ; Enyue YANG ; Uy Dong SOHN ; In Kyeom KIM
The Korean Journal of Physiology and Pharmacology 2009;13(3):201-207
Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300 micrometer) or vehicle. We determined the phosphorylation level of the myosin light chain (MLC20), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of MLC20 phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619-or NaF-induced phosphorylation of MYPT1(Thr855) and CPI17(Thr38), the downstream effectors of Rho-kinase. In regards to the Ca2+-free solution, flavone inhibited the phosphorylation of MYPT1(Thr855) and CPI17(Thr38), as well as vascular contractions induced by U46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
;
Animals
;
Baths
;
Blotting, Western
;
Contracts
;
Endothelium
;
Flavones
;
Myosin Light Chains
;
Myosin-Light-Chain Phosphatase
;
Myosins
;
Phosphorylation
;
Phosphotransferases
;
Protein Kinases
;
Rats
;
rho-Associated Kinases
;
Thromboxane A2
;
Vasodilation
4.Lipid emulsion inhibits vasodilation induced by a toxic dose of bupivacaine by suppressing bupivacaine-induced PKC and CPI-17 dephosphorylation but has no effect on vasodilation induced by a toxic dose of mepivacaine.
Hyunhoo CHO ; Seong Ho OK ; Seong Chun KWON ; Soo Hee LEE ; Jiseok BAIK ; Sebin KANG ; Jiah OH ; Ju Tae SOHN
The Korean Journal of Pain 2016;29(4):229-238
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 ([Ca²⁺]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 [Ca²⁺]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.
Anesthetics, Local
;
Animals
;
Aorta
;
Blotting, Western
;
Bupivacaine*
;
Calcium
;
Fura-2
;
In Vitro Techniques
;
Mepivacaine*
;
Muscle, Smooth, Vascular
;
Myosin-Light-Chain Phosphatase
;
Phorbol 12,13-Dibutyrate
;
Phosphorylation
;
Protein Kinase C
;
Rats
;
Solubility
;
Vasodilation*
5.Lipid emulsion inhibits vasodilation induced by a toxic dose of bupivacaine by suppressing bupivacaine-induced PKC and CPI-17 dephosphorylation but has no effect on vasodilation induced by a toxic dose of mepivacaine.
Hyunhoo CHO ; Seong Ho OK ; Seong Chun KWON ; Soo Hee LEE ; Jiseok BAIK ; Sebin KANG ; Jiah OH ; Ju Tae SOHN
The Korean Journal of Pain 2016;29(4):229-238
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 ([Ca²⁺]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 [Ca²⁺]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.
Anesthetics, Local
;
Animals
;
Aorta
;
Blotting, Western
;
Bupivacaine*
;
Calcium
;
Fura-2
;
In Vitro Techniques
;
Mepivacaine*
;
Muscle, Smooth, Vascular
;
Myosin-Light-Chain Phosphatase
;
Phorbol 12,13-Dibutyrate
;
Phosphorylation
;
Protein Kinase C
;
Rats
;
Solubility
;
Vasodilation*
6.Endothelium Independent Effect of Pelargonidin on Vasoconstriction in Rat Aorta.
Young Sil MIN ; Hyuk Jun YOON ; Hyun Dong JE ; Jong Hyuk LEE ; Seong Su YOO ; Hyun Sub SHIM ; Hak Yeong LEE ; Hyen Oh LA ; Uy Dong SOHN
Biomolecules & Therapeutics 2018;26(4):374-379
In this study, we investigated the effects of pelargonidin, an anthocyanidin found in many fruits and vegetables, on endothelium-independent vascular contractility to determine the underlying mechanism of relaxation. Isometric contractions of denuded aortic muscles from male rats were recorded, and the data were combined with those obtained in western blot analysis. Pelargonidin significantly inhibited fluoride-, thromboxane A2-, and phorbol ester-induced vascular contractions, regardless of the presence or absence of endothelium, suggesting a direct effect of the compound on vascular smooth muscles via a different pathway. Pelargonidin significantly inhibited the fluoride-dependent increase in the level of myosin phosphatase target subunit 1 (MYPT1) phosphorylation at Thr-855 and the phorbol 12,13-dibutyrate-dependent increase in the level of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation at Thr202/Tyr204, suggesting the inhibition of Rho-kinase and mitogen-activated protein kinase kinase (MEK) activities and subsequent phosphorylation of MYPT1 and ERK1/2. These results suggest that the relaxation effect of pelargonidin on agonist-dependent vascular contractions includes inhibition of Rho-kinase and MEK activities, independent of the endothelial function.
Animals
;
Anthocyanins
;
Aorta*
;
Blotting, Western
;
Endothelium*
;
Fluorides
;
Fruit
;
Humans
;
Isometric Contraction
;
Male
;
Muscle, Smooth, Vascular
;
Muscles
;
Myosin-Light-Chain Phosphatase
;
Phosphorylation
;
Phosphotransferases
;
Protein Kinases
;
Rats*
;
Relaxation
;
rho-Associated Kinases
;
Vasoconstriction*
;
Vegetables
7.Comparison of Improving Effects for Diabetic Erectile Dysfunction according to the Anti-Glycemic Agents: Phlorizin and Insulin
Juhyun PARK ; Ohseong KWON ; Sung Yong CHO ; Min Chul CHO ; Jae Seung PAICK ; Soo Woong KIM
The World Journal of Men's Health 2019;37(2):210-218
PURPOSE: To compare the improving effects of diabetic erectile dysfunction with two anti-glycemic agents; phlorizin and insulin. MATERIALS AND METHODS: Sixty Sprague-Dawley rats were divided into four groups (n=15 in each group): normal control (C), untreated diabetic rats (D), and diabetic rats treated by phlorizin (P) or insulin (I). Ten weeks after the diabetic induction using an injection of streptozotocin (55 mg/kg), four weeks of diabetic control was conducted. Erectile response, Western blot, and immunohistochemistry were assessed. RESULTS: During the experiment, the C-group showed continuous weight gain, while the other groups suffered from weight loss. After start of diabetic control, the body weight of I-group was increased; whereas, there was no meaningful change in the P-group. Meanwhile, comparable blood glucose levels were achieved in the P- and I-groups. The erectile response was markedly decreased in the D-group, whereas the P- and I-groups were similar as good as the C-group. In addition, D-group showed the significant decrease in the cavernosal smooth muscle content and increased apoptosis. Platelet endothelial cell adhesion molecule-1 protein expression, phosphorylation of endothelial nitric oxide synthase and myosin phosphatase target subunit 1 were significantly distorted in the D-group, while the P- and I-groups were comparable with the C-group. CONCLUSIONS: Phlorizin treatment resulted in the improvement of erectile function as same as insulin despite the lack of anabolic weight gains. These results suggest that control of blood glucose level rather than a type of anti-glycemic agents is more important for the prevention and treatment of diabetic erectile dysfunction
Animals
;
Antigens, CD31
;
Apoptosis
;
Blood Glucose
;
Blotting, Western
;
Body Weight
;
Diabetes Complications
;
Erectile Dysfunction
;
Immunohistochemistry
;
Insulin
;
Male
;
Muscle, Smooth
;
Myosin-Light-Chain Phosphatase
;
Nitric Oxide Synthase Type III
;
Phlorhizin
;
Phosphorylation
;
Rats
;
Rats, Sprague-Dawley
;
Streptozocin
;
Weight Gain
;
Weight Loss
8.Is It Possible to Recover Erectile Function Spontaneously after Cavernous Nerve Injury? Time-Dependent Structural and Functional Changes in Corpus Cavernosum Following Cavernous Nerve Injury in Rats.
Tae Beom KIM ; Min Chul CHO ; Jae Seung PAICK ; Soo Woong KIM
Korean Journal of Andrology 2012;30(1):31-39
PURPOSE: There has been a scarcity of integrated, long-term (>4 week) studies on structural and functional alterations in the penis according to the period following cavernous nerve (CN) injury. The aim of this study was to investigate time-dependent structural and functional changes in the corpus cavernosum following CN injury in a rat model. MATERIALS AND METHODS: Ninety male Sprague-Dawley rats (10 weeks old) were divided into 4 groups: normal control (C), sham (S), bilateral CN resection (R), and bilateral CN crush injury (I) groups. At 1, 4, and 12 weeks after the procedure, erectile function was assessed by electrostimulation. The terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay was performed for detection of apoptosis. Masson's trichrome staining and immunohistochemistry were performed for detection of alpha smooth muscle actin (alpha-SMA). Western blot analysis was then performed. RESULTS: The R and I groups showed persistent impairment of erectile function at all three points in time. Apoptosis peaked at 1 week after resection or crush injury and then gradually subsided. The smooth muscle cell/collagen ratio and expression of alpha-SMA gradually decreased over time after CN resection or crush injury. Myosin phosphatase target subunit 1 phosphorylation progressively increased over time after CN resection or crush injury. On the other hand, expression of phospho-protein kinase B, phospho-endothelial nitric oxide synthase, and neuronal nitric oxide synthase transiently decreased at 1 week after resection or crush injury and then recovered to the control values. CONCLUSIONS: Our results suggest that persistent up-regulation of the RhoA/Rho-kinase pathway and structural change such as decreased smooth muscle cell and increased cavernosal fibrosis might play an important role in persistent erectile dysfunction following CN injury.
Actins
;
Animals
;
Apoptosis
;
Blotting, Western
;
Caves
;
Deoxyuracil Nucleotides
;
Erectile Dysfunction
;
Fibrosis
;
Hand
;
Humans
;
Immunohistochemistry
;
Male
;
Muscle, Smooth
;
Myocytes, Smooth Muscle
;
Myosin-Light-Chain Phosphatase
;
Nitric Oxide Synthase
;
Nitric Oxide Synthase Type I
;
Penis
;
Phosphorylation
;
Phosphotransferases
;
Prostatectomy
;
Rats
;
Rats, Sprague-Dawley
;
Salicylamides
;
Up-Regulation
9.Screening, cloning and identification of the human endometrial carcinoma-related genes.
Chinese Journal of Oncology 2007;29(8):584-588
OBJECTIVETo screen, clone and identify the cDNA fragments of human endometrial carcinoma-related genes,and explore the molecular mechanism of endometrial carcinogenesis.
METHODSPure endometrial glandular epithelial cells and endometrial carcinoma cells were obtained by laser capture microdissection (LCM). RNA from these cells was isolated, and differentially expressed gene fragments that were specialy relevant to endometrial carcingenesis were identified by using fluorescence differential display reverse transcription polymerase chain reaction (FDD-PCR). The selected fragments were cloned, sequenced and verified by reverse Northern blot analysis, and positive fragments were BLAST analysed and compared with those in Genbank.
RESULTS38 differential fragments were isolated, 3 of which were expressed more abundantly in normal endometrium and 35 were highly expressed in endometrial carcinoma. 10 fragments were recoverd, cloned and sequenced, confirmed by reverse Northern blot analysis, among which 6 fragments were positive. BLAST analysis showed that T1.1 was homologous to cyclin-dependent protein kinase 7 (CDK7, 99%); L1.9 was homologous to protein phosphatase 1 regulatory (inhibitor) subunit 12A (PPP1R12A, 99%); L1.21 and L1.22 were homologous to cellular repressor of E1A-stimulated genes 1 (CREG, 100%); L1.25 and L1.26 were homologous to solute carrier family 39 (zinc transporter) member 10 (SLC39A10, >98%).
CONCLUSIONGene fragments related to endometrial carcinoma have been obtained by applying LCM and FDD-PCR. To our knowledge it is the first time that the correlation between CDK7, PPP1R12A, CREG, SLC39A10 and endometrial carcinoma is discovered at mRNA level, and their role in molecular mechanism of cancinogenesis is discussed. CDK7, CREG, SLC39A10 as new candidate oncogene and PPP1R12A as new candidate anti-oncogene are worthy of being further investigated in the future.
Adenocarcinoma ; genetics ; metabolism ; pathology ; Cation Transport Proteins ; genetics ; metabolism ; Cyclin-Dependent Kinases ; genetics ; metabolism ; DNA, Complementary ; genetics ; Endometrial Neoplasms ; genetics ; metabolism ; pathology ; Female ; Gene Expression Profiling ; Genes, Tumor Suppressor ; Humans ; Myosin-Light-Chain Phosphatase ; genetics ; metabolism ; Oncogenes ; RNA, Messenger ; metabolism ; RNA, Neoplasm ; genetics ; Repressor Proteins ; genetics ; metabolism ; Tumor Cells, Cultured